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Immobilization of Cr(VI) and Its Reduction to Cr(III) Phosphate by Granular Biofilms Comprising a Mixture of Microbes

We assessed the potential of mixed microbial consortia, in the form of granular biofilms, to reduce chromate and remove it from synthetic minimal medium. In batch experiments, acetate-fed granular biofilms incubated aerobically reduced 0.2 mM Cr(VI)
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   A PPLIED AND E NVIRONMENTAL M ICROBIOLOGY , Apr. 2010, p. 2433–2438 Vol. 76, No. 80099-2240/10/$12.00 doi:10.1128/AEM.02792-09Copyright © 2010, American Society for Microbiology. All Rights Reserved. Immobilization of Cr(VI) and Its Reduction to Cr(III) Phosphate byGranular Biofilms Comprising a Mixture of Microbes  Y. V. Nancharaiah, 1,2 C. Dodge, 1 V. P. Venugopalan, 2 S. V. Narasimhan, 2 and A. J. Francis 1 *  Environmental Sciences Department, Brookhaven National Laboratory, Upton, New York 11973, 1  and Water andSteam Chemistry Division, Bhabha Atomic Research Centre Facilities, Kalpakkam 603102, India 2 Received 18 November 2009/Accepted 8 February 2010  We assessed the potential of mixed microbial consortia, in the form of granular biofilms, to reduce chromateand remove it from synthetic minimal medium. In batch experiments, acetate-fed granular biofilms incubatedaerobically reduced 0.2 mM Cr(VI) from a minimal medium at 0.15 mM day  1 g  1 , with reduction of 0.17 mMday  1 g  1 under anaerobic conditions. There was negligible removal of Cr(VI) (i) without granular biofilms,(ii) with lyophilized granular biofilms, and (iii) with granules in the absence of an electron donor. Analyses byX-ray absorption near edge spectroscopy (XANES) of the granular biofilms revealed the conversion of solubleCr(VI) to Cr(III). Extended X-ray absorption fine-structure (EXAFS) analysis of the Cr-laden granularbiofilms demonstrated similarity to Cr(III) phosphate, indicating that Cr(III) was immobilized with phosphateon the biomass subsequent to microbial reduction. The sustained reduction of Cr(VI) by granular biofilms wasconfirmed in fed-batch experiments. Our study demonstrates the promise of granular-biofilm-based systems intreating Cr(VI)-containing effluents and wastewater. Chromium is a common industrial chemical used in tanningleather, plating chrome, and manufacturing steel. The twostable environmental forms are hexavalent chromium [Cr(VI)]and trivalent chromium [Cr(III)] (20). The former is highlysoluble and toxic to microorganisms, plants, and animals, en-tailing mutagenic and carcinogenic effects (6, 22, 33), while thelatter is considered to be less soluble and less toxic. Therefore,the reduction of Cr(VI) to Cr(III) constitutes a potential de-toxification process that might be achieved chemically or bio-logically. Microbial reduction of Cr(VI) seemingly is ubiqui-tous; Cr(VI)-reducing bacteria have been isolated from bothCr(VI)-contaminated and -uncontaminated environments (6,7, 23, 38, 39). Many archaeal/eubacterial genera, common todifferent environments, reduce a wide range of metals, includ-ing Cr(VI) (6, 16, 21). Some bacterial enzymes generate Cr(V)by mediating one-electron transfer to Cr(VI) (1, 4), whilemany other chromate reductases convert Cr(VI) to Cr(III) ina single step.Biological treatment of Cr(VI)-contaminated wastewatermay be difficult because the metal’s toxicity potentially can killthe bacteria. Accordingly, to protect the cells, cell immobiliza-tion techniques were employed (31). Cells in a biofilm exhibitenhanced resistance and tolerance to toxic metals compared with free-living ones (15). Therefore, biofilm-based reductionof Cr(VI) and its subsequent immobilization might be a satis-factory method of bioremediation because (i) the biofilm-bound cells can tolerate higher concentrations of Cr(VI) thanplanktonic cells, and (ii) they allow easy separation of thetreated liquid from the biomass. Ferris et al. (11) describedmicrobial biofilms as natural metal-immobilizing matrices inaqueous environments. Bioflocs, the active biomass of acti- vated sludge-process systems are transformed into dense gran-ular biofilms in sequencing batch reactors (SBRs). As granularbiofilms settle extremely well, the treated effluent is separatedquickly from the granular biomass by sedimentation (9, 24).Previous work demonstrated that aerobic granular biofilmspossess tremendous ability for biosorption, removing zinc, cop-per, nickel, cadmium, and uranium (19, 26, 31, 32, 40). How-ever, no study has investigated the role of cellular metabolismof aerobically grown granular biofilms in metal removal exper-iments. Despite vast knowledge about biotransformation bypure cultures, very little is known about reduction and immo-bilization by mixed bacterial consortia (8, 12, 13, 16, 20, 31, 36).Our research explored, for the first time, the metabolicallydriven removal of Cr(VI) by microbial granules.The main aim of this study was to investigate Cr(VI) reduc-tion and immobilization by mixed bacterial consortia, viz., aer-obically grown granular biofilms. Such biofilm-based systemsare promising for developing compact bioreactors for the rapidbiodegradation of environmental contaminants (17, 24, 29). Accordingly, we investigated the microbial reduction of Cr(VI)by aerobically grown biofilms in batch and fed-batch experi-ments and analyzed the oxidation state and association of thechromium immobilized on the biofilms by X-ray absorptionnear edge spectroscopy (XANES) and extended X-ray absorp-tion fine structure (EXAFS). MATERIALS AND METHODSCultivation of aerobic granular sludge. Aerobic granular biofilms were grownin a 3-liter working-volume laboratory-scale sequencing batch reactor (SBR).SBR setup and operation details have been described previously (26, 27). TheSBR was inoculated with seed sludge collected from the outlet of an aerationtank of an operating domestic wastewater treatment plant at Kalpakkam, India.The reactor was operated at room temperature (30  2°C) at a volumetricexchange ratio of 66% and a 6-h cycle, comprising 60 min of anaerobic static fill,282 min of aeration, 3 min of settling, 10 min of effluent decantation, and 5 minof being idle. The SBR was fed with acetate-containing synthetic wastewater asdiscussed by Nancharaiah et al. (27). Granules, collected 2 months after the * Corresponding author. Mailing address: Brookhaven NationalLaboratory, Environmental Sciences Department, Building 490A, Up-ton, NY 11973. Phone: (631) 344-4534. Fax: (631) 344-7303.  Published ahead of print on 19 February 2010.2433  reactor’s start-up, were washed twice with ultrapure water, and used in thebioreduction experiments. The morphology of the granular biofilms was docu-mented with a DP70 digital camera (Olympus, Japan) connected to a stereozoom microscope SMZ1000 (Nikon, Japan). The particle size and circularity of the granular biofilms were determined using the image analysis software ImageJ 1.99 (26). The settling velocity and dry weight of the aerobically grown granularbiofilms were determined according to standard methods (3). The biofilm density was evaluated following the method of Beun et al. (5). Individual granularbiofilms were fixed in 2.5% glutaraldehyde and dehydrated successively in 3-minsteps with 50%, 80%, and 95% ethanol. Then, the biofilms were sputter-coatedand imaged using scanning electron microscopy (SEM; Philips ESEM). Chromium reduction in batch experiments. Chromate reduction experiments were carried out in acetate minimal medium (AMM) consisting of 1.0 g liter  1 NH 4 Cl, 0.2 g liter  1 MgSO 4  7H 2 O, 3.02 g liter  1 CH 3 COONa, 0.5 g liter  1 KH 2 PO 4 , and 0.5 g liter  1  yeast extract. The pH of medium was adjusted to 7.0 with 0.1 N HCl before autoclaving it. To avoid precipitation, the MgSO 4 stocksolution (50  ) was autoclaved separately and added to the autoclaved media. Astock solution of potassium dichromate [1,000 mg Cr(VI) per liter] was preparedin ultrapure water, filter sterilized, and used as required. Cr(VI) reduction wascarried out under aerobic and anaerobic conditions. For the aerobic reduction of Cr(VI), 250-ml Erlenmeyer flasks containing 100 ml of AMM with chromate andthe granular biofilms (wet weight, 2.5, 5, 10, and 15 g) were incubated on a rotaryshaker at 100 rpm at 30°C. For anaerobic reduction, 100-ml glass bottles con-taining 100 ml of AMM with chromate and microbial granules were bubbled withnitrogen gas and sealed with rubber stoppers incubated at 30°C without shaking.Liquid samples were collected at regular intervals and analyzed for Cr(VI).Batch experiments also were carried out to determine the effect on Cr(VI)reduction, of chromate loading (0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, and 3.0 mM) andgranular biomass content (wet weight, 5, 10, 15, and 20 g 100 ml  1 ). Chromium reduction in fed-batch experiments. For the fed-batch experi-ments, Schott Duran bottles containing 100 ml of AMM amended with 0.25 mMCr(VI) and inoculated with granules (wet weight, 10 g) were used. The bottles were incubated at 30°C without aeration. Samples were collected periodicallyand monitored for Cr(VI). When almost all of the Cr(VI) was removed from themedium, it was replaced with fresh sterile AMM (100% exchange) and amended with Cr(VI). This procedure was repeated up to four times. The Cr(VI) contentof the liquid samples collected at different times during each batch was deter-mined. Cr(VI) analysis. Before analysis, the samples were centrifuged at 10,000 rpmfor 5 min to remove suspended cells. Chromate-reducing ability was estimated asthe decrease in the Cr(VI) concentration in medium detected via the Cr(VI)-specific colorimetric reagent S -diphenylcarbazide (DPC) (3). A 0.25% (wt/vol)solution of DPC was prepared in acetone-H 2 SO 4 to minimize its deteriorationand stored at 4°C. When added to samples containing Cr(VI), a pink colordeveloped; absorbance was measured immediately at 540 nm using a UV-visiblespectrophotometer (Shimazdu, Japan). X-ray absorption near edge spectroscopy analysis. Granular biofilms exposedto Cr(VI) and incubated aerobically or anaerobically were freeze-dried and theCr oxidation state was determined by X-ray absorption near edge spectroscopy(XANES). XANES analyses was performed at the Cr K edge in the fluorescencemode employing a 13-element Ge detector on beamline X10C at the NationalSynchrotron Light Source (NSLS), Brookhaven Laboratory, Upton, NY. Thesamples were placed on an Al sample holder with a cutout geometry of 2 mm(height)  20 mm (length)  1.5 mm (thickness) and sealed with Kapton tape.Standards included Cr 6  (potassium chromate; K  2 CrO 4 ) and Cr 3  [chromiumhydroxide; Cr(OH) 3 ]. Six spectra per sample were collected, from 200 eV belowthe absorption edge to 300 eV above it. Data were acquired in the XANESregion at an energy step of 0.5 eV at 2.0 s per interval. A chromium metal foilsited in the reference channel was run simultaneously with each sample tomonitor shifts in the beamline’s energy due to possible reduction by X rays.The software programs ATHENA and AUTOBACK (34) were used to ana-lyze the XANES data, which included background subtraction and normal-ization of the signal to the edge jump. The oxidation state of Cr was derivedfrom the position of the first derivative of the absorption edge energy. Extended X-ray absorption fine-structure spectroscopy analysis. EXAFSanalyses were performed to determine the association of Cr with its nearestneighbor in the granular biofilms. Samples and standards were prepared as forthe XANES analysis. A Cr(III) phosphate sample was included to obtain fittingparameters for the analysis. The individual scans were averaged followed bylinear pre-edge subtraction, background removal, normalization to the step edge,isolation of the  (  k ) function with a cubic spline function, followed by k 2  weight-ing. Theoretical EXAFS amplitude and phase functions for Cr-O, and Cr-Psingle scattering paths were then generated by FEFF 6.0 (34). Fitted parameterssuch as amplitude reduction factor ( S 02), (   E 0 ), interatomic distance (  R ), andDebye-Waller factor (  2 ) were fitted in R -space. The amplitude reduction value was as 1.0 for all fits. Errors in the overall fits were determined using a goodness-of-fit parameter (34). The spectra were combined and normalized to the edge jump, using programs from the University of Washington. The software pro-grams ATHENA and ARTEMIS were used to process the spectra through amultistep data analysis procedure that included background subtraction andFourier transformation. RESULTS Aerobic granular biofilms. Figure 1 shows the stereo zoommicroscopic images of the morphology of the granular biofilmsformed in the SBR and the scanning electron microscopicimages of the bacteria comprising them. The granular biofilms were dense and nearly circular (Fig. 1A), comparable to thosereported in other studies on aerobic granulation (5, 24). Theaverage size of the biofilms was 1.8 mm, and the averagecircularity was 0.89 mm. The settling velocity and density of aerobic granular biofilms were 55 m h  1 and 40 g liter  1 ,respectively. Visualization by scanning electron microscopy re- FIG. 1. Stereo zoom microscopic (A) and scanning electron microscopic (B) images of aerobic granular biofilms. Scale bar in panel A,1 mm.2434 NANCHARAIAH ET AL. A PPL . E NVIRON . M ICROBIOL .   vealed that rod/coccus-shaped bacteria dominate the acetate-fed granular biofilms (Fig. 1B). Cr(VI) reduction. To determine the mechanism of Cr(VI)removal, the changes in Cr(VI) was monitored in four batchexperiments: (i) without granular sludge; (ii) with lyophilizedgranular sludge; (iii) with intact granular sludge but no elec-tron donor; and, (iv) with intact granular sludge with the elec-tron donor, acetate. There was no decrease in the concentra-tion of Cr(VI) in the cell-free control. Similarly, we found nosignificant change in the Cr(VI) concentration in the media inthe absence of an electron donor or with lyophilized granularsludge (Fig. 2). These results confirm the role of bacterial cellmetabolism in the process. The ability of the microbial gran-ules to reduce Cr(VI) in the presence of the electron donor was revealed by a clear decolorization (yellow to colorless) of the media and verified in Cr(VI)-specific DPC absorbancemeasurements. Cr(VI) reduction was observed under both aer-obic and anaerobic conditions. Although Cr(VI) reduction issimilar in both (0.15 mM day  1 g  1 aerobically versus 0.17 mMday  1 g  1 anaerobically), the Cr(VI) concentration did not fallto zero under aerobic conditions (Fig. 2). In contrast, anaero-bic Cr(VI) reduction was almost complete (Fig. 3). Our sub-sequent work was focused on anaerobic removal of Cr(VI) bygranular biofilms.Figure 3 illustrates the extent of anaerobic reduction of Cr(VI) by granular biofilms at different initial biomass concen-trations: reduction was almost complete at all concentrations,but its rate varied with the content of the granular biofilm inthe medium. Complete removal of 0.2 mM Cr(VI) took ap-proximately 2 to 6 days, depending on the available biomass(Fig. 3). Figure 4 depicts the effect of Cr(VI) loading on itsreduction by the granular biofilms. Cr(VI) content fell at allinitial Cr(VI) concentrations tested and was nearly complete,irrespective of the initial concentration. However, the timetaken for complete removal rose with an increase in the initialCr(VI) concentration. The specific rates of Cr(VI) removal were, respectively, 0.17 mM day  1 g  1 , 0.65 mM day  1 g  1 ,and 1.86 mM day  1 g  1 granular biomass at 0.2, 1.0, and 3.0mM initial Cr(VI) concentration.Figure 5 shows the removal of Cr(VI) by microbial granulesin fed-batch experiments; removal approached completion ineach batch and was sustained in subsequent batches. The av-erage time for complete removal of 0.2 mM Cr(VI) was ap-proximately 2 to 4 days for each batch. Undoubtedly, the mi- FIG. 2. Effect of granular biomass on Cr(VI) removal under aero-bic conditions. The blank lacked granular biofilm. MM (minimal me-dium) received 5 g granular biomass and lacked carbon source. Eachdata point is a mean of three independent experiments. Error barsrepresent  1 standard deviation.FIG. 3. Cr(VI) removal by granular biofilm incubated anaerobi-cally. Each data point is a mean of duplicate experiments. Error barsrepresent  standard deviation.FIG. 4. Cr(VI) removal by granular biofilms under anaerobic con-ditions. The initial granule content was constant (10 g wet weight).Each data point is mean of three independent experiments. Error barsrepresent  1 standard deviation.FIG. 5. Chromium immobilization by aerobic granular sludge inanaerobic fed-batch conditions. The arrows indicate replacement of spent medium with fresh AMM-Cr(VI) medium. The initial concen-tration of Cr(VI) was 0.2 mM.V OL . 76, 2010 GRANULAR BIOFILM Cr(VI) IMMOBILIZATION AND REDUCTION 2435  crobial granules repeatedly can sustain the removal of Cr(VI)in fed-batch experiments. XANES analysis. Figure 6 displays the XANES spectra forthe standards and samples. The first derivatives of the absorp-tion edge energy for Cr(VI) and Cr(III) standards, respec-tively, are at 6005.6 eV and 6003.3 eV. In addition, the Cr(VI)standard exhibited a pre-edge peak at 5993.3 eV due to thetetrahedral coordination (1s  3d transition) of the chromate-oxygen atoms. Under anaerobic conditions, the absorptionedge energy for Cr associated with the granules shifted to6003.8 eV, while under aerobic conditions, it moved to 6003.4eV. This shift to lower energies compared to the Cr(VI) stan-dard and the absence of a pre-edge peak indicate the Cr(VI)predominantly was reduced to Cr(III). EXAFS analysis. Figure 7 and Table 1 give the results of fitting for Cr(III) phosphate- and Cr-containing granules (1mM). The best-fit parameters for Cr(III) phosphate indicatethat 5.7  0.7 O atoms surround the octahedral Cr at 1.97  0.01 Å. A fitting of the second shell shows the presence of 4.0  1.3 phosphorus atoms at a distance of 3.11  0.05 Å,indicating its presence as Cr(III) phosphate. Fitting of theCr-laden granular biofilm is similar to that of the Cr(III) phos-phate standard and shows the presence of 6.3  1.5 O atoms inits inner sphere. The second shell has 4.0  1.3 P atoms at adistance of 3.11  0.03 Å from the chromium. These fittingresults confirm that Cr associated with the granular biofilms ispresent primarily as Cr(III) phosphate; all concentrations of Cr (1.0, 1.5, and 2.0 mM) showed a similar association. EXAFSdata fitted using C, N, and P in the second shell showed a muchbetter fit using P. DISCUSSION Bacterial reduction of Cr(VI) was demonstrated in purecultures and mixed species activated sludge in flowthroughcolumns (37). However, there are no reports on the bioreduc-tion of chromium using microbial granules that offer very spe-cific advantages over activated sludge. Confocal microscopicimaging after staining with nucleic-acid-binding fluorophoresdisclosed that granular biofilms consist of distinct cell clustersseparated by voids (25–27). Microscopic imaging of these clus-ters revealed that rod/coccus-shaped bacteria dominated them(Fig. 1B). Earlier work showed that bacteria in the form of biofilms may be more suitable for chromium bioreduction thanto freely suspended cells. Thus, Morales et al. (23) reportedthat Streptomyces sp. strain CG252 cells grown as biofilms bet-ter removed Cr(VI) than did free cells. Similarly, Ganguli andTripathy (14) reported that Pseudomonas aeruginosa A2Chrcells immobilized in an agarose-alginate film in a rotating bi-ological contactor exhibited significantly higher rates of chro-mate reduction than did planktonic cells.Cr(VI) removal by the granular biofilms was observed bothunder aerobic and anaerobic conditions. However, the incom-plete removal of Cr(VI) under aerobic conditions might reflectcompetition between Cr(VI) and oxygen for electrons andelectron transfer from intermediate Cr(V) to oxygen, resultingin Cr(VI). We observed that Cr(VI) reduction was dependenton the initial content of biomass, as have others (16). Further-more, others found that using pregrown cellular biomass [cul-tivated without Cr(VI)] greatly lowers the time required forcomplete chromate reduction (35). Apparently, employingpregrown aerobic granular biofilms to reduce Cr(VI) avoids themetal’s negative impact on the growth of the microbial bio- FIG. 6. XANES spectra of Cr speciation in granule samples incu-bated aerobically and anaerobically.FIG. 7. The fitted k 2 -weighted (2.5 to 12.5 Å) (A) and Fourier-transformed (B) spectra showing association of 1 mM Cr(III) withbacterial cells. Solid lines represent experimental data, and dashedlines represent fitted data.2436 NANCHARAIAH ET AL. A PPL . E NVIRON . M ICROBIOL .  mass. The microbial species composition of the granular sludge was not identified in the present study; nonetheless, the com-mencement of reduction of chromium immediately after expo-sure to Cr(VI) suggests that bacteria able to reduce chromiumalready were present in the granules (without prior enrich-ment); the lack of a delay demonstrates that the necessaryenzymes are constitutively expressed. Seemingly, previous ex-posure to chromium and subsequent microbial enrichment arenot prerequisites for successful bioreduction. This could bemainly due to the involvement of constitutive chromate reduc-tases, thus corroborating the earlier observation of the rapidreduction of Cr(VI) by Pseudomonas putida unsaturated bio-films (32). Aerobic granular sludge cultivated in an SBR using acetateand lacking prior exposure to chromium efficiently reducedCr(VI) from minimal media. Passive biosorption by the gran-ular biomass was ruled out because Cr(VI) removal was neg-ligible in the absence of a carbon source and by lyophilizedgranules. Analysis of chromium speciation by XANES furtherconfirmed the bioreduction of Cr(VI) to Cr(III), therebypointing to the involvement of cell metabolism. Nonmetabolicreduction of Cr(VI) to Cr(III) by bacterial surfaces undernonnutrient conditions has been reported by Fein et al. (10). Inthis study, no such reduction of Cr(VI) to Cr(III) was observedunder nonnutrient conditions. EXAFS analyses revealed thatthe granular biofilm-bound Cr(III) occurs as Cr(III) phos-phate. Earlier, Neal et al. (28) reported that only Cr(III) wasbound to live Shewanella oneidensis cells. XANES and EXAFSanalyses of a Cr(III)-laden biomass of nonliving seaweed, Eck- lonia , were very similar to spectra from Cr(III) acetate (30).Kemner et al. (18) reported that the speciation of chromiumassociated with Pseudomonas fluorescens cells was consistent with association of Cr(III) with a phosphoryl functional group. A recent study showed reduction of Cr(VI) to Cr(III) by meth-ane-oxidizing bacteria, a ubiquitous group of environmentalbacteria (2). EXAFS analysis showed that Methylococcus cap- sulatus -associated chromium predominantly existed as Cr(III)and most likely associated with phosphate groups. EXAFSspectra of our Cr(III)-laden granular biomass revealed thepresence of Cr(III)-phosphate after Cr(VI) reduction. Overall,our findings suggest the potential use of mixed microbial gran-ules to bioremediate Cr(VI)-containing wastewater or indus-trial effluents.  ACKNOWLEDGMENTS This research was supported by the Department of Atomic Energy,Government of India, and in part by the Environmental RemediationSciences Division, Office of Biological and Environmental Research,Office of Science, U.S. Department of Energy under contract no.DE-AC02-98CH10886. Y.V.N. gratefully acknowledges the AmericanSociety for Microbiology for the Indo-US Visiting Research Profes-sorship Award.We thank Avril D. Woodhead for editorial help. REFERENCES 1. Ackerley, D. F., C. F. Gonzalez, M. Keyhan, R. Blake II, and A. Matin. 2004.Mechanism of chromate reduction by the Escherichia coli protein, NfsA, andthe role of different chromate reductases in minimizing oxidative stressduring chromate reduction. Environ. Microbiol. 6: 851–860.2. Al Hasin, A., S. J. Gurman, L. M. Murphy, A. Perry, T. J. Smith, and P. H. 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Kelly, B. Lai, J. Maser, E. J. O’Loughlin, D. Sholto-Douglas, Z. Cai, M. A. Schneegurt, Jr., C. F. Kulpa, and K. H. Nealson. 2004.Elemental and redox analysis of single bacterial cells by X-ray microbeamanalysis. Science 306: 686–687.19. Liu, Y., S. F. Yang, S.-F. Tan, Y.-M. Lin, and J.-H. Tay. 2002. Aerobicgranules: a novel zinc biosorbent. Lett. Appl. Microbiol. 35: 548–551. TABLE 1. EXAFS fit of Cr(III) with aerobic microbial granules  a Type of atom N R (Å)  2   E 0  F  Cr(III) phosphateCr-O 5.7  0.7 1.97  0.01 0.002  0.001 0.5  1.0 0.038Cr-P 4.0  1.3 3.11  0.05 0.007  0.002 10.5  1.4Bacterial granulesCr-O 6.3  1.5 1.98  0.04 0.001  0.001 1.2  0.8 0.017Cr-P 4.0  1.3 3.11  0.03 0.007  0.002 13.3  2.5  a  N  , coordination number (number of atoms); R , interatomic distances;  2 , disorder parameter;   E 0 , energy shift; F  , goodness-of-fit parameter. V OL . 76, 2010 GRANULAR BIOFILM Cr(VI) IMMOBILIZATION AND REDUCTION 2437
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