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Indian Journal of Engineering & Materials Sciences Vol. 5, August 1998, pp.202-21 0 Biological conversion of sulfide to elemental sulfur P F Henshaw, 1 K Bewtra & N Biswas Department of Civil and Environmental Engineering, University of Windsor, Windsor, Ontario, Canada N9B 3P4 Received 21 February 1997 The use of a biological reactor (bioreactor) as a means of removing hydrogen sulfide from waste water and converting it to elemental sulfur has several advantages over conventional physical/chemical treatment processes. A suspended-growth continuous-flow stirred-tank bioreactor utilizing the green sulfur bacterium Chlorobium limicola was successfully operated at five different sulfide loading rates and three different hydraulic retention times. In all but one of the five experiments, the rate of consumption of the sulfide was equal to its loading rate. The separation of elemental sulfur from the bioreactor contents is essential to realize its value as a chemical industry feedstock. Separations of elemental sulfur by plain settling, settling at elevated pH, filtration and centrifuging were tested at bench scale using the contents of several batch bioreactors. Under plain settling, elemental sulfur and bacteria were removed from suspension to the same degree. Raising the pH to 8.6 or 8.8 resulted in some of the sulfur or bacteria settlingindependently of the sulfur-bacteria floes. Filtration was found to give conflicting results with different batches of bacteria. Centrifugation resulted in the best separation between elemental sulfur and bacteria; 90% of the elemental sulfur and 29% of the bacteria could be removed from suspension. Hydrogen sulfide (H2S) gas is highly toxic and malodorous '. In water, sulfide (S2-) has an oxygen demand/ of 2 mole Oyrnole S2- and thus would consume oxygen and have an adverse effect on aquatic life if discharged into the environment. "Sour" water is produced in petroleum refineries wherever process water comes in contact with gas streams containing hydrogen sulfide. In Canada, refinery waste waters containing sulfide must meet the Federal Refinery Effluent Regulations and Guidelines. Currently the upper limit is 0.3 kg S2-1l 000 m ' of oil refined/day for refineries that commenced operations on or after Nov.l, 1973. Refineries that were operating before that date are subject to the guidelines of 0.6 kg S2-/l000 rrr' of oil refined/day'. In most petroleum refineries, elemental sulfur is recovered from sour water by steam stripping followed by one of many catalytic sulfur recovery processes". Elemental sulfur, So, is nontoxic and is used as a feedstock for the chemical, fertilizer and materials manufacturing industries'. Conventional chemical processes for sulfur recovery from sour water and gas are expensive because .of the need to replace poisoned catalysts, contaminated reactor liquids and corroded reaction vessels". The use of a biological process is potentially less expensive because it acts at low temperatures and pressures, generates its own catalyst (bacteria), and can remove very low concentrations of sulfide. The latter point is especially important if one considers that new environmental regulations tend to be more stringent than their predecessors, which may result in the addition of successive treatment stages to conventional processes. A biological process is being developed to remove H2S from sour water streams. While dissolved in water, H2S is less likely to be an environmental or safety hazard than H2S gas. Chlorobium limicola (A TCC 17092) is a naturally occurring green sulfur bacterium (GSB) capable of oxidizing S2- to So. The SO is produced attached to the outside of the cells. This photosynthetic bacterium requires CO2, inorganic nutrients and sulfide for growth and is strictly anaerobic. Cork?" demonstrated the utility of C. limicola in producing SO from H2S where the sulfide fed into the photosynthetic reactor was in the gas phase. The removal of S2- in the liquid phase by photosynthetic bioprocesses has been demonstrated in some studies, but SOwas either not quan- HENSHA W et al.: BIOLOGICAL CONVERSION OF SULFIDE TO ELEMENTAL tified or not produced+". These authors have measured the production of elemental sulfur from sulfide by GSB in batch9•lo and continuous-flow bioreactors II • A schematic of a continuous-flow sulfide removal/sulfur recovery system employing a suspended-growth bioreactor is shown in Fig. 1. Sour water flows into the bioreactor wherein the sulfide is converted to elemental sulfur. It is desirable to physically remove elemental sulfur from the bioreactor for three reasons. Firstly, SOhas a commercial value which can be realized only if it is separated and purified. Secondly, SO in the bioreactor blocks the available light needed by these photosynthetic bacteria. Finally, SOis an alternate electron donor to S2- in GSB and leaving it in the bioreactor may result in further oxidation of the ele-mental sulfur to sulfate, decreasing the yield of SO from the process. After the elemental sulfur is removed from the reactor effluent stream, most of the bacteria can be separated from the waste water for recirculation back into the reactor. The development of the S2- removal/SO recovery system has proceeded along two tracks. First, the characteristics of a continuous-flow bioreactor employing GSB to remove sulfide have been studied at a bench scale. Second, this paper describes several separation techniques that have been tested at a bench scale to separate SO from the contents of a batch biological reactor. The techniques evaluated were: settling, settling at elevated pH, filtration, and centrifugation. Separation of the elemental sulfur from the bacteria and waste water by gravity settling would be the least expensive separation alternative. Cork" reported that elemental sulfur settled from bioreactor contents within 24 h. Kim and Chang" WQstewater 8IIJIEACHII BacteriQ WQsteWQter. H1Sc ••• Fig. I-Schematic of proposed bioreactor system SULFUR 203 successfully used a settling unit to remove some SO from the recycle stream of a gas-fed bioreactor, claiming it removed 80 to 90% of the elemental sulfur. Jar tests on the GSB/sulfur mixture grown in batch reactors revealed that raising the pH resulted in flocculation and increased settling of the bacteria and SO(Henshaw P F, unpublished data). The use of a centrifuge to produce a settling force several times the force of gravity was suggested as a means of removing elemental sulfur from suspension". Microphotographs of green sulfur bacteria and adjacent elemental sulfur have revealed that the diameters of the SO granules are greater than the short dimension of the ovoid GSB cells IS • Average SOparticle and GSB sizes of 9.4 urn and 1.1 urn, respectively, were reported in a culture of C. limicola", A membrane or fibre filter might utilize this size difference to remove elemental sulfur from suspension by sieving action. Materials and Methods Methods of analysis Sulfide-- The methylene blue colorimetric method was used 17. Elemental sulfur-For settling tests of sulfur in water, about 70 mL of 95% ethanol was added to an aqueous sample of less than 20 mL. The mixture was refluxed for two hours and made up to 250 mL with 95% ethanol, centrifuged, and the optical density measured at 264 nm against 95% ethanol. In settling tests where sulfur and bacteria were present, the colorimetric method was used with 'the addition of mercuric chloride". In filtration and centrifugation experiments, aqueous sam£' 19 . pIes were extracted into chloroform pnor to quantification by high-performance liquid chromatography (HPLC)20. Bacteria--Bacteriochlorophyll (bchl) was extracted into methanol and its absorbance was measured". The concentration ofbchl was taken as an indicator of the bacterial concentration. A 1.00 mL sample was transferred by disposable pipette into 10.00 mL of ACS grade methanol in a centrifuge tube. The tube was capped, swirled by hand for one minute, and centrifuged at 1,400 g's (gravities) for 10 min. The absorbance of the supernatant was measured at 670 nm against an ACS grade methanol blank. 204 INDIAN 1. ENG. MATER. SCt, AUGUST 1998 Growth of bacteria two Philips IR 175 Watt R-PAR bulbs inside the vessel directed into the windows of the first reactor. Both reactors were mounted in a New Brunswick Scientific Co. model FS-314 fermenter drive assembly. This resulted in an average irradiance of 258 W/m2 over both windows. The bioreactor stirsalts solution. The content of a culture tube was ring speed was approximately 200 rpm. Masterflex used to inoculate alL round-bottom flask which (Cole-Palmer Co. ,Chicago) variable speed pumps was, in turn used to inoculate the content of a 15 L with solid state speed controls and standard pump New Brunswick Scientific Co. model F-14 fer- heads were used for all influent and effluent reacmenter as previously described". tor streams. The liquid level in the reactor was kept constant by a level probe inside the 'reactor which Continuous-flow stirred tank reactor actuated the effluent pump. The pH inside the reIn the continuous-flow bioreactor experiments, actor was kept between 6.8 and 7.2 by an Omega the components of the growth medium" were split (Omega Engineering Co., Stamford, CT) PHP-166 amongst several influent streams. In Runs 3, 4 and Chemical Metering Pump controlled by an Omega 5 , there were three influent streams: deionized PHM 55 pH Regulator connected to an Ingold (Inwater, a concentrated nutrient solution and a sulgold Electrodes Inc., Wilmington, MA) 465-35-K9 fide stock solution (Fig. 2). In Trials 9 and 10, combined glass electrode inserted into the reactor. there were only two feeds: a nutrient medium and a Feed pumps were adjusted by timing the revosulfide stock solution. Apart from the subculturing lutions until the proper speed (typically 2 to 10 tubes and 1 L batch reactor, the reactors and rpm) was achieved. For Runs 3, 4 and 5, the conequipment were not sterile. centration of the sulfide stock solution was inA New Brunswick Scientific Co. model F-14 creased in successive Runs while the HRT was fermenter was used as a continuous reactor. The held constant (Table 1). Throughout each Run, a reactor was wrapped in a metallized Mylar film constant sulfide loading rate was maintained reand two 105 mm high by 110 mm wide windows gardless of the condition in the reactor. In Trials 9 were cut, one above the other, into the film so that and 10, HRTs higher than those in Runs 3, 4, and 5 the top of the upper window was at the water level were used and the sulfide loading was adjusted so of the constant temperature (30°C) water bath. A as to have a low but constant sulfide concentration second reactor was used as a submerged light in the reactor. source by removing the stir paddles and clamping In inorganic growth medium" was used for the culture tubes and experimental reactors. The bicarbonate and sulfide solutions were combined and filter-sterilized directly into the autoclaved culture tubes or batch reactor containing sterile mineral :8 '--r----------...., -11r--------------, 'I CO2 GAS [lJTLET III I ~T~Et~-~ ft~. I: '! II 0 w I I I ••uel~ , I , • .., Ii: :r--·-·-·1~~IQ.~.1 I II! _ . L r-;7.-==rt=~ill~I~I~' I I E~r::U£NT . i.o n . n REACTOR EFFLl.O/T iN: =. Nor-nolly (Io~ed Valve .!...~:~~~'lonO:7tVO'vt - - _._.- "' Gas (ondui t • Control line Fig. 2--Schematic of continuous stirred-tank reactor as used in runs 3, 4 and 5 Settling A clear acrylic settling column was used, 90 mm in diameter and approximately 2200 mm tall with four sampling ports, 600 mm apart starting at a depth 130 to 300 mm below the liquid surface. The settling test was performed on: elemental sulfur in water, bacteria in reactor contents and bacteria and elemental sulfur in reactor contents. For testing the settling of bacteria alone, GSB were grown in a 15 L batch reactor and the settling test was performed several days after the sulfide had been completely consumed by the bacteria. In tests where bacteria and SO were required, sulfide was added to the batch reactor one day prior to the column test, and the test was performed as close as possible to the time when sulfide was completely consumed, thus ensuring that elemental sulfur would be at its highest concentration. During the ItU"SItA \V ct al.: BIOLOGICAL CONVERSION Table I-Variables Experiment Run Run Run Trial Trial OF SULFIDE TO ELEMENTAL in continuous reactor operation Vol. Our. [S=]; Load. HRT L h mg/L rng/L h h 13.7 13.7 13.7 12.0 12.0 316 383 283 1680 1220 90 190 260 550 260 2.1 4.4 5.6 3.2 2.7 45 44 46 173 99 3 4 5 9 10 205 SULFUR . pH adj. [S=]", mg/L b 1340 2760 4040 2730 2440 b a a a Vol.= reactor liquid volume, Our.= duration of experiment, [S];= effective inlet sulfide concentration, Load = average reactor sulfide loading, pH adj.= acid (a) or base (b) used to adjust pH in reactor, [S=]sss=sulfide stock solution concentration. tests, the content of the column was stirred for approximately five minutes, then allowed to settle. Samples were collected from all of the sampling ports at various time intervals. The samples were analyzed for elemental sulfur and/or bacteriochlorophyll. The data were analyzed according to the method of Ramalho'". Settling at Elevated pH In this series of tests, a clear acrylic settling column 102 mm in diameter and 1830 mm tall was employed. The sampling ports were 355 mm apart, and the column was covered with aluminum foil to prevent the creation of density currents caused by heating of the column contents by light. GSB were grown in a batch bioreactor until the sulfide had been consumed by the elemental sulphur. One day prior to the test, sulfide was added to the batch reactor so that the SOconcentration would be at its peak. At this point, the contents of the batch reactor were pumped into the column and 700 to 1000 mL of I M NaOH was added while stirring to bring the pH up to the desir~d level. In some cases, additional deionized water needed to be added to the column so that there would be a sufficient liquid level above the upper sampling port throughout the test. The column was slowly stirred for two minutes to encourage flocculation, after which a series of samples was taken at all ports to determine the starting bacteria and elemental sulfur concentration throughout the column. Additional samples were taken 20-25 and 60-70 min after the initial sample. The first 15 mL from each port was drained and discarded prior to collecting each 40mL sample. After collection, the 40 mL samples were stirred with a magnetic stirrer while aliquots were withdrawn for the elemental sulfur (5 to 10 mL) and bacteriochlorophyll (1 mL) assays. The data were analyzed graphically". Filtration Sulfide was added to the bioreactor approximately one day prior to the test to allow the bacteria to produce elemental sulfur. Samples were taken for elemental sulfur and bacteriochlorophyll from a stirred 50 mL grab from the batch bioreactor. The sample was then filtered under vacuum through a 4.7 mm diameter membrane filter of pore size 0.45, 0.8, 1.2, 3.0, 5.0 or 8.0 urn. Preliminary tests included filtration through glass fibre filters with effective pore sizes of 1.2, 1.5 or 2.5 urn. Samples for elemental sulfur and bacteriochlorophyll analysis were taken from the filtrate. The elemental sulfur samples were analyzed by HPLC. The reduction in elemental sulfur and bacteriochlorophyll concentrations were then calculated. The experiment was repeated with other samples of bioreactor contents that were: blended in a commercial blender (Osterizer Galaxie- Ten), adjusted to pH 8.8 with 1 M NaOH or diluted with deionized water. Centrifugation Sulfide was added to the batch reactor approximately one day prior to the test to allow the bacteria to produce elemental sulfur for the test. Samples were taken for elemental sulfur and bacteriochlorophyll from a stirred 50 mL grab from the batch bioreactor. The cap on the sample was then sealed, and the sample centrifuged at 500 or 800 RPM (55.9 or 143.1 g's) in an International Centrifuge Centra-8 centrifuge with a #269 rotor. Centrifuge times were 3, 6, 9, 12, 16, 17 or 30 min and later, confirmatory tests were done for 10 min. 206 INDIAN J. ENG. MATER. SCI., AUGUST 1998 These times were exclusive of a two minute acceleration period for the centrifuge to obtain the set speed. The experiments were also conducted on several samples blended in a commercial blender. consumption equaled S2- loading up to and including a loading of 64 mg/Lh. Therefore, the reactor sulfide loading at which overloading occurs depends upon the reactor configuration. The rate of S2- consumption was not found to be Results and Discussion related to the sulfide or elemental sulfur concentration in the experiments in this study. Continuous-flow stirred tank reactor Fig. 3 shows that up to a sulfide loading of 5 mglLh, the rate of sulfide consumption was equal to the rate of sulfide loading. In other words, all of the sulfide input to the reactor was consumed. The product of the consumption was SOin all cases except Run 3, where the under-loaded reactor produced SO/-. Run 5, where the sulfide loading was above 6 mg/Lh, can be characterized as overloaded in terms of sulfide. One would have expected the consumption/loading curve to level off when the maximum loading had been reached. In fact, the reactor "failed" in Run 5 and S2- consumption decreased, thus allowing sulfide to accumulate in the bioreactor. Had the experiment been allowed to continue, the sulfide concentration would have reached the point of toxicity to the bacteria. Previous researchers" found that sulfide 5 0 •.•. V 0 Settling When allowed to settle independently, GSB and elemental sulfur settle at different rates (Fig. 4). According to the curves for independent settling, SOwould be completely removed from aIm column after 27.8 min (overflow rate = 0.6 mrnls), whereas only 18% of the bacteria would be removed in this time. According to the curve for bacterial settling, the maximum removal of bacteria, even with an infinite settling time, would be about 40%. When settled together, the removal of SOin the presence of GSB and the removal of GSB in the presence of SO were intermediate between the individual removal curves of SOand bacteria. In addition, the removal curves for SO were similar and coincided at an overflow rate of 0.66 mrnls indicating that the two solids settled together. n Run 3 Run 4 Run 5 Triol 9 Trial 10 80 1 o 4 Elemenlal Sulfur alone Bacteria alone Elemental Sulfur in the presence of bacteria .•• Bacteria in the presence of elemental sulfur • V 70 c o ii E •. 3 o > o :l C ., •• Q: "0 :l If) 50 E o u 40 2 -• 30 o "0 20 '" I o R2 = 0.961 LL~~~~~~~~~~~~~~~ 2 o Sulfide Looding (e.eluding Run 5) I 10 o 3 4 5 6 7 Role (mg/h·L) Fig. 3-The effect of sulfide loading on sulfide consumption ~~~~~~~~~-L~~~~~~ 0.0000 0.0005 0.0010 Overflow 0.0015 Role 0.0020 0.0025 (m/s) Fig. 4--R.emoval of elemental sulfur and bacteria by plain settling HENSHA W et af.: BIOLOGICAL CONVERSION OF SULFIDE TO ELEMENTAL SULFUR 207 Forty-four per cent of both the SOand GSB were removed from a I m depth of column after 26 min. The reported elemental sulfur removal values in the combined experiment were lower than the actual SOremoval, since SOwas measured at only one depth in the column. These results show that during simple settling, the association between bacteria and SO remains and therefore this technique cannot be used to separate these two types of suspended solids. is essentially equal to the value at neutral pH in the presence of bacteria. For bacteria, this value is roughly equal to that for bacteria settling alone at neutral pH. From these experiments, it can be concluded that the association between the bacteria and SOis weakened by raising the pH. At a pH of 8.8, elemental sulfur was released from the sulfurbacteria complexes to settle independently. At pH 8.6, bacteria were released to settle at their normally slow rate. Separation of bacteria and elemental sulfur can Settling at elevated pH be achieved by this method. The best separation The settling rates of bacteria and elemental sul- occurred at pH 8.6 and an overflow rate of 0.22 fur were increased by elevating the pH. At pH 8.8, mmls when 70% of the elemental sulfur was setthe removal rate of bacteria at an overflow rate of tled from a mixed suspension compared to 27% of 0.6 mmls was 50% (Fig. 5) as compared to 43% at the GSB. The viability of the bacteria after settling neutral pH (Fig. 4). More importantly though, the at a pH of 8.6 was not tested. rate of removal of SO was different from that of GSB, being 75% at an overflow rate of 0.6 mm/s. Filtration The difference in removal rates between elemental The results of preliminary tests, where a clean sulfur and bacteria is even more pronounced at pH separation of elemental sulfur from the bioreactor 8.6 (Fig. 5) but the overall settling rates are lower contents was achieved, were not reproducible in than at pH 8.8. In fact, the removals of SO and the confirmatory tests. In preliminary tests, all of bacteria at pH 8.6 at an overflow rate of 0.6 mm/s the SObut only 30% of the GSB were trapped on a were 37 and 14% respectively. For So, this removal 5 urn pore size membrane filter (Table 2). Similarly, 100% of the elemental sulfur but only 50% 100r-~--~~----~~==~======~ of the bacteria were removed by a glass fibre filter pH = 8.8 V £Iemental Sulfur that retained particles larger than 1.2 urn. Later •• Bacteria 90 studies using membranes with pore sizes ranging pH = 8.6 o £Iemental Sulfur from 1.2 to 8.0 urn resulted in the removal of all • Bacteria 80 suspended material at all pore sizes. The greater removal in the latter study was attributed to longer 70 GSB chains as a consequence of the longer bacteria retention time in the reactor. Thus, the batch-to..-, 60 ~ batch variation in bacterial characteristics pre'-' 0 cludes filtration as a reliable method of separating 50 > 0 bacteria and elemental sulfur. E • 40 0:: Exploratory studies looked into the possibility of diluting, blending or raising the pH of the reactor contents prior to separation by filtration. None 30 of these techniques resulted in differential separa20 tion of elemental sulfur from the reaction mixture (Table 2). 10 Centrifugation 0 0.0000 0.0005 0.0010 Overflow Rote (m/s) Fig. 5-Removal at elevated pH of elemental sulfur and bacteria by settling In preliminary tests, the use of a centrifuge to produce a strong separating force, was shown to accomplish a good separation of 90: 10 (% SOremoved: % GSB removed) from the contents of a batch bioreactor. Further tests (Fig. 6) achieved 208 INDIAN J. ENG. MATER. SCI., AUGUST 1998 Table 2---Removal of elemental sulfur and bacteria by filtration ~m Treatment of mixture 0.45 0.8 1.2· regular regular regular 1.2 regular 1.5. 2.5· 3.0· 3.0 regular regular regular regular 0 68 20 5.0 regular II 51 56 45 Pore size 5.0 blended 8.0 regular 8.0 regular 8.0 pH=8.82 8.0 diluted Reduction on filtration (%) Preliminary Tests Confirmatory Tests Bacteria Elemental sulfur Bacteria Elemental sulfur 105 99 0 33 28 35 109 117 68 90 91 92 77 39 63 100 100 100 101 99 102 100 100 100 101 105 93 98 98 92 95 96 95 95 94 91 97 100 100 100 100 100 100 100 100 100 100 100 100 100 60 115 79 125 99 99 100 • effective retention similar results, in which the elemental sulfur removal approached 100% as the centrifuging time exceeded 20 min. Conversely, even after prolonged centrifuging, the bacterial removal from suspension was only 35-40%. There is more scatter in the bacteria removal data than the SO removal data. Presumably, unaccounted-for characteristics of the bacteria batches which affected the removal of bacteria were expressed. Good separation of 90:29 was achieved when the sample was spun at 143 gravities for 9 min. Centrifuging at lower speeds (56 gravities) resulted in less removal (65:26) of both elemental sulfur and bacteria from suspension and the difference in removal percentages was less. Extreme agitation of the sample in a household blender to break the SO-bacteria association prior to centrifuging gave similar results to those reported for unblended elemental sulfur removal, but the variability in bacteria removal after blending was much greater than in the unblended case. These results are not shown because the GSB removal variability precluded assigning a statistically-based curve to the bacterial data, as was done for the unblended samples. Even with centrifuging, the low elemental sulfur concentrations used in these experiments resulted in low SOconcentrations in the pellet. To illustrate this, consider that the average elemental sulfur concentration in these experiments was 19 mg/L. If . HENSHAW et al.: BIOLOGICAL CONVERSION 9' 80 :6.- ,, .t:.. P- 70 60 t? ~ D > 0 50 " , ,, , , ••• ,, ... .0 • • ... .•. 30 BOOrpm Bchl. 800 rpm 6.- Sulfur. 500 rpm ••• Bchl. 500 rpm 800 rpm ._-- 500 rpm ••• . ~. . E 0 Sulfur. ••• ,~ ,, 0 • '" -- , ,, ,, ,, • • ••• .. .-~-.---.- -.-.- .. t ••• • 20 .•. 10 • ••• ••• 0 0 10 Time. Fig. ~Removal gation 30 20 SULFUR 209 poor test performance of some of these methods of separation may have been due to the inconsistent nature and low concentration of the bacteria. The tests described were all conducted on bacteria grown in batch reactors. It is hoped in the future that the most promising of these techniques can be repeated on the effluent from a continuous reactor. 100 90 OF SULFIDE TO ELEMENTAL .0 t (min) Acknowledgment This research was supported by funding from Imperial Oil Canada and the Natural Sciences and Engineering Research Council of Canada. The experiments on the separation of elemental sulfur were conducted under the supervision of the authors by Yvette Ly, David Diemer and Brian Dantas for their senior year engineering projects in the Department of Civil and Environmental Engineering at the University of Windsor. References 1 Cadena F & Peters R W, )WPCF, 60 (1988) 1259. 2 Kobayashi H A, Stenstrom M & Mah R A, Water Res, 17 (1983) 579. Losier L, Environmental status report of the canadian petroleum refinery industry (Supply and Services Canada, Ottawa), 1990 Sittig M, Petroleum Refining industry: energy saving and environmental control (Noyes Data Corp., Park Ridge, N.J.),1978 West J R & Duecker W W, in Reigel's handbook of industrial chemistry (7th ed.), edited by Kent J A (Van Nostrand Reinhold Co., Toronto), 1974, 62 of elemental sulfur and bacteria by centrifu- 90% of the SO was removed from aIL sample, 17 mg would end up in the pellet. The average bchl concentration was 39 mg/L. If 29% of this was removed from aIL sample, 11 mg bchl would be in the pellet after centrifuging. Assuming 3% of the VSS in the biomass is bchl!', the VSS in the pellet would be 11 mg/0.03 = 370 mg. Therefore, the percentage of elemental sulfur in the pellet would be about 100(17/(17+370» = 4% on a dry mass basis, 3 4 5 6 7 8 Conclusions A continuous stirred tank bioreactor successfully removed dissolved sulfide at loadings of up to 5 mg S2-/L h. At higher loadings the bioreactor failed and sulfide accumulated within the reactor. Four methods of separating elemental sulfur from a suspension of SOand GSB were tested on a bench scale: settling, settling at elevated pH, filtration and centrifuging. Of these, centrifuging was the most consistent and gave the highest difference in removal of SOand GSB from the bioreactor suspension. Even so, the SO content in the sulfurenriched fraction after separation was low, due to the low SO concentration in the bioreactor. The 9 10 II 12 13 14 Cork D J, Jerger D E & Maka A, Biotechnol Bioeng, 16 (1986) 149. Cork D J, Kinetics of sulfate conversion to elemental sulfur by a bacterial mutualism. a hydrometallurgical application, Ph.D. Thesis, Univ. Arizona, Tuscon, 1978 Maree J P & Strydom W F, Water Res, 21 (1987), 141. Henshaw P F, Bewtra J K, Biswas N & Franklin M, Water Sci Technol, 25 (1992) 265. Henshaw P F, Biological removal of hydrogen sulfide from refinery wastewater and conversion to elemental sulfur, M.A.Sc. Thesis, Univ. Windsor, Windsor, Canada, 1990 Henshaw P F, Biological conversion of hydrogen sulfide to elemental sulfur in a suspended-growth continuous stirred-tank reactor, Ph.D. Thesis, Univ. Windsor, Windsor, Canada, 1995 Cork D J, in Microbial growth on Cl compounds. proceedings of an international symposium, edited by Crawford R L & Hanson R S (Amer. Soc. for Microbiology, Washington, D.C.), 1984,306 Kim B W & Chang H N, Biotechnol Prog, 7 (1991) 495. Cork D J, Patent 4, 666, 852 (to the Institute of Gas Technology), 1987 u.s. 210 INDIAN J. ENG. MATER. SCI., AUGUST 1998 15 Stanier R Y, Ingraham J L, Wheelis M L & Painter P R, The microbial world (5th ed.) (Prentice-Hall, Englewood Cliffs, N.J.), 1986 16 Kim B W, Chang H N, Kim I K & Lee K S, Biotechnol Bioeng, 40 (1992) 583. 17 Truper H G & Schlegel H G, Anton Leeuwenhoek, 30 (1964) 225. 18 Bartlett J K & Skoog D A, Anal Chem, 26 (1954) 1008. 19 Henshaw P F, Bewtra J K & Biswas N, Anal Chem, 69 (1997)3119. 20 Lauren D R & Watkinson J H, J Chromatogr, 348 (1985) 317. 21 Maka A, Control of oxidative sulfur metabolism in Chlorobium, Ph.D. Thesis, Illinois Institute of Technology, Chicago, 1986 22 Madigan M T, in Biology of anaerobic microorganisms, edited by Zehnder A J B (John Wiley & Sons, Toronto), 1988,39 23 Ramalho R S, An introduction to waste water treatment processes (Academic Press, Inc., New York, NY), 1977 24 Metcalf and Eddy, Inc., Waste water engineering (McGraw-Hill Pub!. Co., Toronto), 1991 25 Maka A & Cork D, J Ind Micro, 5 (1990) 337.