Robin Lockington

A Comprehensive Review of Aliphatic Hydrocarbon Biodegradation by Bacteria

Hydrocarbons are relatively recalcitrant compounds and are classified as high-priority pollutants. However, these compounds are slowly degraded by a large variety of microorganisms. Bacteria are able to degrade aliphatic saturated and unsaturated hydrocarbons via both aerobic and anaerobic pathways. Branched hydrocarbons and cyclic hydrocarbons are also degraded by bacteria. The aerobic bacteria use different types of oxygenases, including monooxygenase, cytochrome-dependent oxygenase and dioxygenase, to insert one or two atoms of oxygen into their targets. Anaerobic bacteria, on the other hand, employ a variety of simple organic and inorganic molecules, including sulphate, nitrate, carbonate and metals, for hydrocarbon oxidation.

Enhanced removal of petroleum hydrocarbons using a bioelectrochemical remediation system with pre-cultured anodes

Bioelectrochemical remediation (BER) systems such as microbial fuel cells (MFCs) have recently emerged as a green technology for the effective remediation of petroleum hydrocarbon contaminants (PH) coupled with simultaneous energy recovery. Recent research has shown that biofilms previously enriched for substrate degrading bacteria resulted in excellent performance in terms of substrate removal and electricity generation but the effects on hydrocarbon contaminant degradation were not examined. Here we investigate the differences between enriched biofilm anodes and freshly inoculated new anodes in diesel fed single chamber mediatorless microbial fuel cells (DMFC) using various techniques for the enhancement of PH contaminant remediation with concomitant electricity generation. An anodophilic microbial consortium previously selected for over a year through continuous culturing with a diesel concentration of about 800mgl(-1) and which now showed complete removal of this concentration of diesel within 30days was compared to that of a freshly inoculated new anode MFC (showing 83.4% removal of diesel) with a simultaneous power generation of 90.81mW/m(2) and 15.04mW/m(2) respectively. The behaviour of pre-cultured anodes at a higher concentration of PH (8000mgl(-1)) was also investigated. Scanning electron microscopy observation revealed a thick biofilm covering the pre-cultured anodic electrode but not the anode from the freshly inoculated MFC. High resolution imaging showed the presence of thin 60nm diametre pilus-like projections emanating from the cells. Anodic microbial community profiling confirmed that the selection for diesel degrading exoelectrogenic bacteria had occurred. Identification of a biodegradative gene (alkB) provided strong evidence of the catabolic pathway used for diesel degradation in the DMFCs.

The Biodiversity Changes in the Microbial Population of Soils Contaminated with Crude Oil.

Abstract Crude oil spills resulting from excavation, transportation and downstream processes can cause intensive damage to living organisms and result in changes in the microbial population of that environment. In this study, we used a pyrosequencing analysis to investigate changes in the microbial population of soils contaminated with crude oil. Crude oil contamination in soil resulted in the creation of a more homogenous population of microorganisms dominated by members of the Actinomycetales, Clostridiales and Bacillales (all belonging to Gram-positive bacteria) as well as Flavobacteriales, Pseudomonadales, Burkholderiales, Rhizobiales and Sphingomonadales (all belonging to Gram-negative bacteria). These changes in the biodiversity decreased the ratios of chemoheterotrophic bacteria at higher concentrations of crude oil contamination, with these being replaced by photoheterotrophic bacteria, mainly Rhodospirillales. Several of the dominant microbial orders in the crude oil contaminated soils are able to degrade crude oil hydrocarbons and therefore are potentially useful for remediation of crude oil in contaminated sites.

Microbial diversity and hydrocarbon degrading gene capacity of a crude oil field soil as determined by metagenomics analysis.

Soils contaminated with crude oil are rich sources of enzymes suitable for both degradation of hydrocarbons through bioremediation processes and improvement of crude oil during its refining steps. Due to the long term selection, crude oil fields are unique environments for the identification of microorganisms with the ability to produce these enzymes. In this metagenomic study, based on Hiseq Illumina sequencing of samples obtained from a crude oil field and analysis of data on MG‐RAST, Actinomycetales (9.8%) were found to be the dominant microorganisms, followed by Rhizobiales (3.3%). Furthermore, several functional genes were found in this study, mostly belong to Actinobacteria (12.35%), which have a role in the metabolism of aliphatic and aromatic hydrocarbons (2.51%), desulfurization (0.03%), element shortage (5.6%), and resistance to heavy metals (1.1%). This information will be useful for assisting in the application of microorganisms in the removal of hydrocarbon contamination and/or for improving the quality of crude oil.

A Review on the Genetics of Aliphatic and Aromatic Hydrocarbon Degradation

Because of the high diversity of hydrocarbons, degradation of each class of these compounds is activated by a specific enzyme. However, most of other downstream enzymes necessary for complete degradation of hydrocarbons maybe common between different hydrocarbons. The genes encoding proteins for degradation of hydrocarbons, including the proteins required for the uptake of these molecules, the specific enzyme used for the initial activation of the molecules and other necessary degrading enzymes are usually arranged as an operon. Although the corresponding genes in many phylogenetic groups of microbial species show different levels of diversity in terms of the gene sequence, the organisation of the genes in the genome or on plasmids and the activation mode (inductive or constitutive), some organisms show identical hydrocarbon-degrading genes, probably as a result of horizontal gene transfer between microorganisms.

Multiwall carbon nanotubes increase the microbial community in crude oil contaminated fresh water sediments

Since crude oil contamination is one of the biggest environmental concerns, its removal from contaminated sites is of interest for both researchers and industries. In situ bioremediation is a promising technique for decreasing or even eliminating crude oil and hydrocarbon contamination. However, since these compounds are potentially toxic for many microorganisms, high loads of contamination can inhibit the microbial community and therefore reduce the removal rate. Therefore, any strategy with the ability to increase the microbial population in such circumstances can be of promise in improving the remediation process. In this study, multiwall carbon nanotubes were employed to support microbial growth in sediments contaminated with crude oil. Following spiking of fresh water sediments with different concentrations of crude oil alone and in a mixture with carbon nanotubes for 30days, the microbial profiles in these sediments were obtained using FLX-pyrosequencing. Next, the ratios of each member of the microbial population in these sediments were compared with those values in the untreated control sediment. This study showed that combination of crude oil and carbon nanotubes can increase the diversity of the total microbial population. Furthermore, these treatments could increase the ratios of several microorganisms that are known to be effective in the degradation of hydrocarbons.

The integration of sequencing and bioinformatics in metagenomics

Since most microorganisms in natural environments cannot be cultured in laboratory media, due to low growth rates or their dependency on specific conditions, culture-based systems are unable to estimate the full microbial diversity of an environment. However, molecular techniques can provide this ability by analysing the diversity of macromolecules, such as proteins, RNA or DNA, present in the environment. Metagenomic methods employ sequencing procedures for the determination of the microbial diversity of a community (sequence-driven metagenomic analysis) or for examining a particular functional ability of microorganisms in the environment (function-driven metagenomic gene identification), using genomic DNA obtained directly from environmental samples. Application of metagenomic methods provides a huge amount of data that can be analysed only by using powerful computational bioinformatics tools. Currently, these bioinformatic tools are adequate to allow the ecological structure of a community and the possible functions of its members to be determined. The resulting data can be useful for phylogenetic and biotechnological studies. This paper reviews the emergence of new technologies based on sequencing environmental DNA and on bioinformatics. We assess its potential for application in environmental studies, particularly for developing new biomolecules that can be applied to degrading recalcitrant pollutants in terrestrial and aquatic systems.

A pyrosequencing-based analysis of microbial diversity governed by ecological conditions in the Winogradsky column

The Winogradsky column is used as a microcosm to mimic both the microbial diversity and the ecological relationships between the organisms in lake sediments. In this study, a pyrosequencing approach was used to obtain a more complete list of the microbial organisms present in such columns and their ratios in different layers of this microcosm. Overall, 27 different phyla in these columns were detected in these columns, most (20 phyla) belonged to bacteria. Based on this study, Proteobacteria (mostly Sphingomonadales), Cyanobacteria (mostly Oscillatoriales) and Bacteroidetes (mostly Flavobacteriales) were the dominant microorganisms in the water, middle, and bottom layers of this column, respectively. Although the majority of organism in the water layer were photoautotrophic organisms, the ratio of the phototrophic organisms decreased in the lower layers, replaced by chemoheterotrophic bacteria. Furthermore, the proportion of aerobic chemoheterotrophic bacteria was greater in the higher layers of the column in comparison to the bottom. The green and purple sulfur phototrophic bacteria inhabited the bottom and middle of these columns, with none of them found in the water layer. Although the sulfur oxidizing bacteria were the dominant chemolithotrophic bacteria in the water layer, their ratio decreases in lower layers, being replaced with nitrogen oxidizing bacteria in the middle and bottom layers. Overall, the microbial population of these layers changes from a phototrophic and aerobic chemoheterotrophic organisms in the water layer to a mostly anaerobic chemoheterotrophic population of bacteria in the bottom layers.

Rhodococcus wratislaviensis strain 9: An efficient p-nitrophenol degrader with a great potential for bioremediation

A Gram-positive bacterium, Rhodococcus wratislaviensis strain 9, was isolated from groundwater contaminated with nitrophenolics and trichloroethene following enrichment culture technique. The cells of strain 9 grown on LB broth (uninduced) degraded 720 μM p-nitrophenol (PNP) within 12 h, and utilized as a source of carbon and energy. Orthogonal experimental design analysis to determine optimal conditions for biodegradation of PNP showed that pH had a significant positive effect (P ≤ .05) on bacterial degradation of PNP, while glucose, di- and tri-nitrophenols exhibited significant negative effect. Cell-free extracts obtained from PNP-grown culture that contained 20 μg mL-1 protein degraded 90% of 720 μM PNP within 5 h of incubation. Two-dimensional protein analysis revealed differential expression of the oxygenase component of PNP monooxygenase and an elongation factor Tu in PNP-grown cells, but not in those grown on glucose. The strain 9 remediated laboratory wastewater containing 900 μM PNP efficiently within 14 h, indicating its great potential in bioremediation of PNP-contaminated waters.

Identification of a new operon involved in desulfurization of dibenzothiophenes using a metagenomic study and cloning and functional analysis of the genes

The presence of sulphur-substituted hydrocarbons in fossil fuels are one of main reasons for the release of sulfur oxides into the environment. Dibenzothiophenes (DBT) are organic sulfur-containing molecules in crude oil, which have the potential for biological oxidation, with the sulphur being removed through an enzymatic cleavage of the CS bonds. Therefore, finding new strains that can desulfurize this compound has recently become a point of interest. In this study, three new genes involved in the bacterial desulfurization of Dibenzothiophene, which were sequenced in the course of a metagenomic study, were isolated by PCR amplification in the laboratory. The activities of these genes were then analysed following insertion into an expression vector and cloning in Escherichia coli DH5α cells. Based on the results, all three genes were actively expressed and their products could act on their corresponding substrates.