Mixed Microbial Cultures for Industrial Biotechnology

Chapter 7

Mixed Microbial Cultures for Industrial Biotechnology: Success, Chance, and Challenges Wael Sabraa,b and An-Ping Zenga aHamburg

University of Technology, Institute of Bioprocess and Biosystems Engineering, Denickestr. 15, D-21071 Hamburg, Germany bMicrobiology Department, Faculty of Science, Baghdad Street, Moharram Bey, Alexandria University, Alexandria, Egypt [email protected]

7.1 Introduction The use of mixed cultures or microbial consortia for human purpose has a long history. Long before we ever knew what a microbial cell or a microorganism is, humans were manipulating microbial consortia in some very industrious ways, to produce, for example, foods, chemicals, or improved crops. For several thousand years, natural or spontaneous fermentation has been used to obtain many types of food. Mixed cultures are the rule in nature; therefore, it is not surprising that such conditions are prevailing in fermented food of relatively ancient origin. Food fermentation represents Industrial Biocatalysis Edited by Peter Grunwald Copyright © 2014 Pan Stanford Publishing Pte. Ltd. ISBN 978-981-4463-88-1 (Hardcover), 978-981-4463-89-8 (eBook) www.panstanford.com

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Mixed Microbial Cultures for Industrial Biotechnology

the most ancient biotechnological process used to produce wine, beer, vinegar and bread using microorganisms, primarily yeast. In addition, for ages, yogurt was produced by lactic acid bacteria in milk and molds were used to produce cheese. Currently, these processes are in use and have been optimized for the production of modern foods. Indeed, through the help of modern biochemistry and microbiology techniques, those cultures have been purified and often genetically refined to maintain the most desirable traits and highest quality of the products. Besides the food fermentation and wastewater treatment, there are a few unsterile fermentation processes that involve mixed cultures industrially established. An early work on mixed culture food fermentation was by Macfadyen (1903), in which he referred to mixed-culture fermentations as “mixed infections.” To ensure quality and consistency and to avoid contamination of the fermentation process and the product, the praxis of fermentation industry with mixed cultures was gradually replaced more and more with pure cultures. The establishment of submerged fermentation processes with pure cultures was the prerequisite for the successful development of antibiotic production in the 1940s and 1950s and was viewed as the land marker of biochemical engineering and industrial biotechnology. To date, the multibillion-dollar market values of bulk biotechnological products such as amino acids, organic acids, and antibiotics and high-value products such as vitamins, enzymes, and pharmaceutics are almost exclusively generated by pure cultures of microorganisms or mammalian cells. Focusing on single cultures, however, may neglect important advantages of microbial cultures and consortia derived from interactions among microorganisms. Such interactions can range from synergism to antagonism among microorganisms (Gall, 1970; Sabra et al., 2010; Shah and Dworkin, 2009), and may depend on, besides the microbial groups and species involved, the substrate type and availability. Indeed, the complexities of mixed culture fermentations are usually brought about by the complexity of the substrate composition in nature. This is an especially interesting aspect of industrial biotechnology aiming at the use of biomass (especially cellulosic and lignocellulosic materials) for the production of chemicals and fuels. The breakdown of the complex substrates such as lignocellulosic materials requires various arrays of microorganisms to act upon it. In the process of

Microbial Community and Biochemical Diversity

breaking down the complex substrate with the chemistry of the substrate changing and the environmental parameters changing as results of microbial metabolism, certain species are suppressed while other species will exploit the new conditions. Presently, there are only few exceptions where mixed culture-based bioprocesses are used industrially. However, with the development of industrial biotechnology, it is perhaps time to reappraise the potential of mixed culture systems.

7.2 Microbial Community and Biochemical Diversity

Structurally, prokaryotic organisms are far simpler than eukaryotic cells. Yet, prokaryotes are functionally or metabolically diverse with regard to reactions they mediate or stresses they can endure. Prokaryotes do not possess membrane-bound organelles such as a nucleus, endoplasmic reticulum, mitochondria, or chloroplasts. Therefore, prokaryotes cannot separate metabolically incompatible biochemical processes into discrete compartments (Fig. 7.1). The inability to compartmentalize cellular processes poses a bottleneck for the biochemical and structural evolution of prokaryotes. As a result, prokaryotes have diversified with regard to biochemical abilities while remaining structurally simple. The prokaryotic solution to “compartmentalization” is the formation of associations with other organisms, which gives protections against potentially inhibitory environmental factors. These include exposure to adverse oxygen concentration, ultraviolet radiation, desiccation, and adverse pH value. The steep biogeochemical gradients that exist in mats allow and select for functional diversification such that diverse organisms having requirement for aerobic, microaerophilic, and anaerobic conditions may co-exist and contemporaneously function along a gradient. Such dramatic environmental changes occurring in a small spatial scale set up association that facilitate mutualistic nutrient, gas, and metabolite exchange. Associations reflect synergistic or syntrophic lifestyles where growth and biogeochemical cycling are conducted more effectively and efficiently than on an individual population basis. Such associations are also called microbial consortia. Members of the consortium maintain metabolic and ecological compatibility, as long as biogeochemical

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and environmental gradients allow for individual niches to exist in a close proximity. Microbial mats typify these conditions and, accordingly, are the focus of research on consortial growth strategies in extreme environments.

Figure 7.1

Conversion of substrates (S) to product (P) using pure or mixed microbial cultures respectively. Compartmentalization of metabolic reaction is needed to convert some toxic metabolites (M) within the cells in a pure culture. In a mixed culture, both direct cell–cell interaction and intermediates dependent interaction (substrate/product inhibition) are involved for the development of a stable consortium.

7.3 Advantages of Using Mixed Culture

In environmental biotechnology, e.g., in anaerobic degradation processes of organic waste, mixed cultures of bacteria are often used as a black box to accelerate the degradation of waste materials at low costs. To get insight into the black box, several studies have been made to use synthetic or minimized consortia to degrade complex substrates (Khleifat, 2006; Kim et al., 2009). Despite large efforts done in this field, the use of undefined consortia is still indispensable. On the other hand, pure cultures and/or enzymes in industrial biotechnology are designed to optimize a specific conversion on a

Advantages of Using Mixed Culture

pure substrate, which is mostly a sugar, for maximizing the product formation. Processes for optimal product formation using mixed culture are still under development. The choice between mixed and pure culture processes depends on the complexity of the bioprocess involved. Although mixed culture fermentation offers several advantages over the conventional pure culture fermentations, the predominating bioproduction processes are still based on pure cultures. In the following, we outline some major advantages and disadvantages for the use of microbial mixed cultures.

7.3.1 The Existence of a Food Web for Improved Performance

The product yield or production rate of a pure culture normally decrease with time due to accumulation and inhibition of intermediates or the final product of the desired bioconversion process and quite often also byproducts. In some cases, this can be avoided through the use of a mixed culture in which the inhibiting intermediates or byproducts may be degraded by one of the species of the mixed culture. This is, for example, an important function in the anaerobic food web in the conventional anaerobic degradation in biogas reactors, where specific groups of microorganisms maintain low concentrations of the critical intermediates and promote the flux of carbon and electrons from the feedstock materials to the desired end product (Fig. 7.2). Due to the complex interrelationships that exist in this anaerobic food web, process failure can occur when one group of the organisms results in an inhibition cascade that can cause the entire system to crash. For example, the accumulation of acids in biogas reactors, due to overloading with organic substances, can inhibit acetogenesis and methanogenesis by reducing the pH to less than the optimal range for these microbial groups. A recent work that has been inspired from the food web in anaerobic digesters was the use of a mixed culture consisting of Methanosarcina mazei and Clostridium butyricum for the production of 1,3 propanediol from glycerol (Bizukojc et al., 2010). C. butyricum converts glycerol into 1,3-propanediol (PDO) as a major product, but significant amounts of acetate, formate, and butyrate are also produced as inhibiting by-products. Methanosarcina mazei relieves this inhibition and utilizes the by-products for energy production.

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Figure 7.2

Microbial groups and steps involved in methane production within granules. 1. Fermentative bacteria; 2. obligate hydrogenproducing acetogenic bacteria; 3. hydrogen-oxidizing acetogens; 4. carbon dioxide–reducing, H2-oxidizing methanogens; 5. aceticlastic methanogens.

7.3.2 Stability and Robustness

Robustness is a property that allows a biological system to maintain its functions against internal and external perturbations. Mixed cultures are able to bring about multistep transformations that would be impossible for a single microorganism. Assuming that the main metabolic pathway of an organism has a ﬁnite capacity, an increase in the metabolism of one substrate should down-regulate the uptake of other substrates (Navarette-Bolanos et al., 2007). Mixed cultures can handle variations in substrate composition because alternative metabolic pathways are available among different members of the microbial community. The stability of the microbial community, possibly due to the metabolic flexibility of aerobic biological waste treatment systems has been correlated with increased biodiversity of the microbial community (Gall, 1970). In anaerobic digestion systems, a direct link between functional stability, community stability, and higher biodiversity has not been demonstrated, but functional stability appears to be correlated with community flexibility, i.e., the ability of a system to shift the flow


of electrons and carbon to the same products through alternative pathways. Moreover, metabolites generated by mixed consortia often complement each other and work to the exclusion of unwanted microorganisms and therefore lead to a better process stability. For example, in some food fermentations, yeast will produce alcohol and lactic acid bacteria will produce lactic acid and other organic acids; they can change the environment from aerobic to anaerobic, which excludes most undesirable molds and bacteria. Moreover, in mixed-culture fermentations phage infections are reduced. In pure culture commercial fermentations involving bacteria and actinomycetes, phage infections may occur, and the infection can completely shut down the production. Since mixed cultures have a wider genetic base of resistance to phage, failures do not occur, often because if one strain is wiped out, a second or third phage resistant strain in the inoculum will take over and continue the fermentation. In such processes, especially with a heavy inoculum of selected strains, contamination does not occur even when the fermentations are carried out in open pans or tanks.

7.3.3 Unsterile Open Fermentation Processes

Mixed culture fermentation, especially those with unspecified micro-organisms, do not require expensive sterility procedure for preventing contamination, and such an open culture is the only choice for agricultural waste stream and hence can be operated in a continues mode. In fact, the possibility of utilizing cheaper secondary products (e.g., whey and molasses) or even complex biomass as substrates for biotechnological production of chemicals and fuels represents a further advantage. It was recently shown that processes based on mixed cultures can be established to generate a narrow product spectrum from a mixed substrate (Kleerebezem and van Loosdrecht, 2007). On an industrial scale, working with mixed cultures or natural consortia under unsterile conditions will significantly lower the production costs. On the other hand, with pure culture bioprocessing, the chances of contamination increase as the incubation period increases and therefore, most industrial bioprocesses with pure cultures are performed in batch or fed batch mode. Thus, mixed culture technology could become an attractive addition or alternative to traditional pure culture-

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based biotechnology for the production of chemicals and bioenergy in industrial biotechnology. Typical examples include continuous biogas production in the environmental industrial sector. Efforts were also made for the production of biochemicals such as 1,3 propanediol from glycerin (Chatzifragkou et al., 2011), ethanol using lignocellulosic substrate, polyhydroxybutyrate from glucose (Tan et al., 2011), and lactic and propionic acids (Sabra et al., 2013; Yumoto and Yoshimune, 2012).

7.3.4 Oxygen Removal for Anaerobes

It is known that the biofilm character of plaque allows for population diversity and coexistence of aerobes, anaerobes, and microaerophiles. Overall, the presence of aerobic bacteria is mandatory for the survival of the acid-producing anaerobes. In a mixture of ten bacterial species, Bradshaw et al. (1996) proved that anaerobes were growing in aerated (dO2 of 40–50%, Eh +100 mV) conditions. In their study, they conclude that mixed cultures can protect obligate anaerobes from the toxic effects of oxygen, both in the biofilm and planktonic modes of growth. The same phenomenon was also recognized in anaerobic digestion. It was shown that limited quantities of oxygen can even lead to improved anaerobic reactor performance under certain operating conditions. Coexistence of anaerobic and aerobic cultures in a single-bioreactor environment has been demonstrated. It was even shown that a partial aeration assisted anaerobic digestion and served as a beneficial treatment strategy for simultaneous waste treatment and energy generation (Botheju and Bakke, 2011).

7.3.5 Promoting Growth and Culturing “Unculturable” Bacteria

Fermentation with pure culture depends on bacteria that can be propagated in the laboratory. These bacteria represent only about 1% of the total diversity that exists in nature. At all levels of bacterial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen and other elements, synthesizing novel natural products, and impacting the surrounding organisms and environment. While molecular


techniques such as metagenomic sequencing can provide some information independent of our ability to culture these organisms, it is essentially impossible to learn new gene and pathway functions from pure sequence data (Stewart, 2012). Recent advances in growing these species include co-culture with other bacteria or re-creating the environment in the laboratory. The aim is to identify growth factors that promote the growth of previously “unculturable” organisms. The use of mixed culture approach can provide necessary nutrients for optimal performance. Many microorganisms, such as the cheese bacteria, which might be suitable for production of a fermentation product, require growth factors to achieve optimum growth rates. To add the proper vitamins to production adds complications and expense to the process. Thus, the addition of a symbiotic species that supplies the growth factors is a definite advantage. Evidence includes the fact that many cultured bacterial isolates are lost and no longer viable once their bacterial associations are completely removed.

7.3.6 Use of Complex Substrates

A further advantage of cultivation of mixed cultures is the possibility of utilizing secondary products (e.g., whey, molasses) cheaper than glucose as substrates for biotechnological production of chemicals. Using substrates other than glucose offers potential to develop biological production processes with more competitive costs. Furthermore, co-cultivation processes can help find new substances of industrial interest, because a number of secondary metabolites are produced during co-cultivation (Oh et al., 2007). As an example, Fu et al. (2009) used a co-culture for the simultaneous conversion of glucose by Zymomonas mobilis and xylose by Pichia stipidis to ethanol. Using this co-culture a volumetric ethanol productivity of 1.3 g/L/h and an ethanol yield of 0.50 g/g was recorded, representing more than 96% of the theoretical value. Interestingly, the authors found that viable Z. mobilis cells inhibited the xylose fermentation by P. stipitis and that free cell co-culture fermentations could not completely utilize the xylose, whereas the entrapment of Z. mobilis cells in calcium alginate gel beads resulted in efficient xylose fermentation when co-cultured with P. stipitis free cells. The authors also suggested a modified reactor for their experiment that

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enabled the removal of immobilized Z. mobilis beads from the coculture. Working on lignocellulosic wastes, the same principal was also demonstrated by Maki et al. (2009). They reported the various applications of Clostridium thermocellum together with other strains of Clostridium sp. or Thermoanaerobacterium saccharolyticum. The cellulose- and hemicellulase-producing C. thermocellum can only metabolize glucose, whereas the other micro-organisms can utilize the hemicellulose-derived pentoses. This co-cultivation process therefore, improves product formation because it avoids substrate competition between species. In spite of significant contributions in the fields of microbiology and biochemistry that have led to an improved understanding of natural fermentation phenomena, microorganism selection remains largely based on pure culture evaluation and relies on the use of single strains. However, if the microorganism interactions are evaluated and included in such selection process, a microbial consortium may outperform the results achieved by pure cultures in almost every case. Below we briefly illustrate some examples for the use of mixed cultures (microbial consortia) in established industrial processes. The potential to improve existing processes as well as examples for some novel processes under development are then discussed.

7.4 Established Industrial Bioprocesses

7.4.1 Microbial Consortia in Industrial Wastewater Treatment The global population is expected to increase to approximately 7 billion people by 2050. With this increase in population, sewage and wastewater treatment industries will grow dramatically. For over a century, systems have been designed to increase microbial growth in order to remove organic carbon and other nutrients, while limiting the release of suspended solids into receiving waters. While the concept is very simple (Fig. 7.3), the control of the treatment process is quite complex, because of the large number of variables that can affect it. These include changes in the composition of the bacterial flora of the treatment tanks and changes in the sewage passing into the plant. The influent can show

Established Industrial Bioprocesses

Figure 7.3

The role of mixed culture in aerobic sludge granules in waste water treatment of sewage.

variations in flow rate, in chemical composition and pH, and temperature. Those plants receiving industrial wastewater have to cope with recalcitrant chemicals that the bacteria can only degrade very slowly, and with toxic chemicals that inhibit the function of the bacteria. High concentrations of toxic chemicals can produce a toxic shock that kills the bacteria. When this happens, the plant may pass untreated effluent directly to the environment, until the dead bacteria have been removed from the tanks and new bacterial “seed” introduced. This is why stabilized and robust microbial consortia are needed. In the activated sludge process, the quality of the effluent is to a large extent dependent on how the biological sludge mass can be separated from the treated wastewater (Fig. 7.3). The solid/liquid separation traditionally takes place through a separation of spontaneously aggregated flocks of activated sludge by gravity sedimentation in secondary clarifiers. Moreover, biological oxygen demand (BOD) removal is achieved only after microbial biomass settles down and is removed from the system as sludge. It therefore depends on the settling characteristics of the sludge, which in turn depends on the efficiency of flock formation. Although it is well known that flock-forming microorganisms are the key to the success of the process, the best organisms to perform this function are not well known. Floccules or suspended biofilm has been the aim of various studies (Patil et al. 2011). To achieve good flocculation, one must ensure presence of polysaccharidesand polyhydroxybutyrate-secreting bacteria and a few filamentous microbes in the effluent treatment plant. Kargi and Ozmihci

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(2004) introduced a culture of the alginate-producing bacterium Azotobacter vinelandii for the treatment of nitrogen-deficient wastewater. Alternatively, Patil et al. (2011) suggested that polymer from Azotobacter has a high potential in wastewater treatment as bioflocculant and can be used as a potential alternative to chemical flocculants. In fact, the major problem with many wastewater treatment plants is that they are simply overloaded with organic material. The introduction of a group of natural microbial strains to reinforce the treatment of contaminated soil or water to support other microorganisms that provides the treatment, which is called “bioaugementation,” has been used intensively in the last few years. Fong and Tan (2000) added mixed culture of nine microbial species to domestic water which showed to cause a 60% reduction in the BOD level of wastewater compared to control in which no microorganisms were added. The use of “effective microorganism” was reported to increase the efficiency of wastewater treatment facilities. Similarly, “effective microorganisms,” a mixed culture of coexisting beneficial microorganism predominantly consisting of lactic acid bacteria, photosynthetic bacteria, yeast, fermenting fungi, and actinomycetes was reported to increase the sewage treatment efficiency and thereby reduce environmental impact (Monica et al., 2011; Namsiyavan et al., 2011). In the United States, Terraganix (http://www.teraganix.com/) and SCD Probiotics (http://www.scdprobiotics.com/) are resellers of the “effective microorganism” product. They reported the use of such consortia during land application of sewage or sludge, which can be used for the removal of ammonia and phosphate from wastewater. In the past two decades, we have been confronted with an increasing variety of nonconventional wastewaters in the form of chemical and industrial process effluents and landfill leachates, and hence much work is still needed and new robust consortia have to be developed.

7.4.2 Bioethanol

Although synthetic ethanol production from the petrochemical ethylene was once the predominant source of industrial ethanol, fuel ethanol is currently produced from sugarcane, corn, wheat,

Established Industrial Bioprocesses

and sugar beets by fermentation on a large scale (Jeon et al., 2008). Industrial ethanol fermentations are normally not performed under sterile conditions, and a variety of Gram-positive and Gramnegative bacteria have been isolated from fuel ethanol fermentations (Bischoff et al., 2009). Generally, the natural nutrient media used permit a growth pattern favoring ethanol fermenting yeast and prevent bacterial overgrowth. Nevertheless, contamination can be a serious problem in ethanol fermentation, especially with the fast growing Lactobacillus and hence antimicrobial agents are introduced in such fermentation (Bischoff et al., 2009). The industrial production of bioethanol from starch generally involves liquefaction with α-amylase, enzymatic saccharification, and finally the fermentation of sugars to ethanol. Simultaneous saccharification and fermentation with a mixed culture was suggested to reduce the costs of the process. Still, the high feedstock cost poses a major obstacle to large scale implementation of ethanol as a transportation fuel. Therefore, interest has recently shifted to replacing these traditional feedstocks with the non-food-based lignocellulosic biomass (LB) feedstocks such as agricultural wastes (e.g., corn stover) and energy crops (e.g., switchgrass). Many factors, such as lignin content and crystallinity of cellulose and particle size, limit the digestibility of the hemicellulose and cellulose present in the LB (Abbas et al., 2010; Abulencia et al., 2010). In fact, the carbohydrate composition of the hydrolysate (mainly C6 and C5 sugars) impedes the complete conversion by pure cultures. Genetically modified organisms have been developed. However, on an industrial scale, the production process is still in an early stage as the cost is relatively high with genetically modified organisms. Therefore, the production of ethanol from LB waste was intensively studied and mixed culture approach was thoroughly discussed. Simultaneous saccharification and fermentation of cellulosic material was suggested by Mamma et al. (1996), and a co-culture fermentation with S. cerevisiae and Fusarium oxysporum was performed. More recently, Z. mobilis and Candida tropicalis were evaluated for the production of ethanol from enzymatically hydrolyzed LB and 97.7% of the theoretical yield of ethanol was obtained (Patle and Lal, 2007). Considerable improvement in this area has been observed using co-cultivation of different microorganisms for ethanol production from cellulose (Lin and Hung,

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2008; Steinbusch et al., 2010). The same trend was also followed recently by using the cheese whey powder as a cheap substrate for ethanol production by immobilized mixed culture (Guo et al., 2010).

7.5 Potential Use of Mixed Culture in Industrial Biotechnology 7.5.1 Biofuel and Polymer Production

One of the defining challenges of the 21st century will be shifting our energy supply from fossil to alternative fuels. About 90% of our current energy needs come from three main fossil fuels: petroleum, coal, and natural gas. Within the last few years, the price of petroleum has skyrocketed, straining a global economy whose engine is transportation. Although prices of oil and gases have recently fallen to more reasonable levels, they will undoubtedly rise again as economies recover. Moreover, oil is a limited resource and human consumption will eventually exhaust the global supply. Liquid biofuels offer a promising alternative to fossil fuels. The primary characteristics of a suitable renewable biofuel are that (i) it has the potential to replace a significant portion of fossil fuels but should not affect global food supplies, (ii) it must have a net positive energy balance, and (iii) it should have minimal negative environmental impact. For example, in Brazil, 46% of the energy comes from renewable sources, of which 15% is from sugarcane. Now more than ever, it is vital that carbon-neutral biofuels be widely adopted to eventually replace fossil fuels. Biofuels from botanical biomass maintain relative carbon neutrality, as the CO2 released upon combustion of the biofuel is partially offset by the CO2 fixed originally by the growing organism from which the biofuel was derived and hence not contributing to the increased atmospheric CO2 level as petroleum fuel.

7.5.1.1 Bioelectricity

Electricity production by using microbial consortia in form of biofilms or microbial fuel cells is attracting large attention (Harnisch et al., 2008, 2012). Although microbial fuel cells (MFCs)

Potential Use of Mixed Culture in Industrial Biotechnology

are not new (demonstrated already in 1991 for treating domestic wastewater), research on bioelectrochemical systems with alive and with enhanced power output has blossomed during the last 10 years (Angenent and Rosenbaum, 2013). Generally, an MFC converts energy available in a bio-convertible substrate directly into electricity. This can be achieved when cellular metabolism is switched from the natural electron acceptor, such as oxygen or nitrate, to an insoluble acceptor, such as the MFC anode (Fig. 7.4). In such a system, the survival of microbes depends on the electron transfer to the anode. Because bacteria use the reactions on the anode in their metabolism, they strategically position themselves on the anode surface, forming biofilm and a complex ecosystem where bacteria are living within a self-generated matrix that conducts the electrons.

Figure 7.4

The working principle of MFC in the biodegradation of wastes and generation of electricity. Modified after Hoang (2010).

Based on the calorific content of glucose, a microbial fuel cell can theoretically (at 100% efficiency during fermentation) deliver 3 kWh for every kilogram of organic matter (dry weight) in one single fermentative step (instead of 1 kWh of electricity and 2 kWh of heat per kilogram in hydrogen and biogas production by employing several process steps (Aelterman et al., 2006; Rabaey et al., 2003; Rabaey and Verstraete, 2005). This means that during fermentation

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in MFCs, almost any energy is released in the form of external heat, and that all the biochemical energy in the waste can potentially be converted into electricity. Recent work shows that depending on the experimental conditions employed, overall efficiencies up to 80% can be reached in practice. Microbial fuel cells can be grouped into two general categories, those that use a mediator and those that are mediator-less. Before 2003, almost all researches on bioelectrochemical systems were performed with the addition of artificial mediators to microbial cultures. Although a high current density can be achieved using these mediators, the added cost, especially for the hazardous waste treatment of some toxic mediators renders such a technology not suitable for scaling-up. With the discovery of microbes that can directly deliver electrons to anodes without the requirement of artificial mediators, the interest in MFC resurfaced once more. The circumvention of artificial mediators made inroads to treat organic wastewater in anode chambers with the simultaneous production of electric power (Angenent and Rosenbaum, 2013; Angenent and Wrenn, 2008). Recently, Inoue et al. (2013) reported cassette-electrode microbial fuel cells as efficient and scalable devices for electricity production from organic waste. The power generated in MFC is dependent on both the biological processes, such as the substrate conversion rate, and the electrochemical processes, such as over-potentials on the anode and cathode and the internal resistance of the MFC (Rabaey and Verstraete, 2005). Therefore, the economic scale-up will largely depend on the specific microbial electrocatalytic process. Still, the biggest challenge will be to increase the microbial reaction rates, which are very sluggish at this point. This requires a deep investigation into the microbial physiology and modification of microbial pathways through the use of advanced molecular tools (Angenent and Rosenbaum, 2013; Lovley, 2006, 2008, 2008a).

7.5.1.2 Biohydrogen

Starting in the late 1800s, basic research proved that algae and bacteria could produce hydrogen but it was not seriously considered a practical possibility. Only in the 1990s, when it became apparent that atmospheric pollution by fossil fuels not only is unhealthy locally but also might cause significant climate change


globally, interest in biohydrogen resurfaced. The main limitation of hydrogen production by fermentative bacteria is the maximum electron-based yield that can be established (Angenent and Wrenn, 2008). Owing to biochemical and thermodynamic limitations, the maximum theoretical number of moles of hydrogen that can be generated per mole glucose is four in conventional fermentation processes, which are obtained by glucose oxidation to two moles acetate as “byproduct”:

C6H12O6 + 2H2O  2C2H3O–2 + 2H+ + 2CO2 + 4H2 DG01 = –145 kJ . mol–1

(a)

Figure 7.5

(b)

Mixed microbial culture for the production of hydrogen and methane (a) and butanol using complex substrates (butanol process after Angenent and Wrenn, 2008).

However, the measured hydrogen production per mole glucose, as observed in mixed culture fermentation studies, is much lower and will normally not exceed two moles (Kleerebezem and van Loosdrecht, 2007). This practical limitation seems to be related to a biochemical restriction associated with the electron carriers utilized in the different fermentation pathways. Hydrogen production from different feedstocks by mixed cultures has been well covered in several recent reviews (Kyazze et al., 2008; Mu et al., 2007; Saratale et al., 2008). Because of thermodynamic limitations, hydrogen can only be produced in oxidation reactions in the metabolic pathways coupled to formate production

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or ferrodoxin reduction, and not in the NAD-dependent steps. Fermentative hydrogen production provides only a partial oxidation of the organic substrate. To inactivate most hydrogen scavengers of methanogens or homoacetogens, alkaline, acid and heat pretreatment methods have been investigated. In fact, a nearterm option in this regard is to produce a mixture of hydrogen and methane in a two-stage process (Fig. 7.5). The first step produces hydrogen and organic acids, the latter should be converted to methane in a second fermentation stage. A selling point for this mixture is that hydrogen–methane mixtures significantly reduce air pollutants in internal combustion engines, compared with using pure methanol as fuel (Benemann, 1996). Thermophilic bacteria produce fewer by-products beside hydrogen and the thermodynamic conditions concerning hydrogen partial pressure are more favorable. Heat-treated anaerobic sludge and pure cultures of clostridia and Enterobacter species were used for biohydrogen production in dark fermentation. Recently, Ozmihci and Kargi (2007, 2010) compared different mixed cultures for biohydrogen production by combined dark and light fermentations; a combination of the anaerobic sludge and fermentative bacteria Rhodobacter sphaeroides yielded the highest hydrogen yield (Ozmihci and Kargi, 2010). Similarly, another Japanese group established a two-stage process in pilot scale (Ueno and Goto, 2007). A thermophilic microflora produced hydrogen and methane with garbage and waste paper as substrate at 60°C. Despite the unsterile process, Thermoanaerobacterium species from the inoculated microflora were dominating in the hydrogenotrophic stage. Hence, the thermophilic process strategy reduced the risk for contamination effectively. Nevertheless, the ultimate goal for the R&D hydrogen research still is to achieve high yield of hydrogen. Economic feasibility will not be sustainable until the current yields of 10–20% reach the 60–80% mark. In this context, it is worth mentioning that in a synthetic pathway with enzymes from different microorganisms, a yield of as high as 11 mol H2/mol glucose unit now has been achieved in an in vitro biotransformation system (Zhang et al., 2007). This in vitro system has a theoretical H2 yield of 12 mol H2/mol glucose unit and is of great interest for biohydrogen production and for bioelectricity (Zhang et al., 2010).


7.5.1.3 Butanol and 1,3 propanediol production Glycerol, especially crude glycerol as a byproduct directly from biodiesel production plants, is an interesting substrate for industrial biotechnology. Crude glycerol normally contains impurities such as methanol, fatty acids, salts, and heavy metals, and may need to be purified for certain fermentation processes with pure culture (Hoang, 2008). Recently, mixed culture processes were developed to convert crude glycerol into 1,3-Propanediol (PDO). 1,3-Propanediol is a building block of the polymer polytrimethyleneterephthalate, which represents a new generation of polyester with superior properties (Chatzifragkou et al., 2010; Zeng and Biebl, 2002). Conventionally, microbial production of PDO is carried out by using single microorganism, either natural strains with glycerol as substrate or genetically engineered one with glucose as a substrate (Bizukojc et al., 2010; Friedman and Zeng, 2008). The bioconversion of crude glycerol into PDO is of particular interest as a part of a biorefinery concept either for biodiesel or for bioethanol production. In both cases, glycerol can be obtained as a byproduct with impurities as mentioned above. The microbial conversion of glycerol to PDO is, however, always associated with production of organic acids as byproduct because of the necessity of balancing the reducing power. This results in two major problems. First, organic acids are toxic and limit cell growth and thus limit productivity of the process. Second, only about half of the substrate (glycerol) is converted into PDO, leading to an incomplete use of the substrate. Fermentation with mixed culture was proposed as an interesting and effective solution to these problems (Friedman and Zeng, 2008). Using crude glycerol (80% glycerol) as a carbon source and inocula adapted from a local wastewater treatment plant, PDO can be produced as the main product at concentration as high as 70 g/L in fed-batch cultivation with a productivity of 2.6 g/L h. A high yield between 0.57–0.72 mol PDO/mol glycerol, which is close to the theoretical maximal yield of anaerobic glycerol conversion, has been achieved (Dietz and Zeng, 2013). In comparison to 1,3-PDO production in typical pure cultures, the process developed in our laboratories with a mixed culture achieved the same levels of product titer, yield, and productivity, but has the decisive advantage of operation under complete non-sterile conditions. Moreover, a defined fermentation medium without yeast extract can be used

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and nitrogen gassing can be omitted during the cultivation, leading to a strong reduction of investment and production costs (Dietz and Zeng, 2013). Crude glycerol and glucose containing waste streams can be used to produce butanol. Ramey (1998) patented a system involving two different Clostridia, which produced butyrate and butanol in separate pure culture bioprocesses placed in series. In the first reactor, Clostridium thermobutyricum converts carbohydrates predominantly to butyrate, which is then transferred to the second reactor where it is converted to butanol by Clostridium acetobutylicum. A mixed culture bioprocess for conversion of complex mixtures of carbohydrates to butyrate in the first bioprocess was claimed to be superior alternative to the pure culture process (Fig. 7.5). During the second stage, solventogensis is initiated after the pH of the culture medium decreased due to acid accumulation. Butanol-producing bacteria will rapidly convert butyrate to butanol using electrons derived from oxidation of carbohydrates through glycolysis, and therefore butanol can be efficiently produced by feeding butyrate along with carbohydrates (Angenent and Wrenn, 2008). Recently, it was proved that the addition of butyric acid and to a lesser extent acetic acid enhanced the production of butanol in C. acetobutylicum fermentation (Li et al., 2011). Interestingly, C. pasteurianum was reported to utilize glycerol and convert it into PDO and butanol as the main fermentation products (Jensen et al., 2012). In our laboratories, we demonstrated the simultaneous production of butanol (up to 40 g/L) and 1,3 PDO (up to 60 g/L) in laboratory scale and is being scaled up in pilot plant (results not published). Currently, we are investigating microbial consortia for the simultaneous production of butanol and 1,3 propanediol (unpublished result).

7.5.1.4 Propionic acid

Propionic acid is presently mainly synthesized by petrochemical route, and hence its production is vulnerable to price fluctuations of propane and natural gas (Gu et al., 1998). Indeed, the increasing consumer demand for biological products and the more efficient performance of new fermentation processes have revived industrial interest for a biological production of propionic acid. Many studies and patents have been described for the bioproduction of propionic acid, mainly with strains of Propionibacteria. Despite


the many studies done with these bacteria and media, the biotechnological production of propionic acid has never passed the pilot plant level. Possible reasons include the long duration of batch culture, the end product inhibition, and the high costs of the fermentation and recovery processes (Liang et al., 2012). To overcome the slow growth rate and product inhibition, several strategies such as cell retention systems have been proposed, but the process is still not economically feasible. Usually, cheese whey or whey permeate derived from dairy industries at high concentrations are used as substrates. However, since lactose, the main sugar found in whey, is not the ideal sugar for fermentation with Propionibacteria, a better substrate utility was realized in a co-culture with Lactobacillus plantarum. L. plantarum converts lactose to lactate, which can be converted more rapidly by Propionibacteria. This carbohydrate to propionate fermentation via the intermediate lactate was proposed in the 1980s (Distler and Kronke, 1980). It was reinvestigated recently to ferment whey to propionate with a co-culture of L. zeae and Veillonella criceti and to use the dried fermentation broth as a conservative agent in bread against mould growth. Recently, we studied the interaction of both L. zeae (to convert glucose to lactic acid) and V. criceti (to convert lactate to propionic acid) for propionic acid production from glucose. The defined co-culture was first studied in a dialysis membrane reactor (Fig. 7.6).

Figure 7.6

Dialysis two-chamber bioreactor system used to study the interaction between Veillonella criticae and Lactobacillus zeae.

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Such a reactor was mostly used for high cell density fermentation (Markl et al., 1993). The use and advantages of such a system for the investigation of a defined co-culture have been described (Pestchanker and Ercoli, 1997), but to our best knowledge not realized up to now. In this reactor, the two cell populations are separated through a dialysis membrane and no direct cell–cell contact was established. We showed that the metabolism of L. zeae is markedly stimulated by the co-culture partner independent of nutrient limitations and experimental setup (Dietz et al., 2013). The nature of this interaction is still not clear, but the reduction of the redox potential of the medium by hydrogen produced by V. criceti may be a possible reason. Piveteau et al. (2000) studied the interaction between strains of Lactobacillus and Propionibacterium. They concluded that stimulant (s) produced form Lactobacillus was stable to heating to 121°C for 15 min and eluted in several peaks after chromatography on Sephadex G-25. However, ultrafiltration resulted in a total loss of activity. After detailed kinetic studies of L. zeae and V. criceti in dialysis culture in batch and continuous culture, these strains were used for the production of propionic acid using household flour (Sabra et al., 2013). A substrate limited fed-batch fermentation of hydrolyzed flour was proved to be an efficient production process and a propionic acid final concentration of 30 g/L with a productivity of 0.33 g/L ∙ h was obtained. The co-culture process can be further evaluated for dough formulation for bread manufacturing, especially because flour hydrolysate without the use of expensive constituents was used as the substrate. In comparison, the monoculture approach with P. acidipropionici using the same substrate yielded a concentration of 12 g/L but with a 10-fold decrease in productivity (Sabra et al., 2013).

7.5.2 Antimicrobial Substances

Despite the wide variety of known antibiotics, less than 1% of antimicrobial agents have medical or commercial value. In nature, antibiotics are produced by soil-dwelling fungi and bacteria for the purpose of inhibiting the growth of others microbes. Recently, it was shown that these molecules may be produced not primarily as weapons for competition or combat but as tools of communication between mixed cultures, or even as essential cogs in the producers’ own metabolism. Nisin is an antimicrobial

Challenges Facing the Use of Mixed-Culture Fermentations

peptide, approved in England to be used for microbial stabilization of food, cosmetics, and pharmaceuticals for over 50 years (DelvesBroughton et al., 1996). Nisin acts by increasing permeability of membranes of Gram-positive bacteria resulting in growth inhibition or even cell death. It is produced by Lactococcus lactis, but its production can be increased by co-cultivation of the lactic acid bacterium with S. cerevisiae or Kluyveromyces marxianus (Liu et al., 2006; Shimizu et al., 1999). Voravuthikunchai et al. (2006) reported the production of an antimicrobial peptide by Lactobacillus reuteri, effective against methicillin-resistant S. aureus (MRSA). The induction of plantaricin production by Lactobacillus plantarum NC8 takes place only after co-cultivation with specific Grampositive strains or even the addition of heat-killed cells from some of the inducing strains (Maldonado et al., 2004). More recently, it was proven that Lactacin B production by L. acidophilus was only induced if it senses live target bacteria (Tabasco et al., 2009). The same phenomenon was observed in co-culture of Rhizopus peka and Bacillus subtilis, where co-culture cultivation enhanced the antibiotic activity significantly (Fukuda et al., 2008). Besides the production of antimicrobial peptides, co-culture fermentation processes may lead to the discovery and characterization of new antimicrobial peptides also attributable to the unique induction phenomenon observed in co-cultivation. In this context, Slattery et al. (2001) published the co-cultivation dependent formation of antibiotics by a strain of Streptomyces with different marine bacteria, indicating the importance of co-culture fermentations for the discovery of new antibiotics. It is worth mentioning that the number of antimicrobially active substances produced by strains of the genus Streptomyces is estimated to be as many as 100,000. Today, only 3–5% of these substances are known. Recently, it was shown that the antibiotic activity produced in co-cultures of Rhizopus peka and Bacillus subtilis was higher than that in each of the pure cultures alone (Fukuda et al., 2008).

7.6 Challenges Facing the Use of Mixed-Culture Fermentations

In order to design and develop a successful process, understanding of the precise role and the overall contribution of each microorganism to the fermentation process is required. This knowledge is crucial

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to decide,whether an inoculum should contain a defined co-culture or a mixture of undefined bacterial cultures. However, procedures to reach such understanding have not been established yet. Generally, the major challenges facing mixed culture fermentation are discussed below.

7.6.1 Population Dynamic Determination

Traditional fermented foods use microbial cultures that take over the system and outcompete others. Pickles, cheeses, sauerkraut, and the like may use an inoculum with selected microorganisms, but a useful culture may result from natural selection from the organisms that are always present. Understanding how this works and why it sometimes does not work and leads to contamination can be important. Despite the enormous advances in this field, population dynamics of mixed bacterial culture are still challenging for industrial biotechnologists. Generally the determination of the population dynamics are either through molecular biological, biochemical, or microbiological methods (Spiegelman et al., 2005; Wintermute and Silver, 2010), of which the most promising methods are the molecular biological ones based on the analysis and differentiation of microbial DNA such as sequencing and metagenomics. With them, a great deal of information can be gleaned from even very complex microbial communities. Nevertheless, each physical, chemical, and biological step involved in the preparation and analysis of a mixed culture sample is a source of bias that might give a distorted view of a given ecological niche. Moreover, lysis of the microbial cells during DNA extraction represents a critical step in PCR-mediated approaches (von Wintzingerode et al., 1997). It is often a question of whether there was sufficient or preferential disruption of cells. Rigorous conditions needed to lyse Gram-positive cells may cause excessive shearing of nucleic acids of the Gram-negative cells, potentially biasing the reported diversity of the sample as well as possibly creating artifacts and chimeric PCR products. Moreover, both genome size and copy number differ from one organism to the next, and these properties are unknown in the unculturable fraction of the community. Since both of these variables can alter the number of 16S genes present in a sample, it has been concluded that the PCR amplification of 16S rRNA genes from a complex microbial


community may not give an accurate quantification of the consortium (Spiegelman et al., 2005). This is a factor that must be considered when using methods such as terminal-restriction fragment length polymorphism (T-RFLP), which generate quantitative data (Spiegelman et al., 2005).

7.6.2 Analysis of the Interrelationships between Members of Mixed Cultures

There are several types of microbial interactions that may be involved in a mixed culture fermentation process. Mutualism, synergism, amensalism, food competition, predation, and parasitism are known examples (Bader et al., 2009; Hibbing et al., 2010; Sabra et al., 2010; Wintermute and Silver, 2010). They may influence the functioning of the desired microbial process either individually or in combination. Despite intensive research in this field, elaborating the exact type(s) of microbial interaction(s) involved in a consortium remains a major challenge. One factor hampering the elucidations of these interactions is the difficulty of defining all members included in such complex microflora. Furthermore, it is even more difficult to clarify the roles of each member and the relationships among each of the members of such a community. Constructing a defined mixed culture consisting of microorganisms isolated from the microflora would facilitate our understanding of these mechanisms. Such approaches have often been applied to examine various microbial communities, especially in the field of pollutant biodegradation, but still failed in the wastewater treatment field. A “knockout communities” strategy was used by Kato et al. (2005) to study the role of members of a cellulose degrading community. In their work, they constructed communities in which one of the members was eliminated, and the roles played by each eliminated member in situ were evaluated as well as its impact on the other members of the community. Still there are other types of interaction and behavior that cannot be explained by merely one of the microbial interactions mentioned above. One recent example is the production of plantaricin by L. plantarum NC8, which takes place only after co-cultivation with specific Gram-positive strains or even the addition of heat-killed cells from some of the inducing strains (Maldonado et al., 2004). More recently, it was proven that Lactacin B production by L. acidophilus was only induced if it senses live

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target bacteria (Tabasco et al., 2009). It is a challenge to understand the simultaneous involvement of several different microbial interactions in one and the same industrial process. It is even more challenging and of great importance to control the interactions of microbes.

7.6.3 Engineering Synthetic Microbial Consortia

Brenner et al. (2008) proposed a synthetic biology approach to engineer the interactions of microbes. They reviewed recent efforts to engineer communication among different organisms for the development of synthetic microbial consortia. These synthetic microbial consortia can be used to study the behavior of interacting populations in a minimal microbial consortium or to mimic microbial interactions under controlled conditions. These clearly defined and engineered consortia can be analyzed more easily than natural systems and can even be described through mathematical models. They can thus be used to develop and validate models of more complex systems. Nevertheless, from an application perspective, no examples of such potential uses of synthetic consortia for the production of chemicals and fuels in the context of industrial biotechnology have been reported so far. In fact, engineering microbial consortia with molecular biological tools for industrial application is just in the infancy and faces several challenges such as overcoming the problem of horizontal gene transfer and maintaining homeostasis.

7.6.4 Contamination of Fermentations

Stabilized mixed cultures normally offer more protection against contamination. However, if contamination occurs, it is more difficult to detect or to control it. In the food industries, contamination is a predominant cause of process failure (Lushia and Heist, 2005). It is therefore crucial that industrial fermentations are carefully monitored to detect microbial contamination as early as possible after it occurs. Bacterial contamination is a continual problem in commercial fermentation cultures, particularly in commercial yeast production and fuel ethanol fermentations that are not performed under sterile, pure culture conditions (Bischoff et al., 2009). A variety of Gram positive and Gram-negative bacteria have been


isolated from commercial yeast and fuel ethanol fermentations. Indeed, the quality of commercial yeast production depends not only on the viability and fermentation activity, but also on the absence of gross contamination with other microorganisms. The most common offending microbes, however, are species of Lactobacillus, whose fast growth rate and tolerance to alcohol and low pH allow them to effectively compete with the yeast. L. reuteri can also produce toxic intermediates such as 3-hydroxypropionaldehyde from the byproduct glycerol in ethanol fermentation. 3-hydroxypropionaldehyde is very toxic to bacteria and yeast and can lead to serious problems. In general, chronic bacterial contamination poses a constant drain on sugar available for conversion to ethanol and the bacteria scavenge essential micronutrients required for optimal yeast growth and ethanol production. Acute infections occur unpredictably, and bacterial byproducts such as acetic and lactic acid inhibit yeast growth and may result in “stuck” fermentations that require costly shutdowns of facilities for cleaning. Efforts have been made to prevent contamination of such a mixed-culture fermentation. The most common commercially available products used to control contamination in fuel ethanol facilities are based on the antibiotics virginiamycin and penicillin (Lushia and Heist, 2005). The emergence of drug resistant strains, however, may limit the effectiveness of these agents. Recently, Manitchotpisit et al. (2013) reported the use of Bacillus spp. that showed antibacterial activities against lactic acid bacteria which contaminate fuel ethanol plants.

7.6.5 Consistency of Inoculums of Mixed Cultures

One of the most difficult problems to handle mixed-culture fermentations is the control of the optimum balance among the microorganisms involved. This can, however, be overcome if the behavior of the microorganisms is understood and the respective information is applied to their control. The balance of organisms brings up the problems of storage and maintenance of the cultures. Lyophilization presents difficulties because in the freeze-drying process the killing of different strains’ cells will be unequal. It is also difficult, if not impossible, to grow a mixed culture from liquid medium in contrast to typical fermentations on solid mediums, without the culture undergoing radical shifts in population

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numbers. According to Harrison (1978), the best way to preserve mixed cultures is to store the whole liquid culture in liquid nitrogen below –80°C. The culture, when removed from the frozen state, should be started in a small amount of the production medium and checked for the desired fermentation product and the normal fermentation time. Subcultures of this initial fermentation, if it is satisfactory, may then be used to start production fermentations.

7.7 Concluding Remarks

Microbial consortia play important roles in many traditional industrial bioprocesses such as for food and beverage production, waste material treatment and biogas production (Sabra et al., 2010). The rapid development in the sequencing of microbial consortia opens up both opportunities and challenges for industrial biotechnology with microbial consortia. Here we have illustrated some examples of industrial bioprocesses involving microbial consortia or mixed culture, and pointed out advantages and challenges of working with such bioproduction systems. Generally, the examples described above indicate the importance and the increasing interest to use mixed microbial cultures in industrial biotechnology. Examples include not only cases from the environmental biotechnology (such as wastewater treatment), but also for sustainable energy (butanol, ethanol and hydrogen production) and other valuable products (propionic acid, PDO, antimicrobial agents). Numerous other examples can be found and are discussed in literature (Bader et al., 2009; Sabra et al., 2010). To date, there is an increasing trend toward the use of renewable, cheap, and readily available biomass in the production of a wide variety of fine and bulk chemicals, especially in form of biorefinery. The biorefinery concept offers numerous possibilities to integrate the production of bio-energy and chemicals. The use of complex substrates in a biorefinery approach necessitates the use of mixed bacterial cultures, especially because microbial consortia can perform complicated functions and are more robust to environmental fluctuations than individual populations. We believe that the use of mixed culture technologies that provide improved conversion efficiency—from raw material to final product—will increase the environmental benefits of bio-based

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