Biodegradation Of N-alkanes Research Articles

Biodegradation of crude oil and n-alkanes by fungi isolated from Oman

Ten fungal species isolated from tar balls collected from the beaches of Oman were tested for their abilities to grow and degrade n-alkanes and crude oil. The abilities of Aspergillus niger, A. ochraceus and Penicillium chrysogenum to degrade n-alkanes (C13–C18), crude oil were compared and their mycelial biomass was measured. Significant differences were found in the utilization of C15, C16, C17 and C18 by the three fungi. Similarly, significant differences we found in the amount of biomass produced by the three fungi growing on C13, C17, C18 and crude oil. The correlation coefficient of biomass and oil utilization was not statistically significant for Aspergillus niger, significant for Aspergillus terreus and highly significant for P. chrysogenum.

Alteration of crude oils from the Troll area by biodegradation: Analysis of oil and water samples

Biodegradation of crude oil in reservoirs has been extensively studied and is usually considered to be a quasi-stepwise process. The process is known to increase the acidity of the oil. In this paper, results from the analysis of crude oils and co-produced waters from the reservoir surrounding the Troll C platform in the Norwegian North Sea are presented. Based on the concentrations of light hydrocarbons in the Troll oils, simultaneous biodegradation of n-alkanes, branched alkanes, cycloalkanes, alkyl benzenes, alkyl naphthalenes and methyl phenanthrenes is suggested. Pronounced relative enrichment of methylcyclopentane, cyclohexane and methylcyclohexane provide evidence for a specific recalcitrance of cycloalkanes to biodegradation in this field. Biodegradation can also be seen in the carbon isotopic signature of the lighter hydrocarbons, e.g., heavy signatures for n-pentane and iso-pentane. The composition of the aromatic acids in the produced waters suggests biodegradation to be their predominant source in the reservoir. The occurrence of reduced naphthoic acids previously shown to be specific indicators of anaerobic biodegradation of naphthalene, 2-methylnaphthalene and tetralin indicates that the biodegradation took place under anoxic conditions.

Effects of Spilled Oil on Bacterial Communities of Mediterranean Coastal Anoxic Sediments Chronically Subjected to Oil Hydrocarbon Contamination

The effects of spilled oil on sedimentary bacterial communities were examined in situ at 20 m water depth in a Mediterranean coastal area. Sediment collected at an experimental site chronically subjected to hydrocarbon inputs was reworked into PVC cores with or without a massive addition of crude Arabian light oil ( approximately 20 g kg(-1) dry weight). Cores were reinserted into the sediment and incubated in situ at the sampling site (20 m water depth) for 135 and 503 days. The massive oil contamination induced significant shifts in the structure of the indigenous bacterial communities as shown by ribosomal intergenic spacer analysis (RISA). The vertical heterogeneity of the bacterial communities within the sediment was more pronounced in the oiled sediments particularly after 503 days of incubation. Response to oil of the deeper depth communities (8-10 cm) was slower than that of superficial depth communities (0-1 and 2-4 cm). Analysis of the oil composition by gas chromatography revealed a typical microbial alteration of n-alkanes during the experiment. Predominant RISA bands in oiled sediments were affiliated to hydrocarbonoclastic bacteria sequences. In particular, a 395-bp RISA band, which was the dominant band in all the oiled sediments for both incubation times, was closely related to hydrocarbonoclastic sulfate-reducing bacteria (SRB). These bacteria may have contributed to the main fingerprint changes and to the observed biodegradation of n-alkanes. This study provides useful information on bacterial dynamics in anoxic contaminated infralittoral sediments and highlights the need to assess more precisely the contribution of SRB to bioremediation in oil anoxic contaminated areas.

Synergetic effects of cell immobilization in polyurethane foam and use of thermotolerant strain on degradation of mixed hydrocarbon substrate by Prototheca zopfii

The synergetic effects of an immobilized cell system in an oleophilic polyurethane foam (PUF) and use of a thermotolerant strain on degradation of hydrocarbons by colorless green microalga Prototheca zopfii are reported. Two strains of P. zopfii, i.e. thermotolerant RND 16 and non-thermotolerant ATCC30253, were immobilized in PUF to compare their abilities to biodegrade a mixed hydrocarbon substrate (MHS) containing aliphatic and polycyclic aromatic hydrocarbons (PAHs), as a function of temperature. The thermotolerant strain RND 16 degraded MHS at 35°C, while ATCC30253 did not degrade hydrocarbons at temperatures higher than 30°C. Immobilization of P. zopfii in PUF resulted in shortened lag for growth-associated biodegradation of n-alkanes in MHS, the effect of which was most significant in cultures of RND 16 at 25°C. Nevertheless, the decrease in the amount of degraded PAHs was caused by PUF immobilization and the level of this decrease was marked in the cultures of RND 16, in which rate and extent of n-alkane degradation were higher than for ATCC30253.

Biodegradation of Aliphatic and Aromatic Hydrocarbons by Nocardia sp. H17-1

We investigated the biodegradation of hydrocarbon components by Nocardia sp. H17-1 and the catabolic genes involved in the degradation pathways of both aliphatic and aromatic hydrocarbons. After 6 days of incubation, the aliphatic and aromatic fractions separated from Arabian light oil were degraded 99.0 ± 0.1% and 23.8 ± 0.8%, respectively. Detection of the catabolic genes involved in the hydrocarbon degradation indicated that H17-1 possessed the alkB genes for n-alkane biodegradation and catA gene for catechol 1,2-dioxygenase. However, H17-1 had neither the C23O gene for the degradation of aromatic hydrocarbons nor the catechol 2,3-dioxygenase activity. The investigation of the genes involved in the biodegradation of hydrocarbons supported the low degradation activity of H17-1 on the aromatic fractions.

Accelerated biodegradation of n-alkanes in aqueous solution by the addition of fermented whey

Abstract The effect of fermented whey on the aerobic degradation of n -alkanes by a microbial consortium was investigated in an aqueous system. Microbial degradation of 100 mg n -alkanes l −1 (C 12 , C 14 , C 16 and C 18 ) in mineral nutrient medium was assessed by measuring the decrease in n -alkanes, production of CO 2 and increase in biomass. The addition of fermented whey at a concentration of 5 mg dry weight l −1 to a nutrient medium receiving a small-sized inoculum (10 3.4 CFU ml −1 ) shortened the lag phase from 8 to 3 days, but the degradation rate during the degradation phase was not enhanced. The shortened lag phase at low initial concentration of microorganisms indicates that the fermented whey stimulates growth in the initial phase, without reducing the consortium's capacity for n -alkane degradation.

A new method for the detection of alkane-monooxygenase homologous genes ( alkB) in soils based on PCR-hybridization

An improved method was developed that allowed the specific detection of the gene alkB (coding for the rubredoxin dependent alkane monooxygenase) from bacteria without any obvious strain specific discrimination using a combination of PCR and hybridization. This approach enabled a fast culture-independent monitoring of environmental samples for the occurrence of alkB, and an estimation of the gene copy number and the genetic diversity. Both parameters provide useful informations for an assessment of the intrinsic biodegradation potential that is present at a site. The method was applied to soil samples from different uncontaminated sites. alkB was highly abundant and redundant in all soils tested. Potential biodegradation of n-alkanes was also demonstrated for these soils with substrate utilization assays. Cell numbers of hydrocarbon degraders estimated as MPN varied from 10 3 to 10 6 g − 1 soil (dry weight) for the different soils. Gene copy numbers estimated with MPN-PCR ranged within 1–40 * 10 4 ng − 1 soil DNA. Analysis of the diversity of the alkB sequences obtained from a grassland and an agricultural soil indicated that the alkane degrading microbial populations occurring at these sites were rather diverse. Compared on protein level, three major clusters were distinguishable for both soils that showed highest similarities to AlkB from the Gram-positives Nocardioides and Mycobacterium, and the Gram-negative Alcanivorax. The majority of the cloned AlkB sequences were homologous to proteins from the Gram-positive bacteria. However, significant differences from published sequences were observed; homologies varied from 50% to 90% (identity of amino acids).

Biodegradation of Petroleum Hydrocarbons in Seawater at Low Temperatures (0–5 °C) and Bacterial Communities Associated with Degradation

In this study biodegradation of hydrocarbons in thin oil films was investigated in seawater at low temperatures, 0 and 5 degrees C. Heterotrophic (HM) or oil-degrading (ODM) microorganisms enriched at the two temperatures showed 16S rRNA sequence similarities to several bacteria of Arctic or Antarctic origin. Biodegradation experiments were conducted with a crude mineral oil immobilized as thin films on hydrophobic Fluortex adsorbents in nutrient-enriched or sterile seawater. Chemical and respirometric analysis of hydrocarbon depletion showed that naphthalene and other small aromatic hydrocarbons (HCs) were primarily biodegraded after dissolution to the water phase, while biodegradation of larger polyaromatic hydrocarbons (PAH) and C(10)-C(36) n-alkanes, including n-hexadecane, was associated primarily with the oil films. Biodegradation of PAH and n-alkanes was significant at both 0 and 5 degrees C, but was decreased for several compounds at the lower temperature. n-Hexadecane biodegradation at the two temperatures was comparable at the end of the experiments, but was delayed at 0 degree C. Investigations of bacterial communities in seawater and on adsorbents by PCR amplification of 16S rRNA gene fragments and DGGE analysis indicated that predominant bacteria in the seawater gradually adhered to the oil-coated adsorbents during biodegradation at both temperatures. Sequence analysis of most DGGE bands aligned to members of the phyla Proteobacteria (Gammaproteobacteria) or Bacteroidetes. Most sequences from experiments at 0 degree C revealed affiliations to members of Arctic or Antarctic consortia, while no such homology was detected for sequences from degradation experiment run at 5 degrees C. In conclusion, marine microbial communities from cold seawater have potentials for oil film HC degradation at temperatures < or =5 degrees C, and psychrotrophic or psychrophilic bacteria may play an important role during oil HC biodegradation in seawater close to freezing point.

Middle Distillate Fuel Fingerprinting Using Drimane-Based Bicyclic Sesquiterpanes

Petroleum biomarkers are ubiquitous hydrocarbons that occur in crude oils and are derived from formerly living organisms whose organic materials gave rise to the crude oil over geologic time. Biomarkers' specificity and resistance to weathering renders them useful in chemical fingerprinting of petroleum contamination in the environment. The biomarkers most commonly used in forensic issues, viz., tri- and penta-cyclic triterpanes and regular or rearranged steranes, are too high boiling for use in fingerprinting middle distillate petroleum products. The present article describes a suite of “low boiling” biomarkers (i.e., drimane-based bicyclic sesquiterpanes) that boil within the distillate fuel range (n-C13 to n-C16; ∼ 240°C to 285°C). GC/MS analysis of 24 dispensed (fresh) distillate fuels (Jet A, JP-5, and diesel fuel/fuel oil #2) demonstrate the specificity of sesquiterpane patterns in fuels of different origins. Analysis of fresh diesel fuel #2 and its associated, variably weathered NAPLs demonstrate the stability of the sesquiterpane despite complete biodegradation of n-alkanes. Finally, a case study of a forensic investigation is presented in which the sesquiterpanes are used to further unravel the sources of weathered diesel fuel/fuel oil #2 impacting soils on adjacent properties. Sesquiterpanes were used to demonstrate that the soils from the plaintiff's and defendant's properties contained distinct diesel fuel/fuel oil #2, which implied distinct sources and thereby led to a rapid settlement of the case.

Monitoring of the Degradation Activities and the Diversity of the Microbial Community Degrading Refinery Waste Sludge

The biodegradation of n-alkanes and branched alkanes from waste sludge were observed in landfarming soils of Motor Oil Hellas (a petroleum refinery) and changes in the bacterial communities in the soils were monitored during the remediation. Bacterial 16S rRNA gene (rDNA)-based community fingerprint patterns were obtained from soil samples by terminal restriction fragment length polymorphism (T-RFLP) analysis. Changes in T-RFLP fingerprints, as well as in the petroleum contaminant composition of the polluted soil, correlated with degradation activities in field tests.

Application of a Slow‐Release Fertilizer for Oil Bioremediation in Beach Sediment

A 105-d field experiment was conducted to determine the potential of the slow-release fertilizer, Osmocote (Scotts, Marysville, OH), to stimulate the indigenous microbial biodegradation of petroleum hydrocarbons in an oil-spiked beach sediment on an intertidal foreshore in Singapore. Triplicate microcosms containing 80 kg of weathered sediment, spiked with 5% (w/w) Arabian light crude oil and 1.2% (w/w) Osmocote pellets, were established, together with control microcosms minus Osmocote. Relative to the control, the presence of the Osmocote sustained a significantly higher level of nutrients (NH(4)(+)-N, NO(3)(-)-N, and PO(4)(3-)-P) in the sediment pore water over the duration of the experiment. The metabolic activity of the indigenous microbial biomass, as measured using an intracellular dehydrogenase enzyme assay, was also significantly enhanced over the duration of the experiment in amended sediments. The loss of total recoverable petroleum hydrocarbons (TRPH) and biodegradation of total n-alkanes (C(10)-C(33)), branched alkanes (pristane and phytane), as well as total target polycyclic aromatic hydrocarbons (PAHs) (two- to six-ring), in both the control and Osmocote-amended sediments, followed a first-order biodegradation model. The first-order loss rate of total recoverable petroleum hydrocarbons was 2.57 times greater than that of the control. The hopane-normalized rate constants for total n-alkane, branched alkane, and total target PAH biodegradation in the Osmocote-treated sediments were 3.95-, 5.50-, and 2.45-fold higher than the control, respectively. Overall, the presence of Osmocote was able to significantly enhance and accelerate the biodegradation of aliphatics and PAHs in oil-contaminated sediments under natural field conditions in an intertidal foreshore environment.

Influence of biosurfactants produced by Candida antarctica on surface properties of microorganism and biodegradation of n-alkanes

The objective of this study was to investigate the influence of the biosurfactant BS-UC on surface properties of microbial cells and biodegradation of petroleum hydrocarbons. The biosurfactant BS-UC was produced by Candida antarctica from n-undecane as the substrate. It was found that the addition of BS-UC influenced positively the emulsification and the biodegradation of a variety of n-alkanes substrates. Besides, BS-UC also changed the hydrophobicity and zeta potential of the cell surface, and thus made the microbial cell attach to the hydrophobic substrate easily. The cell transport experiment also indicated that the addition of BS-UC changed both zeta potential of the cell and that of the porous media, and improved the retention of the cells in the media. So it will make BS-UC a promising choice for use in bioremediation of petroleum contamination.

Biodegradation of Dispersed Forties Crude and Alaskan North Slope Oils in Microcosms Under Simulated Marine Conditions

For oil spills in the open sea, operational experience has found that conventional response techniques, such as mechanical recovery, tend to remove only a small fraction of oil during major spills, a recent exception being the Mississippi River spill in Louisiana [Spill Sci. Technol. Bull. 7 (2002) 155]. By contrast, the use of dispersants can enable significant fractions of oil to be removed from the sea surface by dispersing the oil into the water column. It is thought that once dispersed the oil can biodegrade in the water column, although there is little information on the mechanism and rate of biodegradation. Two studies were undertaken on dispersion, microbial colonisation and biodegradation of Forties crude and Alaskan North Slope (ANS) oils under simulated marine conditions. The study using the Forties crude lasted 27 days and was carried out in conditions simulating estuarine and coastal conditions in waters around the UK (15 °C and in the presence of nutrients, 1 mg N-NO 3/l), while the ANS study simulated low temperature conditions typical of Prince William Sound (8 °C) and took place over 35 days. The results of both studies demonstrated microbial colonisation of oil droplets after 4 days, and the formation of neutrally buoyant clusters consisting of oil, bacteria, protozoa and nematodes. By day 16, the size of the clusters increased and they sank to the bottom of the microcosms, presumably because of a decrease in buoyancy due to oil biodegradation, however biodegradation of n-alkanes was confirmed only in the Forties study. No colonisation or biodegradation of oil was noted in the controls in which biological action was inhibited. Oil degrading bacteria proliferated in all biologically active microcosms. Without dispersant, the onset of colonisation was delayed, although microbial growth rates and population size in ANS were greater than observed with the Forties. This difference reflected the greater droplet number seen with ANS at 8 °C than with Forties crude at 15 °C. Although these studies differed by more than one variable, complicating comparison, the findings suggest that dispersion (natural or chemical) changes the impact of the oil on the marine environment, potentially having important implications for management of oil spills in relation to the policy of dispersant use in an oil spill event.

Use of sodium dodecyl sulphate for stimulation of biodegradation of n-alkanes without residual contamination by the surfactant

The capacity of a range of aliphatic alkanes (C 6–C 16), intermediates of n-decane oxidation and sodium dodecyl sulphate (SDS) to induce decane-mineralization activity in the cells of Pseudomonas C12B was compared with that for n-decane. The comparison on quantitative basis had two serious limitations: low solubility of tested inducers in aqueous solutions and their toxicity to bacterial cells. Carbon chain length and the presence of hydroxyl group were the important factors for induction activity. However, presence of hydroxyl groups at both ends of alkyl chain prevented the induction of decane-mineralization activity. Good induction activity by SDS was caused either by the presence of free end of alkyl chain, or by bacterial hydrolysis of sulphate group to yield alcohol, which in turn served as true inducer. The presence of SDS in the culture medium with n-decane as main source of carbon and energy accelerated the growth of Pseudomonas C12B. SDS disappeared from the culture medium in early stages of cultivation suggesting preferential degradation by the bacterium, while the consumption of n-decane was accelerated. This may be associated with the capacity of SDS to induce decane-mineralization system in Pseudomonas C12B and/or with the ability of SDS to stimulate the surface attachment of competent bacteria resulting in the close proximity of the cells with alkane droplets, and thus, enhanced breakdown of the hydrocarbon pollutant.

Enhanced degradation of heavy oil in an aqueous system by a Pseudomonas sp. in the presence of natural and synthetic sorbents

The microbial degradation of the saturated and sulfur aromatic fractions of a heavy oil was examined in aerobic aqueous conditions employing a pure culture of a Pseudomonas sp. in the presence of natural and synthetic sorbents. All three natural sorbents tested (keratin, chitosan and chitin) were capable of adsorbing large amounts of heavy oil as well as enormously increasing the n-alkane biodegradation rate and decreasing the lag phase by up to five-seven times as much as that in the absence of sorbents. In the presence of natural sorbents n-alkane biodegradation was complete after 7 days while in their absence the same result took 40 days. This time decrease was ascribed to an improved interaction between the oil and the microorganisms derived from the formation of a water/cell/oil/sorbent interphase. The values of the accelerating conversion factor, b, and the length of the lag phase, 1/ ab, were determined for the different sorbents using a mathematical expression, and they could represent two important parameters for the description of n-alkane biodegradation kinetics.

Intrinsic biodegradation of diesel fuel in an interval of separate phase hydrocarbons

Emerging acceptance of the limitations of separate phase product recovery has spawned interest in the intrinsic alteration of residual separate phase petroleum products. In this study the geochemical changes in a continuous core through soil containing a separate phase diesel fuel #2 (SPD) in contact with groundwater are investigated. Chemical heterogeneities are shown to exist which can be attributed to weathering, particularly intrinsic biodegradation. The results show that the aliphatic hydrocarbon content is reduced and the δ 13C ratio of the aliphatic hydrocarbons increased from top to bottom in the core. Both changes are thought to be due to preferential biodegradation of (isotopically lighter) n-alkanes. A slight increase in the relative abundance of shorter chain n-alkanes (< n-C 17) was also observed. The distribution of the dominant aromatic hydrocarbons (C 0–C 3 alkyl-naphthalenes) is remarkably consistent throughout the core, although naphthalene is depleted below the oil–water interface. In spite of low oil saturation ( S 0), little or no evidence of biodegradation is noted at the uppermost boundary of the SPD. However, intrinsic biodegradation is evident approximately 0.3 m above the oil–water interface in spite of higher S 0. The extent of the chemical changes attributable to biodegradation (described above) gradually increases below the oil–water interface, eventually reaching a maximum at the bottom of the SPD profile (∼1.2 m below the interface) where S 0 is again reduced. The relatively higher level of biodegradation observed at and below the oil–water interface may be attributed to the reduced S 0 in this zone. An estimate of the mass reduction in diesel fuel between the uppermost and bottommost parts of the core is calculated to be 23% (by weight), due predominantly to the biodegradation of n-alkanes.

Studies on the biodegradation of three groups of pure n-alkanes in the presence of molasses and mineral fertilizer by Pseudomonas aeruginosa

A laboratory biodegradation study of three arbitrary groups of pure n-alkanes chosen according to their carbon chain length was undertaken. The breakdown of n-alkanes was assessed using repirometry and gas chromatography in the presence of Pseudomonas aeruginosa. Molasses or mineral fertilizer were added to the batch culture to investigate their effects on n-alkane biodegradation. The use of molasses enhanced the oxidation rates two fold over a carbon free mineral fertilizer. The percentage reductions for most of the n-alkanes were higher in the presence of molasses than for the mineral fertilizer. For molasses group 1 n-alkanes (C14–C17) and group 2 (C18–C22) were both significantly reduced, whereas in group 3 (C23–C28, and C30) only C23, C24 and C25 showed significant reductions. In the presence of mineral fertilizer only group 2 showed any significant reduction. The significance of using molasses in biodegradation of n-alkanes in relation to oil spills is briefly discussed.

Use of polyurethane foam for the biodegradation of n-alkanes by immobilised cells of Pseudomonas

Pseudomonas strains C12B and A3 were separately immobilised in polyurethane foam and their ability to biodegrade n-decane or n-hexadecane tested and compared with that of freely suspended cells. In the presence of immobilised Pseudomonas C12B or A3 cells about 90% of n-alkane, originally present in liquid medium, was removed, 25%-40% of which was due to biodegradation.

Effects of the addition of organic carbon sources on bacterial respiration and n-alkane biodegradation of Omani crude oil

Respirometry and gas chromatography showed that, in the presence of crude oil, the surfactants Corexit ® 9527 and Corexit ® 9130 were rapidly oxidized by Pseudomonas aeruginosa alone and by a mixed culture of bacteria. The addition of a mineral fertilizer to the oil was ineffective in increasing respiration or alkane breakdown. When cane sugar molasses was added to the oil, respiration increased markedly and was associated with significant n-alkane breakdown. The bacterial counts at the start and end of the respirometry showed no significant variations whichever substrates were metabolized by the bacteria. The significance of the addition of organic substrates to oil in relation to biodegradation of alkanes is discussed.

Biodegradation studies of selected hydrocarbons from diesel oil.

In-vitro biodegradation of aliphatic and aromatic hydrocarbons present in diesel oil by Pseudomonas fluorescens, Texaco was studied in an aqueous medium. Small aliquots of diesel oil and its aromatic fraction were incubated aerobically for periods of up to seven months and analysed by GC-MS. Biotic losses proved to be greater for aliphatic than aromatic compounds. Most biodegradation occurred within the first 20 d of incubation. The most rapid biodegradation, up to 65% in 8 d, was observed for n-alkanes (C14-C18). The same compounds were also shown to be less affected by abiotic losses. Biodegradation of n-alkanes from diesel oil and diesel oil itself showed first order kinetics for the initial incubation period. Aromatic compounds proved to be resistant to biodegradation and only phenanthrene had been degraded (30%) within 6 months.