RESEARCH ARTICLE

Dechlorination of chloroorganics, decolorization,and simultaneous bioremediation of Cr6+ from realtannery effluent employing indigenous Bacillus cereus isolate

Manikant Tripathi & Satyendra Kumar Garg

Received: 19 September 2013 /Accepted: 17 December 2013# Springer-Verlag Berlin Heidelberg 2014

Abstract A native Bacillus cereus isolate has been employed,for the first time, for simultaneous decolorization, dechlorina-tion of chloroorganics, and Cr6+ remediation from the realtannery effluent. Most of the physicochemical variables in 3:1diluted effluent were well above the standard prescribedlimits, which decreased substantially upon microbial treat-ment. The extent of bioremediation was better in diluted(3:1) as compared to undiluted effluent supplemented withnutrients and augmented with B. cereus isolate. Maximumgrowth, effluent decolorization (42.5 %), dechlorination(74.1 %), and Cr6+ remediation (34.2 %) were attained with4.0 % (v/v) inoculum, 0.8 % glucose, and 0.2 % ammoniumchloride in 3:1 diluted effluent at natural pH (8.1) within 72 hof incubation. The efficiency of bioremediation in a bioreactorwas higher as compared to a flask trial during 72 h of incuba-tion: decolorization (47.9 %) was enhanced by 5.4 %, dechlo-rination (77.4 %) by 3.3 %, and Cr6+ removal (41.7 %) by7.5 % at an initial color of 286 Pt-Co units and initial concen-tration of 62 mg chloride ions and 108 mg l−1 Cr6+.Immobilized biomass of Pseudomonas putida and B. cereuscoculture enhanced the extent of Cr6+ remediation (51.9 %) by10.2 % compared to the bioreactor trial. Chromate reductaseactivity and reduced Cr directly correlated and were mainlyassociated with soluble fraction of B. cereus plus effluentnatural microflora. The GC-MS analyses revealed the forma-tion of metabolites such as acetic acid and 2-butenoic acid inbacterially treated effluent. The supplementation of nutrientsalong with B. cereus augmentation is required for efficienteffluent bioremediation.

Keywords Bacillus cereus . Bioreactor . Bioremediation .

Coculture . Immobilization . Tannery effluent

Introduction

Tannery is one of the emerging industrial sectors. In India,there are approximately 3,000 tanneries in existence, whichcollectively employ more than 2.5 million people. Nearly80 % of these tanneries are engaged in tanning processes thatutilize chrome (Rajamani et al. 1995), particularly chromiumsulfate. Approximately 80 million hides and 130 million skinpieces are processed annually. Export of leather goods hasrecently reached a new high of $2.8 billion (Rs 12,500 crores)(Amudeswari et al. 2009). Approximately 50 l of effluent isgenerated per kilogram of skin/hide processed. In India, of thetotal chromium effluent discharged, >50% originates from theleather, iron, and steel industries.

The tannery effluent has very high pollution potential. Itcreates serious threat to the human health. It also makes theland/soil infertile. The ground as well as surface water be-comes unfit for irrigation and drinking. The specific problemassociated with heavy metals is their accumulation in the foodchain and persistence in the environment. Toxic effects ofchromium are valence-dependent. Hexavalent chromium(Cr6+) is highly soluble, mutagenic, and carcinogenic, where-as trivalent chromium (Cr3+) is less soluble and hence lesstoxic (Xu et al. 2009). In India, the standard limit for Cr6+

discharge in inland surface waters is 0.1 mg l−1 (IS: 2296, IS:2490) (Bhide et al. 1996). The similar value established for theUSA is 0.05 mg l−1 (US EPA 1979).

The routine mechanism for mitigation of chromium pollu-tion generally involves reduction of Cr6+ to Cr3+ and subse-quent precipitation of less soluble chromium at or near neutralpH (Sultan and Hasnain 2012). Conventional physicochemi-cal methods such as electrochemical treatment, ion exchange,

Responsible editor: Gerald Thouand

M. Tripathi : S. K. Garg (*)Centre of Excellence, DST-FIST Supported Department ofMicrobiology, Dr. Ram Manohar Lohia Avadh University,Faizabad 224001, Indiae-mail: sk_garg001@yahoo.com

Environ Sci Pollut ResDOI 10.1007/s11356-013-2479-y

precipitation, reverse osmosis, evaporation, and oxidation/reduction for chromium removal from tannery effluent areneither cost-effective nor eco-friendly (Garg et al. 2012).Hence, more practically feasible and economically viablemethods are being explored. In this context, bioremediationis an option that offers the possibility to destroy or rendervarious contaminants harmless using biological activities. Thebioremediation process employs naturally occurring bacteria,algae, fungi, or plants to degrade/detoxify substances that arehazardous to the human health and the environment (Garget al. 2012). Microbial treatment of tannery effluent is pre-ferred over conventional treatment technology because it (1) iseco-friendly, (2) has excellent performance, and (3) is a low-cost technique (Gupta and Rastogi 2008; Viera and Volesky2000). Moreover, the biosorbents can also be employed forchromium removal from tannery wastewater (Daraei et al.2013a, b; Tripathi et al. 2011a).

Furthermore, the leather industries generate complexwastewaters containing not only chromium but also othertoxic compounds, like phenol and its derivatives (Chandraet al. 2011; Paisio et al. 2012). As per Indian Standard Insti-tution (ISI), the permissible limit for phenolic chloroorganicsin inland surface waters is 0.002 mg l−1, whereas the similarlimit in leachates is 1.0 mg l−1 (MOEF). The European Coun-cil Directive has set a limit of 0.5 μg l−1 to regulate theconcentration of phenolic compounds in drinking water. Theyare easily accumulated in various food chains of biologicalsystems, thereby causing profound problems to the humanhealth. Phenolics also contribute to off-flavor problems indrinking and food processing waters (Yang and Humphrey1975). Such compounds are quite inhibitory to environmentalmicrobes also, due to their action on membrane function(Copley 2000) and ability to uncouple oxidative phosphory-lation (Ito and Ohnishi 1982).

During tanning process, chloroorganics such as pentachlo-rophenol (PCP) is used as a biocide (Thakur et al. 2001). PCP isusually present in tanning and other industrial discharges due toits recalcitrance. The USA’s Environmental Protection Agencyhas listed PCP as the priority pollutant due to its toxicity (USEPA 1979). Such a chlorinated compound is known to be stableand difficult to degrade. However, microbes can sometimecatalyze biotransformation reactions in which chloride ions(Cl−) of the chlorinated compounds are displaced by protons(H+). The more chloride ions that are removed, the morereactive the resultant compounds become, thereby renderingthem susceptible to biodegradation (Garg and Tripathi 2011;Haggblom 1990). Therefore, wastewater simultaneously con-taminated with Cr6+ and chlorophenolics should be treatedcarefully before being discharged into receiving water bodiesin order to reduce contaminant levels in the environment.

The deficiency of easily metabolizable nutrients and therequirement of augmentation with native bacterial isolate ne-cessitated us to design the experiments so that the aforesaid

process bottlenecks are properly addressed for the efficientbioremediation of toxicants such as Cr6+ and PCP from realtannery effluent.

This study was, therefore, aimed to judge the efficacy of aBacillus cereus RMLAU1 isolate for simultaneous decolori-zation, dechlorination of chloroorganics, and remediation ofCr6+ from tannery effluent under varied experimental condi-tions at flask as well as bench-scale bioreactor levels. Thephysicochemical changes in the tannery effluent were alsoassessed, before and after B. cereus treatment. Additionally,the tannery effluent bioremediation was attempted withimmobilized biomass of B. cereus and its coculture withPseudomonas putida. The schematic view of the overall pro-cess is depicted in Fig. 1. A correlation was subsequentlyestablished between chromate reductase activity and distribu-tion of reduced chromium in various cell fractions. GC-MSanalysis of tannery effluent samples, before and after bacterialtreatment, was also performed.

Materials and methods

Bacterial culture

A B. cereus RMLAU1 strain (MTCC 9777, NCBI GenBankaccession number FJ959366), isolated previously in our lab-oratory, was employed in this study (Tripathi et al. 2011b).The bacterium was subcultured and maintained on minimalsalt agar (MSA) slants (pH 7.0) and stored at 4±1 °C.

Inoculum preparation

The bacterial inoculum was prepared in 100 ml of sterilizedpeptone water (w/v, 1.0 % peptone and 0.5 % NaCl in distilledwater) in 500-ml Erlenmeyer flasks by transferring a loopfulof B. cereus isolate and incubated at 35±1 °C for 24 h in anincubator shaker (150 rpm). The inoculum size used fordecolorization and bioremediation study was 1.0 % (v/v) ofthe above test culture (OD A600 0.87 containing 2.9×106 cfu ml−1).

Culture conditions

The tannery effluent bioremediation experiments were per-formed with an unsterilized effluent medium. Different exper-imental sets were designed with undiluted and 3:1 diluted(effluent/distilled water) tannery effluent with four combina-tions: set I, nutrients unsupplemented (US) and unaugmented(UA); set II, nutrients unsupplemented (US) but augmented(A) with 1.0% (v/v) B. cereusculture; set III, supplemented (S)with nutrients [w/v, 0.4 % glucose (G) and 0.2 % ammoniumchloride (AC)] but unaugmented (UA) with B. cereus culture;and set IV nutrients supplemented (S) as above and

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augmented (A) with B. cereus culture. The pH of the effluentmedium was adjusted at pre-optimized 7.0 in sets II–IV. TheErlenmeyer flasks were incubated at previously optimized 35±1 °C (Tripathi and Garg 2013) for 120 h in an incubatorshaker (150 rpm). Bacterial growth, decolorization, dechlori-nation, Cr6+ removal, and pH change of the effluent weremeasured periodically at a 24-h interval up to 120 h ofincubation.

Effect of glucose concentration

The effect of glucose cosubstrate as the carbon/energy sourceat 0.4, 0.6, 0.8, and 1.0 % (w/v) with fixed concentration ofnitrogen (ammonium chloride at 0.2 %, w/v) was studied forthe above determinations.

Effect of pH

The bioremediation of diluted (3:1) effluent medium supple-mented with ammonium chloride (at 0.2 %, w/v) and opti-mized glucose (at 0.8 %, w/v) was carried out at naturaleffluent pH (8.1) and adjusted pH (7.0) optimum for theB. cereus isolate.

Effect of inoculum size

The diluted (3:1) effluent medium (optimized natural pH 8.1)inoculated with 1.0–5.0 % (v/v) exponentially growingRMLAU1 strain was incubated at 35±1 °C in an incubatorshaker (150 rpm). The samples were processed as above for allthe determinations.

Bench-scale bioreactor trial

The bioreactor trial for 3:1 diluted unsterilized tannery effluent(1.0 l) was performed under optimized conditions at pH 8.1,35±1 °C and augmented with an optimized 4.0 % (v/v)

inoculum dose of B. cereus with a previously optimized aer-ation rate of 0.6 volume per volume per minute (vvm) andagitation speed of 100 rpm (Tripathi and Garg 2013).

Chromate reductase activity

Preparation of cell-free extract and chromate reductase assaywere performed as per the slightly modified method of Iliaset al. (2011). The bacterial cells grown in tannery effluentbroth (100 ml) were harvested during the exponential growthphase (48 h) and centrifuged at 10,000 rpm (4 °C) for 10 min.The culture supernatant was collected and assayed for solubleprotein and chromate reductase activity. The cells’ pellet wassuspended in 5.0 ml of phosphate buffer (50 mM, pH 7.0),kept in an ice bath (4 °C), and disrupted with an ultrasonicatorat 30-s pulses. The treatment was repeated five times at aninterval of 30 s. After sonication, the cell lysate was centri-fuged at 16,000 rpm (4 °C) for 30 min and cell-free extractwas filtered through a nitrocellulose membrane (pore size0.2 μM) to get a cytosolic fraction, which was then transferredto a fresh tube and kept in an ice bath. Cell debris wasresuspended in 5.0 ml of phosphate buffer and kept in an icebath. Boiled culture supernatant, cell debris, and cytosolicfraction served as controls. The chromate reductase activitywas assayed in a reaction mixture containing 0.5 ml of en-zyme solution (culture supernatant, cell debris, and cytosolicfraction), 0.5 ml of 50 mM phosphate buffer, and K2Cr2O7 asCr6+ at a final concentration of 3.4 μM. The reaction mixturewas incubated at 30 °C for 30 min. The reaction was stoppedby adding 0.2 ml of 20 % trichloroacetic acid (Horitsu et al.1987). Cr6+ reduction was measured by estimating the de-crease in Cr6+ concentration in the reaction mixture using 1,5-diphenyl carbazide method (APHA 1998). One unit of en-zyme activity was defined as 1.0 μM of Cr6+ reduced min−1.Protein was estimated as per Bradford (1976) method usingbovine serum albumin (BSA) as the standard. In this method,a protein solution (0.5 ml) was incubated with 4.5 ml of dye

Fig. 1 Comparative schematicrepresentation of tannery effluentbioremediation processoptimization

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reagent (Commassie Brilliant Blue G-250) for 10 min andabsorbance was recorded at 595 nm against a phosphate buffer(pH 7.0) blank. The calibration curve was plotted using BSAas the standard protein (Bradford 1976).

Gas chromatography and mass spectrometry

GC-MS analysis was performed for the detection and identifica-tion of the compounds present in untreated and bacterially treatedtannery effluent using a GC equipped with an FID detector(Agilent 7890A) and an MS (Jeol AccuTOF GCV). The exper-iment was performed in 3:1 diluted tannery effluent. Control(0 h) and experimental (48 h) cultured broth (100 ml) were takenfrom the bioreactor trial and centrifuged (4 °C) at 10,000 rpm for10 min. In cell-free supernatant fractions, the dechlorinationmetabolites were extracted using equal volume of n-hexane andacetone mixture (1:1 ratio). The organic layer was dried withanhydrous sodium sulfate and solvent evaporated till dryness.The sample residue was diluted with 1.0 ml of n-hexane andanalyzed immediately on GC-MS. In GC, the column and injec-tor temperature was maintained at 240 °C and all injections(5.0 μl each for untreated and treated samples) were carried outon the split mode. The oven program was at 50 °C for 1.0 min,increased to 200 °C at 10 °C min−1, and finally increased to240 °C at 10 °C min−1 and held at 280 °C for 3.0 min. The MSwas operated in the electron ionization (EI) mode (70 eV).Helium was used as the carrier gas with a constant flow rate of1.0 ml min−1. A solvent delay of 3.0 min was selected, and MSwas operated in the total ion current (TIC) mode, scanning from40 to 600 (m/z) at 70 eV (electron energy). The compounds wereidentified by comparing their retention time (RT, minutes) andmass spectra with those of the National Institute of Standard andTechnology (NIST) library.

Immobilization of cell biomass for effluent bioremediation

Sodium alginate suspension (4.0 %, w/v) was prepared as perthe method of Lopez et al. (1997) by suspending 1.0 g ofsodium alginate in 25 ml of hot distilled water, followed byautoclaving at 121 °C for 15 min. The autoclaved slurry wascooled to room temperature and a cell suspension (0.5 mlequivalent to 80-mg cell dry wt of bacterial isolate) was addedand mixed thoroughly by stirring with a sterile glass rod toobtain a uniform mixture. The slurry was filled in a sterilesyringe and added dropwise to chilled 0.2 M CaCl2 solutionwith constant stirring for formation of beads. The beadsformed were kept for curing at 4 °C for 1 h in a refrigerator.The beads were then washed with sterile distilled water threeto four times and preserved in normal saline solution at 4 °C.All processes were performed aseptically under laminar airflow. Further, these immobilized B. cereusbeads were used forunsterilized diluted (3:1) tannery effluent bioremediation

study at a shake flask level. The charging rate of beads was20 beads per 25 ml of effluent.

The effluent bioremediation was also performed with aug-mented coculture containing B. cereus plus P. putida SKG-1isolates. The P. putida strain employed in this study waspreviously isolated in our laboratory (Singh et al. 2011). Theimmobilized beads of coculture were prepared as above. Thecell suspension containing 0.5ml equivalent to 40mgP. putidaplus 0.25 ml equivalent to 40-mg B. cereus cell dry wt wasused for the preparation of immobilized beads of coculture.This bioremediation study employing coculture was per-formed at a bioreactor level.

Analytical determinations

Physicochemical parameters

The physicochemical properties [pH, temperature, total dis-solved solids (TDS), total suspended solids (TSS), oil andgrease, hardness, biochemical oxygen demand (BOD), chem-ical oxygen demand (COD), and heavy metals] were deter-mined as per the standard methods of American Public HealthAssociation (APHA 1998). An average of triplicate for eachexperiment is being reported.

Bacterial growth

The bacterial growth, in terms of absorbance (A600, 1.0-cmcuvette) (Systronics UV-Vis 117), was recorded periodicallyat a 24-h interval in different sets of experiments.

Color units

The intensity of effluent color, before and after treatment, wasmeasured in accordance with the Canadian Pulp and PaperAssociation Standard Method (CPPA 1974). The sampleswere centrifuged at 10,000 rpm for 30 min to removesuspended solids, supernatant was adjusted to pH 7.6, andabsorbance was measured at 465 nm against distilled water.

Chloride ions

The extent of dechlorination was determined by estimation ofchloride ions released in the culture supernatant as per themethod of Bergmann and Sanik (1957).

Chromium

Total chromium in biomass and supernatant The biomass andsupernatant was digested with an acid mixture (6:1 perchloricacid and HNO3) as per the method of APHA (1998). Theconcentrations of total chromium in digested samples were

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analyzed using an atomic absorption spectrophotometer at357.9 nm.

Hexavalent chromium (Cr6+) The estimation of Cr6+ in theculture supernatant was performed following the 1,5-diphenylcarbazide method (APHA 1998).

Statistical analyses

Each experiment was performed in triplicate. The statisticalanalysis was done as per the standard method of Steel andTorrie (1992), and results are expressed as mean ± SD values.

Results and discussion

Physicochemical analyses of effluent

The physicochemical analyses results of diluted (3:1) untreat-ed and bacterially treated tannery effluents revealed that thepH and temperature were within the prescribed limits ofminimum national standards. These are the significant param-eters which affect the enzymatic reactions occurring in variouscellular organisms. The TDS, TSS, oil and grease, BOD, andCOD values were above the recommended permissible limitsin the untreated effluent. All the organic and inorganic con-stituents contribute largely towards high BOD and COD. Theobserved COD was higher than BOD; high COD/BOD5 ratioindicates that the effluent is not totally biodegradable. Thismay be attributed to a large amount of inorganic compoundspresent, which remain unaffected by microflora and henceresult in higher COD. The COD values are also useful inpinpointing toxic condition and presence of biologically re-sistant substances (Kolhe and Pawar 2011). BOD is an impor-tant indicator of organic matter which indicates the presenceof easily biodegradable compounds such as carbohydrates andorganic acids. The high values of BOD and COD indicaterespective higher levels of organic and inorganic constituentsthat are discharged into the water bodies, ultimately contrib-uting to eutrophication. Along with the BOD and COD, TDSare particularly important for the characterization of industrialeffluents and their treatment (Snehal et al. 2002). Higher levelof TDS is one of the major sources of sediments, whichreduces the light penetration into water. This ultimately re-duces the rate of photosynthesis causing a low dissolvedoxygen level, which results in decreased purification of waste-water by microorganisms.

The results also reveal a significant reduction in most of thephysicochemical parameters analyzed during the course ofbacterial treatment of 3:1 diluted tannery effluent. A decreasein TDS (21.8 %), oil and grease (64.1 %), and hardness(32.9 %) was observed after bacterial treatment. However,the decrease in BOD (62.5 %) and COD (78 %) levels

indicates the respective reduction of biologically oxidizableorganic and recalcitrant materials due to joint activities ofaugmented B. cereus and native effluent microflora. A reviewof tannery effluent characteristic in India indicates that thepollutants are of high BOD and COD values and also containsulfide, chromium, and other metals, salts and suspendedparticulate matter, etc. (Jawahar et al. 1998). Chandra et al.(2011) attributed the reduction in BOD and TS to the bacterialdegradation of complex organic pollutants for meeting thenutritional requirements.

The concentration of Cr6+ in 3:1 diluted untreated tanneryeffluent was 108±1.02 mg l−1, which was above the permissiblelimit. The concentration (milligrams per liter) of other heavymetals analyzed in diluted untreated effluent were as follows:iron (4.0), copper (0.39), arsenic (1.27), nickel (3.15), and zinc(2.15 and 1.60), of which Fe, As, and Ni were above thepermissible limits. The level of cadmium was negligible in theeffluent. Copper and zinc were detected negligible in the bacte-rially treated effluent. The levels of Fe, As, and Ni were reducedby 77, 70, and 67.6 %, respectively, after bacterial treatment.According to the Indian standards IS: 2296 and IS: 2490, thestatutory limit for the discharge of total chromium in the inlandsurface waters is 2.0mg l−1 (Central Pollution Control Board). Inour study, the bacterial treatment caused a 51.9 % reduction ofCr6+ concentration in diluted treated effluent.

Basu et al. (1997) described serious and extensive chromatepollution through the tannery effluent. In the environment,heavy metals may disperse both horizontally and vertically asthey migrate (Nikolaidis et al. 1999). Cr6+ is a strong oxidizingagent and may react with organic matter or other reducingagents to form trivalent chromium (Ramteke et al. 2010). Ushaand Kalaiselvi (2009) analyzed chromium, nickel, zinc, andcadmium levels which were found to be 193, 108, 2.5, and0.42 mg l−1 in the tannery effluent. Ramteke et al. (2010) alsoanalyzed heavy metals in tannery effluent. They reported86.98 % Cr, 100 % Cu, 70.07 % Fe, 75 % Mn, 82.57 % Ni,100 % Pb, and 58.97 % Zn removal through microbial treat-ment from initial concentrations (milligrams per liter) of 62.38(Cr), 0.13 (Cu), 6.17 (Fe), 0.26 (Mn), 1.95 (Ni), 0.03 (Pb), and0.21 (Zn), respectively, present in untreated tannery effluent.Chandra et al. (2011) found that the bacterial communitiesgrowing in aeration lagoons I and II of common tannery efflu-ent treatment plant were highly effective to decrease by 87.96,75.45, and 73.71 % of initial Cr (38.9 mg l−1), Zn (1.1 mg l−1),and Fe (7.57 mg l−1), respectively.

Tannery effluent bioremediation study

In this study, the bioremediation of unsterilized as compared tosterilized tannery effluent was performed due to four importantreasons: (1) the efficiency of unsterilized tannery effluent biore-mediation was found to be very fast under any set of conditions,(2) it is practically unfeasible to sterilize the effluent at an

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industrial-level treatment, (3) sterilization of bulk effluent wouldnot only be a tedious but also a highly cost- and energy-intensiveprocess, and (4) the native microflora will be killed after sterili-zation, which plays a significant role in the bioremediationprocess.

Bioremediation of undiluted tannery effluent

TheB. cereusRMLAU1 isolate was employed for the treatmentof tannery effluent under four sets of experimental conditions.The results in Fig. 2a–d depict a continuous increase in thegrowth, decolorization, dechlorination, and Cr6+ removal byeffluent native microflora during 96 h of incubation in tanneryeffluent, which was neither supplemented with exogenous nu-trients nor augmented with B. cereus RMLAU1 test strain (setI). A further increase in incubation time to 120 h slightlyincreased the growth, color, and Cr6+ removal, whereas, innutrient-unsupplemented but B. cereus-augmented effluent(set II), a slightly better response for all the above determina-tions was observed. The decolorization (12.9 %) was enhancedby 1.6 %, dechlorination (23.5 %) by 8.2 %, and Cr6+ removal

(7.8 %) by 2.9 % than the previous set (set I) at 96 h ofincubation. Merely augmentation with test culture in set IIimproved the growth, decolorization, dechlorination, and Cr6+

removal at the expense of indigenous nutrients present intannery effluent. The augmentation of test culture to the effluentenhanced the efficiency of bioremediation simply due to syn-ergistic utilization of natural effluent organic nutrients collec-tively by native plus augmented effluent microflora.

In set III, the effluent was supplemented with the carbon/energy cosubstrate glucose (0.4 %,w/v) and ammonium chlorideas the nitrogen source (0.2 %, w/v) without augmentation of theB. cereus test strain. The growth of native effluent microfloraincreased with time, which directly corresponded with an in-crease in the extent of effluent decolorization, dechlorination, andCr6+ removal during 120 h of incubation. Maximum and en-hanced effluent decolorization (17.6%), dechlorination (31.4%),and Cr6+ removal (10.6 %) with the corresponding highestgrowth (OD 0.079) were observed at 120 h of incubation(Fig. 2a–d, set III). Further, all the above determinations in thisnutrient-supplemented but B. cereus-unaugmented experiment(set III) were slightly higher than those in nutrient-

Fig. 2 aBacterial growth, b decolorization, c dechlorination, and dCr6+

removal of real tannery effluent with four combinations: set I, undilutedeffluent (E) at the natural pH 8.4; set II, nutrient-unsupplemented (US)effluent, but augmented (A) with B. cereus (1.0 %, v/v); set III, B. cereus-unaugmented effluent, but supplemented (S) with nutrients (w/v, 0.4 %

glucose and 0.2 % ammonium chloride); and set IV, effluent with nutri-ents supplemented (S) plus augmented (A) with B. cereus culture at thepre-optimized pH 7.0 (in sets II–IV) and incubated at 35±1 °C for 120 hunder shaking (150 rpm). Error bars are standard deviation

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unsupplemented but B. cereus-augmented (set II) experiment.This reveals that either test culture augmentation or nutrientsupplementation exerted almost a similar inducible effect onbacterial growth and effluent bioremediation, with a slightlybetter efficiency of the latter (set III). This suggests that thesupplemented nutrients stimulated the growth of native effluentmicroflora, which resulted in further enhanced efficiency.

In set IV experiment, all the above determinations werestudied during combined growth of native effluent microfloraplus augmented B. cereus (at 1.0 %, v/v) in undiluted tanneryeffluent supplemented with glucose (0.4 %, w/v) and ammo-nium chloride (0.2 %, w/v) (Fig. 2a–d, set IV). The jointgrowth of combined (natural + augmented) microflora (OD0.113) along with decolorization (19.2 %), dechlorination(33.7 %), and Cr6+ removal (12.7 %) further increasedthroughout the incubation period, which were maximum at96 h and were slightly higher than those in the previous set IIIexperiment. The results indicate that the native microflorapresent in effluent was capable of bioremediation, and theextent of biotreatment was best in nutrient-supplemented plusB. cereus-augmented tannery effluent.

Bioremediation of diluted tannery effluent

Since the efficiency of bioremediation was not much promisingin undiluted tannery effluent, it was diluted, and the determina-tions were evaluated in sets I–IVexperiments. The results in 3:1

diluted tannery effluent revealed that maximum combinedgrowth of native microflora plus augmented B. cereus (OD0.188, Fig. 3a) and decolorization (25.3, Fig. 3b) were observedin the effluent supplemented with glucose (0.4 %, w/v) +ammonium chloride (0.2 %,w/v) at 96 h of incubation, whereaseffluent dechlorination (53.2 %, Fig. 3c) and Cr6+ removal(24 %, Fig. 3d) were maximum at 120 h of incubation. Sincethe bioremediation efficiency in 3:1 diluted tannery effluentwas much better than undiluted effluent, the former was pre-ferred for further biotreatment experiments.

An appreciable extent of growth, decolorization, dechlori-nation, and Cr6+ removal in diluted effluent could be due toreduced toxicity of metals and organic compounds upon dilu-tion. Chandra et al. (2011) reported that most of the organicpollutants in tannery wastewater could be diminished bybacterial communities (Escherichia sp., Stenotrophomonassp., Bacillus sp., Cronobacter sp., and Burkholderiales bacte-rium) of common effluent treatment plant (CETP). The disap-pearance of organic pollutants indicated that bacterial com-munities growing in aeration lagoons I and II of CETP utilizedthese organics as the sole source of carbon, energy, andnitrogen. Contrary to our findings, Paisio et al. (2012) ob-served the highest growth of Rhodococcus sp. CS1 in puretannery wastewater as compared to diluted (25, 50, and 75 %)effluent. They reported complete phenol degradation (from aninitial concentration of 17.5 mg l−1) in tannery wastewater byCS1 strain after 9 h of incubation, whereas only 20 % phenol

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Fig. 3 aBacterial growth, b decolorization, c dechlorination, and dCr6+

removal of diluted (3:1) tannery effluent with four combinations: set I,effluent (E) at the natural pH 8.1; set II, nutrient-unsupplemented (US)effluent, but augmented (A) with B. cereus (1.0 %, v/v); set III, B. cereus-unaugmented effluent, but supplemented (S) with nutrients (w/v, 0.4 %

glucose and 0.2 % ammonium chloride); and set IV, effluent with nutri-ents supplemented (S) plus augmented (A) with B. cereus cultured at thepre-optimized pH 7.0 (in sets II–IV) and incubated at 35±1 °C for 120 hunder shaking (150 rpm). Error bars are standard deviation

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biodegradation was achieved by native tannery effluent mi-croflora in 9 h.

It may be summarized from the results on bioremediation ofundiluted and diluted tannery effluents that dilution of effluent isnecessary for reducing the toxicity exerted on microflora bysome indigenous constituents of the effluent. In general, the orderof maximum growth, decolorization, dechlorination, and Cr6+

removal in all four sets of experiments in undiluted and dilutedeffluent was as follows: effluent (E) + nutrients supplemented (S)+ augmented (A) > E + S > E +A > E (Figs. 2 and 3). The betterresponse of bioremediation in unaugmented effluent supplement-ed with exogenous nutrients (set III) than augmented–nutrient-unsupplemented effluent (set II) was due to the fact that whileutilizing externally added carbon and nitrogen sources, the nativemicroflora cometabolized the organic nutrients present in tanneryeffluent, thereby effecting significant bioremediation. Some bio-remediation effected in unaugmented–nutrient-unsupplemented(set I) effluent reaffirms the role of native effluent microflora inthe metabolism of organic compounds naturally present in tan-nery effluent. The extent of bioremediation was significantlyhigher in nutrient-supplemented plus augmented undiluted ordiluted effluents in all the four sets of experiments, therebyunderlining the requirement of both for efficient bioremediationof tannery effluent. Therefore, diluted (3:1) tannery effluent

augmented with B. cereus RMLAU1 strain and supplementedwith glucose (0.4 %, w/v) and ammonium chloride (0.2 %, w/v)was selected for further bioremediation experiments.

Effect of glucose concentration

In this set of experiment, the effect of varied glucose levels(0.4–1.0 %, w/v) along with fixed 0.2 % (w/v) ammoniumchloride concentration in 3:1 diluted B. cereus augmented (at1.0 %, v/v) effluent was evaluated. The growth, effluent de-colorization, dechlorination, and Cr6+ removal concomitantlyenhanced with increasing glucose concentration (0.4–0.8 %,w/v) during 96 h of incubation. The increase in incubation timefrom 96 to 120 h did not exert any significant effect on abovedeterminations. The maximum growth (OD 0.26, Fig. 4a),decolorization (34.2 %, Fig. 4b), dechlorination (58 %,Fig. 4c), and Cr6+ removal (24 %, Fig. 4d) were achievedwith 0.8 % (w/v) glucose concentration at 96 h, which werenearly similar at 120 h. Furthermore, almost similar responsefor all the above determinations was observed at 1.0 % glu-cose concentration throughout the incubation period (Fig. 4a–d). Therefore, glucose supplementation at 0.8 % (w/v) wasselected for further experiments on tannery effluent bioreme-diation. The supplementation of glucose serves as a carbon/

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optimized 0.2 % (w/v) ammonium chloride, pH 7.0, and incubated at 35±1 °C for 120 h under shaking (150 rpm). Error barsare standard deviation

Environ Sci Pollut Res

energy cosubstrate in the effluent medium that promotesbacterial growth, ultimately leading to enhanced decol-orization, dechlorination, and Cr6+ remediation from thetannery effluent.

Other researchers also reported the requirement of glu-cose supplementation for Cr6+ removal in a syntheticmedium (Garg et al. 2013; Tripathi and Garg 2013). Thechromate-reducing microorganisms may utilize a variety oforganic compounds as electron donors for Cr6+ reduction.Wang and Shen (1995) found that glucose promoted Cr6+

reduction by Agrobacterium radiobacter, B. cereus,Escherichia coli ATCC33456, and Pseudomonasfluorescens LB300. Liu et al. (2006) studied the effect ofglucose supplementation on Cr6+ reduction by Bacillus sp.XW-4 and reported a significant decrease in the level ofCr6+ (from an initial concentration of 40 to 4.24 mg l−1)during 72 h of incubation. This indicates the profound rolethat glucose plays on bacterial metabolism and on Cr6+

reduction. Sau et al. (2008) reported better growth of aBacillus firmus isolate in a glucose-supplemented medium,which, in turn, enhanced Cr6+ reduction. Masood andMalik (2011) reported 72 % Cr6+ removal by Bacillus

sp. FM1 in glucose-supplemented medium from an initialconcentration of 100 mg Cr6+ l−1.

Effect of pH

Two sets of experiments were designed. In set I, the pH of 3:1diluted tannery effluent was adjusted to 7.0 (optimized earlierfor the test culture B. cereus). The set II experiment wasperformed at the natural pH 8.1 of the diluted effluent. Boththe effluents in sets I and II were supplementedwith optimized0.8 % (w/v) glucose and fixed 0.2 % (w/v) ammonium chlo-ride, augmented with B. cereus (1.0 %, v/v) test culture, andincubated at 35±1 °C in an incubator shaker (150 rpm). Thebetter growth (OD 0.343, Fig. 5a) and bioremediation in termsof decolorization (42.3 %, Fig. 5a), dechlorination (66.1 %,Fig. 5b), and Cr6+ remediation (31.4 %, Fig. 5b) were ob-served in the set II experiment performed at the natural pH 8.1of the effluent during 96 h of incubation. Masood and Malik(2012) also employed Bacillus sp. FM1 and reported 97 %Cr6+ bioremediation (from an initial concentration of 100 mgCr6+ l−1) at the natural pH 7.9 of the effluent.

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Environ Sci Pollut Res

It appears that the adjustment of effluent pH to 7.0 may beoptimal for the test strain B. cereus, but may not be suitable forthe natural effluent microflora, ultimately leading to reducedbioremediation efficiency. Furthermore, the pH adjustment ofbulk industrial effluent cannot be an economical option.Therefore, further experiments were performed at the naturalpH 8.1 of the tannery effluent. The pH is an important indexreflecting the microbial activity and is known to modulate thespeciation of ion, cellular metabolism, and sites of interac-tions, leading to changes in accumulation and toxicity ofmetals (Deiana et al. 2007). Since Cr6+ reduction and biodeg-radation of chloroorganics are enzyme-mediated, changes inpH will affect the degree of ionization of the enzyme (s),leading to alteration in protein conformation, thereby affectingthe enzyme activity (Xu et al. 2011).

Effect of inoculum dose

The results in Fig. 6a–d depict a gradual increase in bacterialgrowth, decolorization, dechlorination, and Cr6+ removal dur-ing 72 h, when inoculum size was increased from 1.0 to 5.0 %.At every dose of inoculum under study, the growth responseand bioremediation increased with time during 72 h of incu-bation. When the inoculum size was increased up to 4.0 % (v/

v), the extent of growth, decolorization, dechlorination, andCr6+ removal increased in synchrony with each other at everypoint of time up to 72 h of incubation. However, a furtherincrease in inoculum size to 5.0 % (v/v) resulted in decreasedgrowth and other bioremediation parameters under study.Maximum growth (OD 0.357, Fig. 6a), effluent decolorization(42.5 %, Fig. 6b), dechlorination (74.1 %, Fig. 6c), and Cr6+

remediation (34.2 %, Fig. 6d) were attained with 4.0 % (v/v)inoculum at 72 h, which were close to the results obtained at60 h of incubation. Hence, a 4.0 % (v/v) inoculum dosewas selected for subsequent effluent bioremediationstudies. Further, it is evident that with higher cell den-sities, the extent of dechlorination and Cr6+ remediationwas higher.

Our findings on Cr6+ removal corroborate with theresults of other researchers, who reported a linear rela-tionship between the Cr6+ reduction and biomass con-centration (Chen and Hao 1996). Wang and Xiao (1995)reported enhanced rate of Cr6+ reduction with increasingcell density of P. fluorescens LB300 and Bacillus sp.Benazir et al. (2010) found 1.0 % (v/v) inoculum ofBacillus subtilis, which was satisfactory for a significant99.6 % Cr6+ reduction at an initial concentration of570 mg Cr6+ l−1 during 10 days of incubation.

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Fig. 6 Effect of inoculum size (1.0–5.0 %, v/v) of B. cereuson abacterialgrowth, b decolorization, c dechlorination, and dCr6+ removal of diluted(3:1) tannery effluent (at the optimized natural pH 8.1) supplementedwith

optimized nutrients (w/v, 0.8 % glucose and 0.2 % ammonium chloride)and incubated at pre-optimized 35±1 °C for 72 h under shaking(150 rpm). Error bars are standard deviation

Environ Sci Pollut Res

Bench-scale bioreactor level bioremediation of tanneryeffluent

Tannery effluent bioremediation was also performed in astirred tank bioreactor operating in a batch system, wheregood containment and environmental control may allowa fast and cost-effective treatment. In this experiment, theoptimized dose (4.0 %, v/v) of RMLAU1 strain was usedfor the bioremediation of 3:1 diluted tannery effluent(pH 8.1) supplemented with nutrients (w/v, 0.8 % glucoseand 0.2 % ammonium chloride) at an agitation speed of100 rpm and an aeration rate of 0.6 vvm. The results inFig. 7a, b reveal that the extent of decolorization, de-chlorination, and Cr6+ removal directly correspondedwith the growth of RMLAU1 strain throughout the incu-bation period up to 72 h. The efficiency of bioremedia-tion in bioreactor was higher as compared to the flasktrial during 72 h of incubation: decolorization (47.9 %)was enhanced by 5.4 %, dechlorination (77.4 %) by3.3 %, and Cr6+ removal (41.7 %) by 7.5 % at an initialcolor of 286 Pt-Co units and initial concentration of62 mg chloride ions and 108 mg l−1 Cr6+ concentrations.

Ganguli and Tripathi (1999) evaluated the ability ofPseudomonas aeruginosa strain, isolated from tanneryeffluent, to survive and reduce chromate in effluentsfrom a tannery and electroplating unit. The isolate sur-vived in the native tannery effluent, but the count sharplydeclined in both native and diluted (200×) electroplatingeffluents. Supplementation with a source of carbon, ni-trogen, and phosphorus enhanced bacterial cell numberin both tannery and diluted electroplating effluents andincreased cell numbers directly correlated with enhancedchromate reduction in both the effluents. Srivastava et al.(2007) employed a fungal strain (viz., Aspergillus nigerFK1) and a bacterial culture (viz., Acinetobacter sp.)isolated from pulp paper mill sludge individually forthe bioremediation of chromium and PCP from tanneryeffluent in a sequential bioreactor. The tannery effluenttreated in set 1, initially with Acinetobacter sp., followedby fungus treatment, remediated 90 % of Cr6+ (from aninitial chromium level of 557 ppm) and 67 % of PCP(from an initial PCP level of 15 ppm) in 15 days. In theset 2 sequential bioreactor, wherein the effluent was firsttreated with the fungus and then by the bacteria, only64.7 and 58 % of chromium and PCP was removedwithin 15 days. The higher level of chromium removalin the set 1 bioreactor was attributed to the utilization ofPCP as a food source in step 1 by Acinetobacter sp.,thereby exerting no inhibitory effect of PCP on fungusfor removing Cr6+ in step 2. However, in the set 2bioreactor, the growth of the fungus was inhibited byPCP in step 1, thereby decreasing the extent of chromi-um removal, which led to the bioaccumulation of Cr6+ inthe fungal mycelium. Zhao et al. (2012) used B. cereusto remove Cr6+ from tannery wastewater medium andobserved maximum Cr6+ removal rate at 37 °C andpH 7.0–9.0 with an initial Cr6+ concentration of<50 mg l−1 during 48 h of incubation.

Enzymatic study and Cr distribution

The chromate reductase activity in treated tannery effluent(3:1 diluted) from bioreactor trial was measured to assess theenzymatic conversion of Cr6+ to Cr3+ by augmented B. cereusRMLAU1 plus native effluent microflora. Table 1 reveals thepresence of enzyme activity in culture supernatant, in cytosol-ic fraction, and in the cell debris of augmented B. cereusRMLAU1 strain plus native tannery effluent microflora. Themaximum enzyme activity in cytosolic fraction (57.9 %) wasfollowed by the activity in culture supernatant (39.1 %) andcell debris (3.0 %). This shows that chromate reductase activ-ity was mainly associated with the soluble fraction of bacterialculture. However, no enzyme activity was observed in control(boiled) samples, thereby indicating denaturation and henceinactivation of chromate reductase enzyme.

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Environ Sci Pollut Res

Table 2 reveals the extent of reduced chromium (Cr3+)distribution in culture supernatant and bacterial biomass ofB. cereus plus effluent native microflora. The reduced chro-mium was present both in culture supernatant and bacterialbiomass. The results further indicate that out of the total Cr6+

remediated (45 mg of an concentration of initial 108 mg l−1,41.7 %) from effluent medium at 72 h, 78.2 % Cr6+ wasreduced to Cr3+, of which 18.6 % was detected in the culturesupernatant and 59.6 % in the bacterial biomass. The distri-bution of Cr3+ in the culture supernatant and bacterial biomass(Table 2) correlates directly with the extent of chromate re-ductase activity in culture supernatant and cytosolic fraction(Table 1).

Other researchers also reported chromate reductase activityin soluble cell-free extract during aerobic Cr6+ reduction(Camargo et al. 2003; Pal et al. 2005). Ilias et al. (2011) foundchromate reductase activity in the culture supernatant and celllysate (debris) only, but no activity in the cell extract superna-tant (cytosolic fraction) of Staphylococcus aureus andPediococcus pentosaceus isolates. They reported lesser spe-cific enzyme activity of 0.033 and 0.053 μM Cr6+

reduced min−1 mg−1 protein in cell lysate and culture super-natant, respectively, as compared to specific activity observedin our study (Table 1). Conversely, Rida et al. (2012) reporteda cytosolic fraction of Ochrobactrum intermediumRB-2 cellsmainly associated with chromate reductase activity. It can bededuced from the foregoing that distribution of chromate

reductase enzyme in different cell fractions varies from organ-ism to organism.

GC-MS analysis

The GC-MS analysis of untreated and bacterially treatedtannery effluent revealed the presence of benzene propanoicacid (RT = 17.7), pentachlorophenol (RT = 20.0), 3-methoxybenzaldehyde (RT = 22.0), and octadecadienoic acid (RT =26.6) in untreated tannery effluent, whereas in bacteriallytreated tannery effluent, smaller peaks of pentachlorophenol(RT = 20.0) compared to untreated effluent, acetic acid (RT =15.2), 2-butenoic acid (RT = 23.5), and octadecadienoic acid(RT = 26.6) were detected; the smaller peak of PCP in chro-matogram of bacterially treated sample revealed its degrada-tion after bacterial treatment (not shown).

Chandra et al. (2011) also studied the characteristics oforganic pollutants and their metabolites of untreated and treat-ed tannery effluent. They reported 3-methoxy-4-bezaldehyde,butanedioic acid, lactic acid, propanoic acid, etc. in untreatedeffluent and lactic acid, acetic acid, benzene propanoic acid, 3-hydroxy propanoic acid, benzene, and 2-hydroxy-3-methyl-butanoic acid in bacterially (bacterial communities of aeratedlagoons) treated tannery wastewater. The disappearance ofmost of the organic pollutants during bacterial treatment re-vealed that bacterial communities of aeration lagoons I and IIutilized these pollutants as the sole source of carbon, nitrogen,

Table 1 Chromate reductase activity in different cell fractions of B. cereus plus native microflora in bioreactor trial

Cell fraction Total chromate reductaseactivity (μM min−1)

Chromate reductaseactivity (%)

Total protein (mg) Specific activity(μM min−1 mg−1 protein)

Culture supernatant 2.5 39.1 1.05 2.4

Cytosolic fraction 3.7 57.9 0.58 6.3

Cell debris 0.19 3.0 0.12 1.5

Table 2 Distribution of reduced chromium (Cr3+) between culture supernatant and bacterial biomass during Cr6+ remediation by B. cereus plus nativeeffluent microflora in bioreactor trial

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removalCulture supernatant Biomass Total reduced Cr

(Cr3+) (%)Total Cr (mg l-1) Residual Cr6+

(mg l−1)Reduced Cr(Cr3+)

Total Cr(mg l−1)

Cr6+ Reduced Cr(Cr3+)

mg l−1 % mg l−1 % mg l−1 %

24 20.3 91.5 86.0 5.5 25.0 10.4 ND ND 10.4 47.3 72.3

36 33.3 81.7 72.0 9.7 27.0 15.2 ND ND 15.2 42.2 69.2

48 37.0 74.9 68.0 6.2 15.5 23.7 ND ND 23.7 59.2 74.7

60 39.7 73.2 65.0 8.2 19.0 25.1 ND ND 25.1 58.4 77.4

72 41.7 71.4 63.0 8.4 18.6 26.8 ND ND 26.8 59.6 78.2

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ND not detectable

Environ Sci Pollut Res

and energy (Chandra et al. 2011). Our results also indicate thatB. cereus along with native effluent microflora utilized PCPand other organic pollutants as carbon and energy sources andplayed a vital role in the bioremediation of tannery effluent.

Bioremediation of tannery effluent by immobilized biomass

This experiment was performed to evaluate the tannery efflu-ent bioremediation ability of immobilizedB. cereuscells usingsodium alginate (4.0 %, w/v) as the support material undershake flask trial. Table 3 reveals maximum decolorization(43.5 %), dechlorination (58 %), and Cr6+ removal (44.5 %)at an initial color of 285 Pt-Co units, and initial concentrationof 62 mg chloride ions and 108 mg l−1 Cr6+, with free nativemicroflora plus immobilized B. cereus biomass at 72 h ofincubation with almost similar response of all the determina-tions at 60 h of incubation. The results further reveal that theimmobilized cells were repeatedly used up to four cycles bythe alginate immobilized biomass. The bioremediation ability

decreased after every cycle and has the minimum in the fourthcycle. The decrease in Cr6+ removal efficiency after everycycle (Table 3) could be attributed to the loss of bead integrity,leading to disintegration of matrix. Srinath et al. (2003) alsoevaluated the efficiency of agarose-immobilized Bacilluscoagulans biomass for the removal of Cr6+ from the tanneryeffluent of combined treatment plant (CETP), which resultedin the complete remediation of Cr6+ (from an initial concen-tration of 1.05±0.09 mg Cr6+ l−1) within five cycles. Theimmobilized biomass was contacted with 100 ml of tanneryeffluent in 250-ml Erlenmeyer flasks at pH 2.5 and incubatedat 28 °C (150 rpm) for 2 h (Srinath et al. 2003).

When free cells are employed for bioremediation, theyoften suffer from excessive Cr6+ toxicity and cell damage.Furthermore, in commercial processes, there may be problemsassociated with the physical characteristics of the cells such assmall size, low density, poor mechanical strength/rigidity, andsolid–liquid separation (Garg et al. 2012). Free cells may,therefore, be more prone to toxicity from chromate and othermetals as compared to immobilized cells, the latter of whichmay have some protection from the toxic compoundspresent in the effluent (Poopal and Laxman 2008).

Table 3 Decolorization, dechlorination, and Cr6+ removal in diluted(3:1) tannery effluent (at the natural pH 8.1) supplemented with 0.8 %(w/v) glucose and 0.2 % (w/v) ammonium chloride, augmented withimmobilized biomass of B. cereus, and incubated at 35±1 °C undershaking (150 rpm) conditions

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I II III IV I II III IV I II III IV

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24 238 252 264 261 77 73 75 71 83 90 95 102

36 195 217 228 235 91 85 82 83 67 83 88 97

48 179 194 201 198 94 91 87 85 65 79 84 93

60 168 187 196 193 97 95 90 87 62 77 82 91

72 161 181 188 192 98 94 91 86 60 76 83 88

Standard deviation values of calculated data are <5.0 %

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Environ Sci Pollut Res

However, when immobilized biomass of coculture(B. cereus RMLAU1 plus P. putida SKG-1) was employedfor the bioremediation of tannery effluent in bioreactor, abetter response for all the above determinations was observed(Fig. 8), as compared to single B. cereus immobilized biomass(Table 3). The enhanced extent of decolorization (51 %),dechlorination (69.3 %), and Cr6+ removal (51.9 %) at aninitial color of 286 Pt-Co units and initial concentration of62 mg chloride ions and 108 mg l−1 Cr6+, observed at 72 h,was very close to all the determinations at 60 h of incubation.

Summarily, the comparative statement of outstanding find-ings during shake flask, bioreactor, and immobilized coculturetrials is summarized in Fig. 9. The extent of comparativebacterial growth, decolorization, dechlorination, and Cr6+ re-mediation of 3:1 diluted tannery effluent by B. cereusRMLAU1 strain is arranged in the following order duringrespective flask level, bioreactor level, and immobilized co-culture (B. cereus + P. putida) optimization processes: growthOD 0.357 < 0.448 < 0.482, percent decolorization 42.5 < 47.9< 51.0, percent dechlorination 74.1 < 77.4 > 69.3, and percentCr6+ remediation 34.2 < 41.7 < 51.9. The results on tanneryeffluent remediation reveal that addition of P. putida SKG-1strain enhanced the bioremediation efficiency of B. cereusRMLAU1 strain. There is not even a single report on tanneryeffluent decolorization, dechlorination, and simultaneous Cr6+

removal by immobilized biomass.

Conclusions

In the present study, the bottlenecks in the tannery effluenttreatment process are being addressed. The native B. cereusstrain isolated from tannery effluent has been, for the firsttime, employed for the simultaneous bioremediation ofPCP and Cr6+ from the real tannery wastewater. The avail-ability of easily metabolizable carbon/energy and nitrogensources is scanty in real tannery effluent. Therefore, sup-plementation with such nutrients was expected to increasethe efficiency of bioremediation process; the same hasbeen experimentally proved in this study. The real tanneryeffluent contains many toxicants including variety ofheavy metals and chlorinated phenolic compounds. Thefree bacterial cells in tannery effluent often suffer fromcell damage due to excessive Cr6+ and PCP toxicity. Theimmobilized cells may have protection from toxic com-pounds present in the tannery effluent. Our findings haverevealed, for the first time, that the extent of bioremedia-tion was better with immobilized bacterial biomass ascompared to free cells. A significant decrease in physico-chemical parameters, color, chloroorganics, Cr6+, and othermetals of tannery effluent, after B. cereus treatment, indi-cates its possible use for in situ application.

Acknowledgments The authors express sincere thanks to the Sophis-ticated Advanced Instrumentation Facilities, Indian Institute of Technol-ogy, Bombay, India for the GC-MS analysis. Facilities provided by theGovernment of Uttar Pradesh under Centre of Excellence and Govern-ment of India’s DST-FIST schemes are duly acknowledged.

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