DEGUMMING OF RAW SILK FABRIC WITH HELP OF MARINE...

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American Journal of Biochemistry and Biotechnology, 2013, 9 (1), 12-18 ISSN: 1553-3468 ©2013 Science Publication doi:10.3844/ajbbsp.2013.12.18 Published Online 9 (1) 2013 (http://www.thescipub.com/ajbb.toc)

Corresponding Author: Shaon RayChaudhuri, Department of Biotechnology, School of Biotechnology and Biological Sciences, West Bengal University of Technology, BF-142, Sector-I, Salt Lake, Kolkata-700064, India

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DEGUMMING OF RAW SILK FABRIC WITH HELP OF MARINE EXTRACELLULAR PROTEASE

1Das Sumana, 2Mathummal Sudarshan, 3Ashoke Ranjan Thakur and 1Shaon RayChaudhuri

1Department of Biotechnology, School of Biotechnology and Biological Sciences, West Bengal University of Technology, BF-142, Sector-I, Salt Lake, Kolkata-700064, India

2Inter University Consortium, Sector-III/Plot LB-8, Bidhan Nagar, Kolkata-700098, India 3Vice Chancellors’s Unit, Techno India University, EM-4/1, Sector-V, Salt Lake, Kolkata-700009, India

Received 2012-11-23, Revised 2013-01-03; Accepted 2013-02-04

ABSTRACT

Protease secreting microbe was isolated and characterized on the basis of their morphological, biochemical, physiological and 16S rDNA based molecular properties. The extracellular protease was quantified and characterized. Protease was used for different time (4, 8, 12 and 24 h) at different temperature (RT and 37°C) for optimization of the degumming process for raw silk fabric with enzyme dosage (0.2-1 unit/cm2 of fabric). Post-enzymatic treatment, the fabric quality and texture was compared with conventionally treated as well as untreated fabric in terms of degumming loss, tensile strength and yarn count and colour fastness to light/water. The isolate SM1 (Bacillus thuringensis) was able to grow in Carbon Minimal Salt Medium (CMSM) with jaggery or tamarind as the carbon source (0.3% w/v). Energy Dispersive X-Ray Fluorescense (EDXRF) data showed intracellular accumulation of heavy metal by the isolate. Extracellular protease was able to degum silk fabric within 4 h at RT with enzyme concentration of 0.8unit/cm2 and the maximum degumming loss was 21.72%. Post enzymatic degumming, a shiny texture was observed under Environmental Scanning Electron Microscope (ESEM) and the yarn volume also increased. Utilization of CMSM made the process cost effective during large scale application. Intracellular metal accumulation and growth in a wide range of temperature and pH made the isolate a potential candidate for bioremediation. Extracellular protease with significant degumming property could be used as an eco friendly approach as compared to the conventional chemical treatment. Keywords: Marine Coast, Protease, Bioremediation, Degumming

1. INTRODUCTION

Larva of some insects and arachnids produce silk to fabricate structures such as cocoon, webs, nets and egg stalk. However, silk thread spun by the larva of silk worm, Bombyx mori is commercially important and has a great value in textile industry. Some of the properties like, fitness, strength, elasticity, dye ability, softness, flexibility, smooth feeling, lusture, elegance and grace make silk fiber valuable in textile industry (Trotman, 1970). Silk fiber consists of the two fibrous proteins, fibroin and a gummed amorphous protein named sericin, which cements the fibroin fibers together. Fibroin and sericin proteins are present in

about 75 and 25% of total weight respectively. Fibroin protein is a high molecular weight polypeptide (~ 350KDa), composed of glycine, alanine and serine in molecular ratio 3:2:1, with a six residue repetition of -(Gly-Ser-Gly-Ala-Gly-Ala)n (Zhou et al., 2000). Three types of silk fiber conformations are found in nature: a helical conformation, an antiparallel β-sheet and a random coil without definite order. The most commonly found structure is antiparallel β-sheet (Zhang et al., 2002), where Gly side chains are extended from one surface and it’s Ser and Ala side chains are extending from the other surface. Silk fibroin also has a region where bulk residues such as Tyr, Val, Arg and Asp are present.

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Degumming is a process where sericin is totally removed from the fibroin wall to obtain shine, smoothness and other properties (Freddi et al., 2003). A series of steps are involved in the silk processing: reeling, weaving, degumming, dyeing/printing and finishing. After degumming, the silk fiber becomes shinny and its elasticity improves. The post degumming condition of silk fiber, such as handling, lusture and rubbing behavior is greatly dependant on the quantity of sericin remaining on the silk fibroin. In conventional process silk fiber is boiled in an aqueous solution containing soap, alkali, synthetic detergent and organic acids (Bianchi and Colonna, 1992; Freddi et al., 1996). Nowadays in batch degumming process soap is replaced with synthetic detergent to compensate the acidity of sericin hydrolysis. During conventional silk degumming process associated hydrolytic degradation of fiber leads to change in physical properties such as dull appearance, surface fibrillation and tensile strength (Freddi et al., 2003).

Enzymatic treatment of silk fiber as an alternative of conventional process is now in focus. Alkaline proteases perform better than other proteases (acid and neutral) with respect to uniform sericin removal and improvement of silk quality. In comparison with conventional process there are certain drawbacks which are found in enzymatically degummed silk fiber quality: higher shear and bending rigidity, lower fullness and softness to handle, remnant of the sericin at cross over points between wrap and weft (Chopra et al., 1996). Inspite of lower performance and higher cost of enzyme compared to chemical, enzymatic treatment attract the attention of scientists and technologists for the ecofriendly aspect of the process (Duran and Duran, 2000; Gubitz and Cavaco-Paulo, 2001). Enzymatic degumming process would save the resources in terms of water, energy, chemicals and reduce the cost of effluent treatment.

The present study focuses on the application of marine microbial protease in degumming of raw silk fabric. The aim of the study was: (i) Isolation of an industrially important protease producing strain and its characterization, (ii) Characterization of the extracellular protease, (iii) Studying the degumming kinetics and (iv) Developing an enzyme based degumming process which can compete with conventional treatment in terms of post treatment quality of fabric. Here the quality of silk fabric is reported by degumming loss calculation, tensile strength, colour fastness to light and water, yarn count and Scanning Electron Microscopic (SEM) analysis.

2. MATERIALS AND METHODS

2.1. Sample Collection and Isolation of Extracellular Protease Producing Pure Isolate

Water sample was collected from coastal region of Mandarmani (21° 37.012’N/87° 29.881’E), West Bengal, India and 50 µL of water was spread on milk media (Adarsh et al., 2007) plate to isolate protease secreting strain. The protease producing strain was subsequently purified by repeated streaking. Later the culture was maintained in Luria Bertenni broth (LB) at 37°C with shaking at 150 rpm. It was stored at -80°C as 50% glycerol stock for long term and on LB agar plate for short term.

2.2. Characterization of Isolate

Morphological structures and Gram characteristics were confirmed using light microscopy (1000X magnification on a Zeiss Axiostar Plus microscope) as well as Environmental scanning electron microscopy (FEI QUANTA 200 MARK 2 at 15 kV ) (Adarsh et al., 2007). Gram character was reconfirmed by Real time PCR analysis (Shigemura et al., 2005). Detailed biochemical (DNase, oxidase, lipase, lecithinase, catalase and amylase) and physiological (optimum pH and temperature for bacterial growth, growth kinetics, utilization of different substrates as carbon source, antibiotic sensitivity and metal tolerance ability) characterizations were done following the protocol of (Nandy et al., 2007; Roy et al., 2008).

DNA extracted from the isolate by modified alkali lysis method (RayChaudhuri et al., 2006) was amplified using the following primer: Forward primer -5’ AGA GTT TGA TCA TGG CTC 3’ and Reverse primer-5’ CTA GCG ATT CCG ACT TCA 3’ (RayChaudhuri and Thakur, 2006). The 50 µL reaction mixture was prepared [Template- 30 ng, 0.25 µL (from 50pmole/µL stock) of each of forward and reverse primer, PCR Ready Mix (Sigma)-12.5 µL and triple distilled sterile water to make up the volume to 25 µL] and was subjected to 40 cycles according to the program-Initial denaturation 92°C-2 min then 40 cycles of 92°C -1 min; 50°C-1 min; 72°C-2 min and final hold at 4°C. 16SrDNA PCR product of isolate SM1 was sequenced using Applied Biosystems (ABI) partial sequencing kit and the sequences were subjected to BLAST analysis followed by GenBank submission and phylogenetic tree construction using neighbour joining method.


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2.3. Determination of Enzyme Activity

Using Azocasein as a substrate, effect of temperature and pH on the activity of extracellular protease of the isolate was determined by the method mentioned by (Malathu et al., 2008). One unit of activity was defined as the amount of enzyme required to produce an increase of 0.1 absorbance (OD at 440 nm).

Extracellular cell free supernatant was incubated within the range of 4-60°C for 12 h and enzyme assay was measured using Azocasein method.

For observing the effect of pH, extracellular cell free supernatant was taken and the pH was adjusted with 6N HCl and 4N NaOH within the range of 2-12 and protease activity was measured by Azocasein method at 440 nm.

2.4. Optimization of Silk Fabric Degumming Conditions

Raw silk fabrics were incubated for different time intervals (4, 8, 12 and 24 h) at RT as well as 37°C to observe the degumming loss with enzyme in material-to-liquor ratio 1:200. Inactivation of enzyme was carried out in hot water bath followed by cold water and the sample was air dried. After that, raw silk fabrics were incubated with different enzyme dosage (0.2-1 unit/cm2 of fabric) for 4 h at RT (optimum condition). Optimum pH and temperature for the enzyme function was maintained throughout the tests. All degumming tests were performed in triplicate.

2.5. Comparative Study of Silk Degumming

Raw silk fabric was incubated with enzyme at optimum condition of silk degumming (in terms of time, temperature and enzyme concentration) in material-to-liquor ratio of 1: 200. In case of control sample LB was used instead of enzyme and incubated under same condition. In case of alkaline bath wash, silk fabric was treated with 10 g L−1 soap and 2 g L−1 sodium carbonate in a liquor ratio (1:30) at 90-95°C for 45 min. All of the degummed (enzymatic treatment, control and alkali wash) silks were then washed with hot water followed by cold water and air dried. Weight of the cloths were taken pre and post incubation and weight loss due to degumming was calculated (Nakpathom et al., 2009).

Colour fastness to light and water, tensile strength and yarn count of four silk samples were determined by standard ISO method: BS EN ISO 105 BO2 BW-4, BS EN ISO 105-E01, BS EN ISO 13934:II and ISO 7211-5 respectively.

Raw silk treated with extracellular enzyme of SM1 (as treated), alkali washed, LB broth (as control) and untreated samples were further used for Scanning Electron Microscopic (SEM) analysis. Change in the fabric condition was observed by comparing the Scanning Electron Micrograph (SEM) of treated and untreated samples [FEI QUANTA 200 MARK 2 at 10 and 15kV acceleration].

3. RESULTS

3.1. Isolation and Characterization of Isolate

Depending on the casein degrading ability on the milk media plate, SM1, a protease producing strain was isolated from Mandarmani coastal region, with 99.86% identity with Bacillus thuringensis [FJ377720] at the molecular level (16SrDNA sequence). SM1 was a spore forming, Gram positive diplobacilli. Upon environmental scanning electron microscopy, a connected pattern was observed between two and more cells, which may be due to presence of pilli. SM1 did not possess capsule or flagella. The biochemical characterization of the isolate revealed it to be catalase, oxidase and DNase positive with no growth on lecithinase medium (Hichrome Aureus media). It was able to degrade starch, but did not produce lipase. Thus two industrially important enzymes like protease and amylase were found in SM1.

The pH and temperature profile for the isolate indicated that it was able to grow within a wide range of temperature (20-40°C) and pH (6-12) with the optimum growth at 37°C and pH of 7. Under optimum condition of growth SM1 showed an efficient growth with a lag phase of 2 h, followed by extended logarithmic phase of 8 h and stationary phase. Variation in growth in presence of different substrates was observed, with maximum growth in LB followed by Tamarind and Jaggery (0.3% w/v as carbon source in carbon minimal salt media).

Isolate was resistant to following antibiotics ampicillin, cloxacillin, ceftazidime, metronidazole, polymyxin B, rifampicin and trimethoprin and sensitive to tetracycline, doxycycline hydrochloride, gentamicin, norfloxacin and ciprofloxacin.

Isolate was able to grow in presence of a wide range of metal salts, indicating its tolerance to different metals. SM1 was also able to accumulate five common environmental contaminants, among which it showed highest accumulation in case of Pb (11268.28ppb), followed by Cr (18.33ppb), Ni (9.53ppb), Cu (3.23ppb) and Co (1.95ppb) as evident from the Energy Dispersive X Ray Fluorescence (EDXRF) analysis.


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Table 1. Analysis of silk quality on the basis of different parameter: colour fastness to light and water, yarn count and tensile strength of treated and untreated silk fibers

Yarn count (in Denier) Tensile strength (in Kg) Degumming Colour fastness ---------------------------------------- -------------------------------------- Methods to water a Warp Weft Warp Weft Chemical treatment 4 20.1 34.5 7.7 26.9 Enzymatic Treatment 4 64.5 64.3 11.2 29.6 Treated with LB 4 34.3 28.7 12.1 30.1 Untreated 4 34.2 64.1 11.3 30.3 a; The rating scale of colour fastness to light and water is from 1 (poor) to 5 (excellent)

(a) (b)

Fig. 1. Representing the degumming kinetics: effect of enzyme dosage and time, a. the maximum (21.72%) percentage of

degumming loss was observed within 4 h at RT, when raw silks fabrics were incubated for different time intervals (4, 8, 12 and 24 h) at RT as well as 37°C to observe the degumming loss with enzyme in material-to-liquor ratio 1:200. b. revealed that when silk fabric was incubated at RT for 4 h with different enzyme dosage (0.2-1 unit/gm of fabric), it was found that at dosage of 0.8 units/gm of fabric, degumming loss (%) was maximum.

3.2. Determination of Enzyme Activity

The extracellular protease was stable at wide range of temperature (4-60°C) and pH (4-9) with optimum performance at 40°C and pH 7.

3.3. Silk Degumming 3.3.1. Study of Degumming Kinetics: Effect of

Enzyme Dosage and Time

Degumming loss at different incubation periods clearly indicated the optimum incubation time to be within 4 h (Fig. 1a). Figure 1b reflected 0.8unit/cm2 of enzyme to be optimum for degumming. The bar height corresponded to the degumming loss.

Degumming loss of silk fiber with alkali soap wash and enzymatic treatment was 28.0 and 21.72% respectively, whereas the degumming loss by LB was negligible 4.2%. The efficiency of protease secreted from SM1, in degumming of raw silk was quite satisfactory; it helped to remove the sericin from the raw silk fiber, which was also observed during SEM analysis. SEM analysis revealed that sericin was removed from the fabrics which were treated both conventionally as well as enzymatically (Fig. 2). The gum material stuck on fabric (as observed in untreated sample) was found to be removed in treated fabric and thus it leading to shiny appearance. The yarn also became loose with associated volume increase (Table 1).


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(a) (b)

(c) (d) Fig. 2. Representing the scanning electron micrograph image of (a) untreated; (b) treated with LB only; (C) treated through conventional

method and (d) enzymatic treated raw silk fabric. It was observed that volume of raw silk fiber was increased by enzymatic treatment rather than untreated sample. This is due to removal of sericin by the enzyme and the texture become shiny

4. DISCUSSION

The characterization of the isolate reveals its Gram characteristics and ability to survive under stressful environment like marine coastal saline water. The

presence of enzymes like catalase and oxidase also help them to survive in adverse condition. Amylases especially alkaline amylases are used in detergent industry and also have use in food and beverages (baking), brewing, starch and alcohol industries. As amylase degrades starch into


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amylose and amylopectin, it is also used as digestive aid. Utilization of Tamarind and Jaggery as the carbon sources (0.3% w/v as carbon source in carbon minimal salt media) for microbial growth makes the process economical at large scale. The extent of accumulation of heavy metal makes the isolate a potent bioremedial candidate to remove the toxic metal from the contaminated environmental site. Ability of the isolate to grow in a wide range of temperature and pH makes it suitable for various industrial and bioremedial applications.

Silk fiber contains 30% Sericin. In terms of measurement of degumming loss, conventional method almost reached the target level of sericin removal, whereas the enzymatic treatment was far beneath the target level. This may be due to the temperature in which degumming is performed; sericin needs a higher temperature for removal from the fiber stick. The mechanical agitation could also help in improving the degumming quality which was absent during lab scale enzymatic treatment. Degumming loss was negligible (4.2%) in absence of enzyme, though it is well known that water alone can remove sericin at higher temperature (110-120°C) under high pressure.

The change in volume (increase/shrinkage) of warp and weft is known as take-up, which depends on the fabric texture and treatment. Warp and weft counts depend on the fabric condition. When the fabric was observed under SEM, the yarn width was found to increase after enzymatic treatment, whereas after conventional treatment the width of yarn was found to shrink (decreased in width). This may be due to the higher twist, which was also reconfirmed by the value of yarn count. Tensile strength remained same after enzymatic treatment of silk in comparison with untreated sample, but it decreased for chemical treatment. Using harsh chemicals may be the cause of deterioration of silk quality, which was reflected as shrinking of the weft and tensile strength. There was no difference in colour fastness to water for four processes, whereas the quality of silk after enzymatic treatment decreased in colour fastness to light (data not shown). Use of mild alkali in lower amount with enzyme might improve the quality of colour fastness to light. Inspite of all these points, the ability of this protease for degumming is unquestionable, it could be an environment friendly approach as compared to the use of surfactants for this purpose.

5. CONCLUSION

The isolate SM1 (Bacillus thuringensis) isolated from Mandarnmani coastal region in West Bengal, India could be

used as a potent bioremedial candidate for its intracellular metal accumulating property (Chowdhury et al., 2011) The extracellular protease of the isolate was able to perform degumming of raw silk fabric in significant amount. After the enzymatic treatment, texture of the fabric became shiny and the volume of the yarn increased. The other properties of the fabric like tensile strength, yarn count, colour fastness to water either improved or remained unchanged after the enzymatic treatment in comparison with untreated sample, except for colour fastness to light. With some minor modification in the process, like introduction of mechanical agitation or use of mild alkali, the enzymatic treatment procedure could be improved.

6. ACKNOWLEDGEMENT

The researchers acknowledge the support of the West Bengal University of Technology for the computational facility and the laboratories; University Grant Commision-Inter University Consortium for the student fellowship as well as Department of Atomic Energy, Government of India under the BRNS scheme for financial assistance. The authors would like to thank the World Bank under the TEQIP program for providing the publication fee.

7. REFERENCES

Adarsh, V.K., M. Mishra, S. Chowdhury, M. Sudarshan and A.R. Thakur et al., 2007. Studies on metal microbe interaction of three bacterial isolates from East Calcutta Wetland. Online J. Biol. Sci., 7: 80-88. DOI: 10.3844/ojbsci.2007.80.88

Bianchi, A.S. and G. M. Colonna, 1992. Developments in the degumming of silk. Melliand Textilberichte, 73: 68-75.

Chopra, S., R. Chattopadhyay and M.L. Gulrajani, 1996. Low stress mechanical properties of silk fabric degummed by different methods. J. Textile Inst., 87: 542-553. DOI: 10.1080/00405009608631356

Chowdhury, S., A.R. Thakur and S. RayChaudhuri, 2011. Novel microbial consortium for laboratory scale lead removal from city effluent. J. Environ. Sci. Technol., 4: 41-54.

Duran, N. and M. Duran, 2000. Enzyme applications in the textile industry. Rev. Prog. Coloration, 30: 41-44. DOI: 10.1111/j.1478-4408.2000.tb03779.x

Freddi, G., G. Allara and G. Candiani, 1996. Degumming of silk fabrics with tartaric acid. J. Soc. Dyers Colour, 112: 191-195. DOI: 10.1111/j.1478-4408.1996.tb01817.x


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Freddi, G., R. Mossotti and R. Innocenti, 2003. Degumming of silk fabric with several proteases. J. Biotechnol., 106: 101-112. DOI: 10.1016/j.jbiotec.2003.09.006

Gubitz, G.M. and A. Cavaco-Paulo, 2001. Biotechnology in the textile industry--perspectives for the new millennium. J. Biotechnol., 89: 89-90. PMID: 11500200

Malathu, R., S. Chowdhury, M. Mishra, S. Das and P. Moharana et al., 2008. Characterization and wash performance analysis of microbial extracellular enzymes from East Calcutta Wetland in India. Am. J. Applied Sci., 5: 1650-1661. DOI: 10.3844/ajassp.2008.1650.1661

Nakpathom. M., B. Somboon and N. Narumol, 2009. Papain enzymatic degumming of Thai Bombyx mori silk fibers. J. Microscopy Soc. Thailand, 23: 142-146. www.mstthailand.com/Journals/2009/M19_MN09JMSTp142_146.pdf

Nandy, P., A.R. Thakur and S. RayChaudhuri, 2007. Characterization of bacterial strains isolated through microbial profiling of urine samples. Online J. Biol. Sci., 7: 44-51. DOI: 10.3844/ojbsci.2007.44.51

RayChaudhuri, S. and A.R. Thakur, 2006. Microbial genetic resource mapping of East Calcutta wetlands. Curr. Sci. Ind., 91: 212-217. www.iisc.ernet.in/currsci/dec252006/1697.pdf

RayChaudhuri, S., A.K. Pattanayak and A.R. Thakur, 2006. Microbial DNA extraction from sample of varied origin. Curr. Sci. Ind., 91: 1697-1700.

Roy, S., K. Mishra, S. RayChaudhuri, A.R. Thakur and S. Raychaudhuri, 2008. Isolation and characterization of novel metal accumulating extracellular protease secreting bacteria from marine coastal region of Digha in West Bengal, India. Online J. Biol. Sci., 8: 25-31. DOI: 10.3844/ojbsci.2008.25.31

Shigemura, K., T. Shrikawa, H. Okada, K. Tanaka and S. Kamidono et al., 2005. Rapid detection and differentiation of Gram-negative and Gram-positive pathogenic bacteria in urine using TaqMan probe. Clin. Exp. Med., 4: 196-201. DOI: 10.1007/s10238-004-0056-x

Trotman, E.R., 1970. Dyeing and Chemical Technology of Textile Fibres. 4th Edn., Griffin, London, ISBN-10: 0852641656, pp: 378.

Zhang, H., J. Magoshi, M, Becker, J.Y. Chen and R. Matsunaga, 2002. Thermal properties of Bombyx mori silk fibers. J. Applied Polym. Sci., 86: 1817-1820. DOI: 10.1002/app.11089

Zhou, C.Z., F. Confalonieri, N. Medina, Y. Zivanovic and C. Esnault et al., 2000. Fine organization of Bombyx mori fibroin heavy chain gene. Nucl. Acids Res., 28: 2413-2419.

OnLine Journal of Biological Sciences, 2012, 12 (3), 96-107 ISSN: 1608-4217 ©2012 Science Publication doi:10.3844/ojbssp.2012.96.107 Published Online 12 (3) 2012 (http://www.thescipub.com/ojbs.toc)

Corresponding Author: Shaon RayChaudhuri, Department of Biotechnology, West Bengal University of Technology, BF-142, Sector-I, Saltlake, Kolkata-700064, India

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BACTERIAL ISOLATES OF MARINE COAST AS COMMERCIAL PRODUCER OF PROTEASE

1S. Das, 1I. Mukherjee, 2M. Sudarshan, 3T.P. Sinha, 4A.R. Thakur and 1Shaon RayChaudhuri

1Department of Biotechnology, West Bengal University of Technology,

BF-142, Sector-I, Saltlake, Kolkata-700064, India 2Inter University Consortium, Sector-III/Plot LB-8. Bidhan Nagar. Kolkata-700098, India

3Bose Institute, 93/1, Acharya Prafulla Chandra Road. Kolkata-700009, West Bengal, India 4RCM BioSolutions Private Limited, A-101, Green Woods Premium,

Kaikhali, Shibtala, Kolkata-700136, West Bengal, India

Received 2012-07-30, Revised 2012-08-02; Accepted 2012-08-04

ABSTRACT

The objective of this work was to explore and exploit the extracellular protease secreting marine microbial biodiversity of the eastern coastal region of India. Culture dependent method was applied for isolation of microbes from the marine coast of West Bengal (Digha and Mandarmani) and Andhra Pradesh (Vizag) in India. Six protease secreting isolates were screened using casein hydrolysing property as well as azocasein assay and characterized on the basis of their morphological, biochemical, physiological and 16S rDNA based molecular properties. The enzymes were used for various commercial applications at a laboratory scale. Besides milk media and Luria Bertini broth, all the isolates grew in carbon minimal salt medium with jaggeri or tamarind as the carbon source (0.3% w/v). They showed intracellular metal accumulation when grown in presence of metal salts in the medium as evident from Energy Dispersive X-ray Fluorescense Analysis (EDXRF) data. Maximum accumulation of lead was found in case of Bacillus cereus SM2. It showed equal efficiency of metal removal from solid strip at zero valent state. The isolates were also capable of complete removal of silver from exposed X-ray film after 48 hrs of incubation except for Escherichia coli SD1. Bacillus cereus SM2, isolate SD2 (closest to Bacillus pumilus) and isolate SV1 (closest to Bacillus cibi) were able to enhance the cleaning efficiency of detergent when used as additive. Use of tamarind and jaggeri as carbon source in minimal medium would make the process cost effective during large scale application. The ability to grow in a wide range of temperature and pH and accumulation of heavy metals revealed that these isolates would be potential candidates for bioremediation. Thus the marine diversity for protease production is extremely rich with immense commercial applications. Keywords: Marine Coast, Protease, Bioremediation, Degumming, Detergent-Addtive

1. INTRODUCTION

Bacteria were isolated and cultivated from all possible regions of the earth, on the basis of their habitat, diversity, ecological functions, degree of pathogenicity and biotechnological applications. 70% of the earth’s surface is covered by oceans with rich microbial diversity. About 3.6×1029 microorganisms were found in marine environments, including subsurface and harbour (Sogin et al., 2006). Marine microbes are now being looked upon as a potential source of various compounds; pharmaceutical,

nutritional supplements, agrochemicals, cosmetics and enzymes (Vignesh et al., 2011; Baharum et al., 2010). However compounds from marine sources are often available only in low quantities and hampers their further processing into commercial products (Haefner, 2003). Bioactive screening has also focused on microorganisms associated with such host surfaces and the various natural products isolated from marine invertebrates often show structural similarities to known metabolites of microbial origin (Arpigny and Jaeger, 1999; Haygood et al., 1999). The basic characteristics of the enzymes derived from the marine sources differ from their

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terrestrial counterparts due to their natural habitat. Marine microbial enzymes are reported to be more stable and active than those originating from plant and animal sources since they possess almost all characteristics desired for their biotechnological applications (Bull et al., 2000). Enzymes like protease, lipase, amylase and cellulase have immense industrial demand. Some of the marine microorganisms have enzymes which hydrolyze the polysaccharides like lignin, alginate, agar, cellulase, carrageenan and xylan (Andrykovich and Marx, 1988). They are used in biodegradation, e.g.,: Bacillus cereus, Bacillus sphericus, Vibrio furnisii and Brevundimonas vesicularis are reported to hydrolyze nylon 6 and nylon 66 (Sudhakar et al., 2007). Two γ-proteobacteria; Alcanivorax and Cycloclasticus play an important role in petroleum hydrocarbon degradation posing to be potential candidates for bioremediation at oil spill sites (Harayama et al., 2004). Bacteria attached to the environmental surface by adhesion commonly live as biofilm. Gram positive bacteria like Staphylococcus aureus, Bacillus subtilis and Gram negative organism like Pseudomonas aeruginosa, Escherichia coli are attached to the surface with help of EPS (Karunakaran and Biggs, 2011). Biofilm have both positive and negative impact on normal life, as well as at industrial scale. Biofilm formation helps in remediation of waste water in a bioreactor, degradation of recalcitrant, aquaculture, whereas the formation of biofilm in heat exchanger, pipeline, ship surface, medical and industrial device causes major problem (Kwon et al., 2002). Among all the enzymes, protease dominate the world enzyme market with 60% market share (Rao et al., 1998), 40% of which is of microbial origin (Godfrey and We, 1996). In various industrial sectors, extracellular proteases have multiple applications such as detergent additives (George-Okafor and Odibo, 2011; Kumar et al., 2008; Malathu et al., 2008); in cheese making, baking, preparation of soya hydrolysates and meat tenderization (Rao et al., 1998); extraction of metallic silver from used X-ray films (Gupta et al., 2002), cleaning of contact lenses (Power et al., 2009), degumming of coccon fiber (Arami et al., 2007); in leather industry for soaking, dehairing and bating to avoid pollution caused due to conventional methods (Tork et al., 2010; Hameed et al., 1999). The coastal areas are getting increasingly polluted by domestic, commercial, agricultural and industrial pollutants. The metal contamination of sea water is mainly due to discharge of the chemical load from various industries into the rivers and from the rivers to the sea. Some of the metals like cadmium, arsenic, lead and mercury are toxic in nature. As per reported literature, heavy metals like zinc, copper, nickel, chromium, mercury, cadmium, cobalt, lead and arsenic were found in the coastal regions of the Bay of Bengal

(ftp://ftp.fao.org/docrep/fao/007/ad894e/AD894E06.pdf). The metal accumulating (Cu, Cd, Ni, Vd) property of some marine bacteria make them potential sources for bioremediation (De et al., 2006; Miranda and Rojas, 2006). Bacterial isolates from marine waters demonstrated varying degrees of resistance to antibiotics like Chloramphenicol, ampicillin, streptomycin, tetracycline and kanamycin (Manivasagan et al., 2011; Souza et al., 2006). Certain bacterial isolates from marine coast of West Bengal have already been isolated and characterized. The focus of this study is on isolation and characterization of protease producing marine bacteria for bioremediation with the long term goal of industrial applications.

2. MATERIALS AND METHODS

2.1. Site of Isolation

Water samples were collected from the coastal regions of Digha and Mandarmani in West Bengal and Vizag in Andhra Pradesh, India to screen for extracellular protease producing microbes. The collection was done in plastic containers and transferred to laboratory at room temperature and the process of isolation was initiated immediately.

2.2. Cultivation Medium and Growth Conditions

Since the objective was to isolate extracellular protease secreting bacteria, milk medium plates were used as selective media (Adarsh et al., 2007). 50 µL of water sample collected from the different sites were directly spread on the milk media plates and incubated at 37°C for overnight. Repeated streaking on LB agar was adapted for isolation of pure culture. The maintenance and characterization of the pure isolates was carried out in LB medium whereas the substrate profile was carried out using substrate strips (Himedia Carbohydrate kit, Code No. KB009) as per manufacturers protocol as well as in carbon minimal salt medium using different substrates. Unless mentioned specifically, the general culture condition was maintained at 37°C with shaking at 150 rpm.

2.3. Morphological Characterization

The initial identification of the pure isolates was on the basis of their morphology using light microscopy (1000X magnification on a Zeiss Axiostar Plus microscope) as well as Environmental scanning electron microscopy (FEI QUANTA 200 MARK 2 at 15 kV ) (Adarsh et al., 2007). The Gram nature of the isolate was determined by differential staining as per standard procedure. It was reconfirmed through Real-time PCR as



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reported by Shigemura et al. (2005). Endospore, capsule as well as acid-fast staining was performed using standard protocol followed by observation under a Zeiss Axiostar Plus microscope.

2.4. Biochemical Characterization

The isolates were checked for their ability to produce enzymes like DNase, oxidase, lipase, lecithinase, catalase and amylase. The tests for the first five enzymes were done according to the protocol. The amylase test was done on 3% starch agar plate and incubated at 37°C for overnight followed by addition of iodine solution to the plate.

2.5. Physiological Characterization

The optimum pH and temperature for bacterial growth, growth kinetics and utilization of different substrates as carbon sources was done according the protocol. The biofilm forming ability was checked following the protocol of O'Toole and Kolter (1998).

2.6. Antibiotic Assay

The response of the isolates towards 18 different antibiotics (HiMedia) was checked according to the procedure reported.

2.7. Heavy Metal Tolerance

The Minimum Inhibitory Concentration (MIC) for metal salts; AgNO3, Al(NO3)3.9H2O CdSO4.8H2O, CoCl2.6H2O, Cr2O3, CuSO4.5H2O, FeSO4.7H2O, HgCl2, NiCl2.6H2O, Pb(NO3)2 and ZnSO4.7H2O were determined as per the protocol of Adarsh et al. (2007). Among the eleven metal salts checked for MIC determination, the intracellular accumulation using EDXRF analysis (Adarsh et al., 2007) (Jordan Valley EX 3600 EDXRF system) was carried out only for treatment with common environmental contaminants like Cu, Cr, Co, Ni and Pb.

2.8. Molecular Characterization

The DNA extracted from the pure isolates was used for PCR amplification of 16S rDNA gene (RayChaudhuri and Thakur, 2006). The PCR product was sequenced using ABI (Applied Biosystems) partial sequencing kit. The sequences were subjected to BLAST analysis. The novel sequences were submitted to GenBank and phylogenetic tree was constructed using neighbour joining method.

2.9. Continuous Production of Enzyme by Immobilization on Inert Matrix

Enzyme (amylase and protease) from isolate SV1 were used as additive along with other enzymes to detergent for cleaning efficiency enhancement

(RayChaudhuri S, Novel indigenous sources of enzymes as detergent additives, Indian Patent No 599/KOL/2010 dt June 1 2010; PCT/IB2010/001816 dt July 24, 2010; U.S. Patent Application No.-13/126,109 dt 26-April 2011). To get the continuous supply of enzymes, SV1 was immobilized on two type of inert matrices; corrugated sheet as well as hay. Protease was estimated by azocasein method as reported by Malathu et al. (2008) and amylase production was calculated by DNS method as reported by Anto et al. (2006). Enzyme production at regular intervals of 1 h was compared for immobilized matrix (hay as well as sheet) and shake flask culture (incubated at 37°C with continuous shaking at 150 rpm).

2.10. Application of Crude Extracellular Protease

The extracellular protease from all isolates were checked for different applications; removal of metal from zero valent state, removal of silver impregnated in gelatin layer on exposed X-ray film and wash performance analysis.

2.11. Recovery of Precious Metal

Silver is impregnated within the gelatin layer of X-ray film. The strain with gelatinase activity could be used for recovery of silver. To check the ability of the strains under the current study, 1×1 cm2 exposed X-ray films were incubated with 0.2 units of enzyme for 48 h at room temperature. Proteinase K (0.2 units) was considered as the standard protease under the same conditions whereas distilled water was taken as the negative control. Considering the heavy metal accumulating ability of SM2, the impact of the strain on metal strips (zero valent state) of silver (Ag) and gold (Au) was tested. 1% inoculum of overnight grown SM2 was added to 3 mL of LB in tubes and incubated at 37°C at 150 rpm for overnight. Next Day, the silver (0.7×0.8 mm2) and the gold strip (0.9×0.8 mm2) were dipped into two different sets of SM2 culture and 1ml of 1X LB was added to the respective tubes. To sustain their growth, 1ml of 1X LB medium was replaced every 12 h retaining the cells in each tube. This condition was maintained for 2 months. 109 cells from each condition were harvested, washed with 0.1N HCl thrice followed by Phosphate Buffered Saline (PBS) thrice and finally resuspended in PBS. A second set of gold and silver strip treated cells was prepared by washing with only PBS. Both of these sets were analyzed using EDXRF. The strips were taken out from the respective tubes. The treated and control strips (untreated) were then washed with distilled water, incubated in 1 mL bleach for 15mins and again washed with distilled water thrice before drying. The surface of the strips was observed by SEM (FEI QUANTA 200 MARK 2 at 15 kV) analysis.



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2.12. Wash Performance Analysis

Wash performance analysis was performed using extracellular protease from individual isolates as per the protocol of Malathu et al. (2008). Among the six isolates, extracellular protease from isolate SM2 showed better cleaning efficiency (data not shown). To optimize the enzyme formulation for cleaning efficiency enhancement, two combinations were worked out. 3 mL cell free supernatant (SD2/SD4) as lipase sources was added to 6U protease from SM2 per gram of detergent. Wash performance for these two combinations were tested for stained cloths post washing and drying using densitometric scanning [Molecular analyst (BIORAD)] and the efficiency was expressed as percentage of stain removed.

2.13. Market Survey

As per the densitometric scanning data, the combination of SM2+SD2 was further selected for market survey. Cell free supernatant containing extracellular protease and lipase were concentrated by lyophilization. Each gram of detergent was mixed with 6U protease as well as 2.25U lipase and air dried. The detergent with and without additive were then supplied to 50 different household to get a feedback on the wash performance efficiency as per the supplied questionnaire.

3. RESULTS AND DISCUSSION

3.1. Isolation of Bacterial Strains

Five isolates were obtained with clearing zone around the colony due to casein degradation. Four were isolated from West Bengal marine coast. SM2 was isolated from water of Mandarmani coastal region whereas SD2, SD3 and SD4 were isolated from Digha coast. SV1 was isolated from Rishikonda coastal water (Vizag) in Vishakhapatnam andhra Pradesh, India. SD1, non-casein degrading strain was isolated from Digha coast.

3.2. Characterization of the Isolates

SM2 was diplobacilli; SD1, SD2 and SD4 were bacilli while SD3 and SV1 were bacilli in chain. The Gram nature was also reconfirmed by the Real time PCR based detection method. Except SD1 all other isolates were gram positive while only SD1 showed presence of capsule. One of the important facts observed was the presence of endospores in all the isolates; this finding can be correlated with their survivability in the extreme condition of a marine environment. They all were negative for acid fast staining. They were all catalase and oxidase positive (Table 1). These two enzymes were reported to have important functions in defence system

of an organism, supporting the survivability under adverse conditions. The presence of enzymes like DNase could be important in host prey interaction thus defending the organism. Presence of enzymes like amylase, lipase and protease (Table 1) would be important from the point of application in detergent, leather, pharmaceutical and many other industries. The biochemical characteristics of the isolates were listed in Table 1. To check its protease producing status azocaesin assay was performed using cell free supernatant from each isolate. The result showed extracellular protease activity in case of each isolate in the following descending order of production: SD1 (10.23+0.381 U), SM2 (3.425+0.106 U), SD2 (3.275+0.318 U), SD4 (2.8+0.283 U), SD3 (2.4+0.141 U), SV1 (2.325+0.247 U). This clearly indicated that the method used for analyzing protease at the qualitative level was insensitive for the enzyme produced by isolate SD1. The method of plate clearing reveals only caesinase activity. Any other protease activity would go unnoticed using this method. Scanning Electron microscopy revealed pilli like structure between two or more cells for quite a few isolates (Fig. 1a-f). This structure allows the stacking pattern of cells which are reported in many groups of bacteria (Sattley et al., 2008). The pH profile for isolates indicated that all the isolates except SD1 could tolerate pH variation in the range of 6-12. Isolate SD1 was found to grow in a wider range of pH from 4-12. The optimum pH was at 7 for isolate SM2 and SD1; 7.5 for SD3 and SV1 and 8.5 for SD2 and SD4. Similar condition was observed for the temperature adaptation; the isolates were found to grow in a range between 20-40°C. The optimum temperature was 30°C for isolate SM2 and SD3; 37°C for SD1, SD2 as well as SV1 and 40°C for SD4. This adaptation was an indication for their survival under different environmental conditions and thus their implementation in various aplications. Variation in growth in the presence of different substrates were observed. Maximum growth was observed in LB followed by Tamarind and Jaggery. The later two were used as cheaper sources compared to conventional media for large scale growth thus making the process cost effective during large scale applications. They were able to utilize different carbohydrates for their growth (Table 1). All of the strains showed good biofilm forming ability from the initial day, except for SM2, SD4 and SD1. The biofilm forming ability of SD1, SM2 and SD4 were observed from 2nd, 4th and 5th day onwards respectively (Fig. 2) using standard crystal violet staining method followed by optical density measurement at 620nm. The biofilm forming ability would help in their usage in packed bed bioreactors for bioremedial as well as enzyme production purposes.



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Table 1. Detailed characterization of the six isolates under optimal conditions of growth SM2 SD1 SD2 SD3 SD4 SV1 Morphological Gram Nature + - + + + + characterization Endospore staining + + + + + + Capsule staining - + - - - - Acid Fast staining - - - - - - Biochemical Protease + - + + + + characterization Lipase - - + - + - Amylase + - - + - + DNase + - + No growth + No growth Oxidase - + + + + + Catalase + + + + + + Lecithinase - - - No growth - - Molecular GenBank FJ377719 FJ377721 FJ377718 FJ377722 FJ377724 FJ377723 Accession No. Bacillus Escherichia Bacillus Bacillus characterization Closest Neighbour cereus coli pumilus cibi Bacillus pumilus Bacillus cibi (% similarity) (100) (100) (99.09) (99.95) (99.12) (99.91) Antibiotics Resistant A, Mt, Tr, Mt, Cx, Pb, Mt, Pb, Ca. Mt Mt, Pb, Ca Mt Cx, Pb, Ca, R. Va, R. Sensitive T, Cf, Do, G, Nx. T, Cf, Do, G, Nx, T, Ce, Cf, Do, A, T, Ce, Cf, Do, T, Ce, Cf, Do, A, T, Ce, Cf, C, Ce, Tr, Ca Tr, G, Nx, N, R. A, Tr, G, Nx, N, Tr, G, Nx, N, R, Ro, Do, A, Tr, G, R, Pb, Ca, Ro, C, Cq, Va Nx, N, R, Pb, Ca, Cx, C, Cq, Va Ro, Cx, C, Cq, Va Substrate Carbohydrates Maltose, Fructose, Lactose, Xylose, Lactose, Xylose, α-methyl Fructose, Dextrose, α-methyl Dextrose, Trehalose, Maltose, Fructose, Maltose, Fructose, deglucoside, Trehalose, Sucrose, deglucoside, α-methyl deglucoside, Dextrose, Galactose, Dextrose, Trehalose, Melibiose, L-Arabinose, Esculin. Esculin. utilization Raffinose, Trehalose, Melibiose, Sucrose, Sucrose. Mannose, Melibiose, Sucrose, L-Arabinose, Adonitol, α-methyl L-Arabinose, Mannose, Mannose, deglucoside, Glycerol, α-methyl Glycerol, Salicin, Rhamnose, deglucoside, Rhamnose, Mannitol, Adonitol, Cellobiose, Esculin, Esculin, D-Arabinose, α-methyl deglucoside, D-Arabinose, Citrate, Malonate. Rhamnose, Cellobiose, Malonate. Esculin, D-Arabinose, Citrate, Malonate.

Each isolates were grown under its optimal condition and detailed morphological, physiological (antibiotic sensitivity), biochemical as well as molecular characterization (16SrDNA sequence based phylogenetic analysis) were performed. The sequences being novel were submitted to GenBank and the accession numbers were mentioned above. The abbreviations for the antibiotics were as follows: A for Ampicillin, Cq for Cephadroxil, C for Chloramphenicol, Cx for Cloxacillin, Ce for Cephotaxime, Ca for Ceftazidime, Cf for Ciprofloxacin, Do for Doxycycline Hydrochloride, G for Gentamicin, Mt for Metronidazole, N for Neomycin, Nx for Norfloxacin, Pb for Polymyxin B, R for Rifampicin, Ro for Roxithromycin, T for Tetracycline, Tr for Trimethoprin, Va for Vancomycin.

3.3. Growth Profile

The growth profile determined in enriched medium (LB) indicated that among all the isolates, SM2 and SD1 exhibited more rapid growth with extended logarithmic phase (8 h) in comparison to others (Fig. 3). The remaining four isolates showed a similar growth pattern with about 5 h of logarithmic phase.

3.4. Antibiotic Sensitivity

The complete profile for sensitivity towards different antibiotics would further help in characterizing the isolates and designating them as distinctly different from one another. Since the future objective of this study would be to use these isolates for bioremedial purpose, it might result in a situation where the released microbes from the bioremediation plant cause adverse health effects among the workers there. The knowledge of antibiotic sensitivity would be important in this context. Table 1 indicates the different responses of the six isolates towards various antibiotics. For some antibiotics like tetracycline, ceftazidime, doxycycline, metronidazole, gentamycin and norfloxacine, the isolates exhibited similar responses which could be due to their common site of origin.

3.5. Heavy Metal Tolerance

All the isolates were found to tolerate a wide range of metal salts namely Al, Ni, Pb, Fe, Zn. Among all isolates, SD1 was found to show maximum tolerance towards the above mentioned metals. An important finding was its Minimum Inhibitory Concentration (MIC) of about 14mM for CoCl2.6H2O, 11mM for FeSO4.7H2O and 14 mM for NiCl2.6H2O. All isolates being tolerant to a number of metals, the next step was to find the fate of these metals within the cell. Since the main objective was to apply the isolates for remediation, the primary objective was to investigate their metal accumulating property. For five of the metals viz. Cr, Cu, Co, Ni and Pb, the intracellular accumulation was checked using EDXRF analysis for the six isolates. Here the untreated (normal) cells were taken as negative control in order to assess the intracellular accumulation (Table 2). It was observed that intracellular accumulation of lead was highest in all the isolates as compared to other metals. Copper and chromium were also accumulated in significant amounts in some of the isolates, whereas accumulation of cobalt and nickel was considerably less.



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(a) (b)

(c) (d)

(e) (f)

Fig. 1. Environmental scanning electron microscopic images of the marine isolates visualized using Environmental Scanning

Electron Microscope (FEI QUANTA 200 MARK 2). (a-f) Represents the scanning electron micrographs of the six isolates at the following magnification:, SM2 (20,000X), SD1 (12,000X), SD2 (24,000X), SD3 (6000X), SD4 (24,000X) and SV1 (12,000X) respectively. Figure a, b and d show distinct pilli like structure existing between cells



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(a) (b)

(c) (d)

(e) (f) Fig. 2. Biofilm forming ability of the marine isolates. Figure depicting the biofilm forming abilty of marine isolates on polysterene

plates as determined using standard crystal violet staining method followed by optical density measurement at 620 nm. All of the strains showed good biofilm forming ability (O.D lies between 0.2-0.7) from the initial day, except for SD1, SM2 and SD4. The biofilm forming ability of SM2 and SD4 was observed from 4th and 5th day respectively. SD1 did not show any biofilm forming ability on the 1st day



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Fig. 3. Growth curve of the six isolates under optimum condition. Figure depicting the growth profile of the six isolates in enriched

medium (LB broth). 1% inoculum was transferred to sterile LB broth from confluent overnight grown pure culture. At 0 h of incubation (immediately after adding the inoculum) the O.D was observed to be 0. Then it was incubated under 150 rpm shaking condition at 37°C. At regular intervals of 1 h, the growth was checked by measuring the Optical Density at 660 nm (Beckman UV-Vis spectrophotometer). SD1 and SM2 showed similar patterns of growth with stationary phase onset at the 11th h of growth for the first isolate and at the 13th h for the second. SD2, SD3, SD4 and SV1 show similar growth patterns up to the 7th h of growth with stationary phase at the 8th h for isolates SD3 and SD4 and at 10th h for SV1 and SD2. This variation under optimum growth condition for the isolates points towards their varied identity at the molecular level

Table 2. Table representing the extent of metal accumulation within the cell of the isolates using EDXRF analysis Metal Conc. (in PPb) Co Cr Cu Ni Pb SM2 1.51 11.04 7.00 0.2 36128.85 SD1 2.89 9.09 0.68 0.95 13070.69 SD2 2.83 5.98 34.80 0.68 3214.42 SD3 0.46 0.89 20.82 7.84 4838.24 SD4 0.50 10.04 57.90 0.30 5045.91 SV1 0.60 4.13 0.80 3.47 1989.42 Ceftazidime, Cf for Ciprofloxacin, Do for Doxycycline Hydrochloride, G for Gentamicin, Mt for Metronidazole, N for Neomycin, Nx for Norfloxacin, Pb for Polymyxin B, R for Rifampicin, Ro for Roxithromycin, T for Tetracycline, Tr for Trimethoprin, Va for Vancomycin. Intracellular accumulation of the isolates were measured for five common contaminant metals in parts per billion (ppb). It was found that the accumulation of lead within the cell was highest among all of the isolates. 3.6. Molecular Characterization

The partial 16S rDNA sequence analysis was done to reveal the molecular identity of the isolates. The phylogenetic trees were constructed using neighbour joining method. The partial 16S rDNA sequence could only provide the identification at the genus level. Thus all the sequences being novel were submitted to GenBank, the accession numbers were FJ377718, FJ377719, FJ377721 to FJ377724 for SD2, SM2, SD1, SD3, SV1 and SD4 respectively (Table 1).

3.7. Continuous Production of Enzyme by Immobilized Cells on Inert Matrix

Protease and amylase productions were compared under different conditions of incubation at regular

interval of 1 h for a total span of 12 h. It was found that protease production was maximum from immobilized sheet, whereas the amylase production was maximum in shake flask culture (Fig. 4a and b). The bioreactors were recharged every 12 h and the protease production on sheet (Fig. 4c) reached the highest value after 3rd recharging maintained it till 8th recharging where after is was reduced to half (9th recharging) and maintained as such till the 20th recharge. But on immobilized hay protease production was maximum during 10th recharge and thereafter it slowly decreased till it reduced to half on the 17th recharge. On immobilized sheet and hay the amylase production (Fig. 4d) reached highest value on 9th and 5th recharging respectively.



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3.8. Application of Protease 3.9. Recovery of Precious Metal

The effects of these proteases on exposed X-ray films were also checked after 48 h of incubation where except SD1 all other isolates exhibited complete removal of the gelatin layer (data not shown). This action has already been reported for proteases from different bacterial sources (Gupta et al., 2002). Silver impingement within the gelatin layer comes out with decomposed material which could be recovered further through different chemical methods. The cells from culture containing metal strips that were washed only with PBS showed total accumulation (intracellular as well as adsorbed on the surface) whereas the other samples washed with 0.1N HCl followed by PBS only showed intracellular accumulation. The cell grown normally (i.e., in absence of metal strip) was also analyzed using EDXRF. EDXRF analysis results showed a qualitative difference in count for silver and gold accumulation within the bacterial cells. The actual metal accumulation within the cell was obtained by subtracting the accumulation of the metal within equal weight of untreated cell from that within treated cells. The cell

grown in presence of gold and silver strip only showed silver accumulation. Total accumulation (after only PBS wash) of silver within cells grown in presence of gold and silver strips respectively were 874 and 2232 folds where as the same for intracellular accumulations were 320 and 572 respectively. Thus there was substantial adsorption of metal on the cell surface. But surprisingly in both cases only silver accumulation was visualized. There could be for two reasons, one that SM2 was not able to accumulate gold and second that the gold strip that was incubated was containing mostly silver. So it can be concluded that this strain could be used for testing of silver impurity in alloys. SEM analysis also revealed the surface structure difference between treated and untreated gold and silver strips. The untreated strip surfaces were rough and uneven whereas the surface morphology of the treated samples were smooth. This would be due to leaching of silver from the metal surface of the strips by the isolated strain (Fig. 5). The detection of impurities by help of bacteria is environment friendly, but the drawback of this process is that it is much slower than other conventional processes.

(a) (b)

(c) (d)

Fig. 4. Continuous enzyme production on immobilized matrix. At regular intervals of 1 hour, protease and amylase production on

immobilized matrix (hay and sheet) were compared with shake flask culture (incubated at 37°C, 150 rpm continuous shaking). (a and b) It was found that amylase production was maximum in shake flask culture, whereas the protease production was maximum on immobilized sheet. (c and d)A zig-zag pattern of enzyme (protease and amylase) production was found on both of the inert matrices



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(a) Untreated gold plate (b) Gold plate after treatment

(c) Untreated silver plate (d) Silver plate after treatment Fig. 5. Scanning electron micrographic images of metal strips. Considering the heavy metal accumulating ability of SM2, the metal

strips (zero valent state) of silver (Ag) and gold (Au) were dipped into two different set of SM2 culture and 1ml of 1X LB was added into respective tubes. To sustain their growth 1ml of 1X LB medium was replaced every 12 hours retaining the cells in each tube. This condition was maintained for 2 months. The strips were taken out, washed with distilled water, incubated in 1ml bleach for 15mins and again washed with distilled water thrice before drying. The surface of the strips was observed under SEM (FEI QUANTA 200 MARK 2 at 15 kV). (a and c) depicted a rough surface of untreated gold and silver strip. When it was treated with protease producing metal accumulating bacteria, the surface became smooth for both gold (b) and silver (d) strips

(a) (b) (c)



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(d) (e) (f)

Fig. 6. Pie chart of the market survey of the modified detergent. The combination of SM2+SD2 was used as detergent additive for

market survey. Cell free supernatant containing extracellular protease and lipase was concentrated by lyophilization (Heto Dry Winner, Maxy dry Lyo). Each gram of detergent was mixed with 6U protease and 2.25U lipase before air drying. The detergent with and without additive was then supplied to 50 different households to get a feedback on the wash performance efficiency as per the supplied questionnaire. Pie chart of steel utensil wash performance (a) revealed maximum acceptance (95.74%) followed by 94.59% for glassware (b), 88.24, 92.86, 93.33 and 91.67% for the purpose of kitchen (c), floors (d), commode (e) and sink (f) cleaning respectively

3.10. Wash Performance Analysis

The microbial enzyme formulation using SM2 and SD2 showed better cleaning efficiency. The combined effect of protease and lipase as additive with detergent showed better result than detergent without additive. The enhanced efficiency was backed up by the market survey data. The analysis report revealed acceptance in most of the household for various cleaning purposes (Fig. 6) with maximum acceptance for washing steel utensil (95.74%) followed by glassware (94.59%), washing floors (92.86%) and WC (93.33-91.67%) as well as kitchen cleaning (88.24%). In most cases the modified detergent was needed in same if not less quantity as compared to the control detergent. This analysis indicated that the modified detergent would be industrially appreciated in large scale.

4. CONCLUSION

The complete characterization of the novel isolates revealed their pH and temperature tolerance, antibiotic and heavy metal response. The heavy metal accumulation by the isolates gives rise to the possibility of applying them in remediation of toxic metals. Either the single isolates or their mixed consortia can be applied for bioremediation. The protease secreting property definitely provides an additional advantage for using them as sources of commercial enzymes. The efficiency of proteases as an additive to detergent and X-ray film clearance could also be commercially exploited. Thus the biodiversity screening revealed the existence of biotechnologically important microbes from the coastal regions of India.

5. ACKNOWLEDGEMENT

The researchers acknowledge the support of the West Bengal University of Technology for the computational facility and the laboratories; University Grant

Commision-Inter University Consortium for the student fellowship as well as Department of Atomic Energy, Government of India under the BRNS scheme for financial assistance.

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Shigemura, K., T. Shrikawa, H. Okada, K. Tanaka and S. Kamidono et al., 2005. Rapid detection and differentiation of Gram-negative and Gram-positive pathogenic bacteria in urine using TaqMan probe. Clin. Exp. Med., 4: 196-201. DOI: 10.1007/s10238-004-0056-x

Sogin, M.L., H.G. Morrison, J.A. Huber, D.M. Welch and S.M. Huse et al., 2006. Microbial diversity in the deep sea and the underexplored “rare biosphere”. PNAS, 103: 12115-12120. DOI: 10.1073/pnas.0605127103

Souza, M.J.D., S. Nair, P.A.L. Bharathi and D. Chandramohan, 2006. Metal and antibiotic-resistance in psychrotrophic bacteria from Antarctic Marine waters. Ecotoxicology, 15: 379-384. DOI: 10.1007/s10646-006-0068-2

Sudhakar, M., C. Priyadarshini, M. Doble, P.S. Murthy and R. Venkatesan, 2007. Marine bacteria mediated degradation of nylon 66 and 6. Int. Biodeter. Biodegr., 60: 144-151.

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Corresponding Author: Shaon RayChaudhuri, Department of Biotechnology, School of Biotechnology and Biological Sciences, West Bengal University of Technology, BF-142, Sector-I, Saltlake. Kolkata-700064, India

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Isolation of Nitrate and Phosphate

Removing Bacteria from Various Environmental Sites

1DebRoy, S., 1S. Das, 1S. Ghosh, 1S. Banerjee, 1D. Chatterjee, 1A. Bhattacharjee, 2I. Mukherjee and 1Shaon RayChaudhuri

1Department of Biotechnology, School of Biotechnology and Biological Sciences,

2Department of Natural Sciences, School of Mangement and Sciences; West Bengal University of Technology, BF-142,

Sector-1, Salt Lake, Kolkata-700064, West Bengal, India

Abstract: Problem statement: Nitrate and phosphate are two major pollutants due to anthropogenic activity like excessive use of fertilizers in agriculture. Their contamination has emerged as a global problem and its potential threat is marked on the environmental sustainance as well as on the public health. Approach: The objective of the current study is to isolate efficient nitrate and phosphate removing microbes from various environmental sites that have been selected on the basis of the nature of polutants received by them and their water quality assessment. These well characterized isolates could in future be used for the remediation of waste water. 30 different sites were screened using culture based method. The nitrate and phosphate removing abilities of the microbes were checked in enriched medium (Himedia M439) after 16 h of incubation at 37°C. Results: 7 efficient isolates were obtained from rhizosphere of Water lily, Marine beaches, Paddy field and Raw sewage canal. The highest nitrate removal (88.3%) was shown by isolate (WBUNB009) from raw sewage canal and the highest phosphate removal (82.9%) was shown by isolate (WBUNB004) from rhizosphere of Water lily. Morphologically all the isolates were gram positive bacilli as reconfirmed by environmental scanning electron microscopy. Biochemically as well as physiologically they differ from each other. Conclusion/Recommendation: This study leads to the isolation of efficient nitrate and phosphate removers from environmental origin. The phosphate removing efficiency is much higher than the type strain under identical condition. These native microbes might be responsible for maintaining the phosphate and nitrate levels at the 30 sites investigated inspite of the received pollution load. These isolates could be the potential bioremedial agents for other sites with high nitrate and phosphate contamination level. Key words: Nitrate, phosphate, agricultural runoff, bacteria, Eutrophication, bioremediation, efficient

isolates, phosphate contamination, European Community (EC)

INTRODUCTION Nitrate and Phosphate are recognized as the major nutrients which are required by living organisms for their physiological processes. They are most commonly added as fertilizer to enhance the quality of soil. However they have emerged as most abundant pollutants in the world due to their excess usage. The traditional agricultural practices like dry farming with marginal irrigation, flood plain farming and random application of fertilizers are considered as diffused sources of nitrate and phosphate in soil and aquifers. Besides this, the irregular rainfall during different seasons and the stream flow pattern

causes seepage of these contaminants from soil to surface and ground water (Whitmore et al., 1992; Jorgensen, 1999; Giupponi et al., 1999; Agrawal, 1999; Krishnaswamy et al., 2009). The cultivation patterns like terrace farming results in nitrate leaching into aquifers (Nakasone and Yamamoto, 2004; Kinoshita et al., 2003). Increased levels of nitrate up to 400 ppm have been detected in groundwater (Filintas et al., 2008). Possible sources of nitrate pollution include manure, agricultural fertilizer, industrial effluent, domestic wastewater, septic systems, human waste lagoons, animal feedlots and native soil organic matter, as well as geologic

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sources (Jin et al., 2004) Other point sources of nitrate are municipal sewage canals, septic tanks, sewage dumping grounds (Wakida and Lerner, 2005; 2006). The mining tailings, industrial effluent from nuclear reactors, radioactive waste processing units mainly those dealing with compounds like plutonium or thorium nitrate (Singleton et al., 2005). Nitrate contamination is a global problem and stands as second most dangerous pollutant after the pesticides. High concentration of nitrate in drinking water is a threat especially to infants, causing methemoglobinemia, also called "blue baby syndrome. The carcinogenic effect of nitrate is also reported. Concentration higher than 10ppm in drinking water may also cause stomach cancer in infants (Jin et al., 2004). EPA has demarcated the maximum contaminant level to be 10 ppm for NO3 -N and 45 ppm for NO3 concentration. A similar guideline of 50 ppm as NO3 has been set by the WHO and the European Community (EC). Several conventional technologies adopted for nitrate removal are ion exchange resins, electro dialysis, reverse osmosis and distillation which substantially increase the cost of operation. Therefore the cost-effective alternative lies in the biological denitrification process (Pinar et al., 1997; Eckford and Fedorak, 2002). The addition of phosphorus as phosphate fertilizers in soil in excessive amount causes serious environmental problems in the form of eutrophication which uses up large amounts of oxygen. The main sources of phosphate in aquatic environment is through household sewage water containing detergents and cleaning preparations, agricultural effluents containing fertilizers as well as industrial effluent from fertilizer, detergent and soap industries (Pradyot, 1997). Phosphate is generally present as polyphosphate and orthophosphate. The concentration of phosphate in water bodies vary from 0.005-10 ppm depending on the source of phosphate near the water body. On one hand digestive problems occur from extremely high levels of phosphate, on the other, phosphate levels greater than 1.0 may interfere with coagulation in water treatment plants. The EPA has fixed standard phosphate levels as 0.015 ppm for water supply, 0.025 ppm for aquatic life, 0.05 ppm for lakes and 0.02 ppm for mountain lakes (Kotoski, 1997). Microbial strategies are currently being used for the removal of excess phosphate load in waste water since it is an attractive alternative to chemical processing (Krishnaswamy et al., 2009). The objective of the present study is to isolate efficient nitrate and phosphate reducing microbes from different environmental water bodies. Further

characterization of these isolates would lead to the development of an array of novel organisms which can reduce nitrate and phosphate load in water bodies of various environmental sites leading to bioremediation.

MATERIALS AND METHODS Sampling: Water samples were collected from different environmental sites to screen for nitrate and phosphate removing organisms. The sites were selected on the basis of the pollutants received by them. Main focus was on sites, which according to pollutants received were expected to have high load of nitrate and phosphate but showed low concentrations of these. Presumably these are the sites which host the nitrate and phosphate removers. The list of sites has been given below Table 1. Environmental parameters: Different physical, chemical and biological parameters were assessed for the water samples from each site to understand the different concentration of pollutants in them. The physical and chemical parameters were assessed as per the protocol laid down by Central Pollution Control board of India. The biological parameter of the sites were assessed by studying the normal population of the sites. 10 microbes were selected which are commonly present in waste water (viz Escherichia coli, Enterobacter aerogenes, Shigella flexneri, Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis, Salmonella sp. and Staphylococcus aureus.) and the water from the sites were serially diluted and spread on media specific for the microbes. The different media used were HiChrome E.coli coliform Selective agar base (HiMedia M-1294 for Escherichia coli, Enterobacter aerogenes, Shigella flexneri, Klebsiella pneumonia); HiChrome UTI agar base (HiMedia M-1353 for Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis); Salmonella differential agar base (HiMedia M-1078 for Salmonella sp) and HiChrome Aureus agar base (HiMedia M-1468 for Staphylococcus epidermidis and Listeria monocytogenes). Cultivation medium and growth conditions: Since one of the main objectives of our study is to isolate nitrate reducing microbes therefore the screening for microbes were done in high nitrate containing medium. The water samples of all the above mentioned sampling sites were serially diluted and plated on media containing 2000ppm of nitrate followed by overnight incubation at 37°C to isolate microbes which can survive in high nitrate concentration.

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Table 1: Water samples were collected from the following sites. The sampling sites were selected on the basis of the nature of the pollutatnts received by them. The co-ordinates of the sampling sites have been mentioned below

Types Locations CO-Ords Source of waste Marine coast Udaypur Beach 21°36’31”N Recreational activity, animal waste (West Bengal) 87°28’52”E in low volume, natural waste. Digha Beach 21°36’59”N Recreational activity, animal (West Bengal) 87°30’08”E waste, natural waste. Mandarmani Beach 21°41’N Recreational activity, (West Bengal) 87°33’E animal waste, natural waste. Artificial water Haldiram fountain 22°37’40”N Bathing, natural waste, Bodies 88°26’01”E washing activity. Baguiati fountain 22°36’36”N Natural waste 88°25’41”E River Ganga (Naihati) 22°53’23”N Industrial waste, idol immersion, domestic waste, recreational waste, 88°24’40”E oil spill from transportation, piggery waste, sewage waste from surrounding area. Ichamati (Taki) 22°36’01”N Industrial waste, idol immersion, domestic waste, 88°56’46”E recreational waste, agricultural waste, erosion of banks Churni (Ranaghat) 23°14’35”N Human bathing, domestic and sewage waste, 88°36’23”E transportation by boat, agricultural waste, industrial waste. Damodar (Bardhaman) 23°12’41”N Bathing, recreational waste, domestic waste, 87°50’48”E agricultural waste, fish aquaculture, oil spillage from launch Mayurakshi (Tilpara) 23°57’04”N Fishing, washing, small 87°31’06”E scale agricultural run off Hotspring: Agnikundu, Bakreswar 23°52’51”N Rice and coins thrown by 87°22’32”E devotees, plastic packets, Dudhkundu, Bakreswar 23°52’51”N Natural waste, rice and coins. 87°22’34”E Population of frogs and snails found Jibatsakundu, Bakreswar 23°52’52”N Algal growth seen, natural waste, 87°22’34”E rice and coins by devotees. Swet ganga (Birbhum) 23°52’52”N Washing of hands and feet, 87°22’34”E waste from rituals e.g., flowers, utensil washing. Taptapani (Orissa) 19°29’04”N Human bathing, natural 84°23’37”E wastes, rice and coins thrown by devotees. Paddy field Berunanpukuria (Barasat) 22°44’34”N Fertilizer waste, 88°26’20”E agricultural run off. Rhizosphere Typha sp 22°35’14”N Dumping of wastes from the nearby region. of Swampy Aquatic grass 88°28’31”E (beside the ONGC green building), animal grazing therefore Plants Nymphaeae nouchali animal waste (violet flower, water lily) Water Hyacinth 22°35’40”N Bathing, dumping of waste 88°23’51”E from local shops and residence, washing of utensils, cleaning of buses and trucks, Natural water Under Ultadanga over 22°41’19”N Domestic waste dumping from Bodies bridge (Kolkata) Kalyani 88°25’05”E local residence, erosion of banks. Byepass Expressway (Fordillapur) Site reserved for Himangini 22°43’34”N Aquaculture, dumping of waste from 88°24’12”E local residence, erosion of banks. Jute Retting Site 22°47’12”N Used for jute retting, Thankurbari 88°30’12”E dumping of waste from local shops and residence Kaikhali pond 22°38’03”N Bathing, washing clothes, 88°26’16”E cleaning utensils, idol immersion. Rajarhat pond 22°35’52”N Animal waste, Pisciculture, 88°28’09”E recreational activity, erosion of the banks. Captain Bheri 22°33’10”N Sewage, domestic and garage waste 88°24’43”E passed through a crude filtration for removal of coarsensuspended solids. Primarily used for pisciculture. Bathing and cleaning also found, agricultural run-off from surrounding region. Private Bheri 22°33’19”N 88°24’41”E Raw sewage Kestopur khal 22°35’50”N Domestic and sewage waste, Canal 88°25’37”E waste from agricultural land, oil from garage Bagjola khal 22°37’16”N Domestic and sewage waste, 88°24’10”E waste from agricultural land, oil from garage, effluent from rubber industry. Khal Next to 22°33’17”N Sewage, domestic and garage waste. Captain Bheri 88°24’47”E Dumping from local area.

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Further selection was made on the basis of morphology. The cultures were re-streaked three or more times to obtain pure colonies. The isolates were also grown in Nitrate Broth (Himedia M439-500G) and maintained at 37°C in 150rpm shaking condition. The final selection of the pure isolates was on the basis of their nitrate removing efficiency from the liquid culture. Morphological characterization: The initial morphology of the isolates were determined by using light microscope (1000X magnification on a Zeiss Axiostar Plus microscope) following simple staining using 5% Crystal Violet. The Gram nature of the isolate was determined by differential staining as per standard procedure. Dimensions of the isolates were determined using Environmental scanning electron microscopy (FEI QUANTA 200 MARK 2 at 15 kV) as per the protocol.

Biochemical characterization: The ability of the isolates to produce enzymes like DNase, oxidase, lipase, catalase and amylase was determined. The tests for the first five enzymes were done according to the protocol of Nandy et al. (2007) the amylase test was done on 1% starch agar plate and incubated at 37°C for overnight followed by flooding of the plate with iodine solution. The substrate utilization profile of the isolates were checked as per manufacturer’s protocol using substrate utilization kits (HiMedia KB009). Antibiotic assay: The response of the isolates towards 18 different antibiotics (HiMedia) were checked according to the procedure reported by Nandy et al. (2007) Nitrate removal: The isolates were inoculated (2% inoculum) in Nitrate broth and incubated for 16 h at 37°C in a shaking incubator 150 rpm. The cell free supernatant was taken for estimation of nitrate removal after harvesting the culture at 8609×g for 10min. 200 µL of Salicyalic acid (5% Salicylic acid in H2SO4) and 40 µL of cell free supernatant was added and vortexed. The tubes were incubated in dark for 10 min. The reaction was stopped by addition of 2 mL of 4N NaOH. Optical density of this solution was measured after 20 min at 420 nm. The O.D was then compared to the standard curve prepared with known concentrations of NaNO3 (100-1000 ppm) to determine the concentration of Nitrate remaining in the medium (Cataldo et al., 1975). Phosphate removal: Phosphate can be detected by spectrophotometric method by conversion of the phosphates to Molybdophosphoric acid Complex (MOP) by Ammonium molybdate, followed by reduction of the MOP Complex by Sn2+ of SnCl2 to give

a blue coloured complex. For our study, at first a standard curve of phosphate was prepared by using standard solutions of phosphate from 0.05-0.5 ppm. 10 mL of cell free supernatant of bacterial sample (2% inoculum grown in Nitrate broth for 16hrs at 37°C) was diluted in 60 mL of distilled water in a 250 mL conical flask. 2 mL of Ammonium molybdate reagent was added followed by 4 drops of Stannous chloride. The solution was shaken well and volume was made upto 100 mL. The blue colour which developed indicated presence of phosphate which could be measured spectrophotometrically at 660 nm. The unknown concentration of phosphate was determined by comparing with the standard curve (Krishnaswamy et al., 2009). Statistical analysis: The objective of the study being isolation of efficient nitrate and phosphate removers, the ideal situation would be a single isolate performing both the functions. The relation between nitrate and phosphate removal was investigated by using the Correlation Co-efficient as a measure of the association between the two variables. The correlation co-efficient measures the strength of the linear relationship between the variables.

RESULTS Environmental parameters: The physical parameters assessed were pH, Suspended solids, Turbidity level of the water body, Temperature, Odour and Colour (Table 2). The Chemical parameters include Alkalinity, Hardness, Calcium, Magnesium, Chlorine, Flouride, Nitrate, Ammonia, Phosphate, Total Iron, Residual Chlorine, Biological Oxygen Demand (Table 3). The biological parameters were assessed by the growth of microbes in four different kinds of medium HiChrome E.coli coliform Selective agar base (HiMedia M-1294 for Escherichia coli, Enterobacter aerogenes, Shigella flexneri, Klebsiella pneumonia); HiChrome UTI agar base (HiMedia M-1353 for Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis); Salmonella differential agar base (HiMedia M-1078 for Salmonella sp); and HiChrome Aureus agar base (HiMedia M-1468 for Staphylococcus epidermidis and Listeria monocytogenes) (Table 4). Cultivation medium and growth conditions: The serial dilution of water samples from the above mentioned 30 sites on medium with 2000 ppm of nitrate gave 130 different colonies. Further selection was made on the basis of morphology since most of the nitrate reducers according to literature are bacilli therefore bacilli in long or short chain or isolated bacilli were selected.

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Table 2: Water samples were collected from 28 different environmental sites which were then tested for different physical parameters like pH, suspended solids, turbidity level, Temperature, Odour and Colour

Susp.solid Turbidity Temp SITES pH (gm) level (cm) (°C) Odour Colour Udaypur Beach (West Bengal) 6.30 0.008 Not applicable 15.0 Fishy Normal Digha Beach (West Bengal) 6.10 0.063 Not applicable 15.0 Normal Normal Mandarmani (West Bengal) 5.70 0.056 Not Applicable 15.0 Normal Normal Haldiram fountain 7.02 0.015 Not applicable 24.0 Fishy Normal Baguiati Fountain 7.25 0.030 Not applicable 24.0 Fishy Normal Ganga (Naihati) 7.42 0.010 5.1 21.0 Normal Off white Ichamati (Taki) 6.50 0.005 18.1 21.0 Unpleasant dirty light brown Churni (Ranaghat) 6.50 0.009 55.1 21.0 Normal normal Damodar (Bardhaman) 7.10 0.068 17.75 22.5 Normal Normal Mayurakshi (Tilpara) 7.50 0.013 Not applicable 20.0 Normal Normal Agnikundu, Bakreswar 8.84 0.002 100 72.0 Normal Normal Dudhkundu, Bakreswar 7.40 0.001 100 31.5 Fishy Normal Jibatsakundu, Bakreswar 7.20 0.003 100 22.0 Normal Normal Swet ganga (Birbhum) 7.90 0.017 28.3 34.0 Normal Normal Taptapani (Orissa) 7.30 0.016 100 40.0 Normal Normal West Bengal state university (Barasat) 6.40 0.018 Not applicable 22.0 Normal Light brown Rhizosphere of aquatic plants 7.10 0.003 Not applicable 22.0 Normal Normal Under Ultadanga over bridge 7.20 0.054 20.5 21.0 Fishy Green Byepass Kalyani Highway(Fordillapur) 7.30 0.014 18.1 22.0 Normal Green Site reserved for Himangini 7.60 0.028 49.5 22.0 Fishy Very light green Jute Retting Site Thankurbari 6.50 0.066 5.2 22.0 Unpleasant Black Kaikhali pond 7.80 0.006 49.0 23.0 Normal Normal Rajarhat pond 7.30 0.014 5.1 20.0 Normal Green Captain Bheri 7.20 0.028 28.1 21.0 Normal Green Private Bheri 7.30 0.036 20.5 24.0 Normal Light green Kestopur khal 6.50 0.011 22.0 21.5 Unpleasant Black Bagjola khal 6.80 0.043 18.1 21.0 Unpleasant Grey Khal Next to Captain Bheri 6.50 0.059 20.0 23.0 Unpleasant Grey Table 3: Water samples were collected from 28 different environmental sites which were then tested for different chemical parameters of the

sampling sites like Alkalinity, Hardness, Calcium, Magnesium, Chlorine, Fluorine, Nitrate, Ammonia, phosphate, total Iron, Residual Chlorine and Biological Oxygen Demand in parts per million (ppm)

Alk Hard Ca Mg Cl F NO3 NH3 PO4 Total Iron Res Cl BOD SITES (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (Ppm) (Ppm) (ppm) (ppm) (ppm) Udaypur Beach (West Bengal) 88 7400 4070.4 809.090 15095.320 1.5 17.73 0.5 0.15 0.00 0.00 2.4 Digha Beach (West Bengal) 96 7216 4032.0 773.710 15295.250 1.0 19.02 0.5 0.16 0.00 0.00 2.0 Mandarmani (West Bengal) 88 1248 793.0 110.565 8678.000 1.5 19.30 0.5 0.04 0.00 0.00 2.8 Haldiram fountain 340 360 112.0 60.260 207.080 1.0 0.00 0.0 0.20 0.00 0.10 2.0 Baguiati Fountain 180 240 92.8 35.760 262.410 1.5 8.28 0.0 0.00 0.00 0.10 6.6 Naihati (Ganga) 164 184 54.4 31.490 33.980 3.0 20.89 0.0 1.57 0.20 0.00 0.0 Taki (Ichamati) 180 600 115.2 117.800 1099.659 1.5 22.73 0.0 0.00 0.00 0.00 4.4 Ranaghat(Churni) 348 360 112.0 60.260 19.990 2.0 16.71 0.0 0.00 0.00 0.00 1.2 Bakreswar (Damodar) 108 120 48.0 17.490 45.980 1.5 29.70 0.0 0.609 0.00 0.00 1.2 Tilpara(Mayurakshi) 72 200 44.8 37.710 13.990 1.5 25.60 0.0 0.00 0.00 0.00 2.4 Agnikundu, 124 8 0.0 1.940 52.868 1.0 32.90 0.0 0.00 0.00 0.00 0.8 Dudhkundu, 140 80 16.0 15.550 129.950 1.0 29.76 0.0 1.54 0.20 0.00 1.6 Jibatsakundu, 180 104 48.0 13.60 129.950 0.0 33.93 0.0 0.00 0.20 0.00 2.0 Swet ganga (Birbhum) 140 40 9.6 7.38 121.960 1.0 17.73 0.0 0.14 0.20 0.00 2.8 Taptapani (Orissa) 140 480 41.6 106.53 23.990 0.5 16.15 0.0 0.02 0.00 0.00 1.6 West Bengal state university (Barasat) 228 176 64.0 27.21 154.950 0.5 19.95 0.5 0.00 0.00 0.00 1.2 Rhizosphere of aquatic plants 172 200 51.2 36.15 119.960 1.5 20.23 0.5 0.45 0.20 0.00 2.8 Ultadanga over bridge 404 560 176.0 93.31 624.800 2.0 19.40 0.5 0.00 0.20 0.00 0.4 Byepass Kalyani Highway (Fordillapur) 172 216 76.8 33.82 74.940 1.5 10.78 0.0 0.00 0.00 0.00 2.8 Site reserved for Himangini 340 360 112.0 60.26 187.440 1.5 18.00 0.5 0.00 0.00 0.00 4.4 Jute Retting Site Thankurbari 320 240 96.0 34.99 49.980 2.0 22.00 0.5 2.22 0.20 0.10 2.8 Kaikhali pond 280 224 51.2 41.99 54.980 1.5 18.00 0 0.00 0.20 0.00 7.2 Rajarhat pond 356 360 89.6 65.70 149.950 2.0 27.45 0.5 1.04 0.40 0.10 2.4 Captain Bheri 80 120 96.0 5.83 249.920 1.5 6.80 0.5 0.20 0.40 0.10 2.8 Private Bheri 85 600 96.0 122.47 189.940 0.5 3.10 1.0 0.00 0.40 0.15 3.2 Kestopur khal 168 192 64.0 31.10 87.470 1.5 15.00 0.0 0.33 0.20 0.00 0.4 Bagjola khal 280 280 89.6 46.26 149.950 2.0 26.25 0.0 0.62 0.60 0.00 2.4 Khal Next to Captain Bheri 200 384 131.2 61.43 252.40 1.0 10.00 0.5 1.50 0.80 0.00 2.0 Alk-Alkalinity; Hard-Hardness; Ca-Calcium; Mg-Magnesium; NO3- Nitrate; PO4- Phosphate; Res Cl-Residual Chlorine; BOD- Biological Oxygen demand

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(a) (b) (c)

(d) (e) (f)

(g) Fig. 1: (a) (WBUNB005) (b) WBUNB008 (c) SM2 (d) WBUNB006 (e) WBUNB009 (f) WBUNB004 (g)

WBUNB007 The figures represent the Scanning electron micrographs of the isolates at different magnifications. WBUNB005 (6000X, 2.54µmX966.5nm), WBUNB008 (6000X, 2.26µmX996.5nm), SM2 (20000X), WBUNB006 (3000X, 3.44µmX936.1nm), WBUNB009 (6000X, 1.97µmX922.6nm), WBUNB004 (6000X, 1.95µmX923.3nm), and WBUNB007 (6000X, 3.18µmX791.6nm) These were visualized by Scanning electron microscope (FEI QUANTA 200 MARK 2)

19 such strains were then checked for their efficiency of nitrate removal from liquid culture. 7 bacilli were selected on the basis of nitrate removing efficiency for further characterization. Morphological characterization: Simple staining of the microbes showed different morphology of microbes. The Environmental Scanning Electron Micrographs of the isolates (Fig. 1) show that WBUNB004, WBUNB005 and WBUNB006 (from

rhizosphere of water lily) are bacilli in chain whereas WBUNB008 (from paddy field), SM2 (from marine beach) and WBUNB009 (from raw sewage canal) are short isolated bacilli and WBUNB007 (from marine beach) is long bacilli.Gram staining showed that all the isolates were gram positive in nature. Biochemical characterization: The biochemical characterization of the strains is represented in Table 5. All isolates except WBUNB007 produced oxidase and protease.

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Table 4: Water samples were collected from 28 different environmental sites which were then tested for various microbial populations by incubating diluted water samples on medium specific for the growth of Escherichia coli, Enterobacter aerogenes, Shigella flexneri, Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis, Salmonella sp. and Staphylococcus aureus

E.coli E. S. K. P.aer P.mir E.fae L.monocy SITES It Aerogenes Flexneri Pneumonia uginosa abilis calis Salmonella sp S. epide togenes

Udaypur Beach (West Bengal) 0 20 0 0 0 0 0 0 0 0 Digha Beach (West Bengal) 80 160 740 60 320 0 100 20 20 40 Mandarmani (West Bengal) 0 0 13 40 27 0 40 0 0 13 Haldiram fountain 4000 2480 3120 6000 114000 0 0 0 40 160 Baguiati fountain 2000 200 3600 100 160 0 160 200 0 0 Naihati (Ganga) 300 420 2400 220 960 0 720 80 400 0 Taki (Ichamati) 120 280 800 320 2400 0 160 1040 2480 0 Ranaghat(Churni) 0 0 0 160 60 0 20 80 0 0 Bakreswar (Damodar) 1020 220 0 480 0 0 280 320 1000 0 Tilpara(Mayurakshi) 160 940 120 200 420 0 720 40 0 0 Agnikundu, Bakreswar 0 0 0 0 0 0 0 0 20 0 Dudhkundu, Bakreswar 1200 3680 240 80 800 0 2480 3300 0 0 Jibatsakundu, Bakreswar 2400 4800 800 800 160 400 3280 160 280 140 Swet ganga (Birbhum) 240 0 40 80 420 0 260 60 0 40 Taptapani (Orissa) 0 0 17760 0 20560 0 0 0 0 0 West Bengal state university (Barasat) 0 200 1300 140 900 0 240 2740 0 60 Rhizosphere of aquatic plants 1600 0 0 540 1040 0 160 800 0 20 Under Ultadanga over bridge 150000 1600 0 10000 66000 0 42000 50000 0 1920 Byepass Kalyani Highway (Fordillapur) 440 300 0 140 0 0 0 0 160 0 Site reserved for Himangini 40000 1120 0 800 320 0 0 0 0 1560 Jute Retting Site Thankurbari 312000 40000 112000 48000 56000 0 24000 40000 100 220 Kaikhali pond 80 120 220 100 0 0 380 20 0 120 Rajarhat pond 4000 1740 0 8000 0 0 0 4000 0 0 Captain Bheri 1800 520 8800 160 300 0 2120 0 0 0 Private Bheri 800 1400 500 1200 1200 0 1120 0 0 1440 Kestopur khal 72000 32000 64000 72000 90000 0 192000 48000 140 420 Bagjola khal 240000 464000 88000 16000 208000 0 192000 0 2600 1340 Khal Next to Captain Bheri 328000 0 56000 64000 272000 0 320000 0 2560 5760

Table 5: Morphological and biochemical characterization of 7 isolates. The morphological characterization includes the Gram nature whereas the

Biochemical Characterization includes the capability of the isolates to produce enzymes such as Catalase, Oxidase, Protease, Amylase, lipase and DNase

Strain Gram nature Catalase Oxidase Protease Amylase Lipase Dnase

WBUNB005 Gram positive bacilli + + + + - - WBUNB008 Gram positive bacilli - + + + - + SM2 Gram positive bacilli + + + + - + WBUNB006 Gram positive bacilli + + + + + - WBUNB009 Gram positive bacilli + + + + + + WBUNB004 Gram positive bacilli + + + + + + WBUNB007 Gram positive bacilli - - - + - +

Fig. 2: Graph representing the percentage of nitrate remaining in the medium after incubation with the isolate for 16 h

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Fig. 3: Graph representing the percentage of phosphate remaining in the medium after incubation with the isolate for 16 h Table 6: 7 isolates were tested for the preference of the substrate they required for growth by using Hi-Media Carbohydrate kit (KB 009). The

substrate preference of the isolates are given below

STRAIN SUBSTRATE UTILISED

WBUNB005 Maltose, Fructose, Dextrose, Trehalose, Sucrose, LArabinose, Mannose, Glycerol, Rhamnose, Cellobiose, Melezitose, α Methylmannoside, Xylitol, Esculin, D-Arabinose, Citrate, Malonate, Sorbose WBUNB008 Maltose, Fructose, Dextrose, Galactose, Raffinose, Trehalose, Melibiose, Sucrose, Mannose, L-arabinose, Glycerol, Ribose Rhamnose, Cellobiose, Melezitose, α Methyl mannoside, Xylitol, Esculin, D-Arabinose, Citrate, Malonate, Sorbose SM2 Maltose, Fructose, Dextrose, Trehalose, α Methyl-D-glucoside, Esculin WBUNB006 Maltose, Dextrose, Galactose, Raffinose, Trehalose, Melibiose, Sucrose, LArabinose, Mannose, Glycerol, Ribose Rhamnose, Cellobiose, Melezitose, α Methyl mannoside, Xylitol, Esculin, D-Arabinose, Citrate, Malonate, Sorbose WBUNB009 Maltose, Fructose, Dextrose, galactose, Raffinose, trehalose, Melibiose, Sucrose, Glycerol, Ribose Rhamnose, Cellobiose, Melezitose, α Methyl mannoside, Xylitol, Esculin, D-Arabinose, Citrate, Malonate, Sorbose WBUNB004 Maltose, Raffinose, Melibiose, L-Arabinose, Mannose, Ribose Rhamnose, Cellobiose, Melezitose, α Methyl mannoside, Xylitol, ONPG, Esculin, D-Arabinose, Citrate, Malonate, Sorbose WBUNB007 Dextrose, Trehalose, Glycerol, Ribose Rhamnose, Cellobiose, Melezitose, α Methyl mannoside, Esculin, D Arabinose,

Citrate, Malonate, Sorbose Table 7: The 7 isolates were tested for their resistance and susceptibility to a common range of antibiotics. The antibiotic susceptibility profile of

the isolates is given below

Wbunb Wbunb SM Wbunb Wbunb Wbunb Wbunb Antibiotics Symbol 005 008 2 006 009 004 007

Chloramphenicol (30 mcg) C 30 S S I S S S S Ceftazidime (30 mcg) CA 30 R R R R R R R Ampicillin (10 mcg) A 10 R R R R R R R Methicillin (4mcg) MT 4 R R R R R R R Rifampicin (15 mcg) R 15 R R R R I R R Norfloxacin (10 mcg) N 10 S S S S S S S Roxithromycin Ro S S I S S S S Trimethoprim (5 mcg) Tr R R R R R R R Doxycycline Hydrochloride (30mcg) DO 30 S S S S S S S Vancomycin (30 mcg) VA 30 I I I I I I R Cloxacillin (10 mcg) CX 10 R R R R R R R Polymyxin-B (300 units) PB 300 R R R R R R R Gentamycin (10 mcg) G 10 S S S S S S S Ciprofloxacin (5 mcg) CF 5 S S S S S S S Cefotaxime (30 mcg) CTX 30 R I I R R R R Cephadroxil (30 mcg) CQ 30 S S I S S S S Teicoplanin (30 mcg) TE 30 S S S S S S S Neomycin (30 mcg) N 30 S S I I I R R

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All except WBUNB007 and WBUNB008 produced catalase. Amylase is produced by all the isolates, whereas lipase is produced by WBUNB006, WBUNB009 and WBUNB004 and DNase by WBUNB008, SM2, WBUNB009 and WBUNB004. The substrate utilization profile of the isolates was performed by using different carbohydrate sources (Himedia KB009). This study gives us an array of substrates used by an isolate. Most of them could grow in presence of Maltose, Dextrose, Trehalose, Xylitol, Cellobiose, α-Methyl Mannoside, Melezitose, D-Arabinose, Citrate, Malonate and Sorbose (Table 6). The antibiotic susceptibility profile of the isolates were performed by using different antibiotic discs (Himedia). This study shows that the strains were resistant to antibiotics like Ceftazidime, Ampicillin, Methicillin, Rifampicin, Trimethoprim, Polymixin-B and Cefotaxime and sensitive to antibiotics like Chloramphenicol, Norfloxacin, Roxithromycin, Doxycycline hydrochloride, Gentamycin, Ciprofloxacin, Cefadroxil and Teicoplanin (Table 7). Nitrate removal: The nitrate removal from the medium is the primary step for the reduction of nitrate though after removal the bacteria may use the nitrate by assimilatory or dissimilatory pathway. The result is represented in the form of percentage of nitrate remaining in the medium after incubation with the isolate for 16 h at 37°C (Fig. 2). Phosphate removal: The phosphate removal capacities of the isolates were checked in enriched medium in comparison with a type strain, Acinetobacter baumanii (MTCC 1425) known for phosphate removal obtained from MTCC. The result is represented in the form of percentage of phosphate remaining in the medium after incubation with the isolate for 16hrs in 37°C. The result indicates that all the isolates show better phosphate removal than Acinetobacter baumanii under the given set of conditions (Fig. 3). Statistical analysis: The nitrate removal by the isolates were found to be within 77-88%, the average removal being 85.3%. The phosphate removal by the isolates were found to be within 43.8-82.9%, the average being 63.71% while that of the type strain under similar conditions showed 31.9% removal. The correlation study of nitrate and phosphate showed a negative moderate correlation of (-) 0.5584 which implies that an efficient nitrate remover is not necessarily an efficient phosphate remover.

DISCUSSION In this study we report the isolation of 7 strains with potential for nitrate removal. They could be used for bioremediation of nitrate contaminated sites leading

to environmental protection. Phosphate removers isolated during the study were found to be more efficient than the type strain (Acinetobacter baumanii) under identical conditions. Here we report 7 gram positive bacterial isolates which are highly efficient in phosphate removal. Since the mechanism of phosphate removal in bacteria leads to the intracellular accumulation of polyphosphate granules, these could be used as potential candidates for sequestration of phosphate from environmental sites.

CONCLUSION The study is a successful attempt to isolate efficient nitrate and phosphate removing bacteria from various environmental sites for remediation of waste water by reducing the nitrate and phosphate load. In addition optimization of the waste water treatment parameters by these isolates in future could not only lead to environmental protection but also sequestration of essential plant growth nutrients from the waste which in turn could be re used.

ACKNOWLEDGEMENT The group wishes to acknowledge the financial assistance received from Indian Council for Agricultural Research (ICAR), Govt. of India vide Grant No. GB-2019 dated 24th May 2011 and infrastructural facilities of West Bengal University of Technology, India for carrying out the work. The group is grateful to Mr.Sudip Sen, Mr.Gutam Das and Mr.Sanjib Mondal for their kind support during sampling. The group wishes to thank and acknowledge Prof A.R.Thakur, Prof T.B.Samanta and Dr.K.Ray for their invaluable inputs and suggestions during the work. The authors express their sincere gratitude towards Dr. A Bandopadhyay, ICAR; Dr. A.K. Saxena, Indian Agricultural Research Institute (IARI) and Prof. A.K. Tripathy, Benaras Hindu University, India for their valuable suggestion regarding the selection of environmental sites and parameters of analysis.

REFERENCES Agrawal, G.D., 1999. Diffuse agricultural water

pollution in India. Water Sci. Technol., 39: 33-47. DOI: 10.1016/S0273-1223(99)00030-X

Cataldo, D.A., M. Maroon, L.E. Schrader and V.L. Youngs, 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. Soil Sci. Plant Anal., 6: 71-80. DOI: 10.1080/00103627509366547

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Eckford, R.E. and P.M. Fedorak, 2002. Planktonic nitrate-reducing bacteria and sulfate-reducing bacteria in some western Canadian oil field waters. J. Ind. Microb. Biotechnol., 29: 83-92. DOI: 10.1038/sj.jim.7000274

Filintas, A., P. Dioudis, G. Stamatis, J. Hatzopoulos and T. Karyotis, 2008. Environmental assesment of Groundwater nitrate pollution from agricultural wastes and fertilizers in central Greece watersheds using remote sensing and GIS. Proceedings of the 3rd International Conference AQUA 2008 on: Water Science and Technology with Emphasis on Water and Climate, Oct. 16-19, Athens, Greece, pp: 1-10.

Giupponi, C., B. Eiselt and P.F. Ghetti, 1999. A multicriteria approach for mapping risks of agricultural pollution for water resources: The Venice Lagoon watershed case study. J. Environ. Manage., 56: 259-269. DOI: 10.1006/jema.1999.0283

Jin, Z., Y. Chen, F. Wang and N. Ogura, 2004. Detection of nitrate sources in urban groundwater by isotopic and chemical indicators, Hangzhou City, China. Environ. Geol., 45: 1017-1024. DOI: 10.1007/s00254-004-0962-y

Jorgensen, L.A., 1999. The cycling of nitrogen in the Danish agricultural sector and the loss to the environment. Water Sci. Technol., 39: 15-23. DOI: 10.1016/S0273-1223(99)00028-1

Kinoshita, T., T. Katoh, T. Suji, M. Kanada and A. Inoue, 2003. Downward movement of inorganic nitrogen in tea [Camellia sinensis] fields of yellow soil affected by different N-fertilizer application. Japanese J. Soil Sci. Plant Nut., 74: 637-643.

Kotoski, J.E., 1997. Phosphorus minifact analysis sheet. Spring Harbor Environmental Magnet Middle School.

Krishnaswamy, U., M. Muthusamy and L. Perumalsamy, 2009. Studies on the efficiency of the removal of phosphate using bacterial consortium for the biotreatment of phosphate wastewater. Eur. J. Applied Sci., 1: 6-15.

Nakasone, H. and T. Yamamoto, 2004. The impacts of the water quality of the inflow water from tea fields on irrigation reservoir ecosystems. Paddy Water Environ., 2: 45-50. DOI: 10.1007/s10333-004-0039-2

Nandy, P., A.R. Thakur and S.R. Chaudhuri, 2007. Characterization of bacterial strains isolated through microbial profiling of urine samples. Online J. Biol. Sci., 7: 44-51. DOI: 10.3844/ojbsci.2007.44.51

Pinar, G., E. Duque, A. Haidour, J. Oliva and L. Sanchez-Barbero et al., 1997. Removal of high concentrations of nitrate from industrial wastewaters by bacteria. Applied Environ. Microbiol., 63: 2071-2073.

Pradyot, P., 1997. Hand Book of Environmental Analysis, Chemical pollutants in Air, Water, Soil and Solid Wastes. 2nd Edn., CRC/Lewis Publishers, ISBN-10: 0873719891, pp: 584.

Singleton, M.J., K.N. Woods, M.E. Conrad, D.J. DePaolo and P.E. Dresel, 2005. Tracking sources of unsaturated zone and groundwater nitrate contamination using nitrogen and oxygen stable isotopes at the hanford site, washington. Washington. Environ Sci. Technol., 10: 3563-3570. DOI: 10.1021/es0481070

Wakida, F.T. and D.N. Lerner, 2005. Non-agricultural sources of groundwater nitrate: A review and case study. Water Res., 39: 3-16. DOI: 10.1016/j.watres.2004.07.026

Wakida, F.T. and D.N. Lerner, 2006. Potential nitrate leaching to groundwater from house building. Hydrol. Process., 20: 2077-2081. DOI: 10.1002/hyp.6143

Whitmore, A.P., N.J. Bradbury and P.A. Johnson, 1992. Potential contribution of ploughed grassland to nitrate leaching. Agric. Ecosyst. Environ., 39: 221-233. DOI: 10.1016/0167-8809(92)90056-H

American Journal of Applied Sciences 5 (12): 1650-1661, 2008 ISSN 1546-9239 © 2008 Science Publications

Corresponding Author: Shaon Raychaudhuri, Department of Biotechnology, West Bengal University of Technology, Calcutta, India, 700064

1650

Characterization and Wash Performance Analysis of Microbial Extracellular

Enzymes from East Calcutta Wetland in India

1Ramesh Malathu, 1Sanhita Chowdhury, 1Madhusmita Mishra, 1Sumana Das, 1Prabhat Moharana, 1Joydeep Mitra, 2Ujjal K. Mukhopadhyay, 1Ashoke Ranjan Thakur and 1Shaon Ray Chaudhuri

1Department of Biotechnology, West Bengal University of Technology, BF-142, Sector 1, Salt Lake, Calcutta-700064, India

2West Bengal Pollution Control Board, Paribesh Bhawan, 10A, Block-LA Sector III, Salt Lake, Calcutta-700098, India

Abstract: Extracellular protease from a novel bacterial isolate showing maximum similarity of 98.22% with Microbacterium luteolum was obtained from East Calcutta Wetland, India. It showed compatibility with commercial detergents. The enzyme retains more than 60% of its activity between 6.0 to 10.5 pH. The maximum activity is at pH 7.5 with 71% activity at pH 10.0 and 10.5. The protease retained its activity between 4 to 60°C with maximum activity at 30°C and a residual activity of 74.4% at 60°C after overnight incubation. It was completely inhibited by 5mM PMSF pointing towards the presence of serine group of protease. Its inhibition by EDTA indicates the involvement of metal cations in its catalytic activity. It is not effected by Cu+2, partially inhibited by Pb+2 and Ni+2, while completely inhibited by Co+1, Cr+6, Zn+1, Al+3, Ag+3 and Hg+2. Strong reducing agents like β- merceptoethanol and oxidants like bleach and hydrogen peroxide inactivate the enzyme. The enzyme retains 88% of its activity on being mixed with commercially available detergents while it is inactivated by non-ionic Triton X 100. Its efficiency as an additive with detergent in terms of cleaning stains like grease, burnt mobil, vegetable curry and blood was found to be satisfactory. It could enhance the quality of washing as additive in case of all the ten detergents that were tried. The protease alone was also capable of cleaning but the detergent additive mixture could work better. The enzyme was found to work efficiently on different colors as well as on fabric. On mixing with detergent it was found to retain activity up to 2 months and there after, there was a drop in efficiency of washing. The bacterial cells were immobilized in calcium alginate and the released enzyme was found to be equally effective. Market surveys were carried out and the satisfactory result prompted the use of another additive (extracellular lipase) obtained from yet another bacterial strain from East Calcutta Wetland. The lipase activity was confirmed through degradation of coconut oil analyzed by Gas Chromatography. Thus the combination was observed to be more successful as indicated through the market survey. These observations suggest the suitability of the protease and lipase combination as additive to commercially available detergents. Key words: East Calcutta Wetland, protease secreting bacteria, extracellular lipase, detergent additive

INTRODUCTION

Currently enzymes have attracted the attention of the world over due to their wide range of industrial applications in many fields including organic synthesis, clinical analysis, pharmaceuticals, detergents, food production and fermentation. Enzymes are gradually replacing the use of harsh chemicals in various industrial processes. Since they work under moderate conditions, such as warm temperatures and neutral pH

they reduce energy consumption by eliminating the need to maintain extreme environments, as required by many chemically catalyzed reactions. The reaction specificity of the enzymes lead to minimization of the production of by-products and thereby the application of enzymes offer minimal risk to the environment. Approximately eighty percent of all industrial enzymes are hydrolytic in nature and are used for depolymerization of natural substances i.e. the breaking down of complex molecules into simpler ones. Of these

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enzymes, sixty percent are proteolytic enzymes used by the detergent, dairy and leather industries[1]. Enzymes have been known to be used for improving the cleaning efficiency of detergents and are now well accepted as ingredients in powder as well as liquid detergents and industrial/ institutional cleaning products. Detergent enzymes account for about 30% of the total worldwide enzyme production and represent one of the largest and most successful applications of modern industrial biotechnology[2]. The performance of enzymes in detergents depends on a number of factors, viz the detergent’s composition, type of stains to be removed, wash temperature, washing procedure and wash-water hardness. Besides high temperature and alkalinity, the enzyme often must withstand the presence of detergent additives such as bleaching agents, bleach activators, surfactants, perfumes etc. Out of the vast pool of enzymes, proteases from micro organisms are the most widely exploited enzymes in detergent industries[3]. They can act as minor additives or can become key ingredients in detergents. These enzymes hydrolyze protein based stains in fabrics into soluble amino acids. Among the various proteases, bacterial proteases are the most significant, compared with animal and fungal proteases. Bacterial proteases are mostly extracellular, easily produced in large amount and are often thermostable and active at wider pH range. Alkaline proteases from various strains of Bacillus sp. have been reported[4-6]. There are quite a few patents of enzymatic detergent additive (European Patent EP0581839, United States Patent 4810414). Protease from Nocardiopsis sp. as well as Pseudomonas aeruginosa have also been reported to work as an additive to detergent[7-9]. One important factor to be considered during the selection of the enzyme is its stability and the shelf life of the enzymatic action. For industrial applications, the immobilization of enzyme on a solid support can offer additional advantages like repeated use of the enzyme, ease of product separation and improvement in stability[10]. To improve washing efficiency currently available detergents usually contain enzymes like amylase, cellulase and lipase. Amylase catalyzes the break down of starch based stains into smaller segments of oligosaccharides and dextrins which are water soluble. Lipase is used in detergent formulations to remove fat containing stains such as those resulting from frying fats, butter, salad oils etc. The enzyme hydrolyzes triglycerides into mono and diglycerides, glycerol and free fatty acids which are more soluble than fats. Lipases from different species of Pseudomonas and Acinetobacter have been reported to be used in detergent formulations[11].

Keeping in view the diverse potential of the microbial enzymes, the present study was focused on the characterization and wash performance of detergent compatible protease and lipase from different bacterial isolates obtained from East Calcutta Wetland which is a biodiversity rich ecosystem of Calcutta. The location of this vast low lying area is such that the entire city's waste is drained into it. It in turn act as a sewage treatment plant[12]. The waste dumped into this system is further purified and recycled in activities like agriculture and pisciculture thereby generating products, economy and employment[13-14]. The purification of the waste, mostly heavy metals, is found to be a combined effort of diverse groups of planktons, water hyacinth and microbes[15-16]. Previous studies on microbial resource mapping of this area have revealed the existence of microbes belonging to 12 main bacterial phyla[17]. In that study, certain clones were found to be similar to Streptococcus macedonicus and Acinetobacter lowffi which are known to produce extracellular protease(s) and lipase(s) respectively. The growth conditions of these microbes were replicated in the laboratory to cultivate microbes from environmental samples of East Calcutta Wetland having potential of producing extracellular protease and lipase. Out of 21 isolates obtained, two were selected, one secreting protease and the other lipase with an aim to use the extracellular enzymes as additives to standard detergents.

MATERIALS AND METHODS Microorganisms and culture conditions: The two bacterial isolates, one protease and the other lipase producing were screened out from soil samples collected at two different sites of East Calcutta Wetland, namely Green Zone (oldest portion of solid dumping ground now converted into forest ecosystem) and Captain Bheri (shallow flat bottom waste water fed fishery) respectively. Screening of protease secreting microbes was done on solid milk medium containing 10% double toned milk, 0.3% yeast extract and 1.5% agar. Carbon minimal salt medium (CMS) containing K2HPO4

2.2 g L−1, KH2PO4 0.73 g L−1, (NH4)2SO4 1 g L−1, NaCl 30 g L−1, MgSO4 0.2 g L−1, Oil 15 mL L−1 in distilled water with 1.5% agar was used as the selective medium for isolation of lipase secreting bacteria. Luria Bertani (LB) broth containing tryptone 10 g L−1, yeast extract 5 g L−1, NaCl 5 g L−1 in double distilled water was used for the maintenance of the isolates and their further characterization. The pure isolates were preserved at -80°C in culture medium containing 70% (v/v)

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glycerol. All batch cultivations were carried out at 37°C under shaking at 150 rpm. Characterization of isolates: The two pure isolates were characterized in terms of their morphological, biochemical and molecular nature. The detailed procedure followed for characterization was as reported by Nandy et al.[18]. Enzyme assay Protease assay: Two different quantification procedures were applied. One was spectrophotometric assay using hide powder azure as substrate[19]. 0.5 mL of enzyme was added to 50 mg of hide powder azure in 1.5 mL of assay buffer constituting of 50mM Tris-HCl (pH-8) and 1 mM CaCl2. The reaction mixture was incubated at 28°C for 1 h. The supernatant was collected by centrifugation at 10,000 g for 10 min and absorbance was measured at 595 nm. One unit of activity was defined as the the amount of enzyme giving an increase of 1 absorbance unit/hour. The second assay used azocasein as substrate. 0.003 gm of azocasein was incubated with 600 µL assay buffer (50 mM Tris-HCl (pH-7.5), 5mM CaCl2) and 120 µL of sample at 60°C for 20 min. The reaction was terminated with addition of 480 µL of 15% w/v Trichloroacetic acid (TCA) and the reaction mixture was placed on ice for 10min Supernatant was collected by centrifugation at 12,000 g for 15 min. Ten microliter of 10 M NaOH was added with 900 µL of reaction mixture and absorbance was measured at 440 nm. One unit of activity was defined as the the amount of enzyme required to produce an increase of 0.1 absorbance (OD at 440 nm)[20]. Lipase assay: The preliminary assay for lipase was done on Tributyrin agar medium. The procedure reported by Nandy et al.[18] was followed. The initial quantification was done according to the diameter of the clearance zone on the triglyceride plate. The activity of the extracellular enzyme was further confirmed by Gas Chromatography (GC) taking coconut oil as substrate. From an overnight grown culture, cells were separated by centrifugation at 10,000g for 10 min. To 5 mL of extracellular supernatant 1 mL of coconut oil was added and incubated for 4 h at 35°C with shaking at 150 rpm. Post incubation 2 mL of chloroform was added to the above enzyme substrate mixture for extraction of the free fatty acids. After proper shaking with chloroform, 1.5 mL of organic phase was taken, centrifuged at 10,000 g for 10 min to ensure proper separation of organic and aqueous phase. Finally 1 mL of organic phase was taken, concentrated and analyzed by GC (Perkin Elmer, autosystem excel).

Characterization of Protease: The extracellular protease from one of the bacterial isolate (GZ) was characterized in its crude state. The effect of different physical and chemical factors on the activity of enzyme were checked. Effect of pH on enzyme activity: Supernatant containing the secreted extracellular enzyme was harvested from overnight culture grown at 37°C by centrifugation at 10,000 g for 10 min. The pH of the supernatant was adjusted at various values (2.0-10.5) using HCl and NaOH. The mixture was preincubated for 12 h at 37°C before the hide powder azure assay was performed. To investigate the optimal pH, protease activity at each pH was assayed at 37°C using spectrophotometric method. Effect of temperature on enzyme activity: The enzyme activity was measured in the range of 4-60°C using the standard activity assay (as mentioned above) at respective temperatures. Stability of the enzyme was investigated by measuring the residual activity through hide powder azure assay after incubating the crude enzyme solution at respective temperatures for 12 h. Effect of protease inhibitors on enzyme activity: The effect of various protease inhibitors such as 5 mM Phenylmethylsulphonyl fluoride (PMSF), 100 µg mL−−−−1 Tosyl L−−−−1 phenylalanine chloromethyl ketone (TPCK), 100 µg mL−−−−1 Tosyl L−−−−1 ysine chloromethyl ketone (TLCK) and 100µg mL−−−−1 Leupeptin were determined by preincubation with the extracellular supernatant containing the enzyme at 37°C for 30 min 15 µL of reaction mixture was assayed on milk medium plates by cup assay method. The plates were incubated at 37°C for overnight. The activity was measured according to the diameter of the clearing zone . Effect of various agents on protease activity: The effect of different agents like EDTA, β-mercaptoethanol, hydrogen peroxide, Triton X-100, bleach and detergent on the bacterial protease were investigated. 5 mM concentration of each of the above mentioned chemicals was added with extracellular supernatant, incubated at 37°C for 30 min followed by enzyme assay by cup assay method on milk medium. The activity was quantified according to the diameter of the clearing zone. Effect of metal ions on protease activity: Various metal ions at final concentration of 5 mM were added to the enzyme solution (extracellular supernatant. Metal salts used were Al(NO3)3.9H2O, CuSO4.5H2O, AgNO3 ,

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Pb(NO3), NiCl2.6H2O, HgCl2, CrO3, CoCl.6H2O, ZnSO4.H2O and CdCl. Relative protease activities were measured post incubation at 37°C for 30 min. The assay was done by cup assay method on milk media plates as described previously. Compatibility of the protease as an additive to detergents: From an overnight culture in LB medium, cells were separated by centrifugation at 10,000 g for 10 min and the cell free supernatant was mixed with standard detergent at a proportion of 6 units of enzymes gm−1 of detergent. Four different sets were prepared each having the same concentration of enzyme:detergent i.e. 1.5 units of enzyme to 0.25 gms of detergent 6 mL−1 water. The four different conditions like just detergent and no enzyme additive, detergent with enzyme, only enzyme in water and a control of only water without any detergent or additive were tested for stain removal. Pieces of 1.5 by 1.5 inches cloth were stained with grease and dipped in above formulations followed by incubation for 1 h at room temperature. Post incubation each piece was rubbed using brush, rinsed with water and air dried. Densitometric scanning of the residual stain on the cloth was done using software Quantity 1 from BIORAD to check the efficiency of cleaning. To check the compatibility of enzyme with different types of standard detergents it was mixed at the above mentioned ratio with the popular detergents of Indian market namely surf excel (Hindustan Lever Limited, Mumbai, India), Tide (Procter and Gamble, USA), Aerial (Procter and Gamble, USA), Sunlight (Unilever, South Africa), Nirma (Nirma Ltd., Ahmedabad, India), Sagar (AMOCHEM, Kolkata, India), Blue Bird (Local Make, Kolkata, India), Jet (Hindusthan Chemical Company, Kolkata, India), Soda (local make, Kolkata, India), Vim (Hindusthan Lever Limited, India). The wash performance of the enzyme acting as additive was checked by densitometric scanning of the stained cloths post washing and subsequent drying. Its efficiency on different stains were checked with grease, burnt mobile, food stain and blood. The wash performance was also checked out with different fabrics to look into its effect on the fabric quality and color. In order to check out the washing efficiency of the enzyme with time of incubation, the wash performance was checked at different time interval (10 min to 1 h at an interval of 10 min). For each of the above case the wash performance was checked according to same procedure mentioned above. In order to check the effect of binding of an inert matrix to the enzyme to increase its stability and washing efficiency, the extracellular supernatant

containing enzyme was absorbed onto chalk powder and wash performance was carried out. The ratio of enzyme to chalk powder was the same as that with detergent. The wash performance of the enzyme under this condition was checked in the similar manner as with the normal detergent mentioned earlier. All these tests were carried out with just protease added as enzyme. The wash performance with both lipase and protease added to the detergent were subsequently checked. Shelf life of detergent:Shelf life of the enzyme was important from the point of retention of the activity of the enzyme. The stability of the enzyme mixed with a detergent was checked over a prolonged duration of time. The wash performance of the detergent along with the protease additive was conducted at different time intervals, 15 days followed by 2 months and finally 3 months. Market survey: The lab scale experiments were followed by large scale production of protease by fermentation in a 3 L Biotron fermenter. Fermentation conditions were maintained at 40% Dissolved oxygen content (DO), 150 rpm shaking at 37°C for 16 h The cell free supernatant obtained post centrifugation was added to a standard detergent at a concentration of 6 unit of protease g−1 of detergent. The mixture was placed in trays and air dried. The dried detergent mixture in the form of powder was filled in resealable plastic bags of 500 g each. Two market surveys with protease as additive had been conducted among the families of different socio economic background and different localities of Calcutta. During the 2 trials 34 and 37 families respectively were distributed two 500 gm pack of the detergent and were asked to use the same in place of their previous detergent and compare the performance of the supplied detergent. A questionarie was provided to each family to be filled up after using the detergent for one month. The filled up forms were collected and the results were expressed as pie chart. In the third market survey, lipase from the second bacterial isolate was added along with protease to the detergent with an aim to enhance the washing efficiency. Lipase production was acheived under shake flask conditions at 37°C and overnight shaking at 150 rpm. The final ratio of lipase and protease in the detergent was maintained as 6 unit of protease gm−1 of detergent, 3 mL of crude supernatant containing extracellular lipase g−1 of detergent. The market survey had a sample size of 31 and 500 g of modified detergent was distributed to each and the rest of the analysis was the same.

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Cell immobilization for continuous recycling of cells for enzyme production: The washing efficiency of the enzyme as detergent additive was evident from lab scale findings as well as confirmed by market survey results. These findings guided for a application of immobilization technique which can give way to a cost efficient commercial exploitation. One percent inoculum was added to 3 mL of LB medium and incubated at 37°C for overnight with continuous shaking at 150 rpm. The cells were recovered by centrifugation at 10,000 g for 5 min and the cell pellet was washed with sterile Tris EDTA buffer (TE, pH 8.0). The immobilization of the cell was done as per the protocol reported by Kumar et al.[21] with certain modifications like 8% Na-alginate solution and 1M CaCl2 was used. The beads were then transfered to 4 mL of fresh medium and incubated at 37°C for overnight both with continuous shaking (Set I) and under static condition (Set II). Each 24 h the same beads were transfered to fresh medium as innoculum. After transferring the beads the supernatant of the overnight culture was centrifuged and the cell free supernatant was added to the detergent. The efficiency of the enzyme was checked each time by observing the wash performance and monitoring the units of enzyme secreted through azocasein assay. The process was repeated until the beads were present for Set 1. The relative cleaning and enzyme production for Set I and Set II were compared.

RESULTS AND DISCUSSION Characterization of the microbes: Two bacterial isolates namely GZ (as isolated from green zone at ECW)and BS (isolated from Bheri Soil) were selected for extracellular protease (Fig. 1) and lipase production respectively. Their detailed morphological and biochechemical nature is depicted in Table 1. The isolate GZ is a novel one with 98.22% similarity with Microbacterium luteolum as depicted by the phylogenetic analysis carried out using neighbour joining method (Fig. 2). The isolate BS is as yet uncharacerized at the molecular level. Enzyme assay: The protease assay at 37°C using milk media plate shows a radius of about 8 to 8.5 mm with 15 µL of supernatant while the hide powder azure test shows the supernatant to have an enzyme concentration of 0.38-0.4 U mL−1 on incubation for 1h. The preliminary lipase assay was conducted with the cell free supernatant from overnight culture of isolate BS. The cup assay method on tributyrin plates gave a clearing zone of radius 7.5 mm. The lipase

activity was confirmed through GC analysis of degradation product of coconut oil which was taken as substrate. The degradation pattern of the oil as found from GC analysis was compared with those for other isolates obtained from ECW (mainly Pseudomonas Group and Acinetobacter group) (Fig. 3). The profile for each isolate is indicated with different color. Microbes of two different types, namely Pseudomonas and Acinetobacter were studied and both show distinctly different extent of degradation of the substrate. The read color graph in the figure indicates the degraded products obtained by action of extracellular lipase from isolate BS.

GZ

Fig. 1: Photograph of milk media plate showing

extracellular protease induced clearing zone around the growth for 9 strains of bacteria. The isolate highlighted with a circle is GZ

Fig. 2: Partial 16S rRNA sequence based phylogenetic analysis of isolate GZ constructed using neighbour joining method

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Table 1: Table representing the characterization of the two isolates GZ and BS from East Calcutta Wetland. Morphological Characterization Biochemical characterization Molecular characterization ----------------------- ------------------------------------------------------------------------ ------------------------------------------ Site of Gram Cell Closest neighbor GeneBank Isolate Origin Nature Morph Lipase Catalase DNase Oxidase Protease /percentage similarity Accn. No. GZ Green Gm-ve Bacilli - + - - + Microbacterium EU006695 Zone luteolum (98.22%) BS Soil of Gm+ve Cocci + + - - - Uncharacterized Captain Bheri

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 Fig. 3: GC analysis depicting the comparative action

over coconut oil by extracellular lipase from 7 different isolates of East Calcutta Wetland

The light green, black and dark green graph depicts the pattern generated by Acinetobacter while pink, light blue and dark blue graphs are generated by Pseudomonas sp. from ECW. Protease characterization: For a protease to work as a detergent additive it should be able to perform the function at alkaline pH. Thus the activity of the extracellular enzyme in supernatant was checked at different pH using hide powder azure assay. The protease was checked in pH range of 2 to 10.5 and was found to be active in the range of 4 to 10.5 (Fig. 4a) with maximum activity at pH 7.5. It could retain 71% of its activity at pH 10.5 and thus could be used at alkaline pH as detergent additive. The effect of temperature was checked similarly by preincubating the supernatant for 12 h at different temperature before performing the assay with hide powder azure as substrate. It showed activity along the entire range of 4 to 60°C. It showed maximum activity at 30°C (considered as 100%) (Fig. 4b). It retained 92.3% activity at 2 and 4°C while it retained 97.4, 90 and 74.4% activity at 37, 40-50 and 60°C respectively. This enzyme could work at high temperature retaining maximum activity and thus was suitable for application in detergent industry.

Attempts were made to determine the nature of the protease by using specific protease inhibitors. It (Fig. 4c) showed complete inhibition of activity upon treatment with PMSF (serine protease inhibitor) while partial inhibition in case of TPCK (inhibitor for proteases having Phenylalanine at the P1 cleaving site), TLCK (inhibitor of serine and other proteases) and Leupeptine (inhibitor of serine and other protease). The results indicate the presence of serine protease which is completely blocked by PMSF. The partial inhibition by Leupeptine and TLCK could be because of insufficient/lower concentration of these inhibitors used. A concentration profile with increasing amount of inhibitor could justify the above statement if complete inhibition is obtained at some higher concentration. The inhibition in case of TPCK points towards the possible presence of Phenylalanine at the position P1 of the cleavage site. It needs to be confirmed further. β-merceptoethanol was found to completely inhibit protease activity. It could either be due to destabilization of thiol group at the active site and thus preventing the enzyme from binding the substrate. This could otherwise be due to destabilization of the three dimentional structure of the enzyme due to disulphide bond breakdown at other positions resulting in denaturation of the protein and so inhibition of the function. From here at this stage we could just say that it is sensitive to reducing agents. Hydrogen peroxide and Bleach were found to completely inhibit protease activity. Thus these additive could not be used with detergents containing bleaching agents as it is sensitive to free radical generating/oxidizing agents. The effect of nonionizing and commercial detergents on the protease activity was tested. The nonionic detergent completely inhibited the protease activity while with commercial detergent there was 88% activity. Thus the impact of Triton X 100 treatment might have been such that it impairs the function through disruption of the three dimentional structure of the enzyme. The effect of EDTA (Ethylene diamine tetra acetic acid) on the activity of protease was tested with preincubation in the similar manner and a remnant activity of 2% with

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Fig. 4: Graphs representing the protease characterization in terms of the enzyme activity under conditions of different pH, temperature, enzyme inhibitors, reducing agents and metal cations. (4a) Graph representing the effect of pH on enzyme activity. The activity retained after 12 h incubation at different pH was plotted with pH on X axis and the corresponding enzyme activity along the Y axis. The activity was plotted as the percentage of activity retained considering activity at pH 7.5 to be 100%. (4b) Graph representing the effect of different temperature over enzyme activity. The temperature scales were plotted on X axis while the corresponding enzyme activity in % was depicted along Y axis. (4c) Graph representing the effect of different inhibitors on enzyme activity. Enzyme activity under native condition was considered as 100%. The % of activity retained in response to specific inhibitors was plotted on Y axis with the inhibitors mentioned on the X axis. (4d) Graph representing the effect of different agents on extracellular protease activity. The activity of native protease is taken as 100% and the remaining activity as % after treatment is plotted on the Y axis with agents on the X axis. (4e) Graph representing the effect of different metals on enzyme activity. Enzyme activity under untreated condition was taken as 100%. The activity retained after incubation with metal salts was plotted on the Y axis with the salts listed on the X axis. The result shows except for Cu and to some extent Ni and Pb, other metal ions viz. Co, Cr, Zn, Al, Ag, Hg showed complete inhibition of enzyme activity

5 mM addition was obtained as compared to untreated protease. This points towards the role of divalent cations in the functioning of the enzyme (Fig. 4d). Since metal ions are essential for the activity of the enzyme (as it was inhibited by EDTA), the effect of different salts were tested. There was 99.6% retention of activity in presence of 5mM CuSO4.5H2O, 25% retention of activity in presence of 5mM of NiCl2.6H2O

and Pb (NO3) while complete inhibition of activity for all the rest. Thus the protease is sensitive to metal ions (Fig. 4e). Protease as detergent additive: The protease was used in different combinations to check its effect on the cleaning of grease stain. Stained cloth was washed with plain water, plain detergent, only protease and detergent

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Fig. 5: Graphs representing the efficiency of the protease as detergent additive depicted in terms of its wash performance over various types of stains as well as fabric, time course of the enzyme action over stain removal, compatibility of the enzyme as a additive with different available brands of detergents. (5a) Wash performance analysis with and without addition of the protease on grease stain. The different conditions are indicated on the X axis while the stain intensity was on the Y axis. Prot stands for Protease, Det stands for Detergent while Det+Prot stands for detergent with protease. (5b) Time course of incubation of grease stained cloth with detergent and additive mixture. Stn Clt stands for stained cloth and C stands for control which is wash with just water. (5c) Graphical representation of wash performance of 5 different detergents with protease added as additive. The stain without washing was considered as 100% and there after the stain remaining on washing with detergent and detergent plus protease was compared. In each case there was enhancement of cleaning with addition of protease. Det stands for detergent. The intensity on stained cloths were taken as 100%. The washed cloth with only detergent and those with detergent and protease was compared against the original stain and graphically represented in this Fig. (5d) Graphical representation of effect of the protease additive on cleaning efficiency of various stains like grease, burnt mobil, vegetable curry and blood. In each case there is increase in efficiency of cleaning with addition of protease as compared to just detergent. (5e) Graph representing the washing efficiency of the additive for different types of fabrics. Here cloths of different types and color like blood red silk, white cotton, sky blue and lemon yellow synthetic were used for the trial. The stained (grease) pieces were identically treated with just water, just detergent and detergent with protease for 1 hour and then washed as mentioned above. After drying the cloth pieces were scanned in a densitometric scanner. Y axis represents the % of residual stain post wash as compared to just stained cloth taken as 100%. Det stands for only detergent and Det+Prot stands for wash with detergent containing protease as additive. Stain Cloth represents samples that have not been washed and control represents samples that have been washed only with water. 5f) The shelf life of the detergent additive mixture after 15 days and after 2 months of incubation as indicated by cleaning efficiency are depicted graphically. The efficiency increases on storage up to 2 months and thereafter there is decrease in efficiency

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with protease, dried and scanned using Quantity 1 software (BIORAD) through densitometric scanning. The intensity of stained cloth was taken as 100% and the residual intensity post wash was monitored. The percentage cleaning was the residual activity subtracted from that of stained cloth (i.e. 100). The result (Fig. 5a) indicates that the protease alone can clean better than just detergent but the best efficiency was obtained on mixing both the detergent and the protease. A time course of incubation of the stained cloth with detergent and additive mixture was done. The stained cloths were incubated for 10min, 20min, 30min, 40min, 50min and 60min before washing and the the results were interpreted as above following densitometric scanning (Fig. 5b). Maximum cleaning was observed after 60min incubation. Since the protease was found to work as detergent additive now the next question in hand was to check if it could work similarly for most of the commercially available detergents. This was important to know as the protease was completely inhibited by Triton X 100. Ten different detergents were tried out with all showing satisfactory enhancement of activity. Some of the results are expressed in graphical form in Fig. 5c. Since the additive works for all types of commercial detergent thus the next approach was to test for different kinds of stains. Stains like grease, burnt mobile, vegetable curry and blood were tested. The combination of detergent with additive worked best in all the 4 cases as compared to plain water or only detergent (Fig. 5d). The next attempt was to check the effect of the detergent additive mixture on the color as well as fabric of various kind. The mixture did not affect the color or the nature of the fabric adversely. It was used on Cotton, Silk, Chiffons, Synthetic material, etc. The result of 4 such trials are depicted below in Fig. 5e. Immobilization of enzyme to inert matrix often increases the stability of the enzyme. Thus protease was immobilized on chalk powder and its efficiency of cleaning was checked. It did not show any improvement in performance [data not shown]. The shelf life of the additive post mixing with the detergent was checked at 15 days interval for 3 months and compared with that of the freshly mixture of additive and detergent (Fig. 5f and 6). There was retension of activity comparable to fresh mixture upto 2 months and there after there was decrease in efficiency. Three market surveys were conducted with the first two surveys having detergent with only protease as additive. The population covered under the survey were asked to compare the cleaning efficiency of the mixture with that of their existing household detergent both in

Shelf life

Fig. 6: Photograph showing the cleaning efficiency of

the detergent with enzyme additive in different time intervals in order to check the shelf life or stability of the enzyme

terms of quality and quantity. The results were expressed as acceptable or non acceptable as represented in Fig. 7a. The mixture worked best in case of steel utensils with least preference for washing. In all four cases majority of the population found it acceptable. The distribution among families of different region and economic status ensured that irrespective of the quality of the water in different regions and irrespective of the quality of the household detergent as decided by the socio economic class, this mixture was acceptable to all. Since the two market survey showed encouraging result, attempts were made at the laboratory for adding lipase along with protease as additive to detergent. The laboratory trial yielded positive result and the third market survey with both protease and lipase used as additive was undertaken. It was found to be much more successful as depicted in Fig. 7b. Since the enzymes were found to work effectively as detergent additive in all the market surveys, so attempts were made to simplify the process of obtaining additives at the commercial scale. 1% cells of both the type (isolate GZ and BS) were immobilized in calcium alginate separately and the entrapped cells were used as inoculum for twenty rounds of growth. Results indicate that there is increase in enzyme secretion with shaking as compared to stationary condition. This might be due to the variation in dissolved oxygen concentration as evident from the DO measurement (0% under static condition, 57% immediately after shaking and 72% during shaking). The enzyme production was monitored in each cycle and found to be relatively stable throughout the 20 cycles both for static and shaking condition. There cleaning efficiency of both the additives from static as well as stationary conditions

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Fig. 7: Graphs representing the acceptability of the enzymes, protease and lipase as additives to detergents as

derived from market survey. 7a) Graphs representing the results of market survey with only protease added as additive to detergent. 7b) Graphs representing the results of market survey with protease and lipase added as additive to detergent. In all cases mauve represents acceptable and maroon represents not acceptable

Fig. 8: Photograph showing the cleaning efficiency of

the detergent with enzyme additive under the condition of enzyme production by immobilized cells

(immobilized state) were checked and found to be comparable to that of fresh mixture of detergent and additives from liquid culture (free cells). It is to be noted that the efficiency of cleaning was better under shaking as compared to stationary condition which might be due to higher enzyme production (with activity of enzyme in supernatant under shaking condition as 1 U while that for static as 0.5 U). The comparison of the result of cleaning under free as compared to immobilized condition is provided in Fig. 8.

CONCLUSION In this reserch we report for the first time the detection of laundry detergent compatable extracellular protease from a novel bacterial strain showing maximum similarity with Microbacterium luteolum

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among the cultivable microbes. We also report the presence of detergent compatible extracellular lipase activity from a bacterial isolate showing similarity to Acinetobacter sp as proved by the degradation pattern of coconot oil through GC analysis. Both these isolates are obtained from the unique site of East Calcutta Wetland in Calcutta, India, which happens to be the world's largest dumping ground and treatment site for solid as well as soluble waste covering an area of 12500 Ha.

ACKNOWLEDGEMENT

The authors would like to acknowledge the financial support of Department of Science and Technology, India, Department of Biotechnology, India, Inter University Consortium Calcutta Centre and Board of Research in Nuclear Sciences, Department of Atomic Energy, India. They would thank West Bengal University of Technology for providing the computational as well as Bioinormatics Infrastructure Facility (DBT, GOI supported). We are also thankful to the participants of the Market survey for providing us with the filled in market survey questionaire in time.

REFERENCES 1. Rao, M.B., A.M. Tanksale, M.S. Ghatge and

V.V. Deshpande, 1998. Molecular and Biotechnological aspects of Microbial proteases. Microbiol. Molecular Biol. Rev., 62: 597-635.

2. Research and Application of Microbial Enzymes-India's Contribution, 2003. Advances in Biochemical Engineering/Biotechnology, 85: 95-124.

3. Ganesh Kumar, C. and H. Tyagi, 1999. Microbial alkaline protease: From a bioindustrial viewpoint. Biotechnol. Adv., 17: 561-594.

4. Banik, R.M. and M. Prakash, 2004. Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol. Res., 159 (2): 135-140.

5. Oberoi, R., Q.K. Beg, S. Puri, R.K. Saxena and R. Gupta, 2001. Characterization and wash performance analysis of an SDS stable alkaline protease from a Bacillus sp. World J. Microbiol. Biotechnol., 17 (5): 493-497.

6. Nascimento, W.C.A. and M.L.L. Martins, 2006. Studies on the stability of protease from Bacillus sp. and its compatibility with commercial detergent. Brazilian J. Microbiol., 37: 307-311.

7. Albuquerque, M.K.A., B.F. Teixeira, M.F.S. Porto and A.L.F. Lima Filho, 2002. Application of protease from Nocardiopsis sp. as a laundry detergent additive. World J. Microbiol. Biotechnol., 18 (4): 309-315(7).

8. Najafi, M.F., D. Deobagkar and D. Deobagkar, 2005. Potential application of protease isolated from Pseudomonas aeruginosa PD100. Electronic J. Biotechnol., 8(2): 197-203.

9. Howe, T.R. and B.H. Iglewski, 1984. Isolation and characterization of alkaline protease-deficient mutants of Pseudomonas aeruginosa in vitro and in a mouse eye model. Infect. Immunity, 43 (3): 1058-1063.

10. Ahmed, S.A., S.A. Saleh and A.F. Abdel-Fattah, 2007. Stabilization of Bacillus licheniformis ATCC21415 alkaline protease by immobilization and modification. Aust. J. Basic Applied scie., 1 (3): 313-322.

11. Hasan, F., A.A. Shah and A. Hameed, 2006. Industrial applications of microbial lipases. Enzyme Microbiol. Technol., 39: 235-251.

12. Ray Chaudhuri, S., M. Mishra, P. Nandy and A.R. Thakur, 2008. Waste management: A case study of ongoing traditional practices at East Calcutta Wetland. Am. J. Agric. Biol. Sci., 3: 315-320.

13. Chaudhuri, S.R., S. Salodkar, M. Sudarshan and A.R. Thakur, 2007. Integrated Resource Recovery at East Calcutta Wetland-how safe is these? Am. J. Agric. Biol. Sci., 2: 75-80.

14. Raychaudhuri, S., M. Mishra, S. Salodkar, M. Sudarshan and A.R. Thakur, 2008. Traditional Aquaculture Practice at East Calcutta Wetland: The Safety Assessment. Am. J. Environ. Sci., 4 (2): 140-144.

15. Pradhan, A., P. Bhaumik, S. Das, M. Mishra, S. Khanam, B. Amin Hoque, I. Mukherjee, A.R. Thakur and S.R. Chaudhuri, 2008. Phytoplankton diversity as indicator of water quality for fish cultivation. Am. J. Environ. Sci., 4 (4): 271-276.

16. Chaudhuri, S.R., S. Salodkar, M. Sudarshan, I. Mukherjee and A.R. Thakur. Role of Water hyacinth mediated Phytoremediation in Waste Water Purification at East Calcutta Wetland. Environ. Sci., Accepted.

17. Raychaudhuri, S. and A.R. Thakur, 2006. Microbial genetic resource mapping of East Calcutta Wetland. Curr. Sci., 91: 212-217.

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18. Nandy, P., A.R. Thakur and S.R. Chaudhuri, 2007 Characterization of bacterial strains isolated through microbial profiling of urine samples. Online J. Biol. Sci. 71(1): 44-51.

19. Ching-Hsing, L. and D.E. McCallus, 1998. Biochemical and Genetic Characterization of an extracellular protease from Pseudomonas fluorescens CY091. Applied Environ. Microbiol., 64 (3): 914-921.

20. Koeabryik, S. and B. Erden, 2002. Intracellular alkaline proteasse produced by thermoacidophiles: Detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR). Bioresource Technol., 84 (1): 29-33.

21. Kumar, S.R. and M. Chandrasekharan, 2003. Continuous production of L-glutaminase by an immobilised marine Pseudomonas sp BTMS-51 in a packed bed reactor. Process Biochem., 38: 1431-1436.

American Journal of Environmental Sciences 4 (4): 406-411, 2008 ISSN 1553-345X © 2008 Science Publications

Corresponding Author: Shaon Ray Chaudhuri, Department of Biotechnology, West Bengal University of Technology, BF-142, Sector 1, Salt Lake, Kolkata 700064, India

406

Phytoplankton Diversity as Indicator of Water Quality for Fish Cultivation

1Arunava Pradhan, 1Pranami Bhaumik, 1Sumana Das, 1Madhusmita Mishra,

3Sufia Khanam, 3 Bilqis Amin Hoque, 2Indranil Mukherjee, 1Ashoke Ranjan Thakur and 1Shaon Ray Chaudhuri

1Department of Biotechnology, West Bengal University of Technology, BF-142, Sector 1, Salt Lake, Kolkata 700064, India

2Army Institute of Management, Judges Court Road, Alipore, Kolkata 700027, India 3Environment and Pollution Research Centre, House#242, Road#17, New DOHS,

Mohakhali, Dhaka-1206, Bangladesh

Abstract: Waste water fed fisheries are a common feature in different parts of the world. Yet not all work as efficiently as those operating at East Calcutta Wetland for more than 70 years now. The objective of this study is to unravel the reason for the markedly greater efficiency of the Bheris in fish production compared to other water bodies like rain water ponds or sewage fed fish ponds elsewhere. The study indicates that plankton growth could be an important factor responsible for greater fish production in the Bheris. The architecture of the Bheri itself acts as a facilitator in the process. It is proposed that planktons can act as biomarker for water quality assessment in fish production. Key words: Plankton, bheri, khamar, dighi, east calcutta wetland

INTRODUCTION

The potential of waste water fed aquaculture has long been recognized in Asia, and the history of such systems goes back over several centuries[1]. The traditional systems in China involve the collection of human excreta for use as fertilizer for fish culture. Since 1950s the use of municipal waste water has developed rapidly. In Germany scientists have studied waste water fed aquaculture since the late 19th Century[2] while in India it began around 1930. The Calcutta waste water fish pond system has the advantage of not only the production of fish but also improvement in water quality and reduction of pathogens in the sewage[3]. East Calcutta Wetland (ECW) is a perfect example of wise-use wetland ecosystem where usage of city sewage for traditional practices of fisheries and agriculture is practiced. The wetland ecosystem is a rare example of combination of environmental protection and development management. ECW also happens to be the largest ensemble of sewage fed fish ponds in the world in one place[4,5,6,7]. The tropical regions are more suitable for treating sewage in ponds. Bheri, the flat bottom shallow waste water fed fish ponds, a specialty of the eastern part (mostly West Bengal) of India has been reported to be the best example of integrated

resource recovery. Here the waste water is naturally processed and utilized for the cultivation of fishes. After fish cultivation the water is used for irrigation of the surrounding fields and the effluent at the bottom of the Bheri is used as a fertile soil for the same fields. These waste water fed fish ponds are different from the other waste water fed ponds operating in different countries in certain features. The first among these is their shallow depths (50 to 150 cm) and flat bottoms[8]. This ensures that sunlight penetrates evenly to the bottom of the ponds and results in photosynthetic activity within these ponds which is the basis of natural biological purification. There is a drastic reduction in coliform count between the water of the raw sewage canal and the corresponding Bheri. One of the possible explanations for the purification process operating at Bheri is the alkalinity developed due to processing of the pond bed with lime. This elevated pH is fatal for the coliforms and leads to their reduction. The solar energy that is trapped by a dense population of plankton which in turn are consumed by the fishes may be another cause. The planktons play a significant role in degrading the organic matter. But overgrowth of planktons becomes a problem for pond management since they cause algal bloom. It is at this critical phase of the ecological process that the fish plays an important role by grazing

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on the plankton. The two fold role played by the fishes is indeed crucial – they maintain a proper balance of the plankton population in the pond and also convert the available nutrients in the wastewater into readily consumable form (fish) for humans. Among the diverse variety of planktons, there are some like the cyanobacteria which produce toxins that are detrimental for fish growth and quality but there are the green algae which are known to facilitate fish growth[9,10]. Thus the planktons can themselves be classified as useful and harmful from the angle of waste water fed fish production. The fish farmers of ECW have developed such a mastery of these resource recovery activities that they are easily growing fish at a yield which is 2 to 4 times higher than normal ponds at production costs unmatched by any other freshwater fish ponds of this country[11]. On the other hand the sewage fed fish ponds in the tropical neighboring country of Bangladesh are not showing as much production and even the production that is obtained is not consistent. The pond system at ECW functions in a systematic manner involving five major phases. The first is pond preparation which involves complete draining of the ponds, sun drying of the pond bottom, desilting of silt traps, tilling and repairing of dikes. During this stage there is treatment of the soil with lime which ensures alkalinity of the introduced water. This alkalinity also leads to decreased coliform count. This is mostly in December and January. The second phase is called primary fertilization where waste water is introduced into the Bheri and allowed to undergo natural purification. The pond is stirred intensely to reduce anaerobic conditions in the sediments and promote the development of benthic organisms which would be used as fish feed. This is in the middle of February. The third phase involves fish stocking. This starts in the middle of March. To ascertain the quality of water for fish growth, initially stocking test fish is done in which a small number of fish is stocked as probe species to test water quality. After obtaining satisfactory water quality, proper fish stocking takes place. The fourth phase is that of secondary fertilization in which there is periodic introduction of waste water into the ponds throughout the growth cycle. Sewage is added to the fishpond to stimulate sufficient plankton growth for fish feed. Care is taken to maintain the dissolved oxygen concentration above the threshold level for fish sustenance. The final phase involves the fish harvesting which starts from August and continues to December till the phase one starts. Thus the cycle continues[8]. On the other hand, the waste water fed fish pond (locally called Khamar) preparation at Bangladesh also starts in

November and December where once every year the ponds (300 to 610cm deep) are dried, the top layer of soil is removed and the bed treated with lime and potash which is impure form of potassium carbonate mixed with other potassium salts. The water is treated again with common salt and potash in case of any fish disease out break. As feed they add additionally urea, musted cake, rice dust, wheat brain, boiled rice pellet once every week. The fish growth is best between July to September which is followed by harvesting. This study attempts to analyze the variation in physicochemical as well as plankton profile of the waste water fed ponds bodies from the two countries in order to understand the cause for the variation of fish production. It would also involve comparison of the fresh water ponds with waste water fed fish pond from India to understand the relation between the water source and the other parameters, if any. This study intends to look for biomarkers, if any, as quality indicator for fish production in such ponds.

MATERIALS AND METHODS Collection of water sample: The water samples were collected in sterile plastic containers from a depth of 1.5 to 2.0 cm below the surface of the water and transported to the laboratory for further analysis at 4oC. Samples were collected from four different Bheris at ECW (from India) namely Captain Bheri, Natar Bheri, Nuner Bheri (No.–1) and Charakdanga Bheri; four different Khamars (from Bangladesh) namely: Bawniabad Dhigi 1, Bawniabad Dhigi 2, Bawniabad Dighi 3 and Gulshan Lake as well as four rain water ponds from New Barrackpur, India, a site distant from ECW and thus no way related to Bheri. In addition two other rainwater ponds from India, one from Salt Lake (Central Park Pond) which is close to ECW and another from Ichapore which is further away from New Barrachpur were taken in order to avoid any artifact in the study by ensuring wide spread distribution. Being spread over a large region, thirty aliquots of water samples were collected from different parts of each Bheri and the rain water ponds. Physicochemical analysis: Chloride (kit: Model CD-B chloride test Cat. No. 26018-00, HACH), Nitrate (Model NI-11 Cat. No.1468-03, HACH) and Ammonia Nitrogen (Model NI- 8 Cat. No.2241-00, HACH) were monitored using kits from HACH as per the manufacturers protocol. The sensitivity of these were 0-400mg/l (miligram per liter), 0-50mg/l and 0-3mg/l respectively. The dissolved oxygen (DO-5509, Lutron), conductivity (Model: 6-7004-02, IS-TD Scan 4, Singapore), turbidity (HI 93703, Microprocessor

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Turbidity meter, HANNA) and pH (Wegtech Potatest pH meter) were measured as per the standard protocol. The detection limit for these were 0 to 20.0mg/l, 20 to 200 S (Siemens), 0 to 1000 FTU (Formazin Turbidity Unit) and 0.00 to 14.00 respectively. Faecal Coliform Count: The samples were used for total faecal coliform measurement according to the protocol provided in Standard Method for the examination of Water and Wastewater Analysis by American Public Health Association, Washington, D.C. (1998). The membrane filtration method of measurement of Total faecal coliform bacteria was performed on the samples. This approach estimates the number of FC colonies present. The DIFCO dehydrated mFC Broth base, BDH Agar powder and rosalic acid were used to make the culture medium in 50x12 mm disposable plastic petri dishes. The filtered volumes were determined based on pre-testing of several decimal volumes of a few samples from the first 2 batches of every survey. The petri dishes were placed in an incubator for 24 hours at 44.5oC. The colonies produced by fecal coliform bacteria on mFC medium in various shades of blue color were counted. Then the counts were calculated according to the dilution factor and reported as Colony forming units per 100 ml. Saturation study: Water in each falcon was mixed by inversion and 20µl was observed under the light microscope. The number of observation and the different varieties of microscopic bodies (plankton) observed were noted and a graph was plotted with number of observation on the X axis and the total number of newer varieties observed with each observation on the Y axis. The curve indicates whether saturation in screening the diversity has been obtained as had been shown earlier for a different system[12]. It was done for each water body. Plankton diversity study: Water from different water bodies as mentioned above, were observed under 40X magnification (Phase contrast) of Axiostar Plus fluorescence microscope from Zeiss for identification of different planktons existing there. The different varieties were monitored and identified. The count for different plankton species was taken with haemocytometer.

RESULTS AND DISCUSSION Saturation Curves: The number of planktons were counted and thirty independent sets of observations were taken. When the number of observations was plotted on the X axis and the number of total new varieties observed till a particular observation on the Y axis for each water body, a saturation curve was

0

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80

Number of Observations

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Fig. 1: Figure showing the saturation curve for each

water body. Irrespective of the nature of the water body the saturation in plankton diversity is obtained indicating that no further increase in the plankton diversity would be achieved with further increase in the number of observations

Table1: Comparison of coliform count (cfu 100 mL�1), conductivity

(S) and DO (mg L�1) among Bheri and its corresponding raw sewage canal (RSC) in case of Charakdanga Bheri, Nunar Bheri and Captain Bheri from East Calcutta Wetland, Indi

Site Conductivity DO TTC Charakdanga Bheri 0.90 3.7 5005 Charakdanga RSC 1.00 2.6 Uncountable (>105) Nunar Bheri 0.40 4.3 180 Nunar RSC 0.70 2.9 Uncountable (>105) Captain Bheri 1.6 3.8 1090 Captain RSC 1.7 3.7 23200

obtained (Fig 1). This clearly depicts that the screening was adequate and that no further observation for understanding the diversity was needed. Water quality improvement: Water flows from the Raw sewage canal to the Bheri. A comparison of the profiles of the the two indicates a sharp decrease in coliform count, slight decrease in the conductivity and a slight increase in DO in the Bheri as compared to the corresponding Raw Sewage Canal (Table 1). The results indicate purification of the water in the Bheri. Further the similar parameters were compared among the different water bodies from the two countries (data not shown). There was no major variation in the different physicochemical parameters that were monitored among the different sites that could emphasize the variation in fish production or could act as marker for indicating the fish producing ability of a water body. Plankton Diversity: The different water bodies were analyzed for their total plankton count per

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Table 2: Table indicating the total variety of planktons observed, the total planktons/ml and the planktons useful for fish growth per ml in the

different water bodies Name of Water Body Total variety Total Plankton count/ml Useful Plankton count/ml Captain Bheri (ECW, India) 63 3601200 244000 Nunar Bheri (ECW, India) 57 2018000 831000 Natar Bheri (ECW, India) 55 2380000 950000 Charakdanga Bheri (ECW, India) 58 2369300 905000 Rain water pond 1 (New Barrackpur, India) 47 204600 79400 Rain water pond 2 (New Barrackpur, India) 38 129200 53000 Rain water pond 3 (New Barrackpur, India) 50 208600 83600 Rain water pond 4 (New Barrackpur, India) 38 165800 63200 Rain water pond 5 (Ichapore, India) 61 240815 93165 Central Park Pond (Salt Lake, India) 45 720800 22400 Bawniabad Dighi 1 (Bangladesh) 66 251200 89800 Bawniabad Dighi 2 (Bangladesh) 59 162200 69200 Bawniabad Dighi 3 (Bangladesh) 55 206200 80200 Gulshan Lake (Bangladesh) 53 304400 139800

Table 3: Table enlisting the known bioremedial functions of the different planktons found in the various water bodies studied in this paper Species Role in Bioremediation Chlorella Uptake metals like cadmium, copper, iron, nickel, zinc. Chlamydomonas Removal of lead, copper, chromium, cadmium. Eudorina Biosorption of metals like lead. Chlorococcum Absorb and degrade endocrine disrupting insecticide alpha endosulfan. Scenedesmus Bioremediation of crude oil, n alkanes, polycyclic aromatic hydrocarbons, removal of nitrogen from waste water. Haematococcus Removal of nitrogen and phosphorous from waste water. Spirullina Removal of copper, mercury cadmium ammonia nitrogen. Nostoc Metabolism dependent cellular uptake of copper. Closteridium Transformation of 1,1,1- Trichloroethane, trichloromethane and tetrachloromethane used in pesticides. Navicula Accumulation of cadmium, mercury Oscillatoria Accumulation of cadmium, mercury, lead. Ulothrix Accumulation of cadmium, mercury, lead. Anabena Uptake of radionucleotides such as Co and Cs Stichococcus bacillaris accumulated inorganic lead (Pb) from aqueous solutions extra- and intracellularly. Synedra Intracellular accumulation of mercuric chloride Ankistrodesmus Accumulate cadmium Fragilaria Accumulate Cadmium upto 2.25 of its dry weight Selenastrum Accumulate Uranium upto 1% of its dry weight. Euglena Accumulate Aluminium, zinc, manganese, copper and lead

milliliter of water, number of total varieties found in each and the total number of useful (from the point of fish growth) varieties of Plankton found per milliliter of water for each (Table 2). The useful varieties were determined from the existing literature[9,10]. The Bheries are known for their fish production for more than 70 years now and are showing the highest plankton count (both total as well as useful). The rain water ponds show moderate fish production. Gulshan Lake shows good fish production most of the time including when the sampling was done. It is reflected in its count of useful plankton mL�1. Bawniabad Dhigi 1 shows better production of fish among the water bodies in Bawniabad with Bawniabad Dhigi 3 as the intermediate producer and Bawniabad Dhigi 2 as the worst among them. In all cases these produce much less fish than the Bheris. This is clearly reflected from the useful plankton count mL�1 in these water bodies.

These useful planktons include Chlorella, Crucigenia, Spirulina, Nitzchia, Scenedesmas, Cyclotella, Microcystis, Coelastrum, Melosira, Navicula, Anabaena, Chlamydomonas, Tetraedon, Euglena, Endorina, Ankistrodesmas, Cosmarium, Fragilaria, Pediastrum and Syndera. Plankton diversity in Bheri- putative role in bioremediation: A wide variety of planktons are observed from the analysis of water from different Bheris as well as from rain water bodies. The main aim was to find out the relation, if any, between the existence of differential number of the planktons and their bioremedial activity in the water bodies. The bioremedial function for the planktons observed in these bodies as reported by previous studies are listed in Table 3 [13,14,15,16,17,18,19]. As evident from above studies, planktons have a major role in bioremediation. Several studies have put forward the active role of Chlorella

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vulgaris in removal of heavy metal contamination from water and soil. Chlorella tablets are reported to absorb the mercury released during the replacement of mercury amalgam fillings[20]. The microalgal strains sequester or precipitate metals/radionucleotide either by surface adsorption, intracellular accumulation or by conditioning the surrounding chemical environment, thus bioconcentrating the meta/radionucleotide in a small volume. Organic contaminants are degraded and may be completely mineralized[21,22]. The plankton diversity analysis in bheris, Dhigi (Khamar) and fresh water indicates one primordial observation, i.e. the different species of planktons were found in high numbers (total plankton count per milliliter) in bheris as compared to the other two bodies. The reason could be attributed to the continuous high load of metals, aromatic compounds and other wastes in the Bheris as compared to the fresh water fed pond or Dhigi. The Gulshan Lake receives industrial effluents from time to time and thus shows an elevated quantity of useful planktons as compared to the other rain water ponds and Dhigi. Abundance of species like Chlorella in Bheris indicates the high concentration of metals like Ni, Pb, Cu in the water body .The source of these metals comes from small scale industries involved in electronic good manufacturing, handicraft etc. High load of metals like chromium are drained into Bheris along with effluents of tanneries as is also reflected by the large number of Chlamydomonas sp. Thus the abundance of different planktons in Bheri as compared to the fresh water ponds is in correlation with their active participation in bioremediation process taking place in this complex ecosystem. The metal concentration of water from different Bheris as analyzed through Energy Dispersive X Ray Fluorescence validates the above statement (results not shown).

CONCLUSION The objective of this study was to understand the underlying reason for enhanced fish production in the Bheri as compared to rain water ponds as well as sewage fed fish ponds in other places. From the results of this study it appears that the growth of useful planktons might be an important factor for the fish production. One of the facilitators for this growth is the architecture of the Bheri. The planktons not only enhance fish production but also facilitate bioremediation of heavy metals and other toxic material. In this paper we propose that the above

mentioned useful planktons can act as biomarker for water quality assessment for fish production. It is perceived that the process adopted in the Bheris of East Calcutta Wetland might be employed productively in tropical countries like Bangladesh for enhancing their fish production and managing their liquid waste more efficiently. The process of transfer of this technology would be the subject of our future work.

ACKNOWLEDGEMENT The authors would like to acknowledge the financial support of Department of Biotechnology, India; West Bengal University of Technology, India; Environment and Population Research Centre, Bangladesh as well as Department of Atomic Energy, India under the Board of Research in Nuclear Sciences scheme. They would like to thank Bioinormatics Infrastructure Facility (DBT, GOI supported) of West Bengal University of Technology for its computational facility. Authors from WBUT would also like to acknowledge the assistance of Prof S K Dey, Mrs Indrani Roy and Ms Poonam Nasipuri of Department of Biotechnology, West Bengal University of Technology, Calcutta during the work. Authors from EPRC would like to acknowledge the help extended by Sanowar Hossain, Bikash C. Roy and people of Bauniabad.

REFERENCES 1. Chan, G., L.T. Hung and N.V. Thu, 1993 National

Seminar on Sustainable livestock production on local resources. pp: 2-10

2. Prein, M. 1990 Wastewater-fed fish culture in Germany. In Wastewater-fed aquaculture (Editors: P Edwards and RSV Pullin),. UNDP-World Bank, Water and sanitation Program, Asian Institute of Technology and ICLARM. P:13-41.

3. http://www.utafoundation.org/utacambod/msc99thes/Sophinlr.htm accessed on 23rd October 2007.

4. http://www.wwfindia.org/about_wwf/what_we_do/freshwater_wetlands/our_eork/ramsar_sites/east_calcutta_wetlands_.cfm /ramsar_sites/east_calcutta_wetlands_.cmf accessed on 14th June 2007.

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6. Ray Chaudhuri, S., S. Salodkar, M. Sudarshan and A.R. Thakur, 2007. Integrated Resource Recovery at East Calcutta Wetland how safe is these? Am. J. Agric. Biol. Sci, 2: 75-80.

7. Ray Chaudhuri, S., M. Mishra, P. Nandy and A.R. Thakur, 2008. Waste management: A case study of ongoing traditional practices at East Calcutta Wetland. Am. J. Environ. Sci. 4(2): 140-144.

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8. http://www.cepis.opsoms.org/muwww/fulltext/repind53/calcutta/calcutta.html. Accessed on 20th October 2007.

9. Komarkova, J. 1998. Fish stock as a variable modifying trophic pattern of phytoplankton. Hydrobiologia, 369-370: 139-152.

9. Morabito, G. and A. Oggioni, 1999. Long term evolution (1986-1998) of phytoplankton communities in a shallow manipulated lake (L.Candia, Northern Italy). 2nd European Phycological Congress. Montecatini Terme (Italy) September, 20-26.

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13. Mehta, S.K., 2005. Use of algae for removing heavy metal from waste water : , Crit. re. biotechnol. 25(3): 113-152.

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15. Voltonica, D., H.G. Villa, and G. Correa, 2005. Nitrogen removal and recycling by Scenedesmus obliqqus in semicontinious cultures using artificial wastewater and a simulated light and temparature cycle. Bioresour. Technol., 96 (3): 359- 362.

16. Chojnacka, K., A. Chojnack, and H. Gorecka, 2003. Trace element removal by Spirulina sp. from copper smelter and refinery effluents, Hydrometallurgy, 73 (1-2): 147-153.

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18. Hassett, J.M., J.J. Charles, and J.E. Smith, 1981. Microplate Technique for Determining Accumulation of Metals by Algae. Applied and Environmental Microbiology, 41 (5): 1097-1106.

19. Fujita, M., and K. Hashizume, 1975. Status of uptake of Mercury by the fresh water diatom Synedra ulna. Water research, 9(10): 889-894.

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22. Chan, S.M.N., T. Luan, M. H. Wong, and N. F. Y. Tam, 2006. Removal and biodegradation of polycyclic aromatic hydrocarbons by Selenstrum capricornutum. Environ. Toxicol. Chem., 25(7): 1772-1779.

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