Environmental Technology Removal of humic acid fractions by ...

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Removal of humic acid fractions by Rhizopus arrhizus: Uptake and kinetic studies a

J.L. Zhou & C.J. Banks

a

a

Simon Environmental Technology Centre, Department of Chemical Engineering , UMIST , Manchester, M60 1QD, UK Published online: 17 Dec 2008.

To cite this article: J.L. Zhou & C.J. Banks (1991) Removal of humic acid fractions by Rhizopus arrhizus: Uptake and kinetic studies, Environmental Technology, 12:10, 859-869, DOI: 10.1080/09593339109385080 To link to this article: http://dx.doi.org/10.1080/09593339109385080

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Environmental Technology, Vol. 12. pp 859-869

© Publication Division Selper Ltd., 1991

REMOVAL OF HUMIC ACID FRACTIONS BY RHIZOPUS ARRHIZUS: UPTAKE AND KINETIC STUDIES J.L. ZHOU* AND C.J. BANKS

Simon Environmental Technology Centre, Department of Chemical Engineering, UMIST, Manchester M60 1QD, UK

Downloaded by [East China Normal University] at 16:52 09 January 2014

(Received 18 April 1991; Accepted 19 July 1991)

Keywords: Humic Acid, Fungi, Uptake, Kinetics, Decomposition. ABSTRACT Humic acid components HFO-A and HPI-N were studied for their removal by Rhizopus arrhizus. The removal was stimulated by the presence of sucrose followed by pretreating biomass with formaldehyde or autoclaving. The presence of Cd2+ ions and high ionic strength in solution was found to increase the adsorptive capacity of R.arrhizus for HPO-A and HPI-N. The uptake of HPOA and HPI-N by R.arrhizus was biphasic in nature and generally a much slower process than heavy metal biosorption. The first phase was rapid and independent of metabolic energy while the second phase was slow and dependent on metabolic energy. The infrared spectra showed no chemical reaction has occurred between cell walls and adsorbates. Physical adsorption is therefore the main mechanism of humic removal by inactive R.arrhizus.

INTRODUCTION The accumulation of heavy metals, radionuclides and organic pollutants such as pesticides by a variety of microorganisms including algae, bacteria, yeasts and fungi has been widely reported in the literature (1,2,3,4) and termed biosorption. As the name implies, biosorption is- dependent upon the adsorptive capacity of biological surfaces for the removal of pollutants. As such, biosorption may be an alternative or addition to the traditional processes such as coagulation for removing colour from water, which is due to the presence of humic and fulvic acids of anionic nature (5,6). Initial work has shown that the filamentous fungus Rhizopus arrhizus was able to remove colour from water and the process was via adsorption to the fungal cell wall chitin/chitosan components (7,8). There are however notable differences in the biosorption behaviour of heavy metals and humic acid components such as the equilibration time and pH effect. This implies that the process kinetics of the uptake of anionic humic acid components are different from those

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of metal biosorption, this points to differences in mechanism. The work presented follows on from previous studies (7,8) and supplements this by analysing the adsorptive capacity of R.arrhizus under varying conditions and the process kinetics. Both are important if practical applications of a fungal biosorptive system are to be established as these will govern the overall cost, and hence the competitiveness of an industrial process. Humic acid is a mixture of molecules with a variety of functional groups and molecular weights. It is not a defined chemical entity and hence cannot be described by a precise chemical formula (9). The complexity of structure and variability of the molecule limits the type of analytical technique that can be used to study its interactions with fungal adsorbents. Infrared spectrophotometry has been reported as a useful technique for studying the reaction between R.arrhizus cell walls and uranium ions (10). As such the technique was thought may have potential for studying cell wall reaction with other molecules such as humic acid or its components.

MATERIALS AND METHODS


Chemicals Humic acid (Aldrich Chemical Co. Ltd, UK) was purified and fractionated to give components which were better defined chemically (11). Of the six fractions, hydrophobic acids (HPO-A) and hydrophilic neutrals (HPI-N) were the most colour intensive and environmentally the most important in terms of water quality. Solutions of cadmium nitrate and sodium chloride were prepared by dissolving accurately weighed chemicals of AnalaR grade in deionised water. Solution pH was adjusted to 7 by addition of 4 mM phosphate buffer, dilute sulphuric acid and sodium hydroxide. • Cells and cellular components A stock culture of Rhizopus arrhizus was regularly subcultured on slopes of YMS medium comprising (per litre of water): 10 g yeast extract, 10 g malt extract, 30 g sucrose and 15 g agar. The fungus was grown in a broth culture of the same medium at 25 °C for three days in 500 mL flasks on an orbital shaker. It was then washed first with tap water then distilled water before use. Studies were conducted using the microorganism in a live state as well as after rendering the cells nonviable by autoclaving or formaldehyde treatment. Inactivated cell walls were prepared as described by Zhou and Banks (7). Briefly, the inactivated cells were homogenised in a Waring blender for several minutes, washed with 1% (w/v) sodium dodecyl sulphate in distilled water, followed by washing with distilled water and increasing concentrations of aqueous ethanol. The preparation was then freeze dried. Adsorption studies Isotherm studies were performed in 250 mL Erlenmeyer flasks placed on an orbital shaker at 200 rpm and run for three days at 26°C (7). To each flask was added a known concentration of either HPO-A or HPI-N made up to a total volume of 150 mL and 200 mL, respectively, with 4 mM phosphate buffer. To each flask was then added a known and equal weight of biomass. Two sets of control flasks were also prepared, the first contained only biomass and buffer whilst the second only contained buffered adsórbate solutions. Solutions of different concentrations

of Cd 2+ or NaCl were also introduced to these flasks to study the effects of metal ions and ionic strength on the uptake of humic acid components by R.arrhizus. All the flasks were double sealed with parafilm before shaking to prevent evaporative losses. Flasks containing live biomass were shaken for both one and three days to examine whether humic decomposition had occurred. When an equilibrium position was reached, the flask contents were vacuum filtered through Whatman 0.45 um membrane filters. The filtrate was a n a l y s e d by UV-visible spectrophotometry at 400 ran (12), and the solute adsorbed by the biomass was calculated by material balance. The adsorption of HPO-A and HPI-N has been shown to obey the Freundlich equation (7), as such all the experimental data were modelled according to this equation. The linearised Freundlich equation is: lnQ =lnKf+l/nlnCeq (1) where Q is the amount of solute adsorbed (mg) per gram of biomass and C eq is the equilibrium concentration of solute in solution (mgL*1). Parameters Kf and 1/n are constants and can be calculated by the least squares analysis. Kinetic studies Rates of removal of adsórbate from solution were assessed for both HPO-A and HPI-N fractions. The studies were performed in a well mixed batch reactor with a total volume of 6 L. The impeller agitation rate can be controlled using a Citenco motor. A 0.45 |xm membrane filter capsule was fitted at a bottom outlet; this permitted sampling with the aid of a peristaltic pump. To the reactor was added 4 L of a HPO-A or HPI-N solution of known concentration. The impeller was started following the addition of freshly harvested fungal mycelia. Where required, the adsórbate solution was supplemented with 5 mM sucrose or a combined solution of 5 mM sucrose and 0.5 mM KCN before fungal biomass which had been pretreated in the corresponding solution for 10 minutes was added. Infrared spectrophotometric studies Infrared spectrophotometric studies were useful in detecting interaction products between R.arrhizus cell walls and humic acid fractions as each molecule possesses a unique set of vibrational frequency. The use of infrared

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spectra enabled us to potentially trace any reaction products which may have formed between cell walls and adsorbates. KBr discs were prepared by mixing 200 mg dry KBr with 1 mg of either pure cell wall material which had been previously freeze dried or cell wall material which had been equilibrated with 100 m g L ' 1 adsórbate and dried; these were then, ground in a mortar and subjected to a pressure of about 10 ton in" 2 in an evacuated die. This sintered the mixture and produced a clear transparent disc. The spectra were recorded on Perkin-Elmer-783 infrared spectrophotometer.

RESULTS AND DISCUSSION

Adsorption capacities As shown in Figure 1, the uptake of HPO-A by R.arrhizus was stimulated by various biomass treatments. The presence of 5 mM sucrose showed the greatest effect. Pretreatment of the biomass with 10% formaldehyde or autoclaving significantly increased the uptake over that shown by live cells equilibrated for both one and three days. The adsorption isotherm obtained for live cells in the presence of sucrose was much steeper than the rest of the set; these differences may be due to different mechanism of removal.

Dead cells can only adsorb HPO-A onto surface binding sites, whilst live cells in the presence of an energy source, such as sucrose, may be able to absorb material into the cell protoplasts. Moreover, they may also be able to degrade HPOA into smaller molecules which are easier to absorb or are colourless. Decolourisation of paper industry effluents by many white-rot fungi has been widely reported in the literature, the process involves destruction of chromophores in the polymer and decomposition of the polymer to low-molecular-weight, colourless, soluble/ volatile products (13,14,15,16). The addition of glucose or sucrose has been found to stimulate the decomposition rate of humic acid or kraft mill effluents by a variety of fungi (17,18). It is well established that the degradation of lignin by white-rot fungi is by and large an oxidative process and requires oxygen (19). The fungi cannot grow on lignin as the sole carbon source; they require an additional carbon source as cosubstrates (19,20,21). Lignin metabolism is typical of secondary metabolism as it occurs after the primary growth of fungus (19). Live cells in isolation, did not show a high adsorptive capacity, this may be due to the inability of HPOA solution to support the growth oí R.arrhizus and subsequent degradation of humic compounds. The adsorption isotherms of live cells for contact times of 1 day and 3 days showed this to be the case, their uptake being essentially the same and no degradation having occurred.

ln(Solute adsorbed, mgg ]

O

Figure 1

1 2 _1 InOJquld phase concentration, mgL ]

L(1 day contact)

U3 day contact)

OIHCHO treated)

Uwlth aucroae)

3

D( autoclave d)

Uptake of HPO-A by R. arrhizus (0.67 gL'1) at 26°C, pH 7: metabolic and biomass pretreatment dependence. L » Live; D = Dead.

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Pretreating cells by using either formaldehyde or autoclaving them increased HPO-A uptake; a phenomenon also found for the uptake of metal ions and pesticides by microbial biomass (22,23,24). As adsorption by dead cells is via surface binding only (7), the observed increase in uptake can only be attributed to the exposure of latent binding sites (22). Similar results have been obtained for HPI-N removal by R.arrhizus (Figure 2). However, the presence of sucrose increased uptake of HPI-N to a greater extent than had been shown for the HPOA fraction.

Metal ion effect on uptake Adsorption isotherms of HPO-A in the presence of different concentrations of Cd2* ions were presented in Figure 3 and show Cd2 + increased HPO-A uptake; the higher the metal ion concentration the more pronounced thé effect. Similar stimulatory effect from Cd2+ ions was also found for HPI-N uptake (Figure 4). These results are consistent with previous findings on the positive effect of Cu2+ on humic acid uptake by R.arrhizus (8); and can be explained by the role of metal ions in bridging cell wall surface charge to HPO-A surface charge.


Involute adsorbed, moa' 1

-1

1 2 ., ln[Liqu¡d phase concentration, mgl_ ] Ml day contact)

L(3 day contact)

DfHCHO i reatad)

L(wlth sucrose)

3

D(autoclavsd)

Figure 2 Uptake of HPI-N by R.arrhizus (0.50 gl/ 1 ) at 26°C, pH 7: metabolic and biomass pretreatment dependence. L = Live; D = Dead. InfSolute adsorbed, mgg~ ]

0.5 ln[L¡qu¡d phase concentration. mgL" ] 0 mgL

• 1 mgL

- * - 6 mgL"

10 mgL"1

Figure 3 Adsorption of HPO-A by inactive R. arrhizus (0.67 gL'1) at 26°C, pH 7: Cd2+ ion effect.

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ln[Solute adsorbed, mgg


-0.5

0.5 1 1.5 2 InfLlquld phase concentration. mgL"1] "1 mgC 1

• 0 mgL'

mgL*1

2.5 10 mgL'

Figure 4 Adsorption of HPI-N by inactive R. arrhizus (0.50 gL'1) at 26°C, pH 7: Cd2+ ion effect. Ionic strength effect on uptake It is shown in Figure 5 that the presence of 0.005 M NaCl increased HPO-A uptake by R.arrhizus , with further increase at higher NaCl concentrations. Similar results were observed for HPI-N uptake (Figure 6). Such stimulation on the uptake of humic acid fractions can be analysed according to surface chemistry theory. When two phases, such as R.arrhizus biosorbent and humic acid fraction solution, are in contact, an excess of ions of one type will generally be present on the surface of the non-aqueous phase (i.e. R.arrhizus) and an equivalent amount of

ions of opposite charge will be distributed in the aqueous phase near the interface. The distribution of excess charges on the surface and in solution constitutes an electric double layer (25). Based on the simple Guoy-Chapman theory of the diffuse double layer, the increase in adsorption with rising ionic strength has been explained by a compression of the electric double layer (26). Addition of salt reduces the repulsive electric potential extending from the solid surface into the solution, allowing more ions (i.e. humic acid anions) to approach the surface. Subsequently, a large fraction of all surface sites can overpower the repulsive electric potential with their heterogeneous adsorption potentials.

Involute adsorbed, mgg ]

0.5

1 1.5 Irillquld phaso concentration, mgL 1 ]

- no added NaCl

• 0-005M NaCl

- * - 0.01M NaCl

-O.iMNaCl

Figure 5 Adsorption of HPO-A by inactive R. arrhizus (0.67 gL'1) at 26°C, pH 7: ionic strength effect

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ln[Soluto adsorbed, mgo

0

0.5 1 1.5 2 ln[Uquld phase concentration, mgtT1 ]

- no added NaCl

• 0.005M NaCl

- * - aO1M N»CI

2.5 - S - O.1M Nad

Figure 6 Adsorption of HPI-N by inactive R arrhizus (0.50 gL"1) at 26°C, pH 7: ionic strength effect

Kinetic studies

HPO-A molecules have to be broken down first before being able to penetrate through protoplasts; this involves a process of at least two step reaction.

Figure 7 shows the uptake of HPO-A by R.arrhizus to be biphasic. The first phase, which spanned twenty minutes, was relatively rapid and curvilinear. The second phase was slower but i l i l i shown to be linear. The biphasic nature of HPO-A sorption by fungal cells is similar to that 7.5 reported for heavy metal uptake by microbial biomass (27,28,29). In the case of metals, the first phase usually occupies the first several 6.0 o* minutes whereas for HPO-A it lasted twenty o» minutes; the difference can be explained by the polydisperse nature of HPO-A fraction, i.e. it is 4.5 a mixture of molecules with different molecular size and weight. Polymer adsorption to surfaces has been extensively studied with the general / 3.0 . conclusion that the adsorbent surface is occupied first by the low molecular weight species, due to / their rapid adsorption/diffusion rate, followed by 1 the high molecular weight species; these display, 1.5 T as a rule, preferential adsorbability, and will I eventually substitute themselves for the former • i l l low molecular weight components (30,31,32). The i time required to exchange small polymer 30 60 90 120 150 molecules for larger ones is governed by the Tims (rrin) rates of diffusion and reconformation of the larger components. Such behaviour is one possible reason for the delayed adsorption/diffusion rate of HPO-A molecules. Figure 7 Accumulation of HPO-A by R. arrhizus (0.70 gL'*) from a 10 mgL' * solution at The second phase of HPO-A uptake was also 20°C, pH 7, in the presence of 5mM slower than that of heavy metal uptake because Soliite adsort>ed (0),


-0.5

sucrose.

864

HPO-A uptake in the ñrst phase was highest in the presence of sucrose and only slightly reduced by the absence of sucrose or addition of KCN, a microbial poison (Figure 8). As uptake in the ñrst phase is thought to be by physical binding to cell surface, it is independent of metabolic energy (27,28,29).


W

60

90

120

150

Time (rrin)

Figure 9

Accumulation of HPI-N by R. arrhizus (0.70 gL'1) from a 10 mgL"1 solution at 20°C, pH 7, in the presence of 5mM sucrose.

90 120 150 Tme (min) •, 5mM sucrose (control!; *, no sucrose; 30

60

», 5mM sucrosa + 0.5mM KCN.

Figure 8

Accumulation of HPO-A by R. arrhizus (0.70 gL'1) from a 10 mgL"1 solution at 20°C, pH 7: metabolic dependence.

The second phase exhibited a much slower rate of uptake and was stimulated by the presence of sucrose which yields energy for possible degradation of HPO-A molecules and absorption. The uptake was significantly suppressed by the absence of sucrose or presence of KCN. It appears that the second phase is primarily an energy driven metabolic process. Similar results have been found for HPI-N (Figures 9 and 10). The total uptake of HPI-N was less than that of HPO-A. This may be due to the fact that the two fractions have different molecular weights, composition of functional groups and adsorbability to fungal cells.

0

3C

60

90

120

Time (min) •, 5mM sucrose (control!; *, no sucrose; •. 5mM sucrose + 05mM KCN.

Figure 10 Accumulation of HPI-N by R. arrhizus (0.70 gL'1) from a 10 mgL'1 solution at 20°C, pH 7: metabolic dependence.

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Infrared spectrophotometric studies The infrared spectra of inactive virgin cell walls and cell walls at 1 equilibrium with 100 mgL"1 HPO-A were recorded^ The aim was to detect any possible new bands which might be indicative of chemical interaction between cell


100

Tranamlttance (%)

02

0.7

Figure 11

Transmlttance [%)

12 17 22 2.7 0.2 3.7 Wavenumber/1000 (cm'1)

4.2

0.7

12 17 2.2 2.7 a2 3.7 Wavenumoer/1000 (cm")

4.2