Studies on Metallo-Resistant Microorganisms for Remediation and Monitoring of Heavy Metals in Industrial Waste

Abstract

In the current study, we investigated the role of metallotolerant microorganisms in remediation and detection of heavy metals like cadmium and mercury. Metallotolerant microorganisms were isolated from metal polluted soil. Bacterial isolates were screened for maximum mercury tolerance and isolate exhibiting highest mercury tolerance was selected for mercury bioreporter construction. Fungal isolates were screened for cadmium tolerance and organic acid production to select most putative strains for heavy metals bioremediation through bioleaching and biosorption. Selected bacterial and fungals isolates were identified by 16S rRNA and internal transcribed spacer (ITS) sequence phylogenetic analysis, respectively. Optimal growth conditions like pH and temperature of selected isolates were also studied. Carbon source was optimized to produce maximum amount of various organic acids that were characterized and quantified through high performance liquid chromatography (HPLC) and applied for leachig of heavy metals from metal contaminated soil. Elemental analysis in the entire study was carried out through atomic absorption spectroscopy. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) was performed to evaluate biosorption of cadmium by selected fungal strain. The localization of taken up cadmium by resistant fungi was studied through transmission electron microscopy (TEM) and electron energy loss specroscopy (EELS). The mercury resistant bacterial isolate DWH4 was engineered to serve as a bioluminescent bioreporter for mercury toxicity by linking its merR-like gene and promoter sequence to a reorganized luxABCDE gene cassette from Photorhabdus luminescens through recombinant DNA technology. A total of 10 bacterial and 13 fungal strains were isolated from soil samples collected from Misri Shah and Daroghawala. Out of 10 bacterial isolates, strain DWH4 exhibited highest tolerance to mercury and was selected for construction of mercury bioreporter cell. Among 13 fungal strains, isolate FMS1 was observed to produce maximum concentration of organic acids and was selected for bioleaching of metals. Whereas isolate FMS2, a highly cadmium resistant isolate was selected for cadmium biosorption. Sequence analysis of the 16S rRNA of bacterial isolate DWH4 was 99% similar to Enterobacter cloacae (MK500875). Sequence analysis of internal transcribed spacer regions indicated that fungal isolate FMS1 has 99% similarity to Aspergillus tubingensis (MK026408) and FMS2 was 99% similar to Penicillium chrysogenum (MH865997). The bacterial strain DWH4 could grow at wide range of temperature (20–40°C) and pH (6–9) with optimum growth at 30°C and pH 7.0. The fungal strains could grow at wide range of temperature (15–40°C) and pH (3–12) with optimum growth temperature 30°C and pH 5.0 and 6.0 for A. tubingensis FMS1 and P. chrysogenum FMS2, respectively. Three organic acids were produced with highest concentration of gluconic acid (263 mM) followed by oxalic acid (22mM) and fumaric acid (0.34mM) at day 12 and sucrose was considered as the ideal carbon source. The highest leaching efficiency with organic acids from A. tubingensis strain FMS1 was observed for cadmium (58%), cobalt (53%) and nickel (52%) within 28 days. Cadmium removal capacity of P. chrysogenum strain FMS2 was observed 49% in 15 days. Distinct white patches were seen in SEM with clear peaks of cadmium in EDX analysis of white patches indicating biosorption of cadmium by P. chrysogenum strain FMS2. The TEM and EELS of P. chrysogenum strain FMS2 revealed cytoplasmic localization of cadmium. The constructed bioluminescent bioreporter for mercury detection, designated as E. cloacae strain DWH4lux, detected mercury (HgCl2) at a minimum concentration of 0.2 μg/L with a linear response profile being maintained between a range of 0.4–1600 μg/L (R2 = 0.9604) with a peak bioluminescent response occurring within one hour after exposure. No significant synergistic or antagonistic influences were observed on the bioluminescent response by other contaminating metal elements. E. cloacae strain DWH4lux was also demonstrated to detect mercury effectively in artificially contaminated water sample with linear correlation (R2=0.9623). This study provides new insight into the recruitment of metallotolerant bacteria from environmental samples for mercury bioreporter development and their potential for in-field biosensing applications. The biosorption and bioleaching ability of the newly isolated fungal strains can also be helpful in redemption of polluted soil especially with trace toxic metals such as in case of compost and sewage sludge and bioremediation of industrial effluents containing cadmium to minimize its release to environment.