Bioremediation of hexavalent chromium in soil microcosms P.B. Salunkhe, P.K. Dhakephalkar and K.M. Paknikar* Division of Microbial Sciences, Agharkar Research Institute, G.G. Agarkar Road, Pune 411 004, India. * Fax: 191–212–351542, E-mail: paknikar@giaspn01.vsnl.net.in Pseudomonas mendocina when added in soil microcosms could immobilize 100 μg (2 mM) chromate/g soil in 8 h by converting it into trivalent form. The chromate-contaminated soils, after microbiological treatment, supported growth of wheat seedlings without exerting any toxic effects. The method is potentially useful in the bioremediation of chromate-contaminated sites. Introduction Hexavalent chromium is the major chromium species used in industry and hence is the common pollutant in a variety of industrial wastes, viz. sludge, fly ash, slag, etc. Often such wastes are used as a fill material at numerous locations to reclaim marshlands, for tank dikes, and for backfill at sites following demolition. Cr(VI) levels of more than 75 mg/kg have been reported in the soils at many such sites all over the world. Leaching and seepage of Cr(VI) from the soils into groundwater is a potential health hazard because Cr(VI) is a known carcinogen and mutagen in humans and animals (Roe and Carter, 1969; Enterline, 1974). Cr(VI) in soil can also be dissolved by sweat on exposed skin and such persons could become sensitized to allergic contact dermatitis (Kligman, 1966). Further, it is not possible to grow vegetables at or near Cr(VI)- contaminated soils as chromium has been shown to cause plant growth reduction due to root damage (Anon, 1974). In the light of these concerns, suitable methods need to be developed for the remediation of Cr(VI)-contaminated soils. In our laboratory, a microbiological process for the removal of chromium from industrial waste waters was developed (Bhide et al., 1996; Rajwade and Paknikar, 1997) and licensed to an environmental engineering company for commercialization. The present study attempts to evaluate the potential of this technology for the bioremediation of Cr(VI)-contaminated soils. Materials and methods Chromate reducing culture The chromate reducing bacterial culture used in the pres- ent study, Pseudomonas mendocina MCM B-180 was isolated from a sewage sample by enrichment culture technique. It was grown in EG medium (composition in g/l: NH 4 Cl, 0.03;, K 2 HPO 4 , 0.03; KH 2 PO 4 , 0.05; MgSO 4 .7H 2 O, 0.01; CH 3 COONa, 2.0; yeast extract, 0.15; peptone, 0.5; pH 7.5), unless indicated otherwise (Bhide et al., 1996). Soil microcosms A field soil sample was collected in bulk quantity, sieved (1.25 mm mesh) and stored in plastic bags at 4°C. The physical-chemical properties of the soil were: pH 8.21, total carbon 1.7%, total nitrogen 0.15%, total phosphate 0.01%. Microcosms used in the bioremediation experi- ments consisted of glass test tubes (1 cm diameter, 9.5 cm length) containing 1 g of dried soil. The tubes were plugged with non-adsorbent cotton and autoclaved (121°C, 20 minutes) for three consecutive days. Cr(VI) bioremediation experiment Soil microcosms were dried at 40°C for 24 h. To adjust the desired experimental parameters, addition of nutrient solu- tion, chromate stock solution and inoculum was made in such quantities that the final values of moisture content (w/w, on dry weight basis) ranged between 40–100%; inoculum size of P. mendocina was between 10 5 cells/g soil to 10 8 cells/g soil; and chromate concentration was 2 mM. To determine the suitable additive(s) for achieving desir- able chromate reduction efficiency, the soil microcosms were set up with inoculum size of 10 6 cells/g soil and the moisture content was adjusted to 100% level, with the help of any one of the test media (Table 2). Chromate reduction efficiency was calculated on the basis of residual Cr(VI) content in the soils (as estimated by diphenyl carbazide method, APHA, 1985). All the experiments were carried out in triplicates and repeated twice. Growth of wheat in soil microcosms To assess the effect of chromate bioremediation on the growth of wheat (Triticum vulgare), experiments were per- formed using the soils treated and untreated with P. mendocina in microcosm system described above. A statis- Biotechnology Letters, Vol 20, No 8, August 1998, pp. 749–751 © 1998 Chapman & Hall Biotechnology Letters ⋅ Vol 20 ⋅ No 8 ⋅ 1998 749