Leaching of heavy metals from anaerobic sewage sludge by sulfur-oxidizing bacteria

Leaching of heavy metals from anaerobic sewage sludge by sulfur-oxidizing bacteria D. K. Jain and R. D. Tyagi I N R S - E a u , Universit~ du Quebec, Quebec, Canada

Two sulfur-oxidizing Thiobacillus species, T. thiooxidans ATCC 55127 and T. thioparus ATCC 55128, were isolated by enrichment from anaerobically digested sewage sludge. Under aerobic" conditions, the mixed inoculum of the Thiobacillus species decreased pH of the anaerobic sludge containing sulfur from 7.8 to 2.0 in 40 h. The pH drop was achieved in two steps by the Thiobacillus species. In the first step, T. thioparus decreased the pH to a level which favored the sulfar-oxidizing activity ofT. thiooxidans. In the second step, T. thiooxidans decreased the pH below 2.0, at which Zn and Cu were completely solubilized within 40 and 60 h, respectively.

Keywords: Heavy metals; anaerobic sewage sludge; Thiobacillus species: sulfur-oxidizingbacteria Introduction Sewage treatment plants usually generate millions of tonnes of residual sludges worldwide every year.~ The treatment and final sludge disposal costs represent 50% of the overall cost of the wastewater facility.-" The main concern in the disposal of the sludge is the presence of toxic heavy metals which concentrate during the treatment of sludges due to various physicochemical and biological interactionsf1,4 Generally, the total heavy metal content of sewage sludges is about 0.5 to 2.0% on a dry weight basis. 5 However, in some cases, extremely high concentrations (total metals concentration up to 4% w/w) of metals such as Cd, Cr, Cu, Ni, Pb, and Zn have been reported. 6 Uptake of heavy metals by plants and the subsequent accumulation in the food chain is a potential health hazard. 7 Several chemical and microbial methods for leaching of heavy metals from sewage sludges have been reported, s-23 In chemical methods, mineral and organic acids have been commonly used to solubilize heavy metals. 8-12 A combination of heating and acid treatment improved solubilization significantly.~3-J5 In spite of good metal extractions achieved in the acid treatment method, most often Cu was not solubilized.

Other factors such as cost and operational difficulties in using acids have made chemical methods unattractive. Tyagi et al. 2° observed that the microbial method of metal leaching was 80% cheaper than the acid-requiring chemical method due to a lesser requirement of acid and lime in the former method. Operational difficulties, including the requirement of acid-corrosionresistant apparatus and safe storage and transportation facilities for acid, also put constraints on the utilization of the chemical method. Bacterial leaching of heavy metals from sewage sludges may be a viable alternative. Wong and Henry j8 acidified the sludge with sulfuric acid to pH 4.0 and incubated for 8-10 days to solubilize heavy metals by microorganisms. Other researchers 2°-22,24,25 investigated the role of Thiobacillus ferrooxidans and T. thiooxidans in the leaching of metals by the following processes. Process 1 T. ferrooxidans

8FeSO4 + 202 + 4H2SO4 ~ 4Fe2(SO4)3 + 4H20

(a)

4Fez(SOa)s + 2MS + 4H20 + 202 ~ 2M 2+ + 2SO42- + 8FeSO4 + 4H2SO4 The present address of Dr. Jain is the Biological Research Section, KD 118, Research Division, Ontario Hydro, 800 Kipling Avenue, Toronto, Ontario M8Z 5S4, Canada Address reprint requests to Dr. Tyagi at INRS-Eau, Universit6 du Qu6bec, 2700, rue Einstein, Sainte-Foy, Qu6bec G1V 4C7, Canada Received 1 May 1991; accepted 18 November 1991

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(b)

(MS = metal sulfide, insoluble; M 2+ = free metal ion, soluble) The second reaction does not require bacteria but proceeds chemically. A cyclic process between reactions

© 1992 Butterworth-Heinemann

Leaching of heavy metals by a and b results in more metal solubilization. Formation

of H2804 during the process further enhances the overall efficiency.

Process 2 MS + 202

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M2+ + SO 2

Process 3 T. thiooxidans 2S ° + 302 + 2H20--+ 2H2804 FeSO4" 7H20 is a commonly used substrate in bacterial leaching experiments. ~9-2~,23,24 Sch6nborn and Hartmann j7 added T. thiooxidans and 1% sulfur for the solubilization of heavy metals in municipal sludge. As a result of acidification (process 3), metal sulfides in anaerobically digested sludge were solubilized in 2232 days. The microbial process in terms of chemical requirements is cheaper than chemical methods 2° but requires 8-32 days of bioreaction time to solubilize metals in sewage sludges. Another disadvantage of present microbial technology in metal leaching involving T. ferrooxidans and T. thiooxidans is the requirement of acidic conditions (pH 2.5-4.0) to grow these microorganisms, which makes the microbial process even more expensive. Therefore, the present technology of heavy metals removal by microorganisms needs further improvement to make it a viable process. In this paper, we aimed at developing an improved microbial process of metal leaching which would meet the following criteria: (1) does not require initial sludge pH adjustment; (2) requires small batch bioreaction time (less than 40 h) against 8-32 days required in the existing microbial leaching processes; (3) should be capable of solubilizing all the metals to a recommended level (Table 1); (4) should be inexpensive; (5) should be free from operational difficulties; and (6) operates at room temperature (20-30°C). We isolated two new sulfur-oxidizing Thiobacillus species from sewage sludge. The mixed inoculum of these species decreased the sludge pH from 7.3 to 2.0 and solubilized heavy metals (Cu and Zn). Thus, there was no need to adjust the initial pH of the sludge as required in other microbial methods.

Table 1 Composition of metals of anaerobically digested sewage sludge and their recommended levels38

Metals Cd Cr Cu Fe Ni Pb

Zn

Composition (mg kg ~ dry sludge)

Recommended concentrations

Removal required

2.8 43.4 2,488.1 12,460 8.9 152.7 710.1

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Since most of the heavy metals in an anaerobically digested sewage sludge exist in the reduced and insoluble sulfide form, it would seem logical to isolate sulfide-oxidizing microorganisms that oxidize insoluble metal sulfides to soluble metal sulfates. However, there was no solubilization of metal sulfides in the uninoculated sludge under aerobic conditions, indicating that perhaps sulfide-oxidizing microorganisms were not present in the sludge (personal observation). Therefore, sulfur-oxidizing microorganisms were isolated from anaerobically digested sewage sludge by enrichment, since sulfur is a cheaper energy source for microorganisms than FeSO4 • 7H20, which was used in several bacterial leaching experiments. J8-22,24.25 The solubilization of Cu and Zn was studied for two reasons. First, the level of Cu was above the recommended level (Table 1). Second, the concentration of Zn in sewage sludges of Quebec generally varies between 615 and 1844 mg/kg of sludge and can be above the recommended level of 1750 mg/kg depending on the date of sampling. 25 Moreover, Cu is the most difficult metal to solubilize22: other metals, except Pb, are solubilized before Cu.

Materials and methods

Sludge and inoculum Anaerobically digested sewage sludge was obtained from a wastewater treatment plant in Valcartier, Quebec, Canada. The concentration of metals in sludge samples varied, depending on the sampling dates. The composition of important metals in the sludge which was used in these experiments is shown in Table I. The concentration of sludge solids for this study was 13 g 1-1 , which also varied between 14 and 17 g 1-1 depending on the date of sludge collection. An Erlenmeyer flask (250 ml) containing 100 ml anaerobically digested sewage sludge was acidified with 0.5 M sulfuric acid to pH 4.0 and amended with 1 g 1-I FeSO4.7H20. The flask was incubated aerobically at 25°C while shaking at 250 rev min 1in a gyratory incubator shaker Model 26, New Brunswick Scientific Co., Inc., NJ. The pH of the sludge was maintained at 4.0. Initially we intended to isolate efficient iron-oxidizing microorganisms such as T. ferrooxidans, but we could not enrich iron-oxidizing bacteria more efficiently than the ones reported in the literature (personal observation). Therefore, after 3 months of incubation, the sludge was used as an inoculum source to enrich sulfur-oxidizing microorganisms.

Primary enrichment o f sulfur-oxidizing microorganisms An Erlenmeyer flask (500 ml volume) containing 150 ml sewage sludge and 0.2 g 1-1 powdered elemental sulfur (Fisher Scientific Co., N J) was inoculated with 5% (v/v) of the above-described 3-months-old sludge. The initial pH of the sludge was adjusted with 0.5 M H2804 to pH 4.0. The flask was incubated aerobically at 25°C with shaking at 250 rev min -I. When the pH of

Enzyme

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Papers the sludge decreased to 2.73 in 85 h, this culture was used as inoculum for further enrichment of different groups of microorganisms. All flasks were incubated in the dark to prevent the growth of photosynthetic organisms. In one series, sulfur-oxidizing microorganisms were enriched at pH 4.0, and in another the microorganisms were enriched at pH 7.3 in seven steps. To enrich sulfur-oxidizing microorganisms growing or adapted at pH 4.0, the initial pH of the sludge was adjusted to 4.0 at each step. When the pH dropped to a certain value (Figure 1), 5% (v/v) of the sludge was used as inoculum and was transferred successively to fresh sludge at pH 4.0. To enrich sulfur-oxidizing microorganisms growing or adapted at pH 7.3, the initial pH and sulfur concentration were increased stepwise until adjustment of the sludge pH was not necessary (Figure 2). A 5% (v/v) inoculum was used at each enrichment step. Sulfur concentration, initial pH, and final pH of the sludge at each enrichment step are described in Results and shown in Figures 1 and 2. The pH of the sludge obtained from the Valcartier wastewater treatment plant varied between 7.3 and 7.8. To confirm that we were enriching microorganisms that would oxidize sulfur to sulfate, the enriched mi-

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croorganisms were grown at various sulfur concentrations to observe a relationship between the substrate (sulfur) concentration and the product (sulfate) formation rate. Erlenmeyer flasks (500 ml volume) containing 150 ml sewage sludge were amended with 5% (v/v) inoculum from the enrichment step where initial pH of the sludge decreased from 6.0 to 2.2 in 97 h (enrichment step 3, Figure 2). Sulfur was added at 0.0, 0.5, 1.0, 2.0, 3.0, and 4.0 g l-~ sludge. Uninoculated controls containing 2 g 1 ~ sulphur were also run. Initial pH of the sludge was adjusted to 6.0 with 0.5 M H2SO4. Samples (10 ml each) were drawn from flasks at 0, 9, 22.5, 34.5, 47, 59.5, 71.5 and 94 h, and sulfate concentration was measured by turbidimetric methods 26 on a Hach Turbidimeter, model 2100A. The suspended matter from the samples was removed by centrifugation at 10,000 rev rain J for 20 min at 4°C. To correct for sample color and turbidity, blanks were run to which BaCI2 was not added. Since sulfate production was linear, the sulfate formation was calculated by dividing the net sulfate production (final concentration initial concentration) with time (94 h). -

Selective enrichment of sulfur-oxidizing microorganisms Further steps in the selective enrichment of sulfuroxidizing microorganisms were necessary due to a dominant microflora of non-sulfur-oxidizing fungi, actinomycetes, and bacteria in the enriched culture sludge. The enrichment steps were (i) growth of sulfuroxidizing organisms in a sterile synthetic medium, (ii) addition of selective antibiotics, and (iii) enrichment by serial dilution. Modified 9K medium 27 of the following composition was used to grow sulfur-oxidizing microorganisms: (NH4)2SO4, 3 g; KC1, 0.3 g; MgSO4'7H20, 0.5 g; Ca(NO3)2, 0.01 g; K2HPO4, 0.5 g; powdered sulfur (Sigma Chemical Co.), 10.0 g; water, 1000 ml. Autoclaving of the medium at 121°C and 15 psi resulted in a solid mass of sulfur. Therefore, the medium was sterilized by tyndallization at 100°C (kept under free-flowing steam) for I h on two consecutive days. The powdered form of sulfur was unchanged after tyndallization, as examined visually. The sterility of the medium was checked by streaking nutrient agar plates (Difco). The pH of the medium was adjusted depending on the nature of the microorganisms to be isolated. Microorganisms enriched at pH 7.3 in the sludge were grown in the modified 9K medium at pH 8.0 containing 0.01 g I ~ bromthymol blue as a pH indicator, and microorganisms enriched at pH 4.0 in the sludge were grown in the modified 9K medium at pH 6.0 containing 0.01 g 1-~ bromphenol blue as a pH indicator. The pH of the modified 9K medium was adjusted with 0.1 M N a O H or 0.05 M H2SO4. To identify whether procaryotes or eucaryotes were involved in sulfur oxidation as well as to remove unimportant microorganisms from the enriched culture, selective antibiotics were added in the modified 9K medium. The buffering capacity of the modified medium

Leaching of heavy metals by Thiobacillus spp.: D. K. Jain and R. D. Tyagi was increased by replacing K2HPO4 with 0.05 M Kphosphate buffer. The pH of the medium was adjusted to 8.0 or 6.0 as described above. Filter-sterilized streptomycin sulfate (Sigma Chemical Co.) was used at a concentration of 15 mg 1-1, and cycloheximide (Sigma Chemical Co.) was used at a concentration of 25 mg l -l. Flasks (500 ml) containing antibiotics and 100 ml modified 9K liquid medium were inoculated with 5% (v/v) enriched inoculum and incubated aerobically at 25°C with shaking at 250 rev min J. Sulfur oxidation (H2S04 production) was monitored by observing change in the color of the pH indicator and measuring pH on a pH meter. Procaryotic microorganisms were enriched two times in the modified 9K liquid medium containing cycloheximide. The enriched medium was serially diluted in sterile K-phosphate buffer (0.05 M, pH 8.0 or 6.0), and 0.1 ml of the serially diluted cultures was inoculated in the tubes containing 10 ml modified 9K medium with pH indicator and 0.05 M phosphate buffer. The tubes were incubated at 25°C while shaking. After 6 days of incubation, growth of S°-oxidizing microorganisms in the highest dilution tube was recorded by observing change in color of the pH indicator due to H2SO4 production.

Isolation and purification of bacterial colonies Microorganisms from the highest dilution tube in which the color of the pH indicator changed due to acid production were streaked on modified 9K agar medium (Difco agar 1.5% w/v) plates amended with pH indicators and K-phosphate buffer (0.05 M, pH 8.0 or 6.0). The plates were incubated at 20°C. Single colonies around which the color of the pH-indicator turned yellow were picked up and purified twice. The purified colonies were maintained on modified 9K medium agar slants.

Activity of the isolates in the modified 9K liquid medium Bacterial colonies isolated from "high-pH group" (pH 8.0) and "low-pH group" (pH 6.0) were inoculated together in the modified 9K liquid medium (pH 8.0) amended with 1% (w/v) elemental sulfur and K-phosphate buffer (0.05 M) to screen for a compatible combination of microorganisms that would decrease the pH of the medium from 8.0 to 2.0. We assumed that insoluble metals of the sludge would be solubilized as a result of acidification. A total of 19 combinations were tested to decrease the pH from 8.0 to 2.0. The combination which decreased the pH to 2.0 was used to inoculate the sterile sludge.

Activity of the strains in the tyndallized sewage sludge Anaerobically digested sewage sludge was amended with 0.5% (w/v) elemental sulfur. The sludge was tyndallized twice for 1 h at 100°C on two consecutive days. The initial pH of the tyndallized sludge was ad-

justed with sterile 0.5 M H2804 or 1 M NaOH. The combination of the two isolates which decreased pH of the modified 9K medium containing 1% (w/v) elemental sulfur from 8.0 to 2.0, as described above, was inoculated in the tyndallized sludge. Inocula (in the modified 9K medium, 5% v/v) of the two isolates and their mixture were added in the tyndallized sludge at initial pH 6.1 or 8.3. Erlenmeyer flasks (500 ml) containing 150 ml tyndallized sludge, elemental sulfur, and inoculum were incubated at 25°C while shaking at 250 rev m i n t . Uninoculated controls were run at initial pH 6.4 and 8.1. A drop in pH of the sludge was recorded to observe the sulfur-oxidizing activity of microorganisms.

Activity of the strains in nonsterilized sewage sludge The mixed inoculum prepared in tyndallized sludge was inoculated in nonsterile, anaerobically digested sewage sludge without adjusting the pH. Erlenmeyer flasks containing 150 ml nonsterilized sludge, elemental sulfur (0.5% w/v), and inoculum (5% v/v) were incubated at 25°C with shaking at 250 rev min -~. Two types of controls were run. In the first type, the inoculum was not added but the sludge was amended with sulfur. In the second type, sulfur was not added but the inoculum was added. The samples were taken periodically and the pH was measured (Figure 5). The samples were centrifuged at 10,000 rev min t for 20 min, and metals in the supernatant were determined with a flame atomic absorption spectrophotometer (Varian AA-575) according to standard methods of analysis. 26 To determine metal composition of the sludge, the sludge was digested with conc. HNO3, HF, and HCIO4 as described in Standard Methods. 26Absorption calibration curves for each metal were determined using standardized metal solutions, and the metal analysis was performed in the linear range of the calibration curve.

Results Sulfur-oxidizing microorganisms were enriched in two series. In one series the initial pH of the sludge was maintained at 4.0, and in the other the pH and the concentration of elemental sulfur were increased gradually until adjustment of the pH of the sludge was no longer necessary. At initial pH 4.0, the pH decreased from 4.0 to 2.7 after 3 successive enrichment steps at 0.33 g i ~ sulfur concentration (enrichment steps I-3, Figure 1). Since metals such as Cu are difficult to solubilize at this pH, 2° the concentration of sulfur was doubled to lower the final pH. At 0.66% (w/v) sulfur concentration, the pH of the sludge dropped below 2.0 in 100 h (enrichment step 4, Figure 1). Sulfur-oxidizing microorganisms were enriched three more times at initial pH 4.0 and 0.66% (w/v) sulfur concentration. In the enrichment process, where initial pH and the sulfur concentration were increased gradually, the ini-

Enzyme Microb. Technol., 1992, vol. 14, May

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tial pH of the sludge decreased from 5.0 to 2.6 in 4 days at 0.66 g I-~ sulfur concentration (enrichment step 1, Figure 2). Since we did not know the nature of the microorganisms, a gradual increase in initial pH of the sludge was applied to avoid a lethal pH shock to microorganisms. At initial pH 6.0 and 1.32 g l -t sulfur concentration, the pH decreased to 2.2 (enrichment step 2). H o w e v e r , at initial pH 7.3, the pH first increased to 8.8 and then decreased to 6.5 after 7 days of incubation and below 6.0 after 9 days of incubation (enrichment step 4, Figure 2). To decrease the incubation time, sulfur concentration was increased from 1.2 to 2 g l -~. At initial pH 7.3, the pH of the sludge as it comes out of wastewater treatment plant, and 2.0 g 1 . sulfur concentration, the pH decreased to 5.2 after 3 days of incubation (enrichment step 5). Sulfur-oxidizing microorganisms were enriched two more times at initial pH 7.3 and 2.0 g I-~ sulfur concentration (Figure 2). Sulfur oxidation by enriched microorganisms was confirmed by measuring sulfate formation rates at various sulfur concentrations (Figure 3). A sulfate formation rate of 8.5 mg I ~ h -l was observed in the control samples with inoculum but without any addition of sulfur, probably due to the presence of residual sulfur in the inoculum. In the uninoculated control samples containing 2 g 1-j sulfur, a sulfate production rate of 3.9 mg 1 ~ h ~ was recorded. Uninoculated controls at other sulfur concentrations were not run. As the concentration of sulfur was increased in the sludge, there was a linear increase in the sulfate formation rate (Fig-

ure 3). Antibiotics were added (i) to identify the group responsible for sulfur oxidation and (ii) to remove a dominant microflora of non-sulfur-oxidizing fungi, actinomycetes, and bacteria. The pH of the synthetic medium containing cycloheximide and without antibiotics decreased from 8.0 to 4.5 when inoculated with "high-pH g r o u p " and from 6.0 to 2.0 when inoculated with " l o w - p H g r o u p " enriched inocula in 72 h (data not shown). There was no decrease in pH of the inoculated medium supplemented with streptomycin. These results indicate that procaryotes (not eucaryotes) were responsible for sulfur oxidation and decrease in pH. 380

Enzyme Microb. Technol., 1992, vol. 14, May

Several combinations of purified colonies from "high-pH g r o u p " and " l o w - p H g r o u p " were inoculated together in the synthetic medium. Eighteen out of nineteen combinations decreased the pH of the liquid medium from 8.0 to 6.0 but failed to lower it further (data not shown). H o w e v e r , only one combination of isolates, Nos. 0 and 5, decreased the pH of the synthetic medium from 8.0 to 2.0 in the presence of 1% elemental sulfur within 48 h. No attempts were made to determine the reasons for the failure of the other 18 combinations. Isolate 0 grew well on 9K agar medium containing Na2S203" 5H20. Cells were Gram-negative rods and formed transparent colonies. Isolate 5 did not grow on Na2S203 ' 5HzO but grew well on 9K agar medium containing elemental sulfur. The cells were also Gramnegative rods and formed small white colonies around sulfur particles. The isolates were identified as two Thiobacillus species, T. thioparus (0) and T. thiooxidans (5), and were deposited as ATCC 55128 and ATCC 55127, respectively. The role of the two Thiobacillus species in sulfur oxidation was studied in tyndallized, anaerobically digested sludge containing elemental sulfur. T. thiooxidans ATCC 55127 decreased the pH of the sludge from 6.1 to less than 2.0 in 50 h but did not function at pH 8.3 (Figure 4). T. thioparus ATCC 55128 decreased the pH of the tyndallized sludge from 8.1 to 5.0 in 50 h. The mixed inoculum of ATCC 55127 and ATCC 55128 decreased the pH from 8.05 to less than 2.0 in 40 h. In uninoculated control experiments, there was no drop in the sludge pH (Figure 4). There was no contamination in controlled flasks. The activity of Thiobacillus species was examined in nonsterilized sludge. The mixed strains decreased the pH of the nonsterile sludge from 7.8, the original pH of the sludge as it came out of the wastewater treatment plant, to less than 2 within 44 h of incubation (Figure 5). In the uninoculated control samples containing sulfur there was a slight increase in pH, whereas in the inoculated control samples without sulfur there was a slight drop in pH. Analysis of Cu and Zn by atomic absorption spectrophotometer revealed

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that almost all the Zn was dissolved in 40 h (Figure 6), and Cu (the most difficult metal in sludge to solubilize) within 60 h (Figure 7). There was insignificant solubilization of Zn and Cu in control samples. The initial concentrations of Zn and Cu in the sludge before

Discussion

Two groups of microorganisms were developed after 34 days of enrichment and incubation that oxidized elemental sulfur to sulfuric acid. One group of microorganisms decreased the pH of the sludge from 4.0 to less than 2.0 (Figure 1), at which most of the metals are likely to be solubilized, and the second group decreased the pH from 7.3-7.8 (initial pH of the sludge as it comes out of the wastewater treatment plant) to 5.5 (Figure 2). By the enrichment process, two Thiobacillus species, T. thioparus ATCC 55128 and T. thiooxidans ATCC 55127, were isolated. T. thioparus ATCC 55128 decreased the pH of the nonsterilized anaerobic sludge to a level which was favorable to the sulfur-oxidizing activity of T. thiooxidans ATCC 55127. The mixed inoculum of both the species oxidized sulfur to sulfate and decreased the sludge pH below 2.0 within 44 h, at which most of the Cu and Zn was solubilized. The drop in pH appeared to be due to sulfuric acid production by sulfur-oxidizing microorganisms. Production of organic acids, if any, by these microorganisms seems to be negligible because the pH of the control samples inoculated with T. thioparus ATCC 55128 and T. thiooxidans ATCC 55127 decreased from 7.8 to 7.6 in 65 h, whereas in the presence of 0.5% sulfur, the pH decreased from 7.8 to less than 2.0 (Figure 5). Moreover, a small pH drop may occur because of the residual sulfur present in the inoculum. When microorganisms are introduced into the natural environment, their survival is influenced by several biotic and abiotic factors. 28 Competition between introduced microorganisms and natural microorganisms is one of the most important biotic factors that causes failure of the introduced microorganisms. Since sewage sludges are a rich source of microorganisms, initially we tested the activity of a combination of our isolates in tyndallized sludge so that there was no competition with natural microorganisms. After testing the isolates in tyndallized sludge, we tested them in nonsterile sludge. Both species of Thiobacillus were able to survive and compete well in nonsterilized sludge. Storage stability of microbial strains is one of the main concerns in industrial processes. Storage stability of T. thiooxidans ATCC 55127 and T. thioparus ATCC 55128 was examined in nonsterilized sludge. The mixed species were inoculated in nonsterilized sludge containing 0.5% sulfur. After incubating for 48 h on a shaker at 25°C and 250 rev rain -], the flask was stored at 4°C for 6 months. Six months later, the activity of the strains was checked. A 5% inoculum from this flask was transferred to a flask containing 150 ml sludge and 0.5% (w/v) sulfur. Drop in pH was recorded as sulfur-oxidizing activity. After three successive transfers in nonsterilized sewage sludge containing sulfur, the original activity of the strains could be

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Papers achieved as measured by drop in pH from 7.8 to below 2.0. This shows that T. thiooxidans ATCC 55127 and T. thioparus ATCC 55128 are very stable species upon storage in sludge. Previously, heavy metal removal from sewage sludges by sulfur-oxidizing bacteria has been carried out by Sch6nborn and Hartman. j7 They added T. thiooxidans and 1% sulfur in the municipal sludge. The pH decreased from 5.5 to 1.0 in 22 to 32 days. As a result of acidification, metal sulfides were solubilized. Mixed culture of T. thiooxidans and T. ferrooxidans in the presence of 1% sulfur solubilized more metals than T. thiooxidans alone in more than 32 days of incubation. The mechanism for better solubilization by a mixed inoculum was due to the acidification of sludge by T. thiooxidans (sulfur oxidation) to a pH value (2.5) that favored the growth of T. ferrooxidans which oxidized and solubilized metal sulfides. In our process, a mixed inoculum of T. thioparus ATCC 55128 and T. thiooxidans ATCC 55127 solubilized metals within 40 h at normal pH (7.3-7.8) of the sludge. Thiobacillus strains have been implicated in coal desulfurization with relatively low pH (2-2.5) and long turnaround times. 29-33 T. thiooxidans oxidizes elemental sulfur more efficiently and rapidly than other acidophilic bacteria. Where sulfur is the only energy source, T. thiooxidans predominates and sulfur is oxidized to sulfuric acid. 3° In synthetic medium, T. ferrooxidans oxidized sulfur to reduce the pH from 4.0 to 1.2 in 9 days. 3~ T. acidophilus oxidizes sulfur and can grow at pH value from 1.5 to 6.0 with an optimum at pH 3.0. 32 Recent research has focused on sulfur-oxidizing thermophilic and acidophilic bacteria such as Sulfolobus acidocaldarius. This organism grows on sulfur at pH 2 and 3 and at 55 to 85°C. 33 The high temperature necessary for the optimum growth somewhat hinders the use of S. acidocaldarius as a sulfur-oxidizer because of the high energy cost incurred. All the four acidophilic organisms which are sulfur-oxidizers have been used in coal desulfurization, but the requirement of acid to maintain acidic conditions of coal (pH 2 to 3) and the necessity of unusually long periods (7 to 14 days) have rendered the use of these strains expensive.29-33 Several less acidophilic thiobacilli have been isolated which function at higher and wider pH range and are sulfur-oxidizers. T. capsulatus, which was isolated from soil adjacent to a sulfur block pad in sulfur mines, oxidized sulfur and reduced the pH of the synthetic medium from 6.8 to 3.7 in 20 days. 34 Other sulfur oxidizers include T. kababis, which reduced the pH of the synthetic medium from 3.7 to 2.2 in 9 days35; T. albertis, from 3.8 to 1.8 in 6 days36; T. perometabolis Th 023, from 7.5 to 5.0 in 7 days; and T. perometabolis Th024 and Thiobacillus sp. strain A2 (ATCC 25364), from 7.5 to 7.0 in three successive transfers. -~7The rate of decrease o f p H was 0.3 to 0.4 units per day, which is very slow. Other organisms which are sulfur-oxidizers include T. versutus, T. intermedius, T. neapolitanus, T. thiocynoxidans, and T. thioparus. But these are not faster sulfur-oxidizers than those described above. 34

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In spite of very low buffering capacity of 9K medium, the above-mentioned sulfur-oxidizers took several days to decrease the pH (0.2 to 0.4 units per day). In municipal sludge, which has a strong buffering capacity, 17,19,2°these organisms are expected to decrease the pH at a much lower rate than in 9K medium. This has been proven by the results of Sch6nborn and Hartmann 17who inoculated T. thiooxidans in anaerobically digested sludge (amended with 1% sulfur); the pH of the sludge decreased from 5.5 to 1.0 in 32 to 22 days (0.14 to 0.2 pH units per day). Thus, the microbial species isolated in these studies are superior to those reported in the literature.

Conclusion The process of heavy metals removal from sewage sludges by sulfur-oxidizing T. thioparus ATCC 55128 and T. thiooxidans ATCC 55127 is better than the existing processes, because (i) acid addition to favor the growth of bacteria is not required, (ii) bioreaction time is reduced to as little as 40 h, (iii) the process is simple and operates at room temperature, and (iv) elemental sulfur required in this process is easy to store and transport, unlike in the acid-requiring processes.

Acknowledgments Sincere thanks are due to the Natural Sciences and Engineering Research Council of Canada (grants A4984 and STR 0100710), the Ministry of Education of Qu6bec (grant FCAR 90-AS-9713), and the University of Qu6bec (FODAR) for supporting this research.

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