Microbiol. Biotechnol. Lett. (2019), 47(2), 269–277 http://dx.doi.org/10.4014/mbl.1808.08006 pISSN 1598-642X eISSN 2234-7305 Microbiology and Biotechnology Letters Assessment of Bioremediation Potential of Cellulosimicrobium sp. for Treatment of Multiple Heavy Metals Tushar Bhati, Rahul Gupta, Nisha Yadav, Ruhi Singh, Antra Fuloria, Aafrin Waziri, Sayan Chatterjee, and Ram Singh Purty* University School of Biotechnology, Guru Gobind Singh Indraprastha University, Sec-16C, Dwarka, New Delhi, India Received: August 16, 2018 / Revised: November 4, 2018 / Accepted: November 7, 2018 In the present study, we have studied the bioremediating capability of bacterial strain against six heavy metals. The strain was isolated from river Yamuna, New Delhi which is a very rich repository of bioremedi- ating flora and fauna. The strain was found to be Gram positive as indicated by Gram staining. The strain was characterized using 16s rRNA gene sequencing and the BlastN result showed its close resemblance with the Cellulosimicrobium sp. As each treatment has its own toxicity eliciting expression of different fac- tors, we observed varied growth characteristics of the bacterial isolate and its protein content in response to different heavy metals. The assessment of its bioremediation capability showed that the strain Cellulo- simicrobium sp. has potential to consume or sequester the six heavy metals in this study in the following order iron > lead > zinc > cooper > nickel > cadmium. Thus, the strain Cellulosimicrobium sp. isolated in the present study can be a good model system to understand the molecular mechanism behind its bioreme- diating capabilities under multiple stress conditions. Keywords: Cellulosimicrobium sp, bioremediation, multiple heavy metals tolerant, Yamuna river Introduction cell membrane, disrupt cellular function, and destroy the DNA structure. Metal ions interfere with osmotic Heavy metals are naturally occurring metalloid hav- balance, oxidative phosphorylation process of microor- ing density more than 5 g/cm3 [1, 2]. High level of heavy ganisms, and cellular DNA destruction [6, 7]. metal accumulation, increased bioavailability, and their Heavy metals have adverse effect on human health ever increasing percentage in the atmosphere are major such as Pd metal ion disturb the biological functions of problem to environment. Although they are present nat- cell by replacing other bivalent metal cations such as urally in environment but their presence as contami- Ca2+, Mg2+, and Fe2+. High Pd concentration leads to nants in environment is mainly due to anthropogenic increase in concentration of reactive oxygen species and activities such as mining, tanneries, use of chemical fer- decrease in concentration of antioxidants. High concen- tilizers, etc [3]. Some heavy metals such as Hg, Ag, Cd, tration of cadmium binds to cysteine-rich proteins like and Pb are toxic even at low concentration and does not metallothionein, forming a cysteine-metallothionein have any functional roles (as a metal ions) in organisms complex which is responsible for hepatotoxicity and [4, 5]. They can alter the enzyme specificity, damage the deposition in kidney leads to nephrotoxicity [8]. Arsenic inside the cell undergoes series of biotransformation *Corresponding author reactions and gets converted in methylated inorganic Tel: +91-11-25302311, Fax: +91-11-25302304 arsenic. It is highly toxic and responsible for the induc- E-mail: [email protected] © 2019, The Korean Society for Microbiology and Biotechnology tion of the arsenic carcinogenesis [9]. Chromium exists June 2019 | Vol. 47 | No. 2 270 Bhati et al. in various oxidation states. Cr (VI) is more dangerous for bioremediation [15]. Bacteria can be used for biore- than Cr (III), as Cr (VI) can enter the cells more easily mediation for one metal or mixture of heavy metals. than that of Cr (III). Due to mutagenic properties, the Usually, strains which are resistant to multiple heavy International Agency for the Research on Cancer catego- metals have great advantage over the former one, as rizes Cr (VI) as a group 1 human carcinogen [10]. need for single type of condition only, less time consum- Various techniques are being developed for removal of ing, cost effective as well as less laborious. So isolation of heavy metals from environment but none of them are a bacteria resistant to multiple heavy metals becomes panacea for remediating contaminated soils and often very important. more than one technique may be required to optimize Therefore, in order to isolate bacteria resistant to mul- clean-up efforts. Reverse osmosis, ion exchange, mem- tiple heavy metals it is evident to search for sites which brane technology, filtration, evaporation, and chemical have multiple heavy metals as a pollutant. The Yamuna precipitation are conventional techniques developed for with the catchment area in Delhi serves as a major site removal of heavy metals from environment, but these for drainage of the most of the chemical and biological techniques are inefficient and highly expensive. Most of wastes of the city. The concentration of heavy metals, the above-mentioned techniques are not useful if the namely, Pb, Fe, Cd, Ni, Cu and Co in various effluents concentration of heavy metal is below 100 mg/l [11]. originating from different industries has been reported Most of the metal salts are soluble in water so can’t be to exceed the maximum permissible limits for drinking readily separated by this conventional techniques [12]. in Yamuna water [16]. So, it is expected that there will These physical and chemical conventional methods be a good probability of finding multiple heavy metal become less cost effective and mostly ineffective if the resistant strains in its water sample. Therefore, in the concentration of heavy metals is very low. So there is a present study water sample collected from the Yamuna need for development of innovative treatment tech- was used for isolation of multiple heavy metal resistant niques for remediation of toxic heavy metals from soil bacteria. The isolated bacterial strain can later be used and wastewater [13]. Biological methods like phytoex- as a model species to study the molecular mechanism traction, biosorption, bioaccumulation, phytoremidation behind its bioremediating capabilities as well as multi- and phytostabilization may be most effective alternative ple heavy metal tolerance through transcriptome studies to physico-chemical methods for removal of heavy met- [17]. als [14]. Bioremediation is a biological process that uses the liv- Materials and Methods ing organisms such as bacteria and fungus, to degrade the organic waste present in the environment to a non Chemicals and reagents toxic or at least less toxic form under the controlled con- Stock solutions of 1 M were prepared in sterile deion- ditions. This method is effective when surrounding envi- ised water separately for each heavy metal under study ronment conditions promote microbial growth and using the laboratory grade chemicals. For working solu- optimal activity. Optimization and control of bioremedi- tion preparation, the stock solution was diluted using ation processes are quite complex. In order to be effective sterile deionised water. The metal salts viz. cadmium bioremediation process, there must be manipulation of chloride (CdCl2·H2O) and nickel sulfate (NiSO4·6H2O) the different environment parameters which promote were purchased from Fischer Scientific, ferric sulfate the microbial growth, activity, and ultimately lead to (Fe2(SO4)3·7H2O) from S.D. fine chemicals, Cupric sul- increased rate of degradation. The rate of bioremedia- fate (CuSO4·5H2O), lead acetate (Pb (C2H3O2)2·3H2O) tion is influenced by different factors such as the avail- and zinc sulfate (ZnSO4·7H2O) from SRL. All other ability of contaminants to the microbial population, chemicals and reagents used in this research work were bacterial degeneration ability, environmental factors purchased from SRL unless stated otherwise. such as temperature, pH, the presence of aerobic or anaerobic environment, and nutrients. In the past, sev- Study area and collection of water samples eral bacteria strains have been reported for their ability Water samples were collected from the banks of the http://dx.doi.org/10.4014/mbl.1808.08006 Bioremediation by Genus Cellulosimicrobium sp. 271 Yamuna River, near Monastery market, New Delhi, software using MUSCLE algorithm [20]. Phylogenetic India (28.6726oN, 77.2313oE), in pre-sterilized bottles and molecular evolutionary genetic analysis of the and stored at 4℃ in the laboratory until further analy- sequences was conducted based on Neighbor-Joining ses. Working stock of the water sample was prepared by method. The final figure of the alignment was visualized serial dilution from 10-1 to 10-8 with sterile deionised using Jalview [21]. water. Gram staining Screening of multiple heavy metal resistant bacteria To categorize the isolated multiple heavy metal resis- In order to obtain isolated bacterial colonies, primary tant bacteria as Gram positive or negative, Gram stain- screening for multiple heavy metal resistant microbial ing was carried out following the standard protocol [22]. cultures was carried out with the serial diluted water sample ranging from 10-1 to 10-8. Around 100 µl of the Determination of growth curves each water sample was spread plated on the LB agar To study the effect of different heavy metals of 1 mM plates supplemented with 1 mM of each heavy metals concentration on the bacterial growth,