arsenicresistanceandbiosorptionbyisolated...

Research ArticleArsenic Resistance and Biosorption by IsolatedRhizobacteria from the Roots of Ludwigia octovalvis

Harmin Sulistiyaning Titah 1 Siti Rozaimah Sheikh Abdullah2 Mushrifah Idris3

Nurina Anuar2 Hassan Basri4 Muhammad Mukhlisin5 Bieby Voijant Tangahu 1

Ipung Fitri Purwanti1 and Setyo Budi Kurniawan 1

1Department of Environmental Engineering Faculty of Civil Environmental and Geo EngineeringInstitut Teknologi Sepuluh Nopember (ITS) Kampus ITS Keputih Sukolilo 60111 Surabaya Indonesia2Department of Chemical and Prosess Engineering Faculty of Engineering and Built EnvironmentUniversiti Kebangsaan Malaysia 43600 UKM Bangi Selangor Malaysia3Tasik Chini Research Centre Faculty of Science and Technology Universiti Kebangsaan Malaysia43600 UKM Bangi Selangor Malaysia4Department of Civil and Structural Engineering Faculty of Engineering and Built EnvironmentUniversiti Kebangsaan Malaysia 43600 UKM Bangi Selangor Malaysia5Department of Civil Engineering Politeknik Negeri Semarang Semarang Indonesia

Correspondence should be addressed to Harmin Sulistiyaning Titah harminsulisgmailcom

Received 3 November 2018 Revised 23 November 2018 Accepted 4 December 2018 Published 30 December 2018

Academic Editor Todd R Callaway

Copyright copy 2018 Harmin Sulistiyaning Titah et al )is is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in anymedium provided the original work isproperly cited

Certain rhizobacteria can be applied to remove arsenic in the environment through bioremediation or phytoremediation)is studydetermines the minimum inhibitory concentration (MIC) of arsenic on identified rhizobacteria that were isolated from the roots ofLudwigia octovalvis (Jacq) Raven )e arsenic biosorption capability of the was also analyzed Among the 10 isolated rhizobacteriafive were Gram-positive (Arthrobacter globiformis Bacillus megaterium Bacillus cereus Bacillus pumilus and Staphylococcus lentus)and five were Gram-negative (Enterobacter asburiae Sphingomonas paucimobilis Pantoea spp Rhizobium rhizogenes and Rhi-zobium radiobacter) R radiobacter showed the highest MIC of gt1500mgL of arsenic All the rhizobacteria were capable ofabsorbing arsenic and S paucimobilis showed the highest arsenic biosorption capability (1464 plusmn 234mgg dry cell weight) Kineticrate analysis showed that B cereus followed the pore diffusion model (R2 086) E asburiae followed the pseudo-first-order kineticmodel (R2 099) and R rhizogenes followed the pseudo-second-order kinetic model (R2 093) )e identified rhizobacteria differin their mechanism of arsenic biosorption arsenic biosorption capability and kinetic models in arsenic biosorption

1 Introduction

Currently most research on phytoremediation has emphasizedon the physiological mechanisms of plant transport and tol-erance as well as the plantrsquos storage ofmetal A few informationis available on processes in the hyperaccumulator plant phy-toextraction of metals [1] Rhizobacteria are usually present insoils naturally despite having high amounts of metals

Many bacterial strains contain the genetic determinantsof resistance to heavy metals such as arsenic bismuthcadmium chromium lead mercury nickel silver and

others [2] )e bioavailable and extractable forms of arsenicsignificantly inhibit the soil microbial community althoughbioavailable arsenic exhibits better inhibitory effect thantotal arsenic [3] Some bacteria are resistant to arsenic due totheir ability to remove it from their surroundings [4] ManyGram-negative and Gram-positive bacteria have de-toxification mechanisms [5] Bacteria have various ways ofcoping with high levels of arsenic including reduced uptakemethylation following the reduction of arsenate to arsenitethe adsorption of negatively charged arsenic ions by theoppositely charged amino groups in bacterial cell walls

HindawiInternational Journal of MicrobiologyVolume 2018 Article ID 3101498 10 pageshttpsdoiorg10115520183101498

sequestration by a range of cysteine-rich peptides chelationcompartmentalisation exclusion immobilization and dis-similatory arsenate respiration [5 6] Although arsenic istoxic to many bacteria metal-accumulating bacteria areoften found among metal-resistant bacteria [4] Nanda andAbraham [2] found that the toxicity of arsenic is higher thanchromium magnesium and copper (As gt Cr gt Mg gt Cu)Arsenate is toxic to bacteria because it is analogous tophosphate and can inhibit enzymes such as kinases [7]

Rhizobacteria that are capable of aggressively colonisingplant roots and promoting plant growth are generally knownas plant growth-promoting rhizobacteria (PGPR) [8] PGPRcan invade the interior of the cells and survive inside (in-tracellular PGPR such as nodule bacteria) or remain out-side the plant cells (extracellular PGPR such as BacillusPseudomonas and Azotobacter) PGPR such as Agro-bacterium (Rhizobium) Alcaligenes (Ralstonia) Arthro-bacter Azospirillum Azotobacter Bacillus BurkholderiaSerratia and Pseudomonas are interesting organisms for themetal extraction by plants because they increase the rate ofboth metal and biomass accumulation by plants [9] Con-taminated soils are often poor in nutrients or sometimesnutrient-deficient due to the loss of useful microbesHowever such soils can be made nutrient-rich by applyingmetal-tolerant microbes especially PGPR which not onlyprovide essential nutrients to the plants properties allowplants to remove heavy metals which can then be useful inagricultural production or the phytoremediation of con-taminated soil [11 12] )erefore it is advisable for growersto inoculate plants with growing in the contaminated sitesbut also play a major role in detoxifying heavy metals [8 10])ese rhizobacterial microbes to increase plant biomass andstabilise revegetate and restore heavy metal-contaminatedsoils [9]

)e interactions of rhizobacteria and plants in reme-diating arsenic have been studied According to Titah et al[13] the application of a six-rhizobacterial consortium al-leviated the toxic effects of arsenic in Ludwigia octovalvis andincreased the plant biomass According to Nie et al [14]canola plants inoculated with Enterobacter cloacae havegrown to a significantly higher extent than noninoculatedcanola plants due to the presence of arsenic Glick et al [15]reported the increase in shoot biomass and arsenic con-centration in the shoots of Helianthus annuus after in-oculation with Pseudomonas fluorescens

L octovalvis is a plant species that can survive at a crudeoil-contaminated site [16] and could uptake and accumulatearsenic in its tissues [17] )is study aimed to determine thelevel of arsenic (as arsenate) resistance of the identifiedrhizobacteria that were isolated from the roots of L octo-valvis grown in the greenhouse and contaminated land inMalacca Malaysia It also aimed at determining their arsenicbiosorption capability

2 Materials and Methods

21 Epiphyte Rhizobacterial Isolation from the Root of Loctovalvis )e detailed steps of rhizobacterial isolation wasdescribed by Titah et al [18]

22 Identification of Rhizobacteria Rhizobacterial identifi-cation was conducted using two biochemical methodsBiolog GEN III microbial identification system (Biolog IncUSA) and Vitek2 Compact System (Biomerieux USA) )edetailed steps of rhizobacteria identification was describedby Titah et al [18]

23 Determination of theMinimum Inhibitory Concentration(MIC) ofArsenic )e test was conducted using the modifiedmethod of Guo et al [19] )e MIC of arsenate for eachrhizobacterium species was determined in three replicates ofsterile phosphate buffer saline (PBS) solution with a serialconcentration of 0 (control) 250 500 750 and 1000mgLof arsenic in arsenate form (sodium arsenate dibasic hep-tahydrate (AsHNa2O4middot7H2O)) (FlukaChemika Switzer-land) Two different controls were used for this method aPBS solution with bacteria but without arsenate (negativecontrol) and another PBS solution with arsenate but withoutbacteria (positive control) Based on the study of Harley andPrescott [20] 10 times PBS stock solution contained 80 g NaCl(Merck Germany) 2 g KCl (RampM Chemicals India) 11 gNa2HPO4middot7H2O (RampM Chemicals India) and 2 g KH2PO4(RampM Chemicals India) MIC determination was carriedout in a 250mL Erlenmeyer flask with 50mL workingvolume )e pH of arsenate in sterile PBS solution wasmeasured using Accument Basic AB 15 pH meter (FisherScientific USA) )e average pH readings were 73 74 7576 and 77 in the arsenate concentrations of 0 250 500 750and 1000mgL respectively )e cultures were incubatedusing an incubator shaker (Protech Model SI-100DMalaysia) at 37degC and 150 rpm Bacterial growth was de-termined at an absorbance of 550 nm for 0 3 6 19 and 24 h

24 Arsenic Biosorption Experiment Using Identified Rhizo-bacteria inBatch System An arsenic biosorption experimentwas conducted using a modified published method [21ndash23]Selected rhizobacteria were inoculated onto Trypticase(Tryptic) soy agar (TSA) media without arsenate for 24 hAfter that the selected rhizobacteria were inoculated intosterile Tryptic soy broth (TSB) )e cell suspension of theselected rhizobacteria was prepared by harvesting the cells atthe middle of the logarithmic phase based on the typical ofgrowth rate graph for the selected rhizobacteria At this timethe optical density (OD) at 550 nm was 10 )e cells wereharvested through centrifugation (Eppendorf centrifuge5804 Germany) at 4000 rpm for 15min)e obtained pelletwas then washed twice by using 85 g NaCl1000mLsolution

)e biosorption of arsenic was then tested in a 250mLErlenmeyer flask )e rhizobacterial cell suspension at 20(vv) or 10mg dry cell weight (DCW) was added with 50mLof sterile PBS solution which had an arsenic concentrationof 100mgL by diluting sodium arsenate dibasic heptahy-drate (AsHNa2O4middot7H20) (Fluka Chemika Switzerland) )ecolonyforming unit (CFU) of the initial rhizobacteria wasapproximately 14 times 109 CFUmL A pH of 73 was measuredfor 100mgL arsenate in sterile PBS solution by usingAccument Basic AB 15 pH meter (Fisher Scientific USA)

2 International Journal of Microbiology

All samples were tested in triplicates All the cultureswere incubated using an incubator shaker (Protech ModelSI-100D Malaysia) at 37degC and 150 rpm Samples wereharvested at 0 2 6 17 and 24 h )e OD was measured at550 nm at each sampling by using Genesys 10 UV ()ermoSpectronic USA) Finally these rhizobacterial suspensionswere serially diluted and 01mL of this sample was spreadonto TSA )e number of colonies grown was counted inCFUmL )e dry weight of the rhizobacteria cells wasdetermined by filtering the suspension culture through avacuum filter by using 02 μm membrane filter paper(Whatman Germany) )e DCW was determined afterovernight drying at 105degC [24]

Ten mL of each culture was taken at each sampling timeand the supernatant and pellet were separated throughcentrifugation (Eppendorf Centrifuge 5804 Germany) at10000 rpm for 15min )e residual amount of the totalarsenic present in the supernatant was stored at minus80degC [6]before inductively coupled plasma-optical emission spec-trometer (ICPndashOES) analysis by using Optima 7300DVPerkinElmer (USA) for the total arsenic )e cell pellet wasdried at room temperature for 2 days [25] )en 1mL of69 HNO3 1mL of 30 H2O2 and 3mL of sterile purewater were added to the cell pellet for 24 h )e cell sampleswere stored at minus20degC [24] and the arsenic concentration ofthe cells was determined using ICPndashOES Optima 7300DVPerkinElmer (USA)

25 Stastitical Analysis All statistical tests were performedusing SPSS Version 170 (IBM USA) Two-way analysis ofvariance (ANOVA) at 95 confidence level (plt 005) wasused to evaluate significant changes in the total arsenicconcentration in the supernatant and the biosorption ca-pability of the rhizobacteria Correlation analysis was per-formed to determine the relationship between theconcentration of arsenic in the supernatant and the capa-bility of arsenic biosorption by rhizobacteria by using thePearson correlation

26 Transmission Electron Microscopy (TEM) AnalysisTEM images of selected rhizobacterium which had thehighest biosorption capability were obtained after exposureto arsenate for 48 h )e arsenic concentration was setaccording to the results of the MIC experiment TEM wasperformed to determine the crosswise and longitudinalstructure of the cell after exposure to arsenic compared withthe unexposed cells Objective analysis of spectral elementswas conducted to determine the arsenic content in the cellsof the rhizobacterium which was conducted using a TEMequipment model Philips CM 12 (Netherlands)

27 Biosorption Kinetic Models In the batch experimentskinetic studies were performed to determine the contacttime of the adsorbent with the adsorbate and evaluate thereaction coefficients To analyze the uptake kinetics manymodels such as pseudo-first-order pseudo-second-orderpore diffusion and Elovich equation can be used )e

pseudo-first-order equation of Lagergren is based on thesolid capacity [26] whereas the pseudo-second-order re-action model is based on solid-phase adsorption and impliesthat chemisorption is the rate-controlling step [27])e porediffusion model is based on intraparticle diffusion processes[28] whereas the Elovich equation model is based on thebest fit adsorption mechanisms

271 Pseudo-First-Order Model )e pseudo-first-orderequation is generally expressed as follows

dqt

qt

k1 qe minus qt( 1113857 (1)

where qe and qt are the adsorption capacities at equilibriumand at time t (mgg biomass) and k1 is the rate constant ofthe pseudo-first-order adsorption (h) A linear form of thepseudo-first-order model was described by Lagergren Byintegrating the equation and applying boundary conditionsof t 0 to t t and qe 0 to qt q at t it becomes

log qe minus qt( 1113857 log qe( 1113857minusk1

2303t (2)

A plot of log(qe minus qt) against t gives minus(k12303) as theslope and log(qe) as the intercept

272 Pseudo-Second-Order Model )e pseudo-second-order model is used to describe the sorption kinetics [27])e model assumes that the rate-limiting step may bechemical sorption (or chemisorption) involving valenceforces through sharing or the exchange of electrons betweenthe sorbent and sorbate [29] )is model is represented bythe following equation [30]

qdqt

dt

k2 qeq minus qt1113872 11138732 (3)

where k2 is the rate constant of pseudo-second-order ad-sorption (mgg biomassh) By applying the boundaryconditions t 0 to t t and qe 0 to qt q at t the in-tegrated form of the equation becomes

1qeq minus qt1113872 1113873

1

qeq+ k2t (4)

which is the integrated rate law for a pseudo-second-orderreaction which can be rearranged to obtain the followinglinear form

t

qt

1

k2q2eq1113872 1113873

+1

qeqt (5)

A plot of tqt versus t should provide a linear re-lationship for the applicability of the second-order kinetics)e rate constant (k2) and adsorption at equilibrium (qeq)can be obtained from the intercept and slope respectively

273 Pore Diffusion Order Model In most adsorptionprocesses the uptake varies almost proportionally with t12

International Journal of Microbiology 3

qt Kdt12

(6)

where Kd (mggmin12) is the diffusion rate constant and qt

is the amount of adsorbate adsorbed at t A plot of qt againstt12 yields a slope of Kd )e WeberndashMorris plot versus t12

should be a straight line with a slope Kd and intercept of Cwhen adsorption mechanisms follow the intraparticle dif-fusion processes [31] According to Weber and Morris [28]q sorption processes will be controlled by the slowest of therate-limiting steps

274 Elovich Equation Model )e Elovich equation hasbeen commonly used in determining the kinetics ofchemisorption on gases and solids [29] However someresearchers have applied this model to solid-liquid sorptionsystems especially in the sorption of heavy metals [32 33])e Elovich equation is as follows

qt 1β

1113888 1113889ln(αβ) +1β

1113888 1113889ln t (7)

where qt is the amount of adsorbate adsorbed per unit t andα and β are the Elovich constants β is the initial adsorptionrate of the Elovich equation (mggmiddoth) and α is related to theextent of surface coverage (mgg) and activation energy forchemisorption [34] A plot of qt against ln t yields (1β) asthe slope and (1β) ln(αβ) as the intercept

3 Results and Discussion

31 MIC Ten rhizobacteria from the roots of L octovalvistested for their resistance to arsenate include Arthrobacterglobiformis Bacillus megaterium Bacillus cereus Bacilluspumilus Staphylococcus lentus Enterobacter asburiaeSphingomonas paucimobilis Pantoea spp Rhizobiumrhizogenes and Rhizobium radiobacter Table 1 shows thesummary of the MIC of arsenate for all rhizobacteria Rradiobacter had the highest MIC (gt1500mgL) indicatingthat it was the most resistant rhizobacterium to arsenic (inthe arsenate form) R rhizogenes had an MIC of arsenate of750mgL )e two Rhizobium species were isolated fromthe sand spiked with the highest arsenate concentration(80mgkg) [35] )e growth of R rhizogenes and Sphin-gomonas paucimobilis was inhibited at an arsenate levelof 750mgL )e MIC of arsenate of A globiformisB pumilus Staphylococcus lentus and E Asburiae was500mgL while that of B megaterium B cereus and Pantoeaspp was 250mgL

)e arsenate resistance of A globiformis was higher thanB megaterium and B cereus which agree with the findings ofAchour et al [36] According to Achour et al [36] the MICof arsenate to Arthrobacter sp on a solid medium (LuriandashBertani (LB) agar) was 160mM whereas that of Bacillus spwas 50mM Another study reported the MIC of arsenate forArthrobacter sp and Bacillus sp on LB agar was above75mM [37]

)eMIC for arsenate of Gram-positive rhizobacteria waslower than that of Gram-negative rhizobacteria )is findingindicates that the Gram-negative rhizobacteria are more

inhibited by arsenate or are less resistant to arsenate thanGram-positive rhizobacteria )is condition may be causedby the differences in the cell wall structure between Gram-negative and Gram-positive rhizobacteria )e cell walls ofGram-negative rhizobacteria are more complex than thoseof Gram-positive rhizobacteria )e cell walls of Gram-negative rhizobacteria had outer layers of membrane(OM) which selectively protects rhizobacteria from harmfulsubstances such as antibiotics heavy metals and other toxicchemicals [38] OM contains protein fat and lipopolysac-charide A study reported by Anyanwu and Ugwu [39]shows that 67 of the 12 species were Gram-negativebacteria that were more resistant to arsenic exposure thanGram-positive bacteria

32 Biosorption Capability of the Identified RhizobacteriaFigure 1 shows the concentrations of total arsenic in thesupernatant throughout the biosorption test)e total arsenicconcentrations decreased within the first 2 h increased in Aglobiformis B megaterium and B cereus for 17 h and de-creased in the supernatant of the three rhizobacteria at the endof the exposure (24 h) )e increase in total arsenic con-centrations in the supernatant might be due to the conversionof arsenate to arsenite by the rhizobacteria which is laterpumped out of the cell [5] Botes et al [6] reported that hyper-resistant bacteria can take up 50ndash100 of arsenate andexport approximately 15ndash25 as arsenite

B pumilus showed a different trend Its total arsenicconcentration in the supernatant increased up to 17 h andthen decreased until the end of the exposure Staphylococcuslentus also showed a different trend with increasing anddecreasing arsenic concentration in the supernatant from 2 hto 24 h In E asburiae and Pantoea spp the total arsenicconcentration in the supernatant increased up to 6 h andthen decreased)e arsenic concentration in the supernatantfor Sphingomonas paucimobilis had the highest decrease at2 h whereas those for R rhizogenes and R radiobacter in-creased until the end of the exposure (24 hour) )e arsenicremoval after 24 h of the rhizobacteria can be arranged asfollows Sphingomonas paucimobilis gt B pumilus gt Bmegaterium gt B cereus gt A globiformis gt Staphylococcuslentus gt R radiobacter gt E asburiae gt Pantoea spp gt Rrhizogenes

Arsenate enters the bacterial cell wall through a fastunspecific and constitutive uptake system for phosphate

Table 1 Summary of MIC on arsenate

No Rhizobacteria Gram stain MIC (mgL)1 B megaterium + 2502 B cereus + 2503 B pumilus + 5004 A globiformis + 5005 Staphylococcus lentus + 5006 Pantoea spp minus 2507 E asburiae minus 5008 Sphingomonas paucimobilis minus 7509 R rhizogenes minus 75010 R radiobacter minus gt1500


0

20

40

60

80

100

120

140

160

A gl

obifo

rmis

B m

egat

eriu

m

B ce

reus

B p

umilu

s

Stap

hylo

cocc

uslen

tus

E a

sbur

iae

Sphi

ngom

onas

pauc

imob

ilis

Pant

oea

spp

R rh

izog

enes

R ra

diob

acte

r

Conc

entr

atio

n of

tota

l ars

enic

in

supe

rnat

ant (

mg

L)

0h

24h17h6h2h

Figure 1 Concentration of arsenic in the supernatant throughout the 24 h of exposure with individual rhizobacteria Vertical bars indicateSD of triplicates

0

20

40

60

80

100

120

140

160

180

A gl

obifo

rmis

B m

egat

eriu

m

B ce

reus

B p

umilu

s

Stap

hylo

cocc

us le

ntus

E a

sbur

iae

Sphi

ngom

onas

pauc

imob

ilis

Pant

oea

spp

R rh

izog

enes

R ra

diob

acte

r

Ars

enic

bio

sorp

tion

in ce

ll pe

llet

(mg

g D

CW)

0h

2h

6h

17h24h

Figure 2 Arsenic biosorption capability of A globiformis B megaterium B cereus B pumilus Staphylococcus lentus E asburiaeSphingomonas paucimobilis Pantoea spp R rhizogenes and R radiobacter Vertical bars indicate SD of the triplicates


[40] Arsenate detoxification involves its reduction to ar-senite by arsenate reductase prior to its efflux through amembrane potential driven pump controlled by trans-actingrepressor (ArsR) )e ArsR protein contains a very specificbinding site towards arsenite and can discriminate effectivelyagainst phosphate sulphate cobalt and cadmium AlthoughArsR is specific for arsenite the removal of arsenate mayoccur through the initial conversion of arsenate into arseniteby arsenate reductase and subsequent sequestration byArsR [40]

)e results of ANOVA show that the total arsenicconcentration in the supernatant was significantly differentdepending on the rhizobacteria species and the exposuretime (plt 005) )e total arsenic concentration in the su-pernatant for Sphingomonas paucimobilis was significantlydifferent (plt 005) compared with those of the other ninerhizobacteria at 0 h However at 24 h the total arsenicconcentration in the supernatant for Sphingomonas pauci-mobiliswas significantly different (plt 005) than those of theseven other rhizobacteria but was not different (pgt 005)from R rhizogenes and R radiobacter

Figure 2 shows that each rhizobacterium species hadtotal arsenic concentrations at different cells indicating thatthe arsenic biosorption capabilities of each species differSphingomonas paucimobilis had the highest arsenic bio-sorption (1464 plusmn 234mgg DCW) at 2 h )e maximumarsenic biosorption of B cereus B megaterium and Bpumilus occurred at 24 h with 168 plusmn 42 154 plusmn 31 and 94 plusmn06mgg DCW respectively According to Shakya et al [41]

the accumulation of arsenic by B cereus decreased from 24to 96 h of exposure Another study reported that the max-imum arsenic accumulation of Bacillus spp strain DJ-1which was observed during the stationary phase of growthwas 98 plusmn 05mgg DCW [42] )e arsenic biosorption of Aglobiformis at 24 h was 322 plusmn 50mgg DCW )e arsenicbiosorption of Staphylococcus lentus increased up to 6 h (192plusmn 28mgg DCW) and then decreased up to 24 h (28 plusmn06mgg DCW) )e arsenic biosorption of Pantoea sppdecreased up to 6 h (20 plusmn 02mgg DCW) increased andthen decreased to 24 h (48 plusmn 03mgg cell dry weight) )earsenic biosorption of E asburiae increased up to 17 h (135plusmn 19mgg DCW) and then decreased at 24 h (87 plusmn 37mggDCW) )e arsenic biosorption of R rhizogenes and Rradiobacter declined with arsenic levels of 112 plusmn 01 to 39 plusmn01 and 259 plusmn 04 to 43 plusmn 09mgg DCW respectively )eaverage arsenic biosorption capability after 24 h of therhizobacteria can be arranged as follows Sphingomonaspaucimobilis gt A globiformis gt R radiobacter gt B pumilus gtStaphylococcus lentus gt B cereus gt Pantoea spp gt R rhi-zogenes gt E asburiae gt B megaterium

ANOVA shows that the arsenic biosorption capabilitiesdiffered significantly among rhizobacteria species (plt 005)However the exposure time did not significantly differ(pgt 005) )e LSD analysis on the capability of arsenicbiosorption indicates that the arsenic biosorption capacity ofSphingomonas paucimobilis and A globiformis was signifi-cantly different (plt 005) compared with the other eightrhizobacteria

(a) (b)

K

R BIM

OD

(c)

M

OM

CD

(d)

Figure 3 TEM analysis (a) control 10000times (b) 750mgL arsenate 10000times (c) control 13000times and (d) 750mgL arsenate 17000times


Correlation analysis was conducted to determine therelationship between the concentration of arsenic in thesupernatant and the arsenic biosorption ability of the rhi-zobacteria was tested using the Pearson correlation Resultsshow a correlation coefficient of minus03 at the 001 confidencelevel )e negative value indicates an inverse correlationrelationship As the concentration of arsenic in the super-natant decreased the ability of rhizobacteria to take uparsenic increased

33 TEM Analysis )e TEM analysis shows a significantdifference in Sphingomonas paucimobilis cells exposed toan arsenate concentration of 750mgL and the unexposed(control) cells Figure 3 shows the results of the TEManalysis of Sphingomonas paucimobilis cells with elongated(Figures 3(a) and 3(b)) and transverse pieces (Figures 3(c)and 3(d)) No damage was observed to the unexposed cellsof Sphingomonas paucimobilis Figure 3(c) shows that thecell wall (D) OM boasts a plasma cell (M) cell cytoplasmand its interior ribosomes (R) chromosomes (K) andinclusion bodies (BI) in the control treatment Cells ex-posed to arsenic (in arsenate form AsO4

minus3 or As(V)) hadthickened and wrinkled both the cell wall and the cellplasma membrane Sphingomonas paucimobilis cellsgrown at an arsenate concentration of 750mgL weresmaller than those grown under the control medium(Figures 3(a) and 3(b)))e presence of a capsule (C) couldbe associated with a method of cell protection againstarsenate exposure

34 Biosorption Kinetic Model for Arsenic For the bio-sorption kinetic studies various kinetic equations weretested on the data to determine their fitness Table 2 showsthe summary of the four kinetic models for ten identifiedrhizobacteria

Based on Figure 4 the pseudo-first-order model ofLagergren for arsenic biosorption of E asburiae and B cereusshows a high correlation coefficient (R2) )e plot for both

biosorbents resulted in an R2 of 082 (B cereus) and 091 (Easburiae) )is finding indicates that the pseudo-first-ordermodel of Lagergren best explains the arsenic biosorption of Easburiae and B cereus )e qe and k1 obtained from the

Table 2 Kinetic constants for arsenic biosorption by rhizobacteria

Rhizobacteria

Pseudo-first-orderkinetic model

Pseudo-second-order kineticmodel Pore diffusion model Elovich equation

k1(h)

qe (mg Asgbiomass) R2 k2 (mg Asg

biomassh)qe (mg Asgbiomass) R2 kd (mg Asg

biomassh12) R2 A (mg Asgbiomass)

β (mg Asgbiomassh) R2

B cereus 003 1135 082 0003 2500 048 311 086 482 022 084B pumilus 002 1320 063 008 746 075 mdash mdash mdashB megaterium mdash mdash 009 219 072 182 034 262 040 031A globiformis 006 121 044 002 2326 068 mdash mdash mdashStaphylococcuslentus mdash mdash mdash mdash mdash mdash mdash

Sphingomonaspaucimobilis 001 2014 015 mdash mdash mdash mdash mdash

E asburiae 004 1268 099 001 1408 055 221 070 350 030 073Pantoea spp 001 1164 023 005 617 070 mdash mdash mdashR rhizogenes mdash mdash 035 602 093 mdash mdash mdashR radiobacter mdash mdash mdash mdash mdash mdash mdash

0

02

04

06

08

1

12

14

16

0 10 20 30

log

(qe

ndashq t

)

t

B cereusB pumilusA globiformis

Linear (B megaterium)y = ndash00103x + 1205R2 = 063Linear (A globiformis)y = ndash0026x + 1326R2 = 04372

Linear (Sphingomonaspaucimobilis)y = ndash00069x + 13041R2 = 0148

Sphingomonas paucimobilisE asburiaePantoea sppLinear (B megaterium)y = ndash00118x + 10556R2 = 08166

Linear (E asburiae)y = ndash00173x + 11039R2 = 09927Linear (Pantoea spp)y = ndash00069x + 10666R2 = 0231

Figure 4 Pseudo-first-order of Lagergren plot for arsenic bio-sorption by the rhizobacteria


intercept and slope of equations (2) were 1268mgAsgbiomass and 004h for E asburiae and 1135mgAsg bio-mass and 003h for B cereus )e other four rhizobacteriashowed low R2 values)e summary of the pseudo-first-orderof Lagergren kinetic model is listed in Table 2

Figure 5 demonstrates the pseudo-second-order plot forthe seven rhizobacteria )e R2 of R rhizogenes had thehighest value (093) indicating that the pseudo-second-ordermodel was favourable for the arsenic biosorption by R rhi-zogenes )e obtained qe was 602mgAsg biomass while theobtained k2 from the intercept was 05mgAsg biomassh(Table 2) )e other rhizobacteria showed low R2 values

)e kd obtained from the slope of equation (6) was311mgAsg biomassmin12 for B cereus (Figure 6) )epore diffusion model provided the highest R2 value forarsenic biosorption by B cereus at 086 It indicates that themolecular diffusion of arsenic by B cereus played an im-portant role in the uptake capacity of B cereus )e R2 of theElovich kinetic model for B cereus were much higher thanthe corresponding values of the other rhizobacteria(Figure 7)

)e kinetic model of B cereus could be calculated usingthe four models but R2 was highest under the pore diffusion

model Meanwhile the pseudo-first-order kinetic model forE asburiae had the highest R2 value )e pseudo-second-order kinetic model for R rhizogenes showed a higher R2

0

2

4

6

8

10

12

14

16

18

0 10 20 30

tqt

t

A globiformisE asburiaePantoea sppR rhizogenes

Linear (B cereus)y = 00407x + 05875R2 = 04812Linear (B pumilus)y = 01345x + 02214R2 = 07496Linear (B megaterium)y = 04561x + 22734R2 = 07246

B cereusB pumilusB megateriumLinear (A globiformis)y = 00431x + 00453R2 = 06774Linear (E asburiae)y = 00718x + 0741R2 = 05458Linear (Pantoea spp)y = 01629x + 04838R2 = 06971Linear (R rhizogenes)y = 01663x + 0078R2 = 09311

Figure 5 )e pseudo-second-order plot for arsenic biosorption bythe rhizobacteria

0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6

q t

t12

E asburiaeB cereus

Linear (B megaterium)y = 18209xR2 = 03421

B megateriumLinear (B cereus)y = 31128xR2 = 08583Linear (E asburiae)y = 22137xR2 = 07002

Figure 6 Pore diffusion plot for arsenic biosorption by therhizobacteria

0

2

4

6

8

10

12

14

16

18

0 1 2 3 4

qt

ln t

B megateriumE asburiae

Linear (B megaterium)y = 24945x + 04742R2 = 03056

B cereusLinear (B cereus)y = 4588x + 01565R2 = 08408Linear (E asburiae)y = 33337x + 00149Rsup2 = 07283

Figure 7 Elovich plot for arsenic biosorption by the rhizobacteria


value than that of the other rhizobacteria It can be con-cluded that each species of rhizobacteria has different kineticmodels for arsenic biosorption

4 Conclusions

Ten isolated rhizobacteria from L octovalvis (A globiformisB megaterium B cereus B pumilus Staphylococcus lentusE asburiae Sphingomonas paucimobilis Pantoea spp Rrhizogenes and R radiobacter) have different MICs afterexposure to arsenate R radiobacter was the most resistantrhizobacteria to arsenate with a MIC gt1500mgL All rhi-zobacteria were able to biosorb arsenic Sphingomonaspaucimobilis showed a higher arsenic biosorption (1464 plusmn234mgg DCW) than the other nine rhizobacteria All theresistant rhizobacteria have the potential as PGPR to en-hance the arsenic phytoremediation Based on the kineticrate B cereus E asburiae and R rhizogenes had the highestR2 value of 086 099 and 093 under the pore diffusionpseudo-first-order kinetic and pseudo-second-order kineticmodels It can be concluded that the MIC arsenic bio-sorption capacity and kinetic models in arsenic biosorptiondepend on the rhizobacterial species

Data Availability

)e Excel sheet including the data used to support thefindings of this study is available from the correspondingauthor upon request

Conflicts of Interest

)e authors declare no conflicts of interest regarding thepublication of this paper

Acknowledgments

)e authors would like to thank World Class ProfessorProgram-B by the Ministry of Research Technology andHigher Education 2018 (contract no 12314D23KP2018)

References

[1] Patentstorm ldquoBacterial effects on metal accumulation byplantsrdquo US patent 7214516 2007 httpwwwpatentstormuspatents7214516fulltexthtml

[2] S Nanda and J Abraham ldquoImpact of heavy metals on therhizosphere microflora of Jatropha multifida and their ef-fective remediationrdquo African Journal of Biotechnology vol 10no 56 pp 11948ndash11955 2012

[3] A K Ghosh P Bhattacharyya and R Pal ldquoEffect of arseniccontamination on microbial biomass and its activities inarsenic contaminated soils of Gangetic West Bengal IndiardquoEnvironment International vol 30 no 4 pp 491ndash499 2004

[4] M Takeuchi H Kawahata L P Gupta et al ldquoArsenic re-sistance and removal by marine and non-marine bacteriardquoJournal of Biotechnology vol 127 no 3 pp 434ndash442 2007

[5] S Tsai S Singh and W Chen ldquoArsenic metabolism bymicrobes in nature and the impact on arsenic remediationrdquoCurrent Opinion Biotechnol vol 20 pp 659ndash667 2009

[6] E Botes E Van Heerden and D Litthauer ldquoHyper-resistanceto arsenic in bacterial isolated from an antimony mine inSouth Africardquo South Africa Journal of Science vol 103 no 7-8 pp 279ndash281 2007

[7] L Q Lou Z H Ye and M H Wong ldquoA comparison ofarsenic tolerance uptake and accumulation between arsenichyperaccumulator Pteris vittata L and non-accumulator Psemipinnata L-A hydroponic studyrdquo Journal of HazardousMaterials vol 171 no 1ndash3 pp 436ndash442 2009

[8] M S Khan A Zaidi P A Wani and M Oves ldquoRole of plantgrowth promoting rhizobacteria in the remediation of metalcontaminated soils a reviewrdquo in Organic Farming PestControl and Remediation of Soil Pollutants E Lichtfouse Edvol 1 of Sustainable Agriculture Reviews Springer BerlinGermany 2009

[9] T Lebeau A Braud and K Jezequel ldquoPerformance ofbioaugmentation-assisted phytoextraction applied to metalcontaminated soils a reviewrdquo Environmental Pollutionvol 153 no 3 pp 497ndash522 2008

[10] S Mayak T Tirosh and B R Glick ldquoPlant growth-promotingbacteria that confer resistance to water stress in tomatoes andpeppersrdquo Plant Science vol 166 no 2 pp 525ndash530 2004

[11] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospectsfor utilizationrdquo Soil Biology and Biochemistry vol 42 no 5pp 669ndash678 2010

[12] B R Glick ldquoPhytoremediation synergistic use of plants andbacteria to clean up the environmentrdquo Biotechnology Ad-vances vol 21 no 5 pp 383ndash393 2003

[13] H S Titah S R S Abdullah I Mushrifah N Anuar H Basriand M Mukhlisin ldquoEffect of applying rhizobacteria andfertilizer on the growth of Ludwigia octovalvis for arsenicuptake and accumulation in phytoremediationrdquo EcologicalEngineering vol 58 pp 303ndash313 2013a

[14] L Nie S Shah A Rashid G I Burd D George Dixon andB R Glick ldquoPhytoremediation of arsenate contaminated soilby transgenic canola and the plant growth-promoting bac-terium Enterobacter cloacae CAL2rdquo Plant Physiology andBiochemistry vol 40 no 4 pp 355ndash361 2002

[15] S Glick A Fernandez M Benlloch and E D SancholdquoSunflower growth and tolerance to arsenic is increased by therhizospheric bacteria Pseudomonas fluorescenrdquo in Phytor-emediation of Metal-Contaminated Soils J L MorelG Echevarria and N Goncharova Eds vol 68 of NATOSciences Series IV Earth and Environmental Sciencespp 315ndash318 Springer Berlin Germany 2006

[16] A Rahman A Amelia A Nazri I Mushrifah N S Ahmadand A A Soffian ldquoScreening of plants grown in petrosludge-apreliminary study towards toxicity testing in phytor-emediationrdquo in Proceedings of Colloquium on UKMndashPRSBPhytoremediation Bangi Malaysia August 2009

[17] H S Titah S R S Abdullah I Mushrifah N Anuar H Basriand M Mukhlisin ldquoArsenic toxicity on Ludwigia octovalvis inspiked sandrdquo Bulletin of Environmental Contamination andToxicology vol 90 no 6 pp 714ndash719 2013

[18] H S Titah S R S Abdullah N Anuar M Idris H Basri andM Mukhlisin ldquoIdentification of rhizobacteria from Ludwigiaoctovalvis grown in arsenicrdquo Australian Journal of Basic andApplied Sciences vol 8 no 8 pp 134ndash139 2014

[19] H Guo S Luo L Chen et al ldquoBioremediation of heavymetals by growing hyperaccumulaor endophytic bacteriumBacillus sp L14rdquo Bioresource Technology vol 101 no 22pp 8599ndash8605 2010


[20] J P Harley and L M Prescott Laboratory Exercises inMicrobiology McGrawminusHill Companies Arlington TX USA5th edition 2002

[21] M I Ansari and A Malik ldquoBiosorption of nickel and cad-mium by metal resistant bacterial isolates from agriculturalsoil irrigated with industrial wastewaterrdquo Bioresource Tech-nology vol 98 no 16 pp 3149ndash3153 2007

[22] K M Khleifa M Abboud M Laymun K Al-Sharafa andK Tarawneh ldquoEffect of variation in copper sources andgrowth conditions on the copper uptake by bacterial hemo-globin gene (vgb) bearing E colirdquo Pakistan Jounal of BiologyScience vol 9 no 11 pp 2022ndash2031 2006

[23] Z A Zakaria Z Zakaria S Surif and W A AhmadldquoHexavalent chromium reduction by Acinetobacter haemo-lyticus isolated from heavy-metal contaminated wastewaterrdquoJournal of Hazardous Materials vol 146 no 1-2 pp 30ndash382007

[24] L Cavalca R Zanchi A Corsini et al ldquoArsenic-resistantbacteria associated with roots of the wild Cirsium arvense (L)plant from an arsenic polluted soil and screening of potentialplant growth-promoting characteristicsrdquo Systematic andApplied Microbiology vol 33 no 3 pp 154ndash164 2010

[25] J Kostal R Yang C H Wu A Mulchandani and W ChenldquoEnhanced arsenic accumulation in engineered bacterial cellsexpressing ArsRrdquo Applied and Environmental Microbiologyvol 70 no 8 pp 4582ndash4587 2004

[26] S A Wasay M J Haron A Uchiumi and S TokunagaldquoRemoval of arsenite and arsenate ions from aqueous solutionby basic yttrium carbonaterdquo Water Research vol 30 no 5pp 1143ndash1148 1996

[27] Y S Ho and G McKay ldquoPseudo-second order model forsorption processesrdquo Process Biochemistry vol 34 no 5pp 451ndash465 1999

[28] W J Weber and J C Morris ldquoKinetics of adsorption oncarbon from solutionrdquo Journal of the Sanitary EngineeringDivision ASCE vol 89SA2 pp 31ndash59 1963

[29] M A Lopez-Leal R C Martinez R A C VillenuevaH E M Flores and C D C Penagos ldquoArsenate biosorptionby iron-modified pine sawdust in batch system kinetic andequilibrium studiesrdquo BioResource vol 7 no 2 pp 1389ndash14042012

[30] S Maheswari and A G Murugesan ldquoRemoval of arsenic (III)ions from aqueous solution using Aspergillus flavus isolatedfrom arsenic contaminated soilrdquo Indian Journal of ChemicalTechnology vol 18 pp 45ndash52 2011

[31] G Annadurai R S Juang and D J Lee ldquoAdsorption of heavymetals from water using banana and orange peelsrdquo WaterScience and Technology vol 47 no 1 pp 185ndash190 2003

[32] M Calero F Hernainz G Blazquez M A Martın-Lara andG Tenorio ldquoBiosorption kinetics of Cd (II) Cr (III) and Pb(II) in aqueous solutions by olive stonerdquo Brazilian Journal ofChemical Engineering vol 26 no 2 pp 265ndash273 2009

[33] J C Iqwe and A A Abia ldquoA bioseparation process for re-moving heavy metals from wastewater using biosorbentsrdquondashAfrica Journal of Biotechnology vol 5 no 12 pp 1617ndash11792006

[34] M Mohapatra S Khatun and S Anand ldquoAdsoprtion be-haviour of Pb(II) Cd(II) and Zn(II) on NALCO plant sandrdquoIndian Journal of Chemical Technology vol 16 pp 291ndash3002009

[35] H S Titah S R S Abdullah N Anuar M Idris H Basri andM Mukhlisin ldquoIsolation and screening of arsenic resistantrhizobacteria of Ludwigia octovalvisrdquo African Journal ofBiotechnology vol 10 no 81 pp 18695ndash18703 2011

[36] A R Achour P Bauda and P Billard ldquoDiversity of arsenitetransporter genes from arsenic-resistant soil bacteriardquo Re-search in Microbiology vol 158 no 2 pp 128ndash137 2007

[37] B Chopra S Bhat I Mikheenko et al ldquo)e characteristics ofrhizosphere microbes associated with plants in arsenic-contaminated soils from cattle dip sitesrdquo Science of the To-tal Environment vol 378 no 3 pp 331ndash342 2007

[38] M P Bos V Robert and J Tommassen ldquoBiogenesis of thegram-negative bacterial outer membranerdquo Annual Review ofMicrobiology vol 61 no 1 pp 191ndash214 2007

[39] C U Anyanwu and C E Ugwu ldquoIncidence of arsenic re-sistant bacteria isolated from a sewage treatment plantrdquo In-ternational Journal of Basic amp Applied Science vol 10 no 6pp 64ndash78 2010

[40] M N Chandraprabha and K A Natarajan ldquoMechanism ofarsenic tolerance and bioremoval of arsenic by Acid-ithiobacilus ferrooxidansrdquo Journal of Biochemical Technologyvol 3 no 2 pp 257ndash265 2011

[41] S Shakya B Pradhan L Smith J Shrestha and S TuladharldquoIsolation and characterization of aerobic culturable arsenic-resistant bacteria from surface water and groundwater ofRautahat District Nepalrdquo Journal of Environmental Man-agement vol 95 pp 250ndash255 2012

[42] D N Joshi S J S Flora and K Kalia ldquoBacillus sp strain DJ-1potent arsenic hypertolerant bacterium isolated from theindustrial effluent of Indiardquo Journal of Hazardous Materialsvol 166 no 2-3 pp 1500ndash1505 2009


Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

sequestration by a range of cysteine-rich peptides chelationcompartmentalisation exclusion immobilization and dis-similatory arsenate respiration [5 6] Although arsenic istoxic to many bacteria metal-accumulating bacteria areoften found among metal-resistant bacteria [4] Nanda andAbraham [2] found that the toxicity of arsenic is higher thanchromium magnesium and copper (As gt Cr gt Mg gt Cu)Arsenate is toxic to bacteria because it is analogous tophosphate and can inhibit enzymes such as kinases [7]

Rhizobacteria that are capable of aggressively colonisingplant roots and promoting plant growth are generally knownas plant growth-promoting rhizobacteria (PGPR) [8] PGPRcan invade the interior of the cells and survive inside (in-tracellular PGPR such as nodule bacteria) or remain out-side the plant cells (extracellular PGPR such as BacillusPseudomonas and Azotobacter) PGPR such as Agro-bacterium (Rhizobium) Alcaligenes (Ralstonia) Arthro-bacter Azospirillum Azotobacter Bacillus BurkholderiaSerratia and Pseudomonas are interesting organisms for themetal extraction by plants because they increase the rate ofboth metal and biomass accumulation by plants [9] Con-taminated soils are often poor in nutrients or sometimesnutrient-deficient due to the loss of useful microbesHowever such soils can be made nutrient-rich by applyingmetal-tolerant microbes especially PGPR which not onlyprovide essential nutrients to the plants properties allowplants to remove heavy metals which can then be useful inagricultural production or the phytoremediation of con-taminated soil [11 12] )erefore it is advisable for growersto inoculate plants with growing in the contaminated sitesbut also play a major role in detoxifying heavy metals [8 10])ese rhizobacterial microbes to increase plant biomass andstabilise revegetate and restore heavy metal-contaminatedsoils [9]

)e interactions of rhizobacteria and plants in reme-diating arsenic have been studied According to Titah et al[13] the application of a six-rhizobacterial consortium al-leviated the toxic effects of arsenic in Ludwigia octovalvis andincreased the plant biomass According to Nie et al [14]canola plants inoculated with Enterobacter cloacae havegrown to a significantly higher extent than noninoculatedcanola plants due to the presence of arsenic Glick et al [15]reported the increase in shoot biomass and arsenic con-centration in the shoots of Helianthus annuus after in-oculation with Pseudomonas fluorescens

L octovalvis is a plant species that can survive at a crudeoil-contaminated site [16] and could uptake and accumulatearsenic in its tissues [17] )is study aimed to determine thelevel of arsenic (as arsenate) resistance of the identifiedrhizobacteria that were isolated from the roots of L octo-valvis grown in the greenhouse and contaminated land inMalacca Malaysia It also aimed at determining their arsenicbiosorption capability

2 Materials and Methods

21 Epiphyte Rhizobacterial Isolation from the Root of Loctovalvis )e detailed steps of rhizobacterial isolation wasdescribed by Titah et al [18]

22 Identification of Rhizobacteria Rhizobacterial identifi-cation was conducted using two biochemical methodsBiolog GEN III microbial identification system (Biolog IncUSA) and Vitek2 Compact System (Biomerieux USA) )edetailed steps of rhizobacteria identification was describedby Titah et al [18]

23 Determination of theMinimum Inhibitory Concentration(MIC) ofArsenic )e test was conducted using the modifiedmethod of Guo et al [19] )e MIC of arsenate for eachrhizobacterium species was determined in three replicates ofsterile phosphate buffer saline (PBS) solution with a serialconcentration of 0 (control) 250 500 750 and 1000mgLof arsenic in arsenate form (sodium arsenate dibasic hep-tahydrate (AsHNa2O4middot7H2O)) (FlukaChemika Switzer-land) Two different controls were used for this method aPBS solution with bacteria but without arsenate (negativecontrol) and another PBS solution with arsenate but withoutbacteria (positive control) Based on the study of Harley andPrescott [20] 10 times PBS stock solution contained 80 g NaCl(Merck Germany) 2 g KCl (RampM Chemicals India) 11 gNa2HPO4middot7H2O (RampM Chemicals India) and 2 g KH2PO4(RampM Chemicals India) MIC determination was carriedout in a 250mL Erlenmeyer flask with 50mL workingvolume )e pH of arsenate in sterile PBS solution wasmeasured using Accument Basic AB 15 pH meter (FisherScientific USA) )e average pH readings were 73 74 7576 and 77 in the arsenate concentrations of 0 250 500 750and 1000mgL respectively )e cultures were incubatedusing an incubator shaker (Protech Model SI-100DMalaysia) at 37degC and 150 rpm Bacterial growth was de-termined at an absorbance of 550 nm for 0 3 6 19 and 24 h

24 Arsenic Biosorption Experiment Using Identified Rhizo-bacteria inBatch System An arsenic biosorption experimentwas conducted using a modified published method [21ndash23]Selected rhizobacteria were inoculated onto Trypticase(Tryptic) soy agar (TSA) media without arsenate for 24 hAfter that the selected rhizobacteria were inoculated intosterile Tryptic soy broth (TSB) )e cell suspension of theselected rhizobacteria was prepared by harvesting the cells atthe middle of the logarithmic phase based on the typical ofgrowth rate graph for the selected rhizobacteria At this timethe optical density (OD) at 550 nm was 10 )e cells wereharvested through centrifugation (Eppendorf centrifuge5804 Germany) at 4000 rpm for 15min)e obtained pelletwas then washed twice by using 85 g NaCl1000mLsolution

)e biosorption of arsenic was then tested in a 250mLErlenmeyer flask )e rhizobacterial cell suspension at 20(vv) or 10mg dry cell weight (DCW) was added with 50mLof sterile PBS solution which had an arsenic concentrationof 100mgL by diluting sodium arsenate dibasic heptahy-drate (AsHNa2O4middot7H20) (Fluka Chemika Switzerland) )ecolonyforming unit (CFU) of the initial rhizobacteria wasapproximately 14 times 109 CFUmL A pH of 73 was measuredfor 100mgL arsenate in sterile PBS solution by usingAccument Basic AB 15 pH meter (Fisher Scientific USA)









dqt

qt

k1 qe minus qt( 1113857 (1)



2303t (2)



qdqt

dt

k2 qeq minus qt1113872 11138732 (3)


1qeq minus qt1113872 1113873

1

qeq+ k2t (4)


t

qt

1

k2q2eq1113872 1113873

+1

qeqt (5)




qt Kdt12

(6)





qt 1β

1113888 1113889ln(αβ) +1β

1113888 1113889ln t (7)













0

20

40

60

80

100

120

140

160

A gl

obifo

rmis

B m

egat

eriu

m

B ce

reus

B p

umilu

s

Stap

hylo

cocc

uslen

tus

E a

sbur

iae

Sphi

ngom

onas

pauc

imob

ilis

Pant

oea

spp

R rh

izog

enes

R ra

diob

acte

r

Conc

entr

atio

n of

tota

l ars

enic

in

supe

rnat

ant (

mg

L)

0h

24h17h6h2h


0

20

40

60

80

100

120

140

160

180

A gl

obifo

rmis

B m

egat

eriu

m

B ce

reus

B p

umilu

s

Stap

hylo

cocc

us le

ntus

E a

sbur

iae

Sphi

ngom

onas

pauc

imob

ilis

Pant

oea

spp

R rh

izog

enes

R ra

diob

acte

r

Ars

enic

bio

sorp

tion

in ce

ll pe

llet

(mg

g D

CW)

0h

2h

6h

17h24h








(a) (b)

K

R BIM

OD

(c)

M

OM

CD

(d)










Rhizobacteria



k1(h)








0

02

04

06

08

1

12

14

16

0 10 20 30

log

(qe

ndashq t

)

t













0

2

4

6

8

10

12

14

16

18

0 10 20 30

tqt

t





0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6

q t

t12

E asburiaeB cereus




0

2

4

6

8

10

12

14

16

18

0 1 2 3 4

qt

ln t







4 Conclusions


Data Availability




Acknowledgments


References















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









dqt

qt

k1 qe minus qt( 1113857 (1)



2303t (2)



qdqt

dt

k2 qeq minus qt1113872 11138732 (3)


1qeq minus qt1113872 1113873

1

qeq+ k2t (4)


t

qt

1

k2q2eq1113872 1113873

+1

qeqt (5)




qt Kdt12

(6)





qt 1β

1113888 1113889ln(αβ) +1β

1113888 1113889ln t (7)













0

20

40

60

80

100

120

140

160

A gl

obifo

rmis

B m

egat

eriu

m

B ce

reus

B p

umilu

s

Stap

hylo

cocc

uslen

tus

E a

sbur

iae

Sphi

ngom

onas

pauc

imob

ilis

Pant

oea

spp

R rh

izog

enes

R ra

diob

acte

r

Conc

entr

atio

n of

tota

l ars

enic

in

supe

rnat

ant (

mg

L)

0h

24h17h6h2h


0

20

40

60

80

100

120

140

160

180

A gl

obifo

rmis

B m

egat

eriu

m

B ce

reus

B p

umilu

s

Stap

hylo

cocc

us le

ntus

E a

sbur

iae

Sphi

ngom

onas

pauc

imob

ilis

Pant

oea

spp

R rh

izog

enes

R ra

diob

acte

r

Ars

enic

bio

sorp

tion

in ce

ll pe

llet

(mg

g D

CW)

0h

2h

6h

17h24h








(a) (b)

K

R BIM

OD

(c)

M

OM

CD

(d)










Rhizobacteria



k1(h)








0

02

04

06

08

1

12

14

16

0 10 20 30

log

(qe

ndashq t

)

t













0

2

4

6

8

10

12

14

16

18

0 10 20 30

tqt

t





0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6

q t

t12

E asburiaeB cereus




0

2

4

6

8

10

12

14

16

18

0 1 2 3 4

qt

ln t







4 Conclusions


Data Availability




Acknowledgments


References















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


qt Kdt12

(6)





qt 1β

1113888 1113889ln(αβ) +1β

1113888 1113889ln t (7)













0

20

40

60

80

100

120

140

160

A gl

obifo

rmis

B m

egat

eriu

m

B ce

reus

B p

umilu

s

Stap

hylo

cocc

uslen

tus

E a

sbur

iae

Sphi

ngom

onas

pauc

imob

ilis

Pant

oea

spp

R rh

izog

enes

R ra

diob

acte

r

Conc

entr

atio

n of

tota

l ars

enic

in

supe

rnat

ant (

mg

L)

0h

24h17h6h2h


0

20

40

60

80

100

120

140

160

180

A gl

obifo

rmis

B m

egat

eriu

m

B ce

reus

B p

umilu

s

Stap

hylo

cocc

us le

ntus

E a

sbur

iae

Sphi

ngom

onas

pauc

imob

ilis

Pant

oea

spp

R rh

izog

enes

R ra

diob

acte

r

Ars

enic

bio

sorp

tion

in ce

ll pe

llet

(mg

g D

CW)

0h

2h

6h

17h24h








(a) (b)

K

R BIM

OD

(c)

M

OM

CD

(d)










Rhizobacteria



k1(h)








0

02

04

06

08

1

12

14

16

0 10 20 30

log

(qe

ndashq t

)

t













0

2

4

6

8

10

12

14

16

18

0 10 20 30

tqt

t





0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6

q t

t12

E asburiaeB cereus




0

2

4

6

8

10

12

14

16

18

0 1 2 3 4

qt

ln t







4 Conclusions


Data Availability




Acknowledgments


References















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


0

20

40

60

80

100

120

140

160

A gl

obifo

rmis

B m

egat

eriu

m

B ce

reus

B p

umilu

s

Stap

hylo

cocc

uslen

tus

E a

sbur

iae

Sphi

ngom

onas

pauc

imob

ilis

Pant

oea

spp

R rh

izog

enes

R ra

diob

acte

r

Conc

entr

atio

n of

tota

l ars

enic

in

supe

rnat

ant (

mg

L)

0h

24h17h6h2h


0

20

40

60

80

100

120

140

160

180

A gl

obifo

rmis

B m

egat

eriu

m

B ce

reus

B p

umilu

s

Stap

hylo

cocc

us le

ntus

E a

sbur

iae

Sphi

ngom

onas

pauc

imob

ilis

Pant

oea

spp

R rh

izog

enes

R ra

diob

acte

r

Ars

enic

bio

sorp

tion

in ce

ll pe

llet

(mg

g D

CW)

0h

2h

6h

17h24h








(a) (b)

K

R BIM

OD

(c)

M

OM

CD

(d)










Rhizobacteria



k1(h)








0

02

04

06

08

1

12

14

16

0 10 20 30

log

(qe

ndashq t

)

t













0

2

4

6

8

10

12

14

16

18

0 10 20 30

tqt

t





0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6

q t

t12

E asburiaeB cereus




0

2

4

6

8

10

12

14

16

18

0 1 2 3 4

qt

ln t







4 Conclusions


Data Availability




Acknowledgments


References















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







(a) (b)

K

R BIM

OD

(c)

M

OM

CD

(d)










Rhizobacteria



k1(h)








0

02

04

06

08

1

12

14

16

0 10 20 30

log

(qe

ndashq t

)

t













0

2

4

6

8

10

12

14

16

18

0 10 20 30

tqt

t





0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6

q t

t12

E asburiaeB cereus




0

2

4

6

8

10

12

14

16

18

0 1 2 3 4

qt

ln t







4 Conclusions


Data Availability




Acknowledgments


References















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









Rhizobacteria



k1(h)








0

02

04

06

08

1

12

14

16

0 10 20 30

log

(qe

ndashq t

)

t













0

2

4

6

8

10

12

14

16

18

0 10 20 30

tqt

t





0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6

q t

t12

E asburiaeB cereus




0

2

4

6

8

10

12

14

16

18

0 1 2 3 4

qt

ln t







4 Conclusions


Data Availability




Acknowledgments


References















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







0

2

4

6

8

10

12

14

16

18

0 10 20 30

tqt

t





0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6

q t

t12

E asburiaeB cereus




0

2

4

6

8

10

12

14

16

18

0 1 2 3 4

qt

ln t







4 Conclusions


Data Availability




Acknowledgments


References















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



4 Conclusions


Data Availability




Acknowledgments


References















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


arsenicresistanceandbiosorptionbyisolated...

Documents