Indian Journal of Biotechnology Vol 16, July 2017, pp 380-386

Biophysical remediation of vanadium by sp. E1

Eman A H Mohamed Botany Department, Faculty of Science, Damanhour University, Damanhour, Egypt, Biotechnology Department, Faculty of Science, Taif University, Taif, Saudi Arabia

Received 5 January 2015; revised 10 December 2015; accepted 26 January 2016

Removal of vanadium toxic levels from industrial wastewater is an urgent demand before releasing to the environment. In this study , Shewanella sp. strain E1, an isolate of industrial wastewater body, Riyadh city, KSA, was subjected to molecular identification using partial sequencing of the 16S rDNA and gyrB genes. This bacterium is resistant to vanadium pentoxide up to 15 mg/l and more. The Plackett-Burman statistical design was used for near optimum vanadium biophysical removal conditions and this target was achieved giving that the positive levels of vanadium concentration (a significant factor), pH and cell weight, and the negative levels of solution volume (a significant factor), rotation rate, contact time, and temperature. Protein electrophoresis of active cells in the presence of 15 mg/l vanadium pentoxide showed at least 16 different intracellular proteins. The study suggest that Shewanella sp. E1 can be used for vanadium removal from industrial wastewater to increase its water quality before release to the environment.

Keywords: Vanadium biophysical removal Plackett-Burman design, 16S rDNA, gyrB , Shewanella sp. E1,

Introduction mild induction of some stress-associated genes 6. Although its extensive use in various heavy However, at high concentrations some of these metal industries, the incidence of workers exposure to toxic ions elicit a distinctively different pattern 24, 30 . levels of vanadium increased the concern of Plackett-Burman design 28 comprises one type of toxicologists. The clinical picture of poisoning shows two-level screening design. It is favored to detect the a broad spectrum of toxic effects of vanadium on the significant factors affecting the process before respiratory, circulatory, and central nervous systems, proceeding to the optimization stage of experimental the digestive organs, kidneys and skin 32 . Despite all design 1. In this study, vanadium bioremediation was of its negative environmental effects and its evaluated and near optimized throughout the prevalence as a contaminant, the potential for application of Placket-Burman statistical design using vanadium bioremediation has not been widely studied 34 . cells of the recently isolated Shewanella sp that was However, bacterium Shewanella in the sole genes identified molecularly by the 16S rDNA and gyrB belongs to Shewanella family has attached much sequencing. Besides, total stress proteins of vanadium attention because of its ability to reduce metal ions treated cells have been also examined. including chromium, vanadium, and others 9, 13, 26 Therefore, Shewanella has drawn much attention in Materials and Methods bioremediation, circulation of minerals and Source of Sample and Water Chemical Analysis bioelectricity 14, 22 . The industrial waste water used in this study was Stress studies have focused exclusively on collected in February - 2014 from Wady Haneefa lake, Shewanella oneidensis , the first genome of the Riyadh city, Kingdom of Sandi Arabia (KSA). Shewanellae to be sequenced 17 . An array of assays were isolated, purified and maintained in glycerol. has been carried out to decipher the ability of S. Vanadium analysis of the water sample was performed oneidensis to respond to and survive external stresses. at Soil, Water and Environment Research Institution At low levels, metal ions such as vanadium are taken (SWERI), Giza, Egypt Agriculture Research Centre. as electron acceptors by cells of S. oneidensis causing Cell Morphology and Antibiotic Sensitivity Test ______Routine Gram stain reaction was performed and *Author for correspondence [email protected], [email protected] cells were examined using bright field microscopy at Tel- 00201027737785 100 X magnification. Sensitivity of the isolate toward MOHAMED : BIOPHYSICAL REMEDIATION OF VANADIUM BY SHEWANELLA 381

some commercially available antibiotic discs was Scanning Electron Microscopy (SEM) tested in Muller Hinton agar plates. The plates were Cell morphology was detected using scanning incubated at 30 Cͦ for 24h. The antibiotics were as electron microscopy (SEM) (Electron Microscope follows ( µg): Ampicillin (20), Penicillin G (10), Unit, Taif University, KSA). Cell suspension at the Cephalothin (30), Amoxycillin (10), and basal conditions was centrifuged and harvested cells Sulphamethoxazole (25). were examined using SEM.

Application of the Plackett-Burman Design for Near Optimum Bacterial Resistance to Vanadium Conditions of Vanadium Bioremediation in Aqueous Solutions The pure bacterial cells were cultured in nutrient agar Plackett-Burman experimental design 28 , a plates supplemented with different concentrations of fractional factorial design, was carried out to vanadium pentoxide, 2 to 15 mg/l. The plates were determine the near-optimum vanadium incubated at 30 ͦ C for 24 h. bioremediation capacity by Shewanella sp. E1 cells. Seven independent variables were screened in eight 16S rDNA and gyrB Sequencing different combinations were organized (Table 2). DNA was extracted using Insta Gene Matrix For each variable, a high (+1) and low (−1) level (BIO-RAD). Amplification of the 16S rDNA and were tested. All trials were performed in triplicates gyrB genes was done using 20 ng DNA in 30 µl and the average of vanadium bioremediation results reaction mixture using EF-Taq (SolGent, Korea) as were treated as responses. The batch experiments follows for the former gene: activation of Taq were carried out under aerobic conditions. The polymerase at 95 ͦ C for 2 min, 35 cycles of 95 ͦ C main effect of each variable was simply calculated for 1 min, 55 ͦ C and 72 ͦ C for 1 min, and 10 min as the difference between the average of step at 72 ͦ C. Amplification of gyrB was performed measurements made at the high setting (+) and the according to the following program: activation of average of measurements observed at the low Taq polymerase at 94 ͦ C for 5 min, 35 cycles of 94 ͦ setting (−) of that factor according to the following C for 1 min, 58 ͦ C for 30 sec and 72 ͦ C for 2.5 min, equation: Exi = (ΣMi+ − ΣMi−)/ N, where Exi is the and 7 min step at 72 ͦ C. Amplicons were purified variable main effect, Mi+ and Mi− are the response using multi screen filter plate (Millipore Corp., values in trials, in which the independent variable Bedford, MA, USA). Sequencing was performed at (xi) was present in high and low concentrations, Macrogene Incorporation, Seoul, Korea using respectively, and N is the half number of trials.

PRISM Big Dye Terminator V3. 1 cycle sequencing Bacterial cells obtained from nutrient broth cultures kit. Amplification products were analyzed by ABI grown at 30 ͦ C for 24h with agitation at 100 rpm were PRISM 3730XL DNA analyzer (Applied harvested by centrifugation. For basal conditions, Biosystems, Foster city, CA). The sequences were harvested cells were weighed and 0.1 gm wet cells compared with those in the GenBank data base were used for inoculation of 15 mg/l vanadium using BLAST search 4. The 16S rDNA and gyrB pentoxide solution. The total volume of the sequences were finally deposited in the GenBank vanadium solution was 20 ml in a 100 ml conical and the accession numbers were obtained. Forward flask and the pH was adjusted to 7. Cells were and reverse primers (Macrogene incorporation, suspended in vanadium pentoxide solution for 12 h at Seoul, Korea) used in PCR reactions and 30 ͦ C with agitation rate of 100 rpm. Finally, cells sequencing are illustrated in Table 1. were centrifuged and clear supernatant was isolated in

Table 1 — Forward and reverse primers used in PCR reactions and sequencing. Primer name Primer direction Primer sequence Purpose

For 16S rDNA

27F Forward 5`-AGAGTTTGATCMTGGCTCAG-3` For PCR 1492R Reverse 5`-TAGGGYTACCTTGTTACGACTT-3` 518F Forward 5`-CCAGCAGCCGCGGTAATACG-3` For 800R Reverse 5`-TACCAGGGTATCTAATCC-3` sequencing For gyrB gyrB sp-1 Forward 5`- TTCGTCGATTATTTGAACAGT-3` For PCR and gyrB sp-2r Reverse 5`-TTTTCCAGCAGATAATCGTTC-3` sequencing 382 INDIAN J BIOTECHNOL, JULY 2017

clean tubes for quantitative analysis of vanadium weight and vanadium concentration, and the lower using atomic absorption spectrometer (Conter levels of temperature, solution volume, rotation rate AA700 Graphite, France Analytik Jena AG). A and contact time (Fig. 3). Trial 1 has the highest control was performed using the same basal conditions vanadium removal percentage, while trial 2 has the with no cells. lowest one (Table 5). Main effect of vanadium bioremediation revealed that vanadium concentration Stress Proteins Electrophoresis has the largest positive effect, whereas solution For estimation of stress proteins in the presence of volume and agitation rate have are the largest in vanadium, cells were pre-cultured in nutrient broth over negative effect (Fig. 3). night at 30 ͦC. This pre-culture was used to inoculate fresh nutrient broth medium containing 15 mg/l vanadium pentoxide for 12 h at 30 ͦC. Cells were then separated from the supernatant by centrifugation and sonicated. Intra and extra cellular stress proteins were then electrophoresed. The same protocol was used for cells without vanadium addition. Protein electrophoresis was done according to Laemmli, 1970 20 . Fig. 1 — Gel electrophoresis of the 16S rDNA (a) and gyrB gene (b) from Shewanella sp. E1. M, genetic marker in bp, and gyr B, Results gene encoding the B subunit of the DNA gyrase, topoisomerase A recent industrial wastewater isolate, type II.

Shewanella sp. E1, was subjected to molecular Table 2. — Different levels of the seven independent identification by amplification (Fig.1) and partial variables used in the Plackett-Burman design. sequencing of the 16S rDNA gene. Sequences have Level submitted to the GenBank and the accession Factor Symbol -1 0 +1 number was given, KJ781361. Moreover, partial pH pH 6 7 9 sequencing of the amplified (Fig. 1) molecular Temperatur ( ͦ C) T 35 30 40 chronometer, gyrB gene, was used to confirm this Volume (ml) Vol 30 20 35 molecular identity. The sequences showed 99% Rotation (rpm) R 100 150 200 Contact time (h) CT 2 4 6 similarity with those of Shewanella sp. HRCR02 Cell wet weight (gm) CW 0. 1 0. 2 0. 3 from the GenBank database. However, the Vanidium concentration VC 10 15 20 submitted sequences have an accession number of (mg/l) LC004407. Note: −1, factors at their negative levels; 0, factors at their basal levels; and +1, factors at their positive levels.

The wastewater sample contains 0.32 mg/l Table 3 — Resistance of Shewanella sp. E1 to different vanadium which was chosen for the bioremediation concentrations of vanadium. experiment using Shewanella sp. E1. This Vanadium concentration Sensitive/Resistant bacterium is resistant to vanadium pentoxide from 2 (mg/l) to 15 mg/l (Table 3). Shewanella sp. E1 is also 2 Resistant resistant to ampicillin (20 µg) and penicillin (10 µg) 4 Resistant (Table 4). On the other hand, it showed 6 Resistant 8 Resistant sensitivity towards cephalothin (30 µg), amoxycillin 10 Resistant (10 µg), and sulphamethoxazole (25 µg). Gram stain 15 Resistant reaction showed red long rods (Table 4) and cell morphology was further confirmed by Table 4. — Gram stain and resistance to different antibiotics SEM (Fig. 2). by Shewanella sp. E1. Antibiotics discs ( µg) Sensitive/Resistant The Plackett-Burman design method was applied to Ampicillin (20) estimate the near optimum conditions for vanadium Penicillin G (10) Resistant remediation by Shewanella sp. E1 throughout 8 Cephalothin (30) different runs (Table 5). The predicted near optimum Amoxycillin (10) Sensitive Sulphamethoxazole (25) conditions are found to be the high levels of pH, cell Gram stain Gram- negative rods

MOHAMED : BIOPHYSICAL REMEDIATION OF VANADIUM BY SHEWANELLA 383

Table 5 — Bioremediation of vanadium pentoxide by Shewanella sp . E1 under Plackett-Burman statistical design

Variable Response

Trials pH T Vol R CT CW VC Remaining vanadium concentration % Vanadium remained (mg/l) 1 - - - + + + - 0. 779 5. 2±0. 01 2 + - - - - + + 1. 921 12. 9±0. 05 3 - + - - + - + 1. 548 10. 4±0. 04 4 + + - + - - - 1. 019 6. 8±0. 01 5 - - + + - - + 1. 162 7. 8±0. 02 6 + - + - + - - 0. 9009 6. 1±0. 02 7 - + + - - + - 0. 9102 6. 1±0. 03 8 + + + + + + + 1. 044 7. 0±0. 01 Basal 0 0 0 0 0 0 0 0. 7865 5. 28±0. 02 Control 0 0 0 0 0 Non 0 14. 884 99. 2±5 Note: Results are expressed as mean ± standard error (n = 3).

Fig. 2 — Cell morphology of Shewanella sp. strain E1 using electron scaning microscopy.

Fig. 4 — Electrophoresis of proteins in Shewanell sp. strain E1 cultures. M, protein marker in kDa; C, cells with no vanadium; S, cells supernatant with no vanadium; CV, cells under vanadium stress; and SV, supernatant of cells under vanadium stress. was electrophoresed on gel. Only seven and four proteins are produced intra and extra cellular, respectively in the absence of vanadium. On the other hand, at least 16 proteins are unique to be produced intracellular under vanadium stress. However, 4 of these stress proteins are also produced extracellular (~14, 15, 18, and 22 kDa). Fig. 3 — Main effects of vanadium bioremediation by Shewanella sp. E1 according to Plackett-Burman design. Discussion To investigate the global response of Shewanella Bacteria play a vital role in the removal of heavy sp. EA1 cells to vanadium stress, cell total protein metals from waste water 25 . The main advantage of 384 INDIAN J BIOTECHNOL, JULY 2017

bioremediation over conventional treatment methods 98 upon applying the Plackett-Burman statistical includes low cost, high efficiency and simplicity 15, 16, design. Al-Daghistani 2 has stated that comparison on 23, 29, 33. Shewanella sp. strain E1 cells applied in this using dead and live biomass for the adsorption of study were initially viable, but because they were showed that the dead cells exhibited transferred into aqueous solutions of vanadium higher adsorption potential for Cu, Ni, and Cr. Dead pentoxide, they may act like a trap for vanadium. cells do not depend on growth requirements or Most heavy metals are cations and this determines metabolic energy 27 . In addition, it often requires the their sorption to the negatively charged functional addition of nutrients and this will increase the groups that are present in surfaces which are generally biological and chemical demands of the anionic at a pH between 4 and 8 19 . In this study, treated water 2. My vision would be for the application Shewanella cells adsorbed vanadium with different of Shewanella sp. E1 described here in vanadium efficiencies according to the tested parameters. The bioremediation from wastewater and contaminated most significant parameters are vanadium effluents in order to improve their quality before concentration, solution volume and rotation rate. This release to the environment. conclusion is in agreement with that of Chubar et al. In addition to its high efficiency in vanadium 10 (2008) who stated that the efficiency with which biophysical removal from aqueous solutions, heavy metals can be removed by bacterial cells Shewanella sp. E1 living cells are highly resistant to depends on a variety of factors, including viability of vanadium pentoxide and can tolerate concentrations cells, contact time, pH, aqueous phase and higher than 15 mg/l. Majority of bacteria survived competition among solute species for binding sites. only 100 mM vanadium pentoxide 7. However, the Moreover, they stated that the use of live biomass pollution of the environment with heavy metals has adds further complexity because of the cell response led to the selection of heavy metal resistant to ambient conditions, including the presence of toxic microorganisms in the soil and water of industrial 12 compounds. In addition, Fourest and Roux regions. In many cases, resistance to heavy metals is concluded that the decrease in specific uptake of encoded on , which can be used for the heavy metals is due to the shortage of metal creation of novel microbial strains with high concentration in the solution. This finding explains detoxifying activity against heavy metals 3, 21 . why vanadium concentration is the most significant At least 16 proteins are unique to be produced by positive factor that affects vanadium removal Shewanella sp . E1 under vanadium stress in this efficiency by Shewanella sp. E1 cells. Although study. Yin and Gao (2011) 35 have studied the Plackett-Burman design is seeking for near optimum 1 response of Shewanella oneidensis to acute stresses responses , in this study vanadium bioremediation imposed by a variety of heavy metals. They have reached almost the optimum value (98%). This found that these metals share a common strategy: indicates the usefulness of such design in evaluating 11 survive first and then exert both general and specific the significance of some variables strongly . stress responses. Once the first line of defense is In a study of Kamika and Momba 18 , they stated that initiated, cells employ both general and specific stress Co, Ni and V were present in industrial wastewater at responses that are inseparable from each other to concentrations higher than the UN-FAO permissible recover from the crisis. Alternative sigma factors limits of 0.05, 0.2 and 0.1 mg/l, respectively 5. including RpoS , RpoH , and RpoE , along with stress- Furthermore, all concentrations in industrial response related genes are induced, leading to wastewaters exceeded the national standard limit of induction of a variety of detoxification, resistance, 0.05 mg/l 5. Therefore, scientists are interested in and transport functions. Such coordinated expression bioremediation as a safe solution to get rid of such of stress response and detoxification mechanisms in S. toxic metals. Some studies focused in heavy metal oneidensis may offer an advantage to thrive in anoxic reduction by Shewanella oneidensis 8-9 and others metal reducing conditions in aquatic sediment and concerned with remediation of Au, Co and Fe by submerged soil systems where substantial amounts of Shewanella putrefaciens 31 . In this study, Shewanella heavy metals can be generated 35 . In addition, Brown sp . E1 cells were used for vanadium biophysical et al. (2006) 8 have investigated chromate stress in S. removal instead of metabolism-dependent oneidensis and found that approximately 20% of the remediation and the bioremoval percentage reached genes up-regulated during chromate reduction encode

MOHAMED : BIOPHYSICAL REMEDIATION OF VANADIUM BY SHEWANELLA 385

putative cytochromes, cytochrome synthesis proteins, 12 Fourest E & Roux J C, Heavy metal adsorption by fungal or non-cytochrome consistent with cells being mycelial by-products: influence of pH, Appl Microbiol Biotechnol , 37 (1992) 399-403. potentially limited for under Cr(VI)-shocked 13 Fredrickson J K, Romine M F, Beliaev A S, Auchutung J M, conditions. The chromate shock response of S. Driscoll M E et al. , Towards environmental systems biology oneidensis requires a combination of different of Shewanella , Nature Rev Microbiol , 6 (2008) 592-603. regulatory networks that involve genes with annotated 14 Fredrickson J K & Zachara J M, Electron transfer at the functions in oxidative stress protection, detoxification, microbe-mineral interface: a grand challenge in biogeochemistry, Geobiology , 6 (2008) 245-253. protein stress protection, iron and sulfur acquisition, 15 Goyal N, Jain S C & Banerjee U C, Comparative studies on and DNA repair mechanisms. All of these stress the microbial adsorption of heavy metals, AdVol Environ mechanisms and their complicated pathways affect Res , 7 (2003) 311-319. the efficiency of heavy metal bioremoval and this 16 Hameed M S A, Continuous removal & recovery of by alginate beads free & alginate-immobilized Chlorella makes the maintenance of healthy microbial vulgaris , Afr J Biotechnol , 5 (2006) 1819-1823. population is difficult due to metal toxicity. 17 Heidelberg J F, Paulsen I T, Nelson K E, Gaidos E J, Nelson W C et al. , Genome sequence of the dissimilatory metal ion- References reducing bacterium Shewanella oneidensis , Nat Biotechnol , 1 Abdel-Fattah Y R & Hussein H M, Numerical modeling of 20 (2002) 1118-1123.

petroleum oil bioremediation by a local Penicillium isolate as 18 Kamika I & Momba M N B, Assessing the resistance & affected with culture conditions: application of Plackett- bioremediation ability of selected bacterial & protozoan Burman design, Arab J Biotech , 5 (2002) 165-172. species to heavy metals in metal-rich industrial wastewater, 2 Al-Daghistani H I, Bio-remediation of Cu, Ni & Cr from BMC Microbiol , 13 (2013) 28.

Rotogravure wastewater using immobilized, dead, & live 19 Kuyucak N & Volesky B, Desorption of cobalt-laden algal biomass of indigenous thermophilic Bacillus species, Int biosorbent, Biotechnol Bioeng , 33 (1988) 815-822.

J Microbiol , 10 (2012). 20 Laemmli U K, Cleavage of structural protein during the 3 Aleem A, Isar J & Malik A, Impact of long-term application assembly of the head of bacteriophage T4, Nature (London), of industrial wastewater on the emergence of resistance traits 227 (1970) 680-685.

in Azotobacter chroococcum isolated from rhizospheric soil, 21 Liamas I, Argandon M, Quesada E & del Moral A, Bioresour Technol , 86 (2003) 7-13. Transposon mutagenesis in Halomonas eurihalina , Res 4 Altschul S F, Madden T L, Schaeffer A A, Zhang J, Zhang Z Microbiol , 151 (2000) 13-18.

et al. , Gapped BLAST & PSI-BLAST: a new generation of 22 Lovley D R, The microbe electric: conversion of organic protein database search programs, Nucleic Acids Res , 25 matter to electricity, Curr Opin Biotechnol , 19 (2008) (1997) 3389-3402. 564-571.

5 Ayers O R S & Westcot D W, Water quality for agriculture: 23 Machado M D, Santos M S F, Gouveia C, Soares H M V M & FAO Irrigation & Drainage Food & Agriculture Soares E V, Removal of heavy metal using a brewer’s yeast Organisation, Paper 29 (1985) (http: //www. strain of Saccharomyces cerevisiae : the flocculation as a fao.org/docrep/003/T0234E/T0234E00. HTM). separation process, Bioresour Technol , 99 (2008) 2107-2115.

6 Beliaev A S, Klingeman D M, Klappenbach J A, Wu L, 24 Mugerfeld I, Law B A, Wickham G S, & Thompson D K, A Romine M F et al. , Global transcriptome analysis of putative azoreductase gene is involved in the Shewanella Shewanella oneidensis MR-1 exposed to different terminal oneidensis response to heavy metal stress, Appl Microbiol electron acceptors, J Bacteriol , 187 (2005) 7138-7145. Biotechnol , 82 (2009) 1131-1141.

7 Bell J M L, Philpa J C, Kuyukinab M S, Ivshinab I B, 25 Nanda M, Sharma D & Kumar A, Removal of heavy metals Dunbara S A et al. , Methods evaluating vanadium tolerance from industrial effluent using bacteria, Int J Environ Sci , 2 in bacteria isolated from crude oil contaminated land, (2011) 765-780.

J Microbiol Meth , 58 (2004) 87-100. 26 Nealson K H & Saffarini D, Iron and manganese in 8 Brown S D, Thompson M R, VerBerkmoes N C, Chourey K, : environmental significance, Shan M et al. , Molecular dynamics of the Shewanella physiology, and regulation, Ann Rev Microbiol , 48 (1994) oneidensis response to chromate stress, Mol Cell Proteomics , 311-343.

5 (2006) 1054-1071. 27 Pardo R, Herguedas M, Barrado E & Vega M, 9 Carpentier W, Sandra K, De Smet I, Brig´e A & De Smet L, Biosorption of cadmium, copper, lead and zinc by Microbial reduction & precipitation of vanadium by inactive biomass of Pseudomonas putida , Anal Bioanal Shewanella oneidensis , Appl Environ Microbiol , 69 (2003) Chem , 376 (2003) 26-32. 3636-3639. 28 Plackett R L & Burman J P, The design of optimum 10 Chubar N, Behrends T & Cappellen P V, Biosorption of multifactorial experiments, Biometrika , 33 (1944) 305-325. metals (Cu2+, Zn2+) & anions (F−, H2PO4−) by viable & 29 Tabak H H, Lens P, Hullebusch E D V & Dejonghe W, autoclaved cells of the Gram-negative bacterium Shewanella Developments in bioremediation of soil and sediments putrefaciens , Colloid Surface B , 65 (2008) 126-133. polluted with metals & radionuclides–1. Microbial processes 11 Dutta S & Singh P, The Plackett-Burman model-optimization of and mechanisms affecting bioremediation of metal significant nutritional parameters for petroleum bioremediation contamination and influencing meal toxicity & transport, Rev by Pseudomonas sp, Int J Adv Res , 2 (2014) 898-902. Environ Sci Biotechnol , 4 (2005) 115-156. 386 INDIAN J BIOTECHNOL, JULY 2017

30 Toes A-C M, Daleke M H, Kuenen J G & Muyzer G, 33 Wang J & Chen C, Biosorbents for heavy metals removal Expression of copA & cusA in Shewanella during copper and their future, Biotechnol Adv , 27 (2009) 195-226. stress, , 154 (2008) 2709-2718. 34 Yelton A P, Williams K H, Fournelle J, Wrighton K C, 31 Varia J, Zegeye A, Roy S, Yahaya S & Bull S, Shewanella Handley K M et al. , Vanadate & acetate biostimulation of putrefaciens for the remediation of Au3+, Co2+ & Fe3+ contaminated sediments decreases diversity, selects for metal ions from aqueous systems, Biochem Eng J, 85 (2014) specific taxa, and decreases aqueous V5+ concentration, 101-109. Environ Sci Technol , 47 (2013) 6500-6509. 32 Venkataraman B V & Sudha S, Vanadium toxicity, Asian J 35 Yin J & Gao H, Stress responses of Shewanella , Int J Exp Sci , 19 (2005) 127-134. Microbiol , 2011 (2011) 1-8.