Assessment of Bioremediation Potential of Cellulosimicrobium Sp

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 bioremediating 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 factors, 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 bioremediating 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

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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

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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, growth curves (CdCl2·H2O, NiSO4·6H2O, Fe2(SO4)3·7H2O, Pb analysis was performed. A loop full of freshly grown bac- (C2H3O2)2·3H2O, CuSO4·5H2O and ZnSO4·7H2O). For teria was inoculated in 100 ml of LB broth (control) or control experiment, water sample without dilution was LB broth supplemented with 1 mM of either CdCl2·H2O spread plated on the LB agar plate supplemented with or NiSO4·6H2O or Fe2(SO4)3·7H2O or Pb (C2H3O2)2· 1 mM of each heavy metal. The plates were incubated 3H2O or CuSO4·5H2O or ZnSO4·7H2O, respectively. overnight at 37℃ to obtain the isolated colonies. Multi- Both control and experimental sets were incubated at ple heavy metal tolerance of the bacterial isolates was 37℃ and the absorbance was measured after every 2 h analyzed by streak plating on the LB agar plate supple- interval at 600 nm. mented with 1 mM of either CdCl2·H2O or NiSO4·6H2O or Fe2(SO4)3·7H2O or Pb (C2H3O2)2·3H2O or CuSO4· Effect of different heavy metals on protein content 5H2O or ZnSO4·7H2O, respectively. Bacterial isolate was grown in LB broth supplemented with 1 mM of either CdCl2·H2O or NiSO4·6H2O or Molecular characterization Fe2(SO4)3·7H2O or Pb (C2H3O2)2·3H2O or CuSO4·5H2O Molecular identification has been carried out using or ZnSO4·7H2O, respectively. LB broth without any bacterial 16S rRNA gene sequencing [18]. In order to heavy metal was maintained as control. Both control perform the 16S rRNA gene sequencing, the isolates and experimental sets were incubated at 37℃. In order obtained from 10-8 serial diluted sample were streaked to extract protein from equal number of cells, the growth on a LB agar plate supplemented with 1 mM of each of bacteria was stopped when the absorbance at 600 nm heavy metal (Cd, Fe, Ni, Pb, Cu and Zn) were deposited reached at 0.6 OD. Bacterial cells were harvested by cen- and sequenced at CSIR-National Chemical Laboratory, trifugation at 12,000 rpm for 5 min at 4℃. Using sonica- Pune, India. The chromosomal nucleic acid was tion method, bacterial cells were lysed in lysis buffer extracted using the QIAamp DNA Mini Kit (Cat.No. (50 mM Tris pH-8, 10% glycerol, 0.1% Triton X 100). The 51304, Qiagen). supernatant obtained after centrifugation was used for protein estimation by Lowry’s method [23]. Phylogenetic analysis The 16S rRNA gene sequence obtained after sequenc- Effect of different concentration of heavy metals on ing was compared with the other sequences available in growth GenBank database using BLASTN program [19]. Acces- In order to determine the level of tolerance, bacterial sion numbers of all the hits was tabulated and their isolates was streaked on LB agar plates supplemented sequences were retrieved from the database for further with different concentration of either CdCl2·H2O or analyses. Downloaded sequences were used as tem- NiSO4·6H2O or Fe2(SO4)3·7H2O or Pb (C2H3O2)2·3H2O plates for multiple sequence alignment using MEGA 6 or CuSO4 or ZnSO4·7H2O, respectively. LB agar plate

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supplemented with 1 mM of each heavy metal was grew well in LB agar plate supplemented with 1 mM of maintained as control. The response on growth was each heavy metal (Fig. 1). The bacterial isolates showed observed after incubating the plates overnight at 37℃. growth on all the heavy metal supplemented LB agar plates when their multiple heavy metal tolerance capa- Bioremediation assay bility was analyzed (Fig. 2). To conduct the bioremediation assay, LB broths were supplemented with 1 mM of either CdCl2·H2O or Characterization of bacterial isolates NiSO4·6H2O or Fe2(SO4)3·7H2O or Pb (C2H3O2)2·3H2O In order to identify the isolates, all the three colonies -8 or CuSO4 or ZnSO4·7H2O, was taken as initial concen- obtained from 10 serial diluted water samples were tration. The LB broth is then inoculated with the bacte- molecular characterized using 16S rRNA gene sequenc- rial isolates and incubated overnight at 37℃. After 24 h ing. The 16S rRNA gene sequence obtained was submit- of growth, the culture was centrifuged and the LB broth ted to GenBank with the accession number MH685192. obtained were sent to FICCI Research and Analysis The sequence was later used for BLASTN analysis Centre, New Delhi, for inductively coupled plasma mass which showed that all the three colonies were same and spectrometry (ICP-MS) to determine the final concentra- identified as Cellulosimicrobium sp. (Table 1). There- tion of heavy metals. Percentage bioremediation/accu- fore, for further analysis out of the three isolates, only mulation was calculated using the following formula: one was considered. The sequence of all the BLAST hits that showed 99% identity were retrieved and used for Initial conc.– Final conc. ------×100 Bioremediation (%) = Initial conc. multiple sequence alignment using MEGA 6. Multiple sequence alignment showed similarities with the all sequences of different species obtained from the BLAST Results hits (Fig. S1). Phylogenetic tree analysis showed that the bacterial isolates is closely related to Cellulosimicro- Isolation of multiple heavy metal resistant bacteria bium sp. (Fig. 3). Gram staining showed the bacterial Primary screening for multiple heavy metal resistant strain to be gram positive bacteria (Fig. S2). bacteria resulted in three isolated colonies when plated with 10-8 serial diluted water sample. All the three colo- Growth analysis nies showed resistant to multiple heavy metals as they The growth curve of Cellulosimicrobium sp. was

Fig. 1. (A-I) Spread plates showing primary screening of multiple heavy metal resistant microbial cultures on LB agar plates supplemented with 1 mM of each heavy metals (Cd, Ni, Fe, Pb, Cu and Zn). (A) Control: Without diluted water samples, (B) 10-1, (C) 10-2, (D) 10-3, (E) 10-4, (F) 10-5, (G) 10-6, (H) 10-7 and (I) 10-8 serial diluted water sample.

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Fig. 2. Isolated bacterial strain showing multiple heavy metal tolerance when streak plated on LB agar plates supplemented with different concentration of either CdCl2·H2O or NiSO4·6H2O or Fe2(SO4)3·7H2O or Pb (C2H3O2)2·3H2O or CuSO4·5H2O or ZnSO4·7H2O, respectively. observed under both the conditions, i.e., control and in entered stationary phase after 14 h of incubation and presence of 1 mM concentration of different heavy met- remained for 20 h after that it entered the declined als (Fig. 4). In control, the strain entered the log phase phase. The pattern of the growth in control, zinc and after 4 h of lag phase where it remained for 10 h. The cell lead stress was similar, though the cells in 1 mM stress

Table 1. Table showing various hits obtained after BLASTN analysis. Accession number Strain E value Query cover (%) Identify (%) HM2226651 Cellulosimicrobium sp. strain 0707K4-3 0 98 99 GQ274926.1 Cellulosimicrobium sp. strain TUT 1242 0 98 99 KC466092.1 Cellulosimicrobium sp. strain H2 0 99 99 AB056131.1 Ptomicromonospora sp. strain IFO 16225 0 97 99 JN257084.1 Cellulosimicrobium sp. strain Aq2 0 98 99 X79453.1 Oerskovia xanthineolytica 0 98 99 KF192273.1 Cellulosimicrobium sp. strain KSKE-13 0 96 99 AM992198.1 Cellulosimicrobium cellulans 0 98 99 JN169776.1 Actinoacterium 0 98 99 X79456.1 C. Cartae MSD 0 97 99 KF033112.1 Isoptericola variabilis strain KSR05 0 97 99 EU181237.1 Cellulosimicrobium sp. strain 120-1 0 98 99 KY933465.1 Cellulosimicrobium funkei strain IHB B 0 98 99

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Fig. 3. Neighbor-joining evolutionary tree showing relationship of 16S rRNA gene (Accession No. MH685192) with other sequences used in Multiple Sequence Alignment analysis. grew a little slower in comparison to control. In 1mM total protein content, supernatant obtained after sonica- iron stress, the growth was much better than that of con- tion was estimated for each of the six different metal- trol. The cell entered the log phase only after 3 h of incu- treated cells and calculated using the BSA standard bation and remained for 11 h. The cells entered curve. The protein content in the iron-treated culture stationary phase after 14 h of incubation and decline was found to be maximum followed by control (Fig. 5). phase was seen after 40 h of incubation. The growth rate Protein content decreased or increased upon heavy of bacteria was slower for copper, nickel and cadmium metal stress treatment. In comparison to control, it was treated culture (Fig. 4). In the present study, growth of around 77.27, 16.27, 4.34, and 0.8% decreased under Cellulosimicrobium sp. was observed to be much slower cadmium, nickel, copper and zinc stress, respectively. in cadmium and nickel indicating that 1 mM concentra- Upon iron and lead stress treatment, 19.58, and 1.66% tion of these two metals is more toxic compared to other increase in protein content was noted (Fig. 5). heavy metals tested. Effect of different concentration of heavy metals on Effect of different heavy metal stress on protein content growth To study the effect of different heavy metals on the To study the tolerance level, the isolated Cellulosimi-

Fig. 4. Growth curve of Cellulosimicrobium sp. under 1 mM Fig. 5. Protein content of Cellulosimicrobium sp. under concentration of different heavy metals. 1 mM concentration of different heavy metals.

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Table 2. Bioremediation capability of Cellulosimicrobium sp. bacterial strain from the river Yamuna, New Delhi, Initial Final India. In the recent papers, it has been shown that the Accumulation S No. Metals concentration concentration river Yamuna in contaminated with different heavy (%) (ppm) (ppm) metals due to dumping of industrial waste and various 1 Cadmium 98 90.50 7.65 other human activities [16, 24, 25]. Thus, bacterial 2 Iron 27.95 8.31 70.26 strain isolated from such environment, i.e., Yamuna 3 Lead 31.84 24.16 24.12 River, may possess the mechanism of heavy metal toler- 4 Nickel 54 43.75 18.98 ance. 5 Copper 27.15 21.39 21.21 As expected the growth was observed when the bacte- 6 Zinc 16.12 12.44 22.82 rial isolates was grown in LB agar plates supplemented with 1 mM of different heavy metals. Gram staining crobium sp. strain was grown on LB agar plates supple- showed that the bacteria isolated to be Gram positive. mented with different heavy metals of varying Further, upon molecular characterization using 16s rRNA concentration. The growth of the Cellulosimicrobium sp. gene sequencing, BLASTN and phylogenetic tree was higher on LB agar plate supplemented with iron, analysis, the isolated bacteria was found to be genus where it grew till 7 mM concentration and least was Cellulosimicrobium. It is known that the genus Cellu- observed for cadmium, where its growth was observed losimicrobium is a Gram-positive, yellow-pigmented, till 2 mM (Fig. S3A; S3F). The bacterial strain tolerated non-motile and rod-shaped or coccoid bacterial strain up to 6 mM concentration in lead, 5 mM in zinc and cop- [26, 27]. Cellulosimicrobium sp. which was isolated from per and 3 mM in nickel supplemented LB agar plates the Yamuna in the present study, was earlier reported (Fig. S3B−S3E). from soil [28], tannery waste water [25, 29] and in radio- active wastewater [30]. In one of the study Cellulosimi- Bioremediation assay crobium sp. was also isolated from the patient with The bioremediation capability of Cellulosimicrobium acute renal failure [31]. Recent studies have also shown sp. was studied with the aid of inductive plasma cou- that this may cause bacteremia in bone marrow of trans- pling mass spectrometer. It was observed that isolated plant patients [32]. strain can sequestered/consumed maximum of 70.26% Growth rate of Cellulosimicrobium sp. when exposed iron and least 7.65% for cadmium (Table 2). The bacte- to various treatments cannot be compared as each treat- rial strain has shown the bioremediation capability of ment has its own toxicity and the strain response will sequestering 24.12%, 22.82%, 21.21% and 18.98% of also be different. However, under control conditions the lead, zinc, copper, and nickel from medium, respectively. strain has very short lag phage of 3−4 h followed by 8− 10 h of log phase. In the present study, stationary phase Discussion remained for 20 h followed by decline phase. In the earlier reports it has been noted that growth rate varies Heavy metals are non-essential metals which are not with the treatments and amongst species [33−35]. required by the cell and are toxic to organism even at Under stress condition it is well know that microbes or very low concentration. Higher concentration of both plants tries to express genes that codes for the proteins non-essential and essential metals are toxic to the cells, involves in providing tolerance against the adverse con- they can block the normal function of the cell, and also ditions. In the present study, under different heavy damage the DNA leading to the cell death [6]. However, metal stress the protein content varied with maximum few species have the mechanism to grow and survive during iron stress and least under cadmium and nickel. under extreme environment conditions. In order to iso- The protein content observed can be correlated with the late microbes with multiple heavy metal tolerant capa- growth rate of Cellulosimicrobium sp. indicating that bility, it is important to choose the sites or locations that the strain tried to express various transporters or allows or promote the growth of strains of our interest. enzymes for its survival. Most of the earlier studies are Therefore, in the present study, we have isolated the limited to adaptability of the Cellulosimicrobium sp. in

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chromium and thorium contaminated environment [25, 5. Lima e Silva AAD, Carvalho MA, de Souza SA, Dias PMT, Silva Filho 30]. Various heavy metals can alleviate or suppress the RGD, Saramago CS, et al. 2012. Heavy metal tolerance (Cr, Ag and microbial activity under the mixture conditions. Some Hg) in bacteria isolated from sewage. Braz. J. Microbiol. 43: 1620- 1631. may have a synergistic and some may have an antago- 6. Bruins MR, Kapil S, Oehme FW. 2000. Microbial resistance to met- nistic effect on the overall growth and sustainability of als in the environment. Ecotoxicol. Environ. Saf. 45: 198-207. the organism [36−38]. The present study showed that 7. Valko M, Morris H, Cronin M. 2005. Metals, toxicity and oxidative the bacteria strain is found to be resistant towards mul- stress. Curr. Med. Chem. 12: 1161-1208. tiple heavy metals which include lead, iron, copper, zinc, 8. Jaishankar M, Tseten T, Anbalagan N, Mathew BB, Beeregowda nickel and cadmium. 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Kang CH, Kwon YJ, So JS. 2016. Bioremediation of heavy metals Acknowledgments by using bacterial mixtures. Ecol. Eng. 89: 64-69. 16. Sehgal M, Garg A, Suresh R, Dagar P. 2012. Heavy metal contami- This investigation has been carried out under the Faculty Research nation in the Delhi segment of Yamuna basin. Environ. Monit. Grant Scheme awarded to RSP (Grant No. GGSIPU/DRC/FRGS/2018/ Assess. 184: 1181-1196. 26[1114]L) from GGS Indraprastha University, New Delhi, India. We 17. Volpicella M, Leoni C, Manzari C, Chiara M, Picardi E, Piancone E, also thank GGS Indraprastha University, New Delhi for all the labora- et al. 2017. Transcriptomic analysis of nickel exposure in Sphingo- tory space and encouragement. bium sp. ba1 cells using RNA-seq. Sci. Rep. 7: 8262. 18. Clarridge JE. 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infec- Conflict of Interest tious diseases. Clin. Microbiol. Rev. 17: 840-862. 19. 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June 2019 | Vol. 47 | No. 2