The co-application of biochar with bioremediation for the removal of petroleum hydrocarbons from contaminated soil.

Soil pollution from petroleum hydrocarbon is a global environmental problem that could contribute to the non-actualisation of the United Nations Sustainable Development Goals. Several techniques have been used to remediate petroleum hydrocarbon-contaminated soils; however, there are technical and economical limitations to existing methods. As such, the development of new approaches and the improvement of existing techniques are imperative. Biochar, a low-cost carbonaceous product of the thermal decomposition of waste biomass has gained relevance in soil remediation. Biochar has been applied to remediate hydrocarbon-contaminated soils, with positive and negative results reported. Consequently, attempts have been made to improve the performance of biochar in the hydrocarbon-based remediation process through the co-application of biochar with other bioremediation techniques as well as modifying biochar properties before use. Despite the progress made in this domain, there is a lack of a detailed single review consolidating the critical findings, new developments, and challenges in biochar-based remediation of petroleum hydrocarbon-contaminated soil. This review assessed the potential of biochar co-application with other well-known bioremediation techniques such as bioaugmentation, phytoremediation, and biostimulation. Additionally, the benefits of modification in enhancing biochar suitability for bioremediation were examined. It was concluded that biochar co-application generally resulted in higher hydrocarbon removal than sole biochar treatment, with up to a 4-fold higher removal observed in some cases. However, most of the biochar co-applied treatments did not result in hydrocarbon removal that was greater than the additive effects of individual treatment. Overall, compared to their complementary treatments, biochar co-application with bioaugmentation was more beneficial in hydrocarbon removal than biochar co-application with either phytoremediation or biostimulation. Future studies should integrate the ecotoxicological and cost implications of biochar co-application for a viable remediation process. Lastly, improving the synergistic interactions of co-treatment on hydrocarbon removal is critical to capturing the full potential of biochar-based remediation.

Natural attenuation of legacy hydrocarbon spills in pristine soils is feasible despite difficult environmental conditions in the monsoon tropics.

The Kimberley region of Western Australia is a National Heritage listed region that is internationally recognised for its environmental and cultural significance. However, petroleum spills have been reported at a number of sites across the region, representing an environmental concern. The region is also characterised as having low soil nutrients, high temperatures and monsoonal rain - all of which may limit the potential for natural biodegradation of petroleum. Therefore, this work evaluated the effect of legacy petroleum hydrocarbons on the indigenous soil microbial community (across the domains Archaea, Bacteria and Fungi) at three sites in the Kimberley region. At each site, soil cores were removed from contaminated and control areas and analysed for total petroleum hydrocarbons, soil nutrients, pH and microbial community profiling (using16S rRNA and ITS sequencing on the Illumina MiSeq Platform). The presence of petroleum hydrocarbons decreased microbial diversity across all kingdoms, altered the structure of microbial communities and increased the abundance of putative hydrocarbon degraders (e.g. Mycobacterium, Acremonium, Penicillium, Bjerkandera and Candida). Microbial community shifts from contaminated soils were also associated with an increase in soil nutrients (notably Colwell P and S). Our study highlights the long-term effect of legacy hydrocarbon spills on soil microbial communities and their diversity in remote, infertile monsoonal soils, but also highlights the potential for natural attenuation to occur in these environments.

Can biochar be an effective and reliable biostimulating agent for the remediation of hydrocarbon-contaminated soils?

Petroleum hydrocarbons represent one of the most common soil contaminants, whose presence poses a significant risk to soil biota and human health; for example, in Europe, hydrocarbon contamination accounts for more than 30% of contaminated sites. The use of biochar as a proposed alternative to the conventional remediation of soil contaminated with petroleum hydrocarbons has gained credence in recent times because of its cost-effectiveness and environmentally friendly nature. Biochar is a carbonaceous material produced by heating biomass in an oxygen-limited environment at high temperature. This review provides an overview of the application of biochar to remediate petroleum hydrocarbon-contaminated soils, with emphasis on the possibility of biochar functioning as a biostimulation agent. The properties of biochar were also examined. Furthermore, the mechanism, ecotoxicological impact and possible factors affecting biochar-based remediation are discussed. The review concludes by examining the drawbacks of biochar use in the remediation of hydrocarbon-contaminated soils and how to mitigate them. Biochar impacts soil microbes, which may result in the promotion of the degradation of petroleum hydrocarbons in the soil. Linear regression between bacterial population and degradation efficiency showed that R2 was higher (0.50) and significant in treatment amended with biochar or both biochar and nutrient/fertiliser (p < 0.01), compared to treatment with nutrient/fertiliser only or no amendment (R2 = 0.11). This suggest that one of the key impacts of biochar is enhancing microbial biomass and thus the biodegradation of petroleum hydrocarbons. Biochar represents a promising biostimulation agent for the remediation of hydrocarbon-contaminated soil. However, there remains key questions to be answered.

Impact of necrophytoremediation on petroleum hydrocarbon degradation, ecotoxicity and soil bacterial community composition in diesel-contaminated soil.

Hydrocarbon degradation is usually measured in laboratories under controlled conditions to establish the likely efficacy of a bioremediation process in the field. The present study used greenhouse-based bioremediation to investigate the effects of natural attenuation (NA) and necrophytoremediation (addition of pea straw (PS)) on hydrocarbon degradation, toxicity and the associated bacterial community structure and composition in diesel-contaminated soil. A significant reduction in total petroleum hydrocarbon (TPH) concentration was detected in both treatments; however, PS-treated soil showed more rapid degradation (87%) after 5 months together with a significant reduction in soil toxicity (EC50 = 91 mg diesel/kg). Quantitative PCR analysis revealed an increase in the number of 16S rRNA and alkB genes in the PS-amended soil. Substantial shifts in soil bacterial community were observed during the bioremediation, including an increased abundance of numerous hydrocarbon-degrading bacteria. The bacterial community shifted from dominance by Alphaproteobacteria and Gammaproteobacteria in the original soil to Actinobacteria during bioremediation. The dominance of two genera of bacteria, Sphingobacteria and Betaproteobacteria, in both NA- and PS-treated soil demonstrated changes occurring within the soil bacterial community through the incubation period. Additionally, pea straw itself was found to harbour a diverse hydrocarbonoclastic community including Luteimonas, Achromobacter, Sphingomonas, Rhodococcus and Microbacterium. At the end of the experiment, PS-amended soil exhibited reduced ecotoxicity and increased bacterial diversity as compared with the NA-treated soil. These findings suggest the rapid growth of species stimulated by the bioremediation treatment and strong selection for bacteria capable of degrading petroleum hydrocarbons during necrophytoremediation. Graphical abstract.

A comparative study of biological activated carbon based treatments on two different types of municipal reverse osmosis concentrates.

A study was conducted to understand the impact of reverse osmosis concentrate (ROC) characteristics on the efficacy of biological activated carbon (BAC) based treatments for removing organics and nutrients from two ROC streams (ROCa derived from municipal waste input with high salinity, and ROCb derived from domestic waste plus industrial trade waste with markedly lower salinity). Fluorescence excitation and emission matrix spectra and molecular weight analysis demonstrated that ROCa and ROCb had a significantly different composition of organic compounds due to the petrochemical processing and abattoir waste compounds in ROCb. Although the sequence of coagulation, UV/H2O2 and BAC gave the highest organic removal from the two ROCs (67% DOC for ROCa and 62% for ROCb), UV/H2O2 followed by BAC achieved satisfactory removal (>55%) for both ROC types. Sequential treatment involving coagulation gave better phosphorus removal (>90%) than any single treatment (<65%). Total nitrogen (TN) removal was fairly low (<50%) for all the treatment options and the salinity level had insignificant impact on nitrogen removal. Analysis of bacterial communities suggested that higher phosphorus removal and lower total nitrogen and nitrate removal from ROCb than ROCa was related to the presence of various denitrifying or phosphorus accumulating bacteria in the BAC.

Interfacial separation of concentrated dye mixtures from solution with environmentally compatible nitrogenous-silane nanoparticles modified with Helianthus annuus husk extract.

The capacity of an adsorbent to bind and remove dye from solution greatly depends on the type of functionalization present on the nanoparticles surface, and its interaction with the dye molecules. Within this study, nitrogenous silane nanoparticles were hydrothermally synthesized resulting in the formation of rapid and highly efficient adsorbents for concentrated mixed dyes. The amorphous silane nanoparticles exhibited a monolayer based mechanism of mixed dye adsorption with removal capacities between 416.67 and 714.29 mg/g of adsorbent. Dye removal was predominantly due to the electrostatic attraction between the positively charged silane nanoparticles (13.22-8.20 mV) and the negatively charged dye molecules (-54.23 mV). Addition of H. annuus extract during synthesis resulted in three times the surface area and 10 times increased pore volume compared to the positive control. XPS analysis showed that silane treatments had various nitrogen containing functionalities at their surface responsible for binding dye. The weak colloidal stability of silane particles (13.22-8.20 mV) was disrupted following dye binding, resulting in their rapid coagulation and flocculation which facilitated the separation of bound dye molecules from solution. The suitability for environmental applications using these treatments was supported by a bacterial viability assay showing >90% cell viability in treated dye supernatants.

Petroleum Hydrocarbon Contamination in Terrestrial Ecosystems-Fate and Microbial Responses.

Petroleum hydrocarbons represent the most frequent environmental contaminant. The introduction of petroleum hydrocarbons into a pristine environment immediately changes the nature of that environment, resulting in reduced ecosystem functionality. Natural attenuation represents the single, most important biological process which removes petroleum hydrocarbons from the environment. It is a process where microorganisms present at the site degrade the organic contaminants without the input of external bioremediation enhancers (i.e., electron donors, electron acceptors, other microorganisms or nutrients). So successful is this natural attenuation process that in environmental biotechnology, bioremediation has developed steadily over the past 50 years based on this natural biodegradation process. Bioremediation is recognized as the most environmentally friendly remediation approach for the removal of petroleum hydrocarbons from an environment as it does not require intensive chemical, mechanical, and costly interventions. However, it is under-utilized as a commercial remediation strategy due to incomplete hydrocarbon catabolism and lengthy remediation times when compared with rival technologies. This review aims to describe the fate of petroleum hydrocarbons in the environment and discuss their interactions with abiotic and biotic components of the environment under both aerobic and anaerobic conditions. Furthermore, the mechanisms for dealing with petroleum hydrocarbon contamination in the environment will be examined. When petroleum hydrocarbons contaminate land, they start to interact with its surrounding, including physical (dispersion), physiochemical (evaporation, dissolution, sorption), chemical (photo-oxidation, auto-oxidation), and biological (plant and microbial catabolism of hydrocarbons) interactions. As microorganism (including bacteria and fungi) play an important role in the degradation of petroleum hydrocarbons, investigations into the microbial communities within contaminated soils is essential for any bioremediation project. This review highlights the fate of petroleum hydrocarbons in tertial environments, as well as the contributions of different microbial consortia for optimum petroleum hydrocarbon bioremediation potential. The impact of high-throughput metagenomic sequencing in determining the underlying degradation mechanisms is also discussed. This knowledge will aid the development of more efficient, cost-effective commercial bioremediation technologies.

The impact of lead co-contamination on ecotoxicity and the bacterial community during the bioremediation of total petroleum hydrocarbon-contaminated soils.

The continued increase in the global demand for oil, which reached 4,488 Mtoe in 2018, leads to large quantities of petroleum products entering the environment posing serious risks to natural ecosystems if left untreated. In this study, we evaluated the impact of co-contamination with lead on the efficacy of two bioremediation processes, natural attenuation and biostimulation of Total Petroleum Hydrocarbons (TPH) as well as the associated toxicity and the changes in the microbial community in contaminated soils. The biostimulated treatment resulted in 96% and 84% reduction in TPH concentration in a single and a co-contamination scenario, respectively, over 28 weeks of a mesocosm study. This reduction was significantly more in comparison to natural attenuation in a single and a co-contamination scenario, which was 56% and 59% respectively. In contrast, a significantly greater reduction in the associated toxicity of in soils undergoing natural attenuation was evident compared with soils undergoing biostimulation despite the lower TPH degradation when bioassays were applied. The earthworm toxicity test showed a decrease of 72% in the naturally attenuated toxicity versus only 62% in the biostimulated treatment of a single contamination scenario. In a co-contamination scenario, toxicity decreased only 30% and 8% after natural attenuation and biostimulation treatments, respectively. 16s rDNA sequence analysis was used to assess the impact of both the co-contamination and the bioremediation treatment. NGS data revealed major bacterial domination by Nocardioides spp., which reached 40% in week 20 of the natural attenuation treatment. In the biostimulated soil samples, more than 50% of the bacterial community was dominated by Alcanivorax spp. in week 12. The presence of Pb in the natural attenuation treatment resulted in an increased abundance of a few Pb-resistant genera such as Sphingopyxis spp. and Thermomonas spp in addition to Nocardioides spp. In contrast, Pb co-contamination completely shifted the bacterial pattern in the stimulated treatment with Pseudomonas spp. comprising approximately 45% of the bacterial profile in week 12. This study confirms the effectiveness of biostimulation over natural attenuation in remediating TPH and TPH-Pb contaminated soils. In addition, the presence of co-contaminants (e.g. Pb) results in serious impacts on the efficacy of bioremediation of TPH in contaminated soils, which must be considered prior to designing any bioremediation strategy.

Phytofabrication of Iron Nanoparticles for Hexavalent Chromium Remediation.

Hexavalent chromium is a genotoxic and carcinogenic byproduct of a number of industrial processes, which is discharged into the environment in excessive and toxic concentrations worldwide. In this paper, the synthesis of green iron oxide nanoparticles using extracts of four novel plant species [Pittosporum undulatum, Melia azedarach, Schinus molle, and Syzygium paniculatum (var. australe)] using a "bottom-up approach" has been implemented for hexavalent chromium remediation. Nanoparticle characterizations show that different plant extracts lead to the formation of nanoparticles with different sizes, agglomeration tendencies, and shapes but similar amorphous nature and elemental makeup. Hexavalent chromium removal is linked with the particle size and monodispersity. Nanoparticles with sizes between 5 and 15 nm from M. azedarach and P. undulatum showed enhanced chromium removal capacities (84.1-96.2%, respectively) when compared to the agglomerated particles of S. molle and S. paniculatum with sizes between 30 and 100 nm (43.7-58.7%, respectively) in over 9 h. This study has shown that the reduction of iron salts with plant extracts is unlikely to generate vast quantities of stable zero valent iron nanoparticles but rather favor the formation of iron oxide nanoparticles. In addition, plant extracts with higher antioxidant concentrations may not produce nanoparticles with morphologies optimal for pollutant remediation.

Implications of co-contamination with aged heavy metals and total petroleum hydrocarbons on natural attenuation and ecotoxicity in Australian soils.

The bioremediation of historic industrial contaminated sites is a complex process. Co-contamination, often with lead which was commonly added to gasoline until 16 years ago is one of the biggest challenges affecting the clean-up of these sites. In this study, the effect of heavy metals, as co-contaminant, together with total petroleum hydrocarbons (TPH) is reported, in terms of remaining soil toxicity and the structure of the microbial communities. Contaminated soil samples from a relatively hot and dry climate in Western Australia were collected (n = 27). Analysis of soils showed the presence of both contaminants, TPHs and heavy metals. The Microtox test confirmed that their co-presence elevated the remaining ecotoxicity. Toxicity was correlated with the presence of lead, zinc and TPH (0.893, 0.599 and 0.488), respectively, assessed using Pearson Correlation coefficient factor. Next Generation Sequencing of soil bacterial 16S rRNA, revealed a lack of dominate genera; however, despite the variation in soil type, a few genera including Azospirillum spp. and Conexibacter were present in most soil samples (85% and 82% of all soils, respectively). Likewise, many genera of hydrocarbon-degrading bacteria were identified in all soil samples. Streptomyces spp. was presented in 93% of the samples with abundance between 7% and 40%. In contrast, Acinetobacter spp. was found in only one sample but was a dominant member of (45%) of the microbial community. In addition, some bacterial genera were correlated to the presence of the heavy metals, such as Geodermatophilus spp., Rhodovibrio spp. and Rubrobacter spp. which were correlated with copper, lead and zinc, respectively. This study concludes that TPH and heavy metal co-contamination significantly elevated the associated toxicity. This is an important consideration when carrying out risk assessment associated with natural attenuation. This study also improves knowledge about the dynamics of microbial communities in mixed contamination scenarios.

Large scale treatment of total petroleum-hydrocarbon contaminated groundwater using bioaugmentation.

Bioaugmentation or the addition of microbes to contaminated sites has been widely used to treat contaminated soil or water; however this approach is often limited to laboratory based studies. In the present study, large scale bioaugmentation has been applied to total petroleum hydrocarbons (TPH)-contaminated groundwater at a petroleum facility. Initial TPH concentrations of 1564 mg L-1 in the field were reduced to 89 mg L-1 over 32 days. This reduction was accompanied by improved ecotoxicity, as shown by Brassica rapa germination numbers that increased from 52 at day 0 to 82% by the end of the treatment. Metagenomic analysis indicated that there was a shift in the microbial community when compared to the beginning of the treatment. The microbial community was dominated by Proteobacteria and Bacteroidetes from day 0 to day 32, although differences at the genus level were observed. The predominant genera at the beginning of the treatment (day 0 just after inoculation) were Cloacibacterium, Sediminibacterium and Brevundimonas while at the end of the treatment members of Flavobacterium dominated, reaching almost half the population (41%), followed by Pseudomonas (6%) and Limnobacter (5.8%). To the author's knowledge, this is among the first studies to report the successful large scale biodegradation of TPH-contaminated groundwater (18,000 L per treatment session) at an offshore petrochemical facility.

A review of germination and early growth as a proxy for plant fitness under petrogenic contamination - knowledge gaps and recommendations.

Germination-an important stage in the life cycle of plants-is susceptible to the presence of soil contaminants. Since the early 1990s, the use of germination tests to screen multiple plant species to select candidates for phytoremediation has received much attention. This is due to its inexpensive methodology and fast assessment relative to greenhouse or field growth studies. Surprisingly, no comprehensive synthesis is available of these studies in the scientific literature. As more plant species are added to phytoremediation databases, it is important to encapsulate the knowledge thus far and revise protocols. In this review, we have summarised previously-documented effects of petroleum hydrocarbons on germination and seedling growth. The methods and materials of previous studies are presented in tabulated form. Common practice includes the use of cellulose acetate filter paper, plastic Petri dishes, and low numbers of seeds and replicates. A general bias was observed for the screening of cultivated crops as opposed to native species, even though the latter may be better suited to site conditions. The relevance of germination studies as important ecotoxicological tools is highlighted with the proposed use of root imaging software. Screening of novel plant species, particularly natives, is recommended with selection focussed on (i) species phylogeny, (ii) plant morphological and functional traits, and (iii) tolerance towards harsh environmental stresses. Recommendations for standardised protocols for germination and early growth monitoring are made in order to improve the robustness of statistical modelling and species selection in future phytoremediation evaluations and field programs.

Bioremediation potential of diesel-contaminated Libyan soil.

Bioremediation is a broadly applied environmentally friendly and economical treatment for the clean-up of sites contaminated by petroleum hydrocarbons. However, the application of this technology to contaminated soil in Libya has not been fully exploited. In this study, the efficacy of different bioremediation processes (necrophytoremediation using pea straw, bioaugmentation and a combination of both treatments) together with natural attenuation were assessed in diesel contaminated Libyan soils. The addition of pea straw was found to be the best bioremediation treatment for cleaning up diesel contaminated Libyan soil after 12 weeks. The greatest TPH degradation, 96.1% (18,239.6mgkg(-1)) and 95% (17,991.14mgkg(-1)) were obtained when the soil was amended with pea straw alone and in combination with a hydrocarbonoclastic consortium respectively. In contrast, natural attenuation resulted in a significantly lower TPH reduction of 76% (14,444.5mgkg(-1)). The presence of pea straw also led to a significant increased recovery of hydrocarbon degraders; 5.7log CFU g(-1) dry soil, compared to 4.4log CFUg(-1) dry soil for the untreated (natural attenuation) soil. DGGE and Illumina 16S metagenomic analyses confirm shifts in bacterial communities compared with original soil after 12 weeks incubation. In addition, metagenomic analysis showed that original soil contained hydrocarbon degraders (e.g. Pseudoxanthomonas spp. and Alcanivorax spp.). However, they require a biostimulant (in this case pea straw) to become active. This study is the first to report successful oil bioremediation with pea straw in Libya. It demonstrates the effectiveness of pea straw in enhancing bioremediation of the diesel-contaminated Libyan soil.

Mass culture strategy for bacterial yeast co-culture for degradation of petroleum hydrocarbons in marine environment.

In the present study a metabolically versatile co-culture with two Bacilli and one yeast strain was developed using enrichment culture techniques. The developed co-culture had affinity to degrade both aliphatic and aromatic fractions of petroleum crude oil. Degradation kinetics was established for designing the fermentation protocol of the co-culture. The developed mass culture strategy led to achieve the reduction in surface tension (26dynescm(-1) from 69 dynescm(-1)) and degradation of 67% in bench scale experiments. The total crude oil degradation of 96% was achieved in 4000l of natural seawater after 28days without adding any nutrients. The survival of the augmented co-culture was maintained (10(9)cellsml(-1)) in contaminated marine environment. The mass culture protocol devised for the bioaugmentation was a key breakthrough that was subsequently used for pilot scale studies with 100l and 4000l of natural seawater for potential application in marine oil spills.

Biosurfactant from red ash trees enhances the bioremediation of PAH contaminated soil at a former gasworks site.

Polycyclic aromatic hydrocarbons (PAHs) are persistent contaminants that accumulate in soil, sludge and on vegetation and are produced through activities such as coal burning, wood combustion and in the use of transport vehicles. Naturally occurring surfactants have been known to enhance PAH-removal from soil by improving PAH solubilization thereby increasing PAH-microbe interactions. The aim of this research was to determine if a biosurfactant derived from the leaves of the Australian red ash (Alphitonia excelsa) would enhance bioremediation of a heavily PAH-contaminated soil and to determine how the microbial community was affected. Results of GC-MS analysis show that the extracted biosurfactant was significantly more efficient than the control in regards to the degradation of total 16 US EPA priority PAHs (78.7% degradation compared to 62.0%) and total petroleum hydrocarbons (TPH) (92.9% degradation compared to 44.3%). Furthermore the quantification of bacterial genes by qPCR analysis showed that there was an increase in the number of gene copies associated with Gram positive PAH-degrading bacteria. The results suggest a commercial potential for the use of the Australian red ash tree as a source of biosurfactant for use in the accelerated degradation of hydrocarbons.

Evaluating the efficacy of bioremediating a diesel-contaminated soil using ecotoxicological and bacterial community indices.

Diesel represents a common environmental contaminant as a result of operation, storage, and transportation accidents. The bioremediation of diesel in a contaminated soil is seen as an environmentally safe approach to treat contaminated land. The effectiveness of the remediation process is usually assessed by the degradation of the total petroleum hydrocarbon (TPH) concentration, without considering ecotoxicological effects. The aim of this study was to assess the efficacy of two bioremediation strategies in terms of reduction in TPH concentration together with ecotoxicity indices and changes in the bacterial diversity assessed using PCR-denaturing gradient gel electrophoresis (DGGE). The biostimulation strategy resulted in a 90 % reduction in the TPH concentration versus 78 % reduction from the natural attenuation strategy over 12 weeks incubation in a laboratory mesocosm-containing diesel-contaminated soil. In contrast, the reduction in the ecotoxicity resulting from the natural attenuation treatment using the Microtox and earthworm toxicity assays was more than double the reduction resulting from the biostimulation treatment (45 and 20 % reduction, respectively). The biostimulated treatment involved the addition of nitrogen and phosphorus in order to stimulate the microorganisms by creating an optimal C:N:P molar ratio. An increased concentration of ammonium and phosphate was detected in the biostimulated soil compared with the naturally attenuated samples before and after the remediation process. Furthermore, through PCR-DGGE, significant changes in the bacterial community were observed as a consequence of adding the nutrients together with the diesel (biostimulation), resulting in the formation of distinctly different bacterial communities in the soil subjected to the two strategies used in this study. These findings indicate the suitability of both bioremediation approaches in treating hydrocarbon-contaminated soil, particularly biostimulation. Although biostimulation represents a commercially viable bioremediation technology for use in diesel-contaminated soils, further research is required to determine the ecotoxicological impacts of the intervention.

Assessing the hydrocarbon degrading potential of indigenous bacteria isolated from crude oil tank bottom sludge and hydrocarbon-contaminated soil of Azzawiya oil refinery, Libya.

The disposal of hazardous crude oil tank bottom sludge (COTBS) represents a significant waste management burden for South Mediterranean countries. Currently, the application of biological systems (bioremediation) for the treatment of COTBS is not widely practiced in these countries. Therefore, this study aims to develop the potential for bioremediation in this region through assessment of the abilities of indigenous hydrocarbonoclastic microorganisms from Libyan Hamada COTBS for the biotreatment of Libyan COTBS-contaminated environments. Bacteria were isolated from COTBS, COTBS-contaminated soil, treated COTBS-contaminated soil, and uncontaminated soil using Bushnell Hass medium amended with Hamada crude oil (1 %) as the main carbon source. Overall, 49 bacterial phenotypes were detected, and their individual abilities to degrade Hamada crude and selected COBTS fractions (naphthalene, phenanthrene, eicosane, octadecane and hexane) were evaluated using MT2 Biolog plates. Analyses using average well colour development showed that ~90 % of bacterial isolates were capable of utilizing representative aromatic fractions compared to 51 % utilization of representative aliphatics. Interestingly, more hydrocarbonoclastic isolates were obtained from treated contaminated soils (42.9 %) than from COTBS (26.5 %) or COTBS-contaminated (30.6 %) and control (0 %) soils. Hierarchical cluster analysis (HCA) separated the isolates into two clusters with microorganisms in cluster 2 being 1.7- to 5-fold better at hydrocarbon degradation than those in cluster 1. Cluster 2 isolates belonged to the putative hydrocarbon-degrading genera; Pseudomonas, Bacillus, Arthrobacter and Brevundimonas with 57 % of these isolates being obtained from treated COTBS-contaminated soil. Overall, this study demonstrates that the potential for PAH degradation exists for the bioremediation of Hamada COTBS-contaminated environments in Libya. This represents the first report on the isolation of hydrocarbonoclastic bacteria from Libyan COTBS and COTBS-contaminated soil.

The application of a carrier-based bioremediation strategy for marine oil spills.

The application of recycled marine materials to develop sustainable remediation technologies in marine environment was assessed. The remediation strategy consisted of a shell carrier mounted bacterial consortium composed of hydrocarbonoclastic strains enriched with nutrients (Bioaug SC). Pilot scale studies (5000 l) were used to examine the ability of Bioaug-SC to degrade weathered crude oil (10 g l(-1); initially 315,000±44,000 mg l(-1)) and assess the impacts of the introduction and biodegradation of oil. Total petroleum hydrocarbon mass was effectively reduced by 53.3 (±5.75)% to 147,000 (±21,000) mg l(-1) within 27 weeks. 16S rDNA bacterial community profiling using Denaturant Gradient Gel Electrophoresis revealed that cyanobacteria and Proteobacteria dominated the microbial community. Aquatic toxicity assessment was conducted by ecotoxicity assays using brine shrimp hatchability, Microtox and Phaeodactylum tricornutum. This study revealed the importance of combining ecotoxicity assays with oil chemistry analysis to ensure safe remediation methods are developed.

Potential impact of soil microbial heterogeneity on the persistence of hydrocarbons in contaminated subsurface soils.

In situ bioremediation is potentially a cost effective treatment strategy for subsurface soils contaminated with petroleum hydrocarbons, however, limited information is available regarding the impact of soil spatial heterogeneity on bioremediation efficacy. In this study, we assessed issues associated with hydrocarbon biodegradation and soil spatial heterogeneity (samples designated as FTF 1, 5 and 8) from a site in which in situ bioremediation was proposed for hydrocarbon removal. Test pit activities showed similarities in FTF soil profiles with elevated hydrocarbon concentrations detected in all soils at 2 m below ground surface. However, PCR-DGGE-based cluster analysis showed that the bacterial community in FTF 5 (at 2 m) was substantially different (53% dissimilar) and 2-3 fold more diverse than communities in FTF 1 and 8 (with 80% similarity). When hydrocarbon degrading potential was assessed, differences were observed in the extent of (14)C-benzene mineralisation under aerobic conditions with FTF 5 exhibiting the highest hydrocarbon removal potential compared to FTF 1 and 8. Further analysis indicated that the FTF 5 microbial community was substantially different from other FTF samples and dominated by putative hydrocarbon degraders belonging to Pseudomonads, Xanthomonads and Enterobacteria. However, hydrocarbon removal in FTF 5 under anaerobic conditions with nitrate and sulphate electron acceptors was limited suggesting that aerobic conditions were crucial for hydrocarbon removal. This study highlights the importance of assessing available microbial capacity prior to bioremediation and shows that the site's spatial heterogeneity can adversely affect the success of in situ bioremediation unless area-specific optimizations are performed.

Plant residues--a low cost, effective bioremediation treatment for petrogenic hydrocarbon-contaminated soil.

Petrogenic hydrocarbons represent the most commonly reported environmental contaminant in industrialised countries. In terms of remediating petrogenic contaminated hydrocarbons, finding sustainable non-invasive technologies represents an important goal. In this study, the effect of 4 types of plant residues on the bioremediation of aliphatic hydrocarbons was investigated in a 90 day greenhouse experiment. The results showed that contaminated soil amended with different plant residues led to statistically significant increases in the utilisation rate of Total Petroleum Hydrocarbon (TPH) relative to control values. The maximum TPH reduction (up to 83% or 6800 mg kg(-1)) occurred in soil mixed with pea straw, compared to a TPH reduction of 57% (4633 mg kg(-1)) in control soil. A positive correlation (0.75) between TPH reduction rate and the population of hydrocarbon-utilising microorganisms was observed; a weaker correlation (0.68) was seen between TPH degradation and bacterial population, confirming that adding plant materials significantly enhanced both hydrocarbonoclastic and general microbial soil activities. Microbial community analysis using Denaturing Gradient Gel Electrophoresis (DGGE) showed that amending the contaminated soil with plant residues (e.g., pea straw) caused changes in the soil microbial structure, as observed using the Shannon diversity index; the diversity index increased in amended treatments, suggesting that microorganisms present on the dead biomass may become important members of the microbial community. In terms of specific hydrocarbonoclastic activity, the number of alkB gene copies in the soil microbial community increased about 300-fold when plant residues were added to contaminated soil. This study has shown that plant residues stimulate TPH degradation in contaminated soil through stimulation and perhaps addition to the pool of hydrocarbon-utilising microorganisms, resulting in a changed microbial structure and increased alkB gene copy numbers. These results suggest that pea straw in particular represents a low cost, effective treatment to enhance the remediation of aliphatic hydrocarbons in contaminated soils.