Purification and characterization of dehalogenase from Bacillus cereus SN1 isolated from cow dung

Aims@#This study was aimed to characterize a dehalogenase derived from Bacillus cereus SN1 isolated from cow dung. @*Methodology and results@#Cell-free extract of Bacillus cereus SN1 was purified using ion exchange and gel filtration chromatography. Fraction B2 of gel filtration gave the highest enzyme specific activity (0.155 μmol CI¯/min/mg). The results of SDS-PAGE showed the enzyme was 25 kDa in size. The enzyme reached its optimum activity at 30 °C at pH 6, and was inhibited by Mercury(II) sulfate (HgSO4). The Km and kcat values were 0.2 mM and 1.22/sec, respectively. The partial dehalogenase gene sequence was amplified using Group I dehalogenase primers. The amplified gene sequence was designated as DehSN1. @*Conclusion, significance and impact of study@#Dehalogenase from Bacillus cereus strain SN1 revealed new characteristics of dehalogenase protein. The findings indicated that the DehSN1 dehalogenase is a promising candidate for further studies as a bioremediation agent for agricultural applications.

Isolation and identification of bacteria isolated from ruminant animal waste that able to degrade 2,2-dichloropropionic acid (2,2-DCP)

Aims: A 2,2-dichloropropionic acid (2,2-DCP) naturally degrading bacterial species, strain SN1 was successfully isolated from cow dung capable of utilizing the substance as the sole carbon source and energy. Methodology and results: Strain SN1 was preferred over other strains (SN2, SN3 and SN4) following observations on its rapid growth in 20 mM 2,2-DCP liquid minimal media. Since strain SN1 clearly exhibited tolerance towards 2,2-DCP, its growth in various concentrations (10 mM, 20 mM, 30 mM and 40 mM) of the substance was evaluated. The study found the bacteria grew particularly well in 20 mM 2,2-DCP with the highest chloride release of 39.5 µmole Cl- /mL while exhibiting a remarkably short doubling time of 3.85 h. In view of such notable characteristics, species identification via Biolog GEN III system and 16S rRNA analysis was performed and established strain SN1 as Bacillus cereus. Conclusion, significance and impact of study: Considering the rapid growth of B. cereus strain SN1 in such medium, its employment as a bioremediation agent to treat 2,2-DCP contaminated soils may prove beneficial. Moreover, this is the first reported case of a Bacillus sp. isolated from cow dung capable of utilizing 2,2-DCP. Therefore, further assessment into its ability to degrade other types of haloalkanoic acids merit special consideration.

Identification and characterization of Raoutella ornithilolytica and determination of its herbicide 2,2-dichloropropionate (2,2-DCP) degradation potential

Aims: The goal of the study is to isolate new bacteria species which are capable to utilizing 2,2-dichlropropionic acid (2- 2-DCP) as a sole carbon source from the wastewater sample that was taken from Tioman Island off the coast of Malaysia. Methodology and results: Genomic DNA from unknown bacterial strain (MR1) was extracted and PCR amplification was carried out using universal primers, Fd1 (5’-AGA GTT TGA TCC TGGCTC AG-3’) and rP1 (5’-ACG GTC ATA CCT TGT TAC GAC TT-3’) prior to sequencing. The BLASTn and phylogenetic analysis of the 16S rRNA of the MR1 gene found it shares a 95% homology to an aerobic bacillus Raoutella ornithilolytica. The bacteria which is usually found in an aquatic and hospital environment was found to possess a distinctive feature of being able to utilize 2,2-DCP as sole carbon source. The bacteria has cell doubling time of 23.11 h and maximum release of chloride ion at 0.257 mmol/L in 20 mM 2,2-DCP. Based on morphological and partial biochemical characteristics, the strain was found to be non-motile, Gram negative bacteria with red colonies that gave a positive catalase reaction. Conclusion, significance and impact of study: A better understanding of newly isolated microorganisms from the environment which can potentially be used as bioremediation tools in environmental management. This is the first reported case of Raoutella sp. that has the unique ability to degrade halogenated compound. Hence, the dehalogenation properties and capabilities of the bacteria deserve to be assessed further.

Identification of Serratia marcescens SE1 and determination of its herbicide 2,2- dichloropropionate (2,2-DCP) degradation potential

Aims: The goal of the study is to isolate species of bacteria that capable of utilizing 2,2-dichloropropionic acid (2,2-DCP) as sole carbon source from soil sample collected from surrounding lake water located in Universiti Teknologi Malaysia, Skudai, Johor. Methodology and Results: Genomic DNA from bacterium SE1 was extracted and PCR amplification was carried out using universal primers, Fd1 (5’ - AGA GTT TGA TCC TGGCTC AG - 3’) and rP1 (5’- ACG GTC ATA CCT TGT TAC GAC TT - 3’) before sending for sequencing. The 16S rDNA nucleotide sequences were compared with Basic Local Alignment Search Tool nucleotide (BLASTn) and further analyzed using phylogenetic tree of Neighbour-Joining method (MEGA 5). Phylogenetic analysis indicated that SE1 strain clearly shared 97% homology to the genus of Serratia marcescens and therefore designated as Serratia marcescens sp. SE1. SE1 exhibited the ability to utilize 2,2-DCP as sole carbon source at 20 mM concentration with cell doubling time of 5 h and maximum chloride ion release of 38 μmolCl-/mL. This result suggests that the dehalogenase enzyme present in the bacteria has high affinity towards the substrate. Based on morphological and partial biochemical characteristics, strain SE1 was a non-motile Gram negative bacterium with red colonies, that gave a catalase positive reaction. Conclusion, significance and impact of study: A better understanding of dehalogenases enzyme produce by this S. marcescens sp. SE1 in general will be useful to be used as bioremediation tools for environmental management. This is the first reported case that Serratia sp. has the ability to degrade halogenated compound.