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Q: What is Sulphur Black?

Sulphur black dye (water soluble) was obtained from Chek Tak dyeing factory Ltd. Sodium thiosulfate (Na2S2O3) was obtained from Sigma Chemical Company. Potassium iodide (KI) was obtained from Merck. Starch was obtained from Merck. Iodine (I2) was obtained from BDH Chemical Ltd. Tryptone was obtained from Difco Laboratories. Glucose was obtained from BDH Chemical Ltd. Yeast extract was obtained from Difco Laboratories. Ammonium chloride (NH4Cl) was obtained from Merck. Agar was obtained from Sigma Chemical Company. Dibasic sodium phosphate (Na2HPO4.12H2O) was obtained from Merck. Monobasic sodium phosphate (NaH2PO4) was obtained from Merck. Tris-(hydroxymethyl)aminomethane was obtained from Sigma Chemical Company. Sodium hydroxide (NaOH) was obtained from Merck. Potassium chloride (KCl) was obtained from Merck. Potassium dihydrogen phosphate (KH2PO4) was obtained from Merck. Glycine was obtained from Merck. Hydrochloric acid (HCl) was obtained from Merck. Sodium dodecyl sulfate (SDS) was obtained from Sigma Chemical Company. Triton X-100 was obtained from Sigma Chemical Company. Cetylpyridinium chloride was obtained from CalBioChem. Trypsin was obtained from Merck. Diethyl ether was obtained from Merck. Sodium chloride (NaCl) was obtained from Merck. Activated charcoal was obtained from BDH Chemical Ltd. 

Q: What is Sulphur Black Reactions?

IM medium: 10 g tryptone, 1 g D-glucose, 1 g yeast extract, 1 g NH4 Cl in 1 liter distilled water. IM agar: 15 g agar, 10 g tryptone, 1 g D-glucose, 1 g yeast extract, 1 g NH4 Cl in 1 liter distilled water. Starch solution: 1 g starch in 100 ml boiling water with vigorous stirring. Phosphate buffer: pH 7,39 ml 0.2M NaH 2 PO 4 , 61 ml 0.2M Na 2 HPO 4 .12H2O diluted to 200 ml with H2O. Tris-(hydroxymethyl)aminomethane buffer: pH 9, 50 ml 0.2M tris-(hydroxylmethyl)aminomethane, 5 ml 0.2MHCl diluted to 200 ml with H 2 O. Phosphate-NaOH buffer: pH 11, 50 ml 0.5M Na2 HPO 4 , 4.1 ml 0.1M NaOH diluted to 100 ml with H 2 O. Hydroxide-chloride buffer: pH 13, 25 ml 0.2M KCl, 6 ml 0.2M NaOH diluted to 100 ml with H 2 O. Potassium dihydrogen phosphate-NaOH buffer: pH 7, 50 ml 0.1 KH 2 PO4 , 29.1 ml 0.1M NaOH diluted to 100 ml with H2O. Glycine-NaOH buffer: pH 9, 25 ml 0.2M glycine, 4.4 ml 0.2M NaOH diluted to 100 ml with H2O.

Screening and Isolation of W3

Water samples polluted by waste from textile dyeing facilities in the South China region were screened for sulphur black dye degrading bacterial strains using the following procedure.

Sulphur black agar plates were formed by first dissolving 0.5 g of sulphur black dye powder into 500 ml of distilled water. The resultant solution was autoclaved and then cooled to 50° The solution was then combined with 500 ml of IM agar solution resulting in a 0.05% sulphur black agar solution. 25-30 ml of the sulphur black agar solution was poured into agar plates.

0.1 ml of the polluted water samples were spread evenly on the sulphur black plates in a manner which formed well separated colonies, e.g. 30-300 colonies per plate. The agar plates were then incubated for 3-4 days at 37° C.

Colonies which were surrounded by a clear halo in the agar medium were chosen as indicative of those colonies capable of bio-adsorption, i.e. could adsorb sulphur black dye. These chosen colonies were then diluted and re-spread on additional sulphur black IM agar plates in order to obtain pure colonies and confirm bioadsorption activity of the selected colonies. Form this process, twenty strains were selected.

Strains from these twenty strains which exhibited the greatest bioadsorption activity were selected using the following procedure. Each strain was cultured in IM medium for one day at 37° C.. Sulphur black dye was then added (approximately 3586 ?g/ml) to each culture to obtain an optical density reading of 1.0 at 620 nm (the OD 620 value). The cultures were then spun in a microfuge 13,000 rpm for 5 min. The following results were obtained.

TABLE 1

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Microbial OD at OD at OD at 15 OD at 72 Strain 1 min. 5 min. min. OD at 24 hr. hr.

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W2 .26 .25 .27 .38 .75 W3 .20 .25 .16 .12 .14 W4 1.45 .96 1.03 .58 .54 W6 .77 .69 .68 .45 .51 W7 1.02 1.04 1.03 .78 .74 W8 1.01 1.03 1.02 .96 .84 W9 1.00 1.02 1.00 .93 .75 W10 .44 .41 .43 .74 .69 W11 .26 .26 .23 .46 .47 W12 1.08 1.03 1.04 1.02 .83 W13 .96 .84 .90 .85 .85 W14 .56 .64 .63 .59 .84 W15 .88 .89 .88 .90 .90 W16 .17 .18 .20 .36 .59 W17 .92 .91 .90 .88 .89 W18 1.05 1.00 .99 .96 .97 W19 .80 .78 .52 .81 .33 W20 .89 .89 .91 .87 .92 E. Coli .86 .84 .83 .69 .79 H12 .94 .92 .95 .80 .79 No cells 1.06 1.05 1.05 1.06 1.08

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As indicated in the above results, strain W3, W2, W11 and W16 displayed significant bioadsorption activity. From these strains, W3 was selected for further characterization and study. W3 was shown to reduce the dark color in the agar method by about one half in one day, by comparing the agar plate with a plate inoculated with E. coli and having a 0.3% concentration of sulphur black dye.

C. Characterization of W3

Characterization of the strain W3 by conventional tests provided the following characteristics:

1) Gram positive bacteria (no growth was seen on EMB and tergitol-7 agar plates which are selective for grain negative bacterial)

2) W3 is stainable with crystal violet or safranin and upon staining, appears as a short rod

3) no endospore is observed

4) W3 only grows under aerobic conditions

5) W3 grows best a pH 7, but also at pH 9, but will not grow at pH 3, pH 5 or pH 11

6) generation time for W3 is 43 min. at pH 7, 37° C. and

7) W3 can ferment glucose, xylose and arabinose and is capable of nitrate utilization.

D. Further Characterization of the W3 Strain 1. Growth of bacterial strain W3

The cultures were incubated at 37° C. in IM medium and shaken at 200 rpm (Gyrotory Water Bath Shaker, New Brunswick Scientific). Experimental cultures were harvested after 20 hours incubation, then spun down at 10,000 rpm for 10 minutes at 4° C. (High Speed Centrifuge RC5C, Sorvall Instrument). The centrifuged cells were washed with distilled water once.

The growth curve of W3 was constructed by inoculating 1% overnight culture (18 hours) of W3 into a 250 ml conical flask containing 50 ml IM medium. Incubated at 37° C. and shaken at 200 rpm. The cell density of W3 was determined by the plate count method (viable cell count) and by a haemotocytometer (total cell count).

A very short lag phase, about 1 to 2 hours, was found in both counting methods. Stationary phase was reached at 7 to 9 hours of cultivation. The cell density in stationary phase was 1x10 9 cells/ml by viable cell count and 1.25x10 9 cells/ml total cell count. The culture entered the death phase after 10 hours of cultivation. There was no significant decrease in the total cell density in the death phase. However, a significant decrease of cell density was found in death phase by viable cell count. Viable cells per ml was about 1x10 6 cells/ml at 24 hours of incubation. See FIG. 3.

2. Standard method for measuring the capacity and efficiency of sulphur black dye removed by W3

Cultures of W3 were centrifuged and washed with distilled water prior to use. The dried weight (oven dry) of 20 ml of an overnight culture of W3 is about 10 mg (equivalent to 2.5x10 10 cells). The washed cell pellet was resuspended in a sulphur black dye solution. After incubation under controlled conditions, the cell-dye mixture was centrifuged at 10,000 rpm for 10 minutes (High Speed Centrifuge RC5C, Sorvall Instrument).

The resultant supernatant was then measured at A 625 using the Spectronic 601 from Milton Roy. A control was used which did not comprise cells. Based on the standard curve of sulphur black dye, the amount of sulphur black dye removed by W3 was obtained from the change of absorbance of the supernatant using the following formulas: ##EQU1##

3. Standard Curve of Sulphur Black

A linear relationship was observed between the concentration of sulphur black (range between 0 to 1 mg/ml) and the absorbance at 625 nm. A regression coefficient of r=0.9999 was obtained.

Therefore, the concentration of sulphur black could be calculated from the following equation. sulphur black (mg/ml)=(A 625 -1.79x10 -3)/17.56

4. Sulphur Black Dye Removal Efficiency of W3 under Various Conditions

a. Effect of medium on sulphur black dye removal

The efficiency of sulphur black removal by resting cells of W3 was determined in the following mediums: distilled water, IM medium and 0.1M sodium thiosulfate solution. Resting cells of W3 (10 mg in corresponding dry wt.) were resuspended in 2 ml of medium containing 0.05 mg/ml of sulphur black dye. The cells were then incubated at room temperature for 1 hour. Sulphur black dye removal efficiency was determined using the formula of D.2.

The sulphur black removal efficiency of W3 increased as the medium changed from distilled water to IM medium and to 0.1M sodium thiosulfate. The sulphur black removal efficiency of W3 in 0.1M sodium thiosulfate (about 93% ) was much higher than that in distilled water (about 27%). It was concluded that the presence of sodium thiosulfate positively affected the sulphur black uptake by W3.

b. Effect of the amount of cells used on sulphur black dye removal efficiency and capacity

The sulphur black removal efficiency and capacity of W3 was determined for the following amounts of W3 cells (in corresponding dry wt.): 1 mg, 10 mg, 50 mg and 100 mg. The various amounts of W3 were resuspended in 2 ml of 0.1M sodium thiosulfate with 0.05 mg/ml of sulphur black dye. The resuspensions were incubated at room temperature for 1 hour. The amount of sulphur black removed by W3 was determined by the standard method of D.2.

The sulphur black removal efficiency increased with increasing amounts of W3 (from 1 mg to 100 mg of corresponding dry cells). The increase in sulphur black removal efficiency was much sharper upon addition of 10 mg of W3 than upon addition of 1 mg of W3. The rate of increase of sulphur black removal efficiency decreased as the amount of W3 increased from 10 mg to 100 mg. Sulphur black removal capacity decreased with increasing the amounts of applied W3. See FIG. 4.

c. Effect of sulphur black concentration on sulphur black removal efficiency and capacity of W3

10 mg of W3 (in corresponding dry wt.) was resuspended in 2 ml of 0M sodium thiosulfate solution containing various concentrations of sulphur black ranging from 0.01 to 1.0 mg/ml. The resuspensions were incubated at room temperature for 30 minutes. The amount of sulphur black removed by W3 was determined by the standard method of D.2.

The sulphur black removal capacity of 10 mg of W3 (in corresponding dry wt.) increased gradually as the concentration of sulphur black increased from 0.01 to 0.5 mg/ml. The rate of increase of sulphur black removal capacity slowed as the concentration of sulphur black increased from 0.5 to 1.0 mg/ml. Sulphur black removal efficiency of W3 increased slightly to a maximum (about 99% ) when the concentration of sulphur black increased from 0.01 to 0.2 mg/ml and then decreased when the sulphur black concentration rose to 1.0 mg/ml. See FIG. 5.

d. Effect of concentration of sodium thiosulfate on the sulphur black removal efficiency and capacity of W3

10 mg of W3 (in corresponding dry wt.) were resuspended in 2 ml solution with various concentrations of sodium thiosulfate ranging from 0.05 to 1M, and 0.05 mg/ml of sulphur black. The resuspensions were incubated at room temperature for 1 hour. The amount of sulphur black removed by W3 was determined by the standard method of D.2.

The sulphur black removal efficiency of W3 remained high (about 90%) and was not affected by changes in the concentration of Na 2 S 2 O 3that ranged from 0.05 to 1.0M. See FIG. 6.

e. Effect of temperature on the sulphur black removal efficiency of W3

10 mg of W3 (in corresponding dry wt.) were resuspended in 2 ml of 0.1M sodium thiosulfate with 0.05 mg/ml of sulphur black. The mixtures were incubated at either 4, 25, 37 or 60° C. for 1 hour. The amount of sulphur black removed by W3 was determined by the standard method of D.2.

Over this range of temperatures, 4° to 60° C., sulphur black removal efficiency of W3 remained at about 85 to 92%.

f. Effect of incubation time on sulphur black removal efficiency of W3

10 mg of W3 (in corresponding dry wt.) were resuspended in 2 ml 0.1M sodium thiosulfate containing 0.05 mg/ml of sulphur black. The resuspension was incubated at room temperature for 1, 30, 60 or 120 minutes. The amount of sulphur black removed by W3 was determined by standard method D.2.

The sulphur black removal efficiency of W3 after 1 minute was slightly lower than the removal efficiency observed after 30 minutes. Similar sulphur black removal efficiencies were observed when the incubation time was extended from 30 to 120 minutes.

g. Effect of pH on sulphur black removal efficiency of W3

Two buffer systems covering pH ranges from 7-13 were used. Buffer system I included phosphate buffer pH 7, tris-(hydroxymethyl)aminomethane buffer pH 9, phosphate-NaOH buffer pH 11 and hydroxide-chloride buffer pH 13. Buffer system II included potassium hydrogen phosphate-NaOH buffer pH 7, glycine-NaHO buffer pH 9, phosphate-NaOH buffer pH 11 and hydroxide-chloride buffer pH 13.

10 mg of W3 (in corresponding dry wt.) was resuspended in 2 ml 0.05M buffer containing 0.1M sodium thiosulfate and 0.05 mg/ml of sulphur black. The resuspension was incubated at room temperature for 1 hour. The amount of sulphur black removed by W3 was determined by the standard method of D.2.

The effect of pH was verified by the two buffer systems covering pH 7, 9, 11 and 13. The sulphur black removal efficiency decreased with increasing alkalinity. The sulphur black removal efficiency decreased slightly as the pH increased from 7 to 11, and then decreased dramatically as the pH increased from 11 to 13. See FIG. 7.

h. Effect of different pretreatments on tile sulphur black removal efficiency of W3

10 mg of W3 (in corresponding dry wt.) was exposed to the following chemical or physical pretreatments immediately prior to the sulphur black removal assay. Following pretreatment, the control and the treated samples were harvested by centrifugation at 10,000 rpm for 10 minutes at 4° C. and were washed twice with distilled water. The supernatant were discarded after each centrifugation step. The control and treated cells of W3 were resuspended in 2 ml of 0.1M sodium thiosulfate with 0.05 mg/ml of sulphur black. The resuspensions were incubated at room temperature for 15 minutes. The amount of sulphur black removed by W3 was determined by the standard method D.2. The untreated W3 was used as a control and assigned 100 as relative sulphur black removal efficiency. The relative sulphur black removal efficiency of the various pretreated W3 was about 89 to 102%. The results are found in the table following the descriptions of the various pretreatments that were employed.

Acid treatment--incubate cells in 50 mM HCl at room temperature for 30 minutes or at 100° C. for 10 minutes.

Alkali treatment incubate cells in 50 mM NaOH at room temperature for 30 minutes.

Heat treatment--incubate cells in distilled water at 100° C. for 10 minutes or at 121° C., 15 lb/in 2 for 20 minutes (i.e. autoclave).

Enzyme tmt.--incubate cells in 0.1 mg/ml of trypsin at 37° C. in neutral pH for 30 minutes.

Ether treatment Resuspend cells in diethyl ether at room temperature for 10 minutes.

Control--Cells were kept in distilled water and stored on ice for 10 or 30 minutes.

TABLE 2

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Relative removal Treatment Target component efficiency(%)

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Control -- 100(assumed) HCl(R.T.) non-specific 97.2 HCl(100° C.) non-specific 89.4 NaOH non-specific 40.4 SDS non-specific 101.7 Triton X-100 non-specific 99.7 Cetylpyridinium non-specific 99.2 Chloride 100° C. non-specific 101.5 Autoclave non-specific 101.4 Trypsin protein and 97.2 peptidoglycan Ether primarily lipids 101.2

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i. Removal of sulphur black from the solutions analogous to sulphur black processing solutions comprising sodium thiosulfate

10 mg of W3 (in corresponding dry wt.) were resuspended in a solution having 25% (w/v) sodium thiosulfate (1.0M), 0.1 mg/ml of sulphur black and 5% NaCl (0.86M) in pH 8. The mixture was incubated at room temperature for 30 minutes. The amount of sulphur black removed by W3 was determined by the standard method D.2.

The sulphur black removal efficiency of 10 mg of W3 (in corresponding dry wt.) was about 94% in 2 ml 25% (w/v) sodium thiosulfate (1M), 0.1 mg/ml of sulphur black and 5 (w/v) NaCl (0.86M) at pH 8 after incubation at room temperature for 30 minutes.

j. Complete sulphur black removal from high concentration sulphur black solutions

The concentration of sulphur black used in this experiment was 10 times higher than that in standard method D.2. 10 mg of W3 (in corresponding dry wt.) was resuspended in 4 ml of 0.1M sodium thiosulfate solution containing 0.5 mg/ml of sulphur black. The resuspension was incubated at room temperature for 30 minutes. The supernatant was separated from the cells following centrifugation of the resuspension at 10,000 rpm at 4° C. for 10 minutes. The cell pellet was discarded and the supernatant was retained for determination of sulphur black concentration by the standard method of D.2.10 mg of W3 was then added to the supernatant and the procedure was repeated until 99% of the sulphur black was removed.

99% of the sulphur black was removed from 4 ml of a 3.05M sodium thiosulfate solution containing 0.5 mg/ml of sulphur black (the concentration of sulphur black used was 10 times higher than that used in standard method) following four repeated additions of 10 mg of W3 (in corresponding dry wt.). 98% of the sulphur black was removed after 3 repeated additions of cells. However, the sulphur black removal efficiency decreased as the number of renewals of cells, i.e. repeated additions, increased. See FIG. 8.

k. Sulphur-black removal capacity of resting cells of W3

The maximum sulphur black removal capacity of W3 resting cells was evaluated using the following procedure. 10 mg of W3 (in dry wt.) was resuspended in 2 ml of 0.1M sodium thiosulfate with 0.05 mg/ml of sulphur black. The resuspension was incubated at room temperature for 15 minutes. The resuspension was centrifuged at 10,000 rpm for 10 minutes and the concentration of sulphur black left in the supernatant was measure by the standard method of D.2. The supernatant was then discarded. The cell pellet was saved and resuspended in 2 ml of 0.1M sodium thiosulfate with 0.05 mg/ml of sulphur black. The resuspension was then incubated at room temperature for 15 minutes. This procedure was repeated 15 times. The total amount of sulphur black removed by W3 was determined by the standard method of D.2.

It was observed that 10 mg of W3 (in corresponding dry wt. ) removed 0.5 mg of sulphur black if 2 ml 0.1M sodium thiosulfate with 0.05 mg/ml of sulphur black was renewed 15 times. Since the saturation of sulphur black adsorption had not yet been reached, it was expected that the maximum sulphur black removal capacity of 10 mg W3 should be higher than 0.05 (mg sulphur black removed/mg cells added).

l. Effect of W3 treatment on the concentration of sodium thiosulfate

10 mg of W3 (in corresponding dry wt.) was resuspended in 2 ml of 0.05M sodium thiosulfate with and without 0.05 mg/ml of sulphur black at room temperature for 15 minutes. The initial and final concentration of sodium thiosulfate was determined by the following formula: ##EQU2##

No change was observed in the concentration of either sodium thiosulfate solution after W3 treatment for 30 minutes at room temperature.

m. Adsorption of sulphur black by dead W3 cells

The amount of sulphur black adsorbed is not affected by killing the W3, regardless of how the cells are killed, e.g. by heating, acid, or detergents. It was therefore concluded that sulphur black binding by W3 cells is independent of respiration or other cell metabolic activity. In repeated bioadsorption studies, it was consistently found that the sulphur black binding efficiency of W3 was not influenced by killing the cells.

n. Active agents on the cell surface for sulphur black binding.

Sodium dodecyl sulfate (an artionic denaturing surfactant), Triton X-100 (a nonionic nondenaturing surfactant) and cetylpyridinium chloride (a cationic denaturing surfactant) had neither a positive nor a negative effect on sulphur black binding by W3.

o. Effect of sodium thiosulfate on sulphur black binding.

The presence of Na 2 S 2 O 3 is relevant to sulphur black binding because the sulphur black removal efficiency was reduced in the absence of Na 2 S 2 O 3 . However, a similar amount of sulphur black was adsorbed by W3 in the presence of Na 2 S 2 O 3 in the range of 0.05 to 1.0M. This suggests that a minimal concentration of Na 2 S 2 O 3 (0.05M) is desirable to maintain the high sulphur black binding efficiency of W3.

II. Isolation and Characterization of the Activity of the Biodegradation Strain L5

A. Isolation of L5

Polluted water samples were collected from the South China region. Bacterial strains capable of degrading sulphur black were isolated from these samples by inoculating 100 ml IM liquid aliquots comprising sulphur black with inoculate from the polluted water samples. Aliquots which displayed a color change subsequent to inoculation were deemed to comprise biodegradation bacterial strains.

The isolation of biodegradation strains, as opposed to bioadsorption strains, was confirmed by using the following procedure. 20-30 ml of IM agar was poured onto a petri plate and allowed to solidify for one day. 5 ml of IM agar was combined with bacterial and sulphur black dye at 6% final dilution. This second agar solution was poured onto the surface of the solidified agar on the petri plate. The second layer was allowed to solidify. The petri plate was incubated at 37° C. for one day. A significant color change was observed indicating that the bacteria present in the top agar layer was L5.

B. Characterization of L5

1. Haloforming ability

To determine whether the enzymes responsible for the sulphur black degradation activity of L5 are present within the cells or on the surface of the cells, the halo forming test was employed. A sulphur black IM plate was spotted with an L5 culture. The plate was incubated at 37° C. for one week. Following incubation, no clear halo in the otherwise black color of the plate was observed. It was concluded that either adsorption or absorption was necessary for biodegradation of sulphur black by L5. In other words, the biodegradation enzyme was not secreted by the L5 into the ambient medium.

2. Effect of sulphur black on the growth of curve of L5

It was observed that the presence or absence of sulphur black in the ambient medium of the L5 had no effect on the growth curve of L5. See FIG. 9.

3. Additional characterizations of L5

Using conventional methods, these additional characterizations of the L5 strain were made.

a) L5 is a gram positive cocci

b) L5 is facultatively anaerobic

c) L5 does not possess oxidase activity

d) L5 ferments glucose, arabinose and xylose to produce acid and gas

e) L5 ferments lactose, maltose, sucrose and mannitol to produce gas

f) L5 possesses catalase activity

g) L5 displays positive IMV C activity

h) L5 produced hydrogen sulphide from sulphate

i) L5 hydrolyzes starch, tributyrin and casein but does not hydrolyze gelatin

j) L5 reduces litmus

k) L5 possesses both rennin and pepsin like enzymes and

l) L5 displays resistance to the following antibiotics: ampicillin, bacitracin, neomycin, penicillin G, polymyxin B, rifampin, streptotnycin, tetracycline, oxytetracycline, as well as intermediate resistance to nalidixic acid.

The subject method and microorganisms may be used for treating a wide variety of composites through either bioadsorption or biodegradation. Bioadsorption may find particular use in the treatment of waste streams, such as the thiosulfate waste stream resulting from the production of sulphur black dye. Bioadsorption may also be used for other waste streams, particularly associated with the dying of textile. The method may comprise one or more stages. As already indicated, the cells can be grown and killed and used for adsorption. This may be particularly useful, where one wishes to reduce the concentration level of the sulphur black dye, prior to using viable cells to both adsorb and degrade the sulphur black dye. By reducing the amount of sulphur black dye present in the waste stream one may reduce the time for the processing of the resulting stream by viable cells.

Biodegradation provides further methods for treatment of compositions in which sulphur black dye is a contaminant. In some instances, it may be useful to transform the subject cells to provide for capabilities for degradation of other waste materials which may be present in the common waste stream. Thus, the subject hosts may be modified to provide for multiple capacities. Alternatively, the gene obtained as described above may be used to transform other hosts which may be more convenient as to growth properties, harvesting, safety or the like. In this manner, different bacterial organisms may be employed for one or more purposes.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

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