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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.

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The green algae Botryococcus braunii is widely recognized as a source of oil, including hydrocarbons. However, the slow rate of growth B. braunii hampers its commercial development. This study addresses this by examining the effects of three growth media on biomass and oil production in two B. braunii Race B strains, Kossou-4 and Overjuyo-3. Growth of B. braunii strains in BG11 medium resulted in significantly higher growth (2.3-4.2 and 2.9-6.0 fold increases in Kossou-4 and Overjuyo-3 respectively) compared to the JM and BBM-3N media after 15 days. A similar trend was obtained when biomass was measured indirectly using optical density (OD) and chlorophyll fluorescence. Oil production was also significantly higher in BG11 whether measured as oil weight or absorbance (ODs at 680 and 750 nm). However, the presence of extracellular oil was shown to increase absorbance values making OD measurements less reliable than dry weight assays. Maximum recovery of oil was recorded when hexane was used as solvent compared to hexane-isopropanol and heptane. These results suggest that BG11 is the best growth medium for these two strains under the conditions of this experiment.

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Bioremediation is a widely used environmental friendly treatment method for petrogenic hydrocarbon contaminated soils but its application to the treatment of crude oil tank bottom sludge (COTBS) contaminated soil is limited especially in Mediterranean countries such as Libya. Therefore in this study, the hydrocarbon degrading abilities of three bioaugmentation agents Pseudomonas sp (4M12), Pseudomonas xanthomarina (4M14) and Arthrobacter nitroguajacolicus (1B16A) (isolated from COTBS polluted soils) applied as part of a biostimulation-bioaugmentation (BS/BA) strategy were assessed in COTBS contaminated Libyan soils. Biostimulated (BS) and natural attenuation (NA) microcosms were also set up for comparative purposes. Gas chromatograph mass spectrometer (GC-MS) analysis revealed a total soil petroleum hydrocarbon (TPH) and polycyclic aromatic hydrocarbon (PAHs) content of 30,703 mg kg -1 and 13,816 mg kg -1 respectively. Two carcinogenic fractions (naphthalene and benzenamine, 4, 4`methylenbis [2-methyl-]) and 4 mutagenic fractions (pyrene, phenanthrene, fluorene and anthracene) were detected. Substantial PAH degradation occurred in 4M14 and 4M12 samples within 15 days in contrast to up to 23 days in 1B16A, NA. However, substantial reduction in TPH (> 97%) was only observed in 4M12 and 4M14 inoculated microcosms within 15 days compared to 25-30 days in 1B16A inoculated, BS and NA microcosms. 4M14 inoculated microcosms were most efficient at complete removal (D100) of all carcinogenic and mutagenic fractions; 4M14 (9-10 days), 4M12 (9-15 days), 1B16A (15-23 days), BS (18-21 days) and NA (18-22 days). Pseudomonas xanthomarina was therefore shown as the best candidate for use in a BS/BA approach for treating COTBS contaminated Libyan soils. This study shows the importance of pre-screening bioaugmentation agents for the removal of carcinogenic and mutagenic fractions prior to use; in order to carry out safe, efficient and sustainable COTBS bioremediation in Libya.

Our environment is threatened by thousands of contaminants, mainly as a result of human activities and industrialization. These include both inorganic (e.g. heavy metals) and organic compounds (e.g. polycyclic aromatic hydrocarbons, chlorinated hydrocarbons and herbicides). It has been shown that many of these hazardous chemicals cause health problem such as cancer in living organisms. Therefore, removing them from environments and soils represents a key challenge from both ecosystem and human health perspectives. Among the approaches used to remove these pollutants, microbial remediation or bioremediation represents a promising technology that is cost-effective, environmentally friendly and less disruptive than alternative technologies. Bioremediation involves microbes that are present in or added to the contaminated environment which are capable of degrading contaminants, or reducing them to less toxic forms. There are a number of bioremediation techniques, including natural attenuation, bioaugmentation, biostimulation, phytoremediation/rhizoremediation and necrophytoremediation Many factors, such as soil texture, pH, temperature, levels of oxygen, nutrients and the microbial status of the soils, influence the rate and extent of bioremediation along with the type and bioavailability of the contaminants. In this chapter, we highlight the current status of bioremediation, examining the development of techniques used to assess and optimize the degradation of the contaminants.