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Two case studies are described where partially biodegraded petroleum residues were collected from the marine environment and analysed to investigate the changes in aromatic hydrocarbons with increasing biodegradation.The first of these studies, involved following the weathering of sea-floor residues from drilling discharges from an offshore petroleum exploration and production platform situated off the coast of North Western Australia. During operations, formation cuttings with adhering oil-based drilling muds were discharged into the ocean via a chute into approximately 125n1 of water, forming a substantial mound at the base of the platform. A suite of seabed sediments was collected from 16 sampling sites at various distances from the platform immediately following the cessation of drilling operations. The distribution of hydrocarbons in the sediment directly under tile cuttings chute was consistent with that found in drilling fluids formulated from a kerosene-like fluid. The samples from more remote sites exhibited the successive enhancement of an unresolved complex mixture relative to the n-alkanes, associated with tile presence of residues from petroleum biodegradation processes. In a subsequent sampling some three years later, a 10 cm core was retrieved from the cuttings pile and divided into 1 cm depth intervals. Samples within 6 cm of the surface of the cuttings pile contained biodegraded residues of the drilling mud, where the extent of biodegradation increased with decreasing proximity to the surface, most likely indicative of aerobic biodegradation. Biodegradation was less evident in the underlying sediments, where anaerobic conditions prevailed. / Analysis of the aromatic hydrocarbons in both sets of sediment extracts by using gas chromatography-mass spectrometry (GC-MS) revealed the successive depletion of alkylnaphthalenes, and due to the subtlety of changes in the extent of biodegradation, provided an excellent opportunity to examine the susceptibility of biodegradation towards the individual alkylnaphthalenes in the marine environment. Conventional GC-MS analysis of these mixtures is performed under chromatographic conditions where complete resolution of the mixture is not achieved and several isomers co-elute. The mass spectra of these co-eluting isomers may be so similar that one is unable to differentiate between them, and their abundance may therefore not be determined. Since each isomer has a unique infrared spectrum, however, the abundance of each individual isomer was determined by comparing the infrared spectrum of the co-eluting compounds with the spectrum of each of the isomers. To this end, techniques were developed for the application of direct-deposition gas chromatography - Fourier transform infrared spectroscopy (GCFTIR) to the analysis of the complex mixture of alkylnaphthalenes present in the petroleum. This technique was also extended to discriminate between individual alkylphenanthrene isomers, and to clarify the sorption behaviour of the dimethylphenanthrenes by mordenite molecular sieves. The identification of other compounds of geochemical significance in petroleum is also described. / Analyses of' the aromatic hydrocarbons in the contaminated sea-floor sediments using GC-FTIR enabled the unambiguous identification and quantification of each of the dimethylnaphthalene, trimethylnaphthalene and tetramethylnaphthalene isomers present in the samples, from which the relative extents of depletion of each with increasing extent of biodegradation were determined. It was apparent from the considerable differences in the observed susceptibility to biodegradation that a strong relationship exists between the compound structure and its susceptibility to biodegradation, with 1,6-disubstituted polymethylnaphthalenes being preferentially depleted relative to other isomers that lack this feature. The second case study involved tracking the fate (weathering) of hydrocarbons from an accidental release of condensate from a buried pipeline into intertidal coastal (mangrove) sediments in North Western Australia. Sediment samples were collected on nine occasions over a three-year period. Chemical analysis of the saturated and aromatic hydrocarbon components of the petroleum extracts revealed that both hydrocarbon fractions exhibited an increasingly biodegraded profile with increased residence time in the sediments. In a similar manner to the first case study, detailed analysis of the aromatic hydrocarbons using GC-FTIR techniques was performed to determine the depletion of individual alkylnaphthalene isomers with increasing extent of biodegradation. It was apparent that a relationship similar to that observed for the sea-floor sediments exists between the alkylnaphthalene structure and its susceptibility to biodegradation. / Changes in the distribution of methylphenanthrene and dimethylphenanthrene isomer mixtures were also studied and the susceptibility to biodegradation amongst these determined in a similar manner. These relative susceptibilities to biodegradation of the aromatic hydrocarbons were then related to the established hierarchy of susceptibilities of the saturated hydrocarbons, in effect providing a second parallel system for the assessment of the extent of biodegradation. Finally, a system of ratios calculated from the relative abundances of selected aromatic hydrocarbons was developed and used as indicators to differentiate between several crude oils that have been biodegraded to varying extents. These parameters also offer promise as indicators of multiple accumulation events in oil reservoirs where petroleum fluids biodegraded to differing extents are mixed.

Thesis (Ph. D.)--Florida State University, 2006. / Advisor: Alan G. Marshall, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Sept. 21, 2006). Document formatted into pages; contains xxii, 198 pages. Includes bibliographical references.

A laboratory investigation was conducted to determine the effects of biologically produced surfactants (biosurfactants) on petroleum hydrocarbons and their potential for the removal of hydrocarbons from groundwater systems. Bioaurfactanta have been found to be produced by microorganisms during growth on insoluble substrates for the purpose of increasing substrate solubility so as to promote biological degradation. In this study, three types of biosurfactants were produced by microorganisms grown on gasoline and a mixture of glucose with vegetable oil. Solubilization and biodegradation of selected gasoline compounds in the presence of bioeurfactante were measured in both static batch and flow through column systems. Batch experiments were conducted in culture tubes, using only liquid phases. A clean sand was used in the column system to monitor physical and chemical interactions yet minimize adsorption effects. A mixed culture of gasoline degrading microorganisms along with isolated cultures grown on selected compounds were used in the biodegradation studies. The biosurfactants produced and used in this study acted similarly to synthetic surfactants and increased, to various degrees, the solubility of the monitored gasoline compounds. Biosurfactants produced from growth on glucose and vegetable oil were very effective surfactants, markedly increasing solubility of the gasoline compounds, but inhibiting biological degradation of these same compounds. Biosurfactants produced by microorganisms from growth on gasoline were effective surfactants, but they did not inhibit biodegradation of the gasoline compounds. This indicated that the biosurfactants may be substrate or microorganism specific, produced for growth on a particular insoluble substrate by a specific microorganism. Biosurfactants produced from growth on gasoline or an insoluble hydrocarbon could therefore be used to enhance solubility and subsequent biodegradation of that same hydrocarbon. The effectiveness of the biosurfactants during application by injection or recirculation for groundwater remediation would be limited by the adsorption and removal of the biosurfactant to the soil. The surfactant demand (by adsorption) of the soil would have to be met before the effects of the biosurfactants would become apparent. Biosurfactanta added to groundwater could also create an additional oxygen demand in a system already low in oxygen. / Master of Science

This research was initiated after a 100-year flood caused an oil spill on the San Jacinto River (Houston, Texas) in October of 1994. After the floodwaters subsided the released petroleum floating on the water was deposited on the surrounding lands. The petroleum spill was used as an opportunity to research intrinsic petroleum biodegradation in a 9-acre petroleum impacted estuarine wetland. The first phase of this research (Phase I) began in December 1994, approximately 1.5 months after the spill of opportunity and involved the study and quantification of in-situ petroleum biodegradation. The second phase of the research (Phase II) began in March 1996 with a controlled oil release to study and evaluate the success of two bioremediation treatments versus natural biodegradation. The study of in-situ petroleum hydrocarbon degradation and the evaluation of bioremediation amendments were successfully quantified using GC-MS analytical techniques. However, the GC-MS technique is limited to the analyses of hydrocarbon compounds, a disadvantage that precludes the overall characterization of petroleum degradation. The research presented here details an analytical technique that was used to provide a full characterization of temporal petroleum biodegradation. This technique uses thin layer chromatography coupled with flame ionization detection (TLC-FID) to characterize the saturate and aromatic (hydrocarbon) fractions and the resin and asphaltene (non-hydrocarbon, polar) fractions. Other analysis techniques, such as HPLC-SARA analysis, are available for the full characterization of the four petroleum fractions. However, these techniques do not lend themselves well to the application of large sample set analysis. A significant advantage of the TLC-FID analysis to other petroleum analysis techniques is the ability to analyze several samples concurrently and quickly with relative ease and few resources. For the purposes of the Phase I and Phase II research the TLC-FID analysis method was evaluated, refined and applied to quantify the temporal biodegradation and bioremediation of petroleum. While the TLC-FID analysis produces a full characterization, it cannot supplant the GC-MS analysis for petroleum bioremediation research. However, it can be used in conjunction with the GC-MS to expand the knowledge of petroleum bioremediation and remediation strategies.

In 1990, an oil recycling plant situated in Hammarsdale, South Africa, was decommissioned and a decision was taken by management to rehabilitate the site in preparation for resale. The heavily impacted area covered over two hecatares and oil contamination penetrated soil to depths in excess of three metres, making excavation and removal of the soil very expensive. The options for remediation of the site were limited. No facility for incineration of contaminated soil exists in South Africa, and landfilling was not permitted. The emphasis in developing a remediation strategy, therefore, focussed upon the possibility of in situ remediation with minimal excavation of soil. This study, the first of its kind in South Africa, was subsequently initiated to assess the feasibility of this approach, the results of which would underpin a full-scale cleanup programme. The development of such a strategy involved four key stages of work : (1) a comprehensive site investigation to evaluate and fully understand the particular problems at the site; (2) treatability studies to determine the potential for biological treatment of the contaminated soil and the optimisation of such treatments, particularly in terms of time and cost; (3) the testing of some of the more effective treatments on a pilot-scale; and (4) recommendations for full-scale bioremediation of the contaminated site. various conditions unique to South Africa had to be considered at each stage viz. the lack of funds and remediation experience, which created numerous problems and emphasised the requirement for a simple, "low-tech" approach. Site investigations revealed that in situ remediation may be possible due to the high permeability of the sandy soils and low concentrations of heavy metals. Laboratory experiments also showed that a mixed association of indigenous microorganisms was present which, once stimulated by nutrient supplementation at C:N:P, ratios of between 10:1:1 and 20:1:1, was capable of degrading total petroleum hydrocarbons at an average rate of 11% week -1. Further experimentation, aimed at reducing the cost of remediation and improving the soil quality, focussed on the efficacy of oil solubilisers, a soil ameliorant (composted pine-bark), indigenous fungi and higher plants in the remedial process. Three commercial surfactants (Arkopal N-050, N-060 and E2491) and one natural solubiliser (soybean lecithin) were tested for their biotoxicity, solubilisation and biodegradability at various concentrations (0.01 - 1.0%). Formulation E2491 was able to support a microbial population and was selected as the preferred commercial surfactant if soil washing was to be recommended; however, lecithin was considered to be more useful in situ because of its localised solubilising effect, biological origin and nutritional contribution. The use of fungi was of particular interest in addressing the persistent organic compounds, such as the heavy fractions of oil, for which bacterial remediation methods have been slow or ineffective. While it was not possible, however, to demonstrate in the laboratory that the indigenous fungi contributed significantly towards the degradation of the contaminating oil, the basic trends revealed that the fungal component of the indigenous microbial population was readily stimulated by the addition of nutrient supplements. The bulking-up process was also a success and additional exploratory work was proposed in the form of a larger scale composting design. Finally, the potential for using higher plants and 20% (v / v) composted pinebark (in addition to nutrients) to increase the microbial degradation of the contamination was investigated in both greenhouse and field plot studies. Greenhouse investigations employed soybeans which were postulated to have soil quality and cost benefits. However, although the soybeans were found to significantly enhance the remedial process, the complex soil-contaminant- plant interactions gave rise to strange nutritional effects and, in some cases, severe stunting. In contrast, the field studies employed grasses that had previously established on the site and which ultimately demonstrated a better tolerance for the contaminated conditions. Scanning electron microscopy revealed that there were considerable differences between the root tips of soybean plants which had been grown in contaminated soil and those which had been grown in uncontaminated soil. It was concluded that toxicity symptoms, which are readily observed in the root, could be used as an early indicator for determining the suitability of vegetation for remediation purposes. In both instances, despite the differences, the addition of composted pine-bark and nutrients (nitrogen and phosphorus) resulted in total petroleum hydrocarbon reductions of >85%, illustrating the benefits of plant establishment and oxygen availability. The need to link results from laboratory or pilot-scale experiments to achieve reliable predictions of field-scale behaviour was an essential component of this research. The results of the field study provided evidence, similar to that found in the pot trial, of the accelerated disappearance of organic compounds in the rhizosphere. All experiments incorporated parallel measurements of hydrocarbon residues, microbial activity and pH changes in the contaminated soil, the results of which strongly supported the argument that biodegradation was the dominant component of the remediation process. Thus, after consideration of the significant interactions which dominated the study (time-contaminant-nutrient; time-contaminant-pine-bark; and time-contaminant- pine-bark-plant), it was clear that, aside from these limiting factors, little should preclude the in situ bioremediation of the impacted soil. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1996.

Alcanivorax species are gram negative bacteria that usually require aliphatic hydrocarbon as the sole carbon source for growth. The ability to use petroleum in polluted environments as energy source makes Alcanivorax species biotechnologically relevant in bioremediation. Universal stress proteins confer ability to respond to unfavourable environments, thus the present study was done to analyse the genomic context of genes for universal stress proteins in Alcanivorax genomes. A combination of bioinformatics and visual analytics approaches were used to analyze genome-enabled data including sequences and gene expression datasets. On the basis of transcription unit and adjacent genes, two types of Alcanivorax USP genes observed were (i) adjacent to cyclic nucleotide-binding and oxygen sensing functions; and (ii) adjacent to sulfate transporter function. Both types of genes encode two universal stress protein domains (pfam00582) also referred to as tandem-type universal stress proteins. The sequence and structural characteristics of each of the four USP domains in Alcanivorax needs to be further investigated. This dissertation research evaluated data from Alcanivorax borkumensis cells (grown on either pyruvate or hexadecane as carbon source) that were stressed with 1-octanol and data collected at 15 min, 30 min, 60 min and 90 min after 1-octanol addition. The two genes for Alcanivorax borkumensis SK2 universal stress proteins, ABO_1340 and ABO_1511, had the same direction of expression for adjacent genes. A limitation of this research was that findings based on bioinformatics and visual analytics methods may need confirmation by molecular methods. The differences observed may also reflect the quality of the annotations provided for genes. The sequence and structural characteristics of each of the four USP domains in Alcanivorax needs to be further investigated. Further research is needed on the relationship between number, length and order of genes in operons that include genes for universal stress proteins. Additionally, in vitro studies to confirm the functional prediction made from the genomic context of the universal stress protein in Alcanivorax genome. The knowledge discovered from this genome context analytics research could contribute to improving the performance of Alcanivorax species in bioremediation of environments polluted with petroleum / College of Agriculture and Environmental Sciences / M. Sc. (Environmental Science)

Bioremediation is the process whereby the degradation of organic polluting compounds occurs as a result of biochemical activity of macro- and microorganisms. Bioremediation of hydrocarbon contaminated soils can be practised in situ or ex situ by either stimulating the indigenous microorganisms (biostimulation) or introducing adapted microorganisms which specifically degrade a contaminant (bioaugmentation). This investigation focused on ex situ remediation processes with special attention to the processes and microbiology of landfarming and thermal bioventing. Landfarming was investigated at pilot-scale and full-scale, and thermal bioventing at laboratory and pilot-scale. This study indicated that pilot-scale bioremediation by landfarming was capable of effecting a total petroleum hydrocarbon concentration (TPHC) reduction of 94% (m1m) from an initial concentration of 320 gkg-I soil to 18 gkg-I soil over a period of 10 weeks. Reactors receiving biosupplements showed greater rates of bioremediation than those receiving nutrients. Promotion of TPHC catabolism by addition of a commercial or a site-specific microbial biosupplement was similar. Seedling experiments proved that bioremediation did not necessarily leave the soil in an optimal condition for plant growth. The full-scale landfarming operation reduced the TPHC concentrations from 5 260 - 23 000 mgkg- I to 820 - 2335 mgkg- I soil over a period of 169 days. At full-scale, the larger fraction of more recalcitrant and weathered petroleums. and the less intensive treatment resulted in a slower rate of TPHC reduction than was found in the pilot-scale study. Three distinct decreases in the TPHC were observed during the full-scale treatment. These presented an ideal opportunity to investigate the microbiology of the soil undergoing treatment. The dominant culturable microorganisms were isolated and identified. The bioremediation process was dominated by Bacillus and Pseudomonas species. The method used to study the population was, however, biased to culturable, fast growing microorganisms which represent a small portion of the total microbial population. For this reason, a method to study the total eubacterial population in situ with rRNA targeted oligonucleotide probes was adapted and found to be a valuable technique. Soil microorganisms respiratory activity was investigated at different times in the full-scale treatment. A clear correlation between activity and degradation was recorded. The effect of a supplement. anaerobically digested sludge, was also assessed by this method. Thermal bioventing was investigated as an ex situ in-vessel treatment technology for small volumes of highly contaminated soils. This proved to be a viable technique for the bioremediation of petroleum hydrocarbons at laboratory-scale. Volatilisation contributed to at least 40% of the reduction. Of the two supplements evaluated. dried sludge promoted degradation to a greater extent than chicken manure. The pilot-scale study proved that a chemical contaminant reduction of at least 50% could be achieved in 13 weeks by thermal bioventing. Of the supplemented reactors. the presence of dried sludge and commercial biosupplement etfected the largest contaminant decrease. As a possible supplement to increase the rate of bioremediation. dried anaerobically digested sludge was more effective than chicken manure. A parallel laboratory-scale experiment gave similar results. Gravimetric analyses were found to be conservative indications of the remediation process. The results of this study shed some light on our. still. limited understanding of bioremediation. The gap between the technology in the laboratory and field was narrowed and a better understanding of the soil microbiology was achieved. Due to the limited control of environmental parameters in the case of landfarming. thermal bioventing was investigated and proved to be an effective alternative. The latter technology is novel in Southern Africa. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1996.

The central aim of this study was to determine if indigenous fungi of an oil-contaminated soil could be effectively used in a low-cost bioremediation of the soil. Since some of the contaminant had been present at the site for over two decades, the indigenous microbial species had been subjected to specific selection pressures for a protracted period, thus facilitating key enzymatic capabilities for hydrocarbon degradation. Analysis of the pertinent influential parameters of soil bioremediation indicated that an ex situ technique, utilising the catabolic activities of the indigenous soil fungi, was a feasible low-cost option. Fungi were isolated from the contaminated soil through a variety of techniques. The abilities of these isolates to degrade the contaminant oil and a range of representative hydrocarbon molecules was evaluated by a systematic screening programme. Sixty-two isolates were initially examined for their growth potential on hydrocarbon-supplemented agar. A bioassay, utilising hydrocarbon-impregnated filter paper discs, was then used to examine the abilities of 17 selected isolates to catabolise three representative hydrocarbon molecules (hexadecane, phenanthrene and pristane) in different concentrations. In the same bioassay, the influence of a co-metabolite (glucose) on growth potential was also examined. Eight fungal species: Trichophyton sp.; Mucor sp.; Penicillium sp.; Graphium sp.; Acremoniwn sp.; Chaetomium sp.; Chrysosporium sp.; and an unidentified basidiomycete were then selected. Liquid batch cultures with a hydrocarbon mixture of hexadecane, phenanthrene, pristane and naphthalene facilitated quantitative analysis (HPLC) of the hydrocarbon catabolic abilities of the selected isolates. Ex situ bioremediation was evaluated at laboratory-scale by both bioaugmentation and biostimulation in soil microcosm trials. During the course of the study, total petroleum hydrocarbon (TPH) concentration (U.S. EPA Method 418.1) was used as a simple and inexpensive parameter to monitor hydrocarbon disappearance in response to soil treatments. Soil microbial activities were estimated by use of a fluorescein diacetate hydrolysis bioassay. This was found to be a reliable and sensitive method to measure the activity of respiring heterotrophs as compared with the unreliable data provided by plate counts. In the bioaugmentation trial, the eight selected isolates were individually used to inoculate (30% v/v) the contaminated soil. The highest rate of biodegradation (50.5% > than the non-sterile control) was effected by an Acremonium species after 50 days incubation (25°C). The second highest rate of biodegradation (47% > than the non-sterile control) was achieved with a soil treatment of sterile barley/beer waste only. Comparable rates of hydrocarbon degradation were achieved in simple biostimulation trials. Thus, due to its lower cost, biostimulation was the preferred remediation strategy and was selected for further laboratory investigation. Common agricultural or industrial lignocellulosic wastes such as: wood chips; straw; manure; beer brewery waste; mushroom compost; and spent mushroom substrate were used as soil treatments, either alone or in combination. The effect of the addition of a standard agricultural fertiliser was also examined. The highest level of biodegradation (54.4% > the non-sterile control) was recorded in a microcosm supplemented (40% v/v) with chicken manure. Finally, an ex situ bioremediation technique was examined in a pilot-scale field trial. Wood chips and chicken manure were co-composted with the contaminated soil in a low-cost, low-maintenance bioremediation system know as passive thermal bio venting. Extensive monitoring of the thermal environment within the biopile was made as an indirect measure of microbial activity. These data were then used to optimise the composting process. Three-dimensional graphical representations of the internal temperatures, in time and space, were constructed. From these graphs, it was determined that an inner core region of approximately 500 cm3 provided a realistic simulation of conditions within a full-scale biopile. During this trial a TPH reduction of 68% was achieved in 130 days. The findings of this research suggested that the utilisation of fungal catabolism is applicable to soils contaminated with a wide range of hydrocarbon contaminants. Passive thermal bioventing offers a bioremediation strategy which is highly suitable for South African conditions in terms of its low level of technological sophistication, low maintenance design and, most importantly, its relatively low cost. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.