000_pierwsza_strona Vol.33 Issue 4

Vol. 33

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No. 4

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2012

Chemical and Process Engineering Inżynieria Chemiczna i Procesowa

Polish Academy of Sciences Committee of Chemical and Process Engineering

The Journal is supported by the Ministry of Science and Higher Education

Editorial Board Andrzej Burghardt (Chairman), Polish Academy of Sciences, Gliwice, Poland Jerzy Bałdyga, Warsaw University of Technology, Poland Andrzej Górak, T.U. Dortmund, Germany Leon Gradoń, Warsaw University of Technology, Poland Andrzej Jarzębski, Silesian University of Technology, Poland Zdzisław Jaworski, West Pomeranian University of Technology, Szczecin, Poland Władysław Kamiński, Technical University of Łódź, Poland Stefan Kowalski, Poznań University of Technology, Poland Andrzej Krasławski, Lappeenranta University of Technology, Finland Stanisław Ledakowicz, Technical University of Łódź, Poland Eugeniusz Molga, Warsaw University of Technology, Poland Andrzej Noworyta, Wrocław University of Technology, Poland Ryszard Pohorecki, Warsaw University of Technology, Poland Andrzej Stankiewicz, Delft University of Technology, The Netherlands Czesław Strumiłło, Technical University of Łódź, Poland Stanisław Sieniutycz, Warsaw University of Technology, Poland Krzysztof Warmuziński, Polish Academy of Sciences, Gliwice, Poland Laurence R. Weatherley, University of Kansas Lawrence, United States Günter Wozny, T.U. Berlin, Germany Ireneusz Zbiciński, Technical University of Łódź, Poland

Editorial Office Andrzej K. Biń (Editor-in-Chief) Barbara Zakrzewska (Technical Editor) Marek Stelmaszczyk (Language Editor) Małgorzata Jaworska, DSc, PhD of the Faculty of Chemical & Process Engineering (Warsaw University of Technology) has been involved in preparation of the current issue of Chemical & Process Engineering Address ul. Waryńskiego 1 00-645 Warszawa www.versita.com/cpe Printed in Poland Print run: 200 copies Printing/binding: Polska Akademia Nauk Warszawska Drukarnia Naukowa ul. Śniadeckich 8, 00-656 Warszawa Tel./fax: + 48 22 628 87 77

PROF. KRZYSZTOF W. SZEWCZYK (1952-2011) Prof. Krzysztof Szewczyk was born in 1952 in Warsaw. In 1970 he entered Warsaw University of Technology (WUT) to study Chemical Engineering. He received the MSc (with honours) in 1975, on the basis of a thesis on the plate efficiency in the process of absorption accompanied by chemical reaction. Prof. Szewczyk started his scientific career in 1975 as an assistant in the Institute of Chemical Engineering of the Warsaw University of Technology. In 1981 he received his PhD at WUT with a thesis on the performance of a thermal diffusion column. His second degree (DSc, “habilitation”), obtained in 1987, was granted for another thesis, this time devoted to modelling of kinetics and dynamics of biomass growth in bioreactors. Since that time his scientific interests focused mainly on bioprocesses and bioreactors. During 1989-1990 he conducted research on aerosol emission from fermentation broth at the University of Cincinnati in the USA. In 2002 he became Deputy Director of the Interfaculty Center of Biotechnology of WUT, and in 2007-2008 he was Director of the Center. In 2006 he became Professor of Bioprocess Engineering of WUT, and Head of the Biotechnology and Bioprocess Engineering Unit of the Chemical and Process Faculty of WUT. He was a member of the Biotechnology Committee of the Polish Academy of Science (2004-2007), and Secretary and Vice President (2007-2010) of the Polish Federation of Biotechnology. He was also a member of the editorial council of the Polish journal “Biotechnology”. His scientific interests concerned absorption with chemical reaction, unconventional separation processes, aerosols (mainly bioaerosols generation), and – above all – bioprocess engineering (including modeling of microorganisms growth, kinetics of enzymatic reactions, cultivation of microorganisms on solid substrates, biological methods of wastewater purification, biogas production and biovoltaic cells). He was an author of about 120 articles and conference papers and a holder of 5 patents. He supervised 8 PhD and 70 MSc theses. He was decorated with the Silver Cross of Merit (1995) and the Medal of the Commission of National Education (2005). He was unquestionably an important person in Polish biotechnology. In 2012 the students of biotechnology from three universities (Warsaw University of Technology, The University of Warsaw, and Warsaw University of Life Sciences) inaugurated a session of all-Polish intercollegiate Prof. Szewczyk Biotechnology Symposiums “Symbioza”.

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On a personal note: my collaboration with Prof. Szewczyk dated back to 1975, when I supervised his MSc thesis. In 1987 he joined the Chemical Reactors and Bioprocesses unit of The Chemical and Process Faculty of WUT, of which I was the head. After my retirement he became the head of this unit (under a new name – the Biotechnology and Bioprocess Engineering unit). We also collaborated in the Interfaculty Center of Biotechnology, of which he was first the Deputy Director, and later the Director of the center. We also collaborated in the development of the teaching standards and programs for biotechnology courses. Prof. Szewczyk was always a loyal, reliable, and industrious collaborator. I admired his organisational skills. Whenever anything important should have been done, I could be sure that Prof. Szewczyk would come up with a good idea how to deal with the problem! I shall miss him very much.

Prof. Ryszard Pohorecki Faculty of Chemical and Process Engineering Warsaw University of Technology

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Chemical and Process Engineering 2012, 33 (4), 509-528 DOI: 10.2478/v10176-012-0042-x

LIFE WITH OXIDATIVE STRESS Dorota Gurda1, Anna M. Kietrys1, Aleksandra Szopa2, Tomasz Twardowski* 1 1

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego St.12/14, Poznań, Poland 2

Institute of Technical Biochemistry, Technical University of Łódź, B. Stefanowskiego St. 4/10, Łódź, Poland

Incomplete oxygen reduction gives rise to reactive oxygen species (ROS). For a long time they have been considered unwelcome companions of aerobic metabolism. Organisms using oxygen developed several systems of ROS scavenging with enzymatic and non enzymatic antioxidants, which allow them control the cellular level of oxygen derived from free radicals. It is well established nowadays that ROS are not necessarily negative byproducts, but they also play an important role in cellular mechanisms. They are involved in many regular cellular processes in all aerobic organisms. When the antioxidant system is overcome and the balance between ROS production and scavenging is disrupted, oxidative stress occurs. It has been reported that oxidative stress may be linked to some human diseases and is also involved in biotic and abiotic stress response in plants. Keywords: ROS, oxidative stress, antioxidants, oxidative stress response

1. INTRODUCTION My first meeting with Professor Krzysztof Szewczyk concerned (of course!) biotechnology but not the science. We talked about activities of Polish Federation of Biotechnology and… both of us concluded how much energy we needed for the Federation. Professor Krzysztof Szewczyk was the vice-president of the Federation and he invested a lot of energy and put a lot of his enthusiasm in the establishment of modern biotech in our country. Right then this conversation was very close to the problem of… ecological and sustainable resources of energy, bioenergy and sustainable development. Both of us (basic scientists with a training in chemistry) were enthusiastic about the potential and progress of modern biotechnology. Several different aspects of energy production: using bacteria, plants as well as waste, have been always in the centre of Professor Krzysztof Szewczyk’s interests. So, the basic research carried out with my scientific team dedicated to the oxidative stress in plant system was of interest to him, as well. It was a great privilege to meet and cooperate with Professor Krzysztof Szewczyk. His youthful enthusiasm concerning any form of intellectual activity and optimism were extremely helpful in many fields. I was saddened to learn of Professor Krzysztof Szewczyk passing away on October 20, 2011. I miss him very much.

*Corresponding author, e-mail: [email protected]

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D. Gurda, A. M. Kietrys, A. Szopa, T. Twardowski, Chem. Process Eng., 2012, 33 (4), 509-528

2. LIVING WITH OXYGEN

2.1. ROS chemistry and oxidative stress The environment of most organisms is rarely constant as internal or external stimuli continually disrupt homeostasis inducing stress. Organism responses to stresses are equally complex (Avery, 2011). About ~ 2.7 billion years ago molecular oxygen was introduced in the Earth’s atmosphere by the O2-evoluting photosynthetic organisms (Halliwell, 1981). Oxygen is now the most prevalent element in Earth’s environment (Magder, 2006). Reactive oxygen species (ROS) proved to be unwelcome companions of aerobic life (Gill and Tuteja, 2010; Halliwell, 2006). Ground state triplet molecular oxygen is a radical with its two outermost valence electrons occupying separate orbitals with parallel spins (Cadenas, 1989). The O2 molecule is a free radical, as it has two impaired electrons that have the same spin quantum number. The spin restriction makes O2 prefer to accept its electrons one at a time, leading to a generation of the so-called ROS. In an “oxidative stress” concept ROS appear as unavoidable toxic products of O2 metabolism and aerobic organisms have evolved mechanisms of antioxidant protection (Halliwell, 1981). Many years of studies have indicated reactive oxygen species as markers of oxidative stress and signalling events, which are responsible for induction of adaptive stress response (Mittler et al., 2004; Pucciariello et al., 2012). In eukaryotic organisms ROS are continuously produced as byproducts of many metabolic pathways localised in different cellular compartments (Apel and Hirt, 2004). Moreover, ROS are produced in response to certain environmental changes by activating various oxidases and peroxidases (Allan and Fluhr, 1997). Under physiological steady state conditions ROS are scavenged by components of antioxidative defence (Donahue et al., 1997). The equilibrium between production and scavenging of reactive oxygen species may be perturbed by environmental factors which results in a rapid increase of ROS level. This situation leads to oxidative stress (Feild et al., 1998; Sies, 1991). It is established that organelles (mitochondria, peroxisomes and chloroplasts) with a highly oxidising metabolic activity or with an intense rate of electron flow are the major source of ROS in cells. It has been estimated that 1-2% of O2 consumed by plants is sidetracked to produce ROS and 1-3% of O2 reduced in mitochondria is found in the form of O2•- (Temple et al., 2005; Turrens, 2003). A single electron reduction of O2 results in the generation of O2•- with approximately 2-4 µs of half-life. It has been established that in plants ROS appear during photosynthesis in chloroplasts by partial reduction of O2 or energy transfer to them. O2•- is produced upon reduction of O2 during electron transport along the non-cyclic pathway in the electron transport chain (ETC) of chloroplasts and other compartments, this process can occur at the level of photosystem I (PSI) (Halliwell, 2006; Puntarulo et al., 1988). At low pH conditions, dismutation of O2•- is unavoidable, with one O2•- giving up its added electron to another O2•-, and then with protonation resulting in the generation of H2O2. O2•- can be also protonated to form the HO2•. O2•- in the presence of metals such as Cu, Fe undergoes further reactions and gives up OH•. O2•- donates an electron to iron (Fe3+) to yield a reduced form of iron (Fe2+) which can then reduce H2O2, produced as a result of superoxide dismutase (SOD) led dismutation of O2•- to OH•. OH• is the most reactive form of ROS. The first step of a reaction through which O2•-, H2O2 and iron generate OH• is called the Haber-Weiss reaction. The final step reaction which involves the oxidation of Fe2+ by H2O2 is called the Fenton reaction (Halliwell, 2006; Scarpeci et al., 2008). H2O2 is restrainedly reactive and has a long half-life of 1 ms (Gill and Tuteja, 2010). In mammals O2•- comes from complex NADH dehydrogenase and complex II (ubiquinone-cytochrome c reductase). It was also showed that in stress conditions the active site of xanthine oxidase (XOD), which normally acts as a dehydrogenase, is oxidised and the enzyme acts as an oxidase and produces O2•-. O2•- can be also produced by cytochrome P450 enzymes as a side reaction when they break down target molecules (Magder, 2006). The 1O2 form occurs when an electron is elevated to a higher energy orbital, thereby freeing oxygen that forms its spin-restricted state. 1O2 can also be formed through photoexcitation of chlorophyll and its reaction with O2. Insufficient energy dissipation during photosynthesis can lead to the formation of chlorophyll

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Life with oxidative stress

triple state which can react with 3O2 to give up 1O2. The life time of 1O2 is approximately 3 µs and it is continuously produced by PSII. It has been noted that in plants some species constitutively produce different types of secondary metabolite with photosensitising properties that use 1O2 to increase efficacy as antimicrobial agents (Flors and Nonell, 2006; Gill and Tuteja, 2010).

Fig. 1. Scheme of generation of ROS in cell (Apel and Hirt, 2004)

A significant fact is that ROS are produced as part of normal metabolism (Fones and Preston, 2012). It is noted that ROS are important chemical mediators that regulate the transduction of signals by modulating protein via redox chemistry (Kovacic and Pozos, 2006). It has been proposed that ROS have been conserved throughout evolution as universal second messengers (Schulze-Osthoff et al., 1997). It is well known that ROS also play a crucial role in various pathophysiological processes such as cancer, neurodegenerative disorders, autoimmune disease and in plants in oxidative stress defence or oxidation and damaging of macromolecules (Apel and Hirt, 2004; Takahashi et al., 2011). ROS are strong oxidising agents and can cause lipid peroxidation, protein oxidation and damage of nucleic acids (Fones and Preston, 2012). The function of biological membranes is to constitute selectively impermeable barriers and partake in cellular transport processes. The presence of transition metals and ROS forms such as superoxide anion and hydroxyl radical initiates lipid peroxidation. A single initiation event has the potential to generate multiple peroxide molecules by chain reaction (Evans and Halliwell, 1999; Halliwell, 1999). The effects of lipid peroxidation reaction are decreased membrane fluidity, increase of the leakiness of the membrane to substances that do not normally cross it, damage of membrane proteins and inactivation of receptors, ion channels and enzymes (Moller et al., 2007). It was observed that ROS such as OH• and 1O2 take part in oxidative damage of nucleic acids. RNA damage has not received much attention, because damaged molecules do not accumulate in cell. However, cellular RNA is likely to be more prone to oxidative damage than DNA. RNA damage by ROS results in either the loss or alteration of RNA functions. It has been noted that oxidative lesions alter the structure of RNA or interfere with the interaction between RNA and other cellular molecules (Li et al., 2006). It was shown that ROS can initiate the formation of more than 20 types of oxidative DNA damage the most prevalent of which is the 8-oxoguanine (Avery, 2011). The hydroxyl radical is the most reactive form and causes damage to all components of the DNA molecule (Wiseman and Halliwell, 1996). ROS induce DNA damage which includes base deletion, pyrimidine dimers, crosslinks, strands breaks and bases modification, such as alkylation and oxidation. All these forms of oxidative damage result in various physiological effects, such as protein synthesis reduction, membrane destruction, whose affects disrupt the growth and development of organisms (Britt, 1999; Tuteja et al., 2001). Protein oxidation results in cellular dysfunction and has a negative impact on cellular metabolism (Cecarini et al., 2007). This process is defined as covalent modification of a protein induced by free radicals or byproducts of oxidative stress. Most types of protein oxidative modifications are irreversible, but a few involving sulfur-contaning amino acids are reversible (Ghezzi and Bonetto, 2003). The oxidation of a number of protein amino acids gives rise to free carbonyl groups which can alter or inhibit their activities and increase susceptibility towards proteolytic attack (Moller et al., 2007). An important aspect of ROS toxicity is the localisation in the cell of toxic reaction

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triggered by oxidative modification of a cellular target. Several potential targets may manifest toxic character during oxidative stress (Avery, 2011). ROS signalling is thought to be perturbed by different developmental or environmental signals which can up- or down-regulate their level. Moreover, ROS signalling and its level is controlled by two opposing processes of production and scavenging (Mittler et al., 2004). Scavenging cells from free radicals are formed following non-enzymatic or enzymatic reactions. Superoxide dismutase is the most effective enzymatic antioxidant in all aerobic organisms and all subcellular compartments. SOD removes O2•- by catalysing its dismutation, which can be reduced to H2O2 or oxidised to O2. The enzyme removes O2•- and decreases the risk of OH• formation (Mittler, 2002). Catalase (CAT) dismutates H2O2 into O2 and H2O. CAT is particularly important in the removal of H2O2 in peroxisomes (Gill and Tuteja, 2010). Ascorbate peroxidase (APX) plays a crucial role in scavenging free radicals of H2O2 in water-water and ASH-GSH cycle, since it utilises ASH as the electron donor (Badawi et al., 2004). Glutathione reductase (GR) is a flavoprotein oxidoreductase and it plays a role in defence against ROS by sustaining the reduced status of GSH. This enzyme has been localised in chloroplasts and in small amounts in mitochondria and cytosol (Romero-Puertas et al., 2006). Monodehydroascorbate reductase (MDHAR) is an enzyme present in chloroplasts and cytosol, it has high specificity for monodehydroascorbate (MDHA) as an electron acceptor, preferring NADH as an electron donor (Asada, 1999). Dehydroascorbate reductase (DHAR) is connected with ASH regeneration from oxidised state and regulation of the cellular ASH redox state (Gill and Tuteja, 2010). Glutathione S-transferase (GST) is an enzyme which catalyses the conjugation of electrophilic xenobiotic substrates with tripeptide glutathione (Roxas et al., 2000). Glutathione peroxidases (GPX) comprise a large group of isozymes. GPXs use GSH to reduce H2O2 and lipid and organic hydroperoxides in ROS scavenging process (Noctor et al., 2002). The antioxidant defence system also includes non-enzymatic antioxidants such as vitamins C and E, glutathione, Proline, flavonoids and carotenoids (Gill and Tuteja, 2010). Ascorbic acid – vitamin C is the most important water soluble antioxidant. Vitamin C has the ability to donate electrons in a number of reactions. It can scavenge O2•and OH• and regenerate α-tocopherol from tocopheroxyl radical (Smirnoff, 2000). α-tocopherol (vitamin E) is a lipid soluble antioxidant that takes part in the removal of some ROS such as 1O2 and lipid radicals (Kamal-Eldin and Appelqvist, 1996). Glutathione (GSH) is a scavenger of 1O2, H2O2 and OH and it is considered to be the most important intracellular antioxidant (Mullineaux and Rausch, 2005). Proline was proposed as an osmoprotectant, a protein stabiliser, a metal chelator, an inhibitor of lipid peroxidation and as 1O2, and OH• remover (Verbruggen and Hermans, 2008). Flavonoids scavenge ROS by locating and neutralising damage in the cell, and their antioxidative activity consists in the reduction of radicals. Carotenoids similarly react with peroxyl radicals to form a resonance-stabilised carbon-centred radical within its conjugated alkyl structure, thereby inhibiting chain propagation of reactive oxygen species (Fang et al., 2002). To sum up, the cell defence against ROS and oxidative stress depends on enzymatic and non-enzymatic antioxidants acting to decrease ROS concentrations, repair damaged molecules and eliminate irreparable proteins (Sigler et al., 1999). The oxidative stress caused by ROS is ubiquitous in all organisms. It is a subject of extensive research, particularly in humans and plants, as it is involved in some human diseases and plant stress responses. In this review we focus on those two fields of research.

2.2. Oxidative stress in humans Overproduction of reactive oxygen species evoking oxidative stress can damage the most important cellular molecules such as nucleic acids, proteins and lipids, resulting in serious diseases and dysfunction of living organisms.

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High concentration of ROS in blood vessel results in many pathological consequences. It is well known, that the inflammatory state mediated by ROS causes damage of endothelial and smooth muscle cells. Endothelial dysfunction is represented by disrupting anti-inflammatory, anticoagulant, and vasorelaxation properties as well as activation of monocytes, macrophages, growth factors which leads to the formation of atherosclerotic plaque. It is formed from low density lipoproteins oxidised by free radicals (oxLDL) (Seifried et al., 2007). After oxidation LDL particles are recognised by scavenger receptors on immune system cells (monocytes and macrophages), which subsequently bind to modified LDL. There are eight classes of such receptors, but only SR-A type I and II, CD 36 and LOX1 are able to uptake oxLDL (de Beer et al., 2003), evolving to lipid filled foam cells. LOX1 is thought to be the main receptor for oxLDL expressed on the surface of endothelial cells which are the most susceptible to negative changes during atheroma formation. Foam cells, unable to process oxidised cholesterol, enlarge until rupture resulting in a great amount of cholesterol deposition on the artery wall. As a result of deposition the artery wall becomes inflamed. Various inflammatory cytokines and growth factors released by macrophages cause smooth muscle cells and fibroblast proliferation and thrombus formation (Goyal et al., 2012; Kong and Lin, 2010). ROS-mediated oxidative stress also plays a crucial role in the progression of atherosclerosis. There are studies indicating that nucleic acid undergoes oxidation during the sclerotic plaque formation. Increased levels of oxidative damage in human sclerotic plaque were shown to result in higher amounts of 8-hydroxy-2-deoxyguanosine (8-OHdG), the most frequent oxidative change in mammalian DNA (Martinet et al., 2002). It was also reported that RNA is also prone to oxidative damage in smooth muscle cells and endothelial cells in human atherosclerotic lesions (Martinet et al., 2004, 2005). Brain tissue is extremely sensitive to oxidative degradation, because of its very large demand for oxygen, high polyunsaturated fatty acids and redox-active metals (Cu, Fe) presence. Oxidative stress increases with age and may lead to very serious oxidative injuries that may trigger many neurodegenerative disorders. One of the major neurodegenerative disorders is Alzheimer’s Disease (AD). Oxidative stress is considered to be one of the major factors related to AD pathogenesis as well as dysregulation of amyloid beta precursor protein (APP) functioning and amyloid beta peptide (Aβ) metabolism. AD leads to accumulation of Aβ and neurofibrially tangels. Aβ can be oxidised by metalcalatysed hydroxyl radicals and become more water-insoluble and resistant to protease (Atwood et al., 2004). It is worth mentioning that genes coding Aβ and enzyme beta amyloid precursor protein (BACE) expression is regulated through methylation of its promoter regions. On the basis of experiments on oxidant–transformed cell lines it is suggested that DNA methylation process and DNA oxidative damage interact together, which can alter methylation patterns resulting in transcriptional regulation of gene expression. This way oxidative and methylation changes in the promoter region of APP gene may regulate production of the protein encoded by this gene and its derivatives (Chan and Shea, 2006). The APP promoter sequence consists of 72% CpG islands. A sequence analysis of this revealed at least 13 possible methylation sites. There were 26% of methylated cytosines in the brain tissues from healthy individuals between 36 to 70 years compared to 8% of cytosine methylation in a group AD subjects aged 74 to 90. According to some authors this significant decrease of methylation level related to age may be significant in the Aβ aggregation in brain (Zawia et al., 2009). The second most common neurodegenerative disorder is Parkinson’s Disease (PD). Etiology of this illness is manifested in a loss of dopaminergic neurons in the substantia nigra and in the presence of neuronal inclusions of α-synuclein, called Lewy bodies. Oxidative stress is proposed to be one of the crucial pathogenic factors. Dopamine is a good metal chelator and donor of electrons and is able to react with iron and manganese. Many studies indicate that mutations in α-synuclein gene may result in facilitating dopamine reaction with iron increasing ROS production. Manganese is capable of causing oxidative DNA damage and reducing antioxidants such as GSH, catalase and thiols (Kong and Lin, 2010). Oxidative damage of nucleic acids is also considered to be a factor underlying pathophysiology of neurodegenerative diseases. Among many products of nucleoside oxidation only two, 8-hydroxy-2-deoxyguanosine (8OHdG) and 8-hydroxyguanosine (8OHG), are well characterised.

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In two most frequent neurodegenerative disorders, AD and PD, elevated levels of 8OHG and 8OHdG were found. Their strong abundance in cytoplasm and neurons, suggests that mitochondrial DNA and cytoplasmic RNA in neurons are most prone to oxidative stress (Alam et al., 1997; Nunomura et al., 1999). Similar oxidation of neuronal cytoplasmic RNA was observed in brain samples of patients with other disorders such as Creutzfeldt-Jacob Disease, Down’s Syndrome (Nunomura et al., 2009). Elevated RNA oxidation was also observed in substantia nigra in patients with PD, indicating that those changes may occur in the early stage of this illness (Kong and Lin, 2010). It was demonstrated that up to 50% of mRNA isolated from brains of patients with mild or moderate stage of AD is oxidatively damaged (Shan and Lin, 2006). An analysis of oxidative changes in mRNA revealed that those changes are not random. Some types of polyA (+) mRNA are more vulnerable to oxidative damage than others. For example, mRNA for copper/zinc superoxide dismutase (SOD1) and dynactin 1 is highly oxidised in amyotrophic lateral sclerosis, resulting in a decreased level of proteins encoded by them (Chang et al., 2008). A similar observation was made in post mortem analysis of brains from patients with AD. Some of the mRNA with elevated oxidation encoded for example p21ras or mitogen-activated protein kinase (MAPK), SOD1, but not for amyloid β precursor or tau protein (Shan et al., 2003). Impaired redox state is a feature of many cancer cells. The oxidative DNA damage has been implicated in the etiology of cancer. There are over 100 different products of oxidative nucleic acid damage. These are single or double stranded DNA breaks, purine and thymidine or deoxyribose modifications and DNA cross-links. The oxidative stress plays a role in any stage of carcinogenesis and apoptosis (Valko et al., 2007). For example, oxidation of guanosine resulting in the creation of highly mutagenic particle, 8OHdG, which leads to G:C to T:A transversion, a characteristic feature of oncogenes and tumour suppressor genes (Seifried et al., 2007). There are many observations revealing that oxidative damage of mitochondrial DNA is also involved in carcinogenesis. Mutations and altered expression in mitochondrial genes encoding complexes I, III, IV and V in hipervariable regions have been connected to many human cancers. ROS are implicated in the activation of the nuclear genes involved in mitochondrial biogenesis, transcription and replication of mitochondrial genome (Valko et al 2006). It was also found that fragments of mitochondrial DNA were inserted into nuclear DNA, suggesting its role in the activation of carcinogenesis. It has been also established that lipid peroxidation is also linked to carcinogenesis. Lipoperoxyl radical (ROO•) is prone to cyclisation reaction resulting in the formation of malondialdehyde (MDA). It was observed that MDA is mutagenic in mammalian cells, bacterial, and rats. It can react with guanosine, adenosine and cytosine in DNA creating adducts M1G, M1A, and M1C. It was found that M1G is mutagenic in Escherichia coli, and may cause transversion to T or transitions to A (Valko et al., 2007; Valko et al., 2006). ROS may impair cellular defence mechanisms protecting from carcinogenesis. There are two main mechanisms of carcinogenesis induction. In the first mechanism increased DNA synthesis and mitosis by nongenotoxic carcinogens may lead to mutation in dividing cells through misrepair. In the other mechanism cell divisions mutations may expand leading to serious changes in DNA sequence. The second theory assumes balance between cell death and proliferation. When DNA damage level is too high, cellular mechanisms are activated to specifically direct the cell to apoptosis pathway. During proliferation, the main protein controlling the integrity of cellular DNA is p53 protein. It recognises mutations and initiates repairing mechanisms. If there are too many mutations, the cell is directed to apoptosis. Any change in this specific process may result in carcinogenesis. p53 functions as a transcription factor activating or repressing expression of targeted genes involved in a cell cycle. p53 is very redox sensitive because of its structure. It consists of many cysteine residues that contain redox sensitive thiol groups. Oxidative modifications of this group result in disulfide bonds formation, that induces structural changes, which alter its affinity to sequence specific binding to targeted genes. This suppressor gene is the most mutated gene in human cancers. It was reported, that mutations in p53 may be caused by ROS or by carcinogenic metals. Some reports indicate that mutations in p53 by the

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exposition to NO• and its derivatives are connected to stomach, brain and breast cancer (Valko et al., 2007; Valko et al., 2006; Vurusaner et al., 2012). It is also known that oxidative stress plays a role in the development of hepatocellular carcinoma (HCC). NO may be involved in protection or in development of tumour depending on the environment. It was suggested that lower concentrations of NO (30 – 100 nM) promote antiapoptotic and proliferative AKT and MAPK dependent pathway in cancer cells. In contrast, higher concentrations of NO (over 300 nM) promote apoptosis and antitumour activity. High ROS concentrations lower NO level resulting in a reduction of cell protection mechanisms against carcinogenesis (Marra et al., 2011). The harmful effect of reactive oxygen species is diminished by enzymatic and non-enzymatic antioxidants. The most important enzymatic antioxidants are superoxide dismutase, catalase and glutathione peroxidase. Humans have three different types of superoxide dismutases: cytosolic Cu-, Zn-SOD, mitochondrial Mn-SOD, and extracellular SOD (EC-SOD). Mn-SOD is one of the most efficient antioxidants with anti-tumour activity. Several observations indicated that Mn-SOD elevated level causes cancer retardation. A significant overexpression of Mn-SOD was found in gastrointestinal cancers. EC-SOD acts in a way coordinated by cytokines rather than in an individual way (Fang et al., 2002; Valko et al., 2006). A diminished activity of catalase was observed in the development of a variety of tumours. Glutathione is one of the most essential compounds of antioxidative protection. There are four types of Se-dependent glutathione peroxidases (GPx). They defeat H2O2 or organic peroxide ROOH. GPx also competes with catalase for H2O2 in a low oxidative stress conditions (Valko et al., 2006). The non-enzymatic defence against oxidative stress involves vitamins (such as C, E, and D3), carotenoids, and natural flavonoids. Many vitamins may inhibit production of NO by nitric oxide synthetases, supporting their anti-inflammatory and antiatherogenic properties. They can also directly scavenge ROS and upregulate activity of antioxidant enzymes (Fang et al., 2002). Vitamin C interacts in aqueous environment in human body with radicals and create tricarbonyl ascorbate free radical (AscH•) which is a poorly reactive molecule. It has been also noticed that a high concentration of ascorbic acid exerts a positive effect in reducing the risk of stomach cancer, and plays a protective role in lung and colorectal cancer (Knekt et al., 1991). Vitamin E is fat soluble and exists in several forms and has the greatest antioxidant activity. A reduction of the incidence of colorectal cancer by diet supplementation with vitamin E was observed. It is also suggested that α-tocopherol reacts together with ascorbic acid while preventing from oxidative stress. During antioxidant reaction α-tocopherol is converted to an α-tocopherol radical by donation of a labile hydrogen to lipid or lipid peroxyl radical. The α-tocopherol radical is subsequently reduced to α-tocopherol by ascorbic acid (Kojo, 2004; White et al., 1997). The most widely studied carotenoids connected with human disease preventing potential are β carotene, lycopene crocetin, luthein and zeaxithin. Carotenoids may exert their effects by changing redox status through redox-sensitive cell signalling pathway. At a sufficiently high concentration they can also protect lipids from peroxidation. But carotenoids provide to be effective antioxidants only in low oxidative stress. There are also some studies that indicate their pro-oxidant abilities under the influence of high oxidation conditions (Kennedy and Liebler, 1992; Rice-Evans et al., 1997). Flavonoids are belived to have a great antioxidative potential. They can prevent lipids from peroxidation by donation of hydrogen atom to radicals ROO• resulting in the formation phenoxy radical intermediate. This intermediate is relatively stable so it does not propagate further radical reactions. Flavonoids are able to terminate chain reaction via interactions with other free radicals. They are also very effective peroxyl radicals scavengers. Diet supplementation with flavonoids such as quercetin and polyphenols is connected to lower rates of stomach, pancreatic, and lung cancer (Damianaki et al., 2000; Galati and O'Brien, 2004).

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2.3. Oxidative stress in plants Plants are aerobic organisms and as such they use oxygen. An incomplete reduction of molecular oxygen leads to the emergence of highly reactive intermediates, reactive oxygen species, which are unavoidable byproducts of aerobic metabolism (Asada, 1987). The major sites of ROS production in plant cells are chloroplasts, mitochondria, peroxisomes and apoplasts (Tab. 1). However, these are not the only sources of ROS – they can also be generated in cell walls and plasma membranes (Gill and Tuteja, 2010). Table 1. Sites of ROS production in plants, partially adopted from Mittler 2002

Site

Mechanism

Primary ROI (reactive oxygen intermediates)

References

Photosynthesis ET and PSI or II

O2•-

(Asada, 1987) (Asada, 1999)

Chloroplast Excited chlorophyll

1

O2

(Asada, 1987)

Respiration ET

O2•-

(Dat et al., 2000a) (Maxwell et al., 1999)

Glycolate oxidase

H2O2

(Corpas et al., 2001)

Fatty acid β-oxidation

H2O2


Xanthine oxidase

O2•-


Oxalate oxidase

H2O2

(Dat et al., 2000a)

Amine oxidase

H2O2

(Allan and Fluhr, 1997)

Plasma membrane

NADPH oxidase

O2•-

Cell wall

Peroxidases, Mn2+ and NADH

H2O2, O2•-

Mitochondria

Peroxisome

Apoplast (Grant and Loake, 2000; Hammond-Kosack and Jones, 1996) (Grant and Loake, 2000; Hammond-Kosack and Jones, 1996)

As mentioned before, chloroplasts are major sites of ROS production. They are generated by the reaction centres of PSI and PSII in chloroplasts thylakoids (Asada, 2006). It was discovered that oxygen is photoreduced to hydrogen peroxide in PSI (Mehler, 1951). In a regular way, electron flow from excited photosystem centres is directed to NADP+, which is reduced to NADPH, subsequently entering the Calvin cycle and reducing CO2 (final electron acceptor). When the ETC is overloaded, a part of the electron flow is diverted from ferredoxin to O2, reducing it to O2•- via Mehler reaction (Takahashi and Asada, 1988). Recent research has linked chloroplast-produced ROS with hypersensitive response (HR) (Mur et al., 2008). Chloroplast-produced ROS have been shown to be capable of transmitting the spread of woundinduced PCD through maize tissues (Gray et al., 2002). Plant mitochondria have specific electron transfer chain components and functions in processes such as photorespiration (Douce, 1989; Fernie et al., 2004; Hoefnagel, 1998; Moller, 2001; Noctor et al., 2007; Raghavendra and Padmasree, 2003; Rasmusson et al., 2004). ROS production in mitochondria takes place under normal respiratory conditions but can be enhanced in response to various biotic and abiotic

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stress conditions. Complex I and III of mitochondrial ETC are well know sites of O2•- production, which can be subsequently reduced to H2O2 through SOD dismutation (Moller, 2001; Quan et al., 2008). This hydrogen peroxide can react with reduced Fe2+ and Cu+ to produce highly toxic OH•, which can penetrate membranes and leave the mitochondrion (Greene, 2002; Rhoads et al., 2006; Sweetlove, 2004). Mitochondria may play a central role in cell adaptation to abiotic stress, which is known to induce oxidative stress at the cellular level. It has been found that the energy-dissipating systems of durum wheat mitochondria diminish ROS production in those organelles. It was reported that an early generation of ROS can affect plant mitochondria by impairing metabolite transport, thus preventing further substrate oxidation, membrane potential generation and consequent large-scale ROS production (Pastore et al., 2002). The main metabolic processes responsible for the generation of H2O2 in different types of peroxisomes are photorespiration glycolate oxidase reaction, fatty acid β-oxidation, the enzymatic reaction of flavin oxidases and the disproportionation of superoxide radicals (del Rio et al., 1992; Huang et al., 1983). Xanthine oxidase is responsible for the generation of superoxide radicals in the matrix of leaf peroxisomes (Sandalio et al., 1988). SOD scavenges O2•- radicals and converts them into O2 and H2O2, which can be removed by catalase. Under certain stress conditions the activity of peroxisomal catalase can be strongly depressed (del Rio et al., 1996). There is an NADP(H)-dependent O2•- production site in the peroxisomal membrane, which is apparently formed by a small electron transport chain using O2 as an electron acceptor with the cytosolic production of O2•- radicals (del Rio et al., 1998; Lopez-Huertas et al., 1999). Other important ROS sources in plants are detoxification reactions catalysed by cytochrome P450 in cytoplasm and endoplasmic reticulum (McDowell and Dangl, 2000). ROS are also generated at the plasma membrane level (Bolwell, 1999; Bolwell and Wojtaszek, 1997) or extracellularly in apoplast in plants. pH-dependent cell wall-peroxidases, germin-like oxalate oxidases and amine oxidases have been proposed as H2O2 producers in apoplast of plant cells (Bolwell and Wojtaszek, 1997). Redox signals are involved in all aspects of plant biology. They are key regulators of plant metabolism, morphology and development (Foyer and Noctor, 2003). Redox signals are particularly important in defence responses and cross-tolerance phenomena. One of the earliest events in the hypersensitive response, which is responsible for the activation and establishment of plant immunity to disease (Foyer and Noctor, 2003), is the rapid accumulation of ROS through the activation of enzyme systems (Keller et al., 1998). The oxidative burst is a central component of an integrated HR signalling system, whose function is rapid amplification of the signal (Foyer and Noctor, 2003). The absolute rate of H2O2 production and the rate of scavenging by detoxifying systems are likely to be the main determinants of H2O2 concentration. A possible key determinant of signalling intensity is H2O2 ability to react with signalling compounds. H2O2 mediates intra- and extra-cellular communication during plant reactions to pathogens and several studies have suggested its role in systemic acquired resistance. Antioxidants and/or antioxidative enzymes are likely to take part in the early step of redox signalling (Foyer and Noctor, 2003). Glutathione and ascorbate are also suggested to be involved in this process in plants (Baier et al., 2000; Horling, 2003; Noctor et al., 2000). Specific compartment-based signalling and regulation of gene expression can be achieved via differential compartment-based changes in either the absolute concentrations of ascorbate and glutathione or ascorbate/dehydroascorbate and GSH/GSSG ratios, which are very high and stable in normal state (Noctor et al., 2000). It is suggested that redox signalling is a key function of the cytoplasmic genome found in chloroplasts and mitochondria, along with the exchange of redox information between these organelles and the nucleus (Foyer and Noctor, 2003). An interesting thing is that genes most strongly induced by stress

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conditions are not necessarily those coding antioxidant defence system compounds. Induced genes coding the enzymatic antioxidants often tend to encode cytosolic rather than chloroplastic antioxidative proteins, even if the stress is predicted to be located in chloroplasts (Foyer and Noctor, 2003). Peroxisomes are known to release signals that regulate nuclear gene expression. Signals arising from peroxisomes regulate photomorphogenesis, plant development peroxisomal biogenesis, light signalling and stress responses (Hu et al., 2002). While growing, plants are often exposed to adverse environmental conditions, namely biotic and abiotic stresses. Many of them give rise to the overproduction of reactive oxygen species (ROS). Many stress-related symptoms are exacerbated, if not directly dependent on ROS (Dat et al., 2000a). High-light exposure is one of the most common sources of oxidative stress in plants (Foyer et al., 1994). Expression analysis of antioxidants has provided evidence for accumulation of ROS during high-light exposure. Strong induction of chloroplastic Fe-SOD transcripts has been observed in tobacco plants exposed to high light (Tsang et al., 1991). Fe-SOD mRNA increase was not light dependent but directly responsive to oxidative stress. Activities of most antioxidant enzymes increased in pea seedling after transfer from low to high light (Mishra et al., 1995). A degradation of glutamine synthetase, phosphoglycolate phosphatise, a large subunit of ribulose-1,5-biphosphate carboxylase/oxygenase, and an increased content of carbonyl groups in stromal proteins of pea during high-light treatment is an additional evidence for an involvement of ROS, as all these processes result from oxidative damage (Stieger and Feller, 1997). Drought-induced inhibition of photosynthesis leads to increased ROS production in chloroplasts (Leprince, 1994; Smirnoff, 1993, 1998). The accumulation of ROS during such conditions originates mainly from a decline in CO2 fixation, leading to higher leakage of electrons to O2. Increased thylakoid membrane electron leakage to O2 has been observed in sunflower (Sgherri et al., 1996) and in wheat (Biehler and Fock, 1996) after water deficiency conditions. ROS-dependent changes associated with water deficit (Aziz and Larher, 1998; Gogorcena et al., 1995; Iturbe-Ormaetxe et al., 1998; Moran et al., 1994) as well as levels of antioxidants (Gogorcena et al., 1995; Olsson, 1995; Schwanz et al., 1996) have been reported. Activities of cytosolic and chloroplastic Cu/Zn-SOD and cytosolic APX rise during drought of pea plants (Mittler and Zilinskas, 1992, 1994), and osmotic stress enhance Mn-SOD transcript abundance in maize (Zhu and Scandalios, 1994). Plant response to salt stress often resembles that of the drought-mediated one. The generation of ROS during salt stress is probably similar to that during drought and is mainly attributed to increased leakage of electrons to O2 (Dat et al., 2000a). An increase of H2O2 concentrations in rice shoot tissue has been reported upon salt stress (Fadzilla et al., 1997). The amounts of mitochondrial and chloroplastic SOD and APX isozymes increase during salt stress in pea (Hernandez et al., 1993; Hernandez et al., 1995). Low temperature is one of many studied abiotic types of stress in plants Direct evidence for ROS accumulation during chilling was reported for several plant species. Chloroplast is a major source of ROS during low temperature treatment through the nhibition of CO2 fixation. Mitochondrial ROS accumulation in the same conditions has also been demonstrated (De Santis et al., 1999; GonzalezMeler et al., 1999; Prasad et al., 1995). A transient but significant increase of H2O2 level induced by cold treatment of winter wheat has been showed (Okuda et al., 1991). H2O2 accumulation in chilled cucumber plants (Omran, 1980) and maize seedlings was observed (Prasad et al., 1994). Chilling of callus tissue of Arabidopsis thaliana also resulted in oxidative stress (O'Kane et al., 1996). High-temperature exposure involves ROS accumulation as well. It was shown that an oxidative burst occurred in potato leaf tissues after heat shock. A significant increase in H2O2 level was induced after heat treatment in whole tobacco seedlings (Dat et al., 2000b; Foyer et al., 1997). A similar observation of H2O2 accumulation after heat treatment was made for mustard seedlings (Dat et al., 1998). Changes in antioxidants during high-temperature treatment have also been reported. For example, SOD level

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increases in heat-treated tobacco (Tsang et al., 1991). High activities of APX and GR improve the heat protection of wheat (Kraus and Fletcher, 1994). Heavy metals, like copper, aluminium, cadmium, zinc and iron, may cause oxidative stress. An excessive amount of copper causes ROS formation and lipid peroxidation (De Vos et al., 1992; Halliwell and Gutteridge, 1989; Sandmann and Boger, 1980; Weckx and Clijsters, 1996). ROS accumulation during Cu stress is suggested by differential activation of SOD in tobacco (Kurepa et al., 1997) and soybean (Chongpraditnun et al., 1992) after Cu treatment. Aluminium also causes ROSmediated lipid peroxidation (Kochian, 1995). Aluminium induces glutathione S-transferase (GST), peroxidase, β-1,3-glucanase, PR2, and phenylalanine ammonia lyase in Arabidopsis thaliana (Richards et al., 1998), and several antioxidant gene transcripts in soybean (Cakmak and Horst, 1991). Zinc treatment caused increased levels of H2O2 in roots and lipid peroxidation in primary leaves of Phaseolus vulgaris (Weckx and Clijsters, 1997). Iron excess can also result in oxidative stress (Halliwell and Gutteridge, 1989). It is due to its potential for reacting with H2O2 and O2•- to generate OH•. An increased activity of catalase and APX was observed in tobacco seedlings after iron excess (Kampfenkel et al., 1995). Iron-induced oxidative stress in maize was proposed as a possible trigger for ferritin mRNA and protein accumulation (Lobreaux et al., 1995). Chemical compounds used in plant cultivation are linked to oxidative stress. It has been reported that maize leaves exposed to glyphosate had an increased level of lipid peroxidation, glutathione (GSH), free Proline content, and ion flux, suggesting, that besides the inhibition of its specific target, which is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (Ahsan et al., 2008; Schonbrunn et al., 2001), this treatment leads to oxidative stress in plants (Sergiev et al., 2006). Upregulation of some antioxidant enzymes was identified, including glutathione S-transferase, ascorbate peroxidase, thioredoxin h-type, nucleoside diphosphate kinase 1 (NDPK1), peroxiredoxin, or their precursor such as superoxide dismutase chloroplast precursor after paraquat treatment (Ahsan et al., 2008). A significant upregulation for those enzymes after glyphosate treatment was observed, which supports the idea that plants also utilised antioxidant defence mechanisms to protect against glyphosate stress. Authors also noted different expression for two different isozymes of GST. Two APX isoforms and a Cu/Zn-SOD chloroplast precursor were significantly upregulated by treatment with both glyphosate and paraquat, as well as thioredoxin h and peroxiredoxin (Ahsan et al., 2008). It was suggested that exposure to glyphosate causes oxidative stress resulting in lipid peroxidation and subsequent membrane damage (Ahsan et al., 2008). It was suggested that glyphosate promotes harmful effects on non-target species of Chlorella kessleri through oxidative stress. A significant increase in the amount of malondialdehyde (MDA) was observed (Romero et al., 2011), indicating damage of lipid membranes (Janero, 1990). The level of GSH was also increased, which suggests GSH involvement in the antioxidant defence upon glyphosate exposure in Chlorella kessleri (Romero et al., 2011). The increase of GSH amount in plants treated with glyphosate was reported earlier (Jain and Bhalla-Sarin, 2001; Uotila et al., 1995). UV radiation induces the production of O2•-, which can be subsequently dismutated by SOD to H2O2 (Murphy and Huerta, 1990). In Nicotiana plubaginofolia exposure to UV-B caused a significant increase in Cat2 and GPX mRNA, while APX and SOD transcript levels remain relatively unaltered (Willekens et al., 1994). In Pisum sativum SOD transcript levels decrease after UV-B exposure, but those of GPX are higher (Strid, 1993). Wound-induced ROS accumulation in various species is inhibited by diphenyleneiodonium, an NADPH-oxidase inhibitor, which suggests it is NADP(H) dependent (Orozco-Cardenas and Ryan, 1999). Wounding stimulates H2O2 generation systemically in tomato leaves (Orozco-Cardenas and Ryan, 1999). ROS accumulation resulting from mechanical stress was reported for various species (Schopfer, 1994; Watanabe and Sakai, 1998).

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The active defence of plants against invading pathogens often includes rapid and localised cell death, known as hypersensitive response (Liu et al., 2007). ROS production, altered ion fluxes, protein phosphorylation and dephosphorylation, and gene activation have been implicated in the HR process (Liu et al., 2007). An involvement of protein kinases in HR cell death induced by both avirulent pathogens and pathogen-associated molecular patterns (PAMPs) was suggested (Liu et al., 2007). ROS generation after MAPK activation connection to induction of HR cell death was suggested (Ren et al., 2002). It was demonstrated that the generation of ROS after MAPK activation is preceded by a disruption of metabolic activities in chloroplasts and mitochondria. Scientists found that pathogenresponsive NtMEK2-SIPK/Ntf4/WIPK cascade plays an active role in promoting ROS generation in chloroplasts by inhibiting the carbon fixation (Liu et al., 2007). An alteration in the expression or activity of ROS-scavenging enzymes has been indicated as a key step in the activation of phytopathogen defence (Da Gara et al., 2003). ROS can act directly against phytopathogen attack by killing microorganisms. H2O2 contributes to wall stiffening by facilitating peroxidase reactions catalysing intra- and intermolecular cross-links between structural components of cell walls and lignin polymerisation (Ros Barcelo, 1997), thus hindering and slowing down pathogen penetration in plant tissue, which allows plant cells to arrange defence that requires more time to be activated. H2O2 is diffusible in biological membranes, hence it also acts as intracellular signal, which is able to activate defence responses (Durner et al., 1997). It seems that, in contradiction to most abiotic stress response based on ROS-scavenging system enhancement, pathogen-attack response is dependent rather on ROSscavenging system suppression, which allows plant cells to prevent from pathogen penetration using an extremely oxidative local environment (Da Gara et al., 2003).

3. CONCLUSIONS For many years ROS have been considered as inevitable byproducts of the metabolism of aerobic organisms. However, recently it has become obvious that they are very important and necessary for many cellular processes. ROS participate in regular processes, including growth, development and signalling. When the equilibrium between ROS production and scavenging is disturbed, oxidative stress occurs. It is involved in the aetiology of many human diseases and different stress response patterns in plants. To date, there is extensive knowledge about ROS and oxidative stress in cells, yet much more needs to be elucidated. Hopefully, future research will give us a better understanding of all the mechanisms and components involved in ROS production, usage and function.

This work was supported by grants from Polish Ministry of Science and Higher Education No NN310043738, NN310769840 and NN401579840

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Chemical and Process Engineering 2012, 33 (4), 529-538 DOI: 10.2478/v10176-012-0043-9

ENCAPSULATION OF CHONDROCYTES IN HYDROGEL SYSTEMS EFFECT OF CHITOSAN VISCOSITY AND MICROCAPSULE SHAPE

Iga Wasiak*, Tomasz Ciach Warsaw University of Technology, Faculty of Chemical and Process Engineering, Waryńskiego 1, 00-645 Warszawa, Poland

Alginate – chitosan – alginate multilayer hydrogel encapsulation systems were investigated for encapsulation of chondrocytes. Hydrogel is crosslinked due to ionic interaction between cationic chitosan and anionic alginate, and additionally by calcium ions. Two types of chitosan with molecular weight were investigated. Cells were encapsulated in two shape microcapsules, microbeads with diameter size 300 – 400 and 500 - 600 µm and fibres with diameter 500 - 600 µm. The work provides a detailed examination of the impact of the microencapsulation process on the growth of cells. The viability of chondrocytes can be influenced by the size of produced microcapsules, while the shape of microcapsules has no important significance on cell viability. The applied encapsulation methods do not contain harmful stages and create conducive conditions for cell growth. A possible application area of the developed system is dressing and regeneration of damaged joint cartilage. Keywords: microencapsulation; ACA microcapsule; chondrocytes

1. INTRODUCTION Due to the ageing of societies damage to the articular cartilage caused by the wear, overload, misalignment and accidents became a common and also difficult therapeutic problem. Unfortunately, natural cartilage, probably due to a very low amount of blood capillaries, practically does not heal. Despite the development of many techniques, a selection of the appropriate and effective method for damage treatment is still not a trivial matter. Traditional methods of treatment include the loss of fullthickness debridement of adamaged joint, the stimulation of bone marrow cells through abrasion, drilling and microfracture, the transplantation of bone chondromyxoid pulp and chondromyxoid transplantation - bone in the plasticity of a mosaic. The application of these techniques, results in cartilage substitution of a damaged place by the fibrous cartilage which is not the desired type of cartilage. The resulting improvement is usually short-lived. That is why microencapsulated autologus chondrocytes or differentiated stem cells implantation could be an effective clinical procedure for cartilage repair. To perform an effective local tissue regeneration cells should be kept in place and a proper environment for cells should be also assured. To achieve that microencapsulation of cells in a the proper biomaterial is applied. This creates conditions for the reconstruction of the more valuable, specialised tissue, and also reduces the risk of complications. Three-dimensional scaffold of a suitable shape directs the development of tissue and allows for convenient introduction and adhesion of cells to patients’ joints. Proper encapsulation protects cells from immunological reactions and mechanical stress, providing diffusion of oxygen, nutrients, and metabolic products (Gombotza and Wee, 1999). There are several challenges in the development of encapsulating membranes that have to be solved. *Corresponding author, e-mail: [email protected]

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I. Wasiak, T. Ciach, Chem. Process Eng., 2012, 33 (4), 529-538

The most important problem is reaching an optimum balance between the mechanical strength of the microcapsule and mass transport properties of a membrane. In addition, most of the developed membranes lack appropriate vascularisation, an obstacle to ensuring continuous nutrients and oxygen diffusion to enclosed cells (Fig. 1). The encapsulating material must allow for cell survival and differentiation while maintaining its physio-mechanical properties throughout the required implantation period (Nafea et al., 2011).

Fig. 1. (a) Idea of a semi-permeable membrane for immunoisolation of encapsulated cells; (b) representative chain portion of alginate. Alginates are linear copolymers consisting of blocks of continuous mannuronate residues, guluronate residues or alternating guluronate/mannuronate residues; (c) representative chemical structure of chitosan. Chitosan is produced commercially by deacetylation of chitin

In the conventional methods microcapsules are prepared by dispersing sodium alginate solution into water solution of cross-linking divalent (Ca2+, Ba2+, Sr2+) or trivalent cations (Al3+, Fe3+) to form a jelly, and then coated by a cationic polyelectrolyte (Gombotza and Wee, 1999). The coating slows down the swelling and degradation of microcapsules, but it may also cause immunological reactions and fibrotic growth (De and Robinson 2003; Gaserod et al., 1999a; 1999b). To reduce these undesirable effects microcapsules are coated one more time by alginate which is more biocompatible. Alginate is a copolymer of L-guluronic acid. It is bioadhesive, biocompatible and provides an inert aqueous environment within the matrix; it polymerises in a mild room temperature, encapsulation process is free of organic solvents, it has a high gel porosity which allows macromolecules diffusion. Moreover, it has the ability to control this porosity with simple coating procedures, as well as process of dissolution and biodegradation of athe system under normal physiological conditions (Puppi et al., 2010). The main advantage is the fact that chondrocytes do not dedifferentiate in alginate and produce high levels of GAG (glicosylaminoglycans) and collagen type II (Chia et al., 2005; Domm et al., 2002; Sittinger et al., 1997; Wang et al 2003). After the first study of Lim and Sun (Lim and Sun, 1980) alginate – polylysine- alginate (APA) microcapsules became the most widely used method for cell entrapment. Still, due to the high cost of poly-lysine and its unsuitability for long-term transplantation different polycation methods are worth investigating. The alternative is provided by alginate - chitosan- alginate microcapsules. Chitosan is less immunological, more stable in vivo and has better properties for cryopreservation than lysine (Haque et al., 2005). Chitosan (-1,4-linked N-glucosamine) is a biodegradable cationic aminopolysaccharide derived from naturally occurring acetylated chitin. Chitosan is a homopolymer which has a hydrophilic surface promoting cell adhesion, proliferation and differentiation. Moreover, it is endowed with antibacterial activity and good biocompatibility with acceptable host response (Puppi et al., 2010)].

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Encapsulation of chondrocytes in hydrogel systems effect of chitosan viscosity and microcapsule shape

Here, alginate - chitosan - alagiante microcapsules were studied for their potential use as a scaffolding material for cartilage repair and regeneration. Two types of microcapsules were obtained: micro-beads and micro-fiber. The aim of the presented study was to investigate the cell number and the viability of chicken articular chondrocytes, which were chosen as a model, when cultured in alginate - chitosan alginate microcapsules. Obtained results were compared with non - encapsulated chondrocytes culture. Because of slow proliferation of chondrocytes a long culture period - 30 days, was chosen. In the paper influence of geometrical diffusing properties of different shape of microcapsules on chondrocytes growth were considered. Micro-beads have a bigger surface interface contact than fibre, which inducted a better diffusing ratio of nutrients and oxygen through microcapsule membrane. Also the influence of chitosan molecular weight on the cultured chondrocytes was evaluated.

2. MATERIALS AND METHODS 2.1. Reagents Dulbecco’s modified Eagle’s medium (DMEM), Nutrient mixture F-12 (Ham F-12), chitosan, collagenase type I, fetal chicken serum (FCS), penicillin G, streptomycin, tryptinase, trypan blue were obtained from Sigma – Aldrich. Two types of chitosan with medium viscosity 200 - 800 cP ( 1 wt. % in 1% acetic acid, 25°C, Brookfield) (M) and hight viscosity >1000 cPa (H) were evaluated. Alginate made from brown seaweeds reach in guluronic residues was purchased from Fluka, while Ascorbate acid from Carlo ERBA Reagents. Chemicals were have been used as received, without further purification. 2.2. Chondrocytes isolation Cartilage was harvested from chicken joints, then it was cut into small pieces and subjected to an enzymatic digestion (tryptinase, collagenase) in Ham F-12 followed by digestion in DMEM (supplemented with 50µm/ml gentamicin and 100µm/ml streptomycin) with 0.06% collagenase over night in 37oC, in a humid atmosphere of 5% CO2 incubator. After digestion the solution was filtered (filter porosity 70 µm), washed and centrifuged in order to isolate chondrocytes. Cells were resuspended in 20ml of distilled and aseptic water, counted and their viability was quantified by 0.4% trypan blue in distilled water. 2.3. Microencapsulation and culture conditions Preparation of ACA beads was performed in aseptic conditions. The cell suspension was adjusted to 5.0x106 chondrocytes/ml of sterile 1.5 % alginate solution in 0.9% sodium chloride. Beads were formed by extrusion of the solution in the air 4 cm above the 0.1 M calcium chloride solution. High voltage generator connected to the needle was applied to facilitate liquid jet breakup into small droplets, which is the process of electrostatic micro dripping (Ciach, 2007). Different diameters of microbeads were received by using two potential values (4.5 kV and 6.1 kV), at a constant flow rate (25.5 ml/h) and needle size (27-gauge). Fibres where prepared by forcing out the cell solution in alginate through 30- gauge needle at a constant flow rate 350ml/h to 0.1M calcium chloride. The tip of the needle was immersed below the gelling bath surface. The beads and fibres were hardened for 30 min. Afterwards, both were placed for 30 min in 0.5% (w/v) chitosan solution in 0.1 mol/l sodium acetate – acetic buffer, pH 4.5. In order to investigate the effects of chitosan molecular weight on cell growth two types of chitosan were exanimate. After that period the microcapsules where rinsed with physiological saline to remove excess chitosan and followed by treatment for 10 min in 0,15% alginate solution to contract

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charges on the membrane. Before culturing in full culture media, the microcapsules were rinsed in physiological saline.

Fig. 2. Schematic of microencapsulation process

Cell cultured medium was composed of 1:1 DMEM : Ham F-12 and 10% FCS supplemented white 25 µg/ml ascorbate acid, 100 µm /ml streptomycin and 50µg/ ml gentamycin and changed two times a week. Chondrocytes were cultured for 30 days in 24-well plates. Cultures were done at 37oC in a humidified atmosphere of 97% air and 5% CO2. In order to successfully compare the results, a monolayer culture of chondrocytes was carried out in the same condition. To estimate the size and morphology of capsules a sample of 15 microbeads and microfibers was taken from each type and was measured using a microscope. The average diameter was calculated. A monolayer culture of chondrocytes was cared under this same condition and was used as the control. The structure of microencapsules was investigated using a SEM (Phenom). Samples were lyophilized in a freeze dryer. Samples were not sputtered before examination, to enable observation of the monolayer of chitosan and alginate. 2.4. Cell concentration and viability Every 84h microscopic observations were made (Nikon eclipse 80i). At the same time 0.5g samples of microbead and microfiber were taken. These samples were dissolved in 0.05M sodium citrate in shacked flask for dissolution of alginate followed by adding an acetic acid for dissolution of chitosan. Cell viability was assessed using a trypan blue staining technique. Trypan blue 0.4% was used to distinguish viable cells from non-viable cells. After 2 min of incubation with dye, viable cells appeared round and clear while non-viable were asymmetrical and adsorbed the dye, and their colour turned blue there for appearing blue. Total chondrocytes cell concentration was estimated using a Thoma counting chamber. Cell viability percentage was determined by the following equation. Mean viability and associated standard deviations (n =5) are reported. number of alive cells × 100% number of all counted cells (dead + alive)

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(1)


2.5. Degradation of ACA microcapsules In order to determined the degradation of different microcapsules with two chitosan viscosity values, weight changes were analysed during the culture time period. Samples of each type of microcapsules and microfibers were prepared and were put in PBS saline with pH 7.4. Every 84 h 3 samples of 0.5g (Ws) were taken. Microcapsule dry weight was determined by drying the microcapsules at 70oC for 14h (Wd). The percentage of water content was calculated using the formula. Mean water content and associated standard deviations (n =3) are reported.

Ws - Wd × 100% Ws

(2)

3. RESULTS AND DISCUSSION Based on the literature data and results from previous experiments we decided that the most adequate material for microencapsulation is alginate with high guluronic residues and concentration 1.5%(w/v) (Constantinidis et al., 1999; Moresi and Bruno, 2007; Stabler et al., 1999). This concentration makes microcapsules mechanical strong enough and provides good exchange of substrates and metabolic products. For this reasons the lower concentration 0.1 M of calcium chloride was selected. When the alginate solution was pumped at a rate 25.5 ml/h through a 27 guage needle with a voltage of 4.5 kV, microbeads with a diameter of about 540 ± 40 µm were formed , in a later paragraphs were named as microcapsules from the range of 500 - 600 µm. Smaller microbaeds with diameter between 300 – 400 µm were formed at a higher voltage 6.1 kV and had about 360 ± 50 µm . Only one diameter (560 ± 40 µm) of microfibers was chosen for a culture of chondrocytes, because early research shows that the micro-fibres are easily breakable. Microscopic observation of microcapsules and micro-fibres showed a highly porous structure (Fig. 3a, b). SEM showed that scaffold fibre generally was covered by alginate in one direction. An application of subsequent layers smoothed the surface of microbeads. Taken images also showed, that the imposition of alginate on last layer was not complete, as white spots were visible on the chitosan layer in SEM images. After preparation and cultured for 24h, ACA microcapsules were observed every 84h with optical microscopy. Fig. 3c, d present representative microscope images of the prepared and cultured microcapsules after 24h. It can be seen that ACA microbeads were spherical and intact with smooth surface. The dark line represents the outer layer of microcapsules. There were no differences in microcapsule properties for 28 days of culture. Regardless of molecular weight of chitosan, water content of microcapsules was found to show similar patterns. Water content initially decreased over the first 5 days and then slowly returned to the initial values (Fig. 4). These results are similar to those obtained by Wang and al. The initially decreases were probably caused by molecular rearrangement, while swelling was the result of osmosis caused by unbound carboxyl groups which can cause disintegration of microcapsules. During culturing manipulation and microscopic observations no changes in the appearance of microcapsules or their tendency to disintegrate were noticed.The microfibers confirmed their tendency to tear, which can result in mechanical release of cells to growth medium while the activities associated with culturing. Cell appearance and shape did not change a lot during culture time, they were slightly reduced in size and slightly shrank. During microscopic observation a the secretion of extracellular matrix was observed – which means that cells started to produce cartilage components (Fig. 5). Chondrocytes were encapsulated at 5.0x106 cells/ml, after the immobilisation, the concentration of cells was reduced to about 4.5x106cell/ml (Table 1).

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Fig. 3. Scanning electron microscopic images of (a) microcapsules, (b) micro-fibres. Imagination of chondrocytes culture of (c) microcapsule, (d) micro-fibres after 24h

Fig. 4. Percentage of water content in microbead and microfiber coated by high (H) and medium (M) viscosity chitosan (MV±SD, n =3)

Fig. 5. Microscopic observation of chondrocytes a) after microencapsulation, b) after 24 days of culture, extracellular matrix is shown by the arrow (MV±SD, n =3)

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Table 1. Changes in chondrocyte concentrations before and after microencapsulation in microcapsules and fibres coated by high (H) and medium (M) viscosity chitosan (MV±SD, n =5)

Before [106 cell/ml]

After [106 cell/ml]

microcapsules 300-400µm M

5

4.6 ± 0.09

microcapsules 300-400µm M

5

4.6 ± 0.05

microcapsules 500-600µm H

5

4.5 ± 0.06

microcapsules 500 -600 µm H

5

4.45 ± 0.05

fibres H

5

4.48 ± 0.08

fibres M

5

4.5± 0.06

A reduction in cell concentration was mainly caused by cell release during encapsulation and destruction of them, while they were pumped through a drain and needle. The concentration of the cells subtly varied (Fig. 6).

Fig. 6. Changes in cell concentration during culture in microcapsules and fibres coated by high (H) and medium (M) viscosity chitosan (MV±SD, n =5)

The reduction in cell concentration is autonomous of the type of chitosan applied, but it is dependent on the geometry of microcapsules. There is a clear decrease of cell concentration in the microfibers which is the result of the tearing the fibres and cell release to culture medium. For the microcapsules of diameter 300 - 400 µm a high increase in concentration from 4.6x106 to 4.64x106 cell/ml was observed. For the capsules of diameter 500 - 600 µm concentration increased only from 4.5x106 to 4.52x106 cell/ml. The lower concentration of cell in diameter range 500 - 600 µm was effected by beater substrate and metabolism diffusion. For microcapsules 300 - 400 µm cell concentration increased similarly to the control culture (5.0x106 - 5.05x106cell/ml). The increase of concentration and shrinking of cell suggest that cells were dividing. A comparison of cell viability before and after immobilisation showed that there are no harmful stages in the encapsulation procedure. The encapsulation stage, which was critical for the process and the most harmful for cells, was the stage of microcapsules coating with chitosan which solution pH was 4.5. The previous immobilisation of cells in alginate protected them from destructive environment. Cell

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viability was reduced while conducting culture and it depended on the type of chitosan (Fig. 7). In the control culture viability was decreasing constantly while there were two stages in immobilised cultures. There was no difference in the viability of chondrocytes encapsulated in micro-fibre and micro–beads. Previous studies on model organism Saccharomyces cerevisiae showed an influence of microcapsule shape on cell viability and proliferation. This result could be explained by a difference in the diffusion of substrates inside and metabolites outside microcapsules. Chondocytes are slowly proliferating cells so microcapsule shape has no influence on the examined values. The shape of microcapsules can have an effect in the case of a higher density of cells or in the situation when cells start forming aggregates.

Fig. 7. Change in chondrocyte viability in microcapsules and fibres coated by high (H) and medium (M) viscosity chitosan (MV±SD, n =5)

In the first 252 h cell viability was stable, but after that it was starting to decrease. At the end of the culture cell viability in microcapsules and microfibers, which were both coated by medium viscosity chitosan was 5% higher than that of those coated by high viscosity chitosan (Table 2). Table 2. Chondrocyte viability at the end of the culture (MV±SD, n =5)

high viscosity chitosan

medium viscosity chitosan

microcapsules 300-400 µm

57% ± 8

62% ± 3

microcapsules 500-600 µm

56% ± 4

60% ±5

Fibres

60% ±2

64% ±6

This fact can be explained by differences in chitosan layer thickness (Gaserod et al.,1999b). High chitosan viscosity makes the layer on a alginate thicker, and because of that metabolite diffusion is lower. Finally, cell viability was reduced by about 20% compared to control culture. This small difference shows that immobilisation of chondrocytes in ACA system did not limit their growth.

4. CONCLUSIONS A method of alginate – chitosan – alginate multilayer hydrogel encapsulation system for chondrocytes was investigated. The presented method allows to encapsulate chondrocytes in microbeads and fibres, retaining their viability and promoting extracellular matrix production. Alginate or chitosan were

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prepared from a solution of neutral pH which offered an additional advantage of allowing protein or drug to be uniformly incorporated in its matrix structure with no or minimal denaturisation. Microcapsule coatings make microcapsule mechanically resistant and did not loseng the integrity of their structure within 30 day culture. There is no significant influence of microcapsule shape on slowly proliferating cells. Thanks to all these advantages ACA system appears to be a promising method for the reconstruction of damaged cartilage. To confirm the use of ACA system for cartilage reconstruction further studies on the release of glucosaminoglycans and collagen in long term cultures followed by animal experiments are needed.

This work was supported by ArtiCart project financed by NCBiR

REFERENCES Chandra R., Rustgi R., 1998. Biodegradable polymers. Prog. Polym. Sci., 23, 1273–1335. DOI: 10.1016/S00796700(97)00039-7. Chia S.H., Homicz M.R., Schumacher B.L., Thonar E.J.-M.A., Masuda K., Sah R.L., Watson D., 2005. Characterization of human nasal septal Chondrocytes cultured in alginate. J. Am. College Surg.,, 200, 691–704. DOI: 10.1016/j.jamcollsurg.2005.01.006. Ciach T., 2007, Application of electro hydro dynamic atomization in drug delivery, J. Drug Deliv. Sci. Technol., 17, 367-375. Constantinidis J., Rask J., Long R.C. Jr., Sambanis A., 1999. Effect of alginate composition on the metabolic, secretory, and growth characteristics of entrapped βTC3 muse insulinoma cells. Biomaterials, 20, 2019–2027. DOI: 10.1016/S0142-9612(99)00104-0. De S., Robinson D., 2003. Polymer relationships during preparation of chitosan – alginate and poly-l-lysine – alginate nanospheres. J. Control. Release, 89, 102–112 DOI: 10.1016/S0168-3659(03)00098-1. Domm C., Schünke M., Christesen K., Kurz B., 2002. Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension. Osteoarthr. Cartil., 10, 13–22. DOI: 10.1053/joca.2001.0477. Gåserød O., Sannes A., Skjåk-Bræk G., 1999. Microencapsulation of alginate chitosan. II. A study of capsule stability and permeability. Biomaterials, 20, 773-783. DOI: 10.1016/S0142-9612(98)00230-0. Gåserød O., Smidsrød O., Skjåk-Bræk G., 1999. Microcapsules of alginate chitosan - I: A quantative study of interaction between alginate and chitosan. Biomaterials, 19, 1815–1825. DOI: 10.1016/S0142-9612(98)00073-8. Gombotz W.R, Wee S.F, 1999. Protein release from alginate matrices. Adv. Drug Deliv. Rev., 31, 267–285. DOI: 10.1016/S0169-409X(97)00124-5. Haque T., Chen H., Ouyang W., Martoni Ch., Lawuyi B., Urbańska A.M., Prakash S., 2005. In vitro study of alginate – chitosan microcapsules: An alternative to liver cell transplants for the treatment of liver failure. Biotechnol. Lett., 27, 317-322. DOI: 10.1007/s10529-005-0687-3. Lim F., Sun A.M., 1980. Microencapsulated islets as bioartificial endocrine pancreas. Sci., 210, 908–910. DOI: 10.1126/science.6776628. Moresi M., Bruno M., 2007. Characterisation of alginate gel using quasi-stati and dynamic methods. J. Food Eng., 82, 298–309. DOI: 10.1016/j.jfoodeng.2007.02.040. Nafea E.H, Marson A., Poole-Warren L.A, Martens P.J., 2011. Immunoisolating semi-permeable membranes for cell encapsulation: Focus on hydrogels. J. Control. Release, 154, 110–122. DOI: 10.1016/j.jconrel.2011.04.022. Puppi D., Chiellini F., Piras A.M., Chiellini E., 2010. Polymeric materials for bone and cartilage repair. Prog. Polym. Sci., 35, 403-440. DOI: 10.1016/j.progpolymsci.2010.01.006. Sittinger M., Braunling J., Kastenbauer E., Hammmer C., Gburmester ,Bujie J., 1997. Analysis of the proliferative potential of human nasal chondrocytes for the engineering of cartilage transplants. Laryngo–RhinoOtol., 76, 96 –100. DOI: 10.1055/s-2007-997394. Stabler C., Wilks K., Sambanis A., Constantinidis I., 2001. The effects of alginate composition on encapsulated βTC3 cells. Biomaterials, 22, 1301–1310. DOI: 10.1016/S0142-9612(00)00282-9. Wang L., Shelton R.M., Cooper P.R., Lawson M., Triffitt J.T., Barralet J.E., 2003. Evaluation of sodium alginate for bone narrow cell tissue engineering. Biomaterials, 24, 3475–3481. DOI: 10.1016/S0142-9612(03)00167-4.

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Received 16 May 2012 Received in revised form 22 October 2012 Accepted 22 October 2012

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SUBSTRATE INHIBITION IN LIPASE-CATALYSED TRANSESTERIFICATION OF MANDELIC ACID WITH VINYL ACETATE

Katarzyna Dąbkowska*, Maciej Pilarek, Krzysztof W. Szewczyk Warsaw University of Technology, Faculty of Chemical and Process Engineering, Department of Biotechnology and Bioprocess Engineering, Waryńskiego 1, 00-645 Warszawa, Poland

Kinetic resolution of (R)- and (S)-mandelic acid by its transesterification with vinyl acetate catalysed by Burholderia cepacia lipase has been studied. The influence of the initial substrate concentration on the kinetics of process has been investigated. A modified ping-pong bi-bi model of enzymatic transesterification of (S)-mandelic acid including substrate inhibition has been developed. The values of kinetic parameters of the model have been estimated. We have shown that the inhibition effect revealed over a certain threshold limit value of the initial concentration of substrate. Keywords: kinetic resolution, enantiomers, kinetic model, lipase, substrate inhibition

1. INTRODUCTION The ability of lipases to catalyse many various enzymatic reactions has attracted increasing interest throughout the world. In non-aqueous media they catalyse a wide spectrum of reactions such as: alcoholysis, transesterification and ester synthesis or regiospecific acylation (Sharma et al., 2011; Szewczyk et al., 2001). Lipase-catalysed transesterification is one of the commonly applied methods for kinetic resolution of enantiomers in organic media (Ghanem and Aboul-Enein, 2005). This technique is based on differences in the transformation rate of particular enantiomers with a given acylating agent. Production of enantiomerically enriched compounds has recently become a major research area especially in pharmaceutical and fine-chemical industry. The requirement to prepare pure enantiomers arises from totally different pharmacological activities of enantiomeric pairs (Maier et al., 2001; Stinson, 2001). Pure enantiomers of mandelic acid and their derivatives are recognised as important chiral building blocks, auxiliaries and resolving agents in pharmacochemistry and enzymatic bioprocesses (Kibara et al., 1996; Survivet and Vatèle, 1998; Terreni et al., 2001). Several reports of mandelic acid enantiomers production have been presented so far (Gröger, 2001; Palomo et al. 2002; Yadav and Sivakumar, 2004). One of the easiest and the most promising methods seems to be a kinetic resolution of mandelic acid racemates by lipase-catalysed transesterification (Campbell et al., 2003; Qeiroz and Nascimento, 2002). In our previous papers (Dąbkowska and Szewczyk, 2007; Dąbkowska and Szewczyk, 2009) we have shown that B. cepacia lipase is highly enantioselective towards (S)-substrate during transesterification with vinyl acetate in isopropyl ether used as the solvent. At 25°C only the (S)mandelic acid form was transformed into the product, whilst the conversion of (R)-mandelic acid has


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K. Dąbkowska, M. Pilarek, K.W. Szewczyk, Chem. Process Eng., 2012, 33 (4), 539-536

not been observed. The simple ping-pong bi-bi model has been used to describe the reaction kinetics (Dąbkowska and Szewczyk, 2007). The aims of our present work were to investigate the influence of the initial substrate concentration on the rate of transesterification of (S)-mandelic acid catalysed by B. cepacia lipase and to discuss substrate inhibition in the studied enzymatic reaction. An improved ping-pong bi-bi kinetic model has been used to explicitly determine substrate effects on lipase. To our knowledge this is the first attempt to apply substrate inhibition to completely define the mechanism of mandelic acid transesterification in general.

2. MATERIALS AND METHODS 2.1. Enzyme Lipase from Burkholderia cepacia was purchased from Amano Pharmaceutical Co. (Japan) as Amano PS lipolytic enzyme preparation. It contains enzyme concentrate and diatomaceous earth in the ratio of 50:50 (w/w). The enzyme concentrate could also contain dextrin and some solid constituents originating from the fermentation media. The water and protein content was 25 mg/g and 65 mg/g, respectively. According to the Certificate of Analysis supplied by the producer the activity of enzyme was 31 600 U/g. 2.2. Chemicals All the chemicals which were used in the experiments as well as all HPLC solvents were of analytical grade. (R,S)-mandelic acid (≥ 98%), trifluoroacetic acid (≥ 98%) and vinyl acetate (≥ 99%) were purchased from Sigma-Aldrich whilst n-hexane (99%), isopropyl ether (98%) and ethanol (99.8%) were obtained from POCH S.A. (Gliwice, Poland). 2.3. Analytical methods The concentration of mandelic acid and O-acetylmandelic acid enantiomers was determined by HPLC method. The Varian 65 CL System chromatograph (Varian, USA) supported with Chiralcel OD column (l. 250 mm, i.d. 4.6 mm) from Daicel Chemical Industries, Ltd. (Japan) and with UV detection at 228 nm was used. The mixture composed of n-hexane, ethanol and trifluoroacetic acid at the volumetric ratio of 95:5:0.5 respectively was applied as a mobile phase. The analysis was performed at a temperature of 4°C, whilst the flow rate of the mobile phase was set at 1.0 cm3/min. The retention times were determined as follows: 22.0 min. for (R)-mandelic acid, 19.5 min. for (S)mandelic acid, 12.5 min. for (R)-O-acetylmandelic acid and 11.5 min. for (S)-O-acetylmandelic acid. 2.4. Numerical methods The values of kinetic parameters were estimated numerically by minimising the total error (ε) between measured (j) and calculated (cal) values of (S)-mandelic acid concentrations (CMA), according to Equation 1:

ε = ∑ (C MA, j − C MA,cal )2 j

A fourth order Runge-Kutta method was used to solve the model equations.

540

(1)

Substrate inhibition in lipase-catalysed transesterification of mandelic acid with vinyl acetate

2.5. Transesterification of mandelic acid with vinyl acetate A kinetic resolution of (R)- and (S)-mandelic acid isomers by lipase catalysed transesterification with vinyl acetate was performed in 100 cm3 Erlenmeyer flasks thermostated in a water bath shaker (T = 25 °C, 150 rpm). The reaction mixtures were prepared by dissolving an appropriate amount of (R,S)-mandelic acid and vinyl acetate in 75 cm3 of isopropyl ether. Then 300 mg of B. cepacia lipase (Amano PS) was added to initiate enzymatic process. Depending on the experiment, the initial concentration of each mandelic acid enantiomer in reaction mixture equalled to 0.004, 0.015, 0.025 or 0.035 mol/dm3 (i.e. it corresponded to 2 much higher concentration of racemic mandelic acid in every studied case). Vinyl acetate was used at a 10-fold molar excess to racemic mandelic acid. The 0.2 cm3 samples of reaction mixture were harvested at appropriate time intervals, then they were filtered with single-use syringe filters (0.2 µm; polypropylene housing, nylon membrane) and finally diluted (1:10) with isopropyl ether prior to HPLC analysis.

3. RESULTS AND DISCUSSION 3.1. Effect of initial mandelic acid concentration on transesterification progress The conversion profiles of (S)-mandelic acid transesterification for various initial concentration of substrate have been presented in Figure 1. It can be easily seen that the studied reaction proceeded faster in the case of lower values of the initial mandelic acid concentration. However, no significant differences in the reaction rates were observed for substrate concentrations not exceeding 0.015 mol/dm3.

Fig. 1. The influence of the mandelic acid initial concentration on conversion profiles of (S)-mandelic acid

According to the simple ping-pong bi-bi model, which has been used previously to describe the transesterification progress of (S)-mandelic acid (Dąbkowska and Szewczyk 2007), the rate of the reaction can be expressed as follows:

541


r=

rmax C( S ) − MA C 1 + VA + KVA K ( S ) − MA

(2)

In our experiments the molar ratio of vinyl acetate to (S)-mandelic acid equalled 20:1. In such case the overall concentration of vinyl acetate can be described as:

CVA = 20 ⋅ C 0 ,( S )− MA − (C 0,( S )− MA − C ( S )− MA )

(3)

Taking into account Equation 3, the integration of Equation 2 for the initial conditions (at t = 0 C(S)-MA = C0,(S)-MA) gives the following expression:

t (α MA ) =

 1   20 − α MA  α MA C0,( S )−MA − K VA ln  − K ( S )−MA ln(1 − α MA )  rmax   20  

(4)

where αMA is the conversion of racemic mandelic acid, defined as:

α MA =

C 0, MA − C MA

(5)

C 0 , MA

Only one enantiomer of mandelic acid undergoes the reaction under the applied conditions, thus α MA ∈ 0 ÷ 0.5 and it can be assumed that:

 20 − α MA  ln ≈0  20 

(6)

Additionally, Taylor series expansion gives:

ln(1 − α MA ) ≈ α MA

(7)

Finally, from Equations 4, 6 and 7 the αMA can be defined as:

α MA =

rmax t C0,( S )− MA + K ( S )− MA

(8)

As we previously published (Dąbkowska and Szewczyk, 2009) the enzyme-(S)-mandelic acid affinity constant, K(S)-MA, equals 0.99 mol/dm3. Therefore, for C0,(S)-MA much lower than K(S)-MA value, the conversion of mandelic acid for ping-pong bi-bi mechanism should be practically independent of the initial mandelic acid concentration in the reaction mixture. However, the results of our present study, showed in Figure 1, are not in agreement with the model predictions discussed above. Table 1. Estimated kinetic parameters for ping-pong bi-bi mechanism

C0,(S)-MA mol/dm3 0.004

C0,VA mol/dm3 0.08

rmax·103 mol/dm3·min 6.54

K(S)-MA mol/dm3 0.98

KVA mol/dm3 6.13

relative error % 8.66

0.015

0.30

6.48

0.99

6.02

3.30

0.025

0.50

5.41

0.67

34.66

9.82

0.035

0.70

4.33

0.43

42.91

7.24

0.050

1.00

3.19

0.04

55.82

10.81

The estimated values of the simple ping-pong bi-bi model parameters for various initial mandelic acid concentrations are summarised in Table 1. In the case of a reaction carried out with C0,(S)-MA = 0.004 and

542


0.015 mol/dm3 all the kinetic parameters are independent of the initial substrate concentrations. In the case of higher initial concentrations of the substrate, the maximum reaction rate and the enzymemandelic acid affinity constant decrease, but the enzyme-vinyl acetate affinity constant increases with the increase of initial substrate concentration. Based on the dependence of the presented ping-pong bi-bi kinetic parameters on the initial composition of reaction mixture we hypothesised the presence of an effect which triggered a reduction of the overall reaction rate at relatively high mandelic acid concentrations. 3.2. Ping-pong bi-bi model with substrate inhibition In our next investigations several attempts were made to describe more accurately the kinetics of the studied reaction and a few modifications of simple ping-pong kinetic mechanism were considered. An incorporation of competitive inhibition by acyl acceptor, as the most commonly reported pathway of inhibition for lipase-catalysed reactions (Pilarek and Szewczyk, 2007; Segel, 1993), as well as product inhibition and enzyme deactivation by vinyl alcohol did not produce satisfactory results and did not fit well with our experimental data. Here we propose a modified ping-pong bi-bi kinetics including substrate inhibition of enzyme by mandelic acid to describe accurately the effects of the initial substrate influence on lipase activity observed in the presented experiments. Such an attempt assumes that in the case of a relatively high substrate concentration in reaction mixture, two molecules of (S)-mandelic acid can simultaneously bind to the enzyme active site. Finally, a triple enzyme-substrate-substrate complex is formed which is unable to further convert into the product. According to our considerations, the rate of (S)-mandelic acid conversion can be defined as:

r =

rmax

(9)

C ( S ) − MA C ( S ) − MA ⋅ K ( S ) − MA C 1 + VA + + K VA K ( S ) − MA K i ,( S ) − MA

The estimated values of kinetic parameters for modified ping-pong bi-bi with the substrate inhibition have been showed in Table 2. As can be easily seen all the kinetic parameters are independent of the initial concentration of the substrate. Table 2. Values of kinetic parameters estimated for ping-pong bi-bi kinetics with substrate inhibition by mandelic acid

C0,(S)-MA mol/dm3 0.004

C0,VA mol/dm3 0.08

rmax·103 mol/dm3·min 6.42

K(S)-MA mol/dm3 0.98

KVA mol/dm3 5.98

Ki,(S)-MA·104 mol/dm3 5.03

relative error % 6.18

0.015

0.30

6.78

0.94

5.64

5.99

3.66

0.025

0.50

6.46

1.01

6.07

3.13

9.14

0.035

0.70

6.31

1.00

6.12

4.03

6.28

0.050

1.00

6.51

0.98

6.11

3.75

5.72

6.50

0.98

5.98

4.39

mean

A comparison of experimental data and the modified ping-pong bi-bi model curves have been showed in Figure 2. All the mathematical predictions fitted well the experimental data over the entire range of the initial mandelic acid concentrations used in the tests.

543


Fig. 2. A comparison of experimental data (dots) and the model curves predicted by modified ping-pong bi-bi kinetic model with enzyme inhibition by mandelic acid (continuous lines)

3.3. Threshold limit values of initial (S)-mandelic acid concentration Results of the numerical simulation (based on Equation 9) of (S)-mandelic acid transesterification rate for various initial concentrations of racemic substrate (C0,MA) have been presented in Figure 3. The mean values of the estimated kinetic parameters, taken from Table 2, have been incorporated. It can be seen that the rate of the reaction significantly increased with substrate concentration increase up to reaching the threshold limit point (CMA)lim= 0.042 mol/dm3, and then dropped. In our opinion, for relatively small mandelic acid concentrations, the reaction rate can be considered as practically independent of the initial concentration of the substrate. Based on the presented simulation results of the transesterification process we hypothesise that for the initial concentration of racemic mandelic acid lower than the threshold limit (C0,MA)lim = 0.042 mol/dm3, the simple ping-pong bi-bi mechanism can be applied. Above the (C0,MA)lim value the substrate inhibition should be taken into account to fully describe the kinetics of the studied lipase-catalysed transesterification.

Fig. 3. Results of the numerical simulations of (S)-mandelic acid transesterification rate for the modified ping-pong bi-bi kinetics for various initial concentrations of substrate

544


4. CONCLUSIONS The influence of the initial concentration of substrate on the rate of kinetic resolution of (S)-mandelic acid catalysed by B. cepacia lipase has been discussed in-depth. The modified ping-pong bi-bi mechanism with substrate inhibition of enzyme has been applied to fully describe the progress of mandelic acid transesterification with vinyl acetate. The inhibition effect revealed over the threshold limit value of the initial concentration of substrate, which has been determined as 0.042 mol/dm3. All the predictions of the reaction kinetics fitted well the experimental data over the entire range of the initial (S)-mandelic acid concentrations. To sum up, we conclude that results of such investigations can be useful for more rational design of industrial systems applied for enantiomerically pure mandelic acid production.

SYMBOLS C0,MA C0,(S)-MA CMA C(S)-MA (CMA)lim CVA Ki,(S)-MA KMA KVA r rmax

initial (R,S)-mandelic acid concentration, mol/dm3 initial (S)-mandelic acid concentration, mol/dm3 (R,S)-mandelic acid concentration, mol/dm3 (S)-mandelic acid concentration, mol/dm3 threshold limit value of racemic mandelic acid concentration, mol/dm3 vinyl acetate concentration, mol/dm3 (S)-mandelic acid inhibition constant, mol/dm3 enzyme-mandelic acid affinity constant, mol/dm3 enzyme-vinyl acetate affinity constant, mol/dm3 reaction rate, mol/(dm3min) maximum reaction rate, mol/(dm3min)

Greek symbols αMA conversion of (R,S)- mandelic acid conversion of (S)-mandelic acid α(S)-MA

REFERENCES Campbell R.F., Fitzpatric K., Inghardt T., Karlsson O., Nilsson K., Reilly J.E., Yet L., 2003. Enzymatic resolution of substituted mandelic acids. Tetrahedron Lett., 44, 5477-5481. DOI: 10.1016/S0040-4039(03)01270-X. Dąbkowska K., Szewczyk K.W., 2007. Mathematical modelling of enzymatic transesterification of mandelic acid with vinyl acetate. Chem. Proc. Eng., 28, 795-802. Dąbkowska K., Szewczyk K.W., 2009. Influence of temperature on the activity and enantioselectivity of Burkholderia cepacia lipase in the kinetic resolution of mandelic acid enantiomers. Biochem. Eng. J., 46, 147153. DOI: 10.1016/j.bej.2009.04.023. Ghanem A., Aboul-Enein H.Y., 2005. Application of lipases in kinetic resolution of racemates. Chirality, 17, 1-15. DOI: 10.1002/chir.20089. Gröger H., 2001. Enzymatic Routes to Enantiomerically pure aromatic α-hydroxy carboxylic acids: A further example for the diversity of biocatalysis. Adv. Synth. Catal., 343, 547-558. DOI: 10.1002/16154169(200108)343:6/73.0.CO;2-A. Kibara K., Sakai K., Hashimoto Y., Nohira H., Saigo K., 1996. Design of resolving reagents: p-substituted mandelic acid as resolving reagents for 1-arylalkylamines. Tetrahedron: Asymmetry, 7, 1539-1542. DOI: 10.1016/0957-4166(96)00175-9. Maier N.M., Franco P., Lindner W., 2001. Separation of enantiomers: needs, challenges, perspectives. J. Chromatogr. A, 906, 3-33. DOI: 10.1016/S0021-9673(00)00532-X.

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Palomo M., Fernandez-Lorente G., Mateo C., Ortiz C., Guisan J.M., Fernandez-Lafuente R., 2002. Modulation of the enantioselectivity of lipases via controlled immobilization and medium engineering: hydrolytic resolution of mandelic acid esters. Enz. Microb. Technol., 31, 775-783. DOI: 10.1016/S0141-0229(02)00169-2. Pilarek M., Szewczyk K.W., 2007. Kinetic model of 1,3-specific triacylglycerols alcoholysis catalyzed by lipases. J. Biotechnol., 127, 736-744. DOI: 10.1016/j.jbiotec.2006.08.012. Segel I.H., 1993. Enzyme kinetic. Behavior and analysis of rapid equilibrium and steady-state enzyme systems. Wiley, New York, 827-828. Sharma D., Sharma B., Shukla A.K., 2011. Biotechnological approach of microbial lipase: A review. Biotechnology, 10, 23-40. DOI: 10.3923/biotech.2011.23.40. Stinson C.S., 2001. Chiral pharmaceuticals. Chem. Eng. News, 79, 79-97. DOI: 10.1021/cen-v079n040.p079. Surivet J.-P., Vatèle J.-M., 1998. First total synthesis of (-)-8-epi-9-deoxygoniopypyrone. Tetrahedron Lett., 39, 9681-9682. DOI: 10.1016/S0040-4039(98)02269-2. Szewczyk K.W., Pilarek M., Wrona M., 2001. Enzymatic propanolysis of triacetin. Inż. Chem. Proc., 22, 13511356. Terreni M., Pagani G., Ubiali D., Fernández-Lafuente R., Mateo C., Guisán J.M., 2001. Modulation of penicillin acylase properties via immobilization techniques: one-pot chemoenzymatic synthesis of caphamandole from cephalosporin C. Bioorg. Med. Chem. Lett., 11, 2429-2432. DOI: 10.1016/S0960-894X(01)00463-2. Queiroz N., Nascimento M. G., 2002. Pseudomonas sp. lipase immobilized in polymers versus the use of free enzyme in the resolution of (R,S)-methyl mandelate. Tetrahedron Lett., 43, 5225-5227. DOI: 10.1016/S00404039(02)01057-2. Yadav G.D., Sivakumar P., 2004. Enzyme-catalysed optical resolution of mandelic acid via (R,S)-methyl mandelate in non-aqueous media. Biochem. Eng. J., 19, 101-107. DOI: 10.1016/j.bej.2003.12.004. Received 16 May 2012 Received in revised form 12 November 2012 Accepted 19 November 2012

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CARBON DIOXIDE ABSORPTION INTO AQUEOUS BLENDS OF N-METHYLDIETHANOLAMINE AND 2-ETHYLAMINOETHANOL Władysław Moniuk*, Ryszard Pohorecki, Piotr Machniewski Warsaw University of Technology, Faculty of Chemical and Process Engineering, Waryńskiego 1, 00-645 Warszawa, Poland

Measurements of the absorption rate of carbon dioxide into aqueous solutions of N-methyldiethanoloamine (MDEA) and 2-ethylaminoethanol (EAE) have been carried out. On this basis a mathematical model of the performance of an absorption column operated with aqueous solution of a blend of the above amines at elevated temperatures and pressures have been proposed. The results of simulations obtained by means of this model are described. The work is a part of a wider program, aimed at the development of a new process. Keywords: alkanoloamines, carbon dioxide, absorption, simulation, packed column

1. INTRODUCTION The removal of CO2 from synthesis and flue gases has been gaining increasing interest in the recent years. This is caused by both technological and ecological reasons. The most widespread method of CO2 removal is that of absorption accompanied by a chemical reaction. Among possible reacting solvents, aqueous amine solutions seem to be the most promising. Both single amines and amine blends have been so far proposed for industrial use. The use of mixed amines in gas purification processes is of increasing interest today. The mixed amine systems, which combine higher equilibrium capacity of the tertiary amine with the higher reaction rate of the primary or secondary amine, have been suggested for gas purification processes (Horng and Li, 2002; Liao and Li, 2002; Mandal et al., 2001, 2003; Sun et al., 2005; Ume et al., 2012; Xiao et al., 2000). In many cases the absorption process has to be carried out at elevated temperatures and pressures (e.g. in the case of synthesis gases). The aim of this work was to check the suitability of selected amines for flue and synthesis gas purification. To this end experimental investigations of the rate of absorption into selected amines, and simulations of the column performance using these amines were carried out. The work is a part of a wider experimental program aimed at the development of a new process.

2. REACTION MECHANISMS 2.1. MDEA [CH3CH2(CH2CH2OH)2N] MDEA is a tertiary amine. Reaction mechanisms of primary and secondary amines are different from those of tertiary amines. The following reactions occur during the CO2 absorption into aqueous MDEA solutions (Donaldson and Nguyen, 1980; Rinker et al., 1995). *Corresponding author, e-mail: [email protected]

547

Wł. Moniuk, R. Pohorecki, P. Machniewski, Chem. Process Eng., 2012, 33 (4), 546-561 k21 ,K1 CO2 + R3 N + H 2 O ← → R3 NH + + HCO3−

(1)

k 22 ,K 2 CO2 + OH − ← → HCO3−

(2)

K3 HCO3− + OH − ←→ CO32 − + H 2 O

(3)

K4 R3 NH + + OH − ←→ R3 N + H 2 O

(4)

K5 2 H 2 O ←→ OH − + H 3 O +

(5)

The rate of reactions (1) and (2) can be expressed as:

r1 = k 21 [ CO2 ][ R3 N ] −

k 21 [ R3 NH + ][ HCO3− ] K1

(6)

k 22 [ HCO3− ] K2

(7)

r2 = k 22 [ CO2 ][ OH − ] −

where [ R3 NH + ][ HCO3− ] K1 = [ CO2 ][ R3 N ] K2 =

[ HCO3− ] [ CO2 ][ OH − ]

(8)

(9)

In reactions (3-5) only proton exchange occurs and these reactions can be treated as very fast (“instantaneous”). Equilibrium constants for these reactions are as follows:

K3 =

[ CO32− ] [ HCO3− ][ OH − ]

(10)

K4 =

[ R3 N ] [ R3 NH + ][ OH − ]

(11)

K 5 = [ OH − ][ H 3 O + ]

(12)

Rinker et al. (1995) measured the rate of carbon dioxide absorption into aqueous MDEA solutions in a wetted-sphere absorber. For the interpretation of the obtained results, three different mathematical models, based on Higbie’s penetration theory, were used. The first model is the most general model. It includes reactions (1-5) and treats them as reversible reactions. The second model is almost the same as the first model except that it neglects reaction (2). The third model neglects reactions (2-5) and treats reaction (1) as an irreversible pseudo-first-order reaction. A comparison of the predicted enhancement factor E from models 1-3 is presented in Fig. 1 (Rinker et al., 1995). As it is seen, for high values of interfacial concentration of CO2 (~ 10-2 kmol/m3) the values of the enhancement factor E for these 1-3 models, are very close. For the conditions considered in this work ci = (0.7–1.2)·10-2 kmol/m3, and therefore the simplest, the third model, can be used. Many studies have been performed on the kinetics of the reaction (1) between MDEA and CO2. In Fig. 2 the dependence of the reaction rate constant, k21, on the amine concentration is presented (t = 20 oC). As it is seen, the discrepancies between the results obtained by various authors are very large. The present measurements (Fig. 5) are in agreement with the earlier data of Moniuk and Pohorecki (2000). These data are used in the modelling.

548

Carbon dioxide absorption into aqueous blends of N-methyldiethanolamine and 2-ethylaminoethanol

10 Model 1 Model 2 i 3

8

E [-]

6

4

2

0 1.00E-07

1.00E-06

1.00E-05 1.00E-04 3 ci [kmol/m ]

1.00E-03

1.00E-02

Fig. 1. A comparison of the enhancement factor values, E, obtained for models 1-3 (Rinker et al., 1995)

7

6

k21 [m3/kmol·s]

5

Moniuk and Pohorecki, 2000 4

Rinker et al., 1995 Tomcej and Otto, 1989 Benitez-Garcia et al., 1991

3

Haimour et al., 1987 Versteeget al., 1988 Rangwala et al., 1992

2

1

0 0

0.5

1

1.5

2

2.5

3

cB0 [kmol/m3]

Fig. 2. The dependence of the reaction rate constant, k21, on the amine concentration cBo (t = 20°C)

2.2. EAE [C2H5NHC2H4OH] EAE is a secondary amine. The reactions of CO2 with primary and secondary alkanolamines in aqueous solutions are believed to take place according to the zwitterion mechanism (Danckwerts, 1979). In this

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Wł. Moniuk, R. Pohorecki, P. Machniewski, Chem. Process Eng., 2012, 33 (4), 546-561

mechanism CO2 reacts with amine to form a zwitterion, which further reacts with any base present in the system. k2 → CO2 + R2 NH R2 NH +COO − ← k −2

(13)

kB R2 NH + COO − + B ←→ R2 NCOO − + BH +

(14)

If [z] is the concentration of zwitterion (in quasi-steady state) then the rate of CO2 reaction can be expressed as:

r = k2 [ CO2 ][ Am ] − k− 2 [ z ] = [ z ] ∑ kB [ B ]

(15)

The term ∑ [] indicates the contribution of the various bases present in the system to the rate of removal of protons from the zwitterion. In our case the base (B) may be water of OH- ions. From Eq. (15)

r = [ CO2 ][ Am ] 1 + If

If

k2 k− 2 ∑ kB [ B ]

(16)

∑

k −2 >1 we get k B [B]

r k = 2 ∑ kB [ B ] [ CO2 ][ Am ] k − 2

(17)

and we have third-order kinetics as in the case of diethanoloamine (DEA). There is also an alternative mechanism (termolecular mechanism) proposed by Vaidya and Kenig (2010), but the above (zwitterion) mechanism is more frequently used.

3. EXPERIMENTAL APPARATUS AND PROCEDURE Measurements of the rate of CO2 absorption into aqueous solutions of MDEA and EAE were carried out in a stirred reactor (Autoclave Engineers Inc.) with capacity 1·10-3 m3. The scheme of the experimental apparatus is shown in Fig. 3. At the beginning of the experiment, a mixture of gases (CO2 and N2) was supplied through the tree-way valve (10) to the CO2 analyser (12). During the main part of the experiment the experimental conditions (pressure, temperature and stirrer speed) were kept constant, and the mixture of gases was supplied through the three – way valve to the gas sparger (8) below the stirrer (9). CO2 concentration at the gas outlet from the reactor was measured by the CO2 analyser (12). The measurements were carried out under 1-2 bar pressure at the temperature range: 20 – 70 ºC. The other parameters were as follows: stirred speed 700 rpm; concentration of CO2 in the inlet gas 46-50 %vol.; amines concentration: MDEA: 10, 20 and 30 wt.%; EAE: 1 and 2 wt.%. The MDEA was obtained from Sigma – Aldrich with a purity > 98,5 wt.%, whereas the EAE was obtained from Aldrich with a purity > 98wt.%.

550


Fig. 3. Scheme of the experimental apparatus 1, 2 – gas cylinders; 3, 4 – pressure reducing valves; 5, 6 – gas flow meters; 7 – stirred reactor; 8 – sparger; 9 – stirrer; 10 – three – way valve; 11 – temperature, pressure and stirrer speed regulators; 12 – CO2 analyser (DCS, Model 300)

On the basis of the CO2 concentrations measured at the gas inlet to the reactor and the gas outlet from the reactor, the rate of CO2 absorption, R, was calculated. On the other hand we have:

R = N ⋅a

(18)

In the process of absorption with chemical reaction, the molar flux of absorbed CO2 is equal to

N = k*L ⋅ c Ai

(19)

The concentration of CO2 at the interface was calculated from the Henry's law

p Ai = H ⋅ c Ai

(20)

The partial pressure of CO2 at the interface, pAi, was calculated from the relation

N = k g ( p Ao − p Ai ) = k *L ⋅ c Ai

(21)

The values of the mass transfer coefficient in the gas phase were taken from the Versteeg et al. (1987) data. For the gas flow in the reactor we assumed plug flow. In this case the average value of the partial pressure of CO2 in the gas phase is equal to

p Ao =

( p Ao( inlet ) − p Ao( outlet ) ) ln

p Ao( inlet )

(22)

p Ao( outlet )

4. RESULTS From the CO2 concentrations measured in the gas phase, the rate of CO2 absorption, R and the mass transfer coefficient with chemical reaction, k*L were calculated.

551


Making use of the values of the physical mass transfer in the liquid phase, kL, determined experimentally in our earlier work (Moniuk et al., 1997) the values of the enhancement factor, E, defined as:

E=

k L* kL

(23)

were calculated. On the other hand, enhancement factor E can be determined from the van Krevelen and Hoftijzer diagram (Ramm, 1976)

E = f ( Ha , β )

(24)

where

k 21 ⋅ c B 0 ⋅ D A

Ha =

kc

β=

c B0 ⋅ DB b ⋅ c Ai ⋅ D A

(25)

(26)

For Ha ≥ 2 Porter’s relations can be used:

  − (Ha − 1)   E = 1 + β ⋅ 1 − exp β   

(27)

For a fast pseudo-first-order reaction, when:

k L ≤ k 21 ⋅ c B 0 ⋅ D A Ha ≤

c B 0 ⋅ DB b ⋅ c Ai ⋅ D A

(28) (29)

the enhancement factor is equal to the Hatta number value:

E = H ⋅a

(30)

Enhancement factor E is thus a function of the Hatta number and the parameter β. In our case, depending on the values of enhancement factor E and parameter β, Eqs. (24) or (27) or (30) could be used to determine the values of the enhancement factor, and finally the reaction rate constant, k21 or k2 values were calculated by comparison with Eq. (23). 4.1. System: CO2 – MDEA (tertiary amine) In Fig. 4 the dependence of the reaction rate (1) constant k21 on the amine concentration for various temperatures is presented. In Fig. 5 the Arrhenius plot (logk21 = f(1/T)) is presented. As it is seen the values k21 practically do not depend on the amine concentration. The straight line can be described by the relation:

logk 21 = 10.25 − (2792.3 / T )

552

(31)


160 140

k21 [m3/kmol·s]

120 100 T=293 K

80

T=323 K 60

T=333 K

40

T=343 K

20 0 0

0.5

1

1.5

cB0

2

2.5

3

[kmol/m3]

Fig. 4. The dependence of the reaction (1) rate constant k21 on the amine (MDEA) concentration for various temperatures 2.3 2.1

R² = 0.9999 1.9

log k21

1.7 1.5 cB0=0.84 1.3

cB0=1.68 cB0=2.52

1.1 0.9 0.7 0.5 0.0028

0.0029

0.0030

0.0031

0.0032

0.0033

0.0034

0.0035

1/T Fig. 5. The Arrhenius plot logk21=f(1/T)

4.2. System: CO2 – EAE (secondary amine) The values of the reaction (13) rate constant k2 were calculated in a similar way as the reaction (1) constant, k21. In Fig. 6 the dependence of the reaction (13) rate constant k2 on the amine concentration for various temperatures is presented. In Fig. 7 the Arrhenius plot (logk2 = f(1/T)) is presented. The straight line can be described by the relation:

logk2 = 10.052 − (1929.8 / T )

(32)

The obtained data on the reaction rate constants k21 (CO2–MDEA system) and k2 (CO2–EAE system) were used for simulation of CO2 absorption into aqueous solutions of a blend of both amines.

553


14000 12000

k2 [m3/kmol·s]

10000 8000

T=298 K T=308 K

6000

T=323 K

4000 2000 0 0

0.05

0.1

0.15

0.2

0.25

cB0 [kmol/m3]

Fig. 6. The dependence of the reaction (13) rate constant, k2, on the amine (EAE) concentration for various temperatures

4.1

4

R² = 0.9622

logk2

3.9

3.8

cBo=0.1122 cBo=0.2244

3.7

3.6

3.5 0.003000

0.003100

0.003200

0.003300

0.003400

1/T

Fig. 7. The Arrhenius plot logk2 = f(1/T)

5. MATHEMATICAL MODEL OF CO2 ABSORPTION INTO AQUEOUS ALKANOLAMINE SOLUTIONS In order to estimate the performance of an absorption process in a solvent containing the mixture of both amines, a rate-based mathematical model of a packed column has been used. For the process of absorption with a chemical reaction of the type:

A + bB ↔ dD

(33)

The differential equation for the rate of absorption in a counter-current packed column can be written as follows (Danckwerts, 1970):

R A ⋅ a ⋅ dh = d ( GOM ⋅ x A ) 554

(34)


For a dilute gas phase we have:

GOM dx A 1 − xA

d ( GOM ⋅ x ) ≅

(35)

Hence, from Eq. (34) and (35) we have:

dx A 1 − x A = RA ⋅ a dh GOM

(36)

In the heat balance one should consider heat exchange between phases, heat effects of absorption, reaction and solvent evaporation (or condensation), as well as heat losses to the environment. In the case of heat exchange between phases, one can neglect the heat transfer resistance in the liquid phase (Sherwood and Pigford, 1952). Thus we have:

From the heat balance

dN t = α g ⋅ a (Tg − Tl )dh

(37)

dN t = d (GOM ⋅ C P ⋅ Tg ) ≅ GOM ⋅ C P ⋅ dTg

(38)

Hence

dTg dh

=

αg ⋅ a C P ⋅ GOM

(T

g

− Tl )

(39)

The heat transfer coefficient can be determined from heat and mass transfer analogy

Nu g Sh g

=

A ⋅ Re gB ⋅ PrgC A ⋅ Re gB ⋅ Sc gC

 Pr = g  Sc  g

   

C

(40)

Assuming C = 0.33 (Sherwood and Pigford, 1952) and making use of the definitions of Prandtl and Schmidt numbers we have:

λ α g = k g  g  Dg

   

0 ,66

⋅ (C p ⋅ ρ g )

0 ,33

(41)

Hence

k g ⋅ a  λg  = dh GOM  C p ⋅ D g

dTg

   

0 ,66

⋅ ρ g0 ,33 ⋅ (Tg − Tl )

(42)

In the case of solvent evaporation, one can neglect the mass transfer resistance in the liquid phase. Thus we have:

dN = k gw ⋅ a ⋅ ( p w − pwr )dh = k g ⋅ a ⋅ P ⋅ ( x w − xwr )dh

(43)

 G   x  dxw dxw dN = d (GOM ⋅ x w ) ≅ d  M ⋅ xw  = GM d  w  = GM = GOM 2 1 − xw (1 − xw )  1 − xw   1 − xw 

(44)

and

Hence

555


dxw k gw ⋅ a ⋅ P ⋅ (1 − xw ) (xw − xwr ) = dh GOM

(45)

The heat effect of solvent evaporation is equal to the product of the heat of vaporization (qW) and the amount of evaporated solvent. The heat effect of absorption and reaction is equal to the product of the overall heat of absorption and reaction (qA) and the amount of absorbed component. In these conditions the heat balance can be written as follows (Hobler, 1968)

dTl G = OM dh LO ⋅ cl

dx H 2 O dxCO2 dTg  ⋅  − C p + qw + qr dh dh dh 

 q  + s  Lo ⋅ cl

(46)

The simulations of CO2 absorption into aqueous alkanoloamines solutions in the packed column consisted in simultaneous numerical integrations of the following equations: • Eqs. (36) and (45) describing the concentration profiles of the absorbed component xA and the solvent vapour xW in the gas phase; • Eqs (42) and (46) describing the temperature profiles in the gas (Tg) and liquid (Tl) phases; • Eqs (47) and (48) describing the profiles of gas (Go) and liquid (Lo) flow rates For the dilute gas phase

dGo dx dx = M AGoM A + M W GoM W dh dh dh

(47)

dLo dGo = dh dh

(48)

For the counter-current flow

6. RESULTS OF SIMULATIONS A computer program for the numerical integration of Eqs. (38), (42), (45), (46), (47) and (48) along the column height has been developed in MATLAB environment. The rate of CO2 absorption in packed column can be calculated from the relation (Pohorecki et al., 1978, 1990; Moniuk 2006)

RA = ( x A ⋅ P − p Ar )

H ⋅ D A ⋅ k1 + kl2 H ⋅ R ⋅T 1+ ⋅ D A ⋅ k1 + kl2 kg

(49)

The pseudo-first-order reaction rate constant, k1 is equal to:

k1 = k 21( MDEA ) [ MDEA ] + k2 [ EAE ] [ EAE ]

(50)

The simulations were carried out for the following industrially important conditions (Pohorecki et al., 1979, 1990; Moniuk 2006): • height of column: 20-30 m • column diameter: 2 m • packing of column: steel Pall rings • pressure: 20 - 25 bar • liquid temperature at the inlet to the column (top): 70 - 110 °C • gas temperature at the inlet to the column (bottom): 70 - 110 °C • mole fraction of CO2 in the gas inlet: xA = 0.18 – 0.20 • liquid flow: 345 m3/h

556


• •

gas flow: 79000 m3/h (in normal conditions) amines concentration: MDEA: 30 wt.%, EAE: 2 wt.%.

Mass transfer coefficient in the gas phase, kg, was calculated from Gildenblat correlation (Pohorecki et al., 1978; Ramm, 1976); mass transfer coefficient in the liquid phase kL was calculated from Sherwood and Holloway correlation (Hobler, 1962; Pohorecki et al., 1978). The overall heat of absorption and reaction in aqueous MDEA solution is equal to qR = 48.6 kJ/mol (Critchfield and Rochelle, 1987). The heat of solvent vaporization was calculated from Watson relation (Pohorecki et al., 1978)

T   qw = 40.62 ⋅  2.3615 −  274.1  

0.38

(51)

Some simulation results are presented in Figs. 8 – 11.

Fig. 8. Profiles of CO2 mole fraction in the gas phase, (xA) and gas and liquid temperatures (T) along the column height, h; P=20 bar, Tg =90, TL =90, 30wt.% MDEA + 2wt.% EAE

Fig. 9. Profiles of CO2 mole fraction in the gas phase, (xA) and gas and liquid temperatures (T) along the column height, h; P = 20 bar, Tg = 90, TL =80, 30wt.% MDEA + 0wt.% EAE

557


Fig. 10. Profiles of CO2 mole fraction in the gas phase, (xA) and gas and liquid temperatures (T) along the column height, h; P =20 bar, Tg =90, TL =90, 30wt.% MDEA + 2wt.% EAE

Fig. 11. Profiles of CO2 mole fraction in the gas phase, (xA) and gas and liquid temperatures (T) along the column height, h; P =25 bar, Tg =110, TL =110, 30wt.% MDEA + 2wt.% EAE

In order to determine the most suitable conditions for an industrial process, similar calculations have been carried out for a range of gas and liquid temperatures. It has been found that absorption efficiency of CO2 practically does not depend on the gas temperature but significantly depends on the liquid temperature. It is therefore important to note, that there is a significant temperature increase at the bottom of the column where the rate of CO2 absorption is the highest. It is the result of the high value of the overall heat of absorption and reaction of CO2 in aqueous MDEA solutions. Absorption efficiency significantly depends on EAE concentration. In similar conditions where cEAE = 0 wt.% (Fig. 9) the mole fraction of CO2 at the top of the column decreases only to 8.4% (Fig. 9). For the column height of 30 m (Fig. 10) CO2 concentration decreases to 1%. In conditions similar to the industrial BENFIELD process (CO2 absorption into aqueous potash solutions with DEA, P = 25 bar, tL at the bottom of the column = 110 oC, xA = 0.178) the mole fraction of CO2 at the top of the column decreases to 0.018% (Fig. 11)

558


It has been concluded that the new solvent (aqueous 30% MDEA solutions with 2% EAE) may be promising in the process of gas purification in ammonia production. This solvent has been registered for a patent (Polish Patent Application P391333).

7. CONCLUSIONS In order to develop a new process of synthesis and flue gases purification the measurements of the rate of CO2 absorption into aqueous of MDEA and EAE were carried out in a stirred reactor and the reaction rate constants between CO2 and alkanoloamines were determined. On this basis a mathematical model for the performance of an absorption column operated with aqueous solution of a blend of the above amines at elevated temperatures and pressures has been developed. The results of simulations obtained by means of this model are described. The work is a part of a wider program, aimed of the development of a new technological process.

SYMBOLS A, B, D Am a B b CP c cBo cl DEA DA DB Dg d EAE E Go GM GOM h H Ha Ki kL k *L kg k1 k2 k-2 k21 k22 Lo

components amine interfacial area per unit volume of packing, m2/m3 base stoichiometric coefficient molar heat capacity of the gas, kJ/(kmol⋅K) molar concentration, kmol/m3 amine concentration, kmol/m3 heat capacity of the solution, kJ/(kg⋅K) diethanolamine diffusivity of the absorbed component in the liquid phase, m2/s diffusivity of the liquid component in the liquid phase, m2/s diffusivity of the absorbed component in the gas phase, m2/s stoichiometric coefficient 2 ethylaminoethanol enhancement factor mass gas flow rate per unit column area, kg/(m2⋅s) molar inert gas flow rate per unit column area, kg/(m2⋅s) molar gas flow rate per unit column area, kg/(m2⋅s) packing height, m Henry’s constant, kmol/(m3⋅bar) Hatta number equilibrium reaction (i) constant liquid-film mass transfer coefficient, m/s liquid-film mass transfer coefficient with chemical reaction, m/s gas-film mass transfer coefficient, kmol/(m2 s bar) pseudo-first-order reaction rate constant, 1/s reaction (13) rate constant (from left side to right side), m3/(kmol⋅s) reaction (13) rate constant (from right side to left side), m3/(kmol⋅s) reaction (1) rate constant, m3/(kmol⋅s) reaction (2) rate constant, m3/(kmol⋅s) mass liquid flow rate per unit column area, kg/(m2⋅s)

559


MEA MDEA N Nt Nu P Pr R

r Sc Sh T t x z

monoethanolamine N-methyldiethanolamine molar flux, kmol/(m2·s) heat flux, kJ/(m2·s) Nusselt number total pressure, bar Prandtl number rate of absorption, kmol/(m2·s) gas constant, J/(mol·s) rate of chemical reaction, kmol/(m3·s) Schmidt number Sherwood number absolute temperature, K temperature, oC mole fraction zwitterion

Greek symbols α heat transfer coefficient, kJ/(m2⋅s⋅K) β parameter defined by Eq. (25) λ thermal conductivity, W/(m⋅K) ρ density, kg/m3 Subscripts A g i l w

absorbed component gas interface liquid water

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POLYVINYLPYRROLIDONE-BASED COATINGS FOR POLYURETHANES – THE EFFECT OF REAGENT CONCENTRATION ON THEIR CHOSEN PHYSICAL PROPERTIES

Beata Butruk*, Maciej Trzaskowski, Tomasz Ciach Warsaw University of Technology, Faculty of Chemical and Process Engineering, Department of Biotechnology and Bioprocess Engineering, Waryńskiego 1, 00-645 Warszawa, Poland

A method of manufacturing hydrogel coatings designed to increase the hydrophilicity of polyurethanes (PU) is presented. Coatings were obtained from polyvinylpyrrolidone (PVP) by free radical polymerisation. The authors proposed a mechanism of a two-step grafting – crosslinking process and investigated the influence of reagent concentration on the coating’s physical properties hydrogel ratio (HG) and equilibrium swelling ratio (ESR). A surface analysis of freeze-dried coatings using scanning electron microscopy (SEM) showed a highly porous structure. The presented technology can be used to produce biocompatible surfaces with limited protein and cell adhesive properties and can be applied in fabrication of number of biomedical devices, e.g. catheters, vascular grafts and heart prosthesis. Keywords: polyurethanes, surface modification, surface hydrophilization, hydrogel

1. INTRODUCTION Superhydrophilic surfaces are built of highly hydrophilic polymer chains. Chains are bound to the implant surface in a brush-like manner. In an aqueous medium a significant amount of water is bound between polymer chains (Kim, 2002). Such a surface, consisting of over 90% of water, has very low surface energy, which ensures low protein adsorption adhesion and prevents cell adhesion (Le and Scott, 2010). If a surface is thick enough, proteins protruding from macrophage’s surface do not reach polymer matrix. For instance, a macrophage’s membrane protein CD4 protrudes 14 nm over the cell membrane, while a hydrophilic polymer brush is usually tens of nanometres long (Le and Scott, 2010). Hydrogel polymer surfaces do not activate the immune system, which is a crucial factor in terms of implantable devices (Xu and Siedlecki, 2007). Surfaces of highly water-binding polymers, e.g. polysaccharides and polysaccharide acids, polyethers, polyalcohols and others may be obtained. Negative charge, typical for animal tissues, is favourable. In practice, hyaluronic acid, heparin, polyethylene glycol and polyvinyl alcohol are usually used. Long-lasting implants must be resistant to enzymes and oxidative agents from blood. Polyvinylpyrrolidone (PVP) seems to be a suitable material for this purpose. PVP is characterised by high water-binding properties, high biocompatibility and resistance to mammalian enzymatic apparatus and oxidation (Patel and Mequanint, 2007). Various methods of coating a polymer with a layer of another polymer are known. Generally, physical and chemical methods can be distinguished. Coatings of various thicknesses are obtained easily by most commonly used physical methods. Coatings are bound to the matrix by mechanical forces, Van der Waals forces or polymer chains entanglement. The binding is facilitated by the presence of a *Corresponding author, e-mail: [email protected]

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solvent or higher temperature, which relaxes polymer structure. More sophisticated coatings with a covalently bound polymer network are obtained by chemical methods. These coatings reveal better adhesion to the substrate material and their structure may be precisely designed and produced, regarding both the chemical and physical structure of a polymer network. In this paper the authors present a new method to produce a highly hydrophilic, hydrogel coating for polymer substrates. The presented method is based on free-radical macromolecular polymerisation grafting. Thanks to the combination of slightly water-soluble peroxide absorbed on the substrate and a quick Fenton process (Walling et al., 1974), free radicals are formed mainly on the polymer surface. This assures formation of covalent bonds between polymer substrate and the coating together with a high overall grafting efficiency. The presented coatings are designed to improve hydrophilicity of medical grade polyurethanes (PUs) and can be applied in medical devices that have permanent longterm contact with patients’ tissues. In the previous paper (Butruk et al., 2012) we demonstrated that PVP-based coating significantly improves PU hemocompatibility, which makes the material a potential candidate for use in cardiovascular applications. The aim of this study was to investigate the influence of reagent concentration on the chosen physical properties of the hydrogel layer and to propose the grafting’s mechanism. We also assessed the quality of the fabricated coating in terms of coating’s smoothness, homogeneity and durability.

2. EXPERIMENTAL 2.1. Materials Polyurethane (Chronathane™ P–75A, CardioTech) in a form of discs (40 mm in diameter, 2 mm thick) was used as a polymer substrate for further modifications. Reagents, namely PVP powder with average molecular weight of 360 kDa (PVP360), iron (II) chloride (FeCl2), ascorbic acid (AA), cumene hydroperoxide (CHP), ethylene glycol dimethacrylate (EGDMA) and sodium dodecyl sulfate (SDS) were obtained from Sigma-Aldrich. Toluene was purchased from Chempur, Poland. Phosphatebuffered saline (PBS) was prepared by dissolving 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4 in 1 L of distilled water, pH was adjusted to 7.4. 2.2. The surface modification of polyurethane Hydrogel coatings of PU discs were fabricated in a two-step dip-coating method as described elsewhere (Butruk et al, 2012). Briefly, the PU discs were immersed in a toluene solution containing given amounts of EGDMA and CHP for 10 minutes at 25°C. The samples were then placed in a water solution containing given amounts of PVP, FeCl2 and 0.1% (w/v) AA for 15 minutes at 25°C. After the coating procedure, the polymer discs were washed with 0.1% (w/v) SDS water solution for 5 minutes, followed by washing in water (overnight) and lyophilisation. 2.3. Surface characterisation The materials coated with hydrogel were characterised by the following parameters: the equilibrium swelling ratio (ESR) and the mass of hydrogel grafted to the surface per surface area (HG). Both, ESR and HG were determined gravimetrically using analytical scale (Mettler Toledo) with accuracy 0.0001g. ESR was studied according to ASTM D570 as follows: samples in the form of discs (60 mm in diameter) were dried at 50 °C until a constant weight was reached and weighed. Next, the samples were placed in PBS solution at 37 °C and weighed at different intervals of time until equilibrium swelling was reached. ESR was calculated according to the formula (1), where Weq is the weight of a

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swollen sample at equilibrium, Wd is the weight of a dry sample and W0 is the initial weight of an unmodified dry PU disc. HG was calculated according to the formula (2), where W0 is the initial weight of an unmodified dry PU disc, Wd is the weight of a dry PU disc covered with hydrogel layer and S is the area of a PU disc. Samples for ESR and HG measurements were prepared in triplicates.

ESR =

Weq − Wd Wd − W0

HG =

⋅100%

Wd − W0 S

(1)

(2)

The morphology of hydrogel layers was examined using Scanning Electron Microscopy (Phenom, FEI). Small specimens of the samples were immersed in distilled water for 24 h. To conserve the structure of hydrogel without collapsing, the samples were kept in the temperature of -40°C for 4 h and then freeze dried under vacuum. Finally, the samples were sputtered with gold and the microstructure of hydrogels was depicted.

3. RESULTS AND DISCUSSION 3.1. Mechanism of the coating process Although the presented process is based on a free radical polymerisation, the standard radical-based grafting procedure was modified. To avoid homopolymerisation in the water phase the process was divided into two steps. Thanks to the use of two solutions – organic and water – the reagents were separated, which limited the reaction space (free radicals were formed mainly on the polymer/solution interfacial surface) and increased process effectiveness. In the first step of the process two effects occur: the reagents (CHP, EGDMA) adsorb on the polymer surface and diffuse across the polymer/solution interface, thereafter they accumulate in the thin boundary layer of a polymer matrix. At this stage, time is the limiting factor. The reaction should be long enough for CHP and EGDMA molecules to adsorb. However, its prolongation might lead to their deep penetration inside the polymer. Too deep penetration hinders contact with reagents during the second step of the process and may lead to a potentially toxic effect of the unreacted compounds. In the second step of the process the adsorbed molecules react with the reagents from water solution (iron ions and PVP), which results in the formation of free radicals and chemical bonds between polymer chains. It should be noted that both CHP and EGDMA are slightly soluble in water. For this reason they only partly dissolve in water, while most of the molecules remain adsorbed on the surface and inside the polymer matrix. Ferrous ions present in the water solution diffuse to the material surface where they catalyse the decomposition of hydroperoxide molecule and the formation of free radicals (Fig. 1, reaction 1). A part of radicals formed from CHP decomposition enters into a reaction with water molecules, in which electrons and hydrogen atoms are intermolecularly transferred. As a result highly reactive hydroxyl radicals are formed (Fig. 1, reaction 3). These radicals are capable of capturing protons from polymer molecules, which leads to formation of macroradicals (Fig. 1, reaction 4). A recombination of macroradicals with the formation of carbon-carbon covalent bonds results in (i) the formation of stable chemical bonds between both the modifying (PVP) and the modified (PU) polymer chains, (ii) the cross-linking of modifying polymer and formation of hydrogel structure (Fig.1, reaction 5). The PVP cross-linking is supported by EGDMA adsorbed on the material surface. EGDMA structure contains double bonds that capture unpaired electrons and form ‘bridges’ between adjacent polymer chains. A significant role of regeneration of ferrous ions ‘used’ in the reaction with CHP is played here by ascorbic acid (Fig.1, reaction 2).

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Fig. 1. Radical reactions resulting in formation of hydrogel coating

3.2. Influence of reagent concentrations on coating properties The effect of reagents concentration on the chosen coatings properties was assessed. The equilibrium swelling ratio of a hydrogel coating (ESR) and the mass of hydrogel per surface area (HR) were determined. In our previous studies (Kaźmierska et al., 2010; Paradowska et al., 2010) we have shown that a PVPbased coating can be obtained without the use of free radicals. The mechanism of this process is based on non-covalent hydrogen bonds formed between hydrogen atoms of polyurethane and oxygen atoms present in PVP molecule. However, such bonds are unstable and a limited number of polymer chains can be bound to the modified layer. In the presented study coatings obtained without the use of free radicals (CHP concentration equals 0%) and coatings obtained from solutions containing cumene peroxide (solutions of 2%, 4%, 8%, 12%, 16% v/v concentrations) were compared to prove the positive effect of the radical reaction on hydrogel coating properties. The concentrations of other reagents were constant (0.1% FeCl2, 10% EGDMA, 15% PVP, 1% AA). The obtained values of HG and ESR are shown in Fig. 2.

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Fig. 2. HG and ESR values for various concentrations (0%, 2%, 4%, 8%, 12%, 16%) of cumene hydroperoxide hydrope (CHP) in an organic solution (MV± ±SD, n = 3). The concentrations of other reagents were constant and amounted to: 0.1% FeCl2, 10% EGDMA, 15% PVP, 1% AA

As presented in the diagram, HG values are low (HG = 5.77 g/m2) for coatings obtained by the standard procedure without radical reaction. The swelling ratio for these coatings is also low (ESR ( = 50.14%). This confirms the fact that coatings obtained without free radical species are unstable and the amount bounded PVP is limited. The introduction of of cumene peroxide and the radical formation significantly increased HG and ESR values. Additionally, it was observed that HG and ESR values increased as CHP concentration increased. This correlation is fully understood – the higher the CHP concentration on the material surface, the more free radicals are formed, thereby more PVP chains are bound to material surface. The correlation between CHP concentration and ESR value proves a low degree of hydrogel cross-linking. linking. Thus, it confirms the assumption that radicals radicals are formed mainly on material surface and occur inside the water phase only in a small quantity.

Fig. 3. HG and ESR values for various ethylene glycol dimethacrylate (EGDMA) concentrations (0%, 5%, 10%, 20%, 30%) in an organic solvent (MV±SD, ( n = 3). The concentrations of other reagents were constant and amounted to: 0.1% FeCl2, 8% CHP, 15% PVP, 1% AA

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The correlation between the evaluated parameters and EGDMA concentration is presented in Fig. 3. A set of solutions with five different EGDMA concentrations concentrations (0%, 5%, 10%, 20% and 30%) and a constant concentration of other reagents, namely, 0.1% FeCl2, 8% CHP, 15% PVP and 1% AA was prepared. In the presented method EGDMA is a hydrogel cross-linking cross linking agent. It is noticeable that the presence of EGDMA significantly ificantly affects the properties of coatings. At higher EGDMA concentrations the swelling ratio decreases, which proves a high degree of cross-linking. cross linking. In the absence of crosscross linking agent (EGDMA concentration = 0%) PVP molecules are bound to the material in a limited amount, which results in low HG and ESR values. In the presence of EGDMA, HG values grow significantly; however, a further increase of EGDMA concentration does not affect HG values. As expected, the concentration of cross-linking cross linking agent affects mainly the degree of cross-linking cross and not the coating thickness. The properties of the coatings are also strongly influenced by ferrous ions concentration in water solution. Five ferrous us chloride concentration values were taken into consideration (0,01%, 0,05%, 0,1%, 0,5% and 1% w/v). Every solution contained a constant concentration of CHP (8%), EGDMA (10%), AA (1%) and PVP (15%). The results are presented in Fig. 4. At low Fe2+ concentrations HG and ESR values increase along with the rise of Fe2+ concentration and they reach the highest values for 0.1% concentration (HGmax = 61.69 g/m2) and 0.05% (ESRmax = 445.60%) respectively. A further f increase of Fe2+ concentration results in a significant significant decrease in the values of both parameters. The possible reasons for this correlation are side reactions presented in Fig. 5. The side reactions are catalysed by Fe3+ ions. At a constant concentration of a reducing agent (ascorbic acid), a higher concentration of Fe2+ ions implicates a higher concentration of Fe3+ ions. Fe3+ ions react with radicals, decreasing the amount of radicals in the system, and therefore reducing the effectiveness of hydrogel forming. Another possible explanation assumes that that a high concentration of iron ions provokes fast peroxide decomposition and prevents diffusion of peroxides and radicals into the water phase. Although free radical species are very reactive, they are limited to a narrow space around the polyurethane surface. face. This results in the formation of thin and highly cross-linked cross linked coatings.

Fig. 4. HG and ESR values for various ferrous chloride (FeCl2) concentrations (0.01%, 0.05%, 0.1%, 0.5%, 1%) in a water solution (MV±SD, n = 3). The concentrations of other reagents were constant and amounted to: 8% CHP, 10% EGDMA, 1% AA, 15% PVP

The effect of PVP concentration on coating properties was also assessed. Coatings were obtained using PVP solutions of 0%, 5%, 10%, 15%, 20% and 25% w/v concentrations. The concentrations concentra of other reagents were constant and the values were as follows: 0.1% FeCl2, 8% CHP, 10% EGDMA and 1% AA. The results are presented in Fig. 6. It was observed that PVP concentration highly affected the

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thickness of hydrogel coatings. At higher concentrations more PVP molecules bind to the material surface. Interestingly, too high PVP concentration negatively affects coating’s durability. As the polymer concentration increases, solution density increases significantly. It prevents diffusion of ferrous ions to the material surface; therefore it reduces the effectiveness of polymerisation. Coatings obtained from solutions containing more than 15% PVP were poorly bound to the matrix and were easily removed while rinsing. The degree of cross-linking increases with an increase of solution density. It results in a decrease of adsorbing properties of hydrogel and a decrease of ESR values. A significant decrease of ESR was observed for coatings obtained from over 15% PVP solutions. Furthermore, these coatings showed an unsatisfying quality – too high degree of cross-linking resulted in hydrogel cracking and crushing while drying. The coatings obtained from less concentrated solutions (5%, 10%, 15%) were smooth and strongly bonded to the surface.

Fig. 5. Side reactions decreasing the amount of free radicals in the system

3.3. Surface analysis of the coating The morphology of modified surfaces was analysed by scanning electron microscopy (SEM) The presence of a highly porous structure, which is typical for hydrogels, was confirmed by SEM pictures (Fig. 7a: SEM image of a sample prepared from solutions containing 0.1% FeCl2, 5% CHP, 10% EGDMA, 10 % PVP and 1% AA).

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Fig. 6. HG and ESR values for various PVP concentrations (0%, 5%, 10%, 15%, 25%) in a water solution solutio (MV±SD, n = 3). The concentrations of other reagents were constant and amounted to: 0.1% FeCl2, 8% CHP, 10% EGDMA and 1% AA

Fig. 7. 2D (left) and 3D (right) SEM images of hydrogel-coated hydrogel coated polyurethane (a) and uncoated polyurethane (b)

4. CONCLUSIONS In this paper we proposed a simple method of coating polyurethane materials with hydrogel layer. The modification is based on free radical macromolecular grafting-crosslinking. grafting crosslinking. The fabricated coating is covalently bonded to the polymer substrate, which assures assures a proper durability. The presented results showed that concentrations of all the reagents used in the fabrication process strongly influences the physical properties of the obtained hydrogel layers. The coating was designed to increase hydrophilicity of the implantable polymer materials. Both polymers applied in the presented technology, PU and PVP, are highly biocompatible and widely used in biomedical applications. Thus, the presented technology can be used to produce biocompatible surfaces with limited limited protein and cell adhesive properties and can

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be applied in the fabrication of a number of biomedical devices, e.g. catheters, vascular grafts and heart prosthesis.

The presented work has been supported by the Polish Artificial Heart Project and the European Union in the framework of European Social Fund through the Warsaw University of Technology Development Programme. Patent applications for the presented coating technology are pending.

SYMBOLS ESR HG PU PVP SEM

equilibrium swelling ratio, % mass of hydrogel grafted to the surface per surface area, g/m2 polyurethane poly (vinyl pirrolidone) scanning electron microscope

REFERENCES Butruk B., Trzaskowski M., Ciach T., 2012. Fabrication of biocompatible hydrogel coatings for implantable medical devices using Fenton-type reaction. Mat. Sci. Eng.: C., 32, 1601–1609. DOI: 10.1016/j.msec.2012.04.050. Kaźmierska K., Szwast M., Ciach T., 2010. Determination of urethral catheter surface lubricity. J. Mater. Sci. Mater., 19, 2301-2306. DOI: 10.1007/s10856-007-3339-4. Kim J.H., Kim S.C., 2002. PEO-grafting on PU/PS for enhanced blond compatibility – effect of pendant length and grafting density. Biomater., 23, 2015-2025. DOI: 10.1016/S0142-9612(01)00330-1. Le Y., Scott M., 2010. Immunocamouflage: The biophysical basis of immunoprotection by grafted methoxypoly(ethylene glycol) (mPEG). Acta Biomater., 6, 2631-2641. DOI: 10.1016/j.actbio.2010.01.031. Patel A, Mequanint K., 2007. Novel physically crosslinked polyurethane-block-poly(vinyl pyrrolidone) hydrogel biomaterials. Macromol. Biosci., 7, 727-737. DOI: 10.1002/mabi.200600272. Paradowska A.E., Kaźmierska K.A., Ciach T., 2010. Influence of the coating process parameters on the quality of PUR/PVP hydrogel coatings for PVC medical devices. Pol. J. Chem. Technol., 12, 38-45. DOI: 10.2478/v10026-010-0016-z. Walling C., El-Taliawi G.M., Johnson R.A., 1974. Fenton's reagent. IV. Structure and reactivity relations in the reactions of hydroxyl radicals and the redox reactions of radicals, J. Am. Chem. Soc., 96, 133-139. DOI: 10.1021/ja00808a022. Xu L., Siedlecki C.A., 2007. Effects of surface wettability and contact time on protein adhesion to biomaterial, Biomater., 28, 3273-3283. DOI: 10.1016/j.biomaterials.2007.03.032.

Received 18 May 2012 Received in revised form 16 November 2012 Accepted 16 November 2012

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DESIGNING OF MEMBRANE CONTACTORS WITH CROSS-COUNTER CURRENT FLOW Andrzej B. Kołtuniewicz*1, Szymon Modelski2 and Anna Witek2 1

Warsaw University of Technology, Faculty of Chemical and Process Engineering, Waryńskiego 1, 00-645 Warszawa, Poland

2

Wroclaw University of Technology, Faculty of Chemistry, Norwida 4/6, 50-373, Wroclaw, Poland

The knowledge about membrane contactors is growing rapidly but is still insufficient for a reliable designing. This paper presents a new type of membrane contactors that are integrated with one of the following ways of separation by using absorbents, micelles, flocculants, functionalized polymers, molecular imprints, or other methods that are based on aggregation. The article discusses methods for designing multi-stage cascade, usually counter-current. At every stage of this cascade, relevant aggregates are retained by the membrane, while the permeate passes freely through membrane. The process takes place in the membrane boundary layer with a local cross-flow of the permeate and the retentate. So the whole system can be called a cross-counter-current. The process kinetics, k, must be coordinated with the permeate flux, J, and the rate of surface renewal of the sorbent on the membrane surface, s. This can be done by using ordinary back-flushing or relevant hydrodynamic method of sweeping, such as: turbulences, shear stresses or lifting forces. A surface renewal model has been applied to adjust the optimal process conditions to sorbent kinetics. The experimental results confirmed the correctness of the model and its suitability for design of the new type of contactors. Keywords: membrane contactors, sorption processes, integrated processes of separation, designing, surface renewal theory

1. INTRODUCTION A key concept for the use of membrane in contactors as artificial surface between the phases for the purpose of the processes of mass or heat transfer (Drioli and Giorno, 2005). Although the physical mechanisms are essentially the same, as in classical unit processes, they are generally much less dependent on fluid dynamics and relevant problems. Conventional unit processes, e.g. distillation extraction, absorption have large process constrains. These are high pressure drops, high energy consumption, a narrow range of performance, several disturbances as entrainment, weeping, mixing, emulsification, foaming, etc. Membrane contactors are involved in process intensification defined by Stankiewicz and Moulijn (2004), as "…the development of novel apparatuses and techniques that, compared to those commonly used today, are expected to bring dramatic improvements in manufacturing and processing, substantially decreasing equipment-size/production-capacity ratio, energy consumption, or waste production, and ultimately resulting in cheaper, sustainable technologies." Currently, eight types of well-known contactors found commercial applications, namely: membrane scrubbers, strippers, extractors, distillers (with regard to osmotic distillation), emulsifiers, supported liquid membranes and reactors for phase transfer catalysis. However, many other types have not as yet found practical application in accordance with an “open definition” of contactors by Drioli *Corresponding author, e-mail: [email protected]

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and Giorno (2005): “All operations that are based on the mass transport between two contacting phases can be in principle carried out by membrane contactors”. The importance of integrated membrane processes in sustainable technologies was presented in the literature (Koltuniewicz and Drioli, 2008; Koltuniewicz, 2010; Koltuniewicz, 2011). The new contactors such as membrane adsorption were discussed in detail by (Koltuniewicz et al., 2004) with its specific case of the membrane biosorption (Koltuniewicz and Bezak, 2002). The main idea of the practical application of biosorption is the use of waste materials that can be reused according to the idea of sustainable development and clean technologies. The function of the membrane is a mechanical separation of finely powdered sorbents in aqueous suspensions. Biosorption mechanisms are similar to ordinary adsorption with a high percentage of ion exchange. Another example for membrane sorption was an integrated process using N-methyl glucamine chelating resins with submerged microfiltration membrane for removal of resin-boron complexes (Yilmaza et al., 2004). In a series of articles the effects of micelles supporting the performance of contactors during removal of xenobiotic with membrane extraction (Witek et al., 2006, 2009) as well as membrane absorption (Modelski et al., 2011) were presented. Membrane contactors success is due to the fact that they can be easily retrofitted to conventional industrial technologies leading to considerable reduction of energy consumption and feasibility, or compliance with increasingly stringent environmental requirements. This was made possible through a development of nanotechnology and materials science necessary to produce a membranes (Drioli and Giorno, 2005).

2. DESIGNING OF THE CO-CURRENT AND COUNTER-CURRENT CONTACTORS The Membrane contactor consists of a membrane that two flow regions allows separation between the two phases, where mass transport takes place. The membrane acts as a barrier between the two fluid phases and selectively allows or prohibits the transport of one or more chemical species or particles from one fluid stream to the other. Membranes with specific properties should be used for contactors of membranes, e.g.: microporous with uniform pores and/or composite which can be hydrophobic/hydrophilic, symmetric/asymmetric, and dense/microporous. Pore size distribution should be as sharp as possible in order to protect against exceeding the breakthrough pressure, which can be calculated from a modified Young-Laplace equation:

∆P =

2 ⋅ σ ⋅ cos (θ ) d

(1)

A plant construction containing membrane contactors is very simple because of the modularity and linear scale-up. Assuming that the plant operates uniformly along the entire length, L, and that it uses counter current flow of phases (see Fig. 1), the following balance equation can be written:

v0 ln ci − v0 ln co = k ⋅ a ⋅ L

(2)

Based on this equation, one can precisely calculate the required number of membrane modules to ensure effective separation. A typical system consists of a cascade of membrane modules connected in series counter-currently, to ensure maximum driving force of the process. However, in some cases, system shall be co-current (Fig 1a). Such cases occur when the flow resistances of the media are too large or the membranes are of poor quality (too high wettability, far too large pores or too diffuse distribution of their size). Overall mass transfer coefficient can be calculated after the calculation of the local mass transfer coefficients on both sides of the membrane k1 and k2 as well as in the membrane, according to the following equation (Eq. 3):

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Designing of membrane contactors with cross-counter current flow

1 1 1 K = + + kov k1 km k2

(3)

where K is the equilibrium constant, appropriate for a particular system in which the relevant phase 1 and 2 are defined as gas-liquid (in membrane absorption) or liquid-liquid (for membrane extraction or membrane distillation). An adequate arrangement of the two phases relative to the membrane must be ensured, i.e., which phase flows inside the capillary (inside tube) or outside (shell side). A model takes into account the general case where resistances in both phases are approximately equal. Usually, in specific cases, resistance in one phase is dominant. Then, we consider only the resistance of this phase and the resistance of the membrane, which can be determined by Wilson method discussed in many publications on membrane contactors (e.g. Koltuniewicz, 2010). During calculations, mass transfer coefficient in the membrane must take into account the effective diffusion coefficient, which may differ considerably from physical diffusivity.

km =

Def

δ ef

=

Dm ⋅ ε m δ m ⋅τ m

(4)

This may be due to tortuosity, porosity and incomplete filling volume of membrane pores. Consequently, the diffusion path is not equal to the thickness of the membrane and the surface of mass transfer is smaller than the membrane surface due to its porosity. The pore size distribution in membrane is another reason for relevant inaccuracies. A variable diameter of the pores can lead to excess of the “breakthrough pressure” (see Young-Laplace formula, Eq. 1) in the larger pores of the membrane. This in turn can lead an uneven filling of the pores of phases 1 and 2 depending on the pressure distribution in the module. This phenomenon may be more pronounced at high hydraulic resistance and/or a large number of degrees in a cascade of membrane modules working in a counter current system. It should be noted that during designing, the engineer can (to some extend) affect the value of the overall mass transfer coefficient through the appropriate selection of flow-rates and to proper choice of membrane properties. The rule is to keep the phase in which we expect a larger resistance of mass transfer on the outer membrane (shell side). Membrane resistance is the inverse of the mass transfer coefficient, which may be calculated from one of the two equations presented in the book (Reed et al., 1995): • for shell (outer) side of the membrane: 1/3

 d ⋅ v1  k ⋅d Sh1 = 1 1 = 1.4 ⋅   D1  D1  •

(5)

for the lumen (internal) side of the capillary membrane (Leveque formula): 1/3

 d 2 2 ⋅ v2  k2 ⋅ d 2 Sh2 = = 1.62   D2  L2 ⋅ D2 

(6)

Here, subscripts 1 and 2 refer respectively to the two phases. It should be noted that phase flows can be optimised in terms of kinetics, membrane surface or overall costs of the entire plant. In this case, the hydrodynamic limits as the pressure drops and the associated breakthrough of the membrane must be taken into account. When these optimised flows (v1 and v2) are different than those resulting previously from the feeds assumed at the beginning of the calculations, recirculation is used at each stage of the cascade plant. Then the flow rate required to maximise the performance of the contactor is called an “excess sweep”.

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The cross-counter-current current configuration may be used, for development of the new integrated processes based on the contact of the solid and liquid phases at appropriate membrane surface, which will be discussed in the next part.

a)

b)

c)

Fig. 1. Configurations of membrane modules in the contactors systems: a) co-current co current flow, b) counter-current counter flow, c) cross-counter current flow

3. DESIGNING CROSS-COUNTER-CURRENT CROSS CURRENT CONTACTORS A new type of an integrated process was designed to increase the productivity productivity and efficiency of separation, which involves the creation of larger aggregates prior to their separating on the membrane. During the separation of these larger aggregates, the flux is much larger and the flow resistance is lesser, and consequently, membrane separation becomes more efficient and cheaper. To increase the size of separated particulates or molecules, the following ways of aggregation are used : • adsorption dsorption on pulverised adsorbent, • biosorption iosorption on microorganisms, microorganisms • complexation, chelation, • binding inding on functionalized polymers , • binding inding on ion exchange resins, resins • binding inding on molecularly imprinted materials, materials • coagulation, flocculation, • precipitation, • micellar solubilisation, etc.

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In leaching, physical, chemical or biological sorption; ion exchange, the membrane allows to use grains with a high degree of dispersion, and the only limit is the cost of such disintegration processes. The benefit of much dispersed grains is a larger contact surface (specific surface a = 1/d), which improves orption uptake, an increase of sorption kinetics due to the shortened diffusion paths. Inside the singular stage of a cascade (Fig. 1c), the retentate containing a suspension of such aggregates flows parallel to the membrane surface and permeate passes through the membrane in a direction perpendicular to its surface without particles or aggregate. During a relevant process, the liquid passes through the filter cake layer formed of aggregates retained on the membrane surface. This is the place where the appropriate physical or chemical processes occur much faster than in the bulk of the retentate stream. The reason is the increased concentration of sorbents, catalyst, the micelles, etc., in comparison to the bulk. But the main problem is a small volume of a thin film formed in the vicinity of the membrane and immobility, which facilitates its rapid saturation. However, it is possible to permanently remove aggregates from the surface of the membrane after strictly defined time using well known methods to reduce the polarisation layer. However first one has to check whether membrane s were chosen correctly and whether aggregates form a cake (which is proper), or simply block the membrane pores. This can be done, by using the following formula (Eq. 7) (Koltuniewicz and Field, 2004): 1

J ( t ) =  J 0n −2 − k ⋅ A2−n ⋅ ( n − 2 ) ⋅ t  n−2

(7)

Where A is the membrane surface, J is the permeate flux, t is the current time and the k, and n are the parameters. After an experimental determination of flux decline J(t) in time, under certain process conditions one can identify the parameter n in Eq. (7). If the value of n is close to zero (a good case), then there is a cake on the membrane surface, but when it is close to 2 (poor case), then pores are blocked at membrane and a membrane with smaller pores must be chosen. In the case of “cake” microfiltration, appropriate conditions of retentate flow (tangentially to the membrane surface) can be applied to remove the cake with the specified rate. The rate of renewal of the membrane surface can be determined from the experimental formulas (Koltuniewicz, 1992; Koltuniewicz and Field, 1996):

s = m ⋅ un

(8)

Where u is an average axial velocity of the fluid in membrane module, and the parameters m and n (in Eq. 8) can be determined from Table 1. Table 1. Experimentally determined parameters for calculation of surface renewal rate

Module type

Constant m

Constant n

plate and frame (slot 0.15m x 0.001m)

0.00074

0.75

tubular ceramic (Membralox® 19 tubes)

0.0020

0.80

capillary (Romicon® (d = 1mm)

0.0035

0.66

The rate of surface renewal can be also evaluated using Einstein and Lee (1956) formulae (Eq. 9): s = 0.392 ⋅ f ⋅

τ ρ ⋅µ

(9)

Where: f - fanning factor, τ - shear stress, ρ - liquid density and µ - viscosity. Considering the critical flux model, the sorbent residence time at the membrane surface (on retentate side) can be also controlled. Then flux must be lower than the lateral migration velocity (Eq. 10):

u y 0 = 0.577 ⋅

ρ ⋅ r3 ⋅ γ 2 16µ

(10)

577


Where: r - particle dimension (radius), γ - the shear rate, ρ - liquid density and µ - viscosity. In any case, the back-flushing may also be used to remove sorbent from membrane surface with a given rate to control adequately the kinetics of sorption.

4. EQUILIBRIUM AND KINETICS OF MEMBRANE SORPTION For further analysis there is a need to focus on a particular process, which may be called a membrane sorption process. Equilibrium sorption isotherms are shown in Table 2. The Langmuir isotherm is frequently applied, because of its universal character describing not only the physical adsorption but generally the binding of a solute, to the active sites of sorbent. Sorption isotherms, describing the equilibrium are shown in Table 2. Table 2. Isotherms of equilibrium of the biosorption process (Pagnanelli et al., 2003)

Isotherm Langmuir Freundlich

Equation

q = qmax ⋅

b ⋅C 1+ b⋅C 1

q = k ⋅Cn 1

Langmuir–Freundlich

Radke and Prausnitz Reddlich–Peterson

q = qmax ⋅

b ⋅C n 1

1+ b ⋅C n 1 1 1 = + q a ⋅ C b ⋅ Cn q=

a ⋅C 1+ b ⋅Cn

The kinetics with which the process works is very important for engineering aspects and practical reasons. Usually it is assumed that the rate of saturation of the active sites of sorbent is a reaction of the first order or pseudo-first order. Then one can determine the kinetic constant, k, of the sorbent during the sorption time based on the formulas in Table 3. Table 3. Kinetic equations of the biosorption process (Pagnanelli et al., 2003)

Reaction type

Equation

First-order reaction

q ( t ) = q∗ ⋅ (1 − e− k⋅t )

Pseudo-second order reaction

578

q (t ) =

t 1 t + ∗ ∗ k ⋅q q


A component of the solution is adsorbed during the flow through the sorbent layer situated on the membrane. If this layer is regenerated ( q0 ≠ 0 ), then the concentration of solute in the sorbent can be calculated from the formula: q ( t ) = q ∗ − ( q 0 − q ∗ ) ⋅ e − kt

(11)

The concentration in the permeate flowing out from the sorbent layer can be calculated from the mass balance of the solute in an open system including local permeate flow through the layer in which there is a partial accumulation of the solute system from the formula (Koltuniewicz and Bezak, 2002; Koltuniewicz et al., 2004)

k ⋅ X m ⋅ δ ⋅ ( q∗ − qR )  − kt − J t  ⋅ e − e δ  CP ( t ) = CR − J − k ⋅δ  

(12)

As shown above (see Eq. 12 and Fig. 2), concentration in the permeate decreases initially from initial concentration CR, then after reaching the minimum C Pmin , to grow with the progress of saturation of the

(

)

sorbent, reaching at last the concentration CR at the incoming retentate. Time t C Pmin after which the concentration of the permeate reaches the minimum value, indicates the need to exchange the sorbent layer. Biosorption ceases, when concentration of the solute in biosorbent reaches equilibrium with respect to the concentration in the retentate, CR.

 kδ  ln   J t ( CPmin ) =   J k−

(13)

δ

Equation (Eq. 13) can be used to determine the time between successive cycles of back-flushing. This case assumes that the sorbent layer on the membrane is immobile but homogenous with a constant thickness, δ.

5. RENEWAL OF MEMBRANE BOUNDARY LAYER Membrane sorption can also be carried out without back-flushing due to permanent renewal of sorbent layer at a membrane surface. A proper choice of process conditions enables to select of optimal values of the surface renewal rate (Eq. 18), which maintains a minimum concentration in permeate at a constant level, for a long time (Koltuniewicz et al., 2004). For this purpose the theory of surface renewal by Danckwerts may be used. According to the surface renewal theory, the entire surface of the membrane is covered with infinitely small fragments with different "ages" from zero to infinity. Practically, the age of the oldest element of can never exceed tp the real time of the process itself i.e., from the start. This is due to the stochastic action of eddies that penetrate the membrane surface randomly at any time and place. Then the membrane is covered with a mosaic of elements of the sorbent and the concentration of permeate has an average value of the local concentration C(t) consistent with age distribution of those elements f(t). tP

C = ∫ C ( t ) ⋅ f ( t ) ⋅ dt m P

(14)

0

The local concentration can C(t) is calculated from Eq. 12. The age distribution function after Danckwerts was modified by (Koltuniewicz, 1992), who made the assumption that the age of the oldest element age cannot exceed the duration of the process. Then he gave another form of this function

579


where the age is dependent on the duration of the process (tp) taking into account the possibility of “aging” of surface elements.

s ⋅ e− st f (t ) = − st 1+ e p

(15)

But after infinite time, both functions are converging. After integrating (Eq. 14), followed by substituting (Eq. 12) and (Eq. 15), the equation (Eq. 16) is obtained as a result. This allows to determine the average solute concentration in the permeate passing through the membrane and cake, in the real-time of the process, tp: J   −  + s  ⋅t p  ∗ − ( k + s )⋅t p δ    k ⋅ X m ⋅ δ ⋅ ( q − qR ) − e − e s 1 1 m CP = CR −  − ⋅ ⋅ − s⋅t  J J − k ⋅δ 1− e p +s   k+s δ  

(16)

Eq. (16) describes changes in concentration of permeate during process time, tp, due to “ageing” (i.e., saturating) of the elements’ population on the membrane surface. Since the age distribution approaches the stable distribution after a long-time process, the concentration in permeate will be time independent and it approaches the constant asymptotic value:

CPm = CR − X m ⋅ ( q∗ − qR ) ⋅

k ⋅δ ⋅ s ( J + s ⋅δ ) ⋅ (k + s)

(17)

The best hydrodynamic conditions in the module could be determined using the optimal rate of surface renewal which can be derived from Eq. (18):

sopt =

k⋅J

δ

for CPmin

when

dCPm =0 ds

(18)

The mathematical model has been confirmed many times during membrane biosorption and adsorption studies (Koltuniewicz and Bezak, 2002; Koltuniewicz et al., 2004; Witek et al.. 2006; 2009). But the most spectacular finding was the application of the model to describe the MEU (micelle enhanced ultrafiltration), which can be described as a special kind of integrated process with micellar aggregation. In this case, the model allowed to confirm, two theses. The first was the experimental confirmation of the model suitability for a generalized description of various membrane sorptions. The second objective of this experiment was to document the suitability the membrane in the process of micellar solubilisation of cresol.

6. EXPERIMENTAL The above mentioned apparatus, i.e. membrane contactor can be applied to several different processes that are listed in paragraph 3 of this article. Experimental verification of the model was carried out on the example of micellar solubilisation of cresol, followed by membrane separation of aggregates. The trials were carried out with polysulphone ultrafiltration membrane which does not reject cresol. However, the formation of micelles in this case, explains the visible effect of cresol retention. The micelles were formed at the surface of the membrane due to concentration polarization, although in the bulk of the retentate, surfactant concentration was below the CMC. In this way, it was possible to keep the formation of micellar aggregates within the retentate, based on the presence of cresol within the permeate. The feed solution was prepared by mixing desired amounts of surfactant below CMC and p-cresol (p–cresol, C7H7ClO M = 142.6 POCh Gliwice) with concentration of 0.1% mass. The experiments were conducted in a dead-end system using the surfactant CTAB (C19H42BrN, M = 364.45,

580


CONCENTRATION OF THE SOLUTE IN THE PERMEATE [mM/mL]

CMC = 1 mM/l) with different concentrations (C = 0.113 mM and C = 0.101) but far below CMC = 1 mM. The concentrations of surfactant CTAB were analysed by UV/VIS spectrophotometer (Schimadzu UV-160A) at wavelength of 525 nm. The concentration of p-cresol was analysed using gas chromatography (Schimadzu GC14A). Normally, aggregates are formed after exceeding CMC (critical micelle concentration). In this case, concentration of surfactant is smaller (one order of magnitude lesser than CMC), but at the membrane surface it exceeds the CMC, allowing the formation of micelles and, consequently, the retention of cresol. However, for a higher surfactant concentration the higher separation effect was visible, which indicates a greater number of a micelles. Separation of micellar aggregates of cresol on the membrane, indicate a distinct, but temporary effect of separation, because the "saturation" of surfactant (above CMC concentration) at the membrane surface. Similar observations were reported in many other membrane-assisted integrated processes, such as biosorption, adsorption, ion exchange, etc. The effect of saturation can be seen on the membrane surface in membrane reactors, especially with a deficit of catalyst in the reactor bulk. Here, the role of the membrane may be to increase the concentration of sorbent, resin and catalyst. However, in the case of sorption or reaction that takes place in thin layers near the membrane surface, saturating effect of such small layers is visible. This creates the need for renewal of these layers in a given time and a fixed rate and can be done using the methods and models presented in this article. 0.12

0.10

0.08 experiment (dead-end): C = 0.101 mM

0.06

experiment (dead-edn): C = 0.113 mM

0.04

model model model model

(dead-end, Eq. 12): C = 0.101 mM (dead-end, Eq. 12): C = 0.113 mM (cross-flow, Eq.16): C = 0.113 mM (cross-flow asymptote, Eq. 17 and 18)

0.02

0.00 0

50

100

150

200

250

300

TIME [s] Fig. 2. Effect of membrane separation of micellar aggregates of cresol below CMC in the bulk. J = 10 LMH, k = 2.75 s-1, q* = 0.1 g met/g sorb, Xm = 6 g sorb/l sol, δ = 2.8 µm, sopt = 0.505 s-1

7. CONCLUSIONS •

The new separation processes based on the two steps, i.e.: 1) aggregation of particles with different methods, then followed by 2) membrane separation, may be classified as integrated membrane processes and they can be conducted in membrane contactors. Such processes may include: adsorption on pulverised adsorbent, biosorption on microorganisms or pulverised biomass, complexation, chelation, binding on functionalized polymers, binding on pulverised ion exchange resins, molecularly imprinted materials, and many others such as membrane enhanced coagulation, flocculation, precipitation, crystallization, and micellar solubilisation, etc.

581


•

The new class of separation processes can be performed by means of membrane contactors working in multistage “cross-counter-current” systems. The whole system is counter current whereas the cross-flow is in each stage. The application of very fine particles enhances equilibrium and kinetics of membrane sorption.

•

The usefulness of surface renewal theory for the description of (cross-counter-current) membrane contactors working in the liquid-solid was confirmed in this article (see Fig. 2).

SYMBOLS a C D d f(t) f J K k k L m, n ∆P s q t v X

surface area, m2/m3 concentration of the solute in the fluid, kg/m3 diffusion coefficient, m2/s diameter, m age function, friction coefficient (Fanning factor) permeate flux, m/s = m3/(m2s) equilibrium constant kinetic constant, s-1 mass transfer coefficient, m/s length of the total cascade of contactors. constants at Eq. 7, 8 and Table 1 breakthrough pressure, Pa the rate of surface renewal, s-1 sorption uptake, time, velocity of the fluid, m/s concentration of the sorbent, kg/m3

Greek symbols δ thickness of the membrane, ε porosity, dimensionless γ shear rate, s-1 µ liquid viscosity, kg/(m s) ρ liquid density, kg/m3 σ surface tension of the liquid, N/m τ tortuosity factor, dimensionless τ shear stress, Pa θ contact angle, rad Superscripts (1, 2,…, n) number of stage (*) equilibrium value m mean value Max maximum value Min minimum value Subscripts 1, 2 number of component h hydraulic (diameter) i at the inlet

582


o v m P R

at the outlet overall membrane permeate retentate REFERENCES

Ding H.B., Carr P.W., Cussler E.L., 1992. Racemic leucine separation by hollow-fiber extraction. AIChE J., 38, 1493-1498. DOI: DOI: 10.1002/aic.690381002. Drioli E., Giorno L., 2005. Membrane Contactors: Fundamentals, 1st edition, ELSEVIER. Commercial Brochure of Liqui Cel®, Membrana, 2012. North Carolina, USA. Einstein H.A., Li H., 1956. The viscous sublayer along a smooth boundary. J. Eng. Mech. Div. ASCE, 82, EM2. Koltuniewicz A.B., 1992. Predicting permeate flux in ultrafiltration on the basis of surface renewal concept. J. Membrane Sci., 68, 107-118. DOI: 10.1016/0376-7388(92)80153-B. Koltuniewicz A.B., Field R.W., 1996. Process factors during removal of oil-in-water emulsions with cross-flow microfiltration. Desalination, 105, 79- 89. DOI: 10.1016/0011-9164(96)00063-X. Koltuniewicz A.B., Bezak K., 2002. Engineering of membrane biosorption. Desalination, 144, 219-226. DOI: 10.1016/S0011-9164(02)00315-6. Koltuniewicz A.B., Witek A., Bezak K., 2004. Efficiency of membrane-sorption integrated processes. J. Membrane Sci., 239, 129–141. DOI: 10.1016/j.memsci.2004.02.037. Koltuniewicz A.B., Drioli E., 2008. Membranes in clean technologies, theory and practice. Wiley-VCH. Koltuniewicz A.B., 2010. Integrated membrane operations in various industrial sectors, In: Drioli E., Giorno L. (Eds.), Comprehensive membrane science and engineering. ELSEVIER, chapter 4.05.1. Koltuniewicz A.B., 2011. Process engineering for sustainability, In: Encyclopedia of Life Support Systems. UNESCO EOLSS, chapter 6.34.7.1. Modelski Sz., Kołtuniewicz A.B., Witek-Krowiak A., 2011. Kinetics of VOC absorption using capillary membrane contactor. Chem. Eng. J., 168, 1016–1023. DOI: 10.1016/j.cej.2011.01.075. Kumar P.S., Hogendorn J.A., Feron P.H.M., Versteeg G.F., 2002. New absorption liquids for the removal of CO2 from diluted gas streams using membrane contactors. Chem. Eng. Sci., 57, 1639-1651. DOI: 10.1016/S00092509(02)00041-6. Pagnanelli A.F., Beolchini F., Di Biase A., Veglio V., 2003. Effect of equilibrium models in the simulation of heavy metals biosorption in single and two-stage UF/MF membrane reactor systems. Biochem. Eng. J., 15, 27– 35. DOI: 10.1016/S1369-703X(02)00179-1. Reed B.W., Siemens M.J., Cussler E.L., 1995. Membrane contactors, In: Noble R.D., Sern S.A. (Eds.), Membrane Separations Technology, Principles and Applications. ELSEVIER SCIENCE, Amsterdam, the Netherlands, Chapter 10. Stankiewicz A., Moulin J.A., 2004. Re-engineering the chemical processing plant. Process intensification. Marcel Dekker, Inc., New York. Witek A., Koltuniewicz A.B., Kurczewski B., Radziejowska M., Hatalski M., 2006. Simultaneous removal of phenols and Cr3+ using micellar-enhanced ultrafiltration process. Desalination, 191, 111–116. DOI: 10.1016/j.desal.2005.05.024. Witek A., Szafran R.G., Koltuniewicz A.B., 2006. p-Cresol removal using a membrane contactor enhanced by the micellar solubilization, Desalination, 200, 575–577. DOI: 10.1016/j.desal.2006.03.453. Witek-Krowiak A., Szafran R.G., Koltuniewicz A., 2009. Application of a membrane contactor for a simultaneous removal of p-cresol and Cr(III) ions from water solution, Desalination, 241, 91-96. DOI: 10.1016/j.desal.2007.11.083. Yilmaza I., Kabay N., Bryjak M., Yüksela M., Wolska J., Koltuniewicz A.B., 2006. Submerged membrane–ionexchange hybrid process for boron removal. Desalination, 198, 310–315. DOI:10.1016/j.desal.2006.01.031. Received 16 May 2012 Received in revised form 22 November 2012 Accepted 22 November 2012

583


FERMENTATIVE HYDROGEN PRODUCTION - PROCESS DESIGN AND BIOREACTORS

Małgorzata Waligórska* A. Mickiewicz University, Faculty of Chemistry, ul. Umultowska 89b, 61-614 Poznań, Poland

Substitution of fossil fuels with alternative energy carriers has become necessary due to climate change and fossil fuel shortages. Fermentation as a way of producing biohydrogen, an attractive and environmentally friendly future energy carrier, has captured received increasing attention in recent years because of its high H2 production rate and a variety of readily available waste substrates used in the process. This paper discusses the state-of-the-art of fermentative biohydrogen production, factors affecting this process, as well as various bioreactor configurations and performance parameters, including H2 yield and H2 production rate. Keywords:

biohydrogen production, hydrogen yields

fermentation,

bioreactors,

operational

parameters,

1. INTRODUCTION Energy is a factor significantly influencing the development of civilization. In 2010, its world consumption rose by 5.6 % compared to previous year and equaled 12,002.4 mln tonnes oil equivalent. 87 % of the used energy was generated from fossil fuels (33.5 % crude oil, 29.6 % coal, 23.8 % natural gas) (Statistical Review of World Energy, 2011). If the global population increases by 1.4 billion people over the next 20 years, and assuming an economic growth rate of 3.7%, it has been assessed that the global energy demand will rise by 39 % by 2030 (Statistical Review of World Energy, 2011), whereas in 2100, it can reach a value 3.5 times higher than nowadays (Kruse et al., 2005). Analysing the data concerning fossil fuels, it seems that their reserves are not sufficient to meet the energy demand by the end of 21st century (Ball and Wietschel, 2009). Therefore, it is necessary to research and develop energy production technologies based on alternative sources. An important cause for these actions is also the unequal distribution of fossil fuels. The demand for this type of fuels in developed countries forces them to import these fuels without having any influence over their price. The intention of developed countries to become independent from the uncertain import of energetic materials, along with the fact that fossil fuels are the main source of pollution and global climate changes, has become an additional incentive to search for new, renewable energy sources. In the future, hydrogen can become an important energy carrier as it can be obtained from water electrolysis, pyrolysis, and biomass gasification, methanol and ethanol reforming, as well as biological decomposition of water and organic compounds (Balat, 2008; Demirbas, 2011; Ni et al., 2007; Palo et al., 2007; Stodolny and Łaniecki, 2009; Waligórska and Łaniecki, 2005). Taking into account the current state of the art, hydrogen production by fermentation, using wastewater, cellulose biomass and solid waste from various industry branches is a promising biological method.


585

M. Waligórska, Chem. Process Eng., 2012, 33 (4), 585-594

2. METABOLISM OF FERMENTATIVE HYDROGEN PRODUCTION Fermentation is an anaerobic process, in which hydrogen can be obtained from carbohydrate-rich substrates. This process, easier and cheaper compared to other biological methods, allows to obtain a high rate of hydrogen production. However, its bottleneck is low hydrogen production yield due to the creation of reduced organic compounds (Lee et al., 2008 b). During glycolysis, carbohydrates are decomposed to pyruvate, and its further fermentation depends on the type of bacteria (Hallenbeck, 2009). In the presence of facultative anaerobes (Enterobacter, E. Coli, Klebsiella), pyruvate is converted by means of pyruvate: formate lyase to acetyl-CoA and formate. An increase in the formate concentration leads to a pH drop and induction of the formate: hydrogen lyase, which catalyses its decomposition into CO2 and H2. One glucose molecule can be converted into two formate molecules, so the maximum hydrogen production yield is 2 moles per mole glucose (Hallenbeck, 2009). Strict anaerobes (Clostridium, Ethanoligenens) convert pyruvate into acetyl-CoA and CO2, also producing reduced ferredoxin (Fdred), which transfers electrons to [FeFe] hydrogenase catalysing the generation of hydrogen. It produces 2 moles H2 per mole of glucose. Additional quantities of hydrogen can also be produced thanks to the activity of NADH: ferredoxin oxidoreductase, resulting in the oxidation of NADH and production of reduced ferredoxin, which can be subsequently used for proton reduction (Hallenbeck, 2009). Assuming that glycolysis results in the production of two moles of NADH, one could potentially gain two extra moles of hydrogen from this pathway. Therefore, Clostridium bacteria are capable of producing 4 moles of hydrogen per mole of glucose (Angenent et al., 2004). The redox potential of hydrogen (-0.42 V, at pH 7) is, however, much lower than that of NADH/NAD pair potential (-0.32 V), hence the production of hydrogen using electrons from NADH is possible only at a very low partial pressure of hydrogen (less than 10-3 atm.) (Angenent et al., 2004; Hallenbeck , 2009). It is worth noting that from an evolutionary point of view, fermentation is a process which enables organisms to gather energy (ATP) through substrate-level phosphorylation, a thermodynamically controlled process. It has been concluded that regardless of the environmental pH the three reactions in which ATP is created have large negative values of free energy: glycolysis, acetate production and butyrate production (Lee et al., 2008 b). In fermentation, reducing power (in the form of NADH) is created as well. Oxygenation of NADH also takes place during the production of lactate, propionate, ethanol and butyrate. Therefore, fermentative bacteria often conduct a mixed acid fermentation, during which acetate, butyrate, lactate, ethanol, formate, succinate, as well as acetone, butanol of butanediol can be produced (Hallenbeck, 2009; Lee et al., 2008b; Muraraka et al., 2008). The ratio of products depends on the type of used microorganisms, the substrate and the conditions of the process (Cai et al., 2011). In practice, hydrogen production efficiency is almost two times lower than its theoretical value (Hallenbeck, 2009; Lee et al., 2011; Wang and Wan, 2009). The goal of future research has to be an increase of this efficiency through minimising the amount of organic products and streaming electrons into hydrogen production pathway (Lee et al., 2010).

3. OPERATIONAL PARAMETERS The operational parameters such as inoculum type, pH, temperature, feedstock, H2 partial pressure are crucial to the performance of fermentative hydrogen production and are reviewed in this section.

3.1. Microorganisms The conditions of dark fermentation have been the subject of long-term investigations. Hydrogen can be produced by strict anaerobes from the Clostridiaceae family (e.g. C. butyricum, C. actobutylicum, C.

586

Fermentative hydrogen production - process design and bioreactors

beijerinckii, C. thermocellum), as well as facultative anaerobes: E. coli, Citrobacter or bacteria from the Enterobactericeae family (E. cloacae, E. aerogenes), (Lee et al., 2011; Ntaikou et al., 2010). The process can take place in the presence of a pure or mixed culture, and the choice of a particular microorganism must be done adjusting to their specific requirements (Ntaikou et al., 2010; Wang and Wan, 2009). Each of these culture types has its own pros and cons. A pure culture is characterised by high selectivity, yielding higher hydrogen production efficiency with fewer byproducts. What is more, by changing growth conditions, it is easier than in mixed culture to manipulate the metabolism of microorganisms. However, such a pure culture is susceptible to impurities, which requires aseptic environment and increases the overall cost of the process (Ntaikou et al., 2010). From the engineering standpoint, it is more desirable to deal with a process which uses a mixed culture coming from soil, compost or digester sludge (Howkes et al., 2007; Kleerebezem and van Loosdrecht, 2007; van Ginkel et al., 2001). Such a mixed culture, apart from hydrogen producing bacteria, also hosts microorganisms that, while not being hydrogen producers themselves, increase its production efficiency e.g. by creating biomass granules, using up oxygen, or decomposing complex organic substrates (Hung et al., 2011). A mixed culture of microorganisms can use a wider spectrum of substrates, and the cost of the process can be reduced since no sterilisation is necessary. However, the process also has some downsides. Such a mixture might contain microorganisms which do not produce hydrogen but compete for carbon sources (methanogens, homoacetogenes, lactic acid bacteria). In a mixed culture, domination of hydrogen-generating bacteria can be achieved by maintaining appropriate operation conditions during the process or by getting rid of the competing bacteria in a pretreatment step. For this reason, heat, oxygenation, and methods involving the use of acids, bases, chloroform and acetylene have been proposed (Akutsu et al., 2009; Argun et al., 2009; Hu and Chen, 2007; Kang et al., 2012; Ren et al., 2008; Valdez et al., 2006; Zhu and Beland, 2006). However, due to the different origins of inocula, substrate type and the specifics of the process conditions it is hard to determine unequivocally which of these methods is the most efficient way (Wang and Wan, 2009).

3.2. Substrates The most efficient substrates for hydrogen production by means of dark fermentation are carbohydrates, such as glucose, sucrose and starch (Hawkes et al., 2007; Hallenbeck 2005; Lin et al., 2008), as well as arabinose (Abreu et al., 2012), xylose (Ngo et al., 2012) and glicerol (Seifert et al., 2009). However, pure substrates are very expensive, so in order for industrial-scale hydrogen production to be profitable, cheap waste products such as sewage sludge (Massanet-Nicolau et al., 2010), solid municipal waste (Dong et al., 2009), molasses (Li et al., 2007), and wastewater originating from biodiesel production (Han et al., 2012), olive oil production (Ntaikou et al., 2009) or palm oil production (Vijyaraghavan and Ahmad, 2006) have to be used. Over the past few years, first generation biofuels have been produced from energetic plants, such as oilseed rape or soybean. Nevertheless, their cultivation is controversial due to the fact that it requires high quality soil, which could be potentially used to grow food. This problem could be avoided by using second generation biofuels, e.g. hydrogen generated from plants such as sweet sorghum, switchgrass and miscanthus which can grow on land otherwise less desirable from an agricultural point of view, as well as from lignocellulosic residues produced by the wood processing industry (Magnusson et al., 2008). It is worth remembering, however, that such substrates contain complex polymers: cellulose, hemicellulose and lignin and therefore require pretreatment in order to dispose of lignin and change the structure of cellulose, which facilitates biodegradation of these waste products by cellulolytic microorganisms, but increases the costs of hydrogen production.

587


3.3. Hydrogen partial pressure The bottleneck of dark fermentation is the partial pressure of hydrogen. An increase in hydrogen partial pressure shifts the metabolic activity of the bacteria towards synthesising more reduced products, thus decreasing the overall hydrogen production yield. Purging the bioreactor with nitrogen during the process increases efficiency because it allows to maintain the partial pressure of hydrogen at a low level and to dispose of carbon dioxide, which could potentially take part in acetogenesis, using up the generated hydrogen (Mizuno et al., 2000; Tanisho et al., 1998; Hawkes et al., 2007; Massanet-Nicolau et al., 2010). In a study conducted by Bastidas-Oyanedel et al. (2012) in headspace N2-flushing reactor, hydrogen production yield increased from 1 to 3.25 mol H2/mol glucose at pH 4.5 and N2-flushing of 58.4 l/d. The observed increase in hydrogen yield was explained to be thermodynamically controlled by low hydrogen partial pressure that affected lactate hydrogenase, NADH hydrogenase and homoacetogenesis reactions.

3.4. Temperature Hydrogen production can take place in the presence of mesophilic bacteria (Clostridium, Enterobacter), thermophiles (Caldicellulosiruptor, Thermoanaerobacterium) or hyperthermophiles (Thermotoga). In a mixed culture, a change of the process temperature may affect the dominant bacteria culture. Karadag and Puhakka (2010) observed a change of the dominant culture from Clostridium in mesophilic conditions to Thermoanaerobacterium in thermophilic conditions. A comparison of the parameters suggests that hydrogen generation efficiency, as well as its fraction in biogas, was higher under thermophilic and hyperthermophilic conditions than that in mesophilic conditions (Shin et al., 2004; Valdez-Vazquez et al., 2005). This could be due to the fact that hyperthermophilic bacteria are less inhibited by the partial pressure of hydrogen and that due to high temperature fermentation is less susceptible to contamination with other cultures. This was confirmed by Shin et al. (2004) and ValdezVazquez et al. (2005), who did not notice any activity of methanogenes even though the inoculum was not pretreated. From an engineering point of view, a disadvantage of a thermophilic process is that a reactor must be heated up to desired temperature, and the fact that volumetric production rate of hydrogen is low, which would require an application of much bigger reactors than those used for mesophilic fermentation, and hence increase the costs (Hallenbeck, 2009).

3.5. Culture pH pH is a key parameter to control hydrogen production, which affects the activity of hydrogenase (Dabrock et al., 1992), microbial communities structure, and their metabolism, modifying a spectrum of products (Guo et al., 2010; Ye et al., 2007). In another study, the optimum pH value during fermentative hydrogen production changed in a wide range from 3 to 9 (Gaddhamshetty et al., 2009; Lee et al., 2002), and strongly depended on the type of the subtrate and inoculum (Wang and Wan, 2009). If food wastes were used as the substrate, the optimal pH value was about 5-6, whereas if the substrate were crop residues and animal manure, pH oscillated around 7 (Guo et al., 2010). Investigating C. tyrobutyricum ATCC 25755 (Zhu and Yand, 2004), it was observed that a change in pH affected the expression level of various enzymes. Enzymes responsible for creating butyrate and using up lactate were strongly expressed at pH 6.3, while other enzymes responsible for creating acetate and lactate at pH 5. However, this schema looks different for other organisms, such as C. butyricum CGS5 (Chen et al., 2005), for which an opposite relation was observed: the highest butyrate production rate took place at pH 5.5 and dropped as pH increased to 6.5. Therefore, while applying different microorganisms, it is necessary to check how pH affects the fermentation process (Cai et al., 2011).

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4. BIOREACTORS Bioreactor configuration is of prime importance in hydrogen production process as it influences the microenvironment of the reactor, its established hydrodynamic behaviour, the prevailing microorganism population, and their contact with the substrate. For research purposes, batch reactors are used most frequently, because it is flexible and it is easy to operate. However, industrial-scale hydrogen production requires a continuous-flow bioreactor (Ntaikou et al., 2010). The bioreactor type which is most frequently used is continuously stirred tank reactor (CSTR). Its construction is simple, it is easy to operate, has very effective stirring, and biomass is well suspended in a solution. It provides a good contact between the substrate and microorganisms, and a perfect exchange of mass (Ntaikou et al., 2010; Show et al., 2011). In CSTR, solids retention time (SRT) is the same as hydraulic time retention (HRT). Short HRT is favorable for hydrogen generation. However, using short HRT has both positive and negative aspects. On the one hand, it makes SRT short enough to protect a mixed culture from methanogen growth. On the other hand, if the HRT value is too small, biomass concentration in a bioreactor is limited, and often it may even be washed out from the reactor, which limits hydrogen generation rate and causes fluctuations in the whole functioning of a reactor (Ntaikou et al., 2010; Show et al., 2011). During hydrogen production in CSTR reactors, bacteria can suddenly flocculate and create granules. This phenomenon is promoted by the presence of divalent cations and an increase in carbohydrate concentration in extracellular polymeric substance (EPS) (Jung et al., 2011 a). Moreover, Zhang et al. (2007) claim that a fast creation of granules had to be preceded by incubation in pH 2 for one day, which improved the physicochemical properties of the surface, favoring microbiological granulation, and facilitated biomass retention. Its content increased over 30-fold compared to the amount of biomass in CSTR reactor without granulation, which resulted in an increase of hydrogen generation rate to 3.2 l H2/l h (Zhang et al., 2007). Another example of a reactor with biomass retention is the membrane bioreactor (MBR). There are two types of such reactors: external cross-flow type and submerge type (Jung et al., 2011 a). The advantages of MBRs include higher biomass concentration in the bioreactor, hence a greater usage of organic substrate, a smaller reactor volume due to a higher substrate consumption rate, reduced production of excess sludge due to biomass decay in the reactor, and a lack of microorganisms in the effluent due to their total retention by the membrane (Oh et al., 2004). Lee et al. (2010) concluded, that in a submerged membrane reactor SRT was a key factor deciding about a stable hydrogen production process. In a reactor working on glucose under HRT of 9h, hydrogen production rate increased from 3.9 to 5.8 l/l d as SRT increased from 2 to 12.5 d. The hydrogen production yield changed in the opposite direction and reached the maximum of 1.19 mol H2/molglucose at the SRT of 2 d. An overly high SRT value (90 d) caused a drop in both the rate and efficiency of hydrogen production (Lee et al., 2010). A similar trend was also observed in previous research on an external cross-flow membrane bioreactor (Oh et al. 2004), where excessive biomass concentration increased substrate consumption, resulting in a decrease in hydrogen generation rate. Moreover, lower volatile suspended solids/total suspended solids were observed, as well as the metabolic pathway shift to lactate, and accumulation of extracellular polymeric substance (EPS) (Oh et al., 2004). EPS accumulation is the reason for the fouling of the membrane and it is one of the main reasons limiting the use of such reactors in biological processes (Lee et al., 2008a; Lee et al., 2010; Zheng et al., 2010) . An alternative to CSTR for continuous hydrogen production are upflow packed-bed reactors (PBR), in which the medium, e.g. wastewater, enters at the bottom and exits from the top. In such a reactor, biomass is immobilised either in granules or in biofilms or entrapped in packed media (Kothari et al., 2012). The support material included: sponge, granular activated carbon (GAC), expanded clay, polyethylene-octane elastomer, ceramic ball, alginate gel (Show et al., 2011). Compared to CSTR, PBR has higher mass transfer resistance, resulting in a lower substrate conversion and hydrogen production

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rates (Show et al., 2011). However, Keskin et al. (2012), who compared hydrogen production from sucrose in a bioreactor filled with ceramic balls and CSTR with suspended cells, observed that in an 8 times smaller bioreactor with immobilised microorganisms at optimal HRT (3 h) the hydrogen production yield and rate were both higher (363 ml H2/g sucrose, 2.7 l H2/l d) than those in CSTR (87 ml H2/g sucrose, 0.5 l H2/l d, HRT 24 h). What is more, a packed-bed reactor was more resistant to biomass washing out observed in CSTR at low HRT (Keskin et al., 2012). In a fluidised-bed reactor (FBR), gas or liquid passes through accumulated solid matter, causing its fluidisation. Such reactors are often characterised by good mixing, as well as high hydrogen production efficiency at low HRT and high biomass concentration. The methods for immobilising bacteria are similar to those used in packed-bed reactors. The kind of support material and microorganism immobilisation type has an important influence on hydrogen production parameters. It was confirmed in a study on glucose conducted by Zhang et al. (2008) and Barros et al. (2010). Zhang et al. (2008) used microorganisms immobilised on GAC or in a granulated form, at the same HRT (0.25 h) and similar VSS (35 g/l), achieving a comparable yield of 1.7 mol H2/ mol glucose in both reactors, but the hydrogen generation rate was by 15% higher in GAC bioreactor, equalling 7.6 l H2/l h (Zhang et al., 2008). Barros et al. (2010), by using thermally pre-treated anaerobic sludge and polystyrene and expanded clay as the support material, at HRT 2h, achieved higher efficiency and hydrogen production rate of 2.59 mol H2/mol glucose and 1.21 l H2/l h respectively, in a bioreactor containing expanded clay. This was probably due to better surface characteristics of the expanded clay material for biomass attachment, which allowed to obtain a higher concentration of immobilised biomass on this material (1.1 mg TVS/g expanded clay vs. 0.805 mg TVS/g polystyrene) (Barros et al., 2010). Hydrogen generation was also performed in up-flow anaerobic sludge blanket (UASB) reactor, used hitherto in methane production. This kind of bioreactor contains a gas/ liquid/solid separator on its top, where microbiological granules are formed. Such active biomass sediments easily, creating a thick biomass blanket zone at the bottom. The process in such a reactor proceeds effectively and with high stability (Hawkes et al., 2007; Jung et al., 2011a). For instance, the maximum hydrogen production efficiency and rate, with sucrose as the substrate, equaled 1.33 mol H2/mol hexose i 0.1 l H2/l h respectively (Wang et al., 2007). However, a disadvantage of UASB is its long start-up period i.e. the time necessary to form large enough granules, which in Wang's research was about 5 months. According to Jung et al. (2011b), the granulation rate can be increased by means of a two-stage process. Initially, the process took place in CSTR, after which the mixed liquid was moved to UASB as a seeding source. This strategy shortened the start-up period over 7 times (Jung et al., 2011b).

5. PROSPECTS Hydrogen biotechnologies, in most cases, are not developed enough in order to put them to practical use. In 2009, in techno-economic analysis of biohydrogen production undertaken as a part of the EERE Hydrogen Production Program, the costs of generating 10000 kg/d of hydrogen by various methods were assessed (James et al., 2009). It was concluded that dark fermentation based on lignocellulose substrates can allow for production of 940 cm3 H2/dm3 h for 4.33 $/kg. Given its high cost, this method could be competitive with the standard one, i.e. steam reforming of methane. However, for the costs to be realistic, a significant increase in both the rate and efficiency of fermentation processes needs to take place (James et al., 2009, Brentner et al., 2010). There is a need for further optimisation of the process using complex substances. Research has to be directed towards redirection and/or reconstruction of cellular metabolisms for example, by the elimination of enzymes and carbon pathways interfering or competing with H2 production or the incorporation of non-native metabolic pathways leading to hydrogen production (Oh et al., 2011).

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Another possibility is a development of integrated systems, in which hydrogen production by means of fermentation would be coupled to the next process. Organic compounds from wastewater after fermentation would be converted to methane, hydrogen of electrical energy (Hallenbeck and Ghosh, 2009). Such systems are already being tested in a pilot scale, for instance the coupling of hydrogen and methane fermentation of kitchen waste has allowed to remove 80 % of the organic matter (COD) and obtain 5.4 m3/m3 d of hydrogen and 6.1 m3/m3 d of methane (Ueno et al., 2007). Another process in which effluent from fermentation can be used is photofermentation, during which photosynthetic bacteria convert acids present in wastewater into hydrogen and carbon dioxide. This strategy increases the overall efficiency of hydrogen generation and significantly decreases the amount of organic substances (Chen et al., 2008; Zong et al., 2009; Yang et al., 2010). Electrical energy can be obtained in microbiological fuel cells (MFC) or in microbiological electrolysis cells (MEC), in which organic compounds that occur in post-fermentation wastewater are used (Logan, 2010; Sharma and Li, 2010). However, despite some encouraging results, each approach is still faced with many challenges that need to be addressed in future research before they can be fully and successfully implemented ( Hallenbeck, 2009).

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Received 15 May 2012 Received in revised form 22 October 2012 Accepted 23 October 2012

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ABSORPTION OF CO2 INTO PERFLUORINATED GAS CARRIER IN THE TAYLOR GAS–LIQUID FLOW IN A MICROCHANNEL SYSTEM Paweł Sobieszuk*, Maciej Pilarek Warsaw University of Technology, Faculty of Chemical and Process Engineering, Waryńskiego 1, 00-645 Warszawa, Poland The aim of this study was to determine the solubility of CO2 in perfluorodecalin (PFD) which is frequently used as efficient liquid carrier of respiratory gases in bioprocess engineering. The application of perfluorinated liquid in a microsystem has been presented. Gas-liquid mass transfer during Taylor (slug) flow in a microchannel of circular cross section 0.4 mm in diameter has been investigated. A physicochemical system of the absorption of CO2 from the CO2/N2 mixture in perfluorodecalin has been applied. The Henry’s law constants have been found according to two theoretical approaches: physical (H = 1.22·10-3 mol/m3Pa) or chemical (H = 1.26·10-3 mol/m3Pa) absorption. We are hypothesising that the gas-liquid microchannel system is applicable to determine the solubility of respiratory gases in perfluorinated liquids. Keywords: perfluorodecalin (PFD), liquid gas carrier, solubility, Taylor flow, microreactor

1. INTRODUCTION Liquid perfluorochemicals (perfluorocarbons, PFCs) are characterised by a high solubility of respiratory gases (O2 and CO2) and other non-polar gases. They have raised much interest as fully safe synthetic liquid gas carriers in bioprocess and medical applications (Lowe, 2001; Pilarek and Szewczyk, 2008; Riess, 2006) confirmed by many laboratory studies and clinical investigations. It is assumed that the gas transfer rate in PFCs increases linearly with the partial pressure of a component in the gaseous phase (according to the Henry’s Law) in contrast with sigmoid dissociation curves for biological gas carriers (e.g. haemoglobin or myoglobin) (Krafft and Riess, 1998; Lowe, 2002; Riess, 2001). PFCs are immiscible in aqueous and most other media and they create a separate perfluorinated phase at the bottom of the two-phase (water/PFC) systems due to their relatively high density (about 1.9 kg dm-3) (Lowe, 2002; Riess, 2001). In connection to their various applications it is also important that PFCs added to a reaction or culture system do not change the concentration of its components. Liquid perfluorinated gas carriers are also stable and inert compounds with high resistance to heat sterilisation (e.g. by autoclaving) and possibility for long time-storage at room temperature (Lowe, 2002; Pilarek et al., 2011a). Bioprocess applications of liquid PFCs are mainly related to supplying them into culture systems as gas carriers (mainly O2) as well as scavengers of gaseous by-products (mainly CO2) of cellular metabolism. Many studies have shown that the application of gas saturated perfluorinated liquids can improve efficiency of microbial (Elibol and Mavituna, 1997; Pilarek and Szewczyk, 2008; Pilarek et al., 2011a), plant cell (Pilarek and Szewczyk, 2008) and animal cell cultures (Shiba et al., 1998; Rappaport, 2003; Pilarek et al., 2011b) and thus lead to improved growth or metabolite biosynthesis in cultures of various *Corresponding author, e-mail: [email protected]

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kinds of cells. Generally, applications of PFCs are still limited due to their relatively high cost, which makes their use in large-scale bioprocesses uneconomical. However, PFCs could successfully be used in miniature-scale processes and in microbioreactor cultivations of various kinds of cells to prevent oxygen or carbon dioxide limitation during grow to high cell densities, especially in high throughput screening approaches (Pilarek et al., 2011a; Ukkonen et al., 2011). In view of the unusual mechanism of dissolution of gases in perfluorinated liquid, it would be interesting to determine the nature of the absorption process and the values of CO2 solubility into perfluorodecalin (PFD). A typical apparatus for this type of investigations is the laminar-jet setup (Pohorecki and Moniuk, 1988). However, because of the cost of fluorinated liquids, using such equipment is uneconomical and problematic. We hypothesise that an application of a gas-liquid microreactor system could be more feasible. Mass transfer and hydrodynamics of the Taylor flow in microchannels have widely been investigated (Qian and Lawal, 2006; Sobieszuk et al., 2010; Eskin and Mostowfi, 2012; Sobieszuk et al., 2012). There are lot of experimental data on volumetric mass transfer coefficient (kLa) values in the Taylor flow (Kashid et al., 2011; Sobieszuk et al., 2008; Yue et al., 2007) and kL (Sobieszuk et al., 2011) values . Therefore, a series of experiments involving the study on the rate of CO2 absorption into perfluorodecalin (PFD) were done. On this basis and using the available correlations for mass transfer in the Taylor flow, the values of the Henry’s law constant have been determined. The aim of this study was to determine the solubility of CO2 into perfluorodecalin which is frequently used as an efficient liquid carrier of respiratory gases in bioprocess engineering. To the best of our knowledge this is the first attempt to apply a microreactor system to defining the mechanism of gas absorption in liquid PFC and also the first report of the application of perfluorinated gas carrier in microchannel system in general. 2. EXPERIMENTAL SYSTEM A glass, Y-shaped microreactor, with inlet channels 120° apart from each other and from the main channel has been used. The cross-section of all the channels was circular with 0.4 mm diameter. The main microchannel length was 100 mm. The experimental set-up is shown in Fig. 1.

Fig. 1. Experimental set-up, 1a, 1b – gas bottles with CO2 and N2; 2a, 2b – gas flow regulators; 3 – manometer; 4 - syringe pump; 5 – Y-shaped microreactor; 6 – gas chromatograph ; 7 – separator; 8 - liquid outlet

A physicochemical system of the absorption of CO2 from the CO2/N2 mixture into a pure PFD has been applied. The gases (N2 and CO2) were supplied from cylinders through reducing valves and gas flow

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Absorption of CO2 into perfluorinated gas carrier in the Taylor gas–liquid flow in a microchannel system

regulators. Both gases were mixed before the inlet to the microchannel, and pressure was measured. PFD (cis/trans C10F18; ABCR GmbH, Germany) was supplied by a syringe pump. The liquid flow was measured using a measuring cylinder and a stop watch. The two-phase mixture leaving the microchannel was separated in a separator. The gas was directed to the gas chromatograph (Shimadzu GC 2014). The rate of absorption was measured twice: once for the entire microreactor (Fig. 2a), and for the second time using only the phase contactor and the separator, without the main gas – liquid microchannel (Fig. 2b) in order to eliminate the end effects for each experiment conditions. The properties of PFD (density, viscosity, surface tension) were used based on data presented by Pilarek et al. (2006). The diffusivity of CO2 in perfluorodecalin was calculated based on the Wilke and Chang method. All experiments were conducted at the room temperature. Three concentrations of CO2 at the gas inlet were used: 30 %, 50 % and 70 %, respectively. For each experimental series the gas flow was kept constant and equalled 3.33·10-8 m3/s. The flow ranges of pure PFD were from 1.2·10-8 to 3.1·10-8 m3/s. The CO2 absorption rate was calculated from the gas concentration difference between the inlet and the outlet from microreactor.

Fig. 2. Microchannel set up used in experiments: a) microreactor – separator, b) phase contactor – separator

3. RESULTS AND DISCUSSION The values of CO2 concentration in CO2/N2 mixture at the outlet of both studied microchannel devices have been presented in Fig 3. The significant differences in concentration have been noted for all the studied cases. These results allow to determine the absorption rate only in the microchannel. However, our method could not be effective in the case of gases characterised by lower solubility in PFCs (e.g. O2 or CO) since it could be observed that too large a part of PFC is absorbed in the phase separator. Due to this fact the differences between the measured concentrations of the absorbed gas at the inlet of the microreactor – separator system and the contactor – separator system might be too low in order to ensure sufficiently accurate values of the absorption rate. The absorption rate in microchannel as a function of the superficial velocity of the two-phase flow has been shown in Fig. 4. An expected upward trend of the absorption rate with an increasing two-phase flow velocity (i.e. higher values of the mass transfer coefficient at higher values of two-phase flow velocity) as well as with the increasing CO2 concentration at the gas inlet (i.e. with increased driving force of the absorption process) has been observed. The Henry’s law constant can be determined for the studied CO2 – PFD system based on the estimated values of the CO2 absorption rates in the applied microchannel device. The mechanism of gas dissolution in perfluorinated liquids quoted in the literature (Deschamps et al., 2007) does not explicitly define whether it is chemical or physical absorption. Therefore, two approaches have been attempted for further discussion.

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Fig. 3. Concentration of CO2 in CO2/N2 mixture at outlet in microreactor-separator system and inlet – separator system in the case of various CO2 concentrations at the inlet: a) 30%, b) 50% and c) 70%

Fig. 4. CO2 absorption rate in microchannel as a function of the superficial two-phase flow velocity

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As it is commonly known, the absorption rate is defined as follows

N = k L aVΔc

(1)

The values of the volumetric mass transfer coefficient for the various gas/liquid systems are available in the literature. Sobieszuk et al. (2011) proposed the correlation for kL calculation in the gas-liquid microchannel system. However, in the case of our present study involving a heterogeneous microsystem the interfacial area remains unknown, so the correlation due to Yue et al. (2007) has been used. They proposed the following correlation for the volumetric mass transfer coefficient:

Sh ⋅ a ⋅ d = 0.084 Re G0.213 Re 0L.937 ScL0.5

(2)

The driving force of the process is given by

Δc=

(ci1 − c1 ) − (ci 2 − c2 )

(3)

c −c ln i1 1 ci 2 − c2

The Henry’s law constant is defined as

c=H⋅p

(4)

Since the inlet concentration of CO2 in the liquid phase (PFD) is 0 (c1 = 0) and the gas-side mass transfer resistance can be neglected, Eq. (3) simplifies to

Δc=

Hp1 − (Hp2 − c2 ) Hp1 ln Hp2 − c2

(5)

For physical absorption the outlet concentration of CO2 in the liquid phase can be calculated based on mass balance

c2 =

QG1

p p1 − QG 2 2 RT RT QL

(6)

After rearranging Equations (1), (3) and (5), the absorption rate can be expressed as follows

p p ⎛ QG1 1 − QG 2 2 ⎜ RT RT H p1 − ⎜ H p2 − Q ⎜ L ⎜ ⎝ N = kLa V H p1 ln p p QG1 1 − QG 2 2 RT RT H p2 − QL

⎞ ⎟ ⎟ ⎟ ⎟ ⎠

(7)

In case of chemical absorption it can be assumed that the outlet concentration of CO2 in the liquid phase is 0 (c2=0). Hence

Δc=

p1 − p2 H p ln 1 p2

(8)

and finally the absorption rate is described by a simple equation

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N = kLa V

p1 − p2 H p1 ln p2

(9)

Based on the experimental data of the absorption rate of CO2 into PFD for different values of the twophase superficial flow velocity the Henry’s law constant could be determined. In case of physical absorption the values of H were calculated from Eq. (7) and the average value has been obtained as H = 1.22·10-3 mol/m3Pa with standard deviation of 3.14·10-4 mol/m3Pa. The results estimated for chemical absorption of CO2 into PFD defined by Eq. (9) are shown in Fig. 5.

Fig. 5. Determination of the Henry’s law constant for chemical absorption based on Eq. (9)

As can be seen H = 1.26·10-3 mol/m3Pa with the correlation coefficient R2 = 0.84. It should be emphasised that the obtained values are very close to each other, but are subject to an experimental error. Such errors resulted from the effects of short microchannel used and from relatively low CO2 solubility in PFD. 4. CONCLUSIONS The rates of CO2 absorption in PFD in the Taylor flow in microchannel have been estimated. The Henry’s law constants have been found for the studied microsystem based on two theoretical approaches, physical or chemical absorption processes. Both obtained values of H are very close to each other and are in accordance with previously published literature data of CO2 solubility in PFD (Deschamps et al., 2007; Pilarek and Szewczyk, 2008). Nevertheless, we conclude that the mechanism (physical or chemical) of CO2 absorption in PFD cannot be explicitly defined based on the results of our experiments. It turned out that the applied 100 mm long microchannel was too short to observe any differences between the two examined kinds of absorption. Using microdevices with a longer microchannel enabling greater residence time of media could provide more accurate experimental data. Finally, we can state that the gas-liquid microchannel system is applicable for experimental determination of respiratory gases solubility in perfluorinated liquids.

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This work was supported by the budget sources for The National Centre for Science (Poland), Grant No. N N209 026140.

SYMBOLS a c D d H kL N p Q R ReG=uG⋅d⋅ρG/μG ReL=uL⋅d⋅ρL/μL ScL=μL/(ρL⋅D) Sh=kL⋅d/D T u V

specific interfacial area, m-1 concentration of CO2 in liquid phase, kmol·m-3 diffusivity of CO2 into perfluorodecalin, m2·s-1 microchannel diameter, m Henry’s law constant, mol·m-3·Pa-1 liquid side mass transfer coefficient, m·s-1 absorption rate, mol·s-1 partial pressure, Pa flow rate, m3·s-1 gas constant, J·mol-1·K-1 Reynolds number for gas phase Reynolds number for liquid phase Schmidt number for liquid phase Sherwood number temperature, K superficial velocity, m·s-1 microchannel volume, m3

Greek symbols

ρ μ σ

density, kg·m-3 viscosity, Pa·s surface tension, N·m-1

Subscripts 1 2 i G L

inlet outlet interfacial area gas liquid

Abbreviations PFC PFD

perfluorochemical perfluorodecalin

REFERENCES Deschamps J., Menz D.-H., Padua A.A.H., Costa Gomes M. F., 2007. Low pressure solubility and thermodynamics of solvation of oxygen, carbon dioxide, and carbon monoxide in fluorinated liquids. J. Chem. Thermodyn., 39, 847-854. DOI: 10.1016/j.jct.2006.11.012. Elibol M., Mavituna F., 1997. Characteristics of antibiotic production in a multiphase system. Proc. Biochem., 32, 417-422. DOI: 10.1016/S0032-9592(96)00099-4. Eskin D., Mostowfi F., 2012. A model of a bubble train flow accompanied with mass transfer through a long microchannel. Int. J. Heat Fluid Flow, 33, 147-155. DOI: 10.1016/j.ijheatfluidflow.2011.11.001. Kashid M.N., Renken A., Kiwi-Minsker L., 2011. Gas-liquid and liquid-liquid mass transfer in microstructured reactors. Chem. Eng. Sci., 66, 3876-3897. DOI: 10.1016/j.ces.2011.05.015.

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P. Sobieszuk, M. Pilarek, Chem. Process Eng., 2012, 33 (4), 595-602 Krafft M.P., Riess J.G., 1998. Highly fluorinated amphiphiles and colloidal systems and their applications in the biomedical fields. A contribution. Biochimie, 80, 489-514. Lowe K.C., 2001. Fluorinated blood substitutes and oxygen carriers. J. Fluorine Chem., 109, 59-65. DOI: 10.1016/S0022-1139(01)00374-8. Lowe K.C., 2002. Perfluorochemical respiratory gas carriers: benefits to cell culture systems. J. Fluorine Chem., 118, 19-26. DOI: 10.1016/S0022-1139(02)00200-2. Pilarek M., Szewczyk K.W., Stępniewski J., Anderszewska A., 2006. The use of a perfluorinated oxygen vector in cultures of microorganisms. Przem. Chem., 85, 1131-1133. Pilarek M., Szewczyk K.W., 2008. Effects of perfluorinated oxygen carrier application in yeast, fungi and plant cell suspension cultures. Bio. Eng. J., 41, 38-42. DOI: 10.1016/j.bej.2008.03.004. Pilarek M., Glazyrina J., Neubauer P., 2011a. Enhanced growth and recombinant protein production of Escherichia coli by a perfluorinated oxygen carrier in miniaturized fed-batch cultures. Microb. Cell Fact. 10, art. no. 50. DOI: 10.1186/1475-2859-10-50. Pilarek M., Neubauer P., Marx U., 2011b. Biological cardio-micro-pumps for microbioreactors and analytical micro-systems. Sens. Actuators B: Chem., 156, 517-526. DOI: 10.1016/j.snb.2011.02.014. Pohorecki R., Moniuk W., 1988. Kinetics of reaction between carbon dioxide and hydroxyl ions in aqueous electrolyte solutions. Chem. Eng. Sci., 43, 1677-1684. DOI: 10.1016/0009-2509(88)85159-5. Qian D., Lawal A., 2006. Numerical study on gas and liquid slugs for Taylor flow in a T-junction microchannel. Chem. Eng. Sci., 61, 7609-7925. DOI: 10.1016/j.ces.2006.08.073. Rappaport C., 2003. Review— Progress in concept and practice of growing anchorage-dependent mammalian cells in three dimension. In Vitro Cell Dev. Biol.-Animal, 39, 187-192. DOI:10.1290/1543706X(2003)0392.0.CO;2. Riess J.G., 2001. Oxygen carriers (‘Blood substitutes’) – Raison d’Etre, chemistry and some physiology. Chem. Rev., 101, 2797-2919. DOI: 10.1021/cr970143c. Riess J.G., 2006. Perfluorocarbon-based oxygen delivery. Artif. Cells, Blood Substit. Biotechnol., 34, 567-580. DOI: 10.1080/10731190600973824. Shiba Y., Ohshima T., Sato M., 1998. Growth and morphology of anchorage-dependent animal cells in a liquid/liquid interface system. Biotechnol. Bioeng., 57, 583-589. DOI: 10.1002/(SICI)10970290(19980305)57:53.0.CO;2-D. Sobieszuk P., Aubin J., Pohorecki R., 2012. Hydrodynamics and mass transfer in gas-liquid flows in microreactors. Chem. Eng. Tech., 35, 1346-1358. DOI: 10.1002/ceat.201100643. Sobieszuk P., Pohorecki R., Cygański P., Grzelka J., 2011. Determination of the interfacial area and mass transfer coefficients in the Taylor gas–liquid flow in a microchannel. Chem. Eng. Sci., 66, 6048-6056. DOI: 10.1016/j.ces.2011.08.029. Sobieszuk P., Cygański P., Pohorecki R., 2010. Bubble lengths in the gas-liquid Taylor flow in microchannels. Chem. Eng. Res. Des., 88, 263-296. DOI: 10.1016/j.cherd.2009.07.007. Sobieszuk P., Cygański P., Pohorecki R., 2008. Volumetric liquid side mass transfer coefficient in a gas-liquid microreactor. Chem. Proc. Eng., 29, 651-661. Ukkonen K., Vasala A., Oyamo H., Neubauer P., 2011. High-yield production of biologically active recombinant protein in shake flask culture by combination of enzyme-based glucose delivery and increased oxygen transfer. Microb. Cell Fact., 10, art. no. 107. DOI: 10.1186/1475-2859-10-107. Yue J., Chen G., Yuan Q., Luo L., Gonthier Y., 2007. Hydrodynamics and mass transfer characteristics in gasliquid flow through a rectangular microchannel. Chem. Eng. Sci., 62, 2096-2108. DOI: 10.1016/j.ces.2006.12.057. Received 14 May 2012 Received in revised form 10 November 2012 Accepted 24 November 2012

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Chemical and Process Engineering 2012, 33 (4), 603-610 DOI: 10.2478/v10176-012-0050-x

HARVESTING ENERGY AND HYDROGEN FROM MICROBES Paweł Sobieszuk*, Anna Zamojska-Jaroszewicz, Andrzej Kołtuniewicz Warsaw University of Technology, Faculty of Chemical and Process Engineering, Waryńskiego 1, 00-645 Warszawa, Poland This article presents a critical mini-review of research conducted on bioelectrochemical reactors with emphasis placed on microbial fuel cells (MFC) and microbial electrolysis cells (MEC). The principle of operation and typical constructions of MFCs and MECs were presented. The types of anodes and cathodes, ion-selective membranes and microorganisms used were discussed along with their limitations. Keywords: clean energy, microbial fuel cell, microbial electrolysis cell, hydrogen

1. INTRODUCTION The progress of civilization presents us with many challenges. The growing energy requirements force us to search for cheap, renewable and environmentally friendly methods of its production. Currently, the most commonly used fossil fuels are hard coal, oil and natural gas. Their excavation is associated with land degradation and their treatment causes the release into the atmosphere large quantities of harmful chemicals such as carbon dioxide, sulphur dioxide or nitrogen oxide. Fossil fuels are nonrenewable, and their lifetime is limited. Some forecasts predict that oil reserves will be exhausted within the next few decades, and the only alternative is renewable energy. Electrochemical bioreactors may be one of those "green" energy sources. These devices utilise microorganisms as catalysts for an electrochemical reaction. Bioelectrochemical reactors (BER) reprocess organic matter to electricity using microorganisms. An appropriate final electron acceptor is used, depending on the type of chemical compounds. BERs can be used for different purposes, and their primary function was to produce electricity. Currently, they are used to produce hydrogen and other useful compounds such as methane or ethanol. However, they can also be used in electrochemically supported denitrification. But the greatest advantage of BER is its ability to simultaneously carry out a variety of processes. Sewage and other waste substances can be used as a substrate for microbial growth. This is the technology of the future, because it allows to combine the degradation of waste together with the production of clean energy, which meets all the demands of clean technologies and sustainable development. 2. 2.

TYPES OF BIOELECTROCHEMICAL REACTORS

Schemes of typical bioelectrochemical reactors are presented in Fig.1. The literature usually distinguishes between microbial fuel cells and microbial electrolysis cells. BER is called a fuel cell if it generates power. When an electrochemical reaction requires external energy, we call them electrolytic cells. (Hamelers et al., 2010). Microbial fuel cells (MFC, Fig. 1a) are used for simultaneous wastewater treatment and power generation. In anode chamber anaerobic microorganisms perform substrate *Corresponding author, e-mail: [email protected]

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P. Sobieszuk, A. Zamojska-Jaroszewicz, A. Kołtuniewicz, Chem. Process Eng., 2012, 33 (4), 603-610

oxidation process by which electrons and protons are formed. Released electrons are transferred to the anode and flow through an external circuit to the cathode. Every electron that flows externally to the cathode must be caught by a proton, which flows from the anode to the cathode chamber through the ion exchange membrane. In cathode chamber, electrons and protons are transferred to the final electron acceptor. Microbial electrolysis cells (MEC, Fig. 1b) are used for hydrogen production often with combination with organic substrate degradation.

Fig. 1. Schematic diagram of typical bioelectrochemical reactors: A – microbial fuel cell (MFC), B – microbial electrolysis cell (MEC)

MFCs in contrast to MECs require external power, as in classical electrolysis. Anodic reaction of the MECs is identical with the reaction of MFCs, except that the cathodic reaction leads to the formation of hydrogen in the MFCs. The use of microorganisms allows for more efficient operation of electrolysis with less external energy consumption. Regardless of type, all reactors of this type have in common the anaerobic oxidation of substrates in the anode chamber. Therefore, the overall process within reactor depends on which reaction takes place at the cathode. Currently the following types of BER can be identified: • Microbial fuel cells (MFCs) – where the main objective is to obtain electrical energy (Freguia et al., 2010). Typical designs are shown in Fig. 2. As can be seen different configurations and shapes are possible. • Microbial electrolysis cells (MECs) – which are used for the production of hydrogen (Logan et al., 2006). The apparatus structure is similar to MFC. • Electrosynthesis microbial cells (MESs) – that are used for the synthesis of organic compounds (Nevin et al., 2010). At the cathode, carbon compounds are reduced to more complex forms. Reactors of this type, as well as MEC, require an external input of energy. • Micro-sized microbial fuel cells (μMFCs) – include mL-scale and μL-scale setups. The mL-scale MFCs allow for achieving higher values of current and volumetric power densities compared to macro-scale MFCs. The existing μL-scale MFCs, due to their high internal resistance, exhibit significantly worse characteristic. However, they show a great potential in a rapid screening of electromaterials and electrochemically active microbes (Wang et al., 2011).

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Harvesting energy and hydrogen from microbes

Fig. 2. Schemes of different MFC designs

3. ELEMENTS OF BIOELECTROCHEMICAL REACTORS Satisfactory BER performance depends on the proper selection of components of the apparatus including electrodes, membranes and microorganisms. 3.1. Anodes Lefebvre et al. (2011) show that BER is basically a biofilm reactor and anode materials should meet all the requirements of such applications (to be chemically stable, have a large surface area and high porosity, not be sensitive to impurities and be biocompatible). An additional feature here is the fact that high conductivity is required together with resistance to corrosion. Therefore it makes it impossible to use many metals as the electrode building materials. The surface structure should not impair the bacteria ability for electron transfer - the impact of electron transport mechanism (direct contact, nanowires, and mediators). What is more, a perfect anode material should be cheap and easy to manufacture. The search for a material that meets all these requirements is still on-going. Currently, the most commonly used material for this purpose is carbon in the form of paper, cloths, fibres, meshes and reticulated vitreous carbon (RVC), which is promising owing to its very high conductivity. Graphite rods, felts, foams, plates and sheets are also widely used. Tab. 1 shows typical values of conductivity for some carbon materials mentioned above. The compact structure and smooth surface of plane anodes (plates and sheets) facilitate quantitative measurements per unit of anode surface area. More packed materials such as felts and foams have much more developed surface and biomass concentration can be higher, but the measurement of this concentration is complicated. The highest specific surface area of anodes can be obtained by using graphite fibre brush electrodes. The core of

605


such a brush has to be made from non-corrosive metal e.g. titanium. A carbon plane anode allows to obtain the power per anode area in range: 600÷3290 mW/m2, and per anode chamber volume about 45 W/m3. In the case of packed materials the power per anode chamber volume was in the range 5÷386 W/m3 (Wei et al., 2011). Metal anodes are also used. Because of the non-corrosive requirement many metals were ruled out as anode materials. Only stainless steel, titanium, platinum and gold are proper for these applications. However, metal adhesive properties are insufficient for biofilm formation. Even in the extreme case (an uncoated titanium plate) no current was observed (ter Heijne et al., 2008). There are several surface modification methods for improving bacterial attachment and electron transfer to the anode including: physical or chemical modification (Tang et al., 2011; Wang et al., 2009), addition of conductive or electro-active coatings (Liang et al., 2011), using metal-graphite composite electrodes (Lowy and Tender, 2008). Some preliminary investigations were conducted on conductive polymer coated anodes, although the performance of these systems was unsatisfactory. Some attempts have been made on reducing metabolite electron losses by using dual-anode electrodes (Kim et al., 2011). Two bacterial anodes were jointly installed within one anode chamber. The first one was enriched by glucose utilising microbial population, and the other by propionate utilizing microbial. It allowed for Columbic efficiency improvement up to 59%. Table 1. Conductivity values for different carbon and graphite materials

No.

Material

1

copper

Conductivity [Ω-1cm-1] 10

2

carbon paper

1.250

3

carbon cloth

0.450

4

graphite fibre

0.625

5

conductive polymer sheet

0.008

6

RVC

200

One of the most recent investigations was performed on high-performance mediator-less MFC with graphene/carbon cloth anode. Liu et al. (2012), investigated MFC with anode as: graphene coated carbon cloth and pure carbon cloth. Graphene application delivered 2.7 fold higher power density and even 3.0 fold higher cell voltage than a plain carbon cloth anode (Fig.3).

Fig. 3. Comparison of work of MFC with and without graphene layered anode: A – cell voltage, B – power density (Liu et al., 2012)

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3.2. Cathodes The most commonly used MFC cathodes are air-cathodes, aqueous air-cathodes and bio-cathodes. Aircathodes allow to obtain the power per cathode area in range: 331 - 1610 mW/m2. In the case of aqueous air-cathode the range of power per electrode area is: 33 - 788 mW/m2 (Wei et al., 2011). Cathodes in MFCs are made of similar materials to those of anodes. Additionally cathodes contain a catalyst to improve the reduction process (Lefebvre et al., 2011; Logan, 2010). The MFC catalyst that gives the best results is platinum. It reduces energy losses and increases the pace of reduction of oxygen, both dissolved in water and contained in the air. However, platinum is too expensive to use it on a larger scale. In order to reduce costs, its content may be reduced to 0.1 mg per cm2 of the cathode (Logan et al., 2006). New catalysts that contain no precious metals seem to be much more promising. They are based on metallo-organic compounds containing cobalt and iron. Examples of such catalysts are ferricyanide and anthraquinone-2,6-disulfonate. The cathode material for oxygen reduction may also be activated carbon (Logan, 2010). It has a poorer oxygen reduction rate compared with other carbon materials with platinum addition, but has a much larger surface area. It is applied in combination with a metal sponge, which serves as current collector. Platinum also gives way to new catalysts in MFCs. Electrodes made of nickel or stainless steel achieve comparable or even better results. Electrodes made of stainless steel brushes give similar hydrogen production rates to a flat carbon electrode with platinum addition. However, there is a problem with gas bubbles that remain attached on the surface of the cathode. Good results are obtained with alloys of nickel and molybdenum. Tungsten carbide may also be useful although the problem is its high susceptibility to corrosion in phosphate buffer at neutral pH (Logan, 2010). Microorganisms can also play the role of catalysts. Their presence alleviates the need for using expensive metal, reduces overvoltage and allows for greater efficiency of the process (Freguia et al., 2010; Nevin et al., 2010; Rozendal et al., 2008; Yang et al., 2011). Conductive polymers, which were not yet used as building blocks of anodes, can be a good material for building cathodes. They show good catalytic properties for oxygen reduction and are suitable as support for biofilm development. They contain a functional group -OH, which makes them less sensitive to pH changes in the cathode chamber. They also contain -NH3 groups which help in a rapid colonisation of the cathode surface by microorganisms (Li et al., 2012). 3.3. Membranes Cation exchange membranes are the most commonly used separators in electrochemical bioreactors. As noted above, a typical bioelectrochemical reactor BER is composed of two compartments separated by a selective proton exchange membrane. In the anode chamber, there is decomposition of organic material by anaerobic bacteria with the release of carbon dioxide, protons and electrons. While the membrane should pass only protons, electrons migrate to the cathode chamber by an external electrical circuit. In the cathode chamber, oxygen reduction takes place with the participation of electrons, and then reaction with protons, to form water. In both chambers, typically, there are additives which are used to facilitate these reactions. A properly functioning proton-exchange membrane is not permeable to these substances along with the growth substrates, oxygen or electrons between the two chambers. Such membranes should also be resistant to biofilm obliteration. To keep the membrane transport process in proper condition, the flow of electrons in an external circuit must be compensated by an equal number of protons transported at the same time through the membrane. Common problems of proton-exchange membrane are as follows: • diffusion of oxygen and buffers with cathode to the anode chamber must be eliminated, • do not allow diffusion through the membrane of chemical oxidants such as ferricyanide or MN (IV), which must be constantly replenished, • do not allow diffusion of substrates and carbon dioxide from the anode to the cathode chamber,

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• • •

do not let a membrane lose its separation properties due to the preferential saturation of sulfonic groups, cations other than protons, do not let a membrane lose its separation properties due to the aging of the membrane, degree of fouling and biofilm overgrowing, do not let a membrane lose its separation properties due to the pH reduction in the anode chamber, with a corresponding increase of pH in the cathode chamber.

The most popular membranes for BER are made of Nafion (Rozendal et al., 2006), which is sulfonated tetrafluorethylene copolymer, consists of a hydrophobic fluorocarbon backbone (–CF2–CF2–). A high cation conductivity of Nafion can be explained by a high concentration of these negatively charged sulfonate groups in the membrane ([-SO3-] 1.13 mole/L). When in contact with water, the hydrogen proton (H+) detaches and flights from one sulfonic molecule (SO3-) to another and thus acts like an electrolyte in the presence of water. Therefore, Nafion-117 transfers H+ across the PEM to the cathode, but does not allow electrons to cross. In order to effectively meet all of the above requirements during durable conditions of work and to ensure membrane efficiency it should be stored in an appropriate manner. For example, Nafion membranes are stored in deionised water. Their regeneration requires boiling in 30% H2O2 and then soaking in 0.5 M H2SO4, and then again soaking with deionised water, for 1 hour, to prevent swelling of the membrane before use. Typical membranes are Nafion-117 which has a pore size of 50 Å (10-10 m) and power density 7.63 mW/m2 and I.C.E. 450 or Hybond™-N, which has power densities between 12-14 mW/m2 pore size of 0.45 microns. There are other commercially available membranes such as Ultrex CMI-7000 (a copolymer of polystyrene and divinylbenzene) (Yang et al., 2011). Currently available membranes are still too expensive to be profitable on industrial scale. But the search for new, inexpensive materials for production of membranes is still going on. 3.4. Microbial community The operation of BER is based on the efficient transport of electrons produced by microorganisms in catabolic processes. Three different mechanisms of electron transport were identified: direct electron transfer, transport via nanowires and external mediator supported transport. Microorganisms that use metals as electron acceptors must have developed mechanisms of direct electron transfer to an insoluble solid. The first microorganisms used in BER were Shewanella putrefaciens (Kim et al., 1999). These bacteria are electrochemically active, because their cytochromes are located on the outer cell wall. Direct transport of electrons requires an establishment of a biofilm on the electrode surface. However, biofilm formation on the electrode results in na decrease of substrates and reaction products diffusion rates (Franks and Nevin, 2010). The accumulation of toxic products (such as butanol) may inhibit the metabolism of cells (Rabaey et al., 2011). Electrochemical activity of biofilms appears to be much more complex than expected. Transport of electrons via nanowires is also realised by biofilm creation. Microorganisms forming the outer layers of the biofilm must produce nanopiles, nanowires for the transfer of electrons to the electrode. Conductive nanopiles and nanowires are present in many strains, but their share in the transport of electrons is different in different microorganisms (Lovley, 2008). In the biofilm of Geobacter sulfurreducens nanowires are the only mechanism used by bacteria at large distances from the electrode surface. The strain of Geobacter sulfurreducens KN400 forms a homogeneous biofilm, where nanowires are the dominant form of transport (Franks and Nevin, 2010). On the contrary in Shewanella oneidensis nanowires play a marginal role (Rabaey et al., 2011). External mediator supported transport is realised through cyclic mediator oxidation and reduction. Bacteria pass electrons to the mediator, which is being reduced. The mediator transports electrons to the anode. As a result, the mediator returns to its oxidised form capable to accept more other electrons. Electron carrier’s origin can be exogenic (e.g. methylene blue, neutral red) or endogenic: (sometimes the electron mediator can be synthesised; e.g. in bacteria of the genus the riboflavin is capable of

608


mediator synthesis (Lovley, 2008). Populations of bacteria in BER can be very complex. This allows to utilise a wide variety of substrates. Microbes that do not exhibit electrochemical activity can also be present in BER. Some play a positive role, transforming complex substances into easily digestible compounds. But others can provide a competition for electricity-producing bacteria. Examples of such microorganisms are bacteria that carry out methanogenic fermentation. This problem can be solved by introducing methanotrophs into reactor, which will decompose formed methane and transfer electrons to the electrode (Yang et al., 2011). 4. CONCLUSIONS Although bioelectrochemical reactors are still in the early stages of development, numerous attractive applications are of particular interest to both researchers and practitioners. Microorganisms are ubiquitous in the environment and can use various types of substrates, which increases the range of raw materials from which energy can be obtained. Most of BERs do not require expensive noble metals, high prices of which can be a barrier in the dissemination of conventional fuel cells. Bacteria can be treated as self-renewing and self-reproducing catalysts, which ensure sustainability of BERs use. A development of BERs requires both fundamental research and a development of new processes and equipment. Mathematical models can help to better understand the metabolism of microorganisms capable of direct transfer of electrons to the electrode, while on the other hand, they help to optimise performance of BERs. At present the practical use of BERs is limited as they are not capable of generating high energy outputs. As can be seen in Fig. 3 typical values of electrical parameters of MFCs are not sufficient enough to be an independent power source. Therefore, they are used in combination with other non-conventional energy sources (solar panels, etc.). An example of such an application is the Leaf, the latest concept car designed by Shanghai Automotive Industry Corporation (SAIC), where the on-board microbial fuel cells recharge lithium-ion batteries. The example for their use as independent power source is a large scale microbial fuel cell using bottom sediment to power marine measuring and navigation devices, which is working for more than ten years. (Reimers et al., 2001). This study was supported by the National Centre for Research and Development (Poland) through grant NR 15-0049-10.

REFERENCES Franks A.E., Nevin K.P., 2010. Microbial fuel cells, a current review, Energies, 3, 889-919. DOI: 10.3390/en3050899. Freguia S., Tsujimura S., Kano K., 2010. Electron transfer pathways in microbial oxygen biocathodes. Electrochimica Acta, 55, 813–818. DOI: 10.1016/j.electacta.2009.09.027. Hamelers H.V.M., Ter Heijne A., Sleutels T.H.J.A., Jeremiasse A.W., Strik D.P.B.T.B., Buisman C.J.N., 2010. New applications and performance of bioelectrochemical systems. Appl. Microbiol. Biotechnol., 85, 1673–1685. DOI: 10.1007/s00253-009-2357-1. Kim B.H., Kim H.J., Hyun M.S., Park D.H., 1999. Direct electrode re action of Fe(III)-reducing bacterium, Shewanella putrefaciens. J. Microbial. Biotech., 9, 127-131. Kim K.-Y., Chae K.-J., Choi M.-J., Ajayi F.F., Jang A., Kim C.-W., Kim I.S., 2011. Enhanced Coulombic efficiency in glucose-fed microbial fuel cells by reducing metabolite electron losses using dual-anode electrodes. Bioresour. Tech., 102, 4144-4149. DOI: 10.1016/j.biortech.2010.12.036. Lefebvre O., Uzabiaga A., Chang I.S., Kim B., Ng H.Y., 2011. Microbial fuel cells for energy self-sufficient domestic wastewater treatment – a review and discussion from energetic consideration. Appl. Microbiol. Biotechnol., 89, 259–270. DOI: 10.1007/s00253-010-2881-z.

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P. Sobieszuk, A. Zamojska-Jaroszewicz, A. Kołtuniewicz, Chem. Process Eng., 2012, 33 (4), 603-610 Li C., Ding L., Cui H., Zhang L., Xu K., Ren H., 2012. Application of conductive polymers in biocathode of microbial fuel cells and microbial community. Bioresour. Technol., 116, 459-465. DOI: 10.1016/j.biortech.2012.03.115. Liang P., Wang H.Y., Xia X., Huang X., Mo Y. H., Cao X.X., Fan M. Z., 2011. Carbon nanotube powders as electrode modifier to enhance the activity of anodic biofilm in microbial fuel cells. Biosens. Bioelectron., 26, 3000-3004. DOI: 10.1016/j.bios.2010.12.002. Liu J., Qiao Y., Guo C.X., Lim S., Song H., Li C. M., 2012. Graphene/carbon cloth anode for high-performance mediatorless microbial fuel cells. Bioresour. Technol., 114, 275-280, DOI: 10.1016/j.biortech.2012.02.116. Logan B. E., Hamelers B., Rozendal R., Schröder U., Keller J., Freguia S., Aelterman P., Verstraete W., Rabaey K., 2006. Microbial fuel cells: methodology and technology. Environ. Sci. Technol., 40, 5181–5192. DOI: 10.1021/es0605016. Logan B.E., 2010. Scaling up microbial fuel cells and other bioelectrochemical systems, Appl. Microbiol. Biotechnol., 85, 1665–1671. DOI: 10.1007/s00253-009-2378-9. Lovley B.E., 2008. The microbe electric: conversion of organic matter to electricity. Current Opinion Biotechnol., 19, 564-571. DOI: 10.1016/j.copbio.2008.10.005. Lowy D.A., Tender L.M., 2008. Harvesting energy from the marine sediment-water interface: III. Kinetics activity of quinone- and antimony-based anode materials. J. Power Sources, 185, 70-75. DOI: 10.1016/j.jpowsour.2008.06.079. Nevin K.P., Woodard T.L., Franks A.E., Summers Z.M., Lovley D.R., 2010. Microbial electrosynthesis: Feedin microbes electricity to convert carbon dioxide and water to multicarbon extracellular organic compounds. mBio, 1, e00103-10. DOI: 10.1128/mBio.00103-10. Rabaey K., Girguis P., Nielsen L.K., 2011. Metabolic and practical considerations on microbial electrosynthesis. Current Opinion in Biotechnol., 22, 371-377. DOI: 10.1016/j.copbio.2011.01.010. Reimers C.E., Tender L.M., Ferting S., Wang W., 2001. Harvesting energy from the marine sediment-water interface. Environ. Sci. Technol., 35, 192-195. DOI: 10.1021/es001223s. Rozendal R.A., Hamelers H.V.M., Buisman C.J.N., 2006. Effects of membrane cation transport on pH and microbial fuel cell performance. Environ. Sci. Technol., 40, 5206–5211. DOI: 10.1021/es060387r. Rozendal R.A., Jeremiasse A.W., Hamelers H.V.M., Buisman C.J.N., 2008. Hydrogen production with a microbial biocathode. Environ. Sci. Technol., 42, 629–634. DOI: 10.1021/es071720+. Tang X., Guo K., Li H., Du Z., Tian J., 2011. Electrochemical treatment of graphite to enhance electron transfer from bacteria to electrodes. Bioresour. Technol., 102, 3558-3560. DOI: 10.1016/j.biortech.2010.09.022. ter Heijne A., Hamelers H.V.M., Saakes M., Buisman C.J.N., 2008. Performance of non-porous graphite and titanium-based anodes in microbial fuel cells. Electrochimica Acta, 53, 5697-5703. DOI: 10.1016/j.electacta.2008.03.032. Wang H-Y., Bernarda A., Huang C-Y., Li D-J., Chang J-S., 2011. Micro-sized microbial fuel cell: A mini review. Bioresour. Technol., 102, 235-243. DOI: 10.1016/j.biortech.2010.07.007. Wang X., Cheng SA., Feng Y.J., Merrill M.D., Saito T., Logan B.E., 2009. Use of carbon mesh anodes and the effect of different pretreatment methods on power production in microbial fuel cells. Environ. Sci. Technol., 43, 6870-6874. DOI: 10.1021/es900997w. Wei J., Liang P., Huang X., 2011. Recent progress in electrodes for microbial fuel cells. Bioresour. Technol., 102, 9335-9344. DOI: 10.1016/j.biortech.2011.07.019. Yang Y., Sun G., Xu M., 2011. Microbial fuel cells come of age. J. Chem. Technol. Biotechnol., 86, 625–632. DOI: 10.1002/jctb.2570. Received 14 May 2012 Received in revised form 10 October 2012 Accepted 24 October 2012

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LACCASE IMMOBILISATION ON MESOSTRUCTURED SILICAS Jolanta Bryjak*1, Katarzyna Szymańska2, Andrzej B. Jarzębski2, 3 1

Wrocław University of Technology, Faculty of Chemistry, Department of Bioorganic Chemistry, Norwida 4/6, 50-373 Wrocław, Poland 2

Department of Chemical Engineering, Silesian University of Technology, M. Strzody 7, 44-100 Gliwice, Poland 3

Institute of Chemical Engineering, Polish Academy of Sciences, Bałtycka 5, 44-100 Gliwice, Poland

Extracellular laccase produced by the wood-rotting fungus Cerrena unicolor was immobilised covalently on the mesostructured siliceous foam (MCF) and three hexagonally ordered mesoporous silicas (SBA-15) with different pore sizes. The enzyme was attached covalently via glutaraldehyde (GLA) or by simple adsorption and additionally crosslinked with GLA. The experiments indicated that laccase bound by covalent attachment remains very active and stable. The best biocatalysts were MCF and SBA-15 with Si–F moieties on their surface. Thermal inactivation of immobilised and native laccase at 80°C showed a biphasic-type activity decay, that could be modelled with 3parameter isoenzyme model. It appeared that immobilisation did not significantly change the mechanism of activity loss but stabilised a fraction of a stable isoform. Examination of time needed for 90% initial activity loss revealed that immobilisation prolonged that time from 8 min (native enzyme) up to 155 min (SBA-15SF). Keywords: Laccase, immobilisation; mesostructured enzyme carrier; thermal stability

1. INTRODUCTION Oxidising enzymes such as laccases (EC 1.10.3.2) attract considerable attention because they offer unique solutions to selective oxidation reactions in aqueous, organic and biphasic systems that have been extensively reviewed (Burton, 2003, Duran et al., 2002; Witayakran and Ragauskas, 2009). These enzymes are of particular interest not only because they are easily available at scale, but because their copper-containing redox sites catalyse oxidation of important substrates with concomitant reduction of molecular oxygen to water, without expensive co-factors. As the array of substrates for laccases includes alkenes, phenolics, polyamines and lignin-related molecules, these enzymes can be used in bioremediation processes, as adjuvant in textile or paper industries, biosensing elements and also as catalysts in organic synthetic chemistry and in biotransformations of natural compounds (Burton, 2003; Duran et al., 2002; Mikolasch and Schauer, 2009; Witayakran and Ragauskas, 2009). However, operational stability of native enzymes is rather limited. Therefore, immobilisation is very often applied in order to ease this limitation. The main advantage of immobilised enzymes is their easy recovery from reaction mixture by simple filtration or application in continuous processes. The enzymes are usually attached to water or organic solvent insoluble carriers, and that can additionally improve both their thermal and operational stability. Amongst many immobilisation techniques, covalent binding and simple adsorption of laccases seem to be the most popular and effective methods (Duran et al., 2002). *Corresponding author, e-mail: [email protected]

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J. Bryjak, K. Szymańska, A.B. Jarzębski, Chem. Process Eng., 2012, 33 (4), 611-620

Typically, the selection of an immobilisation method suitable for a particular enzyme, is achieved by surveying a broad database of performances of different immobilised preparations. With this in mind we undertook a systematic study of Cerrena unicolor laccase immobilisation onto typical enzyme carriers: acrylic beads (Bryjak et al., 2007), cellulose-based carriers (Rekuć et al., 2008), commercial silica gels (Rekuć et al., 2010) and onto a relatively new class of carriers - silica mesoporous cellular foams (MCFs) (Rekuć et al., 2009) beads, all modified with various anchor groups, to find that only aminated MCFs could offer very active preparations. Mesoporous silicas reveal significant advantages as enzyme supports: i. particles are fairly small (20-30 µm) with a very open structure of mesopores which and this relaxes considerable diffusional constrains; ii. pore sizes are even over 20 nm, thus similar or larger than most enzyme molecules; iii. large internal surface area is easily accessible to enzymes; iv. high chemical, thermal, mechanical and biological resistance and environmental inertion (Avnir et al., 2006; Chaudhary et al., 2007; Hudson, et al. 2008; Pierre, 2004). Although laccase covalently bound to MCFs carriers appeared to be extremely active, none of the preparations was stable enough at elevated temperatures. In fact, some were more stable than a native preparation, but the improvement was not deemed satisfactory. Therefore, the present research was focused on the covalent and adsorptive immobilisation of laccase onto aminated mesocellular materials, some of which were additionally modified with Si-F moieties. For this purpose MCF with ultra large mesopores was synthesised as well as three other ordered mesoporous silicas with hexagonal pore arrangement (SBA-15): a conventional SBA-15, SBA-15L with same mesopore structure as MCF, yet not using NH4F during synthesis, SBA-15S and SBA-15SF were more conventional silicas with medium sized mesopores but differed in presence of NH4F in synthetic protocol (SBA-15SF). The effect of carrier structure and additional crosslinking with glutaraldehyde on the activity and stability of immobilised enzyme was examined and after that, selected very active enzyme-carrier preparations were tested at elevated temperature. Special attention was given to thermal stability as it was the first sign that immobilised preparations could be sufficiently stable under process conditions.

2. MATERIALS AND METHODS 2.1. Materials Trihydroxymethylaminomethane (tris), glutaraldehyde (GLA), 2,2’-azino-bis(3-ethylbenzothiazoline-6sulfonate) sodium salt (ABTS) were purchased from Sigma. Tetraethoxysilane (98%, TEOS), 1,3,5trimethylbenzene (TMB), 1-chloro-2,3-epoxypropane, chloroacetic acid and 2-aminoethyl-3aminopropyltrimethoxysilane (AEAPTS) applied as donors of amine groups, were obtained from Aldrich. Pluronic PE 9400 was purchased from BASF. Other reagents, all of analytical grade, were supplied by POCh (Poland). 2.2. Synthesis and functionalization of SBA-15 and MCF materials The synthesis of SBA-15L (with ultra large mesopores) was as described by Szymańska et al. (2009). In brief, 6 g Pluronic P123 was dissolved in 2 M HCl solution (180 cm3) at 40 °C (Lei C et al., 2006) and then 10.5 cm3 of TMB and 13.5 cm3 of TEOS were added and stirred for 12 h at the same temperature. The mixture was heated up to 40 °C for 8 h and next to 100 °C for 24 h, without stirring. The white precipitate was collected by filtration, dried in air, and finally calcined at 550 °C for 6 h. Preparation of SBA-15S and SBA-15SF (with medium size mesopores) was done as described Zhao et al. (1998). Pluronic P123 (4 g) was dissolved in 30 cm3 of water and 120 cm3 of 2 M HCl solution with stirring at 35 °C (in case of SBA-15SF 0.04 g of NH4F was added). Then 9.1 cm3 of TEOS was added into that solution with stirring at this temperature for 20 h. The mixture was aged at 80 °C overnight without stirring. The solid product was recovered, washed, and air dried at room temperature.

612

Laccase immobilisation on mesostructured silicas

Calcination was carried out by increasing temperature from 23 to 500 °C for 8 h and heating at 500 °C for 6h. Preparation of MCF (with ultra large mesopores) was done as described by Szymańska et al. (2009). In a typical procedure Pluronic P123 (4 g) was dissolved in 1.6 M HCl (75 cm3) at room temperature and then TMB (5.8 cm3) and NH4F (0.023 g) were added under vigorous stirring at 40 °C. After 1 h stirring TEOS was added (4.7 cm3), stirred for 1 h and stored at 40 °C for 20 h and then at 100 °C for 24 h. The precipitate was filtered, dried at room temperature for 4 days and calcined at 500 °C for 8 h. Amino groups were grafted onto pristine carriers surface by reacting suitable amounts of organosilanes (AEAPTS dissolved in toluene) under reflux (24 h, 40 °C) with silanols present on the silica surface to obtain a load of functional moiety of about 1.5 mmol/g of silica (Rekuć at al., 2009). 2.3. Characterisation of MCF and SBA-15 materials The values of specific surface area (SBET), pore volume (VpN2), diameter of cells (dp), and that of interconnected windows (dw) of pristine (calcined) carriers were determined from the analysis of nitrogen adsorption/ desorption isotherms measured at -196 ºC using a Micromeritics ASAP 2000 instrument and applying BJH (Barret–Joyner–Halenda) method. Prior to measurements the samples were outgassed at 150 °C for 4 h. The texture of materials was also examined using transmission electron microscopy (TEM, Tecnai G2 apparatus operating at 200 kV). 2.4. Enzyme preparation and activity assays The wood-rotting fungus Cerrena unicolor (Bull.ex.Fr.) Murr, No 139, was obtained from the culture collection of the Department of Biochemistry, UMCS University (Lublin, Poland). Microorganism cultivation and laccase production was performed according to the method described by Al-Adhami et al. (2002). The laccase containing culture fluid was purified according to the procedure proposed by Bryjak and Rekuć (2010). The enzyme activity was determined from the change of optical density in time and calculated from the initial reaction rate region, using 0.207 mM ABTS in 0.1 M citratephosphate buffer, pH 5.3, as a substrate (Childs and Bardsley, 1975). The enzyme activity unit (U) was defined as the amount of the enzyme required to change absorbance (420 nm) of 1.0 per min at 30 °C. The mean analytical error was less than ± 2.0%. Protein concentration was determined by the Lowry’s method (Lowry at al., 1957) using bovine serum albumin as a standard. The mean analytical error was less than ± 2.5%. The activity of immobilised enzymes was measured in a well-mixed (200 rpm) reactor in batch regime. The immobilised enzyme was suspended in the buffer (pH 5.3), placed into reactor the temperature of which was maintained at 30 °C. Then, preheated substrate in the buffer was added (1.25 mM end ABTS concentration) and several samples were taken from the reactor at one-minute intervals. After absorbance (420 nm) measurement the samples were returned to the reactor to ensure a constant ratio of carrier to substrate volume. Immobilised enzyme activity was recalculated per 1 cm3 of freely sedimented carrier. The mean analytical error was less than ±4.5%. 2.5. Laccase immobilisation For immobilisation, the functionalised carrier (4-5 cm3) was washed by centrifugation with distilled water and 0.1 M phosphate buffer (pH 7) and then activated with 2.5% (v/v) GLA for 2 h. The same but not activated carriers were used for adsorptive immobilisation. All the carriers were suspended in 10 cm3 of laccase solution (pH 7) and left at 4 °C overnight. After that the excess protein was removed

613


by washing under different pH and ionic strength of buffers. Next, all immobilised preparations were divided into two parts and one of them was additionally crosslinked with 2.5% (v/v) GLA solution (pH 7; 1 h). Active aldehyde groups remaining on the carrier were blocked in 0.5 M tris–HCl buffer, pH 8.2. Finally, the obtained preparations were rinsed with 0.1 M phosphate-citrate buffer of pH 5.3 and activities were measured. The immobilised preparations were stored at 4 °C in an appropriate buffer and they were washed several times with the buffer shortly prior to experiments. All the eluates were collected and analysed for the presence of protein and activity. The amount of bound protein (activity units) was calculated from a difference between the amount used for immobilisation and that washed off (protein/activity mass balance). 2.6. Thermal inactivation of a native and immobilised enzyme Thermal inactivation was tested at 80 °C as the enzyme was previously found to be very stable at temperatures ranging from 18 up to 55 °C (Bryjak et al., 2007). For the inactivation experiments a settled immobilised preparation (0.1 cm3) in the buffer was introduced into thin-layer probes and the excess of the buffer was removed. Then 2 cm3 of the buffer (80 °C) was added to the probes, tightly closed and placed into a water bath of 80 °C. At certain time intervals the samples were taken out, cooled rapidly in an iced-water bath and stored therein prior to activity measurement conducted after 1.5 h of storage. A control experiment was conducted using native enzyme of similar reactivity. Kinetic parameters of thermal inactivation model were estimated using nonlinear regression (parameter estimation software OriginPro 8).

3. RESULTS AND DISCUSSION 3.1. Properties of the supports Table 1 lists the properties of surface structure of the pristine supports obtained from nitrogen adsorption experiments and Fig. 1 shows TEM images of the supports. The specific surface area (Table 1) was almost the same in all carriers, whereas the pore volume and pore diameter for MCF and SBA-15L were respectively, two and three times larger than those for SBA-15SF and SBA-15S. It can be seen that only SBA-15S and SBA-15SF showed an ordered (hexagonal) structure (Fig. 1). TEM studies confirmed a significant similarity in pore structure of both MCF and SBA-15L, that indeed, appeared to be much more open than that of SBA-15S or SBA-15SF and it was predominated by large spherical mesopores (dp) connected by windows (dw). A modification of pristine silica carriers with organosilanes resulted in a slight decrease of surface parameters (results not shown) but had little effect on pore sizes. Table 1. Structure parameters of MCF and SBA carriers

Carrier

SBET [m2/g]

VpN2 [cm3/g]

dp [nm]

dw [nm]

MCF

620

2.50

27.0

14.0

SBA-15L

635

2.16

25.2

10.7

SBA-15SF

638

1.11

8.5

-

SBA-15S

645

0.71

6.3

-

614


Fig. 1. TEM images for: MCF (A), SBA-15L (B), SBA-15SF (C) and SBA-15S (D)

3.2. Laccase immobilisation It is clear that activity of a biocatalyst experimentally determined is the most important single parameter of an immobilised enzyme. Its value is strongly influenced by the amount of bound protein, enzyme affinity to a carrier surface and accessibility of the active center for substrates. In the case of adsorption (Table 2) very weak dependence of the protein content on the pore size was observed. Protein load varied from 0.615 to 0.775 mg/ cm3 of the carriers and, that probably reflected similarity in their specific surface area. Interestingly, when laccase was covalently bound to the carriers, the protein content was about a half of that value. This phenomenon can be explained by a change in surfaceprotein interactions, caused by activation of amino groups with GLA, which reduced affinity of the enzyme molecules to the carriers’ surface, and also concluded from the proportionally lower value of expected activity of covalently bound laccase. But, surprisingly enough, this significantly lower protein content and expected activity values resulted in actual (measured) catalysts activities even five-fold larger than those obtained for the enzyme bound by adsorption. This clearly demonstrates that the covalent bonding via amino groups with GLA activation is exceptionally effective for immobilisation of laccase. Moreover, closer examination shows that the observed phenomena are more complex. Two couples of aminated samples, respectively: SBA-15L and MCF or SBA-15S and SBA-15SF had a similar pore structure and laccase was immobilised on their surface in the same way. Yet, the activities of SBA-15L and SBA-15S were by ca. 14% lower than those of MCF and SBA-15SF and this can hardly be explained by minor differences in porosities of the corresponding materials. As the sole difference lay in the synthesis of pristine MCF and SBA-15SF or SBA-15L and SBA-15S, respectively, and more specifically in that ammonium fluoride was applied in the synthesis of the former and not in the latter group, we may speculate that its presence changed the intrinsic properties of the silica surface in MCF and SBA-15SF materials, perhaps due to formation of small but strongly hydrophobic Si–F entities. Their presence could positively affect protein orientation on the biocatalysts surface to afford higher activity. It is noteworthy that the same trend was more recently observed also in the hydrolysis of sucrose using biocatalysts with invertase immobilised on identical silica carriers (Szymańska et al, 2009). This suggestion can be supported by activities of immobilised preparations in which the proteins were additionally crosslinked after their immobilisation (Fig. 2). In this case the activities of MCF and SBA-15SF catalysts were about 11% lower than those of SBA-15S and SBA-15L, respectively. As can be seen from Figure 2, an additional crosslinking of proteins already bound to the carriers by adsorption

615


or covalent attachment brought about activity increase from 14 up to 33%. It is likely, that it was caused by a reduction in desorption of laccase bound unspecifically. Table 2. Activity of laccase immobilised on mesoporous silicas by adsorption and covalent attachment

Carrier

MCF

SBA-15L

SBA-15SF

Expected activity [U/cm3] 4755

Measured activity [U/cm3] 2599

Active in bound protein [%] 54.7

Activity Yield [%] 28.8

adsorption

0.774

6926

420

6.1

5.8

covalent

0.297

3396

2250

66.3

25.9

adsorption

0.615

6165

420

6.8

6.5

covalent

0.239

3346

2584

77.2

44.6

adsorption

0.651

6140

450

7.3

7.2

covalent

0.354

4737

2215

46.8

27.4

adsorption

0.775

8118

495

6.1

5.5

SBA-15L

SBA-15SF

Method

Measured activity [U/cm3]

SBA-15S

covalent

Bound protein [mg/cm3] 0.248

3500 3000 2500 2000 1500 1000 500 0

SBA-15S

MCF

Fig. 2. Activities of laccase immobilised on different carriers: white bars – adsorption; black bars – covalent attachment; dotted bars – as the previous ones but additionally crosslinked with GLA

To summarise, it appears that biocatalysts obtained by adsorption of laccase on MCF and SBA-15 type of mesoporous silica are not very active. For that reason, thermal stability tests were performed only for the preparations with laccase attached by the covalent method. 3.3. Thermal stability of immobilised laccase Temperature is a critical variable in enzyme reactor operation. It produces two contradictory effects; an increase in reaction rate, but also an increase in the rate of enzyme inactivation. Then, an experimental analysis of the inactivation process is a prerequisite to most applications of enzymes. Our previous study showed that thermal stability of laccase immobilised on MCF carrier, incubated at 80 °C, was only marginally better than that of a free enzyme (Rekuć, 2009). To improve this stability by synthesis of new sol-gel carriers was the main objective of the present work. For this purpose all the preparations with the enzyme attached covalently with or without additional crosslinking were incubated at this

616


temperature. A typical example of obtained results is presented in Figure 3. As can be seen from Figure 3a, both the native and immobilised laccase inactivation patterns are quite similar and they feature a rapid activity loss in the first phase followed by its significantly slower decay in the second one, typical for a biphasic inactivation (see Fig. 3b). Interestingly, Michniewicz et al. (2006) reported before that laccase from Cerrena unicolor consists of 2 isoforms which differ in thermal stability. For that reason, a simple isoenzyme mechanism (Sadana, 1991) of inactivation was adopted: k1 E1 → I1

(1a)

k2 E 2 → I2

(1b)

to obtain a final expression for activity:

A = a exp( − k1t ) + b exp(− k 2 t ) A0

(2)

where: E1 and E2 are native isoforms, I1 and I2 are inactive forms, A and A0 are activities at given time and at time t = 0, k1 and k2 are the reaction rate constants, a and b are stable and labile fraction of isoenzymes, respectively.

Fig. 3. Thermal inactivation of laccase at 80 °C. Symbols: yellow points - native enzyme; blue points – laccase immobilised on SBA-15SF; green points – laccase immobilised on the same carrier with additional crosslinking; lines display predictions from the isoenzyme model (2). b) ln(A/A0) vs. heating time demonstration of the biphasic inactivation behaviour of the native and immobilised laccase

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When a sum of a and b fractions is normalised this equation can be simplified to a three parameter expression with b = 1-a. From the values given in Table 3 it can be seen that the initial fraction of a stable form of the native enzyme was about 31% and it did not change notably after immobilisation. The values of k1, corresponding to the stable fraction, showed a stabilisation of this isoform by immobilisation, except for MCF. However, the labile isoform was not stabilised (c.f. k2 constants), except for SBA-15L without GLA crosslinking. Surprisingly, no evident positive stabilisation effect of post-immobilisation protein crosslinking was observed. To determine the type of silica and protein immobilisation method that afford most stable catalysts, we calculated the time needed for a 90% loss of the initial activity (Fig. 4). It can be seen that immobilised laccase was more stable than its native form, and among various mesoporous silica supports that of SBA-15SF appeared to give the most stable preparations. As it appeared earlier to be nearly as active as MCF, it is the best catalysts among the investigated mesoporous silica materials. But of considerable interest may also be quite a surprising observation that post-attachment protein crosslinking strongly destabilised the enzyme, probably by modification of aminoacid(s) involved in maintaining the enzyme’s compact structure. Table 3. Comparison of kinetic parameters of inactivation of the native and immobilised laccase at 80 °C. Symbols: k1 and k2 - reaction rate constants for stable and labile fraction of isoenzymes, a – fraction of stable isoenzyme

Enzyme/Carrier

Glutaraldehyde crosslinking

a [-]

k1 [1/min]

k2 [1/min]

Native

-

0.305±0.066

0.0672±0.012

0.92±0.155

No

0.343±0.067

0.083±0.020

0.800±0.162

Yes

0.295±0.057

0.065±0.020

0.835±0.133

No

0.376±0.058

0.018±0.06

0.599±0.101

Yes

0.448±0.021

0.045±0.005

1.153±0.125

No

0.234±0.022

0.0054±0.0017

0.784±0.088

Yes

0.284±0.021

0.026±0.005

1.153±0.125

No

0.320±0.021

0.047±0.006

1.068±0.093

Yes

0.318±0.034

0.059±0.018

0.768±0.099

MCF

SBA-15L

SBA-15SF

Time of 90% activity loss [min]

SBA-15S

160

120

80

40

0

Native

MCF

SBA-15L SBA-15S SBA-15SF

Fig. 4. Time needed for 90% activity loss of laccase incubated at 80 °C. Preparations with post-attachment crosslinking are marked by dotted bars

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4. CONCLUSIONS Siliceous mesostructured materials (MCF and SBA-15) show a great potential as carriers for laccase immobilisation to provide very active biocatalysts. The final activity of preparations depends on their structure and the applied method of protein binding. Regardless of the type of silica carrier, the covalent attachment of the enzyme via glutaraldehyde appeared to be about five times more effective than the adsorption-based method. This observation contradicts a common view that harsh conditions of covalent immobilisation strongly denature enzymes’ superstructure and significantly reduce their activity. It is also supported by the fact that the additional crosslinking of already immobilised laccase had no detrimental effect on the activity of the catalysts. The most active preparations were those immobilised covalently on MCF and SBA-15SF carriers and that can be linked with the presence of Si– F moieties on the surface of these materials. Thermal inactivation of covalently-bound laccase, performed at 80°C, revealed that its activity decays in a biphasic way that can be modelled by a simple isoenzyme equation, typical for the native enzyme. It was found that immobilisation did not affect the fraction of a stable isoform, and except for MCFbased catalysts, it reduced the rate of its inactivation. The performed studies of a deep activity decay (up to 10% of the initial value) indicate that: i. post-attachment protein crosslinking destabilises the enzyme; ii. immobilisation of laccase onto MCFs appeared to be not effective at all; iii. immobilisation of laccase on SBA-15L and SBA-15SF carriers significantly prolonged the time of activity decay, from 8 min (for native enzyme) to 75 and 155 min, respectively. Thus, taking into account the highest activity and thermal stability of the immobilised laccase, aminated and GLA activated SBA-SF emerges as the carrier of choice.

The support for this work by the Polish State Committee for Science and Research under grant N N209 119337, 2009-2012 is gratefully acknowledged.

REFERENCES Al-Adhami A.A.J.H., Bryjak J., Greb-Markiewicz B., Peczyńska-Czoch W., 2002. Immobilization of woodrotting fungi laccases on modified cellulose and acrylic carriers. Process Biochem., 37, 1387-1394. DOI: 10.1016/S0032-9592(02)00023-7. Avnir D., Coradin T., Lev O., Livage J., 2006. Recent bio-applications of sol-gel materials. J. Mater. Chem., 16, 1013-1030. DOI: 10.1039/b512706h. Bryjak J., Kruczkiewicz P., Rekuć A., Peczyńska-Czoch W., 2007. Laccase immobilization on copolymer of butyl acrylate and ethylene glycol dimethacrylate. Biochem. Eng. J., 35: 325-332. DOI: 10.1016/j.bej.2007.01.031. Bryjak J., Rekuć A., 2010. Effective purification of Cerrena unicolor laccase using microfiltration, ultrafiltration and acetone precipitation. Appl. Biochem. Biotechnol., 160, 2219-2235. DOI: 10.1007/s12010-009-8791-9. Burton S.G., 2003. Laccases and phenol oxidases in organic synthesis – a review. Curr. Org. Chem., 7, 13171331. Chaudhary Y.S., Manna S.K., Mazumdar S., Khushalani D., 2008. Protein encapsulation into mesoporous silica hosts. Micropor. Mesopor. Mat., 109, 535-541. DOI: 10.1016/j.micromeso.2007.06.001. Childs R.E., Bardsley W.G., 1975. The steady-state kinetics of peroxidase with 2,20-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid) as chromogen. Biochem. J., 145, 93–103. Durán N., Rosa M.A., D’Annibale A., Gianfreda L., 2002. Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review. Enzyme Microb. Technol., 31, 907-931. DOI: 10.1016/S0141-0229(02)00214-4. Hudson S., Cooney J., Magner E., 2008. Proteins in mesoporous silicates. Angewandte Chemie. Int. Ed., 47, 8582-8594.

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Lei C., Shin Y., Magnusom J.K., Fryxell G., Lasure L.L., Elliott D.C., Liu J., Ackerman E.J., 2006. Characterization of functionalized nanoporous supports for protein confinement. Nanotechnol., 17, 5531-5538. DOI: 10.1088/0957-4484/17/22/001. Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J., 1951. Protein measurement with the Foulin phenol reagent. J. Biol. Chem., 193, 265-275. Michniewicz A., Ullrich R., Ledakowicz S., Hofrichter M., 2006. The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties. Appl. Microbiol. Biotechnol., 69, 682-688. DOI: 10.1007/s00253-005-0015-9. Mikolasch A., Schauer F., 2009. Fungal laccases as tools for the synthesis of new hybrid molecules and biomaterials. Appl. Microbiol. Biotechnol., 82, 605-624. DOI: 10.1007/s00253-009-1869-z. Pierre A.C., 2004. The sol-gel encapsulation of enzymes. Biocatal. Biotransform., 22, 145-170. DOI: 10.1080/10242420412331283314. Rekuć A., Kruczkiewicz P., Jastrzembska B., Liesiene J., Peczyńska-Czoch W., Bryjak J., 2008. Laccase immobilization on the tailored cellulose-based Granocel carriers. Int. J. Biol. Macromol., 42, 208-215. DOI: 10.1016/j.ijbiomac.2007.09.014. Rekuć A., Bryjak J., Szymańska K., Jarzębski A.B., 2009. Laccase immobilization on mesostructured cellular foams affords preparations with ultra high activity. Proc. Biochem., 44, 191-198. DOI: 10.1016/j.procbio.2008.10.007. Rekuć A., Bryjak J., Szymańska K., Jarzębski A.B., 2010. Very stable silica-gel-bound laccase biocatalysts for the selective oxidation in continuous systems. Biores. Technol., 101, 2076-2083. DOI: 10.1016/j.biortech.2009.11.077. Sadana A., 1991. Biocatalysis. Fundamentals of enzyme deactivation kinetics. Prentice Hall, Englewood Cliffs, New Jersey. Szymańska K., Bryjak J., Jarzębski A.B, 2009. Immobilization of invertase on mesoporous silicas to obtain hyper active biocatalysts. Top. Catal., 52, 1030-1036. DOI: 10.1007/s11244-009-9261-x. Witayakran S., Ragauskas A.J., 2009. Synthetic applications of laccase in Green Chemistry. Adv. Synth. Catal., 351, 1187-1209. DOI: 10.1002/adsc.200800775. Zhao D., Huo Q., Feng J., Chmelka B.F., Stucky G.D., 1998. Nonionic triblock and star diblock copolymer and oligomeric surfactant syntheses of highly ordered, hydrothermally stable, mesoporous silica structures. J. Am. Chem. Soc., 120, 6024-6036. DOI: 10.1021/ja974025i. Received 18 May 2012 Received in revised form 06 November 2012 Accepted 08 November 2012

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HYDRAULIC MIXING MODELLING IN REACTOR FOR BIOGAS PRODUCTION

Andrzej G. Chmielewski* 1,2, Aleksandra Berbeć1, Michał Zalewski2, Andrzej Dobrowolski2 1

Warsaw University of Technology, Department of Chemical and Process Engineering, Waryńskiego 1, 00-645 Warsaw, Poland 2

Institute of Nuclear Chemistry and Technology, Dorodna16, 03-195 Warsaw, Poland

Two-stage biogas production plant consists of two reactors: a hydrolyser and a fermentor. The bioreactor construction has to meet three requirements: low cost and simplicity of construction and good biomass mixing conditions with an application of appropriate method. This paper reports CFD modelling of hydraulic mixing in the tank to be applied in a two-stage industrial installation. Keywords: biogas, biomass mixing, bioreactor, CFD modelling

1. INTRODUCTION Different solutions are applied for biogas production. One of them is a two-stage system in which hydrolyse is separated from methanogenesis. Such a solution has been developed at a pilot plant constructed at Szewnia Wielka, Poland (Kryłowicz et al., 2008 ). High biogas productivity and high methane concentration in this product have been achieved (Kryłowiecz et al., 2011). Therefore industrial plants are being constructed. The best engineering solution for construction of a plant is reinforced concrete, which however requires simple casing board geometry and a horizontal rectangular cuboid seems to readily meet all the chemical and engineering requirements. Applied hydraulic mixing has to meet requirements of the process and therefore Computer Fluid Dynamics (CFD) has been applied to find a proper localisation of inlet – outlet biomass transportation pipes.

2. DESIGN OF THE BIOREACTOR The analysed fermentor is a horizontal rectangular cuboid with the following dimensions: • length - 10 m, • height of liquid in a rectangular section – 3.75 m, • width - 5 m, • radius of curvature at the bottom – 0.25 m. The biogas plant consists of two such tanks in series, therefore, the analyses are valid for both units – hydrolyser and fermentor. A semibatch process is applied and feed is delivered in regular time intervals. It is well known that performance of anaerobic digesters is affected primarily by the retention


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time of substrate in the reactor and the degree of contact between incoming substrate and a viable bacterial population (Karim et al., 2005). In order to provide efficient mixing, a set of circulation pumps will be used. Cylindrical tubes with a diameter of 0.1 m will be used to provide inlet and outlet streams.

3. APPLIED CFD METHODOLOGY To conduct the simulation, ANSYS FLUENT package was used, that refers to a finite volume method for numerical solutions of momentum balance equations. Calculations assume that the suspension is a homogeneous liquid with a density of water. The viscosity of the suspension was taken as 1.25 of the water viscosity (Rudniak, 2010). Fully developed turbulent flow was assumed and a modified k-ε turbulence model was used. The geometry of the fermentor was discretised using tetrahedron elements, the number of these was about 1.2 million for each case. Free surface of the liquid was modelled as a condition of symmetry (no normal component of the velocity vector and the derivative of the other components of the velocity vector in the normal direction is equal to zero). No slip on the walls of the fermentor was assumed. Based on these assumptions and boundary conditions, a numerical simulation of suspension flow in a fermentor was carried out. It consisted of a numerical solution of momentum balance equation and additional two equations for the model k-ε turbulence.

4. RESULTS A hydraulic mixing system was analysed using the circulation in two dimensions: horizontal and vertical. The first simulation - horizontal circulation - was to find a system that will provide the movement of the material throughout the length of the reactor and allow to recycle the biomass. Six different cases were examined. The location of the inlet pipe axis from the bioreactor bottom was variable, while the difference of 0.8 m between the inlet and outlet maintained the same for all the cases. Table 1. presents a summary of the analysed cases. Table 1. Summary – horizontal circulation

1

Inlet pipe axis height from bottom [m] 0.2

Outlet pipe axis height from bottom [m] 1.0

2

0.4

3

Mass flow [kg/s]

Stream range [m]

Fraction of height affected by stream

11.07

7

1/3

1.2

11.07

5

2/3

0.6

1.4

11.07

4

1

4

0,8

1.6

11.07

2

1

5

1.0

1.8

11.07

1

1

6

2 x 0.4

2 x 1.2

2 x 11.07

10

1

Case No.

The inlet stream impact range was evaluated on the basis of the flow charts presenting velocity as a function of the distance from the inlet (Fig. 1., Fig. 3., Fig. 5., Fig. 7., Fig. 9., Fig. 11.). It was observed that the range of the stream impact increased with a decreasing height of the installation of the inlet tube from the bottom of the reactor. In addition, due to the flow structure charts (Fig. 2., Fig. 4., Fig. 6.,

622

Hydraulic mixing modelling in reactor for biogas production

Fig. 8., Fig. 10., Fig. 12.), the movement of the liquid in the entire height of the th fermentor was analysed. It was observed that a fraction of liquid height affected by the stream increased while increasing the height of the inlet tube installation from the bottom of the vessel. The results showed that in order to ensure horizontal circulation culation two inlet streams located at the same height of 0.4 m distance from the bottom should be applied (case 6). Moreover, to mix the entire volume it is required to use an additional vertical circulation loop.

Fig. 1. Flow chart – Case 1

Fig. 2. Flow structure chart – Case 1


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624






625



The second simulation – vertical circulation - was to find a system proving efficient mixing in the entire volume of the fermentor. The analysis started from one-loop circulation, and then it was developed with other loops, until a complete mixing was achieved in the analysed fermentor. An analysis of the impact range from one-loop case (Fig. 13.) showed that the solution does not accomplish the task of obtaining the forced movement of the suspension in the entire volume of the fermentor. The two - loop case (Fig. 14.) increased the area of mixed fluid, but only the use of three - loop model proved to be a solution ensuring the suspension movement in the entire volume of the analysed reactor (Fig. 15).

Fig. 13. Flow chart – one-loop circulation

Fig. 14. Flow chart – two-loop circulation

626


Fig. 15. Flow chart – three-loop circulation

These streams play a very important role regarding foaming reduction and forced submergance of floating part of biomass. A schematic structure of mixing in the fermentor is shown in Fig. 16.

Fig. 16. Schematic structure of the mixing process in the fermentor

This solution is a new advanced solution in comparison to previously used systems, including plug flow reactors (Jagadish et al., 1998) or batch mode system in mixed biofermentors (Raposo et al., 2011). The proposed mixing system assures effective mixing and a good contact between biomass flocks and the substrate.

5. CONCLUSIONS The study on hydraulic mixing leads to the following conclusions: •

Horizontal circulation allows to recycle the leachate with the respective cultures of bacteria to the anaerobic digestion process. For this purpose, two inlet streams located symmetrically at the same height at a distance of 0.4 m from the bottom of the tank should be applied in the fermentor.

•

Sections of vertical circulation are designed to mix the material in the entire volume of the reactor. In the presented reactor, it is advised to use three recirculation loops spaced at equal intervals of 2.5 m.

•

A course of the mixing cycle in the analysed fermentor consists of the following stages: activation of the recirculating pump, deactivation of the recirculating pump, activation of the first section of the mixing for a certain time, deactivation of the first section, activation of the second mixing section for a limited time, deactivation of the second section, activation of the third section of mixing for a certain time, deactivation of the third section.

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The reported work has been partly financed by National Centre for Research and Development, Poland in the frame of strategic program “Advanced Technologies for Energy Generation”; Task 4.”Elaboration of Integrated Technologies for the Production of Fuels and Energy from Biomass as well as from Agricultural and other Waste Materials”, Subtask 2.1.B ” Design and construction of biogas plant”.

REFERENCES Jagadish K.S., Chanakya H.N., Rajabapaiah P., Anand V., 1998. Plug flow digestors for biogas generation from leaf biomass. Biomass Bioenergy, 14, 415 – 423. DOI:10.1016/S0961-9534(98)00003-8. Karim K., Hoffman R., Klasson Th., Al-Dahhan M.H., 2005. Anaerobic digestion of animal waste: Waste strength versus impact of mixing. Bioresource Technol., 96, 1771 – 1781. DOI: 10.1016/j.biortech.2005.01.020. Kryłowicz A., Chrzanowski K., Usidus J., 2008. The method and generation system of methane, electricity and heat. Polish patent 197595 (in Polish). Kryłowicz A., Chrzanowski K., Usidus J., 2011. The method, transport and mixing system of the biomass suspension in the fermenter and hydrolizer. Polish patent 395860 (in Polish). Kryłowicz A., Chrzanowski K., Usidus J., 2011. New polish technologies of the production of biomethane, electricity and heat. IX Conference: Dla miasta i środowiska. Problemy unieszkodliwiania odpadów, Warszawa, 2011 (in Polish). Raposo F., De la Rubia M.A., Fernández-Cegrí V., Borja R.. 2012. Anaerobic digestion of solid organic substrates in batch mode: An overview relating to methane yields and experimental procedures. Renew. Sustain. Energy Rev., 16, 861 – 877. DOI: 10.1016/j.rser.2011.09.008. Rudniak L., 2010. The numeric simulations of the flow of the suspension forced by circulation pumps in the fermenter. Work of the Chemical and Process Engineering Department of Warsaw University of Technology, No. 501E/1070/0072/000, Warsaw. Received 16 May 2012 Received in revised form 14 November 2012 Accepted 19 November 2012

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LOW BOD DETERMINATION METHODS: THE STATE-OF-THE-ART Łukasz Górski*, Kamil F. Trzebuniak, Elżbieta Malinowska Warsaw University of Technology, Faculty of Chemistry, Institute of Biotechnology, Department of Microbioanalytics, 00-664 Warsaw, Poland

Biochemical Oxygen Demand (BOD) is an important factor used to measure water pollution. This article reviews recent developments of microbial biosensors with respect to their applications for low BOD estimation. Four main methods to measure BOD using a biosensor are described: microbial fuel cells, optical methods, oxygen electrode based methods and mediator-based methods. Each of them is based on different principles, thus a different approach is required to improve the limit of detection. A proper choice of microorganisms used in the biosensor construction and/or sample pre-treatment processes is also essential to improve the BOD lower detection limit. Keywords: biochemical oxygen demand, biosensor, wastewater, microbial sensor

1. INTRODUCTION Biochemical Oxygen Demand (BOD) is an agreed indicator used to quantify the amount of organic substance possible to be degraded by microorganisms present in a water sample which is proportional to the water pollution (ISO 5815-1:2003). IUPAC defines BOD as: “the amount of oxygen divided by the volume of the system used by the microorganisms growing on organic compounds present in the sample (e.g. wastewater, or sludge) during incubation over period of time and given temperature. BOD is a way to measure organic impurities present in the water sample undergoing biological degradation. Usually BOD is expressed in milligrams O2 per litre” (Nagel et al., 1992). For practical purposes it can be assumed that BOD is the difference between the dissolved oxygen (DO) present in the sample at the beginning and the end of the measurement in fixed conditions (ISO 5815-1:2003). Microorganisms oxidise organic impurities in 2 stages. First, carbon-compounds are oxidised and after about 10 days the nitrification process begins. During the first 5 days about 68% of organic compounds are oxidised and after 20 days nearly 99%. (Penn et al., 2004; Miksch and Sikora, 2010). It should be noted, however, that the extend of biodegradation depends largely on the structure of compounds present in the sample and microbial community. There are many standards concerning BODn estimation e.g. (ISO 58151:2003) and (ISO 5815-2:2003). The traditional method starts with diluting a sample in different ratio using water saturated with oxygen and adding aerobic, heterotrophic microorganisms. The sample is incubated for 5 or 7 days (different countries prefer different periods) in fixed temperature of 20°C in the absence of light with the pH value fixed between 7 and 8. Next, the amount of DO in the sample is measured by titration or electrochemically (ISO 5815-1:2003; Miksch and Sikora, 2010). It is possible to measure BOD in undiluted samples, but this method is less accurate (ISO 5815-2:2003). Active sludge (biomass produced in the process of wastewater treatment during the growth of bacteria and other microorganisms in the presence of DO) is most widely used as the biological material (ISO 61071:2004). The D-glucose and L-glutamic acid solution (GGA) is most often used as a reference standard (ISO 5815-1:2003). Some authors prefer the use of artificial wastewater (AWW) as a reference *Corresponding author, e-mail: [email protected]

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standard. It is reasonable when developing a BOD sensor to be used in specific conditions e.g. rubber latex industry (Kumlanghan et al, 2008) or a distillery (Oota et al., 2010). Traditional methods of BOD estimation have many drawbacks, notably very long period of time needed to perform the measurement (at least 5 days). Furthermore, the used microorganisms are very sensitive to toxins, bactericides, heavy metal ions, or chlorine which inhibit their respiration process and may lead to the death of the microorganisms (ISO 5815-1:2003). Traditional methods are neither very sensitive nor precise (Bourgeois et al., 2001). Finally, the measurement procedure must be made soon after acquiring the sample i.e. during the first 24 hours. Many of those flaws may be eliminated by using biosensors (Miró et al., 2004; Pasco et al., 2011; Ponomareva et al., 2011). IUPAC defines a biosensor as a device which uses specific biochemical reactions, mediated by isolated enzymes, tissue, organelle or whole cells, to detect chemical compounds, usually using electrochemical, thermal or optic methods (Nagel et al., 1992). A biosensor consists of biological material directly connected with a transducer, which allows to process the analytical signal nto a form that enables its detection (Thevenot et al., 2001). This definition of a biosensor is based on the type of receptor layer, rather than the sample origin (Brzózka and Wróblewski, 1999; Brzózka, 2009). Biosensors were first used as a BOD sensor by Karube et al. in 1977 (Karube et al., 1977) using a MFC which allowed the time of the measurement to be reduced from 5 days to 40 minutes. Biosensors are still being improved nowadays, with research focused mostly on shortening the response time and lowering the limits of detection and quantification. This review is focused on the second field of modern research.

2. METHODS OF ESTIMATION There is no fixed, arbitrary and widely agreed BOD value below which the method is considered a “low BOD determination method”. Furthermore, some BOD estimation methods e.g. optical methods are able to measure lower BOD values than oxygen-sensitive electrode based methods, but they have some drawbacks e.g. they are harder to implement in flow analysis. There are many strategies used to lower the limit of detection. Some researchers concentrate on finding the optimal microorganism or a consortium of microorganisms most sensible in fixed conditions, other researchers focus on the oxidation of organic compounds using photocatalytic methods (Chee et al., 1999b; Chee et al., 2007) or introducing ozone to the sample (Chee et al., 2001; Chee et al., 2005). 2.1. Microbial fuel cells Microbial Fuel Cells are devices converting chemical energy into electric current by a reaction catalysed by microorganisms (Kumlanghan et al., 2007). Typical MFCs are divided into two compartments: anaerobic with a negatively charged electrode and aerobic with a positively charged electrode. Those two sections are connected through a proton exchanging membrane. In the anaerobic compartment, organic substances are oxidised which leads to proton production. The protons migrate through the membrane to the cathode. The electrons produced during the oxidation process migrate through the anode by the external electric circuit to the cathode where the reduction of oxygen occurs (Grzebyk and Poźniak, 2005; Seo et al., 2009). Anode: Cathode:

C6H12O6+6H2O → 6CO2+24H++24e-

(1)

4H++O2+4e- → 2H2O

(2)

Produced electric current is proportional to the concentration of the organic compounds being oxidised in the anaerobic compartment. Research is being conducted in order to standardise MFCs so that comparison between laboratories is possible (Higgins et al., 2011). Original Karube research was

630

Low BOD determination methods: The state-of-the-art

focused on shortening the BOD measurement time by usage of the bacteria Clostridium butyricum and the limit of detection was not determined (Karube et al., 1977). Present studies (Moon et al., 2005) use oligotrophic microbial consortium instead of copiotrophic microorganisms. Although oligotrophes respond to changes in the BOD less eagerly than other microorganisms and require very long time to reach the previous steady state when the sample is removed (about 15 hours when the BOD concentration was changed from 10 to 20 mg O2 L-1), they are capable of responding to much lower concentrations of organic substances. Enrichment by feeding with AWW for 8 weeks further improves their LOD. Moon et al. tried also to minimise the background noise generated during the measurement. They investigated the influence of the basal inorganic salt solution and buffer concentration on the MFC response. It was shown that these factors indeed do affect signal generation, but only in specific conditions. A trace mineral solution concentration also affected the measured current. The feeding rate was raised in order to saturate the MFC when the BOD value of the sample was about 10 mg O2 L-1. This further improved the LOD and sensitivity at the cost of the upper limit of detection. The dynamic linear range of the calibration curve was between 2 mg O2 L-1 and 10 mg O2 L-1. Kang et al. (2003) tried a different approach. They identified oxygen leaking through a cation specific membrane to the anaerobic compartment as the main reason of lowering the coulombic yield. Calculations showed that decreasing the size of the membrane may lead to lowering the LOD. It was determined that membrane area of 5.2 cm2 is sufficient for undisturbed flow of protons while minimising the leaking oxygen. They used an oligotropic consortium enriched for 8 weeks by feeding with AWW or river surface water. Also the performance of a carbon rod electrode was compared with a platinum coated electrode which proved to give better results. Decreasing the size of the membrane from 26 cm2 to 5.2 cm2 lead to ca. 4 fold higher coulombic yield and the possibility of determining BOD values of 2 mg O2 L-1. 2.2. Oxygen electrode based methods Measuring changes of DO using an oxygen-sensitive electrode is the most straightforward method of BOD estimation. Oxygen electrode based biosensors use the Clark electrode (Clark et al. 1953; Severinghaus and Freeman-Bradley, 1958; Severinghaus and Astrup, 1986) covered with an immobilised microbial layer. LOD can be improved by different approaches: screening for a sensitive microorganism or microorganism consortium, photocatalitic pre-treatment or introducing ozone to the sample. Typically, GGA solution is used for standardisation of BOD determination methods. However, river water samples usually show low BOD values, caused mostly by highly stable organic compounds, such as humic acid or lignin. For such samples, a different solution, namely artificial wastewater (AWW) was proposed for biosensor calibration. It usually contains the following compounds: nitrohumic acid, tannic acid, sodium ligninsulfonate, gum arabic and sodium lauryl sulphate. This solution (Chee et al., 1999a) imitates more closely real river water samples, as compared to a traditional GGA solution. One of the proposed microorganisms are bacteria Pseudomonas putida (Chee et al., 1999a) or salt tolerant yeast Arxula adeninivorans LS3 (Renneberg et al., 2004). Using these microorganisms provided good LOD, as they are not influenced by many interfering ions. There are many papers concerning the use of microorganism consortiums to broaden the spectrum of oxidised compounds. Authors of those publications used a very wide array of microorganisms e.g. activated sludge for monitoring treatment of wastewater from a rubber latex industry (Kumlanghan et al., 2008); commercial activated sludge (Liu et al., 2011); Trichosporon cutaneum and Bacillus subtilis immobilised in sol-gel derived composite matrix (Jia et al., 2003); Trichosporon cutaneum and Bacillus licheniformis (Suriyawattanakul et al., 2002); Enterobacter cloaca, Citrobacter amalonaticus,

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Pseudomonas aeruginosa, Yersinia enterocolitica, Klebsiella oxytoca, Enterobacter sakazaki and Serratia liquefaciens for industrial waste-water monitoring (Rastogi et al., 2003). Chee et al. tried a different approach to low BOD estimation (Chee et al., 2001). It was assumed that low sensitivity of BOD estimation methods might be caused by a low rate of assimilation of large chemical compounds by microorganisms in a relatively short period of time. They proposed sample pre-treatment by irradiation with UV light on TiO2 catalyst. This allows degradation of stable organic compounds and facilitates their biodegradation. The influence of irradiation time, sample pH and TiO2 concentration on sensor response was investigated. It was found that after optimisation of working parameters, sensor response in river water was higher after photocatalysis than that for samples without UV pre-treatment. The method was further improved and applied in a FIA system (Chee et al., 2005). H2O2 was also introduced to the sample, which when excited with UV light, produces free radicals, decomposing large organic compounds even more aggressively. One potential problem in such a system is killing the microbial consortium with the H2O2, but it was proven that the lifetime of O2-· radicals is about 2.5 s which is too short to reach the measurement cell. Such an approach improved the sensitivity of the sensor 1.4 fold and eliminated the problem with many biological contaminants of the sample. It is also possible to use ozonation rather than photocatalytic reaction. Ozone is a powerful oxidising agent that decomposes organic matter both directly and indirectly through the creation of hydroxyl radicals (Staehelin and Hoigné, 1982). Excess ozone has to be removed before the measurement e.g. by intense stirring of the sample. Such an approach was proposed by Chee et al. (Chee et al., 1999b) leading to a 2 fold signal increase and a detection limit of 0.2 mg O2 L-1. It was shown that BOD values measured by the proposed method correlate well with those determined by the conventional BOD5 method. This method was further improved and applied in a stopped-flow system (Chee et al., 2007). In this automated system, 1.6 fold signal increase was observed and the lower detection limit was 0.5 mg O2 L-1. 2.3. Optical methods Optical methods of BOD estimation are generally characterised by low limits of detection. This can be attributed to the fact that oxygen is not consumed during the measurement, as it is in the case of amperometric detection. Accordingly, more oxygen is available to the process of pollutant oxidation by microorganisms. The process of further lowering this parameter is performed by screening for the most sensitive microorganisms. Some papers prove that Pseudomonas putida (Chee et al., 2000) or Saccharomyces cerevisiae (Nakamura et al., 2007; Nakamura et al., 2008) provide the necessary parameters, but other researchers tend to use mixed cultures (Jiang et al., 2006; Lin et al., 2006; Xin et al., 2007). Using mixed consortiums of different types of bacteria broadens the array of organic compounds oxidised by the biofilm which leads to improved limits of detection and, more importantly, to more precise results. An important problem that spawned many interesting articles is the determination of BOD in seawater samples (Jiang et al., 2006; Nakamura et al., 2008; Renneberg et al., 2004; Xin et al., 2007). This is a relative difficult task, as the BOD of seawater is usually low and the microorganisms used in typical BOD sensors are not tolerant enough to high salt concentration. Therefore, various microorganisms of increased tolerance towards NaCl were used in proposed BOD sensors, including Bacillus licheniformis, Dietzia maris and Marinobacter marinus consortium obtained from seawater. The sensing film of proposed sensors consisted of an organically modified silicate (ORMOSIL) film doped with an oxygen-sensitive ruthenium complex. It was shown that with the use of this set-up, the minimum measurable BOD was 0.18 mg O2 L-1 in natural seawater. Saccharomyces cerevisiae ARIF KD-003 acquired from cold regions of Japan, showing excellent characteristics in salty conditions, were also employed for BOD determination in seawater samples (Nakamura et al., 2008). In this work,

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Low BOD determination methods: The state-of-the-art

2,6-dichloroindophenol spectrophotometric method was used to estimate yeast respiration activity. A remarkably low detection limit of 0.07mg O2 L-1 BOD was obtained using GGA-containing artificial seawater. Measurements of low BOD values, caused by persistent organic pollutants, pose a serious problem in river water samples. This issue was addressed by Chee et al. with the use of commercial optical fibre oxygen sensor and immobilised Pseudomonas putida bacterium (Chee et al., 2000). The minimum value of BOD that could be measured using this biosensor was 0.5 mg O2 L-1. A very interesting approach to low BOD determination was proposed by Sakaguchi and co-workers (Sakaguchi et al., 2003). The proposed system is based on bacterial luminescence from recombinant Escherichia coli that contains lux A-E genes from Vibrio fischeri. The luminescence of these bacteria depends on their metabolic activity, which can be strengthened by the presence of carbon source in the incubation medium. The increase of light emission by recombinant E. coli was correlated with the raised BOD value of tested samples. Bacterial luminescence was measured using a charge coupled device camera and a photomulti-counter (consisting of a photon-counter and a photomultiplier). Using this relatively simple system, the lower BOD detection limit was estimated at 1 mg O2 L-1. 2.4. Mediator-based methods One of the major drawbacks of DO sensors is the necessity of sample pre-treatment which includes saturation with oxygen. This process is strongly dependent on the sample temperature and achieving good repeatability is difficult. Replacement of oxygen with a mediator solution solves this problem. Mediators are chemical compounds able to accept electrons in place of oxygen in the metabolism of microorganisms. They are able to transport electrons to the electrode surface where a reduced form of the mediator is oxidised resulting in electron transfer to the electrode, which leads to electric current production. The use of mediators in BOD sensors is mainly motivated by the extension of linear calibration range towards higher BOD values. However, the lower detection limits are also usually improved. This was the case in the work conducted by Trosok et al (Trosok et al., 2001), where different hypothetical mediators were investigated, with potassium ferricyanide(III) and hydroxymethylferricinium proven to be the most useful ones. The 2 mg O2 L-1 BOD detection limit was achieved using SPT1 yeast strain which showed a marked similarity to the genus Candida. 2.5. Carbon dioxide-based method All the methods described above are based on changes in the oxygen (or mediator) concentration which is the result of microbial respiration. However, it is also possible to develop a sensor based on the measurement of carbon dioxide produced during this process. Recently, Cortón et al. (Cortón et al., 2010) described such a sensor, based on a modified Severinghaus electrode (Severinghaus and Freeman-Bradley, 1958) with immobilised yeast Saccharomyces cerevisiae. The BOD detection limit of 1 mg O2 L-1 was reported for this device. Such a low LOD was possible owing to the fact that the potentiometric CO2 electrode does not consume the analyte in contrast to the amperometric oxygen electrode.

3. CONCLUSIONS Biochemical Oxygen Demand (BOD) is a well-established method for evaluation of water quality. BOD values are most often used to assess the effectiveness of wastewater treatment plants. One of the disadvantages of the classical methods of BOD determination is a relatively high detection limit, preventing analysis of pristine river water or sea water samples. In this review, articles describing

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methods of determination of low BOD values are collected and discussed. Various measurement modes are mentioned, including microbial fuel cells, optical methods, oxygen electrode based methods and mediator-based methods. It is shown that a proper choice of a microorganism or microorganisms consortium is of fundamental importance in the design of low BOD measurement devices. Moreover, certain sample pre-treatment processes can be employed to decompose persistent organic pollutants, making them more bioavailable. Attention is also drawn to artificial wastewater, used for standardisation of low BOD determination methods instead of traditional GGA solution. Table 1. Comparison of the methods for determination of low BOD values

Response time, minutes 40

Dynamic range, mgO2 L-1 ?-250

2

60

1,A

3 1 1

LOD, mgO2 L-1

Microorganism

Reference

6*

Cl. Butyricum

Karube et al, 1977

2-10

4

ND