Optimisation for Enhancement of Phenol Degradation by Arthrobacter sp. Strain AQ5-15 from Antarctic Soil Using Conventional and Statistical Approach

The Tenth Symposium on Polar Science/Ordinary sessions : [OB] Polar Biology, Wed. 4 Dec. / Entrance Hall (1st floor) , National Institute of Polar Researc

Statistical assessment of phenol biodegradation by a metal-tolerant binary consortium of indigenous Antarctic bacteria

Since the heroic age of Antarctic exploration, the continent has been pressurized by multiple anthropogenic activities, today including research and tourism, which have led to the emergence of phenol pollution. Natural attenuation rates are very slow in this region due to the harsh environmental conditions; hence, biodegradation of phenol using native bacterial strains is recognized as a sustainable remediation approach. The aim of this study was to analyze the effectiveness of phenol degradation by a binary consortium of Antarctic soil bacteria, Arthrobacter sp. strain AQ5-06, and Arthrobacter sp. strain AQ5-15. Phenol degradation by this co-culture was statistically optimized using response surface methodology (RSM) and tolerance of exposure to different heavy metals was investigated under optimized conditions. Analysis of variance of central composite design (CCD) identified temperature as the most significant factor that affects phenol degradation by this consortium, with the optimum temperature ranging from 12.50 to 13.75 °C. This co-culture was able to degrade up to 1.7 g/L of phenol within seven days and tolerated phenol concentration as high as 1.9 g/L. Investigation of heavy metal tolerance revealed phenol biodegradation by this co-culture was completed in the presence of arsenic (As), aluminum (Al), copper (Cu), zinc (Zn), lead (Pb), cobalt (Co), chromium (Cr), and nickel (Ni) at concentrations of 1.0 ppm, but was inhibited by cadmium (Cd), silver (Ag), and mercury (Hg)

Biodegradation potential of phenol by pure and defined mixed cold-adapted bacterial consortia from Antarctica

The risk of phenol pollution from daily waste discharge and accidental oil spillage is ever-present due to increasing activities in the Antarctic continent, mainly related to the supply and operation of research stations and field expeditions, tourism, marine and air transportation. Increased levels of phenol concentration in the Antarctic environment bring significant risk to both aquatic and terrestrial biota due to its highly toxic properties and persistence. Sustainable human presence and activity in Antarctica require effective remediation technologies to be developed and their rapid application when required. The main purpose of the present study was to isolate new taxa of pure phenol-degrading bacteria from Antarctic soil and, both as a pure isolate and together with previously isolated phenol-degrading bacteria as a consortium, will be capable of rapid degradation of phenol at low temperatures (0-15°C). In addition, this study also focuses on identification of phenol-degrading pathway(s) of the pure culture, conventional and statistical optimisations of phenol degradation by both pure and mixed cultures, and the effect of heavy metals on phenol degradation by pure and mixed cultures. This thesis reports the isolation of a potential phenol-degrading bacterial strain (AQ5-15) from soil from King George Island, South Shetland Islands, Antarctica. This strain was identified as a member of the genus Arthrobacter based on 16S rRNA gene sequence analysis. Based on whole arest identified relative was suggested to be Paeniglutamicibacter sulfureus (99.38% similarity). Preliminary screening showed that strain AQ5-15 was capable of completely degrading 0.5 g/L phenol within 108 h at 10°C and it was selected for a detailed study. The genomic analysis identified the presence of genes encoding a complete pathway of aromatic compound metabolism in strain AQ5-15, consistent with the ability of the strain to utilise phenol as the sole carbon source. The genomic analysis was validated using enzyme assays of catechol 1,2-dioxygenase and catechol 2,3- dioxygenase, which confirmed the presence of the enzyme catechol 1,2- dioxygenase, consistent with the genes identified in the WGS. A study of the influence of parameters including nitrogen source, salinity, pH, and temperature was conducted to optimise the conditions for phenol degradation using onefactor- at-a-time (OFAT) and response surface methodology (RSM). Based on the results from OFAT, strain AQ5-15 showed the highest phenol degradation at 0.5 g/L (NH4)2SO4, 0.1 g/L NaCl, pH 7 and 20°C, proving that this strain is a psychrotolerant and prefers low salinity and near-neutral conditions. Statistical analysis of the results obtained from RSM showed that the strain could degrade phenol optimally at 0.5 g/L (NH4)2SO4, 0.13 g/L NaCl, pH 7.25 and 15°C, with pH and temperature identified as significant factors. This strain was mixed with two other previously isolated phenol-degrading strains (AQ5-06 and AQ5-07) in different combinations to further enhance degradation efficiency. The data obtained showed that mixture of strains AQ5-06 and AQ5-15 together could completely degrade 0.5 g/L phenol within 48 h at 10°C while mixture of strains AQ5-06, AQ5-07 and AQ5-15 together could completely degrade 0.5 g/L phenol within 60 h at 10°C. RSM analysis showed that the combination of strains AQ5- 06 and AQ5-15 could degrade phenol optimally at 0.4 g/L (NH4)2SO4, 0.13 g/L NaCl, pH 7.25 and 12.5°C, with only temperature as a significant factor. RSM analysis showed that the combination of strains AQ5-06, AQ5-07 and AQ5-15 can degrade phenol optimally at 0.4 g/L (NH4)2SO4, 0.13 g/L NaCl, pH 7.25 and 12.5°C, with ammonium sulphate concentration, sodium chloride concentration and temperature being significant factors. The tolerance levels of pure and mixed cultures towards different heavy metals that are widely present in Antarctic soils was studied by exposing strains AQ5-06, AQ5-07 and AQ5-15 individually as well as in consortia to the heavy metals Arsenic (As), Aluminum (Al), Copper (Cu), Zinc (Zn), Lead (Pb), Cobalt (Co), Cadmium (Cd), Chromium (Cr), Nickel (Ni), Silver (Ag) and Mercury (Hg) at an initial concentration of 1.0 ppm. Phenol degradation by strain AQ5-15 was inhibited when exposed to Cd, Ag and Hg while strain AQ5-06 was inhibited when exposed to Ag and Hg, and strain AQ5- 07 was inhibited when exposed to Cd and Hg. Consortia containing strains AQ5- 06 and AQ5-15 and all three strains were inhibited when exposed to Hg, Cd and Ag. In a nutshell, the attempt to develop highly efficient phenol-degrading bacterial consortia for significant inclusion in cold region bioremediation, specifically Antarctica was successful with a few limitations in the event of the co-occurrence of some heavy metals such as Hg, Cd, and Ag

Effects of zinc (Zn) and chromium (Cr) on the phenol-degrading bacteria growth kinetics

Heavy metals occur naturally within the earth crust; however, anthropogenic activities can artificially introduce these elements into the environment. Despite being the foremost isolated continent, Antarctica is not free from human contamination. Heavy metals are well-known to be the powerful inhibitors of xenobiotics biodegradation activities. A microbial growth model was presented for bacteria cell growth in the biodegradation of phenol containing heavy metals such as zinc (Zn) and chromium (Cr) ions. The Gompertz model was used to estimate three main growth parameters namely lag phase (λ), maximum growth rate (μmaz), and maximum cell number at the stationary phase (Nmax). Bacterial growth for both heavy metals was shown to be properly fit towards the curve with a high value of R2 and low square root of the variance of residuals (RSME) value. The effect of heavy metals at 1.0 ppm showed that Cr has a considerable effect on bacteria consortium, inhibiting the degradation of phenol, while Zn has no effect, removing 100% of phenol. The predicted biokinetic from this model suggests the suitability of the bacteria consortium to be used in phenol removal

Optimisation of Various Physicochemical Variables Affecting Molybdenum Bioremediation Using Antarctic Bacterium, Arthrobacter sp. Strain AQ5-05

The versatility of a rare metal, molybdenum (Mo) in many industrial applications is one of the reasons why Mo is currently one of the growing environmental pollutants worldwide. Traces of inorganic contaminants, including Mo, have been discovered in Antarctica and are compromising the ecosystem. Bioremediation utilising bacteria to transform pollutants into a less toxic form is one of the approaches for solving Mo pollution. Mo reduction is a process of transforming sodium molybdate with an oxidation state of 6+ to Mo-blue, an inert version of the compound. Although there are a few Mo-reducing microbes that have been identified worldwide, only two studies were reported on the microbial reduction of Mo in Antarctica. Therefore, this study was done to assess the ability of Antarctic bacterium, Arthrobacter sp. strain AQ5-05, in reducing Mo. Optimisation of Mo reduction in Mo-supplemented media was carried out using one-factor-at-a-time (OFAT) and response surface methodology (RSM) approaches. Through OFAT, Mo was reduced optimally with substrate concentration of sucrose, ammonium sulphate, and molybdate at 1 g/L, 0.2 g/L, and 10 mM, respectively. The pH and salinity of the media were the best at 7.0 and 0.5 g/L, respectively, while the optimal temperature was at 10 °C. Further optimisation using RSM showed greater Mo-blue production in comparison to OFAT. The strain was able to stand high concentration of Mo and low temperature conditions, thus showing its potential in reducing Mo in Antarctica by employing conditions optimised by RSM

Statistical asssessment of phenol biodegradation by a metal-tolerant binary consortium of indigenous Antarctic bacteria

Since the heroic age of Antarctic exploration, the continent has been pressurized by multiple anthropogenic activities, today including research and tourism, which have led to the emergence of phenol pollution. Natural attenuation rates are very slow in this region due to the harsh environmental conditions; hence, biodegradation of phenol using native bacterial strains is recognized as a sustainable remediation approach. The aim of this study was to analyze the effectiveness of phenol degradation by a binary consortium of Antarctic soil bacteria, Arthrobacter sp. strain AQ5-06, and Arthrobacter sp. strain AQ5-15. Phenol degradation by this co-culture was statistically optimized using response surface methodology (RSM) and tolerance of exposure to different heavy metals was investigated under optimized conditions. Analysis of variance of central composite design (CCD) identified temperature as the most significant factor that affects phenol degradation by this consortium, with the optimum temperature ranging from 12.50 to 13.75 °C. This co-culture was able to degrade up to 1.7 g/L of phenol within seven days and tolerated phenol concentration as high as 1.9 g/L. Investigation of heavy metal tolerance revealed phenol biodegradation by this co-culture was completed in the presence of arsenic (As), aluminum (Al), copper (Cu), zinc (Zn), lead (Pb), cobalt (Co), chromium (Cr), and nickel (Ni) at concentrations of 1.0 ppm, but was inhibited by cadmium (Cd), silver (Ag), and mercury (Hg)

Optimization of phenol degradation by Antarctic bacterium Rhodococcus sp.

This study focused on the ability of the Antarctic bacterium Rhodococcus sp. strain AQ5-14 to survive exposure to and to degrade high concentrations of phenol at 0.5 g l-1. After initial evaluation of phenol-degrading performance, the effects of salinity, pH and temperature on the rate of phenol degradation were examined. The optimum conditions for phenol degradation were pH 7 and 0.4 g l-1 NaCl at a temperature of 25°C (83.90%). An analysis using response surface methodology (RSM) and the Plackett-Burman design identified salinity, pH and temperature as three statistically significant factors influencing phenol degradation. The maximum bacterial growth was observed (optical density at 600 nm = 0.455), with medium conditions of pH 6.5, 22.5°C and 0.47 g l-1 NaCl in the central composite design of the RSM experiments enhancing phenol degradation to 99.10%. A central composite design was then used to examine the interactions among these three variables and to determine their optimal levels. There was excellent agreement (R2 = 0.9785) between experimental and predicted values, with less strong but still good agreement (R2 = 0.8376) between the predicted model values and those obtained experimentally under optimized conditions. Rhodococcus sp. strain AQ5-14 has excellent potential for the bioremediation of phenol

Effects of heavy metals on bacterial growth parameters in degradation of phenol by an Antarctic bacterial consortium

Antarctica has often been perceived as a pristine continent until the recent few decades as pollutants have been observed accruing in the Antarctic environment. Irresponsible human activities such as accidental oil spills, waste incineration and sewage disposal are among the primary anthropogenic sources of heavy metal contaminants in Antarctica. Natural sources including animal excrement, volcanism and geological weathering also contribute to the increase of heavy metals in the ecosystem. A microbial growth model is presented for the growth of a bacterial cell consortium used in the biodegradation of phenol in media containing different metal ions, namely arsenic (As), cadmium (Cd), aluminium (Al), nickel (Ni), silver (Ag), lead (Pb) and cobalt (Co). Bacterial growth was inhibited by these ions in the rank order of Al < As < Co < Pb < Ni < Cd < Ag. Greatest bacterial growth occurred in 1 ppm Al achieving an OD600 of 0.985 and lowest in 1 ppm Ag with an OD600 of 0.090. At a concentration of 1.0 ppm, Ag had a considerable effect on the bacterial consortium, inhibiting the degradation of phenol, whereas this concentration of the other metal ions tested had no effect on degradation. The biokinetic growth model developed supports the suitability of the bacterial consortium for use in phenol degradation