Bioremediation of Phthalate Esters by Bacillus Species Isolated from Agriculture Soil

Abstract

Phthalate acid esters (PAEs) is a class of refractory organic compounds, widely used as additives or plasticizers in plastic industry and their release in the environment during the manufacturing, use, and disposal has caused serious environmental health concerns, since some of them are suspected to be mutagens, hepatotoxic agents, and carcinogens. This class of organic compound has been classified as an endocrine disruptor by several countries. As endocrine disruptors, PAEs mimic estrogenic activities in animals and humans. PAEs are ubiquitous endocrine-disrupting pollutants and can be degraded by microorganisms. The present study is divided into three phases describes the bioremediation of PAEs. In first phase of the study new isolate were enriched from the agriculture soil (Cotton field, Tomato field and Chilli field) and identified as Bacillus thuringiensis and Bacillus cereus. Subsequently biodegradation potential of these isolates was checked against the dimethyl phthalate (DMP) and diethyl phthalate (DEP). These phthalates are low molecular weight PAEs and widely used in cosmetics, perfumes, and plasticizers. The results of DMP degradation by a novel aerobic bacterium, enriched from soil samples of cotton field utilizing DMP as the sole carbon and energy source. The enriched isolate was recognized as Bacillus thuringiensis based on the biochemical and morphological appearances as well as genetic analysis. Mineral salt medium was found as the growing medium for Bacillus thuringiensis at pH 7.0. after 48 hrs (h) of incubation and 30 ºC. Bacillus thuringiensis can degrade DMP (400 mg/L) under aerobic environments with 99 % biodegradation potential. A cooperation of GC and GC-MS analysis exposed a comprehensive DMP biodegradation pathway. The outcomes specify that Bacillus thuringiensis can be a proficient biocatalyst for DMP biodegradation at a viable scale. In another attempt, DMP degrading bacterial culture was isolated from tomato field soil by enrichment culture technique and identification using standard protocol. Biochemical and gene sequence analysis reveals that the new isolated aerobic bacterium is Bacillus cereus. Morphology of Bacillus cereus was studied by Atomic force microscopy. Optimum growth of isolated bacterium was achieved at pH 7 after 48 h. Percent degradation potential was also investigated by using VII different parameters like, inoculum size, temperature, substrate concentration and kinetics. The isolated bacterium was able to biodegrade the DMP under aerobic environments with 99 % biodegradation potential. The degradation was monitored on GC and LC- MS/MS techniques. Present work is supportive to the development of bacterial resources for biodegradation. For DEP degradation Bacillus thuringiensis strain, isolated from chilli field soil, was utilized for biodegradation constitutively before the completion of log period, and its kinetics followed a first order model. In the presence of mineral salt medium, whole cells rapidly degrade DEP at high concentrations (up to 500 mg/L for DEP). The optimum biodegradation temperature, pH, inoculum age, inoculum size and concentration were found as 30 ºC, 7, 24-30 h, 15 mL and 500 mg/L respectively. The isolated microbial strain was capable to degrade the DEP in aerobic environment with >98% degradation capacity. The biodegradation of DEP by newly isolated Agro field bacterium was studied with the help of GC-FID and LC-MS/MS techniques. Current study substantiated that Bacillus thuringiensis strain has potential application for bioremediation of PAEs. In second phase of the study, degradation potential of new isolates was checked for biodegradation of DPP, DBP and mixture of PAEs i.e. DMP, DEP, dipropyl phthalate (DPP) and dibutyl phthalate (DBP). DPP is one of PAEs which are broadly used as plasticizer in commercial and industrial products and due to its toxic effects on immune mediator expression, genotoxicity and developmental toxic effects without teratogenicity in vivo. The outcomes of the biodegradation of DPP by an aerobic bacterium, Bacillus thuringiensis showed that pH 7.0, 30 °C, 13 mL inoculum size and substrate concentration 200 mg/L were optimal for degradation. Biodegradation of DPP followed first-order kinetics. The half-life of degradation was about 12.16 h when the concentration of DPP was 200 mg/L. The degradation efficacy was monitored on GC and LC-MS/MS techniques. Thus, significant concern over its extensive distribution and potentially harmful effects on the environment has prompted its remediation through environmental friendly processes. Further biodegradation of DBP was investigated with Bacillus thuringiensis and found encouraging biodegradation potential. The effect of DBP concentrations on degradation was investigated between 50 and 500 mg/L. The VIII results showed that the degradation fits a first-order kinetic model, and the half life was about 10.83 h at 400 mg/L substrate concentration. The major degradation products were identified as monobutyl phthalate and phthalic acid. The optimal pH, temperature and inoculum size for biodegradation were 7.0, 30 ºC and 8 mL respectively. This was the first report about DPP and DBP degradation and the metabolic pathway by Bacillus thuringiensis and showed that this microbe is novel bacterial specie and it has potential application for bioremediation of DPP and DBP containing wastes. Finally, the assimilation of four PAEs mixture (DMP, DEP, DPP and DBP) by two Bacillus species; Bacillus thuringiensis and Bacillus cereus and their consortium were studied. Among which the optimal degradation of 82–96 % was achieved by Bacillus thuringiensis. This was the first report on the metabolic breakdown of four basic PAEs mixture. The optimum conditions for biodegradation were found to be pH 7, temperature 30 °C, inoculum size 10 mL and concentration 400 mg/L. Moreover, the respective biodegradation obeyed the first-order kinetic equation. Our results proffered supplementary confirmation of the wide spectrum of PAEs utilization by this strain and suggest the possibility of applying Bacillus thuringiensis for the remediation of PAEs contamination waste. In third phase, the applicability of more PAEs tolerant species i.e. Bacillus thuringiensis was monitored for biodegradation of leached PAEs. Firstly, leaching of PAEs from different drinking stuffs (water cooler, mineral water bottles) exposed to sunlight and baby feeders subjected to different heating treatments (boiling, autoclave and oven) was studied. Results showed that a total of 10 PAEs were leached and identified. Among them, DMP, bis(2-methoxyethyl) phthalate, DEP, and DBP were the major leached PAEs found in the range 9-112.50 μg/L. Boiling treatment was found safer for baby feeders as PAE leaching was ~26-54 % less as compared to other two treatments. The leached PAEs in water samples were then subjected to biodegradation experiment with Bacillus thuringiensis strain at optimized conditions (time 72 h, 30 °C, pH 7 with 75-96 % degradation of PAEs). Hence, leaching of hazardous PAEs from different water stuffs is alarming and needs immediate attention.