Herbicide Bioremediation Using Hyperbutachlor Tolerant Microorganisms in Batch and Continuous Systems

Abstract

In the present thesis, an attempt has been made towards the development of an efficient biotreatment technology for handling herbicide contamination in industrial effluents and agricultural soils. Butachlor, the most complex structured and extensively used herbicide in India is of primary focus. Three hyper-butachlor tolerant bacterial strains identified as Serratia ureilytica strain AS1, Enterobacter cloacae strain FP2 and Pseudomonas putida strain G3, based on the 16S rRNA gene sequencing analysis were isolated from three different contaminated sites. To maximize the removal efficiency, the factors affecting the metabolic activity of any microorganism were optimized by the application of various statistical design of experiments. Initially the factors were screened as per the Plackett Burman design of experiment to determine the significant factors which were further optimized using the Response Surface Methodology to identify the optimum conditions where the isolated strain will yield the maximum butachlor removal efficacy. Biodegradation of butachlor and other herbicides such as alachlor and glyphosate, by the isolated strains at various initial concentrations (100 – 1000 mg/L), was studied at the optimum conditions obtained in the previous study. It was observed that with increase in the butachlor concentration, the rate of biodegradation decreases which indicates the substrate inhibition phenomenon. The degradation kinetics of the bacterial strains were fitted with various substrate inhibition models available in the literature which are able to predict the experimental data fairly. To elucidate the plausible metabolic pathway followed during the biodegradation process, the intermediate metabolites were identified. To overcome the substrate inhibition at higher butachlor concentrations, various strategies have been adopted to investigate the enhancement of the remediation efficiency. Firstly, the bacterial strains were immobilized within Ca-alginate beads and batch biodegradation experiment was carried out. The study revealed that on being immobilized, the microbial strains are able to tolerate and degrade higher butachlor concentration as compared to their freely suspended cell counterparts. Secondly, to overcome the microbial toxicity of butachlor, the bacterial strains were co-cultured to formulate a defined synthetic microbial consortium termed as SMC 1. The consortium displayed the potentiality to be a promising candidate for the remediation of butachlor and other herbicides at higher concentration both in aqueous medium as well as in the soil. viii After establishing the batch biodegradation of butachlor and other herbicides in shake flasks studies, experiments were performed in continuous mode to treat synthetic wastewater containing butachlor in packed bed bioreactors. The combined effect of external mass transfer and the biochemical reaction on the mass transfer correlation was investigated in a re-circulated packed bed bioreactor using the microbial consortium SMC1 immobilized in Ca-alginate beads. It is observed that the rate of bio-removal of butachlor from the medium is dependent on the operating parameters; feed flow rate and substrate concentration. The effect of the external mass transfer was investigated by calculating the mass flux (G), Reynold’s Number (NRe) and the mass transfer coefficient (km) for varying feed flow rates. The constants obtained from the plot of ln km vs ln G is used to determine the external mass transfer correlation between the Colburn factor (JD) and Reynolds number (NRe) which can predict the experimental data precisely. Effect of various operating parameters such as HRT, inlet loading rates on the performance of the bioreactor was investigated in another up-flow packed bed biofilm reactor filled with ceramic raschig rings immobilized with the microbial consortium SMC1. The study proposes the immobilized system of demonstrating excellent biodegradation efficacy of the immobilized microbial consortium in treating herbicide contaminated synthetic wastewater in continuous mode