Potency of Indigenous Bacteria of Mt. Merapi, Arthrobacter chlorophenolicus for Chromium (VI) Bioremediation

Chromium (VI) in the production process, such as textile, tannery, and electroplating industry, produce hazardous waste when disposed of directly into the aquatic environment. Several chromium pollutions cases, not only in water but also in the aquatic organism, occurred in some regions in Indonesia. Various methods can reduce the Chromium (VI) waste. One of them is the biological method by employing such kinds of bacteria. Arthrobacter chlorophenolicus is a pioneer bacterium of Mt. Merapi, which can survive in the minimum conditions of the bacterial primary nutrients, carbon, and nitrogen. This study aims to investigate the ability of A. chlorophenolicus to remove Cr (VI) at various concentrations. The research was carried out by growing the A. chlorophenolicus into two nutrient media conditions, minimal and rich-nutrient media containing different concentrations of Cr (VI) (5, 10, 20 ppm) for eight days. The results showed that the A. chlorophenolicus were grown on both minimal and rich-nutrient media. The A. chlorophenolicus could reduce for about 80% of 10 and 20 ppm chromium in eight days. Our results indicate that A. chlorophenolicus, the pioneer bacteria of Mt. Merapi, has a grand promise for use in Cr (VI) remediation even under minimum nutrients conditions.

Co-metabolic biodegradation of 4-bromophenol in mixture of pollutants system by Arthrobacter chlorophenolicus A6

Brominated phenols are listed as priority pollutants, and are the key components of paper pulp wastewater together with nitrophenol and chlorophenol. However, the biodegradation of bromophenol in a mixed substrate system is very scanty. In the present investigation, simultaneous biodegradation kinetics of three substituted phenols (4-bromophenol, 4-BP; 4-nitrophenol, 4-NP; and 4-chlorophenol, 4-CP) were investigated using Arthrobacter chlorophenolicus A6. A 23 full factorial design was applied with varying 4-BP and 4-CP from 75–125 mgl− 1and4-NP from 50–100 mgl− 1. Almost complete degradation of this mixture of substituted phenols was achieved at an initial concentration combination of 125, 125, and 100 mgl− 1of 4-CP, 4-BP, and 4-NP, respectively in 68 h. Statistical analysis of the results revealed that among the three variables, 4-NP had the most prominent influence on both degradations of 4-CP and 4-BP. While the concentration of 4-CP had a strong negative interaction effect on the biodegradation of 4-NP. Irrespective of the concentration levels of these three substrates, 4-NP was preferentially biodegraded over 4-CP and 4-BP. Further, 4-BP biodegradation rates were found to be higher than that of 4-CP followed by 4-NP. Besides, the variation of biomass yield coefficient of the culture was investigated at different initial concentration combinations of these substituted phenols. Although the actinomycetes consumed 4-NP at a faster rate, the biomass yield was very poor. This revealed that the microbial cells were more stressed when grown on 4-NP compared to 4-BP and 4-CP. Overall, this study revealed the prospective of A. chlorophenolicus A6 for the degradation of 4-BP in mixed substrate systems.

Kinetic of 4-Chlorophenols Biodegradation by Arthrobacter Chlorophenolicus A6

Chlorophenols are listed as priority pollutants both by European community and US EPA. Biodegradation of pchlorophenol (4-CP) was investigated in batch shake flasks by Arthrobacter chlorophenolicus A6 at initial 4-CP concentrations between 25 to 350 mgl-1. The rate of 4-CP removal decreased with increasing initial 4-CP concentrations due to toxic effects on the microorganisms. The growth and biodegradation kinetic of the culture was evaluated. The batch growth profile of the A chlorophenolicus A6 followed substrate inhibition kinetics with the estimated biokinetic parameters of Ksi = 272 mgl-1, Ks = 65 mgl-1 for 4-CP respectively. High inhibition constant (KSI = 272 mg -l) with a KSI/KS ratio of 4.18 indicates superior 4-CP biodegradation potential of the A chlorophenolicus A6. The maximum rate of 4-CP degradation has been achieved at an optimum substrate concentration of Smax = (KSKSI)1/2 = (65×272)1/2 = 133 mgl−1.

Function Analysis of Chlorophenol Monooxygenase for Chlorophenol Degradation

4-Chlorophenol is well-known as an extensively used antiseptic, and it may cause severe damage to the environment and human health. 4-Chlorophenol can be biologically degraded by Arthrobacter chlorophenolicus A6, which can be attributed to the cphC-I and cphB genes of the microorganism that encode for a two-component flavin-diffusible monooxygenase (TC-FDM), composed of oxygenase and reductase components. This study reports the cloning, overexpression, purification, and function analysis of the oxygenase and reductase components from the genes cphC-I and cphB. The genes were cloned into vector pET-24a, and 4 different strains of Escherichia coli were transformed with these recombinant genes. The optimization of expression conditions indicated that cphC-I is best overexpressed in E. coli BL21(DE3) incubated for 24 hours at 15°C with 0.5 mM of isopropyl-β-D-1-thiogalactopyranoside. However, cphB was not expressed into soluble form enzyme in any of the conditions, and therefore fre of E. coli was used instead to analyze the function of CphC-I. CphC-I was able to degrade approximately 13.86% of 4-chlorophenol, indicating that it is indeed a reduced flavin-dependent monooxygenase and utilizes 4-chlorophenol as a substrate. The results of this study are expected to establish the basic understanding of TC-FDM for its application to enzymatic bioremediation of phenol-contaminated environment.