and Research (SIBER), University Road, Kolhapur-416 004, Maharashtra, India. ABSTRACT. A chemoheterotrophic .... Stratford et aI. (1994) have proposed the ...
Nature Environment and Pollution © Technoscience Publications
Technology
ISOLATION OF THIOCYANATE BACTERIAL CONSORTIUM
pp. 135-138
DEGRADING
2006
CHEMOHETEROTROPHIC
Yogesh B. PatH
P. G. Department of Environmental Management, Chh. Shahu Central Institute of Business Education and Research (SIBER), University Road, Kolhapur-416 004, Maharashtra, India ABSTRACT A chemoheterotrophic bacterial consortium, capable of degrading thiocyanate, was isolated from sewage slurry by the enrichment culture technique. The bacterial consortium was composed of three bacterial species. Preliminary characterisation of microorganisms was carried out by microscopic examination and by studying colony characteristics. Investigations under laboratory conditions showed that the isolated bacterial consortium could degrade 50 mglL of thiocyanate within a period of 40 hours with an efficiency
of more than 99.9%.
INTRODUCTION Anionic thiocyanate (SCN-) in significant amount (5-]00 mg/L) is frequently encountered in the effluents emnated from the industries such as coal processing, extraction of precious heavy metals, photographic units, herbicide and insecticide production, dyeing, acrylic fibre production, manufacturing of thiourea, and electroplating industries. From most of these industries, thiocyanate is found in wastewaters along with other toxic chemicals like free cyanide and metal complexed cyanides. Since thiocyanate, cyanide and metal-cyanides are chemically related species, they are toxic to all classes of living organisms (Westley 1981). It is, therefore, imperative for the industries using and/or generating thiocyanate to adequately detoxify the effluents before discharge, as it may pose a major environmental, ecological and health hazard. Although chemical methods of treating these toxic compounds are known, bacterial detoxification of thiocyanate and its related species is of interest both, in order to understand how thiocyanate may be dealt within the environment and to evaluate the economic viability of bacterial systems for thiocyanate detoxification. Microorganisms, capable of utilising one-carbon containing compounds like free cyanide, metal-cyanides and thiocyanate have been reported in the recent past (Karavaiko et al. 2000, Patil & Paknikar 2000, Sorokin et al. 2001). Few attempts have also been made to set-up large-scale biological treatment plants for cyanide, metalcyanides and thiocyanate containing wastes (Mudder & Whitlock] 984). The present paper reports the isolation of thiocyanate detoxifying chemoheterotrophic bacterial consortium, which is the first necessary step towards development of microbial technology for tackling the industrial effluents contain ing th iocyanate. MATERIALS AND METHODS Enrichment medium M-9 minimal salts medium (MSM) was used to set-up enrichment and growth of bacterial consortium. The ingredients of medium were: Na2HP04.2HP - 3.0 g/L; KHl04 - 1.5 g/L; NaCl- 0.25 g/L and I milL trace metal solution. pH of the medium was adjusted between 7.0 and 7.5 using I M NaOH/HCI. Glucose (] 0 mM) was supplemented to the medium as a sole source of carbon and energy, while thiocyanate (50 mg/L) acted as the sole nitrogen source.
136
Yogesh B. Patil
Enrichment, isolation and characterisation cultures
of thiocyanate
degrading bacterial
To set-up enrichment culture, sewage slurry was obtained from the sewage treatment plant, Bawada, Kolhapur, which was used as the source of microorganisms for the isolation of thiocyanate degrading bacterial cultures. Enrichment culture was carried out in one-litre capacity borosilicate glass reactor in unsterilised conditions. 100 mL of sewage slurry was added into 900 mL MSM containing thiocyanate and glucose as the sole nitrogen and carbon source, respectively. After addition, the final concentration of thiocyanate' and glucose obtained in the enrichment medium was 50 mglL and 10 mM, respectively. The pH of the medium was adjusted to 7.0 to 7.5. The glass reactor was incubated at room temperature (30±2°C). Air was sparged continuously at the bottom of the medium at the rate of 1000±50 mUmin using electrical aerator unit. Five to six successive transfers of 10% (v/v) enrichment/acclimatized culture were given periodically in the fresh MSM containing thiocyanate (50 mg/ L), The enrichment cultures, as obtained after five to six successive transfers, were streaked on the nutrient agar and MSM agar (containing thiocyanate and glucose) plates and incubated at 35°C for 48-96 h. The bacterial colonies appeared on the petri plates were further transferred to MSM agar and nutrient agar slopes. Preliminary characterisation of the isolated bacterial cultures was carried out by microscopic examination (Gram staining and motility) and by studying colony characteristics on the nutrient agar plates. Biodegradation
of thiocyanate
by enriched bacterial consortium
Batch mode studies on biodegradation of th iocyanate were conducted using accl imatized bacterial consortium isolated from sewage slurry. Experimental conditions for conducting the studies were kept similar as mentioned earlier. Final concentration of thiocyanate in the medium was 50 mg/L. which served as the sole nitrogen source. Glucose (10 mM) was supplemented in the medium as a source of carbon and energy. The medium was inoculated with ] 0% of the inoculum (having cell densityapprox. 108 cells/mL) previously grown on the same medium. Appropriate uninoculated controls were run simultaneously along with the experiments, to detect air stripping of thiocyanate, if any, during incubation. The experiments were conducted twice to confirm the results. Parameters such as pH, thiocyanate content and bacterial cell count were checked periodically. Chemicals, glassware and analyses All the chemicals used in the experiments were of analytical grade. The glassware used during the study were of borosilicate material. Thiocyanate estimation was carried out using ferric nitrate colorimetric method (at 460 nm) as described in Standard Methods (APHA, AWWA, WEF 1998). pH was measured using digital pH meter (Systronics, India). Microscopic bacteria] cell count was carried out using Neubauer's chamber. RESULTS Enrichment and isolation of thiocyanate degrading cultures: During the process of enrichment for the isolation of bacterial consortium, each successive transfer of the acclimatized culture (10% v/v) was given in fresh MSM (containing thiocyanate) soon after the thiocyanate present (50 mg/L) was completely degraded. It was observed that during each subsequent transfer cycle the time taken by bacterial consortium for degradation of thiocyanate was reduced. For example, in the first cycle of enrichment culture transfer, a lag period of72 h was observed, which was further followed by thiocy-
ISOLATION OF THIOCYANATE DEGRADING BACTERIAL CONSORTIUM
137
anate degradation. Complete degradation of thiocyanate in the said cycle took place in 120 hours at a rate of 0.4 mg/Llh. Further transfer cycles showed the decreased lag period and an increased thiocyanate biodegradation rate. During the fifth cycle, the lag period and log period (i.e. the actual period of thiocyanate biodegradation) were ] 0 and 41 h respectively, and rate of biodegradation obtained was 1.2 mglL. The bacterial count during each successive transfer was more than 108 cells/mL. Characterization of thiocyanate degrading microbial cultures: From enrichment culture, three bacteria] colony types were obtained. The bacterial cultures were further purified by streak plate technique on the same medium. All the three bacterial isolates were Gram-negative rods and motile. Table I shows the colony characteristics of thiocyanate degradingbacterial isolates. Thiocyanate biodegradation by bacterial consortium: Batch mode studies revealed that the bacterial consortium isolated from sewage slurry could degrade 50 mg/L thiocyanate within 40 h with an efficiency of greater than 99.9% (Table 2). During the process of degradation, the bacterial population increased from ] 05 to 108 cells/mL with concomitant decrease in thiocyanate concentration. There was no significant change in the pH after biodegradation (final pH range 7.4-7.6) as against the initial pH of7.5. In uninoculated controls, the thiocyanate concentration remained unchanged throughout the experimental period. DISCUSSION Development of a microbial process for detoxification of thiocyanate containing industrial effluents is being developed in our laboratory. For this purpose, a chemoheterotrophic bacterial consortium (composed of three bacterial cultures) capable of utilizing thiocyanate as a sole source of cellular nitrogen was isolated from sewage slurry using enrichment technique. Preliminary characterization showed that all the cultures were Gram negative and motile. The cultures are being identified further. During enrichment culture studies, it was found that after each subsequent transfer cycle, the time taken by the bacterial consortium for degrading thiocyanate was s'ignificantly reduced (from 120 h to 40 h). This could be explained by the fact that microorganisms were getting acclimatised to the toxic compound present in the system during each subsequent transfer cycle. In batch mode laboratory testing, thiocyanate at the level of 50 mg/L was degraded completely by the isolated bacterial consortium within 40 h with more than 99.9% efficiency. The decrease in thiocyanate concentration of the medium was concomitant with the increase in bacterial population. The fact that the final cell density obtained was considerably high (> 108 cells/mL) indicated the use of well-acclimatised, thiocyanate tolerant culture having high thiocyanate removal efficiency. Uninoculated controls did not show any decrease (i.e., auto oxidation) in thiocyanate concentration confirmed that biodegradation ofthiocyTable 1: Colony characteristics 3 sec for 24 h.
of thiocyanate
Colony
Isolate
characteristics
I
Colour
Colourless
Opacity Size
Opaque 2 -3 mm Circular Entire convex Soft Motile
Shape Margin and elevation Consistency Motility Gram's reaction
degrading bacterial isolates grown on nutrient agar plates after incubation at
Gram negative short rods
Isolate 2 Colourless Translucent 1-3 m
Isolate 3 Yellow
Irregular Entire flat Soft Motile
Opaque mm Circular Entire convex Soft Motile
Gram negative long rods
Gram negative short rods
I
138
Yogesh B. Patil
Table 2: Degradation of thiocyanate by bacterial consortium size 105 cells/mL and aeration rate 1000 ± 50 mLlmin). Incubation time (h)
Thiocyanate MSM + Glucose + Thiocyanate Bacterial consortium
0
(Conditions:
concentration
+
pH 7.5, temperature
30 ± 2°C. inoculum
(mglL)
MSM + Glucose + Thiocyanate (Un inoculated control)
47.33 47.04 44.37 50.89 5118 51.18 50.79 36.98 3934 31.95 27.21 24.85 50.67 5192 52.42 50.43 52.91 5118 3.5 0.5 49.11 48.81 50.62 1508 51.67 52.67 51.18 0.0
(All the values are the average of two readings)
anate was the predominant reaction taking place during thiocyanate degradation by the bacterial consortium. Stratford et aI. (1994) have proposed the conversion of thiocyanate to carbon dioxide and ammonia via cyanate by an inducible enzyme; while the sulphur moiety from thiocyanate gets oxidised to sulphates. It might be possible that the bacterial cultures, isolated in the present study, also have similar thiocyanate tolerant/removal mechanism. Details regarding development of microbial process for thiocyanate removal from industrial effluents are being investigated further. ACKNOWLEDGEMENT The author thanks the Director, Founder Director and Managing Trustee, and Head, Dept. of Env. Mgt., SIBER, Kolhapur for the laboratory facilities provided. Help offered by the laboratory staff is gratefully acknowledged. The author is thankful to University Grants Commission (UGC) for providing the minor research grant. REFERENCES APHA, AWWA, WEF 1998. Standard Methods for the Examination of Water and Wastewater. 20th Ed., American Public Health Association, Washington DC. Karavaiko, G.!.. Kondrat'eva, T.F., Savari. E.E., Grigor'eva, N. V and Avakyan, Z.A. 2000. Microbial degradation of cyanide and thiocyanate. Microbiology, 69(2): 167-173. Mudder, T.!. and Whitlock, J.L. 1984. Method for the biological removal of free and complex cyanides and thiocyanates from water. US Patent No. 4.440, 644 (to Home Stake Mines). Pati!, YB. and Paknikar, K.M. 2000. Development ofa process for biodetoxification of metal cyanides from waste waters. Process Biochemistry, 35: 1139-1151. Stratford, .1.. Dias, A.E.X.O and Knowles, C.J. 1994. The utilization of thiocyanate as a nitrogen source by a heterotrophic bacterium: The degradative pathway involves formation of ammonia and Microbiology, 140: 2657-2662. Westley, J. 1981. Cyanide and sulfane sulfur. In: Vennesland, B. (eds.) Cyanide in Biology, Academic pp. 20 I.
tetrathionate Press, London,
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