Evaluation of Cr(VI) Reducing Capability of Shewanella putrefaciens (MTTC8410) and Optimization of Operational Parameters

Bioremediation is an important technology to remediate the chromium (Cr) contaminated soil and water. In this study, Shewanella putrefaciens (MTTC8410) was used to investigate the influence of carbon concentration, pH, and temperature on reduction of hexavalent chromium [Cr(VI)] into trivalent chromium [Cr(III)]. The increased bacterial growth rate was significantly reduced the Cr(VI) concentration. In batch mode experiments, 1% starch recorded the highest reduction of Cr(VI) (90%) followed by 1% glucose (88% reduction) and a reduction of 77% was by 1% cellulose. By using various pH conditions the maximum Cr(VI) reduction was achieved at pH 7.0. In this experiment the maximum Cr(VI) reduction (75%) was observed at 35°C, followed by 30°C with 62% of Cr(VI) reduction. Bioreactor analysis revealed the highest reduction of Cr(VI) (88%) in unsterile tannery effluent. The significant levels of physicochemical parameters were reduced in unsterile tannery effluent, as compared to the sterile tannery effluent. The experimental results revealed that the S. putrefaciens (MTTC8410) could be used as a potential bacterial strain for reduction of Cr(VI) from contaminated groundwater.

previously successfully isolated a variety of possible microorganisms, minimizing effectiveness with Cr(VI). Some bacterial and fungal species such as Escherichia coli, Shewanella oneidensis, Bacillus firmus, Aspergillus niger and Pleurotus ostreatus were used to transform the Cr(VI) into Cr(III) [18][19][20][21][22] . The novelty of this study is to evaluate the ability of live cells of a bacterium namely S.putrefaciens (MTTC8410) to reduce the Cr(VI) into Cr(III). The batch process and bioreactor study was carried out for Cr reduction from chromium contaminated ground water.
The present study was aimed at evaluating S.putrefaciens' Cr(VI) reduction capability. Studies of the batch mode were performed by living bacterial cells. Under optimized conditions, the bacterial strain on Cr contaminated tannery effluent is checked via bioreactor analysis. The toxicity of treated wastewater samples from the bioreactor was examined using seed germination of on black gram.

Bacterial strain
The S. putrefaciens (MTTC8410) utilized in this study was obtained from MTCC Chandigarh in India. It was sub cultured in the laboratory using Luria broth medium. The bacterial strain was maintained by nutrient agar slant. The bacterial strain was subcultured every month and preserved using a glycerol solution 20% and stored at 4°C for further studies.

Media and chemicals
The optimization of potential bacterial strain was performed by using mineral salt medium. All analytical grade chemicals and reagents were purchased from Loba Chemie Laboratories (Mumbai, India) and Hi-media Laboratories (Mumbai, India).

Influence of Cr(VI) on bacterial growth
An influence of different concentrations of Cr(VI) on the bacteria's growth rate was investigated. Shewanella putrefaciens strain (MTTC8410) was grown at 120 rpm and pH 7.0 in a rotary shaker (model: OrbiTech LETT, India), whereas the temperature in LB broth medium was 37°C supplemented by Cr(VI) range 10 to 30 mg/ L 23 . Every 4 hrs time interval 5 ml samples were obtained from the conical flasks. Followed by the samples were collected and centrifuged for 10 min at 3000 rpm. The pellet was then dissolved with sterile distilled water and calculated growth rate was measured at 600 nm using UVspectrophotometer (model: Cyber lab-UV-100, USA) 24 .

Effect of carbon sources on reduction of Cr(VI)
Shewanella putrefaciens (MTTC-8410) was used to evaluate the influence of various carbons sources on the reduction of Cr(VI). So far, 100 ml mineral salt medium (MSM) was prepared in 250 ml conical flasks with various concentrations of carbon sources (0.5, 1 and 1.5 per cent) and amended with 10 mg Cr(VI) and all flasks were sterilized. After the sterilization individually, 1% of S. putrefaciens (MTTC-8410) was transferred by aseptically 33 . Flasks were then kept for 10 days in shaker at 37°C at125 rpm. Then 5 ml of sample were taken from all conical flasks at every 24 hrs time interval. After this the collected samples were spun at 3000 rpm for 10 min. Then aqueous phase was withdrawn from the centrifuge tube and chromium was estimated by DPC (diphenylcarbazide) method using in UVspectrophotometer at 540 nm (model: Cyber lab-UV-100, USA). The culture growth rate was measured simultaneously, at 600 nm.

Effect of different pH conditions on Cr(VI) reduction
S. putrefaciens was used to assess the effectiveness of different pH conditions on the Cr(VI) reduction. In which various pH of (5 to 9) 100 ml (MSM) medium were prepared with optimized carbon source 0.5 per cent starch in 250 ml conical flasks and adjusted with 10 mg Cr(VI) and all flasks were sterilized. After the sterilization 1% of S. putrefaciens (MTTC-8410) was transferred to individual conical flask by aseptically 34 . Flasks were then kept for 10 days in shaker at 37°C with 125 rpm. Then 5 ml of sample were taken from all conical flasks at every 24 hrs time interval. After this the collected samples were spun at 3000 rpm for 10 min. Then aqueous phase was withdrawn from the centrifuge tube and Cr(VI) was estimated by DPC method in UVspectrophotometer at 540 nm (model: Cyber lab-UV-100, USA). Simultaneously the culture growth rate was measured at 600 nm.

Influence of temperature on Cr(VI) reduction
Shewanella putrefaciens (MTTC-8410) was used to assess the influence of different temperatures on reduction of Cr(VI). Hence, in 250 ml conical flasks 100 ml (MSM) medium was prepared with optimized carbon source 0.5% starch and amended with 10 mg Cr(VI) and all flasks were sterilized. After the sterilization 1% of S. putrefaciens was transferred to individual conical flask by aseptically. Then conical flasks were kept in shaker under different temperatures (25 to 45°C) at 125 rpm for 10 days 35 . Followed by 5 ml of sample was taken from all conical flasks at every 24 hrs time interval. After this the collected samples were spun at 3000 rpm for 10 min. Then aqueous phase was withdrawn from the centrifuge tube and Cr(VI) was estimated by DPC method in UV-spectrophotometer at 540 nm (model: Cyber lab-UV-100, USA). Simultaneously the culture growth rate was measured at 600 nm.

Analytical method
The [Cr(VI)] was determined using the (DPC) procedure using a UV-spectrophotometer (model: cyber lab-UV-100, USA) 36 . Around 0.025 g of DPC was mixed with 9.67 ml of acetone and 330 ml of 3 M H 2 SO 4 . The absorbance was calculated at 540 nm and all assays were performed in triplicates, and mean values were recorded.

Bioreduction of chromium from tannery effluent using lab scale bioreactors
The tannery effluent treatment method was planned and the setup was made as shown in (Fig. 1). This set up was prepared based on the pilot scale water treatment plant 37 . The tannery effluent was obtained from tannery industry in Periyakulam, district of Erode, Tamil Nadu. The sample was collected in sterile container and was taken to the laboratory and analyzed for various physicochemical parameters 38 . The established lab scale bioreactors have a capacity of 10 liters and are made of tarson. Mechanical stirrers were installed in the reactor tank and the settling tanks. Artificial aerator was connected to both bioreactors for aeration purpose. About 10 liters of tannery effluent containing 20 mg of Cr(VI) was subjected to primary (bioprocess) treatment and inoculated with S.putrefaciens. Then 5 ml of sample was taken from all conical flasks at every 24 hrs time interval. After this the collected samples were spun at 3000 rpm for 10 min. Then aqueous phase was withdrawn from the centrifuge tube and Cr(VI) was estimated by DPC method using UV-spectrophotometer at 540 nm (model: Cyber lab-UV-100, USA). Simultaneously the culture growth rate was measured at 600 nm. Further, all physic-chemical parameters were analyzed before and after the treatment of tannery effluent.

Reactor 1 (R1)
Unsterile tannery effluent + 0.5% starch + 1% inoculum Reactor 2 (R2) Sterile tannery effluent + 0.5% starch + 1% inoculum Phytotoxicity assay Pot culture experiments were performed to evaluate the toxicity of the treated and untreated tannery effluent. The fertile soil was collected without any contamination near Omalur Taluk of Salem district. The Cr(VI) amended soil was filled in sterile plastic cups with the dimensions of 5 x 15 cm. The black gram seeds obtained from the licensed agency and surface sterilized by mercury chloride (HgCl 2 ) (0.1%, w/v). Four seeds were sowed in each cup and the set up was kept for phytotoxicity assay. In each pot containing black gram (Vigna mungo) the treated and untreated tannery effluents at varying percentages viz 25, 50, 75 and 100% were irrigated and allowed to germinate the plants. Control pot was irrigated with tap water to compare the efficiency of the treatment. Germination of the seeds was noted after 3 rd day of irrigation and calculated 39 .

Statistical Analysis
All tests were performed in triplicates and reduction of Cr(VI) was evaluated using error bars 40 . Data were statistically analyzed using the Microsoft ® Excel (Version 2010) statistical package.

Effect of Cr(VI) on bacterial growth rate
The influence of different Cr(VI) (10 and 30 mg/L) concentrations on the growth of S.putrefaciens has been performed and presented in (Fig.2). The interaction between cell growth and varying Cr(VI) concentration was studied at 24 hrs time of incubation. The rate of bacterial growth in LB media without Cr(VI) was very significant as compared to the LB medium containing Cr(VI) 10 and 30 mg / L containing. In which 10 mg/L of Cr(VI) has a minor effect on the development of bacterial cells. Cr(VI) at a concentration of 10 to 30 mg/L have negatively effect on the growth of the bacteria. The previous study reported that concentration of Cr(VI) in the range of 10 and 20 mg/L would have a small impact on Bacillus sp. growth, while 100 mg/L of Cr(VI) was greatly inhibit the bacterial growth 41 . The present study reported a decrease in Escherichia coli ATCC 33456 growth rate during lag phase due to the increased concentrations of Cr(VI) 42 .

Biochemical tests of S. putrefaciens
The enzymatic characteristic of S. putrefaciens was presented in Fig. 3. The strain developed a clear zone around the culture in starch agar medium, but in the case of cellulose, chitinase and pectinase enzyme assay, the culture could not develop the clear zone around the colonies due to the lack of enzyme producing gene. The S. putrefaciens (MTTC-8410) can produce enzymes like gelatinase, tryptophanase, citrase, L-Asparginase, protease, oxidase and catalase and it was confirmed by the strain developed a clear zone in respective media. Similar results were reported by Cr reducing Pseudomonas species 43 .

Effect of different concentrations of glucose on Cr(VI) reduction
The effect of different doses of glucose (0.5, 1 and 1.5 percent) on reduction of Cr(VI) by S. putrefaciens was presented in Fig. 4a. In which, by using 0.5% of glucose, no significant Cr(VI) reduction was observed from 1 st to 5 th day of experiment. After the 5 th day of experiment, slight significant Cr(VI) reduction (82%) was observed. By using 1% of glucose, the maximum Cr(VI) reduction (88%) was observed in 8 th day to 10 th of day of experiment. The previous study reported that glucose was donor of electrons, which could easily be oxidized glucose 44 . Some previous research reported that glucose can play a good carbon source while act as a efficient electron donor to reduce Cr(VI) by bacteria 45-47 . By   Fig.3. Biochemical tests of Shewanella putrefaciens (MTTC8410) using 1.5% of glucose, 80% of Cr(VI) reduction was observed. Overall in this different concentration of glucose optimization, 1% of glucose achieved very significant Cr(VI) reduction. Previously many researchers reported that glucose is a known electron donor to several bacterial strains for Cr(VI) reduction 48 . Fig. 4b showed the growth rate of S. putrefaciens by various doses of glucose in MSM medium containing 10 mg of Cr(VI). In this study by using 0.5% of glucose, there was no significant growth observed from 1 st day to 10 th day of experiment and it does not influence the Cr(VI) reduction. But by using 1 and 1.5% of glucose the significant Cr(VI) reduction was observed. In which both 1 and 1.5% of glucose can influence the Cr(VI) reduction as well as growth rate of S. putrefaciens. Different concentrations of cellulose on Cr(VI) reduction.
The effect of different doses of cellulose (0.5, 1 and 1.5 percent) on reduction of Cr(VI) by Fig. 4. Influence of various carbon sources on the reduction of hexavalent chromium S. putrefaciens was presented in (Fig.4c). In this experiment, by using 0.5% of cellulose, there was very poor Cr(VI) reduction was observed. By using 1% of cellulose, 77% of Cr(VI) reduction was observed in 6 th to 8 th day of experiment. Followed by using 1.5% of cellulose, there was 75% of Cr(VI) reduction from 5 th to 10 th day of experiment. Fig.  4d showed the growth rate of S. putrefaciens under various concentrations of cellulose (0.5, 1 and 1.5%) in MSM medium containing 10 mg of Cr(VI). In this study by using 0.5% of glucose, better significant growth was observed from 1 st day to 10 th day of experiment and it also contribute poor influence on Cr(VI) reduction. But by using 1 and 1.5% of cellulose, significant chromium reduction was observed. In which both 1 and 1.5% of glucose can influence the Cr(VI) reduction as well as growth rate of S. putrefaciens. Previous study reported that a number of electron donors play an important role for the reduction of Cr(VI) by Ganoderm lucidum 49 .

Effect of different concentrations of starch on Cr(VI) reduction
The effect of different doses of starch (0.5, 1 and 1.5 percent) on reduction of Cr(VI) by S. putrefaciens was presented in (Fig.4e). In this study, by using 0.5% starch, there was considerable Cr(VI) reduction was observed in first two days, after that on the 3 rd day of experiment it loss the Cr(VI) capacity. Using 1% of starch, 90% of Cr(VI) reduction was observed in 6 th to 9 th day of experiment. Followed by using 1.5% of starch, there was 79% of Cr(VI) reduction was achieved from 7 th to 8 th day of experiment. The electron donors of carbon sources influenced the Cr(VI) reduction by using Brevibacterium casei 50 . Fig.4f was showed the growth rate of S.putrefaciens under various concentrations of starch (0.5, 1 and 1.5%) in MSM medium containing 10 mg of hexavalent chromium. In this study, by using 1% of starch was can influenced the maximum level of Cr(VI) reduction and growth rate of S.putrefaciens when compared to 0.5 and 1.5% of starch.

Influence of different concentrations of pH (5,6,7,8 and 9)
The reduction of Cr (VI) under various pH conditions by using S.putrefaciens was shown in Figure 5a. The pH has an influence on Cr(VI) bioreduction. The effects of incubation period on reduction of Cr(VI) at various pH (5, 6, 7, 8 and 9) was analyzed (The operation conditions; 10 mg of Cr (VI)/100 ml medium, temperature 37°C and 1ml of one OD inoculums). In this study, all pH from 5 to 9 was very significantly reduced the Cr(VI) from the aqueous solution. In this experiment the maximum Cr(VI) reduction was observed at pH7. The similar result was observed that the maximum removal of Cr(VI) (88%) by PCP3 strain at pH 7.0 51 . So the further studies were carried out at pH7. The reduction of Cr(VI) during low pH may be due to the relationship of bioaccumulation and functional group dissociation 52 . The (Fig. 5b) was showed the growth rate of S. putrefaciens under various pH conditions. All pH levels significantly influenced the growth rate.

Influence of different temperature of pH (25, 30, 35, 40 and 45°C)
Temperature is one of the important key for microbial growth Cr(VI) reduction. The effectiveness of various temperatures on reduction of Cr(VI) by S. putrefaciens were presented in (Fig.  5c). In this study, the maximum Cr(VI) reduction (75%) was observed at 35°C. Followed by 30°C was showed the 62% of Cr(VI) reduction. The previous study was reported that hat 30°C was significantly encourage the growth of Bacillus KCH3 and growth was inhibited by below15°C and above 40°C 53 . The significant bacterial growth achieved by 30°C and 35°C when compared to other temperatures (Fig. 5d). The similar result was observed that the maximum removal of Cr(VI) (85.87%) by Brevibacillus sp at 35°C 54 .

Bioreduction of chromium contaminated tannery effluent through lab scale bioreactor
Bioreduction of chromium contaminated tannery effluent through lab scale bioreactor results were presented in (Fig. 6a). In sterile effluent, the maximum Cr(VI) reduction (84%) was observed from 4 th day to throughout the study. In unsterile effluent, the maximum Cr(VI) reduction (88%) was observed from 5 th day to throughout the study. The significant Cr(VI) reduction was obtained in bioreactor study, due to more biochemical reactions to convert raw materials to products through the action of biocatalyst and enzyme of microorganisms. The growth rate of S.putrefaciens was presented in (Fig. 6b). In unsterile effluent, growth rate was very significantly high because of unsterile effluent have native microbes, hence those native microbes also influence the Cr(VI) reduction and growth rate.

Physicochemical parameters of untreated and treated tannery effluent
The physicochemical parameters of the raw tannery effluent, treated sterile tannery effluent and treated unsterile tannery effluent were described in Table 1. The tannery effluent

Phytotoxicity assay by pot culture method
The data regarding phytotoxicity assay of treated and untreated tannery effluent studies has been presented in (Table 2). In treated sterile tannery effluent (25 to 100%), significant black gram seed germination was observed every day. In treated unsterile tannery effluent (25 to 100%), very significant growth was observed when compared to treated sterile effluent. The similar results were reported by using bacterial strain pv 26 59 . But in the case of raw untreated tannery effluent very poor growth rate was observed by using 25 and 50% of raw untreated effluent and there was no growth observed when using 75 and 100% raw untreated tannery effluent. At higher concentrations (80 and 100%) tannery effluent inhibited germination of black gram due to toxicity and stress by using untreated effluent 60 . The previous study reported that the higher tannery effluent concentration reduces the dehydrogenase activity of the enzyme, which may be changing to disrupt germination and seedling growth 61 . This study was revealed that after the treatment of tannery effluent by using S.putrefaciens the treated effluent was did not showed toxic effects on black gram plants.

CONClUsiONs
S h ew a n e l l a p u t r e fa c i e n s st ra i n (MTTC8410) was investigated for its an ability to reduce Cr(VI) into Cr(III). In this study the maximum Cr(VI) reduction was achieved at pH 7.0 and temperature 37°C. In the bioreactor test, S.putrefacien maximum Cr(VI) reduction capacity was 97 %. Consecutive experiment for the toxicity of treated tannery effluent on black gram showed no toxicity in plant growth parameters. The bacterially treated tannery effluent has very less amount of Cr, due to this nature its harmless for the agricultural crops. Hence, it was recommended that the S.putrefaciens (MTTC8410) have potential to reduce Cr(VI) from aqueous media.
with human participants or animals performed by any of the authors.