July 2015 Isolation, characterization and identification of heavy … · 2016. 1. 19. · July 2015...

European Journal of Biotechnology and Bioscience

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Volume: 3, Issue: 7, 17-22 July 2015 www.biosciencejournals.com ISSN: 2321-9122 Impact Factor: 3.742 Sonali Patil Birla College of Arts, Science and Commerce, Kalyan-421301, Dist. Thane, MS. Geetha Unnikrishnan Birla College of Arts, Science and Commerce, Kalyan-421301, Dist. Thane, MS. Correspondence: Sonali Patil Birla College of Arts, Science and Commerce, Kalyan-421301, Dist. Thane, MS.

Isolation, characterization and identification of heavy metal tolerating thermophiles from hot water spring

Sonali Patil, Geetha Unnikrishnan Abstract Many industries such as metal finishing / metal plating, gold and silver refining, petroleum refining, iron and steel use lead (Pb), cadmium (Cd), zinc (Zn),mercury (Hg), arsenic (As), silver (Ag) chromium (Cr),copper (Cu) iron (Fe), platinum group elements and other heavy metals as important ingredients to develop various types of metal alloys which are the major contributors to water as well as soil pollution as they create more subtle effects on behavior, reproduction or even survival of biotic communities. An enrichment culture technique and molecular biology technique was used to isolate bacterial strains from hot water springs of Vajreshwari and Ganeshpuri which can tolerate high concentrations of the selected heavy metals. Five pure bacterial cultures, named SZP 4, SZP 8, SZP 12, SZP 16, SZP 18 were isolated and subsequently characterized by 16S rRNA gene sequencing and biochemical tests. The optimum temperature, pH and NaCl concentration was determined for them. Tolerance to high levels of metal salts (Cd, Cr, Cu, Fe, and Zn) was seen in all five bacterial isolates. By SDS – PAGE analysis it was found that there is presence of specific proteins indicating that the resistant trait observed was specifically operon mediated. Thus such findings may be useful in designing a multi resistant bacterium that can be used to return the altered environment to its original condition. Keywords: Enrichment, heavy metals, 16s r RNA, SDS-PAGE, Operon. Introduction Environmental pollution has become a key focus of concern for all the nations worldwide, as not only the developing countries but developed nations as well are affected by and suffer from it. Among all the pollutants, heavy metals are most dangerous one as these are non – biodegradable and persist in environment [8]. At low concentrations some of the heavy metals stimulate some biological processes, but at threshold concentration these become toxic. Being nonbiodegradable, these metals accumulate at various trophic levels through food chain and can cause human health problems [1]. Several physico-chemical methods like chemical precipitation, electrodialysis, ion-exchange, ultra-filtration, reverse osmosis etc. are commonly employed for stripping toxic heavy metals from waste waters [2, 3]. The above stated methods have some disadvantages such as incomplete metal removal, limited tolerance to pH change, expensive equipment and monitoring system requirements, high reagent or energy requirements and generation of toxic sludge or other waste products that require disposal [6]. Further, these techniques may be ineffective or extremely expensive when metal concentration in waste water is in the range 1-100 ppm [5]. To overcome some of the limitations of physicochemical treatments, there is a need for inexpensive and efficient technology for the treatment of metal containing wastes so that metal concentration can be reduced to environmentally acceptable levels [9]. Use of microorganisms for metal removal/recovery is considered to be a viable alternative to conventional methods. The current study involves bioremediation by thermophiles isolated from Vajreshwari and Ganeshpuri hot water springs for different heavy metals which are common pollutant viz., Cd, Cr, Cu, Fe, Zn. And also the isolation of protein that are responsible for tolerating high concentration of heavy metals and also to decreae the concentration of the same. Materials and Methods Sampling for isolation of thermophiles Water samples were collected from seven different hot springs (fromVajreshwari&Ganeshpuri, Thane) Mumbai. Surface water samples were taken from the Hot Springs using a grab sampler. A 500-ml plastic cup attached to a 2-m pole was dipped into the water twice to rinse it. The sample was then transferred to a clean, new, polyethylene container with a snap-on lid. The temperature of

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the sample was taken with a laboratory thermometer and recorded. All samples were taken on the same day to prevent discrepancies due to sample date. Samples were kept cool during transport to the laboratory and processed within 12 h of collection (Vieille C. 1996). Media & growth Conditions Bacillus Medium described by Postgate (1969) was used for routine stock maintenance and all enrichment culture studies. Bacillus Medium contained (g/ Lit.: Soluble starch – 30.0 g, Agar – 20.0 g, Peptone – 5.0 g, Yeast Extract – 5.0 g, Distilled Water – 1000 ml, pH 7.5 ± 0.2 (45˚C). Colonies were isolated from anaerobic roll tubes (Hungateet. al. 1969) containing Medium and 4% (w/v) purified agar. Stock cultures of all strains were prepared from single isolated colonies that proliferated on transfer in Media. All stock cultures were incubated at 50°C. Cultures were routinely checked for contamination (Zeikuset, al. 1979). Bioremediation All Strains isolated during the course of study were investigated for their bioremediation activity. The screening was done by using 100ppm of heavy metals (Control) and effluent from industry containing 5 specific heavy metals i.e. Cd, Cr, Cu, Fe, Zn (5 heavy metals were chosen as these are common pollutant in industrial wastewater). Characterization and identification of the isolates The isolates showing bioremediation by biogenic volatile compound were further investigated for their cellular, biochemical and molecular properties. I) Morphological Studies Morphological properties were investigated by using 18 hour old bacterial cultures. These included the wet mount preparations using light microscope & Gram staining to confirm Gram reaction. Motility was determined by hanging drop method. II) Biochemical Tests Two thermophilic isolate was identified by the use of conventional methods for the presumptive identification of physiological and biochemical tests. These tests were; Gram reaction, catalase production, hydrolysis of protein, starch and lipid, and acid production from sugar (Campos,et.al. 1995). The species was reconfirmed by using automated BiomerieuxVitek 2 System (Nucleus Diagnostic Centre, Kalyan). Bioremediation All Strains isolated during the course of study were investigated for their bioremediation activity. The screening was done by using 100ppm of heavy metals and by calculating MTC (Maximum Tolerance Capacity) for the isolate showing tolerance to 5 specific heavy metals i.e. Cd, Cr, Cu, Fe, Zn (5 heavy metals were chosen as these are common pollutant in industrial wastewater).

Strain identification by 16s rRNA Method The isolated colonies showing high tolerance to heavy metals were sequenced for its conserved sequences and analysed for partial 16s rRNA by geneOmbio, Pune, Maharashtra. The predicted 16S rRNA sequences from this study were compared with 16S rRNA sequences in a BLASTable database constructed from sequences downloaded from the Ribosomal Database Project (release 8.1; http://rdp8.cme.msu.edu). Comparisons were made using the program BLAST (ftp://ftp.ncbi.nih.gov/BLAST/executables/LATEST/) and a FASTA-formatted file containing the predicted 16S rRNA sequences. All the sequences were deposited into NCBI database. Mechanisms involved in Bioremediation Isolation of Proteins and identification by SDS-PAGE The bacterial cell of both the strains were grown at 45 oC for 24 hrs. and the cells were harvested by centrifugation at 13,000 rpm for 10 mins. Later the cell pellet was washed with phosphate buffer (pH 7.0) to remove the traces of remaining media and again centrifuged at 10,000 rpm for 10 mins. The supernatant was discarded. The cell pellet obtained was mixed with 1ml of 2X sample buffer (0.5% SDS, 25% beta mercaptoethanol, 0.03% bromophenol blue, 2.5% Glycerol, 15mM trisHCl (pH 6.8)). The samples were vortexed and incubated in a boiling water bath for half an hour. The samples were used directly for SDS-PAGE analysis (Maiti et al., 2009). The same method of isolation was performed with and without the treatment of heavy metals (control and Test). The most probable protein matching according to molecular weightwas carried out by insilico method using by http://web.expasy.orgDatabase. Results and Discussion Characterization of in situ Bioremediation: Microbial heavy metal reduction at high temperatures was studied at several sites in Vajreshwari & Ganeshpuri hot springs. Enrichment cultures were initiated with Bacillus Medium. After incubation of all enrichments for 6 d at in situ temperature, the cultures formed a dense colonies. Repeated transfer of enrichments revealed small rod-shaped bacteria in all cultures. All strains appeared morphologically identical with one being mucoid colony (SZP 1) and one with smooth colony (SZP 2) when cultured on st. media at 41°C. Stock cultures of both maintained on St. Media and transferred monthly. Cellular properties Cells appeared as very tiny straight rods. Motility was not observed. Exponential phase cells stained Gram-positive. Other biochemical properties (As per Berge’s manual) are described in Table 1-4. Species was again confirmed by using automated system of Biomerieux System (Done at Nucleus diagnostic Centre, Kalyan) stated as in Table 5 - 9.

Table 1: Colony characteristics of selected isolates

Isolate colony characteristics

Size Shape Colour Consistency Opacity Elevation Gram nature Motility SZP 4 1 MM Circular Colour less Dry opaque Flat Gram Negative rods NM

SZP 8 Pin point Irregular Pale white Butyrous Trans lucent

Flat Gram Negative rods M

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SZP 12 7 mm Irregular Colour less Dry Opaque Convex Gram Negative Bacilli M SZP 16 Pin point Irregular Colour less Butyrous opaque Flat Gram Positive bacilli NM

SZP 18 3 mm Irregular Dull white Butyrous Trans parent

Convex Gram Positive Cocci NM

Table 2: Acid Production and fermentation of Sugar of selected isolates

Isolate Biochemical Test (Acid Production and fermentation of Sugar) L-Ara Glu Gal Suc Mal Fru Raf Star Cel Ino Mann

SZP 4 - + + + + + + - - + + SZP 8 + + + + + + + + + + +

SZP 12 + + - + + + + + - + + SZP 16 + - - - - + - + + + +SZP 18 + + + + + + - - - -

Table 3: Biochemical Test of selected isolates

Isolate Biochemical Tests Nitrate Cellulose Gelatin Casein H2S Catalase PAD MR VP Oxidase Citrate I

SZP 4 - - - - - - - + + - + - SZP 8 + - - - + + + + - - + - SZP 12 + - + - - + + - - + - + SZP 16 - + - - + + + - - - + - SZP 18 - + + - + + - - - - - -

Table 4: Physiological properties of selected isolates

Isolate Physiological properties pH Temperature (o C) NaCl (%)

SZP 4 5 45 5 SZP 8 7 45 2

SZP 12 6 45 10 SZP 16 5 45 7 SZP1 8 4.5 45 5

Table 5: Biochemical Tests of SZP 4 by automated method

Biochemical Details for SZP 4 2 APPA - 3 ADO - 4 PyrA - 5 IARL - 7 d CEL - 9 BGAL -

10 H2S - 11 BNAG - 12 AGLTp - 13 d GLU + 14 GGT - 15 OFF + 17 BGLU - 18 d MAL + 19 dMAN + 20 d MNE + 21 BXYL + 22 BAlap - 23 ProA - 26 LIP - 27 PLE - 29 TyrA - 31 URE - 32 dSOR - 33 SAC - 34 d TAG - 35 d TRE - 36 CIT + 37 MNT - 39 5KG - 40 ILATk + 41 AGLU - 42 SUCT - 43 NAGA - 44 AGAL - 45 PHOS - 46 GlyA - 47 ODC - 48 LDC - 53 IHISa - 56 CMT - 57 BGUR - 58 0129R - 59 GGAA - 61 IMLTa - 62 ELLM - 64 ILATa +


Biochemical Details for SZP 8 2 APPA - 3 ADO - 4 PyrA - 5 IARL - 7 d CEL - 9 BGAL - 10 H2S + 11 BNAG - 12 AGLTp - 13 d GLU + 14 GGT - 15 OFF - 17 BGLU - 18 d MAL + 19 dMAN - 20 d MNE - 21 BXYL - 22 BAlap - 23 ProA - 26 LIP - 27 PLE - 29 TyrA - 31 URE + 32 dSOR - 33 SAC - 34 d TAG - 35 d TRE - 36 CIT + 37 MNT - 39 5KG - 40 ILATk + 41 AGLU - 42 SUCT + 43 NAGA - 44 AGAL - 45 PHOS - 46 GlyA - 47 ODC - 48 LDC - 53 IHISa - 56 CMT - 57 BGUR - 58 0129R - 59 GGAA - 61 IMLTa - 62 ELLM - 64 ILATa +


Biochemical Details for SZP 12 2 APPA - 3 ADO - 4 PyrA - 5 IARL - 7 d CEL - 9 BGAL + 10 H2S - 11 BNAG - 12 AGLTp - 13 d GLU + 14 GGT - 15 OFF - 17 BGLU - 18 d MAL - 19 dMAN - 20 d MNE - 21 BXYL - 22 BAlap -23 ProA + 26 LIP + 27 PLE - 29 TyrA - 31 URE - 32 dSOR - 33 SAC - 34 d TAG - 35 d TRE - 36 CIT - 37 MNT - 39 5KG - 40 ILATk - 41 AGLU + 42 SUCT - 43 NAGA - 44 AGAL + 45 PHOS - 46 GlyA - 47 ODC - 48 LDC - 53 IHISa - 56 CMT - 57 BGUR - 58 0129R - 59 GGAA - 61 IMLTa - 62 ELLM - 64 ILATa -

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Biochemical Details for SZP 16 2 AMY + 3 CAT + 4 ADH1 - 5 PyrA - 7 BGAL - 9 AGLU -

10 APPA - 11 ADO - 12 BGAR - 13 AGLTp - 14 AMAN - 15 PHOS - 17 LeuA - 18 BNAG - 19 AGAL - 20 dMAN - 21 PyrA - 22 BGUR - 23 AlaA - 26 d MAL - 27 URE - 29 dGLU + 31 POLYB - 32 dGAL - 33 dRIB - 34 TyrA - 35 NAG - 36 d TRE - 37 dMAL - 39 BACI - 40 NOVO - 41 ILATk - 42 dMNE - 43 SUCT - 44 MBdG - 45 PUL - 46 dRAF - 47 NC6.5 - 48 SAC - 53 LDC - 56 dNTRE - 57 ADH2S - 58 OPTO - 59 0129R - 61 dARA - 62 IMLTa - 64 OXI +


Biochemical Details for SZP 18 2 AMY - 4 PIPLC - 5 dXYL + 8 ADH1 - 9 BGAL + 11 AGLU +13 APPA - 14 CDEX - 15 AspA - 16 BGAR - 17 AMAN - 19 PHOS - 20 LeuA - 23 ProA - 24 BGURr - 25 AGAL - 26 PyrA - 27 BGUR - 28 AlaA - 29 TyrA - 30 dSOR - 31 URE - 32 POLYB - 37 dGAL + 38 dRIB - 39 ILATk - 42 LAC + 44 NAG - 45 dMAL - 46 BACI - 47 NOVO - 50 NC6.5 - 52 dMAN - 53 dMNE - 54 MBdG - 56 PUL - 57 dRAF - 58 0129R - 59 SAL - 60 SAC - 62 dNTRE - 63 ADH2S - 64 OPTO +

Bioremediation The MTC for both the strains for bioremediation of heavy metals were found to be above 1000 ppm, Table 10.

Table 10: MTC for heavy Metals

Isolate Maximum Tolerance Capacity for Heavy metal

(concentration in ppm) Cd Cr Cu Fe Zn

SZP 4 4355 3090 2540 2893 4300 SZP 8 4389 2356 4430 2977 3972 SZP 12

3900 2804 3899 3797 3897

SZP 16

4998 4387 3456 2446 4299

SZP 18

4778 3900 3889 2787 4209

16s rRNAAnalysis The sequences were aligned for comparison with the existing databases by using in-silico tool BLAST. The sequence of isolated strain were deposited in NCBI database with specific accession number respectively as shown in table 11.

Table 11: NCBI Accession number

Denotation Organisms name Accession number

SZP 4 Acidithiobacillus ferrooxidans

ABHAY KM527215

SZP 8 Thiobacillus acidophilus

POPATNP KM527210

SZP 12 Pseudomonas fluorescens

SONALIZANKAR KM527212

SZP 16 Geobacillus stearothermophilus

BHALPRAVIN KM527211

SZP 18 Streptococcus thermophiles

ROHANMANALI KM527213

Protein Isolation The gel showed that in presence of heavy metals some of the protein/enzyme for both the strains are over expressed. These protein were identified by insilico method and most probable protein mathching with the observed results are shown in figure 1 – 5 and table 12.

Fig 1: Protein separation from Acidithiobacillus (SZP 4) by Sds-Page

Fig 2: Protein separation from Acidiphilum (SZP 8) by SDS-PAGE

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Fig 3: Protein separation from Pseudomonas (SZP 12) by Sds-Page

Fig 4: Protein separation from Geoacillus (SZP 16) by Sds-Page

Fig 5: Protein separation from Streptococcus (SZP18) by Sds-Page

Table 12 : The expasy results showed the probable proteins present may be

Acidithiobacillus ferrooxidans POPATNP (SZP 4) : 1. Zn Transporter - 49.55 KD 2. Permease – 60.46 KD 3. Metal transporter – 49.51 kD 4. Iron transporter – 38.4 KD 5. Cadmium Transporter – 67.06 KD

Thiobacillus acidophilus ABHAY (SZP 8). 1. Zinc permease - 34 KD 2. Iron permease – 60.04 KD 3. Metal ABC transporter- 30.16 KD 4. Heavy metal Bnding Protein – 26.48 KD Pseudomonas fluorescens SONALIZANKAR (SZP 12). 1. Cadmium transporter ATPase - 65.93 KD 2. Copper Transporter - 42.07 KD 3. Ferrous iron transporter - 31.88 KD 4. Periplasmic iron transport protein - 42.96 KD 5. Zinc transporter - 28.21 KD 6. Cobalt- Zinc- Cadmium resistant protein - 9.04 KD Geobacillus stearothermophilus BHALPRAVIN (SZP 16). 1. Cadmium accessory protein - 14.01 KD 2. Cadmium transporter - 76.04 KD 3. Metal ion transporter periplasmic protein - 35.67 KD 4. Ferrodoxin - 37.42 KD 5. Zinc Transporter - 28.8 KD 6. Chromate Transporter - 43.17 KD 7. Fe- S Oxidoreductase - 42.42 KD 8. Zn/Fe transporter - 25.75 KD Streptococcus thermophiles ROHANMANALI (SZP 18). 1. Cadmium resistance regulatory Protein - 13.38 KD 2. Cadmium putative efflux system regulator protein - 12.53 KD 3. Copper transport operon regulator - 15.64 KD 4. Iron uptake transporter substrate binding protein - 38.03 KD 5. Zinc transport transcriptional repressor - 16.89 KD 6. zinc Transporter substrate binding protein - 58.14 KD Discussion The present study extends the known niche for bioremediation in nature to extreme (> 60 °C) thermal environments. Thermophilic organisms at high temperatures appears widespread in Vajreshwari & Ganeshpuri hot springs was found to be associated with Bioremediation. Biochemical test (Done by Manual and automated method) and also 16s rRNA analysis was done to identify the strain. All the five strains showed tolerance level of heavy metals above 2000 ppm. Protein isolation and characterization showed that the specified proteins were over expressed during the bioremediation which confirms that various operons are responsible for the process of bioremediation. Further studies are required for identification and characterization of the same and for the use of these organisms for bioremediation of other heavy metals. As, the bioremediation of inorganic material is important for health of the people & for monitoring environment, in situ remediation by thermophilic species is one of the best ways for the treatment of industrial effluent containing high levels of heavy metals.

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References 1. Ajaz M, Jabeen N, Ali T A and Rasool S A. Pakistan

Journal of Botany, 2009, 41, 4. 2. Buchanan RE, Gibbons N.Bergey’s manual of

determinative bacteriology. (Eighth edition), The Williams and Wilkins Co., Baltimore, 1974, 747-842.

3. Bunge M, Araghipour N, Mikoviny T, Dunkl J, Schnitzhofer R, Hansel A, Schinner F, Wisthaler A, Margesin R, Mark TD. Appl Environ Microbiol, 2008, 74:2179–2186

4. Essa AM, Macaskie LE, Brown NL. Biotechnol.Lett, 2005, 27(21): 1649-55.

5. Gadd G M. Micrbiol. 2010, 156: 609-643. 6. Ilhan S, Cabuk A, Filik C, Calikan F.Trakya. Univ. J.

Sci., 2004, 5: 11-17. 7. Kesselmeier J, Staudt M. J. Atmos Chem., 1999, 33:23–

88. 8. Lanyi B. Methods Microbiol. 1987, 19: 1-67. 9. Perry RS, Mcloughlin N, Lynne BY, Sephton MA,

Oliver JD, Perry CC, Campbell K, Engel MH, Farmer JD, Geol., 2007, 201: 157-179.

10. Vajiheh K, Naser B et al. African Journal of Biotechnology 2003, 2, 10.

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