Microbial degradation of 3-chloropropionic acid

Abstract

A wide variety of contaminants have been introduced into the environment by human activity, through processes such as the manufacture and use of chemicals, petroleum products, herbicides and pesticides. Pollution not only affects human and animal health but also severely hampers the ecosystem by altering and destroying the habitats of flora and fauna. Globally, many environments including surface water, subsurface aquifers, sediments and soils have been affected. Contaminants such as hydrocarbons and halogenated compounds are significant because they have been identified as mutagenic, carcinogenic and teratogenic. Bioremediation can be successfully employed in the sustainable and harmless clean-up of many contaminated environments. The attenuation of pollutants can be achieved under aerobic or anaerobic conditions using bacteria present in the impacted site or by the introduction of organisms capable of biodegradation. Successful bioremediation has been achieved in the aerobic degradation of petroleum hydrocarbons. However, relatively little is known about the potential for using bacteria in the clean-up of halogenated aliphatic compounds. Consequently, this study investigated the isolation and identification of bacterial and fungal strains that could degrade 3-chloropropionic acid (3-CP) as well as profiles of bacterial communities degrading 3-CP. Three pure 3-CP degrading bacteria were isolated from Oxley Creek in Brisbane and designated strains O1, O2, and O3. Analysis of 16S rRNA gene sequences showed that the strain O1 was most related to Lysinibacillus fusiformis (99.8% similarity), strain O2 matched 100% with both Curtobacterium luteum and Curtobacterium oceanosedimentum, while strain O3 was equally related to Cytobacillus firmus and Cytobacillus oceanosediminis (both 99.3% similarity). One 3-CP degrading bacterium, designated strain CX, was isolated from a bioremediation facility at Caltex Oil Refinery in Brisbane and was most related to Rhodococcus zopfii (99.8%). Two 3-CP degrading fungi isolated from Toohey Forest also in Brisbane and were designated as M1 and M2. Analysis of ITS/18S rRNA gene sequences, revealed that strain M1 was most related to Mucor variicolumellatus (99.1% similarity), and strain M2 to Trichoderma afroharzianum (99.4% similarity). Bacterial strains O1, O2, O3, and CX all grew well with 20 mM 3-CP as a sole source of carbon. High-performance chromatography (HPLC) revealed that the 3-CP was being degraded as the cells grew. All strains grew steadily over the first 2 days and reached maximum growth after 2-4 days incubation. With each strain, 3-CP had been almost completely degraded after 6 days incubation. Concomitant dechlorination of the 3-CP, the initial stage of degradation, was confirmed over a similar time period using ion exchange chromatography. Optimal 3-CP degradation occurred with 10-20 mM concentrations. In addition, strains O2, O3 and CX were able to grow anaerobically, while strain O1 did not. Fungal strains M1 and M2 grew much slower with 3-CP as sole carbon source than the bacterial strains. Maximum growth levels were not achieved until around 12 days for both fungal isolates and complete degradation of 3-CP took 20 days for strain M1 and slightly longer for strain M2. While the pure bacteria were isolated and tested for 3-CP degradation in single substrate laboratory tests, it would be expected that in situ mixed consortia would largely contribute to degradation in natural environments. Here, target contaminants would be mixed with many other organic compounds and soil particles making degradation more difficult. Oxley Creek and Caltex microcosms spiked with 3-CP were established to replicate natural environments. Degradation and microbial communities were tracked over time. As expected, degradation was much slower than with the pure bacteria in liquid medium, taking up to 4 weeks before most of the 3-CP was degraded. Bacterial profiles changed significantly while the 3-CP was degraded. In both Oxley Creek and Caltex microcosms, there was a substantial reduction in diversity over time while degrading population established. Dominant populations in the later stages of the microcosms were Sphingomonadaceae family members in the Oxley Creek microcosms and Alicyclobacillus, Dyella, Cellulomonas genera in the Caltex microcosms. Whole genome sequencing (WGS) was done to provide a deeper understanding of bacteria and insights into the link between physiological and genomic properties of the two best 3-CP degrading bacterial isolates, strains O2 and CX. WGS is an approach to give a more definitive identification of isolates and also is used to identify key genes of interest that a specific organism possesses, including antibiotic resistance, metabolic pathways and excretory system. From average nucleotide identity (ANI) analyses, both strains were determined as novel strains of Curtobacterium (strain O2) and Rhodococcus (strain CX) which differed to the single gene 16S rRNA phylogeny. Potential metabolic pathways were proposed and predicted dehalogenases genes within the isolates were identified and investigated. A number of dehalogenases were revealed in both isolates, eleven in strain O2 and six in strain CX. The common genes in the isolates were haloalkanoic acid (haloacid) dehalogenases and haloalkane dehalogenases. The amino acid sequences in the dehalogenases of both isolates shared homology with corresponding genes in Rhodococcus species suggesting common evolutionary origins. Information provided in the current study have built upon our understanding of the degradation of chlorinated alkanoic acids, particularly 3-CP. The research has shown that there is clear potential in using microorganisms in the bioremediation 3-CP contaminated environments.