Microbial Degradation of Chlorophenols in Batch and Continuous Bioreactors: Kinetic Study and Optimization of Process Parameters

Abstract

Chlorophenols are known to be detrimental to the environment owing to their physiochemical properties, which result in a higher persistence and subsequent bioaccumulation. Chlorophenols have been reported for their carcinogenicity, immunogenicity, mutagenicity and fatality to humans and other organisms. Biodegradation is an eco-friendly, energy efficient and potential alternative to various physicochemical treatment methods used for environmental pollution abatement with promising removal efficiency. The main objective of the study is to propose a scheme for efficient treatment of chlorophenol containing wastewater. Effective implementation of biological methods have limitations and requires the understanding of the complex interaction between substrate and microorganisms. Wastewaters, both domestic as well as industrial, commonly encounter a mixture of different recalcitrant synthetic organic compounds (SOC) along with biogenic substrates. The common treatment plants are unable to treat recalcitrant SOC effectively and they generally pass through without complete treatment. Hence a complete understanding of the biological methods and various other parameters involved is highly necessary for achieving the anticipated results. In the first part of the study, eight different bacterial strains were isolated from a two different possible sources which has high chance of chlorophenol contamination. Out of these eight strains, four pure strains were selected for further study based on their high tolerance and degradation capacity for chlorophenols. The isolated strains were characterized morphologically, biochemically as well as genetically and they were identified as Bacillus
cereus, Bacillus endophyticus, Kocuria rhizophila and Pseudomonas aeruginosa. The 16S rDNA sequences have been indexed in GenBank database for public acknowledgement. Process optimization could enhance the treatment of recalcitrant compounds efficiently. In the second part of the research, pure strains were tested for degradation of six different chlorophenols (2-CP, 3-CP, 4-CP, 2,4-DCP, 2,4,6-TCP, and PCP). Using Response Surface Methodology, four experimental parameters were optimized and proven to be effective to achieve maximum degradation and tolerance for chlorophenols. The biodegradation kinetics and growth kinetics of the strains were also evaluated. The ability of the microbes to cometabolize 2,4-DCP and mono-chlorophenol has also been assessed in this part. Next, the biodegradation of chlorophenols and its co-metabolism (multi-substrate degradation) by the two different mixed microbial consortia, one defined consortium and the other one undefined consortium was carried out. The biodegradation kinetics was studied using Andrews’s substrate inhibition model for biodegradation of chlorophenols by the mixed consortium. The final part of the study focused on the continuous degradation of chlorophenols in three customized bioreactors: two packed bed biofilm reactor (PBBR) and one airlift inner loop reactor (ALR). In the first packed bed biofilm reactor (PBBR-1), the biodegradation of 2,4- dichlorphenol by two different isolated strains was studied. In the second packed bed biofilm reactor (PBBR-2), the biodegradation of 3-chlorophenol and 4-chlorophenol by the defined mixed microbial consortium was studied individually. In the third bioreactor i.e., airlift inner
VI loop reactor (ALR), two different studies have been conducted. First, the biodegradation of 4- chlorophenols by the mixed microbial consortium (undefined) was explained with the metabolic pathway. Second, the biodegradation of mixture of different chlorophenols including 2-chlorophenol, 3-chlorpohenol, 4-chlorophenol, 2,4-dichlorophenol, and 2,4,6- trichlorophenol was conducted. In all the three bioreactor studies, the effect of hydraulic retention time, initial substrate concentration or loading rate and effect of biogenic substrate concentration on removal efficiency of chlorophenols in bioreactors during continuous mode were evaluated extensively. From the experimental results obtained, the general hypothesis considered for the present work were been evaluated. The results indicated that, acclimatization of a microbe to a certain compound has a profound effect on its tolerance towards the compound. The pure strains were able to degrade only 2-CP and 2,4-DCP but unable to degrade 3-CP, 4-CP, 2,4,6- TCP and PCP. Next, the biodegradation results obtained by mixed microbial consortia makes them a potential option not only for complete mineralization of the recalcitrant compounds but also for their ability to treat various compounds simultaneously. Among the two different consortia studied, the defined mixed consortium showed excellent prospective for degradation of lower to higher chlorophenols in single and mixture as compared to the undefined one. The biodegradation kinetics of chlorophenols gives an important insight for comparison of the degradation process and can help in designing and implementing a bioreactor. Finally, the bioreactor studies and effect of various parameters proven very effective for understanding the degradation of chlorophenols and its complete removal. The effect of biogenic substrate showed that, the presence of higher concentration of biogenic substrate do not affect the removal efficiency of chlorophenols in bioreactors except 3-CP. As a matter of fact, at lower biogenic substrate concentration, the removal efficiency and biodegradation rate increased for chlorophenols. The effect of hydraulic retention time and loading rate indicated that, there are two different optimized conditions that can be applied based on the selected outcome i.e. maximum volumetric removal and maximum biodegradation of the chlorophenols. This study is important as it will provide the information which has direct relevance towards the applicability of microbes for degradation of organic pollutant in the environment. Knowledge acquired from the biodegradation processes and their mechanisms for degradation of chloro-aromatic compounds provides a better understanding of fate and cycling of chlorophenolic compounds in the environment.