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3 Vice Chancellor's Office, West Bengal State University (Barasat, North 24 Parganas),. Berunanpukuria, P.O. Malikapur, North 24 Parganas, Calcutta-700126, ...
HEAVY METAL ACCUMULATING AND ENZYME SECRETING NOVEL PSEUDOMONAS SP. FROM EAST CALCUTTA WETLAND: IMPLICATIONS FOR ENVIRONMENTAL SUSTAINANCE Madhusmita Mishra1, P. R. Rout1, S. Mohapatra1, M. Sudarshan2, A. R. Thakur3 and Shaon Ray Chaudhuri1* ABSTRACT Microbial enrichments from soil and water samples collected from different sites of East Calcutta Wetland (ECW) produced three bacterial isolates which were identified as Pseudomonas based on 16S rRNA sequence analysis. They were further examined for their ability to tolerate heavy metals like lead (Pb+), chromium (Cr +++), copper (Cu++) etc.. Energy Dispersive X Ray Fluorescence analysis, Transmission Electron Microscopy and Scanning Electron Microscopy were used for understanding the metal microbe interaction. The presence of Super Oxide Dismutase (SOD) gene explains the defense mechanism against the oxygen radical and the metal induced stress. From the point of environmental sustainance and commercial application, the extracellular enzymes protease and lipase from one of the isolates were tried out as dehairing agents for the treatment of goat hide for possible replacement of the conventional method of using harmful chemicals. Key words: East Calcutta Wetland, heavy metal, super oxide dismutase, extracellular enzymes, dehairing. 1

Department of Biotechnology, West Bengal University of Technology, BF 142, Salt Lake, Sector 1, Kolkata-700064 2 UGC-DAE Consortium for Scientific Research, Calcutta Center, III/LB-8, Bidhan Nagar, Calcutta-700064. 3 Vice Chancellor’s Office, West Bengal State University (Barasat, North 24 Parganas), Berunanpukuria, P.O. Malikapur, North 24 Parganas, Calcutta-700126, India Author for correspondence : [email protected], +91-33-23370731, +9133-23341030.

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1. INTRODUCTION The aqueous as well as surface discharges of heavy metals have introduced a toxicological risk to all life forms on earth (Goyer, 1997; Barceloux, 1999; Cervantes, 2001). The biological agents like microbes, are of considerable interest for the development of safe, economical and environment friendly methods for the cause of environment detoxification and sustainance. Several aerobic bacteria belonging to genus Pseudomonas, Shewanella, Bacillus, Cellulomonas have been reported to influence the remediation of several toxic metals like Pb and Cr (Ekundayo and Killham, 2001; Mattuschka et al., 1994; Ackerley et al., 2004; Camargo et al., 2003; Chowdhury et al., 2008). Significant research has been made towards understanding the cellular mechanism of metal uptake and the role of cellular design in metal adsorption. (Beveridge and Grahm, 1991; Gadd, 2000). It is not only the microbes that were applied for the environment reclamation from toxic wastes, but also many microbial enzymes are presently used in production process with an objective to reduce energy and raw material consumption, avoid the use of harmful chemicals as well as reduce the load of waste generated (Alcade et al., 2006). In the study reported here, the bacterial isolates were obtained by enrichment of soil and water samples collected from different sites of a wetland ecosystem of Kolkata, viz. East Calcutta Wetland. It is worth mentioning that this wetland which covers an area of about 12500 hectares function as the sewage dumping ground for the entire city. At the same time it acts as a resource recovery system where waste is recycled and used in production of paddy, vegetables as well as fish (Ghosh 1999; Ghosh 2005; Bhattacharyya et al., 2003; Ray Chaudhuri et al., 2008a, 2008b). The vast population of microbes, planktons as well as plants existing in this ecosystem operate the natural mechanism of remediation (Ray Chaudhuri et al., 2008; Pradhan et al., 2008; Ray Chaudhuri and Thakur, 2006). The presence of diverse bacterial population in the wetland system has been demonstrated by culture independent approaches (RayChaudhuri and Thakur, 2006) and the significant finding was the close resemblance of majority of the novel sequences to groups like Actinobacteria, Proteobacteria and Firmicutes. These data reinforce the idea of exploring the rich microbial resource for selective screening of potential microbes and most importantly the development of a strategy for their application in environmental sustainance. The main objectives of this study were i) to investigate the heavy metal tolerance and accumulation efficiency of the isolates ii) to quantify the extent of intracellular accumulation followed by the detection of their internal localization by the methods of Energy Dispersive X ray Fluorescence (EDXRF) and Transmission Electron Microscopy (TEM) respectively iii) to evaluate the effect of metal induced stress on the cytoskeletal structure of the cells exposed to different heavy metals by the Scanning Electron Microscopy iv) to analyze the efficiency of extracellular enzymes protease and lipase in dehairing of goat hide, the strategy was to develop an alternative method to chemical mediated conventional process.

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2. THE ORIGIN SITE OF THE MICROBES The soil and water samples were obtained from three different sites of ECW, i) the old solid dumping grounds that were later converted to the recreational center with forest like ecosystem and was thereby designated as green zone, ii) the Charakdanga Bheri, a swallow flat bottomed sewage fed fish pond and iii) its associated Raw Sewage Canal. 3. CULTURE CONDITIONS FOR SELECTIVE ENRICHMENT OF MICROBES FROM ENVIRONMENTAL SAMPLES The study aimed at isolation of microbes which can have significant application in bioremediation, the main focus was for organic and heavy metal pollutants. Isolation and purification were performed by dilution plating of samples on carbon minimal salt (CMS) medium [K2HPO4 2.2 g L-1, KH2PO4 0.73 g L-1, (NH4)SO4 1 g L-1 , NaCl 30 g L-1 , MgSO4 0.2g L-1 , Oil 15 ml L-1]. This selective medium was employed to screen microbes having potential of utilizing different oils (both vegetative and mineral) as their primary growth substrates. The cultivation conditions and the regular maintenance of the isolates were carried out as previously described (Adarsh et al., 2007). 4. CHARACTERIZATION OF THE PURE ISOLATES The preliminary characterization (morphological, biochemical, physiological, molecular nature) of the pure isolates were carried out using methods previously described by Sarkar et al., (2008). Three pure bacterial isolates were obtained which appeared as gram negative rods having about 1-2.5 m length and 0.4-0.6 m diameter. The details regarding the source of isolation are presented in Table 1. Table 1: Table represents the preliminary characterization of the pure bacterial isolates. 1.i. The nomenclature of the bacterial isolates designated according to their site of isolation. 1.ii. The details of the physiological parameters supporting the growth of the isolates. 1.iii. The biochemical features of the isolates in term of the presence or absence of enzyme. 1.iv. The antibiotic sensitivity of the isolates as evaluated on the basis of the diameter of the clearance zone as compared to the standard chart provided by National Committee for Clinical Laboratory Standard’s (NCCLS). 1.v. The molecular identification of the isolates based on 16S rDNA analysis. The sequence was subjected to BLAST analysis and the identity was deciphered on basis of the closest neighbor within the existing database showing maximum % of identity. Isolate GZN showed 100% identity at the partial sequence level. The partial sequence can reveal the identity only upto the genus level.

Microbial Biotechnology: Methods and Applications

219 Isolates Source of isolation

Temperature tolerance range Optimum temp pH tolerance range Optimum pH Protease Lipase DNase Oxidase Catalase Lecithinase

Resistance profile

GZN 1. i. Nomenclature Green zone

RSCO

BWO

Raw Sewage Canal adjacent to Charakdanga Bheri

Bheri water (Charak danga bheri)

1.ii.Physiological characterization 15-50oC 15-45oC

15-45oC

40 oC 5-14

40 oC 5-12

30 oC 5-14

6

8

7.5

1.iii. Biochemical characterization + + + + + + + + 1.iv. Response towards antibitics Vancomycin, Vancomycin, Ampicillin, Ampicillin Polymyxin B, Chloramphenicol, Doxycycline, Polymyxin B, Tetracycline, Rifampicin Rifampicin

+ + -

Vancomycin, Ampicillin, Polymyxin B, Rifampicin

Sensitive profile

Gentamycin, Ceftazidime, ciprofloxacillin, Norfloxacillin

Gentamycin, Neomycin, Ceftazidime, Cefotaxime, Doxycycline, Norfloxacillin, Ciprofloxacillin,

Gentamycin, Neomycin, Ceftazidime, Tetracycline, Ciprofloxacillin, Norfloxacillin, Trimethoprim, Doxycycline

Intermediate response

Neomycin, Cefotaxime, Trimethoprim, Chloramphenicol

Tetracycline, Trimethoprim

Cefotaxime, Chloramphenicol

1.v. Molecular Characterization FJ788518 EU 006702

EU 006700

Maximum identity with organism

Pseudomonas aeruginosa

Pseudomonas pseudoalcaligenes

Pseudomonas mendocina

% of similarity

100%

99.53%

99%

GenBank Accession no

* ‘+’ indicates the presence of enzyme while ‘-’ stands for the absence of enzyme.

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The physiological characterization of the isolates (Table 1) indicated their survivability under wide range of temperature and pH. Isolate GZN was found to grow best at a slightly acidic pH while for the other two, neutral pH was found to support maximum growth. The pH and temperature tolerance would have significant implications for the strategic development for in situ bioremediation. As can be seen from Table 1, all the isolates were oxidase positive which is a typical property of Pseudomonas sp. The presence of enzymes like lipase and protease would open up the avenues for commercial application of the isolates as well as their enzymes. In one of the isolates GZN, the presence of DNase enzyme explains the probable defense against invaders and similarly presence of catalase implies the microbial mechanism to fight against oxygen free radicals generated during aerobic oxidation pathway. The antibiotic response profile (Table 1) indicated that all the isolates were sensitive to gentamicin, ceftazidime, ciprofloxacillin and norfloxacillin. As certain reports state, the co transmission of antibiotic and metal resistance occurs in high proportion because of the selective pressure of one property on other (Ramteke, 1997; Spain, 2003). The isolates demonstrated growth in substrates like glucose, fumarate and malonate which confirmed their aerobic oxidation pathway. Growth was proficiently found in case of complex substrate like jaggery which could be exploited for large scale growth of microbes as well as production of enzymes. All the isolates exhibited a sigmoidal growth pattern. The initial lag phase was short (1-2 hours) followed by a logarithmic phase (3-6 hours). The growth cycle would be significant for the implication of the microbial interaction with the contaminants. Moreover the growth efficiency of all the isolates varied with exposure to light and darkness with GZN showing maximum growth with exposure to alternate cycles of light and darkness; RSCO in complete darkness while BWO in complete light during its growth phase (Table 2). Table 2: The growth efficiency of the isolates under different conditions of illumination Illumination condition

GZN

RSCO

BWO

Growth in terms of OD at 660nm Light

0.642 ± 0.004 (SD)

0.636 ± 0.013 (SD)

0.605 ± 0.011 (SD)

Dark

0.917 ± 0.002 (SD)

0.651 ± 0.025 (SD)

0.498 ± 0.072 (SD)

Light/Dark

1.022 ± 0.006 (SD)

0.558 ± 0.025 (SD)

0.563 ± 0.002 (SD)

The growth was checked in LB medium with 1% inoculation under optimum cultivation conditions. Three sets were subjected to complete dark, complete light and alternative light and dark conditions respectively and the growth efficiency was determined in terms of OD at 660 nm.

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The oil utilizing ability of the isolates were tested using both vegetative (coconut and mustard oil) and mineral oils (like petrol, diesel, mobil and burnt mobil) through turbidity measurement after 72hours. The isolates were found to utilize vegetative oil like coconut oil and mineral oils like diesel, mobil and burnt mobil as its carbon source, this property would be exploited for the application in crude oil remediation. The gram nature of the isolates was reconfirmed using Real time PCR analysis as per the protocol of Shigemura et al. (2005). The pathogenecity test for the isolates was conducted using TaqMan kit (Applied biosystems) [Detection Kit for Staphylococcus aureus (PN 4368606) and Pseudomonas aeruginosa (PN4368604)] as per manufacturers protocol and none of the three isolates were found to possess either of the pathogenic gene. Based on 16S rRNA gene sequence analysis, the isolates were identified as novel Pseudomonas sp. Partial sequences (~500bp) were analyzed by BLAST-N to provide information regarding the closest neighbors. Identities of closest matching strains, percentage identity and GenBank Accession numbers of the 16S rRNA sequences are provided in Table 1. The phylogenetic position were determined by neighbor joining method and the relationship between these isolates and those of the type strains within the family were presented in Fig.1. (Fig 1a, 1b, 1c).

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Figure 1: Phylogenetic analysis based on neighbour joining method depicting the identity of the three isolates at the molecular level. The branch lengths were provided at the top of the tree. The partial 16S rDNA sequence analysis could confirm the identity up to the genus Level

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5. TOLERANCE TOWARDS HEAVY METALS Nine different heavy metal salts like Al(NO3)3.9H2O, CuSO4.5H2O, AgNO3, Pb(NO3), NiCl2.6H2O, HgCl2, CrO3, CoCl.6H2O and CdCl3 were used for screening of heavy metal tolerance. The determination of Minimum Inhibitory Concentration (MIC) of the respective metals was done as described previously (Adarsh et al., 2007). All the isolates were found to grow in presence of heavy metals like nickel (Ni), copper (Cu), silver (Ag), aluminium (Al), iron (Fe), chromium (Cr), lead (Pb) upto different extent. The metal accumulation was tested using Energy Dispersive Xray Fluorescence (EDXRF) technique in a Jordan Valley EX 3600 system as reported elsewhere (Adarsh et al., 2007). The relative accumulation of different metals as obtained from EDXRF analysis was presented in Table 3. As expected the maximum accumulation of Pb corresponded to highest Minimum Inhibitory Concentration of Pb. Table 3: Tabular representation of the efficiency of different isolates in the accumulation of various heavy metals Isolates  Metals salts 

GZN

RSCO

BWO

0.641

1.504

Metal Concentration in ppb

Ni

1.589

Co

9.921

Cu

28.39

16.31

52.028

Cr

249.89

669.1

424.29

Pb

2688.99

1970.4

1504

* The data for metal accumulation has been checked in three sets and the mean result is presented in table.

Prior to analysis by EDXRF, the metal treated cells were washed with 0.1N HCl to remove the adsorbed metal. The cell suspension was concentrated by vaccum filtration through Whatman Filter membrane (0.22 mm) so as to detect only the intracellular metal concentration which was represented in ppb. The total accumulation would be much higher. Exact intracellular localization was determined through Transmission Electron Microscopy under unstained condition and through and through accumulation of metal salt was observed in most of the isolates while in some cases there were patches of intracellular deposition. Certain representative micrographs were presented in Fig 2. Cytoskeletal changes post metal treatment was observed through Scanning Electron Microscopy as reported elsewhere (Adarsh et al. 2007 and Chowdhury et al. 2008). The distinct response observed was the shrinkage in cell size (Fig 3.) post metal treatment. The cell shortening would be essential to increase the surface area to volume ratio so as to increase the interaction interface of the microbes with the metals.

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Figure 2: Transmission Electron Micrographs displaying intracellular metal accumulation in the metal treated cells.(i) Intracellular accumulation of Ag salt observed in isolate GZN at a magnification of 3500X, (ii) Electron micrograph (magnification 880X) depicting metal accumulation throughout in the isolate GZN treated with Cu salt, (iii) Isolate RSCO treated with Cu salt showing accumulation throughout as visualized at a magnification of 3500X, (iv) Localized accumulation of Ag salt in isolate BWO, the electron micrograph represented was captured at a magnification of 2800X

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Figure 3: Scanning Electron Micrographs (Magnification 9000X) representing the effect of metal treatment on the cell morphology and dimension. (i) Isolate GZN without metal treatment, (ii) GZN treated with Cr salt depicting a shortening in cell. Fig 3.iii. Isolate RSCO under untreated condition, (iv) Isolate RSCO exhibiting cell shrinkage post treatment with Ag salt 5.1. Rate of metal uptake Among the metal tolerant isolates, the one showing maximum accumulation as per EDXRF data (isolate GZN) was selected to study the rate of metal uptake. The isolate was grown in presence of the highest concentration of metal that it could tolerate. Equal weight of cells (100 mg) was harvested at regular time intervals (4 hr, 8 hr and 10 hr) along the growth phase. The cell pellet was resuspended in 10 ml of 0.125M EDTA and was gently treated at 30 rpm (Stuart Orbital Shaker Incubator SI50) for 30 minutes at room temperature in order to ensure that the adsorbed metal was released to EDTA solution. Post wash, the cell pellet was harvested by centrifugation at 10000 rpm (Eppendroff Centrifuge 5810R, rotor no FL 121) for 15 minutes. The cell pellet and the EDTA supernatant were treated with concentrated HNO3 and 70% HClO4 in a microwave prior to analysis by Atomic Absorption Spectroscopy (Perkin elmer-A-Analyst 700). The extent of metal adsorbed (EDTA wash) and accumulated intracellularly (cell pellet) were determined on comparison with the control cells (without any metal treatment). Metal uptake was found both by the mechanism of adsorption as well as intracellular accumulation, both being equally efficient. Maximum uptake was demonstrated in the logarithmic phase of growth cycle while in stationary phase the efficiency of metal uptake reduces drastically (Table 4.). The rate of adsorption and

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accumulation being similar, it indicates the existence of dynamic state of metal uptake. As observed in Fig 4.ii. and 4.iii., cell disruption and distortion in cell surface were observed in later stages of growth in case of metal treated cells. This could be a probable reason for the stunted growth in presence of metal (Fig.4.i.) as well as reduced rate of adsorption post logarithmic phase. Table 4. Table representing the rate of Pb uptake by cells (accumulation and adsorption) as determined by AAS. The initial concentration of Pb in the medium was 940 ppm. The supernatant post EDTA wash of the cells provided the quantity of adsorbed metal while cell pellet analysis provided intracellular metal accumulation Accumulation

Adsorption

Metal concentration in ppb + SD 4 hour

3317.01 + 296.64

3260.76 + 40.32

8 hour

1040.06 + 288.50

483.16 + 52.48

12 hour

424.38 + 21.23

422.26 + 38.59

Figure 4: Figure displaying the alteration in the growth profile of the isolate. (i) post treatment with different concentration of Pb salt, (ii) and (iii) Scanning electron micrographs representing the morphological features of the isolate pre and post treatment with 4 mM of Pb salt. The cells treated with Pb exhibited a short and stout shape with distorted cell surface

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5.2. Detection of SOD gene Super Oxide Dismutase is a major component of biological defense mechanism against oxygen toxicity and is also reported to exhibit defense in presence of metal stress where oxygen radical is a main byproduct (Roy et al. 2008). Presence of SOD as a defensive tool would be essential in these isolates which are aerobic in nature as well as showing metal tolerance. With universal SOD primers as reported by Zolg and Schulz (1994), a 480 bp fragment was lifted from the genomic DNA of the three isolates (Fig 5). The 451 bp sequence was obtained for that of isolate GZN and the BLAST-N analysis of the sequence provided 97% similarity with Pseudomonas Mn SOD gene. The sequence being novel was submitted to GenBank (GenBank Accession no FJ788516).

Figure 5: Figure showing the SOD gene amplification products resolved on a 2% agarose gel. Lane M represents the 500bp ladder (Fermentus), L1 is the negative control, L2, L3 and L4 represents the SOD amplicons obtained from the isolate GZN, RSCO and BWO respectively 6. MICROBIAL ENZYMES AS AN AID IN DEHAIRING OF HIDE The objective of employing protease and lipase enzymes in dehairing was to achieve effective degreasing by the simultaneous action of proteolysis, lipolysis, and emulsification. The enzyme production was achieved using shake flask under

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optimum growth conditions. The culture was harvested after 8 hours of growth and the lipase and protease activity was measured by the spectrophotometric method reported by Sarkar et al. (2008) and Chowdhury et al. (2008) respectively. The enzymatic treatment of hide was checked in presence of whole cells as well as with cell free extracellular enzymes. As represented in Fig 6, the dehairing starts at 8th hour of incubation and complete dehairing was achieved by the 10th hour of incubation.

Figure 6: Figure depicting the effect of enzymatic dehairing with respect to different incubation time. Hide pieces were dipped in GZN culture that was harvested post 8 hours of growth. The effectiveness in dehairing was checked at regular intervals by manual method of scrubbing Table 5. Table representing the dehairing efficiency demonstrated post treatment of hide under different conditions. (i) Treatment with GZN culture containing whole cells ii. Cell free supernatant containing protease and lipase, (iii) the conventional method containing 5% lime and 5% sodium sulfide in water, (iv) dehairing efficiency under different concentration of enzyme when diluted with water or jaggery. Hides of uniform size were dipped in each mixture for 10hours and the dehairing efficiency was assessed on the basis of the hair removal by scrubbing as well as change in size and decrease in weight Condition

Pre treatment

Post treatment

Length Brea dth Thick ness Weight

Length Brea dth Thick ness Weight

Length, breadth and thickness in cms, weight in gms Control

6.82

3.68

0.17

5.62

6.8

3.6

0.14

5.56

Comparative anaysis of enzymatic and conventional deharing technique i.

Whole cell

14.15

4.3

0.14

6.49

8.1

3.34

0.1

4.17

ii. Suspension

11.50

3.89

0.18

5.19

6.2

3.9

0.1

3.26

8.4

3.95

0.24

5.19

6.75

3.7

0.12

3.44

iii. Conventional process

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229 iv.

Deharing efficiency under different concentrations of enzyme 10% enzyme + water

6.1

3.98

0.23

5.46

6.54

2.83

0.14

4.68

75% enzyme + jaggery

8.00

3.3

0.15

6.03

9.7

3.00

0.1

4.04

50% enzyme + jaggery

6.94

3.13

0.14

5.44

7.25

3.15

0.14

3.91

25% enzyme + jaggery

8.74

3.25

0.15

6.13

7.95

3.4

0.2

5.64

10% enzyme + jaggery

6.5

3.7

0.16

5.06

6.2

3.00

0.14

5.45

The enzyme efficiency was compared with the conventional technique which involves treatment with 5% lime and 5% sodium sulfide. The effectiveness of the enzymatic treatment over the conventional process was observed by the marked decrease in hide weight. Dehairing efficiency of hides dipped in cell culture was at par with cell free enzyme (Table 5) and this step overcomes the time lag as well as expenditure necessary for cell removal. The optimum enzyme concentration required for effective dehairing was determined by using supernatant containing protease and lipase enzyme diluted with water and jaggery upto different percentage. The significant finding was that 75% of enzyme (diluted with 25% water) showed complete dehairing while 25% of enzyme also showed indication of partial dehairing. In case of jaggery with enzyme, 50% enzyme showed dehairing while 75% enzyme concentration was most efficient for dehairing. 6.1. A comparative statement of growth and enzyme production under shake flask and fermentation condition Since the enzymes were used in applications like dehairing, the next objective was to scale up the cell as well as enzyme production. Isolate GZN producing both the enzymes was further studied for optimization of different parameters under both the cultivation conditions. For shake flask culture, 1% inoculum was added to the enriched medium [Luria Bertani (LB) broth, 1% tryptone, 0.5% yeast extract and 0.5% NaCl] and cultivation was allowed at the respective optimum temperature and pH. In case of batch fermentation, 5% inoculum was seeded into 3 lit LB medium (working volume capacity) in 5lt BioG Micom, BIOTRON fermenter. The fermentation was carried out at optimum growth condition with different agitation speed (100-300 rpm) and with maximum air flow (1-3 vvm). The growth was measured in terms of turbidity (optical density at 660nm measured in Beckman Coulter DV– 530, UV Vis spectrophotometer) while the enzyme (lipase and protease) production was determined by the quantitative assay method reported by Sarkar et al. (2008) and Chowdhury et al. (2008) respectively. The impact of agitation speed on the growth efficiency was clearly depicted in Fig 7. The lower shaft speed facilitates microbial growth while growth was inhibited at higher agitation speed of 300rpm. The higher agitation could provide a shearing

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force on the cell causing the cellular rupture and thus inhibition of growth. The production of lipase enzyme was influenced by agitation speed, with 150 rpm showing higher production than at 200 rpm. Likewise with scale up from 150 ml in shake flask to 3 lt, the production of lipase enzyme was hiked up relatively 2.5 times in the late logarithmic phase (Fig 8). Protease production was found at consistent concentration in both flask culture as well as fermenter.

Figure 7: Graphical representation of the growth efficiency of isolate GZN under shake flask and batch fermentation conditions. LB was used as the cultivation medium and the fermentation was carried out with 5% initial inoculum in 5 lt Biotron fermenter. The growth efficiency under fermentation conditions was checked at different agitation speeds as mentioned in the legend on the right side of the graph

Figure 8: Graph representing the enzyme (protease and lipase) production profile of isolate GZN under shake flask and fermentation conditions. Fermentation was carried out in LB medium (pH 7.5) seeded with 5% inoculum and carried out in 5 lt BIOTRON fermenter. Enzyme production was checked under variable agitation speed

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7. CONCLUSION The detailed characterization of these isolates exposes the untapped potential of these for the cause of environmental sustainance. The heavy metal accumulating property would cause the concentration of the toxic waste within minimal volume of a cell thereby reducing the load of metal toxicity in the environment. The study also initiates the probability of application of the microbe as well as its products i.e. the enzyme for industrial bioremediation. The enzyme application in dehairing process could replace the conventional method involving harmful chemicals and in overall sense would make the commercial process environment friendly leading to “Green Technology”. 8. ACKNOWLEDGEMENTS The authors would like to acknowledge the financial assistance from Department of Atomic Energy, Government of India under the Board of Research in Nuclear Studies scheme and Department of Science and Technology, Government of India under the DST Fast Track Scheme. They would acknowledge West Bengal University of Technology, Calcutta, India for their computational facility. 9. REFERENCES Ackerley, D.F., Gonzalez, C.F., Park, C.H., Balke, R., Keyhan, A. and Matin, A. (2004). Chromate reducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia coli. Appl. Environ. Microbiol. 70, 873-888. Adarsh, V.K., Mishra, M., Chowdhury, S., Sudarshan, M., Thakur, A.R. and Ray Chaudhuri, S. (2007). Studies on metal microbe interaction of three bacterial isolates from East Calcutta Wetland. OnLine J. Biol. Sci. 7, 80-88. Alcalde, M., Ferrer, M, Plou, F.J. and Ballesteros, A. (2006). Environmental biocatalysis: from remediation with enzymes to novel green processes. Trends Biotechnol. 24, 281-287. Barceolux, D.G. (1999). Chromium. J. Tox. Clin. Tox. 37, 173-194. Beveridge, T.J. and Graham, L.L. (1991), Surface Layers of Bacteria. Microbiol Rev. 55, 684-705. Bhattacharyya, S., Santra, S.C. (2003). Environmental Status of East Calcutta Wetlands and Strategies for Sustainable Management, In Khasnabis, R. (ed.), Ecology, Economy and Society. DRS Pha se-II Progra mm e, En vi ron ment al Management, Department of Business Management, University of Calcutta, Kolkata, India, pp. 65-94. Camargo, F.A.O., Okeke B.C., Bento FM. and Frankenberger, W.T. (2003), In vitro reduction of hexavalant chromium by a cell free extract of Bacillus sp. ES 29. stimulated by Cu2+. Appl Microbiol Biotechnol. 62, 569-573. Cervantes, C., Campos Gracia, J., Devras, S., Guttierez Corona, F., Loza Tavera, H., Torres Guzman, J.C. and Moreno Sanchez, R. (2001), Interactions of chromium with microorganisms and plants. FEMS Microbiol Rev. 25, 335- 347.

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