Minimisation of biomass in an extractive membrane bioreactor

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Wal. Sci. T~clt. Vol. 34, No. 5-6, pp. 273-280, 1996. Copyright C 1996IAwQ. Publi511ed by Elsevier Science I.ld Prinled in Great Britain. All riglltsreserved, 0273-1223196$15'00 + 0-00

Pergamon

PH: S0273-1223(96)00655-5

MINIMISATION OF BIOMASS IN AN EXTRACTIVE MEMBRANE BIOREACTOR L. F. Strachan, L. M. Freitas dos Santos, D. J. Leak and A. G. Livingston Departmenr of ChemicalEngineering and ChemicalTechnology, ImperialCollegeof Science. Technology and Medicine. LondonSW72BY. UK

ABSTRACf Many Iraditional biological methods for the treatment of wastewatercope poorly with toxic, volatileorganic compounds. The extractive membrane bioreactor is a novel process for the treatment of industrial wastewaters containing such compounds which combines extractionacross a siliconerubbermembrane with biodegradation. Previous work has shown that there is a problem in this system with excess biofilm growtll on the membrane surface. resulting in reduced flux of organic substrate across the membrane. The work presented here shows that addition of sodium chloride to the biomedium increases the maintenance energy requirement of the degradative microorganisms and results, in a carbon-limited situation, in a reduction in biofilm growth. Flux of organic subsIrate was shown to remain high under reduced biofilm growth conditions. Copyright (C) 1996IAWQ. Published by ElsevierScienceLtd.

KEYWORDS Biofilm; biomass minimisation; extractive membrane bioreactor; volatile organic compounds; wastewater treatment.

INTRODUCfION Biological processes are widely used in the treatment of both domestic and industrial wastewaters. Toxic organic compounds. especially those which are also highly volatile (Volatile Organic Compounds, VOCs) can be a problem in such systems since they often pass through the treatment plant untouched, or else volatilise and become pollutants in the atmosphere (Petrasek et al. 1983). The extent of stripping of VOCs is a function of both the physicochemical properties of the compound and the wastewater treatment plant design and operation. The treatment of individual wastewater streams containing these molecules is one way in which specific bacteria could be applied directly to the compounds which they are able to degrade. However biological treatment of such streams can be adversely affected by the extremes of pH and ionic strength commonly found in such 'point source' wastewaters. The Extractive Membrane Bioreactor, or EMB, overcomes this problem. The EMB is a novel process designed to remove toxic. poorly water-soluble organic compounds from waste streams through a combination of membrane extraction an~ ~iodegradation (Livingston. 1993 a, b). The wastewater stream and the biomedium are separated by a SIlicone rubber membrane as shown in Fig, 1. 273

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Poorly water-soluble organic compounds can pass rapidly through the non-porous membrane whilst inorganic species in the wastewater stream cannot. Having passed through the membrane, the organic compounds can be degraded by microorganisms which have been individually chosen for the particular organics present in the wastewater. The ionic components of the wastewater thus have no effect on the degradative microorganisms. The reactor consists of a conventional bioreactor linked to a shell-and-tube membrane module in which the extraction step takes place. The biomass grows principally as a biofilm on the surface of the membrane tubes. This causes problems as the biofilm grows both hydraulically. since the biomedium can no longer flow freely around the shell side of the module, and through its effect on the mass transfer of the organic compound from the wastewater to the biomedium. Previous work in this department has shown that whilst very thin biofilms enhance the flux of organic compound across the membrane. thicker biofilms significantly reduce it (Freitas dos Santos and Livingston. 1995). At larger scales. the production of superfluous biomass is undesirable since the resulting sludge must be treated and disposed of.

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Figure I. Principle of the ExtractiveMembraneBioreactor.

Preliminary experiments suggested that the addition of sodium chloride to the biomedium reduced the growth of a chlorobenzene-degrading Pseudomonas species. It was proposed that sodium chloride addition affected the maintenance energy requirement of the microorganisms through the increased energy required for pumping sodium out of the cell. Sodium ions diffuse readily into the bacterial cell and. owing to their toxicity. energy must be expended in order to pump them out against a concentration gradient. This additional expenditure of energy manifests itself as an increased maintenance energy requirement. In a carbon-limited situation this results in less carbon being available for cell growth and biomass production. At very high concentrations of sodium ions, the bacterial cell can no longer pump out sufficient sodium to survive, resulting in the inactivation of the cell .

In light of the above, the following research was split into two parts. Microorganisms were initially grown in continuous culture in an chemostat at different dilution rates and sodium chloride concentrations in order to measure the effect of sodium chloride concentration on maintenance energy requirement. A single tube EMB (STEMB) and a projection technique for biofilm thickness measurement. developed in this laboratory. were subsequently used to investigate the effect of biomedium sodium chloride concentration on biofilm growth. Three different microorganisms were used in these experiments; a pseudomonad and a Xanthobacter species were used in both the chemostat and the STEMB experiments; a Klebsiella species was also used in the chemostat experiments in order to verify the effect of sodium chloride on maintenance energy for as many different species as possible.

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MATERIALS AND METHODS Pseudomonas sp. strain JSI50 is capable of utilising monochlorobenzene (MCB) as a sole carbon and energy source and was obtained as freeze-dried culture from J.C. Spain of the Air Force Civil Engineering Suppon Agency. Florida, USA (Haigler et al.• 1992). The nutrient salts medium composition is as folIows: MgS04.7H20 (300 mg 1. 1). CaCI2.2H20 (66 mg 1.1). Na2Mo04.2H20 (190 mg 1.1). FeCI3.6H20 (4.0 mg I' I), CuS04 (0.2 mg 1.1). KH2P04 (26.0 mg 1.1). K2HP04 (21.0 mg 1. 1). (NH4)2S04 (20.0 mg 1.1), Na2EDTA (1.0 mg 1.1). ZnS04.7H20 (4.4 mg 1.1) . MnS04.H20 (0.6 mg 1.1). CoCI3.7H20 (0.15 mg 1.1). Boric acid (0.05 mg 1. 1) . In chemostat culture. 5 g I" potassium acetate was used as the carbon and energy source owing to the problems of volatility associated with using MCa. Xanthobacter autotrophicus GJlO (Janssen et al., 1985) is capable of using 1.2-dichloroethane (DCE) as a sole carbon and energy source and was obtained from Dr D. Janssen of the University of Groningen. The nutrient salts medium composition is as given by Janssen et al. (1984). As with the pseudomonad. in chemostat culture carbon for energy and growth was provided by 5 g I" potassium acetate. Klebsiella pneumoniae subsp. pneumoniae NCTC 418 was used in chemostat culture only. This microorganism was chosen due to its well-documented ease of use. The nutrient salts medium used was as described for Pseudomonas strains but containing no nitrilotriacetic acid. The carbon source was 2.5 g 1.1 Dglucose.

Maintenance energy requirement was calculated according to the equations detailed in Pin (1975). MCB and DCE concentrations in the wastewater. exit gas and biomedium were analysed using a Perkin Elmer Gas Chromatograph with a flame ionisation detector and a megabore column 25 m long and 0.23 rom i.d. with BPI (SGE, Australia) as the stationary phase. 1 J.Ll samples were injected directly onto the column . The temperature program ran from 40'C to 120·C. Peak areas were compared with those of solutions of known MCB or DCE concentration. The uncertainty in this assay (quoted as the standard deviation of three separate determinations at the 100 mg 1.1 level) was 4.2%. The detection limits were 0.2 mg 1.1 MCB and 0.1 mg I-I DCE. The exit gas sample was also analysed in this way with a sample volume of I ml, Chloride release during MCB and DCE degradation was followed using the colorimetric chloride ion assay as described by Iwasaki et al. (1956). Chloride concentration was calculated using a calibration curve. The uncertainty in this assay (quoted as the standard deviation of five separate determinations at the 20 mg 1-' level) was 4%. Carbon dioxide concentration in the exit gas from the EMB and STEMB was determined using an infra-red CO 2 analyser (Servomex PA404. Range 0-1%). This gave a C?2 .concentration as a weight of CO:!volume of air. The accuracy of this assay (quoted as the standard deviation of five separate determinations at the 2000 ppm level) was 5%. Potassium acetate and glucose concentrations in the feed and biomedium of the chemostat were analysed using High Performance Liquid Chromatography. 10 ~I samples were injected onto the column with sulphuric acid as the liquid phase. A standard, 1.5 I working volume chemostat with pH and temperature control was used for the continuous culture experiments. Stirrer speed was maintained ~t 9~ rpm and dissolved oxygen concen~ation Was kept above 20% by varying the air flow rate. pH was maintained at 7.0±0.05 and temperature at 30 C±O.I. The lay-out of the STEMB is shown in Fig. 2. An airlift bioreac:tor~as coupled to a memb~ane module via a recirculating biomedium flow. The square gl~s module used In th~s wor~.had external dimensions of 200 rom height x 60 mrn x 60 mm, with a glass thkkness of 5 nun. A single stllco.ne rubber tube (2.0 rom i.d. x 0.5 nun wall thickness). supplied by Esco Rubber (UK) Ltd. was fixed vertically along the centre of the

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module. Wastewater was pumped through the insides of the tube and biomedium was recirculated around the shell side of the module.

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Tubing throughout the system was constructed from teflon in order to minimise the losses of MeB through the tubing walls. Connections between sections of teflon were made using flexible viton tubing. Nutrients were provided continuously to the biomedium at a rate of 0.069 I h-I and the excess biomedium left the reactor at the same rate. A pH controller held the pH in the biomedium at pH 7.0±0.05. A temperature controller with a thermistor and a heating element kept the temperature in the biomedium constant at

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30"C±O.1. Biofilm thickness measurements were obtained using the novel in-situ measurement technique described by Freitas dos Santos and Livingston (1995). The error in thickness measurements is estimated to be ±1Oum. RESULTS AND DISCUSSION Figure 3 shows the effect of sodium chloride concentration on maintenance energy in Pseudomonas sp. JS150. A steady increase in maintenance energy requirement with increasing sodium chloride concentration can be observed. This would be expected if, as proposed, the presence of high concentrations of sodium ions results in an increased energy requirement by the bacterial cell for pumping.

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Figure 4 shows the effect of sodium chloride on maintenance energy in Klebsiella pneumoniae subsp. pneumoniae. As in Fig. 3, when sodium chloride concentration is increased an increase in maintenance energy can be seen. Although this experiment was only performed for two different sodium chloride concentrations the effect is again clear. In Fig. 5 the effect of sodium chloride on X. autotrophicus sp. GJIO can be seen; as expected, again there is an increase in maintenance requirement when sodium chloride is present. Figure 6 shows MCB flux and biofilm thickness over time for Pseudomonas sp. strain J5150. Over the time span of this experiment, no reduction in MeB flux can be discerned. This seems to be because of the slow growth rate of the Pseudomonas biofilm, which never reached sufficient thickness to cause flux reduction. The addition of sodium chloride to the system would therefore show no improvement and thus the experiment was repeated using X. autotrophicus GJIO.

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Minimisalion of biomassin a bioreactor

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Figures 7 and 8 show the DCE flux and the biofilm thickness over time in the STEMB for X. autotrophicus OJ 10. The experiment was run at 0 g I-I and 30 g I-I sodium chloride. Afterapproximately 200 hours with no

sodium chloridepresent in the biomedium the flux of DCE across the membrane had dropped to around 12 mg C h-I (Pig. 7)- When sodium chloride was present in the biomedium at 30 g I-I, the DCE flux across the membrane after 200 hours remained close to 20 mg C h-I (Fig. 8). These resultssuggest that the addition of sodiumchlorideto the biomedium can indeedcontrolthe rate of growthof a biofilm.

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CONCLUSIONS The experiments performed in the chernostat showed that for three different microorganisms. in a suspended growth system, increased sodium chloride concentration resulted in increased maintenance requirement. This was expected owing to the increase in energy required to pump unwanted sodium ions out of the bacterial cells. This result is of significance not only to wastewater treatment applications, but to any situation where the growth of a suspended bacterial culture needs to be limited. It is also important to consider the effect of sodium ion concentration when designing growth media for microorganisms. since an inadvertentincreasein concentration may significantly affect the energeticsof the cellular growth. The results of the X. autotrophicus experiments in the STEMB supported the chemostat results. since although maintenance energy could not be measured directly. an increase in sodium chloride concentration resulted in a markeddecreasein biofilm formation. This would be expected in a carbon-limited systemsince if more carbon is used to provide energy for pumping sodium ions. less carbon is available for cell. and therefore biofilm, growth. Further experiments are necessary to determine whether these results are purely due to the effect of sodium chloride on growth rate. or whether there are also other effects. for example on exopolysaccharide (BPS) production. EPS is an important structural component of biofilms but its production may decrease under sodium chloride stress. since energy will instead be diverted into sodium pumping. In the absence of a biofilm on the surface of the membrane tubes. an additional problem of air stripping of the organic compound occurs. since the compound is de~rade.d f?r the most part in ~he aerated region of the bioreactor, A trade-off is therefore necessary between air stnppmg and flux reduction. Current research in this group is investigating the use of sodium chloride .to preve?t further growth in a biofilm already present on the surfaceof the membrane- that is, to hold the biofilm thickness at a level where air stripping and flux reductionare both low.

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The slow growth rate of Pseudomonas JSI50 biofilm suggests that the addition of sodium chloride may not always be necessary in order to maintain a high flux in an EMB system. However at larger scales. running for significantly longer periods, it is likely that excessive growth of this organism will also eventually cause problems in the EMB. These results suggest that sodium chloride addition may indeed be a cheap, non-toxic, effective method for controlling biofilm growth in a full-scale Extractive Membrane Bioreactor. This could greatly enhance the performance of such a reactor, owing to the maintenance of a high transmembrane flux of organic pollutant. It would also increase the time for which the membrane module could be in use before cleaning of its surface became necessary. Application of this technique to other wastewater treatment systems, both biofilm and suspended growth, could also lead to important enhancements of treatment processes through the reduction of sludge production. Further work is necessary to determine whether this biomass reduction technique is applicable to mixed populations of bacteria. Different bacteria will tolerate different maximum concentrations of sodium chloride and therefore a salt concentration which reduces the growth rate of one strain may be sufficient to result in the death of another strain, so altering the balance of populations in the mixed culture. ACKNOWLEDGMENTS The authors wish to acknowledge the Biotechnology and Biological Sciences Research Council for their financial support. S. Nishino and J. C. Spain for providing Pseudomonas sp. JS150 and D. Janssen for providing Xanthobacter autotrophicus GJlO. REFERENCES Freitasdos Santos.L. M. and Livingston. A. G. (1995). Membrane attached biofilmsfor VOCtreatment I: A novelin-situbiofilm thickness measurement technique. Biotechnol. Bioeng.•47.82-89. Haigler. B. E.• Pettigrew. C. A. and Spain, 1. C. (1992) Biodegradation of mixtures of substituted benzenes by Pseudomonas sp, strainIS 150.Appl. Environ. Microbiol. 58. 2237-2244. Iwasaki. I.. Utsumi. S.• Hagino, K. and Ozawa. T. (1956) A new spectrophotometric method for the determination of small amounts of chloride usingthe mercuric thiocyanate method. J. Chern. Soc. Japan29. 860. Janssen. D. B.• Scheper. A. and Witholt. B. (1984) Biodegradation of 2-chloroethanol and 1.2-dichloroethane by pure bacterial cultures.In: Innovations in Biotechnology. ElsevierSciencePublishers. Netherlands. pp. 169-179. Janssen. D. B., Scheper. A.• Dijkhuizen, L. and Witholt, B. (1985) Degradation of halogenated aliphatic compounds by Kamhobacter autotropnicus 0110. Appl. Environ. Microbiol. 49. 637-677. Livingston. A. G. (l993a) A novel membrane bioreactor for detoxifying industrial wastewater (I): Biodegradation of phenol in a synthetically concocted wastewater. Biotechnol. Bioeng.•41. 915-926.1993. Livingston. A. G. (l993b) A novel membrane bioreactor for detoxifying industrial wastewater (Il): Biodegradation of 3chloronitrobenzene in an industrially produced wastewater. Biotechnol. Bioeng.• 41. 927-936. Petrasek, A. C., Kugelman. I. I .• Austern, B. M.• Pressley, T. A., Winslow. L. A. and Wise. R. H. (1983) Fate of toxic organic compounds in wastewater treatment plants.J. WaterPoll. Cont. Fed.•55. 1286. Pitt, S. J. (1975)Principles of Microbe and Cell Cultivation. Blackwells, Oxford.