Chioma Blaise Chikere

Monitoring of microbial hydrocarbon remediation in the soil

Bioremediation of hydrocarbon pollutants is advantageous owing to the cost-effectiveness of the technology and the ubiquity of hydrocarbon-degrading microorganisms in the soil. Soil microbial diversity is affected by hydrocarbon perturbation, thus selective enrichment of hydrocarbon utilizers occurs. Hydrocarbons interact with the soil matrix and soil microorganisms determining the fate of the contaminants relative to their chemical nature and microbial degradative capabilities, respectively. Provided the polluted soil has requisite values for environmental factors that influence microbial activities and there are no inhibitors of microbial metabolism, there is a good chance that there will be a viable and active population of hydrocarbon-utilizing microorganisms in the soil. Microbial methods for monitoring bioremediation of hydrocarbons include chemical, biochemical and microbiological molecular indices that measure rates of microbial activities to show that in the end the target goal of pollutant reduction to a safe and permissible level has been achieved. Enumeration and characterization of hydrocarbon degraders, use of micro titer plate-based most probable number technique, community level physiological profiling, phospholipid fatty acid analysis, 16S rRNA- and other nucleic acid-based molecular fingerprinting techniques, metagenomics, microarray analysis, respirometry and gas chromatography are some of the methods employed in bio-monitoring of hydrocarbon remediation as presented in this review.

Bioremediation techniques–classification based on site of application: principles, advantages, limitations and prospects

Environmental pollution has been on the rise in the past few decades owing to increased human activities on energy reservoirs, unsafe agricultural practices and rapid industrialization. Amongst the pollutants that are of environmental and public health concerns due to their toxicities are: heavy metals, nuclear wastes, pesticides, green house gases, and hydrocarbons. Remediation of polluted sites using microbial process (bioremediation) has proven effective and reliable due to its eco-friendly features. Bioremediation can either be carried out ex situ or in situ, depending on several factors, which include but not limited to cost, site characteristics, type and concentration of pollutants. Generally, ex situ techniques apparently are more expensive compared to in situ techniques as a result of additional cost attributable to excavation. However, cost of on-site installation of equipment, and inability to effectively visualize and control the subsurface of polluted sites are of major concerns when carrying out in situ bioremediation. Therefore, choosing appropriate bioremediation technique, which will effectively reduce pollutant concentrations to an innocuous state, is crucial for a successful bioremediation project. Furthermore, the two major approaches to enhance bioremediation are biostimulation and bioaugmentation provided that environmental factors, which determine the success of bioremediation, are maintained at optimal range. This review provides more insight into the two major bioremediation techniques, their principles, advantages, limitations and prospects.

Dynamics of indigenous bacterial communities associated with crude oil degradation in soil microcosms during nutrient-enhanced bioremediation.

Bacterial population dynamics were examined during bioremediation of an African soil contaminated with Arabian light crude oil and nutrient enrichment (biostimulation). Polymerase chain reaction followed by denaturing gradient gel electrophoresis (DGGE) were used to generate bacterial community fingerprints of the different treatments employing the 16S ribosomal ribonucleic acid (rRNA) gene as molecular marker. The DGGE patterns of the nutrient-amended soils indicated the presence of distinguishable bands corresponding to the oil-contaminated–nutrient-enriched soils, which were not present in the oil-contaminated and pristine control soils. Further characterization of the dominant DGGE bands after excision, reamplification and sequencing revealed that Corynebacterium spp., Dietzia spp., Rhodococcus erythropolis sp., Nocardioides sp., Low G+C (guanine plus cytosine) Gram positive bacterial clones and several uncultured bacterial clones were the dominant bacterial groups after biostimulation. Prominent Corynebacterium sp. IC10 sequence was detected across all nutrient-amended soils but not in oil-contaminated control soil. Total heterotrophic and hydrocarbon utilizing bacterial counts increased significantly in the nutrient-amended soils 2 weeks post contamination whereas oil-contaminated and pristine control soils remained fairly stable throughout the experimental period. Gas chromatographic analysis of residual hydrocarbons in biostimulated soils showed marked attenuation of contaminants starting from the second to the sixth week after contamination whereas no significant reduction in hydrocarbon peaks were seen in the oil-contaminated control soil throughout the 6-week experimental period. Results obtained indicated that nutrient amendment of oil-contaminated soil selected and enriched the bacterial communities mainly of the Actinobacteria phylogenetic group capable of surviving in toxic contamination with concomitant biodegradation of the hydrocarbons. The present study therefore demonstrated that the soil investigated harbours hydrocarbon-degrading bacterial populations which can be biostimulated to achieve effective bioremediation of oil-contaminated soil.

Culture-independent analysis of bacterial community composition during bioremediation of crude oil-polluted soil.

Aim: To use cultivation-independent techniques based on DGGE of PCR-amplified 16S rRNA gene and to evaluate bacterial community composition during bioremediation of crude oilpolluted soil. Study design: Molecular fingerprints of bacterial populations involved in the active phase of crude oil biodegradation were generated with DGGE after 16SrRNA gene amplification. Place and Duration of Study: Department of Microbiology and Plant Pathology, University of Pretoria, South Africa, between March and August 2008. Methodology: Crude oil-degrading bacteria in soil microcosms contaminated with 4% crude oil and then biostimulated with nitrogen-phosphorus-potassium inorganic fertilizer (NPK: designated PN soil), calcium ammonium nitrate (designated PU soil) and poultry droppings (designated PP soil) respectively were characterized with PCR of the gene for the small subunit (SSU) of the bacterial ribosome. Total culturable heterotrophic and hydrocarbon utilizing bacteria were enumerated using plate count and Bushnell Haas media. Total organic carbon content was measured throughout the study period to indirectly determine the effect of microbial activity on carbon content in biostimulated treatments as against controls. Gas chromatography was used to monitor hydrocarbon degradation with time while electron microscopy examined community richness during hydrocarbon degradation. Reamplified dominant DGGE bands (550bp) were cleaned up and sequenced using an ABI 3130XL genetic analyzer. Electropherograms were inspected with Chromas Lite 2.01. Sequence identification was performed using BLAST. Results: Dendogram of the DGGE bands constructed using Jaccard coefficient algorithm revealed that communities from PU and PP-amended soils each formed distinct clades whereas PN treated soil showed less association when compared with PU and PP respectively. Fifty distinct bands were excised, reamplified by PCR and sequenced. Sequence analysis revealed the presence of phylogenetically distinct known hydrocarbon degrading bacteria like Corynebacterium spp., Dietzia spp., Janibacter sp. low G+C Gram positive bacterial clones Nocardioides spp., Rhodococcus erythropolis and uncultured bacterial clones. Forty successful sequence data obtained from the excised DGGE bands were submitted to GenBank database under accession numbers GU451069 to GU451108. Chromatograms of the residual hydrocarbons in test treatments and controls showed that biodegradation occurred markedly in treated soils in this order PN>PU>PP while no signification loss was observed in the oil-contaminated control on days zero and 42 respectively. Bacterial counts increased significantly in PN, PU and PP treatments and not in controls PC and OC. total organic carbon increased appreciablly in PN, PU and PP respectively from day zero to dayElectron micrographs of microbial consortia in the nutrient-amended soils revealed presence of active populations induced by biostimulation as against the sparsely populated controls.

Application of Molecular Microbiology Techniques in Bioremediation of Hydrocarbons and Other Pollutants

Molecular microbiology techniques have revolutionized microbial ecology by paving the way for rapid, high -throughput methods for culture -independent assessment and exploitation of microbial communities present in complex ecosystems like crude oil/hydrocarbon polluted soil. The soil microbial community is relatively diversewith a high level of prokaryotic diversity. This soil species pool represents a gold mine for genes involved in the biodegradation of different classes of pollutants. Currently, less than 1% of this diversity is culturable by traditional cultivation techniques. The application of molecular microbiology techniquesinstudyingmicrobial populations in polluted sites without the need for culturing has led to the discovery of novel and unrecog nized

Molecular Assessment of Microbial Species Involved in the Biodegradation of Crude Oil in Saline Niger Delta Sediments Using Bioreactors

Purpose: At elevated salinities conventional microbiological processes are not very effective, therefore clean up of contaminants using bioremediation strategy will involve the use of halophilic and halo-tolerant bacterial species. This research therefore aimed at isolating and identifying potential halophilic and halo-tolerant bacterial species capable of hydrocarbon degradation during bioreactor based treatment with exogenous nutrients. Methods: The diversity of indigenous bacterial species with potential to degrade hydrocarbons was investigated using both culture-dependent and independent techniques. Bioremediation of hydrocarbon contaminated saline sediments was carried out using seven 2.5 liter bioslurry bioreactors operated over a 64-day period. Physicochemical parameters monitored were pH, nitrate, phosphate, total petroleum hydrocarbon (TPH), polycyclic aromatic hydrocarbon (PAH), temperature, salinity, and total organic carbon (%TOC). Results: The baseline TPH, PAH and pH of the sediments were 19 ppm, 3.1 ppm and 7.0 respectively. The baseline salinity of the sediment was 10% thus the sediment was adjudged moderately saline. TPH ranged from 97 ppm-105 ppm on day zero and decreased to an average of 5.62 ppm on day 64, while PAH ranged from 56 ppm-61 ppm on day zero and decreased to an average of 4.02 ppm on day 64. The bacterial species identified as potential hydrocarbon degraders includes Halomonas lutea, Achromobacter spp, Aquitalea magnusonii, Bacillus sp, Sphingobacterium sp, Shewanella sp, Brevundimonas naejangsanensis, Pseudomonas pseudoalcaligenes, Pseudomonas aeruginosa, unidentified bacterium BH23 and Gordonia sp. The genus Pseudomonas formed majority of the isolates successfully sequenced and exhibited similarity values ranging from 91% to 100% with sequences deposited in GenBank. A combination of both molecular and culture based technique allowed the identification to species level of twelve isolates. One isolate could not be identified while the remaining isolates were identified to their generic level. Treatment BCD recorded highest total culturable heterotrophic bacteria (TCHB) count (7.1 × 108 cfu/g) and total culturable hydrocarbon utilizing bacteria (TCHUB) count (6.7 × 108 cfu/g). There was a significant difference at P<0.05 in TCHUB bacteria counts between the unamended bioreactor slurries and those amended with organic and inorganic nutrients. There were also significant differences in TCHUB counts when the bioaugumented slurry was compared with those amended with NPK, Urea and cow dung using one way ANOVA and Tukey’s multiple comparison tests. Conclusion: This study revealed potentially novel bacterial species and previously described hydrocarbon degrading bacterial species that can be characterized further to determine their role in hydrocarbon degradation as well as their salt tolerance level prior to application in bioremediation of saline environments.

Culture-dependent characterization of hydrocarbon utilizing bacteria in selected crude oil-impacted sites in Bodo, Ogoniland, Nigeria

This investigation was carried out to characterize microbial communities in selected crude oil polluted sites in Bodo community, Gokana Local Government Area of Rivers State, Nigeria. Total heterotrophic bacterial counts ranged from 0.7 to 1.37x107 cfu/g and 0.2 to 5.9x106 cfu/ml while counts of hydrocarbon utilizing bacteria ranged from 0.1 to 8.0 x 106 and 0.2 to 7.5 x 105 cfu/ml for soil, sediment and water, respectively. Physiochemical parameters of all samples were determined. The ranges obtained were temperature 31-33°C, pH 7.5-8.2, conductivity 1134 - 7680 µs/cm, total nitrogen 792.4 - 886.3 mg/kg, nitrate 36.55 - 42.70 mg/kg, total organic carbon 2.06 - 2.18%, total petroleum hydrocarbon 1007 - 1104 mg/kg, vanadium 0.001 - 0.007 mg/kg, iron 3.772 - 4.889 mg/kg, chromium 52.40 - 66.20 mg/kg, nickel 40.02 - 41.62 mg/kg, lead 17.30 - 19.40 mg/kg and zinc 35.10 - 39.50 mg/kg for soil and sediments while water had total nitrogen 868 mg/l, nitrate 40.6 mg/l, total organic carbon 3.1 mg/l, total petroleum hydrocarbon 768 mg/l, nickel 39.2 mg/l, lead 17.3 mg/l and turbidity 250 NTU. Bacteria isolates characterized belonged to these genera Bacillus, Proteus, Pseudomonas, Flavobacterium, Corynebacterium, Serratia, Micrococcus, Klebsiella, Enterobacter and Azotobacter. The findings reveal that there is a high population of active indigenous hydrocarbon utilizing bacteria which can be monitored and enhanced to bring about bioremediation in the study area. Key words: Hydrocarbon pollution, soil, water, sediments, hydrocarbon utilizing bacteria, Bodo, Ogoniland.