Indian Journal of Experimental Biology Vol. 41 , September 2003 , pp.986-990

Biosorption and elution of chromium from immobilized Bacillus coagulans biomass

T Srinath I , S K Garg2 & P W Ramteke3*

I Environmental Microbiology Section, Industria l Toxicology Research Centre, Lucknow 22600 I , Indi a. 20epartment of Microbiology , Or. R. M. L. Avadh University , Faizabad 224 00 I, India.

30epartment of Biotechnology, Allahabad Agricultural Institute, Allahabad 211007, India

Bacillus coaguial1s, a tannery wastewater isolate, previously shown to bind di ssolved Cr(VI), retained its ability to biosorb Cr(VI) in different matrices. Polymeric materials like agar, agarose, calcium alginate and polyacrylamide were screened. Agarose emerged as the suitable candidate for biomass immobilization mainly due to its higher sta bility and integrity in acidic pH. Aptness of agarose as the matrix for B. coaguial1s biomass was re vealed during Cr(VI) biosorptio n from natural wastewater.

Keywords : Bacillus coaguial1s, Biosorption, Chromium (VI ), Elution, Immobilized biomass

Studies during past two decades have shown that metal-microbe interaction for bioremediation of heavy metals contaminants are broadly of three types. First is biosorption , where metal uptake is metabolism­independent I and it is performed by both living and dead cells. Second is bioaccumulation, which often compri ses two stages2

. The initial phase involves rapid physical adsorption or ion exchange at cell surface. In the subsequent phase via active metabolism, metal is transported into cells. Third is resistance detoxification mechani sms, involving ox idati on/reducti on to a less toxic form 3

. Recently, genetic engineering of cells to alter morphological and phys iolog ical features have widened the scope of microbes based bioremediation4

.5

.

Chromium is considered an essenti a l nutrient in trace amo unt , however it is toxi c above permissible limits6

. Chromium is s table in trivalent (III) and hexavalent (Vi) states and the former is 1000 times less toxic to latter due to low solubility. Standard limit for di scharge of Cr(VI) in the inl and surface waters is 0.1 mg rl in India (IS: 2296 and IS: 2490{ In tanning industry, chromium is extensively used to convert raw sk in/hide to non-putrefying leather and a large amount of chromium-laden effluent is di scharged into the environment8

. Use of methods like reduction , prec ipitati on and ion exchange have economic and technical constra ints in a developing natio n like Indi a. Thus, conventi onal aerobic/anaerobic, common ---_._--*Corrcspondenl aUlhor: E-mai l: pwramteke @yahoo.com.

Fax : +9 1-0532-2684295

effluent treatment plants (CETPs) are used to reduce Cr(VI) concentration. However, the achieved level is still toxic to flora and fauna.

Previous research9.,o

has demonstrated that under acidic conditions (PH 2.5) , Bacillus coagulans biomass biosorbed maximum of 62.11 mg Cr g. 1 dry weight. Selective removal of Cr(VI) from multi-metal solution, broad range of operational temperature and e lution of chromium with regeneration of biomass makes B. coagulans biomass a suitable candidate for biosorption of Cr(Vl) from tannery effluent. However, to bioremediate effluent economically in large scale, immobili zation of biomass is essential for easy separation of biomass from effluent and easy operation of repeated cycles of bi osorption and elution ' I. ' 2. The objective of the present study was to investigate the kinetics of Cr(V I) uptake by immobili zed biomass and its operational stability with CETP effluent.

Materials and Methods Preparation of chemicals and biomass - The

chromium (VI) stock solution (10,000 mg r' ) was prepared by the di ssolution of K2Cr20 7 in di stilled and de ionized water. Stock solution was diluted with de ioni zed water to obtain working solutions and des ired pH was obtained by adju sting with 0.1 M NaOH and O. IM HN03. For B. coagu/a/1s biomass preparation, late exponential phase cells were collected by centrifugation (10,000 rpm, 30 min) and then the pelleted biomass was washed thoroughl y

I ~

SRINATH et al.: BIOSORPTION & ELUTION OF CHROMIUM 987

with distilled water. This biomass was washed with distilled water acidified to pH 2.5 (H2S04) until the pH of the washed water showed no change9

. Biomass was then dried in oven (80°C) and later crushed in a blender and sieved through a 14-16 Briti sh standard size mesh (1.2-1.6 mm) .

Metal analysis-Chromium (VI) contents in aqueous samples were determined by measuring optical den sity of the purple complex of Cr(VI) with 1,5-diphenylcarbahydrazide at 540 nm by UV spectrophotometer l3. Chromium content in biomass­matrix was determined using Perkin-Elmer 5000 atomic absorption spectrophotometer (AAS) at 357.9 nm after digestion of samples with a mixture of concentrated nitric (six parts) and perchloric (one part) acid. The AAS was calibrated using Cr(VI) working standards in the linearity range prepared from stock Cr(VI) standard of 1000 mg rl. Blank and spiked samples were also processed simultaneously and analyzed.

Immobilization- Various polymeric materials with or without biomass were evaluated for Cr(VI) biosorption. For each immobilized preparation, 0.2 g of biomass was entrapped in 1 g of matrix used. The preparations were obtained as follow s:

Calcium alginate - The ionotropic method of Lopez et a1. 14

, where sodium alginate (2%) was dissolved in hot distilled water with constant stirring. At room temperature , B. coagulans biomass was added under stirring condition for even dispersal. The slurry solution was dispersed drop wise into 0 .5 M CaCI2. Instantaneous spherical gel beads formation occurred at the drop-solution interface as the alginate was cross-linked by Ca2

+. The gel beads (3-4 mm diam.) were allowed to cure for 2 hr at 4°C and were washed thoroughly with distilled water.

Agar- The agar solution (2%) was prepared by dissolving it in distilled water at 90°C I4. The biomass was added at room temperature and evenly dispersed by stirring. Spherical beads were obtained on dropwise addition of slurry into a hydrophobic liquid phase (sunflower oil, Saffola) over distilled water. The beads were collected and then washed with 0 .01 % Triton X-lOO to eliminate residual oil phase ls.

Agarose - 6% of agarose was prepared by dissolving in distilled water at 90°C, cooling to room temperature 16. The biomass was evenly distributed in it to make slurry and then rapidly poured into petri plates kept on ice. Later on, 3 x 3 x 3 mm3 cubes were cut and were washed with distilled water.

Polyacrylamide - Stock solution of acrylamide/ [N,N'-methylenebis (acrylamide)] (MBA) was prepared by dissolving 60 mg of acrylamide and 1.6 mg MBA in 100 ml of distilled water l7. From thi s stock solution, 0 .833 ml was added to 3 .984 ml of degassed water. Then 0.083 ml of ammonium persulfate solution (0.5 g mrl degassed water) and 0.1 ml of N,N,N',N'-tetramethylethylenediamine (TEMED) was added and mixed. Thi s slurry was rapidly poured into petri pl ates kept on ice. Later on , 3 x 3 x 3 mm3 cubes were cut and were washed with distilled water.

These washed matrices were pH-conditioned with repeated washing with acidified deionized water (PH 2.5, H2S04) until the pH of the washed water showed no change. These matrices were stored at 4°C till use.

Metal sorption studies-A batch equilibrium method was used to determine the biosorpti on of

. Cr(VI) by immobilized biomass of B. coagulans. Matrix (l g) was contacted with 100 ml of Cr(VI) solution ( 100 mg rl) in Erlenmeyer flask s for 24 hr at 28°C at 150 rpm. The Cr(VI) concentration in bulk solution was monitored with time. In order to obtain the sorption kinetics data, the chromium uptake value was calculated using the following equation:

- I where, Qeq is the biosorption capacity (mg g biomass), V is the volume of solution (I) , C j is the initial concentration of metal in solution (mg r l), Ceq is the residual concentration of metal in solution (mg rl), and M is the weight of matrix-biomass (g) .

The kinetic rate constant k (min-I) data was obtained from Lagergren's equation I8.19:

log (Qeq-Q) = log Qeq-[ktl2 .303]

Chromium biosorptionleiution cycles-After 2 hr of batch equilibration, the matrices were collected in a screen sieve. The external surface liquid on beads was removed with a paper towel. For elution, the beads were contacted with 500 ml of 0 .1 M H2S04 for 24 hr in shaking ( 150 rpm). The chromium content eluted was analysed by AAS . Following the elution , the immobili zed biomass was washed with 0 .1 M CaCh + 0.1 M MgS04 for 15 min 10.20. Then the immobilized biomass was reconditioned by repeated washing with deionized water, acidified to pH 2.5 by H2S04. This regenerated immobilized biomass was used for next biosorption-elution cycle.

Biosorption of Cr(VI) from tannery effluent- The CETP effluent (1.05 ± 0.09 mg Cr(VI) rl ) was used to

988 INDI AN J EX P BIOl , SEPTEMBER 2003

assay the potentia l of immo bili zed bio mass of B. coagulans for biosorption of Cr(YI) . The immobili zed biomass (l g of matri x) was contacted with 100 ml of eftl uent in 250 ml of Erlenmeyer fl asks. Before additi on of the matrix, the p H of the e fflu ent was adju sted to 2.5 . The fl asks were ag itated ( 150 rpm) for 2 hr at 28°C. For next round of biosorption, matri ces were sieved from bulk solutio n, rinsed with deioni zed water and re introduced into another set of 100 ml of effl uent. The concentratio n o f C r(YI ) in bulk solution was analyzed after each round of ex posure. This reintroducti on step was repeated till the Cr(YI) concentrati on in bulk solutio n was equivalent to the in iti al leve l.

Results and Discussion Screening of a suitable matrix fo r immobilization

of B. coagulans biomass- The present study was to identi fy a suitable matri x fo r the immobilization of B. coagulans bio mass. Biosorption C r(YI) by free B. coagulans bio mass was compared with biomass immobili zed in diffe rent matrices (Table I). Biosorptio n of Cr(YI) is hi ghly influenced by pH and max imal biosorption was found at pH 2.59

, thus stabi lity of the matri x during bi osorpti on was essential wi thout hampering Cr sorption effi c iency. Immobi­li zation o f biomass in agarose and po lyacry lamide had least effec t on C r(Y I) sorption (Fig. I ) and matri x was highl y stable (T able 1). Similarl y, Uchiyama et al. 16

fo und that agarose mainta ined its integrity at acidic pH . However, agar and calcium alginate showed low stab ility and Cr(Y I) biosorption. Owing to its catio nic cross-lin k nature, a lginate lost its integrity due to the presence of anion like chromate and ac idic conditions 17 .

Cr (VI) biosorption/ellition cycles by bion!{[ss ­agarose - The p H stability of agarose and polyac rylamide and retention o f Cr(YI) bi osorption

Table I - Chrom ium sorption ( 100 mg Cr(V I) 1" ) by B. coagulans biomass in free (0.2 g) and immobili zed

Slate (0.2 g biomass g- ' matri x) at p H 2.5 for 2 hr

Matrix Cr biosorbed k (min" )" Correlati on

(mg g" dry wt) coeffic ient (r2)

Free biomass 40.6 1 0.065 0.94 Agar 33.43 b 0.048 0.89 Calc ium alg in ate 35.43 b 0.0976 0.6 1 Agarose 40.56 0.057 0 .90 Polyacry lam ide 40.58 0.085 0.89

, Lagergren rate constant val ues were calcu lated from linearised equation of the model. b Matrix parti al ly broken.

effici ency made them a suitable candidate for bi omass immobili zation. Agarose and po lyac ry lamide a re known to provide good res istance to hydrostati c pressure and mechanical degradation21

, polyacryl­amjde is comparatively less res istant to the mechanical stress 17. Thus, agarose was opted as immobili zation matrix for further studies. After 24 hI' equi libratio n o f bio mass-agarose with Cr(YI) solution, around 70% of bound chromium was e luted (Fig. 2) whereas the e lution effic iency of free bio mass was 86 . 11 %10. The low interacti on of bound Cr(Y I) with e luent and slow reduction of Cr(YI ) may cause thi s lowering in e lution to Cr(IJI)22. It is also poss ible that this reduced chromium was not leave matrix due to obstruction or low solubility, During second cycle o f biosorption­e lution, C r(YJ ) sorptio n and elutio n was comparable to that of first ex posure. Around 80% of Cr(Y l) sorption effi ciency was retained by biomass-agarose but Cr e lution was less than 50%. Durin g the fifth cycle, the matrix started to loose its integrity .

Biosorption of Cr(VI) by biomass-agarose from CETP ejjluenl - Evaluation o f the efficiency of the agarose immobili zed B. coagulans biomass fo r remo val of Cr(YJ) from CETP treated effluent is depicted in Fig. 3. These immobilized bi omass were able to remove C r(YI) complete ly for five rounds. Compl ete removal o f C r(YJ) sixth round o nwards not seen although the matri x was stable up to e ighth round

50

40

... ~ 20 S - Free bio mass

-0- Agar --- Calcium a lg inate ~ Agarose --- Po lyacrylam ide

o +---- -,------,- - -----. o 50 100 ISO

Time (min)

Fig. I - Time course of Cr(V I) biosorption ( 100 mg 1" ) at pH 2.5, 150 rpm, 28°C by B. coagu/mls biomass in free (0.2 g) and immobilized state (0.2 g biomass g - , matri x).

SRINATH el al.: BIOSORPTION & ELUTION OF CHROMIUM 989

o o First Second Third Fourth Fifth

Cycles

Fig. 2 - Influence of biosorpt ion/elut ion cycles on Cr(VI) bio­sorption by B. coagu!ans biomass-agarose, matrix stability and elution effic iency in each cycle. Immobili zed biomass (0.2 g bio­mass g.1 agarose) was equilibrated wi th 100 mg Cr(VI) 1"1 for 2 hr at 150 rpm, 28°C. Chromium in solu tion and eluent (0.1 M H2S04,

500 ml ) after 24 hr contact with immobili zed biomass loaded with Cr. These immobili zed biomass were washed with 0. 1 M CaCI2 + 0.1 M MgS04 for 15 min . The regenerated immobilized biomass was pH conditioned (2 .5 by repeated washing with acidified de­ioni zed water) and then subjected for next round of biosorption­elution cycle. + : matrix intact; ± : matrix partially broken .

120

+ + + + + 100

"C

" ~ 0 C 80 "

+

.. ~ 60 .. + u ..... 0

C 40 " ± '" ... OJ

Q.,

20

± 0

Fig. 3 - Biosorption of Cr(VI) from CETP e ffluent by B. coaguians biomass-agarose (0.2 g biomass g.1 agarosc). I g of biomass-agarose was equilibrated with 100 ml of e fflu ent (pH 2.5) for 2 hr at 28°C, 150 rpm . For nex t round of exposure, the biom:1ss-agarose was sieved out, rinsed with deioni zed water and subjected to another round of exposure + matrix intact; ± : matri x p:1rtially broken .

of biosorption . Dias et al .23 used a commercial ion­exchange resin, Dowex-50WX2 (Aldrich) to immobilize a distillery waste mass from stainless steel industry effluent. In comparison to the free biomass, which biosorbed 39.5 mg Cr g.t, the biomass in Dowex biosorbed 24.5 mg Cr g, t dry wt. When chitin was used as the matrix, only 2.5 mg Cr g' ) of chitin was biosorbed. In the contrary to their observations, Muzzarelli24 and Masri et al?5 found that chitin exhibited chromium biosorption property due to protonation of nitrogen-containing functional groups (amines in free or substituted forms) in an ac idic solution26

.

As evident from the present studies, use of agarose as the suitable matrix for B. coagulans biomass immobilization for biosorption of Cr(Vl) from effluent. Further tests on removal of Cr(VI) from organic and inorganic effluents containing higher concentration of Cr(Vl) will evaluate the broader applicability of B. coagulans biomass-agarose.

Acknowledgement The authors would like to thank Dr. P. K. Seth,

Director, Industrial Toxicology Research Centre, Lucknow for his constant encouragement. T. Srinath is thankful to Department of Biotechnology, Government of India, for financial assistance.

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