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Removal of Hydrogen Sulfide by Chlorobium thiosulfatophilum in Immobilized-Celland Sulfur-Settling Free-Cell Recycle Reactors Byung Woo Kim and Ho Nam Chang' Department of Chemical Engineering and Bioprocess Engineering Research Center, Korea Advanced Institute of Science and Technology, Daeduk Science Town, Taejon 305-701, Korea
Bioconversion of hydrogen sulfide to elementary sulfur by the photosynthetic bacterium Chlorobium thiosulfutophilum was studied in immobilized-cell and sulfur-settling freecell recycle reactors. The cells immobilized in strontium alginate beads excreted elementary sulfur and accumulated it as crystal in the bead matrices, which made it possible that the reactor broth remained clear and the light penetrated the reactor deeper than with the free cells. In comparison with the free cells, the immobilized cells required 30% less light energy at a H2S removal rate of 2 mM/(L.h) and showed an activity of 2.4 times that of the free cells. However, in 40 h after the reaction the deterioration of the H2S removal efficiency became significant due to the accumulation of sulfur in the beads. The scanning electron micrograph (SEM)and energy-dispersive X-ray spectrometer (EDS)studies showed that the sulfur in the beads existed within a layer of 0.4 mm from the bead surface. In the sulfur-settling free-cell recycle reactor, about 80% of the sulfur excreted by the free cells could be removed in a settler. The 4-L fed batch reactor with the settler improved the light transmission to result in a H2S removal rate of 3 pmol/ (mg of proteinoh), 50% higher than that without it. The settling recycle reactor was much better in the removal of H2S than the immobilized-cell reactor because the former was a continuous system with the constant removal of sulfur particles by settling and of spent medium by supplying fresh medium a t the same rate as the filtering rate of the reactor broth, while the latter was essentially a batch system where toxic metabolites and produced sulfur could not be removed.
Introduction The toxicity and corrosiveness of hydrogen sulfide gas has made it necessary that its release into environments be strictly regulated. The human threshold exposure limit to H2S is 10ppm for 7-8-h periods (Buchman and Gibbons, 1974), and humans recognize H2S odor associated with anaerobic sewage decomposition in concentrations as low as 0.0005 ppm (Price and Cheremisinoff, 1978). The discharge limit is set at less than 5 ppm in most countries. Recoveriesof sulfur up to 97 % have been achieved using multiple-stage treatment in the traditional Claus plant. However, the current air pollution regulations require a better than 99% recovery, which has forced industries to add an additional system to clean Claus tail gas (Berry, 1980). Several physicochemical methods are available for treating the tail gas from a sulfur recovery plant (Cadena and Peters, 1988;Loran and O'Hara, 1978). But in addition to high capital and operational costs, incineration of hydrogen sulfide of 200-500 ppm carried over from the preceding stage produces sulfur dioxide in the Clam process, also a key atmospheric pollutant. Sulfate or thiosulfate is produced in the Stretford process (Cork and Ma, 1982), which should be treated further with calcium chloride. The byproduct gypsum also causes a problem in disposal. Biological oxidations were also considered as alternatives to the physicochemical methods (Buisman et al., 1990; Cork et al., 1983; Sublette and Sylvester, 1987). Buisman et al. (1990) cultured aerobic Thiobacillus in a Na2S broth, but this required deliberate oxygen control to suppress the accumulation of sulfate in the reactor medium. Sub-
* To whom correspondence should be addressed. 8756-7938/91/3007-0495$02.50/0
lette and Sylvester (1989) adopted facultative Thiobacillus denitrificans not requiring oxygen control and demonstrated a stable operation at a reactor loading of as high as 4-5 mmol of H2S h-l (g of cells)-l. But in a 1.4-L reactor the sulfate accumulated up to 24 mM while no elementary sulfur was produced during the initial 17 h. It is desirable to recover sulfur as much as possible, rather than as sulfate that requires another wastewater treatment process. The problem of biooxidation of H2S is light transmission into the medium (Sublette, 1987). As the reaction proceeds, the number of cells and sulfur increases to such a level that the light transmission is no longer effective. Thus carrying out the reaction with free cells in suspension will not be effective soon because of poor transmission of light due to the cells and sulfur particles. Kim et al. (1990) immobilizedwhole cells of Chlorobium thiosulfatophilum to obtain 30% light energy saving in comparison with a free cell reactor. However, the accumulation of sulfur in the beads results in intraparticle mass transfer resistance, which eventually deteriorates the H2S removal capability. In this study we intend to compare the H2S removal performances of free-cell suspensions and immobilizedcell and free-cell reactors with sulfur settling. Especially for the immobilizedcells, the scanning electron micrograph (SEM) and the energy-dispersive X-ray spectrometer (EDS) were used to study sulfur deposition in the gelbead matrices.
Materials and Methods Bacterial Strain, Media, and Growing Conditions. The bacterial strain used was Chlorobium limicola forma
0 1991 American Chemical Society and American Institute of Chemical Engineers
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thiosulfatophilum (ATCC 17092), commonly known as Chlorobium thiosulfatophilum. The medium consisted of KH2P04 (0.74 g/L), NHdC1 (0.74 g/L), M g S 0 ~ 7 H z 0 (1.8 g/L), CaCl2.2H20 (0.05 g/L), NaCl (7.4 g/L), B12 (0.0002 mg/L), and 100 mL of 0.05 M NaH2P04 and 200 mL of 0.05 M Na2HP04 per liter of medium, including 1 mL of trace solution where Na2 EDTA (5.2 g), FeC1~4H20 (1500 mg), ZnCl2 (70 mg), MnC1~4Hz0(100 mg), H3B03 (62 mg), CoC1~6H20(190 mg), CuCb2H20 (17 mg), NiClr6H20 (24 mg), and Na2Mo0~2H20(36 mg) were dissolved in a l-L solution of distilled water in the order given. NazS.9H20 (0.1 g/L of reactor solution) was added at the start to ensure an anaerobic environment by removing dissolved oxygen in the reactor, which was as low as possible so as not to cause inhibition by sulfide (Larsen, 1952).The feed gas mixture consisted of H2S (4.16 mol % ), CO2 (9.34 mol 5% ), Nz (86.01 mol % ), and H2 (0.49 mol % ), equivalent to 46 385 ppm of H2S in the state of gas mixtures. The solubilities of HzS in water are 92.1 mM at 30 "C and 83.2 mM at 40 OC (Wilhelmet al., 1977). The pH of the medium was adjusted to 6.8 with 0.1 N NaOH or HC1. Sterilization of the medium and the feed gas was not necessary because of the inhibition of H2S to other microorganisms. Assays. Elementary sulfur produced during the photosynthesis was excreted by the cells and suspended as particles in the reactor broth. Sulfur removal from the sampled solution was necessary for determining the dry or wet weight of the cells. Since this was a difficult task, we measured protein concentrations for the cell density determination. An optical absorbance of 0.1 at 578 nm by spectrophotometer corresponded to 0.1 g of dry cell/L without sulfur (Fuchs et al., 1980). The correlation of the cell protein with the dry weight showed that protein was about 24% of the dry cell weight. It was shown that significant amounts of synthesized carbon were stored in the cells as polyglucose and chlorophyll (Sirevag and Ormerod, 1977). Protein was determined by Bradford's CBB (Coomassie Brilliant Blue; Bradford, 1976) method. A sample of 10mL volume taken from the reactors was centrifuged at 18000g,4 "C, and then the recentrifuged precipitate, after the extraction of chlorophyll and sulfur (Cork and Cusanovich, 1978), was boiled with 10-20 mL of 1N NaOH in a water bath. The CBB solution was added and the absorbance was read at 595 nm. Protein concentration was determined from a standard curve prepared with bovine serum albumin. To analyze total sulfide concentration, 1 or 2 drops of 2 N zinc acetate and 0.2-0.6 mL of 6 N HCl were added to the supernatant after centrifugation. After it was mixed with 6 mL of 0.025 N iodine solution (20-25 g of KI in 10 mL of water, followed by 3.2 g of iodine, and water to a final volume of 1L) and 1or 2 drops of 2 % starch solution, titration was done with 0.025 N Na2S203 solution (Greenberg et al., 1981). Sulfate concentration in the supernatant was determined turbidimetrically at 420 nm from a standard curve prepared with sodium sulfate solution. Sulfur in the precipitate was mixed with acetone, and then residual water was evaporated with acetone in water bath. After evaporation, dried sulfur was dissolved in chloroform to determine its concentration with a spectrophotometer at 290 nm from a standard curve (Koch, 1949; Cork and Cusanovich, 1978). Immobilization. Cells to be immobilized were harvested as concentrated paste and stirred into a 1.5%
h Figure 1. Settling recycle setup with filtration of toxic accumulations in reactor broth. Settler, metal filter, and supply of fresh medium were not placed in an immobilized-cell reactor.
solution of sodium alginate (pH 6.8 in the medium with Tris-HC1 buffer). The cell alginate suspension was then extruded dropwise by a syringe with a needle of 0.2 mm (i.d.) into a bath containing a solution of 0.1 M strontium chloride at 10"C. One day was allowed for complete gelling of strontium alginate beads. The beads were washed 2-3 times with distilled water, and then loaded in the fedbatch reactor. Microscopic Techniques. For the microscopic examination of accumulation patterns of sulfur excreted extracellularly, the cross sections of the strontium alginate beads were prepared. The segments of the sectioned beads were fixed for 1 day using 2% glutaraldehyde and 1% paraformaldehyde solution in a 0.1 M Tris-HC1 buffer (pH 6.8) and postfixed for 1 h in an aqueous solution of 1%uranyl acetate. The stained sections were freeze-dried for 1day, and remaining water was dehydrated further in a vacuum drier for 1day. Following the dehydration, the gold-coatedspecimen was examined in a scanning electron microscope (Model 840-A, Jeol Co.) with an accelerating voltage of 20 kV. In addition, nuclide analysis of the coated specimens was done with the energy-dispersive X-ray spectrometer (Model 10 000, Link System Co.). Reactor System and Operation. Two fed-batch reactors of 2- and 4-L capacity (SY-250 and SY-500, Korea Fermentor Co., Inchon, Korea) were used. From a standard gas cylinder, hydrogen sulfide and carbon dioxide were fed into the reactor through a gas distributor with the feed rate control by a two-stage gas regulator and a flow meter. Medium was stirred with two disc-turbine type impellers at 500 rpm. The pH and temperature of the medium were maintained at 6.8-6.9 and 30 "C by controllers, respectively. Two sets of zinc acetate traps were placed in an effluent line to scrub hydrogen sulfide gas, and a wet gas meter was used to measure the volumetric flow rate. The average light intensity (Lee et al., 1987) from two incandescent light bulbs was determined with a lux meter (IM-2D, Topcon Co., Tokyo, Japan). Figure 1shows the schematic diagram of the reactor system with the sulfur settler made of Pyrex glass (4.5-cm i.d. X 25-cm length). Cells were grown up on the described medium in which gas mixtures were fed and dissolved as electron and carbon sources for the photosynthesis and then harvested by centrifugation. Usually initial cell loadings were about 0.5-0.8 g of dry cells/L. The feed rate in the 2-L reactor was increased stepwise with the cell growth, corresponding to about 0.106 pmol of HzS/min-' (mg of protein)-' L-l, which was the H2S removal rate expected for the C. thiosulfatophilum cells. If light energy is transferred to P840, a reaction center in bacteriochlorophyll, P840 is activated to accept electrons from H2S oxidation (eq 1). If more light is available than necessary for eq 1, elementary sulfur is oxidized further
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to sulfate (eq 2). The optimal conditions of operation
+
-2s' + + -
2H2S CO, H2S
+ 2C0,
2H20
(CH20) + H 2 0
(1)
2(CH20) + H2S0,
(2)
were established to minimize sulfate production as well as sulfide accumulation by adjusting the light intensity and the feed rate. In the case of the settling recycle reactor, produced sulfur particles were removed from the settler (Pyrex glass, 4.5cm i.d. X 25-cm length), in which a vertically dividing plate was attached in order to reduce a resuspension effect of sulfur particles by influent flow. Then the old medium, after removing sulfur particles, was sent as a cross-flow to the metal filter (stainless steel 316, 6.4-cm 0.d. X 25-cm length) with pore size of 13 pm. The old medium was filtered at a rate of 3 mL/min with a supply of the fresh medium at the same rate, and the remaining medium was recycled to the fed-batch reactor at a flow rate of 70 mL/ min.
Results and Discussion Removal Rate of Hydrogen Sulfide. Figure 2 shows that the removal rate of hydrogen sulfide is proportional to the cell concentration shown as protein concentration. The experiments were performed with the stepwise increases of feed rate and illuminance according to an optimal operational condition (Kim et al., 1990) which did not accumulate sulfide or sulfate. Sulfate could be generated by eq 2 if the available light intensity was higher than the optimal condition or hydrogen sulfide was fed insufficiently. If the light intensity is not sufficient or hydrogen sulfide gas is supplied excessively, sulfide is accumulated to cause substrate inhibition. The inhibition occurred at 6.7 mM sulfide in a stirred-batch reactor with a sulfide source of NazS.9H20. The H2S removal rates at 10-15 h of the reactor operation were chosen for comparison. This was a time when the bacterial activities did not decrease considerably and the sulfur accumulation maintained a growth-associatedpattern of production with a yield of 0.3-0.7 g of sulfur/g of dry weight of cells. Light intensity was increased stepwise with the cell growth in order to compensate the illuminance decrease due to the turbidity increase. The cells contributed 10times as much to the total turbidity of the reactor medium as the sulfur of the same concentration did (Kim, 1991). The H2S removal rates per unit concentration of protein, shown as the slopes in Figure 2, were 0.106, 0.136, and 0.205 pmol of H2S min-' (mg of protein)-' L-l for the freecell reactors of 2 and 4 L and the sulfur-settling recycle reactor of 4 L, respectively, which were determined during 10-15 h (Figure 2). The 4-L fed-batch reactor with the free cells had a removal rate of only 1.3-fold that of the 2-L reactor because light was supplied less efficiently in the 4-L reactor. The previous study shows that light attenuation in the reactor was attributed to light absorption by the cells and light scattering by the excreted sulfur from the cells (Kim et al., 1991). The H2S removal rate per unit concentration of protein in the 2-L immobilized-cell reactor was 0.259 pmol min-l (mg of protein)-' L-',which was improved 2.4 times over 0.106 pmol min-' (mg of protein)-' L-l of the free-cell 2-L reactor, if the grown cells in the loaded beads of 1.2 L were assumed to be suspended in the reactor solution as in the free-cell reactor.
CONCENTRATION OF PROTEIN(mg/L)
Figure 2. Comparison of HzS removal rates in 2- and 4-Lfreecell reactors and 4-Lfree-cell membrane recycle reactor with
sulfur settler.
In the settling recycle, sulfur particles aggregated, settled, and were removed in the settler. Thus the light attenuation due to the scattering of sulfur particles decreased considerably. About 80-90 96 of the sulfur particles generated in the fed-batch reactor could be removed by the settler when the recycle rate was 33 mL/ min and the average residence time in the settler was about 9 min. Sulfur Recovery. The sulfur concentrations with time for the three reactor systems are shown in Figure 3. The feed rate was 20 pmol of HzS/min and the average light intensity was 15 000 lux in the free-cell reactor where the initial cell concentration was 308 mg of protein/L. The feed rate was 67 pmol of HzS/min and then increased to 88 pmol of HaS/min with light intensity of 15 000 lux in the immobilized-cell reactor. The cell concentration in the beads increased from an initial loading of 600 mg of protein/L to 2200 mg of protein/L at 236 h. In the recycle reactor the feed rate was increased stepwise from 77 to 88 (in 5 h), 128 (22 h), and 171 (32 h) pmol of HzS/min and the average light intensity was increased from 13 000 to 22 000 (6 h) and 30 000 (52 h) lux. A relatively lower concentration of elementary sulfur was maintained in the immobilized-cell reactor and the settling recycle reactor than in the free-cell reactor. Excreted sulfur by the C. thiosulfatophilum cells had an orthorhombiccrystal shape (Truepper, 1967)and tended to agglomerate. In contrast to the sulfur particles removed from the settler in the recycle reactor, those in the immobilized-cell reactor accumulated in the matrices of the strontium alginate beads. The maintenance of low sulfur concentration in the reactor broth enhanced the light availability in the reactor broth and the removal rate of hydrogen sulfide. In the settling recycle, 80-90% of sulfur particles in the reactor broth were recovered from the settler and these can be used as a raw material in the fertilizer industry (Cork, 1985). Most of the physicochemicalmethods in sulfur recoveries produce thiosulfate and sulfate requiring additional treatments instead of elementary sulfur, which is valued at about U.S. $300/sulfur ton in Korea in 1991. In the chemical oxidation of hydrogen sulfide with oxidizing agents such as KMn04, Cl2, and HzO2, the pH should be below 7.5 and NaSH should be recovered immediately so that the formation of sulfate can be suppressed. The Claus process can convert 95 % of hydrogen sulfide to elementary sulfur in the presence of Fe203. However, the equipment investment is very high and additional treatment of the tail gas is necessary. Sulfur Accumulation in the Bead Matrices of StrontiumAlginate. The immobilization of whole cells
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d iai
,
Figure 3. Sulfur concentrations in the reactor broths for freecell, free-cell membrane recycle with sulfur settler, and immobilized-cell reactors.
in the strontium alginate beads improvedthe H2S removal rate owing to better light availability as well as the enhancements of cell viability and stability. When both reactors were illuminated by 200-W incandescent light bulbs with an efficacy of 20 W/(m24ux) and the H2S removal rate was 2 mM/(L*h),the light energyrequirement was 600 W/m2 in the immobilized cell reactor, a 30% reduction in comparison with 850 W/m2 in the free cell reactor. Microscopic analyses were made to observe the accumulation pattern of elementary sulfur. Figure 4 (top) shows a piece of the strontium alginate bead that shrank from 3.5 mm in diameter to 2.1 mm during the staining and dehydration. The reactor was operated for 213 h with an initial cell loading of 600 mg of protein/L. The average final cell and sulfur concentrations were 2200 mg of protein/L of beads and 26 900 mg of sulfur/L of beads when the beads were dissolved in the dibasic phosphate solution. Since elemental sulfur, converted to alkaline compounds in NaOH solution, acted as the interfering substance with the Coomassie Brilliant Blue dye a t 595 nm, an increment of optical absorbance of 0.1 OD unit (100 mg of sulfur)-l L-l was subtracted from the total absorbance. The average accumulation rate of sulfur in the beads was 2.2 mg/(L*min). The sulfur accumulated in the outer region of the bead appeared denser than that in the inner region. An interface where sulfur particles existed very sparsely formed a t a distance of 0.4 mm from the bead surface, which corresponded to 38% of the bead radius and also shows that 80 90 of the bead volume was utilized for the H2S removal. The size of sulfur aggregated in the outer region was about 10 pm (Figure 4, middle). A t a 2000 fold magnification of Figure 4 (middle), the sulfur aggregation looked like a crystal grown to an averagesizeof 10pm (Figure4, bottom), similar to an average size of 9.4 pm with a Gaussian-like distribution ranging from 1to 17 jtm in the free cell reactor. Rod-shaped bacteria with widths of 0.7-1.1 pm and lengths of 0.9-1.5 pm in the form of free cells (Buchnan and Gibbons, 1974) were seen with the sulfur aggregates. Qualitative analyses of chemical elementsby the energydispersive X-ray spectrometer in the regions corresponding to the regions of A and C in Figure 4 (top) are shown in Figure 5, top and bottom, respectively. The peak area correspondingto every nuclide existing in a sample target can be compared relatively in order to investigate the sulfur accumulation in the bead. Sulfur, labeled S, that was excreted from the cells and aggregated in the bead and strontium, labeled U, that made cross-links with alginate are shown as peaks in the spectrum. Low peaks of strontium, labeled U, occurred due to stepwise electron
6
Figure 4. (Top) Electron micrographof the central cross section of a strontium alginate bead immobilized with C. thiosulfatophilum cells after 7-day operation (X21). The diameter of the spherical bead shrank from 3.5 to 2.1 mm. (Middle) A magnified view of the outer region (A) of the bead (X140). Dense aggregates of sulfur with a size of about 10 pm were shown. (Bottom) A magnified view of sulfur aggregates and cells in the outer region of the bead (X1400). Rod-shaped bacteria with widths of 0.7-1.1 pm and lengthsof 0.+1.5pm wereshownamongsulfur aggregates.
losses in the orbitals 4s4p4d and 5s5p5d in the outer subshells by the electronic beam energy. The ratio of S to Sr was much higher in the outer region of the bead (Figure 5,top) than in the inner region of the bead (Figure 5, bottom). This was because the light availability for the photosynthesis was better in the outer region, and furthermore excreted sulfur in the outer region made the mass transfer of H2S and the light transmission into the inner region worse.
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__ _ _ _ _ _ _ ~ _ _ _ _ I
I
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il
..inlet ... .for...free . . .cell .....................................................
inlet for immobilized
20 I
o
I
1
1
40
20
60
ao
100
120
1
140
1
1
160
180 200
T I M E (HRS)
Figure 6. H2Sremoval rates in 2-Lfed-batch reactors with free
cells (X) and immobilized cells (A)in strontium alginate beads. (--) Feed rates of H2S for each case. Initial cell loading was 310 mg of protein/L of medium in the free-cell reactor, and initial bead volume was 0.8 L with cell loading of 600 mg of protein/L of beads in the immobilized reactor.
t Figure 5. (Top)Qualitative analysisof A region (Figure 4, top) by the energy-dispersive X-ray spectrometer. Note S- and
U-labeled peaks stand for sulfur and strontium, respectively. Full scales of x - and y-axes are the energy level of 7.96 keV and count number of 1500. (Bottom)Qualitative analysisof C region (Figure4,top) by the energy-dispersiveX-ray spectrometer. Full scales of x - and y-axes are the energy level of 8.44keV and count number of 500.
Variation of Removal Rate of Hydrogen Sulfide with Time. Even if the removal rates of H2S indicated an identical fashion (Figure 6), the cell concentrations were different. The initial cell loadings in the free-cell reactor and the immobilized one with 0.8-L beads were 310 mg of protein/L of medium and 600 mg of protein/L of beads. Also, the averagespecificgrowth rates were 0.006 and 0.04 h-l in the free-cell reactor medium and in the immobilized beads. The immobilization has advantages in maintaining higher cell concentration and activity and in improving light transmission in the reactor medium over free-cell systems. The viability and stability of immobilized cells could be kept longer than those of free cells. Cell growth and sulfur accumulation in the beads made the reactor medium remain clear for better light transmission to maintain a higher H2S removal rate than the free-cell reactor. The removal rate in the reactor was 2.4 times that in the free-cell reactor, as explained beforehand. However, sulfur recovery from the beads is almost impossible. If dissolving agents such as dibasic phosphate are used to recover the sulfur from the beads, sulfur particles react with the agents to make oxidized sulfur compounds instead of elementary sulfur. Also, dissolved alginate is difficult to reuse. Effect of Continuous Filtering of Toxic Metabolites in Settling Recycle Reactor. Sulfur recovery was done with the settler in the 4-L free-cell recycle reactor, and toxic metabolites accumulated in the reactor which may inhibit the cell growth were filtered by the metal filter (stainless steel 316, 6.4-cm 0.d. X 25-cm length) with a pore size of 13 pm. The light transmission could be
20
40
60
80
100
120
140
TIME (HRS)
Figure 7. H2S removal rate (A)in membrane recycle reactor with sulfur settler. (-) Feed rate of H2S.
improved considerably by removing 80-90 % of the sulfur in the reactor from the settler. The H2S removal rate per unit concentration of protein increased to 3 pmol of H2S h-l (mg of protein)-l L-l, 50% higher than that of the free-cell reactor without the settler and the filter. In contrast to Figure 6, Figure 7 shows that the removal rate of hydrogen sulfide did not decrease with time when the H2S feed rate increased stepwise from 83 to 110 (50 h) to 154 (70 h) pmollmin because toxic accumulations in the reactor were continuously filtered out at a rate of 3 mL/min by supplying fresh medium at the same rate. When the medium was recycled at a rate of 70 mL/min from the settler and the filtration rate was 3 mL/min, after 40 h a semisteady cell concentration of 800 mg of protein/L was maintained by discharging 10% of the cells continuously in the filtrate. A semisteady state of removal rate could be also obtained after 70 h when the feed rate of hydrogen sulfide was 154 pmol/min. Figure 8 shows that the relative activity of the cells did not decreasein the filtration recycle reactor with the settler, unlike in the free-cell reactor. This result clearly shows a possibility of a steady-state operation in the photosynthetic reactor.
Conclusions (1)The fed-batch system with the free cells did not provide long-term operation because of the light attenuation by the increasing number of cells and sulfur particles. Also, the light transmission was affected by the
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Buisman, C. J. N.; Geraats, B. G.; Ijspeert, P.; Lettinga, G. Optimization of sulphur production in a biotechnologicalsulphide-removingreactor. Biotechnol. Bioeng. 1990,35,5+56. Cadena, F.; Peters, R. W. Evaluation of chemical oxidizers for hydrogen sulfidecontrol. J.-WaterPollut. Control Fed. 1988, 60,1259-1263.
0.2
1 0
I
‘9 40
80
120
180
200
240
T I ME (HRS)
Figure S. Comparison of relative activities between free cell fed-batch reactor ( 0 )and membrane recycle reactor with sulfur settler ( 0 ) .Reference activities per unit protein concentration based on Figure 2 were 2.0 pmol/(mg of protein-h) for the former and 3.0pmol (mgof protein-h)for the latter. The reactor volume was 4 L for 0th cases.
b
reactor configuration since it decreased exponentially with the distance from a light source and the total turbidity of the reactor solution. As a result, H2S removal performance per unit concentration of protein increased only 28%, not in proportion to the reactor volume in the 4-L reactor in comparison with the 2-L reactor. (2) The immobilized-cell system was better than the free-cell system since it kept the cells and the sulfur particles in the beads. This reactor required 30% less light energy and the volumetric removal rate was 2.4 times than that with the free-cell reactor. The analyses by the scanning electron micrograph and the energy-dispersive X-ray spectrometer showed that the sulfur deposition was limited to the region 0.4 mm from the bead surface. The accumulation of sulfur and cells in the beads made the system effective only for a limited period. (3) On the other hand, the continuous system with the removal of sulfur and cells from the reactor achieved a semisteady state operation of H2S removal. Also, the removal efficiency was up to 50% higher than that of the free-cell system. Thus, it can be concluded that the continuous system showed a prospect of long-term operation of hydrogen sulfide removal.
Acknowledgment We express our thanks to Mr. J. M. Kim in the Lucky Central Research Institute for the preparation of the SEM and the EDS and Mr. I. K. Kim in the Korea Atomic Energy Research Institute for his helpful comments. Literature Cited Berry, R. I. Treating hydrogensulfide: when Claus is not enough. Chem. Eng. 1980 (October), 92-93. Bradford, M. M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein dye binding. Anal. Biochem. 1976, 72, 248-254.
Buchnan, R. E., Gibbons, N. E., Eds. Bergey’s Manual of Determinative Bacteriology, 7th ed.; Williams and Wilkins Co.: Baltimore, MD, 1974; pp 52-54.
Cork, D. J. Microbial conversion of sulfate to sulfur-An alternative to gypsum synthesis. In Advances in Biotechnological Processes; Mizrahi, A., van Werel, A. L., Eds.; Alan R. Liss, Inc.: New York, 1985; Vol. 4, pp 183-209. Cork, D. J.; Ma, S. Acid-gas bioconversion favors sulfur production. Biotechnol. Bioeng. Symp. 1982, 12, 285-290. Cork, D. J.; Cusanovich, M. A. Sulfate decomposition: A microbiological process. In Metallurgical applications of bacterial leaching and related microbiological phenomena;
Murr, L. E., Torma,A. E., Brierley,J. A,, Eds.;AcademicPress, New York, 1978; pp 207-221. Cork, D. J.; Garunas, R.; Sajjad, A. Chlorobium limicola forma thiosulfatophilum: Biocatalyst in the production of sulfur and organic carbon from a gas stream containing H2S and C02. Appl. Environ. Microbiol. 1983, 45, 913-918.
Fuchs, G.; Stupperich,E.; Jaenchen, R. Autotrophic COz fixation in the Chlorobium limicola. Evidence against the operation of the Calvin cycle in growing cells. Arch. Microbiol. 1980, 128,5643.
Greenberg, A. E., Connors, J. J., Jenkins, D., Eds. Standard Method for the Examination of Water and Wastewater;
American Public Health Association: Washington, DC, 1981; pp 439-440. Kim, B. W. Kinetics of H2S oxidation by the Chlorobium thiosulfutophilumin photosyntheticreactors. Ph.D. Dissertation, Korea Advanced Institute of Science and Technology, -. Seoul, Korea, 1991. Kim, B. W.; Kim, I. K.; Chang, H. N. Bioconversion of hydrogen sulfide by free and immobilized cells of Chlorobium thiosulfatophilum. Biotechnol. Lett. 1990, 12, 381-386.
Kim. B. W.: Chane. H. N.: Kim. I. K.: Lee. K. S. Growth kinetics of photosynthetic green sulfur bacteria in fed batch reactor. Biotechnol. Bioeng. 1991, submitted for publication. Koch, H. P. Absorption spectra and structure of organic sulfur compounds. J. Chem. SOC.1949, 394-401. Larsen, H. On the culture and general physiology of the green sulfur bacteria. J . Bacteriol. 1952, 64, 187-196. Lee, H. Y.; Erickson, L. E.; Yang, S. S. Kinetics and bioenergetics of light limited photoautotrophic growth of Spirulina platenis. Biotechnol. Bioeng. 1987, 29, 832-843.
Loran, 0. H.; O’Hara,J. B. A clean coal conversion technology. Environ. Sci. Technol. 1978, 12, 1258-1263.
Price, E. C.; Cheremisinoff,P. N. Sewagetreatment plants combat odor pollutionproblems. WaterSewage Works 1978 (October), 64-69.
Sirevag, R.; Ormerod, J. G. Synthesis, storage and degradation of polyglucose in Chlorobium thiosulfatophilum. Arch. Microbiol. 1977, 111, 239-244.
Sublette, K. L. An experimentin autotrophic fermentation.Chem. Eng. Educ. 1989 (Winter), 32-37. Sublette, K. L.; Sylvester, N. D. Oxidation of hydrogen sulfide by Thiobacillus denitrificans: Desulfurization of natural gas. Biotechnol. Bioeng. 1987,29, 249-254.
Trueper, H. G.; Hathway, J. C. Orthorhombic sulfur formed by photosynthetic sulfur bacteria. Nature 1967, 215, 435-436. Wilhelm,E.;Battino, R.; Wilcock,J. Low-PressureSolubility of Gases in Liquid Water. Chem. Rev. 1977, 77, 219-262. Accepted September 10,1991. Registry No. SHz, 7783-06-4; S, 7704-34-9.
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Removal of Hydrogen Sulfide by Chlorobium thiosulfatophilum in Immobilized-Celland Sulfur-Settling Free-Cell Recycle Reactors Byung Woo Kim and Ho Nam Chang' Department of Chemical Engineering and Bioprocess Engineering Research Center, Korea Advanced Institute of Science and Technology, Daeduk Science Town, Taejon 305-701, Korea
Bioconversion of hydrogen sulfide to elementary sulfur by the photosynthetic bacterium Chlorobium thiosulfutophilum was studied in immobilized-cell and sulfur-settling freecell recycle reactors. The cells immobilized in strontium alginate beads excreted elementary sulfur and accumulated it as crystal in the bead matrices, which made it possible that the reactor broth remained clear and the light penetrated the reactor deeper than with the free cells. In comparison with the free cells, the immobilized cells required 30% less light energy at a H2S removal rate of 2 mM/(L.h) and showed an activity of 2.4 times that of the free cells. However, in 40 h after the reaction the deterioration of the H2S removal efficiency became significant due to the accumulation of sulfur in the beads. The scanning electron micrograph (SEM)and energy-dispersive X-ray spectrometer (EDS)studies showed that the sulfur in the beads existed within a layer of 0.4 mm from the bead surface. In the sulfur-settling free-cell recycle reactor, about 80% of the sulfur excreted by the free cells could be removed in a settler. The 4-L fed batch reactor with the settler improved the light transmission to result in a H2S removal rate of 3 pmol/ (mg of proteinoh), 50% higher than that without it. The settling recycle reactor was much better in the removal of H2S than the immobilized-cell reactor because the former was a continuous system with the constant removal of sulfur particles by settling and of spent medium by supplying fresh medium a t the same rate as the filtering rate of the reactor broth, while the latter was essentially a batch system where toxic metabolites and produced sulfur could not be removed.
Introduction The toxicity and corrosiveness of hydrogen sulfide gas has made it necessary that its release into environments be strictly regulated. The human threshold exposure limit to H2S is 10ppm for 7-8-h periods (Buchman and Gibbons, 1974), and humans recognize H2S odor associated with anaerobic sewage decomposition in concentrations as low as 0.0005 ppm (Price and Cheremisinoff, 1978). The discharge limit is set at less than 5 ppm in most countries. Recoveriesof sulfur up to 97 % have been achieved using multiple-stage treatment in the traditional Claus plant. However, the current air pollution regulations require a better than 99% recovery, which has forced industries to add an additional system to clean Claus tail gas (Berry, 1980). Several physicochemical methods are available for treating the tail gas from a sulfur recovery plant (Cadena and Peters, 1988;Loran and O'Hara, 1978). But in addition to high capital and operational costs, incineration of hydrogen sulfide of 200-500 ppm carried over from the preceding stage produces sulfur dioxide in the Clam process, also a key atmospheric pollutant. Sulfate or thiosulfate is produced in the Stretford process (Cork and Ma, 1982), which should be treated further with calcium chloride. The byproduct gypsum also causes a problem in disposal. Biological oxidations were also considered as alternatives to the physicochemical methods (Buisman et al., 1990; Cork et al., 1983; Sublette and Sylvester, 1987). Buisman et al. (1990) cultured aerobic Thiobacillus in a Na2S broth, but this required deliberate oxygen control to suppress the accumulation of sulfate in the reactor medium. Sub-
* To whom correspondence should be addressed. 8756-7938/91/3007-0495$02.50/0
lette and Sylvester (1989) adopted facultative Thiobacillus denitrificans not requiring oxygen control and demonstrated a stable operation at a reactor loading of as high as 4-5 mmol of H2S h-l (g of cells)-l. But in a 1.4-L reactor the sulfate accumulated up to 24 mM while no elementary sulfur was produced during the initial 17 h. It is desirable to recover sulfur as much as possible, rather than as sulfate that requires another wastewater treatment process. The problem of biooxidation of H2S is light transmission into the medium (Sublette, 1987). As the reaction proceeds, the number of cells and sulfur increases to such a level that the light transmission is no longer effective. Thus carrying out the reaction with free cells in suspension will not be effective soon because of poor transmission of light due to the cells and sulfur particles. Kim et al. (1990) immobilizedwhole cells of Chlorobium thiosulfatophilum to obtain 30% light energy saving in comparison with a free cell reactor. However, the accumulation of sulfur in the beads results in intraparticle mass transfer resistance, which eventually deteriorates the H2S removal capability. In this study we intend to compare the H2S removal performances of free-cell suspensions and immobilizedcell and free-cell reactors with sulfur settling. Especially for the immobilizedcells, the scanning electron micrograph (SEM) and the energy-dispersive X-ray spectrometer (EDS) were used to study sulfur deposition in the gelbead matrices.
Materials and Methods Bacterial Strain, Media, and Growing Conditions. The bacterial strain used was Chlorobium limicola forma
0 1991 American Chemical Society and American Institute of Chemical Engineers
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thiosulfatophilum (ATCC 17092), commonly known as Chlorobium thiosulfatophilum. The medium consisted of KH2P04 (0.74 g/L), NHdC1 (0.74 g/L), M g S 0 ~ 7 H z 0 (1.8 g/L), CaCl2.2H20 (0.05 g/L), NaCl (7.4 g/L), B12 (0.0002 mg/L), and 100 mL of 0.05 M NaH2P04 and 200 mL of 0.05 M Na2HP04 per liter of medium, including 1 mL of trace solution where Na2 EDTA (5.2 g), FeC1~4H20 (1500 mg), ZnCl2 (70 mg), MnC1~4Hz0(100 mg), H3B03 (62 mg), CoC1~6H20(190 mg), CuCb2H20 (17 mg), NiClr6H20 (24 mg), and Na2Mo0~2H20(36 mg) were dissolved in a l-L solution of distilled water in the order given. NazS.9H20 (0.1 g/L of reactor solution) was added at the start to ensure an anaerobic environment by removing dissolved oxygen in the reactor, which was as low as possible so as not to cause inhibition by sulfide (Larsen, 1952).The feed gas mixture consisted of H2S (4.16 mol % ), CO2 (9.34 mol 5% ), Nz (86.01 mol % ), and H2 (0.49 mol % ), equivalent to 46 385 ppm of H2S in the state of gas mixtures. The solubilities of HzS in water are 92.1 mM at 30 "C and 83.2 mM at 40 OC (Wilhelmet al., 1977). The pH of the medium was adjusted to 6.8 with 0.1 N NaOH or HC1. Sterilization of the medium and the feed gas was not necessary because of the inhibition of H2S to other microorganisms. Assays. Elementary sulfur produced during the photosynthesis was excreted by the cells and suspended as particles in the reactor broth. Sulfur removal from the sampled solution was necessary for determining the dry or wet weight of the cells. Since this was a difficult task, we measured protein concentrations for the cell density determination. An optical absorbance of 0.1 at 578 nm by spectrophotometer corresponded to 0.1 g of dry cell/L without sulfur (Fuchs et al., 1980). The correlation of the cell protein with the dry weight showed that protein was about 24% of the dry cell weight. It was shown that significant amounts of synthesized carbon were stored in the cells as polyglucose and chlorophyll (Sirevag and Ormerod, 1977). Protein was determined by Bradford's CBB (Coomassie Brilliant Blue; Bradford, 1976) method. A sample of 10mL volume taken from the reactors was centrifuged at 18000g,4 "C, and then the recentrifuged precipitate, after the extraction of chlorophyll and sulfur (Cork and Cusanovich, 1978), was boiled with 10-20 mL of 1N NaOH in a water bath. The CBB solution was added and the absorbance was read at 595 nm. Protein concentration was determined from a standard curve prepared with bovine serum albumin. To analyze total sulfide concentration, 1 or 2 drops of 2 N zinc acetate and 0.2-0.6 mL of 6 N HCl were added to the supernatant after centrifugation. After it was mixed with 6 mL of 0.025 N iodine solution (20-25 g of KI in 10 mL of water, followed by 3.2 g of iodine, and water to a final volume of 1L) and 1or 2 drops of 2 % starch solution, titration was done with 0.025 N Na2S203 solution (Greenberg et al., 1981). Sulfate concentration in the supernatant was determined turbidimetrically at 420 nm from a standard curve prepared with sodium sulfate solution. Sulfur in the precipitate was mixed with acetone, and then residual water was evaporated with acetone in water bath. After evaporation, dried sulfur was dissolved in chloroform to determine its concentration with a spectrophotometer at 290 nm from a standard curve (Koch, 1949; Cork and Cusanovich, 1978). Immobilization. Cells to be immobilized were harvested as concentrated paste and stirred into a 1.5%
h Figure 1. Settling recycle setup with filtration of toxic accumulations in reactor broth. Settler, metal filter, and supply of fresh medium were not placed in an immobilized-cell reactor.
solution of sodium alginate (pH 6.8 in the medium with Tris-HC1 buffer). The cell alginate suspension was then extruded dropwise by a syringe with a needle of 0.2 mm (i.d.) into a bath containing a solution of 0.1 M strontium chloride at 10"C. One day was allowed for complete gelling of strontium alginate beads. The beads were washed 2-3 times with distilled water, and then loaded in the fedbatch reactor. Microscopic Techniques. For the microscopic examination of accumulation patterns of sulfur excreted extracellularly, the cross sections of the strontium alginate beads were prepared. The segments of the sectioned beads were fixed for 1 day using 2% glutaraldehyde and 1% paraformaldehyde solution in a 0.1 M Tris-HC1 buffer (pH 6.8) and postfixed for 1 h in an aqueous solution of 1%uranyl acetate. The stained sections were freeze-dried for 1day, and remaining water was dehydrated further in a vacuum drier for 1day. Following the dehydration, the gold-coatedspecimen was examined in a scanning electron microscope (Model 840-A, Jeol Co.) with an accelerating voltage of 20 kV. In addition, nuclide analysis of the coated specimens was done with the energy-dispersive X-ray spectrometer (Model 10 000, Link System Co.). Reactor System and Operation. Two fed-batch reactors of 2- and 4-L capacity (SY-250 and SY-500, Korea Fermentor Co., Inchon, Korea) were used. From a standard gas cylinder, hydrogen sulfide and carbon dioxide were fed into the reactor through a gas distributor with the feed rate control by a two-stage gas regulator and a flow meter. Medium was stirred with two disc-turbine type impellers at 500 rpm. The pH and temperature of the medium were maintained at 6.8-6.9 and 30 "C by controllers, respectively. Two sets of zinc acetate traps were placed in an effluent line to scrub hydrogen sulfide gas, and a wet gas meter was used to measure the volumetric flow rate. The average light intensity (Lee et al., 1987) from two incandescent light bulbs was determined with a lux meter (IM-2D, Topcon Co., Tokyo, Japan). Figure 1shows the schematic diagram of the reactor system with the sulfur settler made of Pyrex glass (4.5-cm i.d. X 25-cm length). Cells were grown up on the described medium in which gas mixtures were fed and dissolved as electron and carbon sources for the photosynthesis and then harvested by centrifugation. Usually initial cell loadings were about 0.5-0.8 g of dry cells/L. The feed rate in the 2-L reactor was increased stepwise with the cell growth, corresponding to about 0.106 pmol of HzS/min-' (mg of protein)-' L-l, which was the H2S removal rate expected for the C. thiosulfatophilum cells. If light energy is transferred to P840, a reaction center in bacteriochlorophyll, P840 is activated to accept electrons from H2S oxidation (eq 1). If more light is available than necessary for eq 1, elementary sulfur is oxidized further
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to sulfate (eq 2). The optimal conditions of operation
+
-2s' + + -
2H2S CO, H2S
+ 2C0,
2H20
(CH20) + H 2 0
(1)
2(CH20) + H2S0,
(2)
were established to minimize sulfate production as well as sulfide accumulation by adjusting the light intensity and the feed rate. In the case of the settling recycle reactor, produced sulfur particles were removed from the settler (Pyrex glass, 4.5cm i.d. X 25-cm length), in which a vertically dividing plate was attached in order to reduce a resuspension effect of sulfur particles by influent flow. Then the old medium, after removing sulfur particles, was sent as a cross-flow to the metal filter (stainless steel 316, 6.4-cm 0.d. X 25-cm length) with pore size of 13 pm. The old medium was filtered at a rate of 3 mL/min with a supply of the fresh medium at the same rate, and the remaining medium was recycled to the fed-batch reactor at a flow rate of 70 mL/ min.
Results and Discussion Removal Rate of Hydrogen Sulfide. Figure 2 shows that the removal rate of hydrogen sulfide is proportional to the cell concentration shown as protein concentration. The experiments were performed with the stepwise increases of feed rate and illuminance according to an optimal operational condition (Kim et al., 1990) which did not accumulate sulfide or sulfate. Sulfate could be generated by eq 2 if the available light intensity was higher than the optimal condition or hydrogen sulfide was fed insufficiently. If the light intensity is not sufficient or hydrogen sulfide gas is supplied excessively, sulfide is accumulated to cause substrate inhibition. The inhibition occurred at 6.7 mM sulfide in a stirred-batch reactor with a sulfide source of NazS.9H20. The H2S removal rates at 10-15 h of the reactor operation were chosen for comparison. This was a time when the bacterial activities did not decrease considerably and the sulfur accumulation maintained a growth-associatedpattern of production with a yield of 0.3-0.7 g of sulfur/g of dry weight of cells. Light intensity was increased stepwise with the cell growth in order to compensate the illuminance decrease due to the turbidity increase. The cells contributed 10times as much to the total turbidity of the reactor medium as the sulfur of the same concentration did (Kim, 1991). The H2S removal rates per unit concentration of protein, shown as the slopes in Figure 2, were 0.106, 0.136, and 0.205 pmol of H2S min-' (mg of protein)-' L-l for the freecell reactors of 2 and 4 L and the sulfur-settling recycle reactor of 4 L, respectively, which were determined during 10-15 h (Figure 2). The 4-L fed-batch reactor with the free cells had a removal rate of only 1.3-fold that of the 2-L reactor because light was supplied less efficiently in the 4-L reactor. The previous study shows that light attenuation in the reactor was attributed to light absorption by the cells and light scattering by the excreted sulfur from the cells (Kim et al., 1991). The H2S removal rate per unit concentration of protein in the 2-L immobilized-cell reactor was 0.259 pmol min-l (mg of protein)-' L-',which was improved 2.4 times over 0.106 pmol min-' (mg of protein)-' L-l of the free-cell 2-L reactor, if the grown cells in the loaded beads of 1.2 L were assumed to be suspended in the reactor solution as in the free-cell reactor.
CONCENTRATION OF PROTEIN(mg/L)
Figure 2. Comparison of HzS removal rates in 2- and 4-Lfreecell reactors and 4-Lfree-cell membrane recycle reactor with
sulfur settler.
In the settling recycle, sulfur particles aggregated, settled, and were removed in the settler. Thus the light attenuation due to the scattering of sulfur particles decreased considerably. About 80-90 96 of the sulfur particles generated in the fed-batch reactor could be removed by the settler when the recycle rate was 33 mL/ min and the average residence time in the settler was about 9 min. Sulfur Recovery. The sulfur concentrations with time for the three reactor systems are shown in Figure 3. The feed rate was 20 pmol of HzS/min and the average light intensity was 15 000 lux in the free-cell reactor where the initial cell concentration was 308 mg of protein/L. The feed rate was 67 pmol of HzS/min and then increased to 88 pmol of HaS/min with light intensity of 15 000 lux in the immobilized-cell reactor. The cell concentration in the beads increased from an initial loading of 600 mg of protein/L to 2200 mg of protein/L at 236 h. In the recycle reactor the feed rate was increased stepwise from 77 to 88 (in 5 h), 128 (22 h), and 171 (32 h) pmol of HzS/min and the average light intensity was increased from 13 000 to 22 000 (6 h) and 30 000 (52 h) lux. A relatively lower concentration of elementary sulfur was maintained in the immobilized-cell reactor and the settling recycle reactor than in the free-cell reactor. Excreted sulfur by the C. thiosulfatophilum cells had an orthorhombiccrystal shape (Truepper, 1967)and tended to agglomerate. In contrast to the sulfur particles removed from the settler in the recycle reactor, those in the immobilized-cell reactor accumulated in the matrices of the strontium alginate beads. The maintenance of low sulfur concentration in the reactor broth enhanced the light availability in the reactor broth and the removal rate of hydrogen sulfide. In the settling recycle, 80-90% of sulfur particles in the reactor broth were recovered from the settler and these can be used as a raw material in the fertilizer industry (Cork, 1985). Most of the physicochemicalmethods in sulfur recoveries produce thiosulfate and sulfate requiring additional treatments instead of elementary sulfur, which is valued at about U.S. $300/sulfur ton in Korea in 1991. In the chemical oxidation of hydrogen sulfide with oxidizing agents such as KMn04, Cl2, and HzO2, the pH should be below 7.5 and NaSH should be recovered immediately so that the formation of sulfate can be suppressed. The Claus process can convert 95 % of hydrogen sulfide to elementary sulfur in the presence of Fe203. However, the equipment investment is very high and additional treatment of the tail gas is necessary. Sulfur Accumulation in the Bead Matrices of StrontiumAlginate. The immobilization of whole cells
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d iai
,
Figure 3. Sulfur concentrations in the reactor broths for freecell, free-cell membrane recycle with sulfur settler, and immobilized-cell reactors.
in the strontium alginate beads improvedthe H2S removal rate owing to better light availability as well as the enhancements of cell viability and stability. When both reactors were illuminated by 200-W incandescent light bulbs with an efficacy of 20 W/(m24ux) and the H2S removal rate was 2 mM/(L*h),the light energyrequirement was 600 W/m2 in the immobilized cell reactor, a 30% reduction in comparison with 850 W/m2 in the free cell reactor. Microscopic analyses were made to observe the accumulation pattern of elementary sulfur. Figure 4 (top) shows a piece of the strontium alginate bead that shrank from 3.5 mm in diameter to 2.1 mm during the staining and dehydration. The reactor was operated for 213 h with an initial cell loading of 600 mg of protein/L. The average final cell and sulfur concentrations were 2200 mg of protein/L of beads and 26 900 mg of sulfur/L of beads when the beads were dissolved in the dibasic phosphate solution. Since elemental sulfur, converted to alkaline compounds in NaOH solution, acted as the interfering substance with the Coomassie Brilliant Blue dye a t 595 nm, an increment of optical absorbance of 0.1 OD unit (100 mg of sulfur)-l L-l was subtracted from the total absorbance. The average accumulation rate of sulfur in the beads was 2.2 mg/(L*min). The sulfur accumulated in the outer region of the bead appeared denser than that in the inner region. An interface where sulfur particles existed very sparsely formed a t a distance of 0.4 mm from the bead surface, which corresponded to 38% of the bead radius and also shows that 80 90 of the bead volume was utilized for the H2S removal. The size of sulfur aggregated in the outer region was about 10 pm (Figure 4, middle). A t a 2000 fold magnification of Figure 4 (middle), the sulfur aggregation looked like a crystal grown to an averagesizeof 10pm (Figure4, bottom), similar to an average size of 9.4 pm with a Gaussian-like distribution ranging from 1to 17 jtm in the free cell reactor. Rod-shaped bacteria with widths of 0.7-1.1 pm and lengths of 0.9-1.5 pm in the form of free cells (Buchnan and Gibbons, 1974) were seen with the sulfur aggregates. Qualitative analyses of chemical elementsby the energydispersive X-ray spectrometer in the regions corresponding to the regions of A and C in Figure 4 (top) are shown in Figure 5, top and bottom, respectively. The peak area correspondingto every nuclide existing in a sample target can be compared relatively in order to investigate the sulfur accumulation in the bead. Sulfur, labeled S, that was excreted from the cells and aggregated in the bead and strontium, labeled U, that made cross-links with alginate are shown as peaks in the spectrum. Low peaks of strontium, labeled U, occurred due to stepwise electron
6
Figure 4. (Top) Electron micrographof the central cross section of a strontium alginate bead immobilized with C. thiosulfatophilum cells after 7-day operation (X21). The diameter of the spherical bead shrank from 3.5 to 2.1 mm. (Middle) A magnified view of the outer region (A) of the bead (X140). Dense aggregates of sulfur with a size of about 10 pm were shown. (Bottom) A magnified view of sulfur aggregates and cells in the outer region of the bead (X1400). Rod-shaped bacteria with widths of 0.7-1.1 pm and lengthsof 0.+1.5pm wereshownamongsulfur aggregates.
losses in the orbitals 4s4p4d and 5s5p5d in the outer subshells by the electronic beam energy. The ratio of S to Sr was much higher in the outer region of the bead (Figure 5,top) than in the inner region of the bead (Figure 5, bottom). This was because the light availability for the photosynthesis was better in the outer region, and furthermore excreted sulfur in the outer region made the mass transfer of H2S and the light transmission into the inner region worse.
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__
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__
__ _ _ _ _ _ _ ~ _ _ _ _ I
I
I
I
I
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il
..inlet ... .for...free . . .cell .....................................................
inlet for immobilized
20 I
o
I
1
1
40
20
60
ao
100
120
1
140
1
1
160
180 200
T I M E (HRS)
Figure 6. H2Sremoval rates in 2-Lfed-batch reactors with free
cells (X) and immobilized cells (A)in strontium alginate beads. (--) Feed rates of H2S for each case. Initial cell loading was 310 mg of protein/L of medium in the free-cell reactor, and initial bead volume was 0.8 L with cell loading of 600 mg of protein/L of beads in the immobilized reactor.
t Figure 5. (Top)Qualitative analysisof A region (Figure 4, top) by the energy-dispersive X-ray spectrometer. Note S- and
U-labeled peaks stand for sulfur and strontium, respectively. Full scales of x - and y-axes are the energy level of 7.96 keV and count number of 1500. (Bottom)Qualitative analysisof C region (Figure4,top) by the energy-dispersiveX-ray spectrometer. Full scales of x - and y-axes are the energy level of 8.44keV and count number of 500.
Variation of Removal Rate of Hydrogen Sulfide with Time. Even if the removal rates of H2S indicated an identical fashion (Figure 6), the cell concentrations were different. The initial cell loadings in the free-cell reactor and the immobilized one with 0.8-L beads were 310 mg of protein/L of medium and 600 mg of protein/L of beads. Also, the averagespecificgrowth rates were 0.006 and 0.04 h-l in the free-cell reactor medium and in the immobilized beads. The immobilization has advantages in maintaining higher cell concentration and activity and in improving light transmission in the reactor medium over free-cell systems. The viability and stability of immobilized cells could be kept longer than those of free cells. Cell growth and sulfur accumulation in the beads made the reactor medium remain clear for better light transmission to maintain a higher H2S removal rate than the free-cell reactor. The removal rate in the reactor was 2.4 times that in the free-cell reactor, as explained beforehand. However, sulfur recovery from the beads is almost impossible. If dissolving agents such as dibasic phosphate are used to recover the sulfur from the beads, sulfur particles react with the agents to make oxidized sulfur compounds instead of elementary sulfur. Also, dissolved alginate is difficult to reuse. Effect of Continuous Filtering of Toxic Metabolites in Settling Recycle Reactor. Sulfur recovery was done with the settler in the 4-L free-cell recycle reactor, and toxic metabolites accumulated in the reactor which may inhibit the cell growth were filtered by the metal filter (stainless steel 316, 6.4-cm 0.d. X 25-cm length) with a pore size of 13 pm. The light transmission could be
20
40
60
80
100
120
140
TIME (HRS)
Figure 7. H2S removal rate (A)in membrane recycle reactor with sulfur settler. (-) Feed rate of H2S.
improved considerably by removing 80-90 % of the sulfur in the reactor from the settler. The H2S removal rate per unit concentration of protein increased to 3 pmol of H2S h-l (mg of protein)-l L-l, 50% higher than that of the free-cell reactor without the settler and the filter. In contrast to Figure 6, Figure 7 shows that the removal rate of hydrogen sulfide did not decrease with time when the H2S feed rate increased stepwise from 83 to 110 (50 h) to 154 (70 h) pmollmin because toxic accumulations in the reactor were continuously filtered out at a rate of 3 mL/min by supplying fresh medium at the same rate. When the medium was recycled at a rate of 70 mL/min from the settler and the filtration rate was 3 mL/min, after 40 h a semisteady cell concentration of 800 mg of protein/L was maintained by discharging 10% of the cells continuously in the filtrate. A semisteady state of removal rate could be also obtained after 70 h when the feed rate of hydrogen sulfide was 154 pmol/min. Figure 8 shows that the relative activity of the cells did not decreasein the filtration recycle reactor with the settler, unlike in the free-cell reactor. This result clearly shows a possibility of a steady-state operation in the photosynthetic reactor.
Conclusions (1)The fed-batch system with the free cells did not provide long-term operation because of the light attenuation by the increasing number of cells and sulfur particles. Also, the light transmission was affected by the
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Buisman, C. J. N.; Geraats, B. G.; Ijspeert, P.; Lettinga, G. Optimization of sulphur production in a biotechnologicalsulphide-removingreactor. Biotechnol. Bioeng. 1990,35,5+56. Cadena, F.; Peters, R. W. Evaluation of chemical oxidizers for hydrogen sulfidecontrol. J.-WaterPollut. Control Fed. 1988, 60,1259-1263.
0.2
1 0
I
‘9 40
80
120
180
200
240
T I ME (HRS)
Figure S. Comparison of relative activities between free cell fed-batch reactor ( 0 )and membrane recycle reactor with sulfur settler ( 0 ) .Reference activities per unit protein concentration based on Figure 2 were 2.0 pmol/(mg of protein-h) for the former and 3.0pmol (mgof protein-h)for the latter. The reactor volume was 4 L for 0th cases.
b
reactor configuration since it decreased exponentially with the distance from a light source and the total turbidity of the reactor solution. As a result, H2S removal performance per unit concentration of protein increased only 28%, not in proportion to the reactor volume in the 4-L reactor in comparison with the 2-L reactor. (2) The immobilized-cell system was better than the free-cell system since it kept the cells and the sulfur particles in the beads. This reactor required 30% less light energy and the volumetric removal rate was 2.4 times than that with the free-cell reactor. The analyses by the scanning electron micrograph and the energy-dispersive X-ray spectrometer showed that the sulfur deposition was limited to the region 0.4 mm from the bead surface. The accumulation of sulfur and cells in the beads made the system effective only for a limited period. (3) On the other hand, the continuous system with the removal of sulfur and cells from the reactor achieved a semisteady state operation of H2S removal. Also, the removal efficiency was up to 50% higher than that of the free-cell system. Thus, it can be concluded that the continuous system showed a prospect of long-term operation of hydrogen sulfide removal.
Acknowledgment We express our thanks to Mr. J. M. Kim in the Lucky Central Research Institute for the preparation of the SEM and the EDS and Mr. I. K. Kim in the Korea Atomic Energy Research Institute for his helpful comments. Literature Cited Berry, R. I. Treating hydrogensulfide: when Claus is not enough. Chem. Eng. 1980 (October), 92-93. Bradford, M. M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein dye binding. Anal. Biochem. 1976, 72, 248-254.
Buchnan, R. E., Gibbons, N. E., Eds. Bergey’s Manual of Determinative Bacteriology, 7th ed.; Williams and Wilkins Co.: Baltimore, MD, 1974; pp 52-54.
Cork, D. J. Microbial conversion of sulfate to sulfur-An alternative to gypsum synthesis. In Advances in Biotechnological Processes; Mizrahi, A., van Werel, A. L., Eds.; Alan R. Liss, Inc.: New York, 1985; Vol. 4, pp 183-209. Cork, D. J.; Ma, S. Acid-gas bioconversion favors sulfur production. Biotechnol. Bioeng. Symp. 1982, 12, 285-290. Cork, D. J.; Cusanovich, M. A. Sulfate decomposition: A microbiological process. In Metallurgical applications of bacterial leaching and related microbiological phenomena;
Murr, L. E., Torma,A. E., Brierley,J. A,, Eds.;AcademicPress, New York, 1978; pp 207-221. Cork, D. J.; Garunas, R.; Sajjad, A. Chlorobium limicola forma thiosulfatophilum: Biocatalyst in the production of sulfur and organic carbon from a gas stream containing H2S and C02. Appl. Environ. Microbiol. 1983, 45, 913-918.
Fuchs, G.; Stupperich,E.; Jaenchen, R. Autotrophic COz fixation in the Chlorobium limicola. Evidence against the operation of the Calvin cycle in growing cells. Arch. Microbiol. 1980, 128,5643.
Greenberg, A. E., Connors, J. J., Jenkins, D., Eds. Standard Method for the Examination of Water and Wastewater;
American Public Health Association: Washington, DC, 1981; pp 439-440. Kim, B. W. Kinetics of H2S oxidation by the Chlorobium thiosulfutophilumin photosyntheticreactors. Ph.D. Dissertation, Korea Advanced Institute of Science and Technology, -. Seoul, Korea, 1991. Kim, B. W.; Kim, I. K.; Chang, H. N. Bioconversion of hydrogen sulfide by free and immobilized cells of Chlorobium thiosulfatophilum. Biotechnol. Lett. 1990, 12, 381-386.
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