Evaluation of heavy metal tolerant bacterial isolates for effluent treatment

Establishment of numerous small and medium-scale industries in Punjab has lead to environmental pollution with toxic heavy metals that may have negative consequences on the hydrosphere. A collective treatment of this waste at a centralized facility known as Common Effluent Treatment Plants (CETPs) can go a long way in minimizing the level of heavy metal contaminants. Metal remediation through conventional technologies in these CETPs is often inefficient or very expensive, whereas, bioremediation by using heavy metal resistant microorganisms has received a great deal of attention for its potential application in industry. The present study was envisaged with the objective to isolate indigenous heavy metal tolerant bacteria from effluents collected from CETPs and their subsequent utilization for heavy metal uptake or metal reduction from more toxic oxidation state to the less toxic one. Effluent samples were collected from two working CETPs located in Ludhiana (sample 1) and Jalandhar (sample 2) and were analyzed for their physico-chemical parameters (dissolved oxygen (DO), biological oxygen demand (BOD), chemical oxygen demand (COD) and pH) and heavy metal profile using Inductively Coupled Argon Plasma-Emission Spectroscopy (ICAP-AES). In both the samples Cr was found to be the dominant metal contaminant. Out of twenty one bacterial isolates obtained from effluent samples, ten morphologically distinct isolates were tested for minimum inhibitory concentration (MIC) against six different heavy metals i.e. As, Cr, Hg, Ni, Pb and W by supplementing the Luria Bertani (LB) agar media with increasing dose of heavy metal salts (5-100 ppm). Four isolates (HM 2, HM 3, HM 15 and HM 16) showed maximum tolerance to selected five different heavy metals except mercury (Hg). These isolates were biochemically characterized, whereby, HM 3 and HM 16 were closely related to Enterobacter, and HM 2 was found to be related to Klebsiella. Optimum growth of selected isolates was obtained at pH 9.0 and temperature 40°C. Of these four isolates, only HM 2 and HM 16 showed comparable growth rate and in-vitro compatibility and were selected as potent candidate for consortium. Heavy metal uptake by these isolates was determined by measuring the concentration of heavy metals in effluents with and without inoculations after 5 and 10 days of growth. There was no active uptake of Cr and other heavy metals in sample 1 with or without adjustment of pH and supplementation with nutrients (DGlucose, MgSO4 and KNO3). Though, heavy metal uptake was observed in sample 2. Both the isolates were tested for their ability to reduce Cr (VI) to Cr (III) and compared with a standard strain Shewanellaputrefaciens(MTCC 8104). Chromium reduction efficiency was determined in LB media containing Cr (VI) @ 20ppm by S-diphenyl-carbazide (DPC) method, whereby complete reduction was observed with the standard culture followed by 76.66% by HM 16 and 46.76% by HM 2 after 7 hours of incubation. Maximum Chromate Reductase activity (HM 2 – 0.161 U, HM 16 – 0.163 U and S. putrefaciens– 0.175 U) was reported in cell extract supernatant in both the isolates and in standard culture. This study elucidated the potential of resident microbes of effluents/sludge for heavy metal removal.