- Email: [email protected]
Degradation of thiocyanate by immobilized cells of mixed and pure cultures
819
Degradation of thiocyanate by immobilized cells of mixed and pure cultures 9
a
L. H. Rosa a, E. M. Souza-Fagundes a, M. H. Santos a, J. C. T Dias b, P. F. Plmentel, N. C. M. Gomes a,c, aDepartment of Biotechnology and Chemical Technology- CETEC, Belo Horizonte, MG, Brazil bGraduate Program in Microbiology, Federal University of Minas Gerais, Brazil ~Institute of Microbiology Prof. Paulo de G6es, Federal University of Rio de Janeiro, Brazil
Thiocyanate is frequently found in industrial wastewater such as those from coal, gold and silver mining. Although the microbial degradation of thiocyanate has been well documented, scarce information is available on the heterotrophic degradation of thiocyanate. A pure bacteria strain (BMV8) and a mixed culture of bacteria and fungi were isolated by an enrichment method from a fixed bed bioreactor for cyanide and thiocyanate removal and they were able to metabolize thiocyanate heterotrophically as a nitrogen source. The efficiency of thiocyanate biodegradation decreased when the cells were immobilized in citric pectin. The immobilized mixed culture was able to degrade thiocyanate more efficiently than the BMV8 strain.
1. INTRODUCTION Cyanide, a well-known metabolic inhibitor, is frequently used in industrial processes, including synthetic production and gold and silver extraction [ 1, 2]. Thiocyanate is commonly found in mining wastewater as a result of the interaction of free cyanide and sulfur. Unlike cyanide, thiocyanate presents low toxicity at low concentrations, but its toxicity increases at high concentration [ 1]. Chronic absorption of thiocyanate can cause dizziness, skin eruption, running nose, vomiting and nausea [3], so it must be removed before disposal procedures. Traditionally, cyanide and related compounds were detoxified by alkaline chlorination, hydrogen peroxide and SO2/air methods. These conventional techniques are characterized by their universal applicability because of their insensitivity towards the nature of the waste material and by their relatively low costs. However, their main handicaps are need of hazardous reagents like chlorine and their potential for creating toxic residues requiring a post *CX Postal. 2306, 31.170-000,BeloHorizonte,Brasil. Fax (031)489 2200. e-mail:[email protected]
820 treatment [4]. After Homestake Mine (Lead, SD) began operating a biological treatment plant in 1984, a lot of interest was generated in biodegradation of cyanide and thiocyanate. However, biological treatment as an alternative to those chemical methods is still in its infancy [5]. Although microbial treatment has been used for removing thiocyanate, the efficiencies of the process are variable. This is particularly observed when the treatment also involves mixtures of pollutants such as phenol and cyanide. Thiocyanate is known as an aggravating ion for the low efficiency in these process. Therefore, a better understanding of the biochemistry and microorganism interactions involved in thiocyanate degradation in bioprocess would be of fundamental importance to create consistent systems with efficient designs for the biotreatment of thiocyanate containing wastes [6]. The application of immobilized cells in the treatment of wastewaters offers the possibility of degrading higher concentrations of toxic pollutants that can be achieved with free cells [7]. Immobilized cells have advantages over free cells because they do not suffer cell loss during washing and also provide higher population densities at any flow [8]. This paper reports the results obtained on thiocyanate degradation by free and citric pectin immobilized cells of a mixed culture and a bacterial strain isolated from a fixed bed bioreactor for cyanide and thiocyanate removal. 2. MATERIAL AND METHODS 2.1. Culture enrichment and isolation Liquid samples were collected from a laboratory scale fixed bed bioreactor used for mining effluent treatment studies, and inoculated into Erlenmeyer flasks containing 25 ml of modified minimal salts medium M9 as described by Miller [9]. The M9 medium composition in (g/l) was Na2HPO4 0.6, KH2PO4 0.3 and NaCI 0.05, pH adjusted to 7.0. To this medium were added 5 mM of thiocyanate and 10 mM of glucose as a nitrogen and carbon source, respectively. The M9 medium was supplemented with 100 ~M of calcium chloride, 1.0 mM of magnesium sulfate and 0,1% (v/v) of trace metal solution [10] containing (g/l): MgO, 10.75, CaCO3, 2.0; FeSO4.7H20, 4.5, ZnSO4.7H20, 1.44, MnSO4.5H20, 1.12, CuSO4.5H20, 0.28, COSO4.7H20, 0.28; H3BOa, 0.06, 3 ml HC1 5 M. The thiocyanate degrading microorganisms were isolated by spreading the growth onto Petri dishes containing agar M9 medium after incubation at 30~ for 3 to 5 days. The colonies were replica-plated onto the same medium and three successive replicates were performed. Isolated bacteria were characterized by microscopic examination and Gram staining. 2.2. Cells immobilization (pectin-microorganism) Citric pectin (4 % w/w) (Citrus Colloids - low methoxy - amide gel) was dissolved in distilled deionized water and mixed with a magnetic stirrer for 1 hour at room temperature. To this solution was added the mixed bacterial culture or BMV8 strain, containing 10% (w/v) dry weight. This final solution (pectin-microorganism) was dropped into a 0.2 M solution of BaClz. A spontaneous cross-linking reaction occurred resulting in spherical beads with an average diameter of about 3 ram. These beads were stored at 4~ for 24 hours. After that period the beads were washed with distilled deionized water to remove the excess of BaC12.
821
2.3. Kinetic study The thiocyanate degradation profile was studied by culturing the mixed culture and the BMV8 strain in M9 medium supplemented with 5 mM of thiocyanate. Erlenmeyer flasks containing 25 ml of M9 medium were inoculated with 1% (v/v) inoculum of a 48 - hour culture grown in the same medium. At 12 hours intervals, the cultures were harvested by centrifugation at 27 200 x g for 10 minutes. The supernatants were retained for thiocyanate analysis and the cell pellets were dried at 60~ until constant weight, for dry weight determination. The mixed culture and BMV8 strain immobilized in pectin citric (20 g wet weight), were incubated in Erlenmeyer flasks containing 25 ml of M9 medium supplemented with 5 mM of thiocyanate and 10 mM of glucose. At 12 hours intervals, the supernatant were harvested by centrifugation at 27 200 x g for 10 minutes and retained for thiocyanate, glucose, sulfate, nitrite and nitrate analysis. In all steps, non-inoculated controls containing the M9 medium with thiocyanate and glucose were run as control. A negative control was accomplished (M9 medium with citric pectin). Thiocyanate was determined by the method of Stafford & Callely [ 11 ]. Nitrite, nitrate and sulfate were determined by the method of APHA [ 12]. 3. RESULTS AND DISCUSSION Continued growth and complete thiocyanate removal were observed following several serial transfers to fresh medium, from which the mixed culture was obtained. Three bacterial strains were isolated from these mixed culture and named BMV7, BMV8 and BMV9. These strains are in taxonomic classification tests and they were initially characterized as Gram-negative, rod shaped bacteria, and were catalase positive and oxidase negative. In the non-inoculated control M9 medium, thiocyanate levels did not decrease during the incubation period. This eliminated the possibility of thiocyanate removal due to a chemical reaction with other medium components. The mixed culture and the isolated strains were able to degrade thiocyanate heterotrophically using this compound as a nitrogen source. Most of the thiocyanate utilizing microbes reported in the literature are autotrophs, and only few heterotrophic with ability to degrade thiocyanate have been described [13-16]. Although differences in the thiocyanate removal by each strain isolated were observed, the strains were physiologically very similar and probably they belong to the same species, therefore, only the isolated BMV8 was examined in detail due to its greatest ability to remove thiocyanate from growing culture (data not shown). The kinetics of growth of the BMV8 strain and the mixed culture in presence of 5 mM of thiocyanate showed that both cultures were able to grow and remove thiocyanate from the medium in 36 hours of incubation (Figure 1 a, b). The thiocyanate utilization occurred mainly during the exponential phase of growth. Similar results were previously reported for Arthrobacter species by Betts et al. [ 17] and for an isolated strain by Stratford et al. [ 16]. In BMV8 strain cultures, unlike the mixed culture, was observed formation of 1,46 mM of ammonia after 36 hours of incubation. This amount decreased to 1,23 mM in 48 hours. Youatt [13] and Katayama et al. [18] suggested ammonia as a possible product from thiocyanate consumption. In a similar way Happold et al. [ 19] also observed that ammonia is originated by thiocyanate degradation. The oxidative pathway of thiocyanate has been known to proceed as
822 follows. First thiocyanate is hydrolyzed to cyanate and sulfide, then the cyanate is hydrolyzed to ammonia and bicarbonate, whereas sulfide is oxidized to sulfate. However some authors did not found ammonia in degrading bacterial cultures and it can be attributed to a rapid utilization of ammonia by the bacterial cells for further metabolism during growth [6]. Nitrite, nitrate and sulfate were not identified as end products of thiocyanate degradation.
~ 18
18
100
100 80
80
~
6
40 20 ~ !
16 ~
0
20 40 TIME(l-r
(a)
60
~
40~! 60
2o ~
o
o 0
20
40
60
TIME (hours)
(b)
Figure 1: Kinetic of growth (-tl--) and thiocyanate degradation ( ~ ) of BMV8 (a) and the mixed culture (b) on 5 mM thiocyanate as a nitrogen source and 10 mM glucose as a carbon and energy source. The kinetics of thiocyanate degradation by citrate immobilized cells of BMV8 strain and the mixed culture in presence of 5 mM of thiocyanate showed that both cultures kept the capacity of removing this compound from the medium (figure 2). However, there were a decrease of 12% (mixed culture) and 45% (BMV8) in comparison to the end of the incubation period. Numerous authors have reported differences in specific metabolic activity between free and immobilized microorganisms. The cause of these changes of cellular activity is still unclear and could be attributed to a modification of the physical and chemical environment of immobilized cells, or actual change of cellular physiology induced by immobilization [20]. So, more studies should be done in order to understand the mechanism of thiocyanate degradation in the immobilized system and optimization of the parameters as well for determining its efficiency as internal and external mass transfer, cell density, effects on metabolic state and osmose pressure. Some authors reported an increase in the efficiency of cyanides removal by immobilized cells suggesting that this technology can be employed in the environment for the remediation of inorganic cyanides [21 ].
823 lOO
N2 "~ 80 4o 2o
"~
0
I
12
I
I
I
1
I
24
36
48
60
72
TIME (hours)
Figure 2 - Kinetics of thiocyanate degradation by immobilized BMV8 strain (--I--) and mixed culture (---D---) on 5 mM thiocyanate as a nitrogen source. 4. CONCLUSIONS From results presented in this work, it can be seen that a mixed culture of microorganisms and the pure culture strain (BMV8) were able to metabolize thiocyanate heterotrophically as a nitrogen source. Despite of similar kinetics of thiocyanate degradation in the growing cultures, the mixed culture presented better efficiency when immobilized than BMV8 strain. This advantage could be useful in bioprocesses for the treatment of thiocyanate containing effluents and provides further information about the physiological conditions and biochemical mechanism of thiocyanate degradation. ACKNOWLEDGEMENTS We thank the "Minera~o Morro Velho" for the laboratory and field facilities. This work was supported by Funda~ao de Amparo/l Pesquisa de Minas Gerais - FAPEMIG. REFERENCES 1. C. Boucabeille; A. Bories; P. Olliver, Biotechnol. Lett., 16 (1994) 425. 2. K. D. Chapatwala,; G.R.V Babu, E. R. Armstead, E. M. White, J. H. Wolfram, Appl. Biochem. Biotechnol., 51/52 (1995) 717. 3. Y.L. Paruchuri, N. Shivaraman, P. Kumaran, Environ. Poilu., 68 (1990) 15. 4. S. Basheer, O. M. Kut, J. E. Prenosil and J. R. Bourne, Biotecnol. Bioeng. 39 (1992) 629. 5. J.B. Mosher, L. Figueroa, Minerals Engineering, 9 (1996) 573. 6. E.X. Oliveira Dias, Thiocyanate degradation by a novel isolate. Ph.D. Thesis. Faculty of Natural Sciences. University of Kent, 1993.
824 7. D. A. Kunz, O. Nagappan, J. Silva-Avalos and G. T. Delong, Appl. Environ. Sc. Technol. 58 (1992) 2022. 8. S.K. Dubey and D. S. Holmes, W. J. Microbiol. Biotechnol., 11 (1995) 257. 9. J. H. Miller, Experiments in molecular genetics. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York, (1972) 431. 10. T. Bauchop & S. R. Elsden, J. Gen Microbiol., 23 (1960) 457. 11. D. A. Stafford and A. G. Callely, J Gen Microbiol., 55 (1969) 285. 12. APHA. Methods for the examination of water and wastewater. Standard Methods, 19 ed. Washington, 1995. 13. J. B. Youatt, J.Gen.Microbiol., 11 (1954) 139. 14. T. I. Mudder and J. L. Whitlock, United State Patent 4,461,834 (1983). 15. V. Andreoni, A. Ferrari, A. Pagani, C. Sorlini, V. Tandoi, and V. Treccani, Anal. Microbiol., 38 (1988) 193. 16. J. Stratford, A.E.X.O. Dias, and C. J. Knowles, Microbiol. 140 (1994) 2657. 17. P. M. Betts, D. F. Rinder, and J. R. Fleeker, Can. J. Microbiol., 25 (1979) 1277. 18. Y. Katayama, Y. Narahara, Y. Ioue, F. Amano, T. Kanagawa,. and H. Kuraishi, J. Biol .Chem., 267 (1992) 9170. 19. F. C. Happold, K. I. Johnstone, H. J. Rogers and J. B Youatt, J. Gen. Microbiol., 10 (1954) 261. 20. S. Norton and T. Dte Amore, Enzyme Microb. Technol., 16 (1994) 365. 21. K. D. Chapatwala, G. RI V. Babu and J. H. Wolfram, J. Ind. Microbiol., 11 (1993) 69.
Degradation of thiocyanate by immobilized cells of mixed and pure cultures 9
a
L. H. Rosa a, E. M. Souza-Fagundes a, M. H. Santos a, J. C. T Dias b, P. F. Plmentel, N. C. M. Gomes a,c, aDepartment of Biotechnology and Chemical Technology- CETEC, Belo Horizonte, MG, Brazil bGraduate Program in Microbiology, Federal University of Minas Gerais, Brazil ~Institute of Microbiology Prof. Paulo de G6es, Federal University of Rio de Janeiro, Brazil
Thiocyanate is frequently found in industrial wastewater such as those from coal, gold and silver mining. Although the microbial degradation of thiocyanate has been well documented, scarce information is available on the heterotrophic degradation of thiocyanate. A pure bacteria strain (BMV8) and a mixed culture of bacteria and fungi were isolated by an enrichment method from a fixed bed bioreactor for cyanide and thiocyanate removal and they were able to metabolize thiocyanate heterotrophically as a nitrogen source. The efficiency of thiocyanate biodegradation decreased when the cells were immobilized in citric pectin. The immobilized mixed culture was able to degrade thiocyanate more efficiently than the BMV8 strain.
1. INTRODUCTION Cyanide, a well-known metabolic inhibitor, is frequently used in industrial processes, including synthetic production and gold and silver extraction [ 1, 2]. Thiocyanate is commonly found in mining wastewater as a result of the interaction of free cyanide and sulfur. Unlike cyanide, thiocyanate presents low toxicity at low concentrations, but its toxicity increases at high concentration [ 1]. Chronic absorption of thiocyanate can cause dizziness, skin eruption, running nose, vomiting and nausea [3], so it must be removed before disposal procedures. Traditionally, cyanide and related compounds were detoxified by alkaline chlorination, hydrogen peroxide and SO2/air methods. These conventional techniques are characterized by their universal applicability because of their insensitivity towards the nature of the waste material and by their relatively low costs. However, their main handicaps are need of hazardous reagents like chlorine and their potential for creating toxic residues requiring a post *CX Postal. 2306, 31.170-000,BeloHorizonte,Brasil. Fax (031)489 2200. e-mail:[email protected]
820 treatment [4]. After Homestake Mine (Lead, SD) began operating a biological treatment plant in 1984, a lot of interest was generated in biodegradation of cyanide and thiocyanate. However, biological treatment as an alternative to those chemical methods is still in its infancy [5]. Although microbial treatment has been used for removing thiocyanate, the efficiencies of the process are variable. This is particularly observed when the treatment also involves mixtures of pollutants such as phenol and cyanide. Thiocyanate is known as an aggravating ion for the low efficiency in these process. Therefore, a better understanding of the biochemistry and microorganism interactions involved in thiocyanate degradation in bioprocess would be of fundamental importance to create consistent systems with efficient designs for the biotreatment of thiocyanate containing wastes [6]. The application of immobilized cells in the treatment of wastewaters offers the possibility of degrading higher concentrations of toxic pollutants that can be achieved with free cells [7]. Immobilized cells have advantages over free cells because they do not suffer cell loss during washing and also provide higher population densities at any flow [8]. This paper reports the results obtained on thiocyanate degradation by free and citric pectin immobilized cells of a mixed culture and a bacterial strain isolated from a fixed bed bioreactor for cyanide and thiocyanate removal. 2. MATERIAL AND METHODS 2.1. Culture enrichment and isolation Liquid samples were collected from a laboratory scale fixed bed bioreactor used for mining effluent treatment studies, and inoculated into Erlenmeyer flasks containing 25 ml of modified minimal salts medium M9 as described by Miller [9]. The M9 medium composition in (g/l) was Na2HPO4 0.6, KH2PO4 0.3 and NaCI 0.05, pH adjusted to 7.0. To this medium were added 5 mM of thiocyanate and 10 mM of glucose as a nitrogen and carbon source, respectively. The M9 medium was supplemented with 100 ~M of calcium chloride, 1.0 mM of magnesium sulfate and 0,1% (v/v) of trace metal solution [10] containing (g/l): MgO, 10.75, CaCO3, 2.0; FeSO4.7H20, 4.5, ZnSO4.7H20, 1.44, MnSO4.5H20, 1.12, CuSO4.5H20, 0.28, COSO4.7H20, 0.28; H3BOa, 0.06, 3 ml HC1 5 M. The thiocyanate degrading microorganisms were isolated by spreading the growth onto Petri dishes containing agar M9 medium after incubation at 30~ for 3 to 5 days. The colonies were replica-plated onto the same medium and three successive replicates were performed. Isolated bacteria were characterized by microscopic examination and Gram staining. 2.2. Cells immobilization (pectin-microorganism) Citric pectin (4 % w/w) (Citrus Colloids - low methoxy - amide gel) was dissolved in distilled deionized water and mixed with a magnetic stirrer for 1 hour at room temperature. To this solution was added the mixed bacterial culture or BMV8 strain, containing 10% (w/v) dry weight. This final solution (pectin-microorganism) was dropped into a 0.2 M solution of BaClz. A spontaneous cross-linking reaction occurred resulting in spherical beads with an average diameter of about 3 ram. These beads were stored at 4~ for 24 hours. After that period the beads were washed with distilled deionized water to remove the excess of BaC12.
821
2.3. Kinetic study The thiocyanate degradation profile was studied by culturing the mixed culture and the BMV8 strain in M9 medium supplemented with 5 mM of thiocyanate. Erlenmeyer flasks containing 25 ml of M9 medium were inoculated with 1% (v/v) inoculum of a 48 - hour culture grown in the same medium. At 12 hours intervals, the cultures were harvested by centrifugation at 27 200 x g for 10 minutes. The supernatants were retained for thiocyanate analysis and the cell pellets were dried at 60~ until constant weight, for dry weight determination. The mixed culture and BMV8 strain immobilized in pectin citric (20 g wet weight), were incubated in Erlenmeyer flasks containing 25 ml of M9 medium supplemented with 5 mM of thiocyanate and 10 mM of glucose. At 12 hours intervals, the supernatant were harvested by centrifugation at 27 200 x g for 10 minutes and retained for thiocyanate, glucose, sulfate, nitrite and nitrate analysis. In all steps, non-inoculated controls containing the M9 medium with thiocyanate and glucose were run as control. A negative control was accomplished (M9 medium with citric pectin). Thiocyanate was determined by the method of Stafford & Callely [ 11 ]. Nitrite, nitrate and sulfate were determined by the method of APHA [ 12]. 3. RESULTS AND DISCUSSION Continued growth and complete thiocyanate removal were observed following several serial transfers to fresh medium, from which the mixed culture was obtained. Three bacterial strains were isolated from these mixed culture and named BMV7, BMV8 and BMV9. These strains are in taxonomic classification tests and they were initially characterized as Gram-negative, rod shaped bacteria, and were catalase positive and oxidase negative. In the non-inoculated control M9 medium, thiocyanate levels did not decrease during the incubation period. This eliminated the possibility of thiocyanate removal due to a chemical reaction with other medium components. The mixed culture and the isolated strains were able to degrade thiocyanate heterotrophically using this compound as a nitrogen source. Most of the thiocyanate utilizing microbes reported in the literature are autotrophs, and only few heterotrophic with ability to degrade thiocyanate have been described [13-16]. Although differences in the thiocyanate removal by each strain isolated were observed, the strains were physiologically very similar and probably they belong to the same species, therefore, only the isolated BMV8 was examined in detail due to its greatest ability to remove thiocyanate from growing culture (data not shown). The kinetics of growth of the BMV8 strain and the mixed culture in presence of 5 mM of thiocyanate showed that both cultures were able to grow and remove thiocyanate from the medium in 36 hours of incubation (Figure 1 a, b). The thiocyanate utilization occurred mainly during the exponential phase of growth. Similar results were previously reported for Arthrobacter species by Betts et al. [ 17] and for an isolated strain by Stratford et al. [ 16]. In BMV8 strain cultures, unlike the mixed culture, was observed formation of 1,46 mM of ammonia after 36 hours of incubation. This amount decreased to 1,23 mM in 48 hours. Youatt [13] and Katayama et al. [18] suggested ammonia as a possible product from thiocyanate consumption. In a similar way Happold et al. [ 19] also observed that ammonia is originated by thiocyanate degradation. The oxidative pathway of thiocyanate has been known to proceed as
822 follows. First thiocyanate is hydrolyzed to cyanate and sulfide, then the cyanate is hydrolyzed to ammonia and bicarbonate, whereas sulfide is oxidized to sulfate. However some authors did not found ammonia in degrading bacterial cultures and it can be attributed to a rapid utilization of ammonia by the bacterial cells for further metabolism during growth [6]. Nitrite, nitrate and sulfate were not identified as end products of thiocyanate degradation.
~ 18
18
100
100 80
80
~
6
40 20 ~ !
16 ~
0
20 40 TIME(l-r
(a)
60
~
40~! 60
2o ~
o
o 0
20
40
60
TIME (hours)
(b)
Figure 1: Kinetic of growth (-tl--) and thiocyanate degradation ( ~ ) of BMV8 (a) and the mixed culture (b) on 5 mM thiocyanate as a nitrogen source and 10 mM glucose as a carbon and energy source. The kinetics of thiocyanate degradation by citrate immobilized cells of BMV8 strain and the mixed culture in presence of 5 mM of thiocyanate showed that both cultures kept the capacity of removing this compound from the medium (figure 2). However, there were a decrease of 12% (mixed culture) and 45% (BMV8) in comparison to the end of the incubation period. Numerous authors have reported differences in specific metabolic activity between free and immobilized microorganisms. The cause of these changes of cellular activity is still unclear and could be attributed to a modification of the physical and chemical environment of immobilized cells, or actual change of cellular physiology induced by immobilization [20]. So, more studies should be done in order to understand the mechanism of thiocyanate degradation in the immobilized system and optimization of the parameters as well for determining its efficiency as internal and external mass transfer, cell density, effects on metabolic state and osmose pressure. Some authors reported an increase in the efficiency of cyanides removal by immobilized cells suggesting that this technology can be employed in the environment for the remediation of inorganic cyanides [21 ].
823 lOO
N2 "~ 80 4o 2o
"~
0
I
12
I
I
I
1
I
24
36
48
60
72
TIME (hours)
Figure 2 - Kinetics of thiocyanate degradation by immobilized BMV8 strain (--I--) and mixed culture (---D---) on 5 mM thiocyanate as a nitrogen source. 4. CONCLUSIONS From results presented in this work, it can be seen that a mixed culture of microorganisms and the pure culture strain (BMV8) were able to metabolize thiocyanate heterotrophically as a nitrogen source. Despite of similar kinetics of thiocyanate degradation in the growing cultures, the mixed culture presented better efficiency when immobilized than BMV8 strain. This advantage could be useful in bioprocesses for the treatment of thiocyanate containing effluents and provides further information about the physiological conditions and biochemical mechanism of thiocyanate degradation. ACKNOWLEDGEMENTS We thank the "Minera~o Morro Velho" for the laboratory and field facilities. This work was supported by Funda~ao de Amparo/l Pesquisa de Minas Gerais - FAPEMIG. REFERENCES 1. C. Boucabeille; A. Bories; P. Olliver, Biotechnol. Lett., 16 (1994) 425. 2. K. D. Chapatwala,; G.R.V Babu, E. R. Armstead, E. M. White, J. H. Wolfram, Appl. Biochem. Biotechnol., 51/52 (1995) 717. 3. Y.L. Paruchuri, N. Shivaraman, P. Kumaran, Environ. Poilu., 68 (1990) 15. 4. S. Basheer, O. M. Kut, J. E. Prenosil and J. R. Bourne, Biotecnol. Bioeng. 39 (1992) 629. 5. J.B. Mosher, L. Figueroa, Minerals Engineering, 9 (1996) 573. 6. E.X. Oliveira Dias, Thiocyanate degradation by a novel isolate. Ph.D. Thesis. Faculty of Natural Sciences. University of Kent, 1993.
824 7. D. A. Kunz, O. Nagappan, J. Silva-Avalos and G. T. Delong, Appl. Environ. Sc. Technol. 58 (1992) 2022. 8. S.K. Dubey and D. S. Holmes, W. J. Microbiol. Biotechnol., 11 (1995) 257. 9. J. H. Miller, Experiments in molecular genetics. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York, (1972) 431. 10. T. Bauchop & S. R. Elsden, J. Gen Microbiol., 23 (1960) 457. 11. D. A. Stafford and A. G. Callely, J Gen Microbiol., 55 (1969) 285. 12. APHA. Methods for the examination of water and wastewater. Standard Methods, 19 ed. Washington, 1995. 13. J. B. Youatt, J.Gen.Microbiol., 11 (1954) 139. 14. T. I. Mudder and J. L. Whitlock, United State Patent 4,461,834 (1983). 15. V. Andreoni, A. Ferrari, A. Pagani, C. Sorlini, V. Tandoi, and V. Treccani, Anal. Microbiol., 38 (1988) 193. 16. J. Stratford, A.E.X.O. Dias, and C. J. Knowles, Microbiol. 140 (1994) 2657. 17. P. M. Betts, D. F. Rinder, and J. R. Fleeker, Can. J. Microbiol., 25 (1979) 1277. 18. Y. Katayama, Y. Narahara, Y. Ioue, F. Amano, T. Kanagawa,. and H. Kuraishi, J. Biol .Chem., 267 (1992) 9170. 19. F. C. Happold, K. I. Johnstone, H. J. Rogers and J. B Youatt, J. Gen. Microbiol., 10 (1954) 261. 20. S. Norton and T. Dte Amore, Enzyme Microb. Technol., 16 (1994) 365. 21. K. D. Chapatwala, G. RI V. Babu and J. H. Wolfram, J. Ind. Microbiol., 11 (1993) 69.