Microbial degradation kinetics of solid alkane dissolved in nondegradable oil phase

An Acinetobacter species was isolated and found to be able to grow on crude oil n-alkanes and solid alkanes at room temperature as the sole carbon source. The growth of the isolate on n-heneicosane dissolved in non-biodegradable pristane has been studied. A kinetic model of the growth of microorganism on the hydrophobic substrate dissolved in non-biodegradable oil droplet assuming direct contact of cell with oil droplet was developed and validated with a model system of crude oil biodegradation. The model was focused on the substrate transport to the cell being contact with the surface of droplet. The high value of saturation constant of n-heneicosane, K_s = 0.086 kg m⁻³, and the maximum specific growth rate, μ_m = 0.60 h⁻¹, were obtained. The transport limitation was considered and estimated. The high value of attached cell fraction was reasonable to explain the observed growth rate by the direct contact model and varied with time till it reached a plateau at the stationary growth phase. By considering the direct contact of the cells with the surface of pristane and the transport of n-heneicosane to the cell, the degradation of hydrophobic substrate in the oil phase could be elucidated.     

Utilization of gas oil by a yeast culture

A mixed yeast culture (Culture 4) was grown on representative gas oil samples as well as paraffin wax. Culture 4 was found to utilize n-paraffinic hydrocarbons almost quantitatively from most gas oil fractions; significant alteration of other hydrocarbon components was not detected. Generation times of 4.0–9.0hr. were typical during the exponential growth phase in fermentations with various gas oil fractions. Cell yields were 70–90% based on n-paraffin utilization. The culture appeared to exhibit maximum efficiency of n-alkane removal in the C₁₉ to C₂₄ range. The cells recovered from the fermentations contained 8.8–9.3% nitrogen. Paraffin wax also served as a suitable carbon source when dissolved in 2,6,10,14-tertramethylpentadecane (pristane). However, substrate utilization appeared to be incomplete.     

Variability in Pseudomonas aeruginosa Lipopolysaccharide Expression during Crude Oil Degradation

Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.     

Microbial degradation of petroleum hydrocarbons in a polluted tropical stream

Summary Crude oil degradation was observed in water samples from three sites along the course of a polluted stream in Lagos, Nigeria. Consistent increase and decrease in the total viable counts (TVCs) of indigenous organisms occurred in the test and control experiments, respectively. Enrichments of the water samples with crude oil resulted in the isolation of nine bacteria belonging to seven genera. A mixed culture was developed from the assemblage of the nine species. The defined microbial consortium utilized a wide range of pure HCs including cycloalkane and aromatic HCs. Utilization of crude oil and petroleum cuts, i.e., kerosene and diesel resulted in an increase in TVC (till day 10) concomitant with decreases in pH and residual oil concentration. Crude oil, diesel and kerosene were degraded by 88, 85 and 78%, respectively, in 14 days. Substrate uptake studies with axenic cultures showed that growth was not sustainable on either cyclohexane or aromatics while degradation of the petroleum fractions fell below 67% in spite of extended incubation period (20 day). From the GC analysis of recovered oil, while reductions in peaks of n-alkane fractions and in biomarkers namely n-C₁₇/pristane and n-C₁₈/phytane ratios were observed in culture fluids of pure strains, complete removal of all the HC components of kerosene, diesel and crude oil including the isoprenoids was obtained with the consortium within 14 days.     

Degradation of crude oil by an arctic microbial consortium

The ability of a psychrotolerant microbial consortium to degrade crude oil at low temperatures was investigated. The enriched arctic microbial community was also tested for its ability to utilize various hydrocarbons, such as long-chain alkanes (n-C₂₄ to n-C₃₄), pristane, (methyl-)naphthalenes, and xylenes, as sole carbon and energy sources. Except for o-xylene and methylnaphthalenes, all tested compounds were metabolized under conditions that are typical for contaminated marine liquid sites, namely at pH 6–9 and at 4–27°C. By applying molecular biological techniques (16S rDNA sequencing, DGGE) nine strains could be identified in the consortium. Five of these strains could be isolated in pure cultures. The involved strains were closely related to the following genera: Pseudoalteromonas (two species), Pseudomonas (two species), Shewanella (two species), Marinobacter (one species), Psychrobacter (one species), and Agreia (one species). Interestingly, the five isolated strains in different combinations were unable to degrade crude oil or its components significantly, indicating the importance of the four unculturable microorganisms in the degradation of single or of complex mixtures of hydrocarbons. The obtained mixed culture showed obvious advantages including stability of the consortium, wide range adaptability for crude oil degradation, and strong degradation ability of crude oil.     

Differential protein expression during growth on linear versus branched alkanes in the obligate marine hydrocarbon-degrading bacterium Alcanivorax borkumensis SK2T

Alcanivorax borkumensis SK2^T is an important obligate hydrocarbonoclastic bacterium (OHCB) that can dominate microbial communities following marine oil spills. It possesses the ability to degrade branched alkanes which provides it a competitive advantage over many other marine alkane degraders that can only degrade linear alkanes. We used LC–MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on linear (n-C₁₄) or branched (pristane) alkanes. During growth on n-C₁₄, A. borkumensis expressed a complete pathway for the terminal oxidation of n-alkanes to their corresponding acyl-CoA derivatives including AlkB and AlmA, two CYP153 cytochrome P450s, an alcohol dehydrogenase and an aldehyde dehydrogenase. In contrast, during growth on pristane, an alternative alkane degradation pathway was expressed including a different cytochrome P450, an alcohol oxidase and an alcohol dehydrogenase. A. borkumensis also expressed a different set of enzymes for β-oxidation of the resultant fatty acids depending on the growth substrate utilized. This study significantly enhances our understanding of the fundamental physiology of A. borkumensis SK2^T by identifying the key enzymes expressed and involved in terminal oxidation of both linear and branched alkanes. It has also highlights the differential expression of sets of β-oxidation proteins to overcome steric hinderance from branched substrates.     

Effectiveness of surfactants in the microbial degradation of oil

Nonionic surfactants increase the rate of selective hydrocarbon utilization by Acinetobacter SL1. Within an homologus series of nonionic surfactants, growth on and utilization of a model oil by Acinetobacter SL1 is dependent upon the surfactant hydrophile‐lipophile balance (HLB). Biological effectiveness of the surfactants apparently is related to the degree of micelle formation by the surfactant in the aqueous phase. A simple algebraic expression describing the response of Acinetobacter SL1 to surfactant concentration gives a measure of the biological effectiveness of an individual surfactant. A cationic and an anionic surfactant inhibited the growth of Acinetobacter SL1 and Pseudomonas SL6 on hydrocarbon substrates. These results are discussed in relation to the selection of suitable detergents for increasing the effective biodegradation of pollutant oil in aquatic habitats.     

Factors influencing hydrocarbon degradation in three freshwater lakes

The mixed microbial flora of 3 lakes in Ohio with differing histories of hydrocarbon pollution was examined in relation to the ability to use hydrocarbons. Weathered kerosene was spiked with naphthalene, pristane, 1,13-tetradecadiene, andn-hexadecane and added to water-sediment mixtures from the 3 lakes, and utilization of the 4 marker hydrocarbons was measured. Each of the marker hydrocarbons was metabolized; naphthalene was the most readily used and pristane was the most resistant. Values for dissolved oxygen suggest that oxygen did not limit hydrocarbon degradation in the water column at any site examined. Nutrient addition studies indicated that nitrogen and phosphorus limited hydrocarbon degradation at all sites examined. Maximum numbers of heterotrophic bacteria were detected when the water temperature was 10°C or higher. The data indicate that temperature limits hydrocarbon degradation in the winter, except at a site which had been impacted by an oil spill and which received chronic inputs of hydrocarbons and nutrients. In samples from that site, all 4 marker hydrocarbons were degraded at 0°C. Results of temperature and nutrient-addition experiments suggest that different seasonal populations of hydrocarbon users are selected at that site, but not at other lake sites.     

Utilization of normal alkanes by yeasts

A stable mixed yeast culture designated as Culture 4, consisting of Candida intermedia and Candida lipolytica was investigated. The culture was judged stable based on uniformity of fermentation results and the nearly constant ratio of the two organisms at the completion of fermentations. However, the ratio of the two organisms at different times during the fermentation was not determined. The mixed culture grew more rapidly on n-alkanes than did C. intermedia; C. lipolytica did not grow on unsupplemented mineral salt–n-alkane medium. Solid n-alkanes were dissolved in 2,6,0,14-tetramethylpentadecane (pristane) for investigation as carbon sources. With Culture 4, on n-alkanes ranging from pentadecane (C₁₅) through octacosane (C₂₈), cell yields were 74.2–89.5%; generation times were 3.0–8.0 hr. during the exponential growth phase. The fastest growth rates and highest cell yields were obtained with docosane (C₂₂) as substrate. The cells obtained contained 6.75–8.81% nitrogen and 1.9–13.4% lipid. Crude protein yields were 34.4–47.6%. The oxidation of n-alkanes by C. intermedia was studied manometrically with resting whole cells. The alkaneoxidizing system of this organism appears to be constitutive and nonspecific for alkane substrates.     

Cell wall adaptations of planktonic and biofilm <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> cells to growth on C5 to C16 <Emphasis Type="Italic">n</Emphasis>-alkane hydrocarbons

Rhodococcus erythropolis was found to utilize C5 to C16 n-alkane hydrocarbons as sole source of carbon and energy when growing as planktonic or biofilm cells attached to polystyrene surfaces. Growth rates on even numbered were two- to threefold increased relatively to odd-numbered n-alkanes and depended on the chain length of the n-alkanes. C10-, C12-, C14-, and C16-n-alkanes gave rise to two- to fourfold increased maximal growth rates relative to C5- to C9-hydrocarbons. In reaction to the extremely poor water solubility of the n-alkanes, both planktonic and biofilm cells exhibited distinct adaptive changes. These included the production of surface active compounds and substrate-specific modifications of the physicochemical cell surface properties as expressed by the microbial adhesion to hydrocarbon- and contact angle-based hydrophobicity, as well as the zeta potential of the cells. By contrast, n-alkane-specific alterations of the cellular membrane composition were less distinct. The specificity of the observed autecological changes suggest that R. erythropolis cells may be used in the development and application of sound biocatalytic processes using n-alkanes as substrates or substrate reservoirs or for target-specific bioremediation of oils and combustibles, respectively.     

Bacterial metabolism of long-chain <Emphasis Type="Italic">n</Emphasis>-alkanes

Degradation of alkanes is a widespread phenomenon in nature, and numerous microorganisms, both prokaryotic and eukaryotic, capable of utilizing these substrates as a carbon and energy source have been isolated and characterized. In this review, we summarize recent advances in the understanding of bacterial metabolism of long-chain n-alkanes. Bacterial strategies for accessing these highly hydrophobic substrates are presented, along with systems for their enzymatic degradation and conversion into products of potential industrial value. We further summarize the current knowledge on the regulation of bacterial long-chain n-alkane metabolism and survey progress in understanding bacterial pathways for utilization of n-alkanes under anaerobic conditions.     

Biosurfactant Production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SNP0614 and its Effect on Biodegradation of Petroleum

An efficient biosurfactant-producing strain was isolated and cultured from Dagang oil field (China) using crude oil as sole source of carbon. Based on partial sequenced 16S rDNA analysis, the isolated strain was identified as Pseudomonas aeruginosa SNP0614. The bacterium P. aeruginosa SNP0614 produced a type of biosurfactant with excessive foam-forming properties. After microbial cultivation at 37°C and 150 rpm for 12 h, the produced biosurfactant was found to reduce the surface tension to 25.4 mN/m with critical micelle concentration (CMC) of 45.0 mg/L. After 20 days of incubation, the biosurfactant exhibited 90% emulsification activity (E₂₄) on crude oil. FTIR spectroscopy of extracted biosurfactant indicated the biosurfactant as lipopeptide. The significant synergistic effect between P. aeruginosa SNP0614 and the mixed oildegrading bacteria resulted in increasing n-alkanes degradation rate by 30%. The strain P. aeruginosa SNP0614 represented as a promising biosurfactant producer and could be applied in a variety of biotechnological and industrial processes, particularly in microbial enhanced oil recovery and the bioremediation of oil pollution.     

生物竞争抑制作用对渤海J油田微生物降解驱油聚合物功能的影响

【目的】揭示可降解驱油用聚合物的油藏内源微生物群落组成,分析生物竞争抑制作用(bio-competitive exclusion,BCX)对微生物聚合物降解功能的影响。【方法】通过室内培养实验,观察BCX对驱油用聚合物黏度的影响,随后借助高通量测序技术分析渤海J油田中与聚合物降解相关的微生物菌种,并探寻样本中丰度较高的聚合物降解功能基因─酰胺酶、加氧酶、硫化氢生成酶基因。之后,比对测序结果,采用实时荧光定量法验证上述功能基因在样本之间的含量差异,最后进一步注释携带上述功能基因的微生物群落组成。【结果】BCX可有效地延缓驱油聚合物黏度的损失。油田中与聚合物降解相关的微生物有Acetomicrobium、 Tepidiphilus、Thermoanaerobacter、Fervidobacterium、Ralstonia、Halomonas、Roseovarius、Deferribacteraceae和Comamonadaceae等9类菌种。高通量测序分析得到样本中BCX可显著下调丰度的聚合物降解功能基因共计有7种,其中酰胺酶基因ansB、加氧酶基因ssuD在样本之间的含量经定量验证,发现...     

The <Emphasis Type="Italic">Prestige</Emphasis> oil spill: bacterial community dynamics during a field biostimulation assay

A field bioremediation assay using the oleophilic fertilizer S200 was carried out 12 months after the Prestige heavy fuel-oil spill on a beach on the Cantabrian coast (north Spain). This assay showed that S200-enhanced oil degradation, particularly of high-molecular-weight n-alkanes and alkylated PAHs, suggesting an increase in the microbial bioavailability of these compounds. The bacterial community structure was determined by cultivation-independent analysis of polymerase chain reaction-amplified 16S rDNA by denaturing gradient gel electrophoresis. Bacterial community was mainly composed of α-Proteobacteria (Rhodobacteriaceae and Sphingomonadaceae). Representatives of γ-Proteobacteria (Chromatiales, Moraxellaceae, and Halomonadaceae), Bacteroidetes (Flavobacteriaceae), and Actinobacteria group (Nocardiaceae and Corynebacteriaceae) were also found. The addition of the fertilizer led to the appearance of the bacterium Mesonia algae in the early stages, with a narrow range of growth substrates, which has been associated with the common alga Achrosiphonia sonderi. The presence of Mesonia algae may be attributable to the response of the microbial community to the addition of N and P rather than indicating a role in the biodegradation process. The Rhodococcus group appeared in both assay plots, especially at the end of the experiment. It was also found at another site on the Galician coast that had been affected by the same spill. This genus has been associated with the degradation of n-alkanes up to C₃₆. Taking into account the high content of heavy alkanes in the Prestige fuel, these microorganisms could play a significant role in the degradation of such fuel. A similar bacterial community structure was observed at another site that showed a similar degree of fuel weathering.     

Progressive Degradation of Crude Oil n-Alkanes Coupled to Methane Production under Mesophilic and Thermophilic Conditions

Although methanogenic degradation of hydrocarbons has become a well-known process, little is known about which crude oil tend to be degraded at different temperatures and how the microbial community is responded. In this study, we assessed the methanogenic crude oil degradation capacity of oily sludge microbes enriched from the Shengli oilfield under mesophilic and thermophilic conditions. The microbial communities were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes combined with cloning and sequencing. Enrichment incubation demonstrated the microbial oxidation of crude oil coupled to methane production at 35 and 55°C, which generated 3.7±0.3 and 2.8±0.3 mmol of methane per gram oil, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that crude oil n-alkanes were obviously degraded, and high molecular weight n-alkanes were preferentially removed over relatively shorter-chain n-alkanes. Phylogenetic analysis revealed the concurrence of acetoclastic Methanosaeta and hydrogenotrophic methanogens but different methanogenic community structures under the two temperature conditions. Candidate divisions of JS1 and WWE 1, Proteobacteria (mainly consisting of Syntrophaceae, Desulfobacteraceae and Syntrophorhabdus) and Firmicutes (mainly consisting of Desulfotomaculum) were supposed to be involved with n-alkane degradation in the mesophilic conditions. By contrast, the different bacterial phylotypes affiliated with Caldisericales, “Shengli Cluster” and Synergistetes dominated the thermophilic consortium, which was most likely to be associated with thermophilic crude oil degradation. This study revealed that the oily sludge in Shengli oilfield harbors diverse uncultured microbes with great potential in methanogenic crude oil degradation over a wide temperature range, which extend our previous understanding of methanogenic degradation of crude oil alkanes.     

Sequencing and analysis of plasmids pAV1 and pAV2 ofAcinetobacter venetianus VE-C3 involved in diesel fuel degradation

Acinetobacter venetianus strain VE-C3 was isolated in the Venice lagoon (Italy) as a strain able to degrade diesel fuel oil. This strain possesses genes of the alkane monoxygenase complex responsible forn-alkane degradation and carries two plasmids, pAV1 (10820 bp) and pAV2 (15135 bp), which were supposed from the analysis of Alk mutant strains to harbour genetic determinants for hydrocarbon degradation. In this work we determined the nucleotide sequence of both plasmids and showed the presence of a putative aldehyde dehydrogenase gene, essential for hydrocarbon degradation, on plasmid pAV2, and of an ORF similar toalkL gene present on pAV1 plasmid. These data, combined with genetic reports indicating that strains lacking one of the two plasmids or carrying transposon insertion on pAV1, are defective inn-alkane degradation, suggest a complex genomic organisation of genes involved in alkane degradation inA. venetianus VE-C3. In this bacterium these genes are carried by both the chromosome and the plasmids, while inAcinetobacter sp. strain ADP1 and M1 all the genes for alkane monoxygenase complex are located only on the chromosome.     

Production of Dicarboxylic Acids by Candida cloacae Mutant Unable to Assimilate n-Alkane

Strain MR-12 which was derived from Candida cloacae M-l as a mutant unable to assimilate n-alkane showed marked increase in dicarboxylic acid (DC) productivity from n-alkane.

Resting cells of strain MR-12 produced 42.7g/liter of n-tetradecane 1,14-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) after 72 hr’ incubation. DC degradation activities of strain M-1 and MR-12 were found to be markedly reduced and their activities against DC-16 decreased to 40% and 10% of that of the parent strain, respectively.

Strain M-1 and MR-12 produced DC from the various oxidized derivatives of n-alkane such as alcohol, diol, aldehyde, fatty acid and methyl- or ethylester of fatty acid other than n-alkane.

The carbon balance in n-C₁₆ oxidation was determined by using resting cells of strain MR-12 and about 60% of utilized carbon was recovered as DC-16 and about 40% was recovered as CO₂.     

Microbial Production of Long-chain Dicarboxylic Acids from n-Alkanes

Microorganisms which produced n-alkane ω,ω′-dicarboxylic acid (DC) from n-alkane were selected from natural sources. It was found that the best three producers thus obtained belonged to yeast. All of the stock cultures which are able to assimilate n-alkane and are belonged to genus Candida and Pichia were also found to produce DC from n-alkane.

Candida cloacae 310, a representative strain selected from natural source, was able to produce DCs having 5 to 16 carbon atoms from various n-alkanes. Among them, DCs with 5 to 9 carbon atoms were more heavily accumulated than those with more than 9, except those with the same number of carbon atoms as the substrates which were the main products from the substrates with less than 15 carbon atoms. It was also clearly demonstrated that DCs with odd carbons alone were produced from n-alkanes with odd carbons, while DCs with even carbons alone from n-alkanes with even carbons.

Then, cultural conditions of Candida cloacae 310 were studied for the production of DC-12 from n-dodecane (n-C₁₂) which showed the highest yield among the observed accumulation.

Under the optimum conditions, 2.28 g/liter of DC-12 was obtained together with 1.86 g/liter of DC-6 and 0.82 g/liter of DC-8 after 72 hr’ cultivation in a synthetic medium containing 100 ml of n-C₁₂ per liter.