Screening of hydrocarbon-degrading bacterial isolates using the redox application of 2,6-DCPIP

Petroleum hydrocarbons remain as the major contaminants that could be found across the world. Remediation approach through the utilisation of microbes as the bioremediation means is widely recognised due to their outstanding values. As a result, scientific reports on the isolation and identification of new hydrocarbon-degrading strains were on the rise. Colourimetric-based assays are one of the fastest methods to identify the capability of hydrocarbon-degrading strains in both qualitative and quantitative assessment. In this study, the hydrocarbon-degrading potential of nine bacterial isolates was observed via 2,6-dichlorophenolindophenol (DCPIP) test. Two potent diesel-utilising isolates show a distinctive tendency to utilise aromatic (ADL15) and aliphatic (ADL36) hydrocarbons. Both isolates prove to be a good candidate for bioremediation of wide range of petroleum hydrocarbon components

Bioremediation of petroleum hydrocarbon in Antarctica by microbial species: an overview

The increase of anthropogenic activities and growth of technology in Antarctica is fuelled by the high demand for petroleum hydrocarbons needed for daily activities. Oil and fuel spills that occur during explorations have caused hydrocarbon pollution in this region, prompting concern for the environment by polar communities and the larger world community. Crude oil and petroleum hydrocarbon products contain a wide variety of lethal components with high toxicity and low biodegradability. Hydrocarbon persistence in the Antarctic environment only worsens the issues stemming from environmental pollution as they can be long-term. Numerous efforts to lower the contamination level caused by these pollutants have been conducted mainly in bioremediation, an economical and degrading-wise method. Bioremediation mainly functions on conversion of complex toxic compounds to simpler organic compounds due to the consumption of hydrocarbons by microorganisms as their energy source. This review presents a summary of the collective understanding on bioremediation of petroleum hydrocarbons by microorganisms indigenous to the Antarctic region from past decades to current knowledge

The growth of the Rhodococcus sp. on diesel fuel under the effect of heavy metals and different concentrations of zinc

Co-contamination of diesel fuel and heavy metals can be challenging for microbial remediation due to the complex composition of the fuel and the inhibitory effect of heavy metals. There is an urgent need to study this interaction to improve the pollutant removal efficiency in the Polar Regions. The growth of an Antarctic bacterium, Rhodococcus sp. was studied by comparing the growth at the logarithmic phase under the effect of selected heavy metals (Pb, Cr, As, Cd, Cu, Zn, Ni, Hg and Co). The selected heavy metals inhibited the growth of the Rhodococcus sp. on diesel fuel in an order from highest to lowest of: Hg > Zn > Cd > Cu > Co > Ni > As >Pb> Cr. Growth on diesel fuel co-contaminated with Hg and Zn were 2.95% and 5.71%, respectively compared to the no-metal control. A further experiment with various Zn concentrations was conducted. The specific growth rate of Rhodococcus sp. co-contaminated with different concentrations of Zn showed a correlation coefficient (r) of 0.916, and was modelled with an exponential decay model. Additional investigation is needed to determine the effect of low concentration of Zn on hydrocarbon degradation. It is important to understand the relationships between microbes, hydrocarbons and heavy metals, especially in the Polar Regions because this interaction might be promising in treating hydrocarbon-polluted sites containing heavy metals. The data and results also provide baseline tools of bioremediation processes at low temperatures and the knowledge of the ecological roles of Rhodococcus sp. in Antarctica

Comparative screening methods for the detection of biosurfactant-producing capability of Antarctic hydrocarbon-degrading Pseudomonas sp.

Four preliminary screening methods for biosurfactant synthesis - drop collapse assay, oil displacement activity, microplate assay and emulsification index (E24) were compared and evaluated for their reliability and ease of use. All screening methods showed positive indications for the synthesis of biological surface-active agents. Nevertheless, partial collapse of the supernatant and low emulsification index (E24) of Pseudomonassp. might signify a low production of biosurfactants. Based on our observation, both drop collapse and oil displacement assay is the fastest, easiest and most reliable analytical routine to be suggested to screen for biosurfactant producing strains. In the extent for a high throughput screening (HTS), drop collapse assay is the best method for an accurate screening of biosurfactant producers

Production of lipopeptide biosurfactant by a hydrocarbon-degrading antarctic Rhodococcus

Rhodococci are renowned for their great metabolic repertoire partly because of their numerous putative pathways for large number of specialized metabolites such as biosurfactant. Screening and genome-based assessment for the capacity to produce surface-active molecules was conducted on Rhodococcus sp. ADL36, a diesel-degrading Antarctic bacterium. The strain showed a positive bacterial adhesion to hydrocarbon (BATH) assay, drop collapse test, oil displacement activity, microplate assay, maximal emulsification index at 45% and ability to reduce water surface tension to < 30 mN/m. The evaluation of the cell-free supernatant demonstrated its high stability across the temperature, pH and salinity gradient although no correlation was found between the surface and emulsification activity. Based on the positive relationship between the assessment of macromolecules content and infrared analysis, the extracted biosurfactant synthesized was classified as a lipopeptide. Prediction of the secondary metabolites in the non-ribosomal peptide synthetase (NRPS) clusters suggested the likelihood of the surface-active lipopeptide production in the strain’s genomic data. This is the third report of surface-active lipopeptide producers from this phylotype and the first from the polar region. The lipopeptide synthesized by ADL36 has the prospect to be an Antarctic remediation tool while furnishing a distinctive natural product for biotechnological application and research

Phenol degradation and molecular validation of phenol hydroxylase gene of Alcaligenes faecalis

Currently, phenol pollution has caused some environmental concerns as it causes severe toxicity towards human health and environmental conditions. Intensive efforts to reduce the contamination of pollutants have been done especially in bioremediation techniques. Many microbial species have been introduced to be utilised for contamination clean-up and one of them is Alcaligenes faecalis. A study on the phenol degrading ability and molecular analysis of A. faecalis was conducted. To study the phenol degrading ability of A. faecalis, the bacteria was incubated in six different concentrations of phenol – 0.1, 0.4, 0.5, 0.9, 1.0, and 1.5 g/L. The growth of bacteria and phenol degradation in each phenol concentration were monitored. Among all the concentrations studied, phenol concentration of 0.9 g/L showed the highest degradation rate. Meanwhile, the molecular analysis of the bacteria was carried out by isolating the phenol hydroxylase gene which is responsible for degrading phenol by using the designated primer. The gene was amplified by using PCR technique with an annealing temperature of 56.4°C. The expected size of the gene was between 300 - 400 bp. After DNA sequencing, molecular analysis was done and the DNA fragment obtained had a length of 337 bp. Next, BLAST search was used to confirm the sequence obtained was phenol hydroxylase gene isolated from A. faecalis. The BLAST result showed that the phenol hydroxylase gene was successfully amplified from the bacteria. These studies showed the hypothetical use of this bacteria to treat phenol-contaminated environment

Biodegradation of diesel fuel by two psychrotolerant strains isolated from Southern Victoria Island, Antarctica

Hydrocarbon contamination in Antarctica poses a great threat to the delicate and unique ecosystems of this continent. Bioremediation of hydrocarbon pollutants via utilisation of the indigenous hydrocarbon-degrading bacteria, has been proposed as an environmentally friendly method to clean-up contaminated soils in Antarctica. This study focused on diesel-degrading Pseudomonas and Rhodococcus species isolated from pristine soils located at the Southern Victoria Island, Antarctica. Isolates were assessed for their ability to grow on diesel as a sole carbon source on solid media at 4°C. Nine isolates showed significant growth in enriched agar after 14 days of incubation. Isolates were then screened to obtain the most promising diesel-degrading strains through colourimetric assay. Two potent isolates that possess rapid utilisation of 0.5% (v/v) diesel were selected and identified as Pseudomonas sp. strain ADL15 and Rhodococcus sp. strain ADL36. The factors that contribute to the growth of both strains were characterised initially using the conventional ‘one-factor-at-a-time’ approach. During this stage, the optimal condition for the growth of both ADL15 and ADL36 were at pH 7.0, 20°C, 1.0% (w/v) NaCl, and 1.0 g/L NH4NO3. However, strain ADL36 favoured a higher amount of diesel (2.0% (v/v)) for bacterial growth by comparison to ADL15 (1.0% (v/v)). Percentage of dodecane mineralisation was used as the mean to indicate diesel reduction through gas chromatographic analysis. While strain ADL36 showed 83.75% of dodecane mineralisation, the reduction of dodecane by AD15 is merely at 22.39%. Response surface methodology (RSM) based on central composite design (CCD) was used to improve and optimise the effect of significant factors toward the biodegradation of diesel. RSM proved to enhance the reduction of experimented hydrocarbon (dodecane) with a 15% and 16% increment of mineralisation for isolate ADL15 (38.32%) and ADL36 (99.89%), respectively. The results also demonstrated that addition of salt to culture media was the limiting factor in hydrocarbon degradation. Whole genome sequencing showed that ADL15 and ADL36 were closely related to the Pseudomonas fluorescens and Rhodococcus erythropolis grouping, respectively. Metagenomic analyses revealed the presence of alkane hydroxylases systems which was responsible for alkane degradation in ADL36 but not in ADL15. This founding corresponds to the gas chromatographic analysis in which ADL36 proved to be a better alkane degrader than ADL15. Detection of the complete pathway of aromatic compound degradation in the latter strain indicates a stronger inclination of the strain to utilise aromatic components in diesel as the carbon source. The presence of putative monooxygenases may also suggest that this strain may utilise specific alkane for their growth. The results from this study showed that strain ADL15 and ADL36 have an excellent potential in bioremediation of aromatics and aliphatics, respectively

Biodeterioration of Untreated Polypropylene Microplastic Particles by Antarctic Bacteria

Microplastic pollution is globally recognised as a serious environmental threat due to its ubiquitous presence related primarily to improper dumping of plastic wastes. While most studies have focused on microplastic contamination in the marine ecosystem, microplastic pollution in the soil environment is generally little understood and often overlooked. The presence of microplastics affects the soil ecosystem by disrupting the soil fertility and quality, degrading the food web, and subsequently influencing both food security and human health. This study evaluates the growth and biodegradation potential of the Antarctic soil bacteria Pseudomonas sp. ADL15 and Rhodococcus sp. ADL36 on the polypropylene (PP) microplastics in Bushnell Haas (BH) medium for 40 days. The degradation was monitored based on the weight loss of PP microplastics, removal rate constant per day (K), and their half-life. The validity of the PP microplastics&rsquo; biodegradation was assessed through structural changes via Fourier transform infrared spectroscopy analyses. The weight loss percentage of the PP microplastics by ADL15 and ADL36 after 40 days was 17.3% and 7.3%, respectively. The optimal growth in the BH media infused with PP microplastics was on the 40th and 30th day for ADL15 and ADL36, respectively. The infrared spectroscopic analysis revealed significant changes in the PP microplastics&rsquo; functional groups following the incubation with Antarctic strains

Evaluation of conventional and response surface level optimisation of n-dodecane (n-C12) mineralisation by psychrotolerant strains isolated from pristine soil at Southern Victoria Island, Antarctica

Abstract Background Biodegradation of hydrocarbons in Antarctic soil has been reported to be achieved through the utilisation of indigenous cold-adapted microorganisms. Although numerous bacteria isolated from hydrocarbon-contaminated sites in Antarctica were able to demonstrate promising outcomes in utilising hydrocarbon components as their energy source, reports on the utilisation of hydrocarbons by strains isolated from pristine Antarctic soil are scarce. In the present work, two psychrotolerant strains isolated from Antarctic pristine soil with the competency to utilise diesel fuel as the sole carbon source were identified and optimised through conventional and response surface method. Results Two potent hydrocarbon-degraders (ADL15 and ADL36) were identified via partial 16S rRNA gene sequence analysis, and revealed to be closely related to the genus Pseudomonas and Rhodococcus sp., respectively. Factors affecting diesel degradation such as temperature, hydrocarbon concentration, pH and salt tolerance were studied. Although strain ADL36 was able to withstand a higher concentration of diesel than strain ADL15, both strains showed similar optimal condition for the cell’s growth at pH 7.0 and 1.0% (w/v) NaCl at the conventional ‘one-factor-at-a-time’ level. Both strains were observed to be psychrotrophs with optimal temperatures of 20 °C. Qualitative and quantitative analysis were performed with a gas chromatograph equipped with a flame ionisation detector to measure the reduction of n-alkane components in diesel. In the pre-screening medium, strain ADL36 showed 83.75% of n-dodecane mineralisation while the reduction of n-dodecane by strain ADL15 was merely at 22.39%. The optimised condition for n-dodecane mineralisation predicted through response surface methodology enhanced the reduction of n-dodecane to 99.89 and 38.32% for strain ADL36 and strain ADL15, respectively. Conclusions Strain ADL36 proves to be a better candidate for bioaugmentation operations on sites contaminated with aliphatic hydrocarbons especially in the Antarctic and other cold regions. The results obtained throughout strongly supports the use of RSM for medium optimisation