Microbial degradation of crude oil in sea water in continuous culture

Summary The degradation of crude oil in continuous culture of a mixed bacteria population has been studied. The degradation percentage reaches 83 % with a 0.05 h^-1 dilution rate and a 6 g 1^-1 crude oil concentration. The different crude oil compounds : saturated, aromatic, polar hydrocarbons and asphaltenes are degraded at 97 %, 81 %, 52 % and 74 % respectively.     

Production of biosurfactants by a mixed bacteria population grown in continuous culture on crude oil

Summary Large amounts (3 g.l^–1) of tensio-active substances can be produced continuously and recuperated using a tangential-flow filtration device. These compounds can emulsify crude oil and the emulsions, obtained with a crude oil over biosurfactants ratio of 500, remain stable for hours.     

The importance of weathered crude oil as a source of hydrocarbonoclastic microorganisms in contaminated seawater

This study investigated the hydrocarbonoclastic microbial community present on weathered crude oil and their ability to degrade weathered oil in seawater obtained from the Gulf St. Vincent (SA, Australia). Examination of the native seawater communities capable of utilizing hydrocarbon as the sole carbon source identified a maximum recovery of just 6.6 × 10(1) CFU/ml, with these values dramatically increased in the weathered oil, reaching 4.1 × 10(4) CFU/ml. The weathered oil (dominated by >C30 fractions; 750,000 +/- 150,000 mg/l) was subject to an 8 week laboratory-based degradation microcosm study. By day 56, the natural inoculums degraded the soluble hydrocarbons (initial concentrations 3,400 +/- 700 mg/l and 1,700 +/- 340 mg/l for the control and seawater, respectively) to below detectable levels, and biodegradation of the residual oil reached 62% (254,000 +/- 40,000 mg/l) and 66% (285,000 +/- 45,000 mg/l) in the control and seawater sources, respectively. In addition, the residual oil gas chromatogram profiles changed with the presence of short and intermediate hydrocarbon chains. 16S rDNA DGGE sequence analysis revealed species affiliated with the genera Roseobacter, Alteromonas, Yeosuana aromativorans, and Pseudomonas, renowned oil-degrading organisms previously thought to be associated with the environment where the oil contaminated rather than also being present in the contaminating oil. This study highlights the importance of microbiological techniques for isolation and characterisation, coupled with molecular techniques for identification, in understanding the role and function of native oil communities.     

Volatile hydrocarbon biodegradation by a mixed-bacterial culture during growth on crude oil

Volatile hydrocarbon biodegradation by a mixed-bacterial culture during growth on Bow River crude oil was investigated using solid phase microextraction (SPME). Inoculum treatments were examined in relation to C₅–C₁₁ hydrocarbon degradation. Up to 1600 mg/l biomass (dry weight) was tested without achieving significant volatile hydrocarbon partitioning and affecting analysis. Inoculum age rather than concentration had the most profound impact on biodegradation. When late log phase crude oil-grown inocula were used, C₅–C₁₁ biodegradation reached 55–60%; methylcyclohexane and other branched compounds eluting before n-C₈ were recalcitrant. Increasing the late log inoculum concentration from 0.63 to 63 mg/l resulted in a twofold increase in degradation rate without improving the substrate range. Methylcyclohexane recalcitrance was correlated with reduced levels of hydrocarbon-degrading bacteria and volatile hydrocarbon evaporation from the inoculum flasks. A decreased lag phase prior to degradation was observed when using early stationary phase cultures as inocula and most compounds up to C₁₁, including methylcyclohexane, were biodegraded. Journal of Industrial Microbiology & Biotechnology (2001) 26, 356–362. Received 16 November 2000/ Accepted in revised form 17 March 2001     

Application of a Doehlert experimental design to the optimization of microbial degradation of crude oil in sea water by continuous culture

Summary Crude oil was degraded by a mixed bacterial community grown in continuous culture on sea water. The fermentation process included an emulsification step prior to the introduction of the substrate in the reactor, with external cell recycling by a tangential-flow filtration system. Optimization of the fermentation technique was achieved by using the surface response methodology (Doehlert experimental design). Besides reducing the number of experiments, this approach allowed optimal experimental conditions to be chosen, for the particular goal: percent degradation of crude oil (80%), biomass (7.6 g·l^-1) and degradation rate (0.73 g·l^-1·h^-1). This biodegradation process could be used as a tool to fight against pollutions by petroleum products.     

Bioremediation of crude oil polluted seawater by a hydrocarbon-degrading bacterial strain immobilized on chitin and chitosan flakes

In this laboratory-scale study, we examined the potential of chitin and chitosan flakes obtained from shrimp wastes as carrier material for a hydrocarbon-degrading bacterial strain. Flakes decontamination, immobilization conditions and the survival of the immobilized bacterial strain under different storage temperatures were evaluated. The potential of immobilized hydrocarbon-degrading bacterial strain for crude oil polluted seawater bioremediation was tested in seawater microcosms. In terms of removal percentage of crude oil after 15 days, the microcosms treated with the immobilized inoculants proved to be the most successful. The inoculants formulated with chitin and chitosan as carrier materials improved the survival and the activity of the immobilized strain. It is important to emphasize that the inoculants formulated with chitin showed the best performance during storage and seawater bioremediation.     

Survival of the North American strain of viral hemorrhagic septicemia virus (VHSV) in filtered seawater and seawater containing ovarian fluid, crude oil and serum-enriched culture medium

The North American strain of viral hemorrhagic septicemia virus (NA-VHSV) could be recovered for up to 40 h in natural filtered seawater (27 ppt) with a 50% loss of infectivity after approximately 10 h at 15 degrees C. Addition of 10 ppb North Slope crude oil to the seawater had no effect on virus survival. However, when various concentrations of teleost ovarian fluid were added to seawater, virus could be recovered after 72 h at 0.01% ovarian fluid and after 96 h at 1.0%. When cell culture medium supplemented with 10% fetal bovine serum was added to the seawater, 100% of the virus could be recovered for the first 15 d and 60% of the virus remained after 36 d. These findings quantify NA-VHSV infectivity in natural seawater and demonstrate that ovarian fluid, which occurs naturally during spawning events, significantly prolongs the survival and infectivity of the virus. The extended stabilization of virus in culture medium supplemented with serum allows for low titer field samples to be collected and transported in an unfrozen state without significant loss of virus titer.     

Influence of chemical surfactants on the biodegradation of crude oil by a mixed bacterial culture.

The effects of surfactant physicochemical properties, such as the hydrophile-lipophile balance (HLB) and molecular structure, on the biodegradation of 2% w/v Bow River crude oil by a mixed-bacterial culture were examined. Viable counts increased 4.6-fold and total petroleum hydrocarbon (TPH) biodegradation increased 57% in the presence of Igepal CO-630, a nonylphenol ethoxylate (HLB 13, 0.625 g/L). Only the nonylphenol ethoxylate with an HLB value of 13 substantially enhanced biodegradation. The surfactants from other chemical classes with HLB values of 13 (0.625 g/L) had no effect or were inhibitory. TPH biodegradation enhancement by Igepal CO-630 occurred at concentrations above the critical micelle concentration. When the effect of surfactant on individual oil fractions was examined, the biodegradation enhancement for the saturate and aromatic fractions was the same. In all cases, biodegradation resulted in increased resin and asphaltene concentrations. Optimal surfactant concentrations for TPH biodegradation reduced resin and asphaltene formation. Chemical surfactants have the potential to improve crude oil biodegradation in complex microbial systems, and surfactant selection should consider factors such as molecular structure, HLB, and surfactant concentration.     

Fermentation of glycerol by Clostridium pasteurianum--batch and continuous culture studies

The fermentation of glycerol by Clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions. In the majority of batch cultures, butanol was the main fermentation product, but a varying fraction of glycerol was also converted to 1,3-propanediol, butyric and acetic acids and ethanol. More than 60 g/l glycerol was utilized, and up to 17 g/l butanol was produced. Fed-batch cultures did not offer an advantage. When molecular nitrogen was used as a nitrogen source, the fermentation time was prolonged by a factor of 1.5. Fermentations at constant pH values between 4.5 and 7.5 did not reveal significant differences in product formation except for an increase in the ethanol content starting at pH 6.5. Chemostat cultures also yielded predominantly n-butanol, but in some fermentations, the 1,3-propanediol fraction was relatively high. The pH auxostat cultures, which were operated at a glycerol excess, contained 1,3-propanediol as the main product. As a whole, the fermentations were characterized by a certain variability in product formation under seemingly equal or slightly varied conditions. It appears that the regulation of the numerous fermentation pathways occurring in this organism is not very strict. Journal of Industrial Microbiology & Biotechnology (2001) 27, 18–26. Received 25 September 2000/ Accepted in revised form 07 April 2001     

Effect of keratinous waste addition on improvement of crude oil hydrocarbon removal by a hydrocarbon-degrading and keratinolytic mixed culture

The aim of this work was to evaluate the effect of keratinous waste addition on oil-hydrocarbon removal, through a mixed culture of oil-degrading bacteria, with the ability to secrete keratinases. The mixed culture was grown in the media with oil, or oil supplemented with chicken-feathers as the keratinous waste. Residual oil-hydrocarbons were determined as total petroleum hydrocarbons (TPHs) and oil fractions and then quantified by GC–FID and GC–MS.Results showed that in presence of the keratinous waste, the removal of oil-hydrocarbons was 57,400 mg l^?1, meanwhile the treatment without waste presented an oil-hydrocarbons removal of 35,600 mg l^?1. The aliphatic fraction was the most removed in both treatments. In addition, chromatographic profiles indicated that the aliphatic fraction showed different degradation pattern; in the presence of keratinous wastes, the C₁₈ to C₂₈ compounds were preferably removed over the C₁₀ to C₁₇. The addition of keratinous waste not only improved the oil-hydrocarbons removal but, it changed the removal pattern of the target hydrocarbons.     

Biodeterioration of crude oil and oil derived products: a review

Biodeterioration of crude oil and oil fuels is a serious economic and an environmental problem all over the world. It is impossible to prevent penetration of microorganisms in oil and fuels both stored in tanks or in oilfields after drilling. Both aerobic and anaerobic microorganisms tend to colonise oil pipelines and oil and fuel storage installations. Complex microbial communities consisting of both hydrocarbon oxidizing microorganisms and bacteria using the metabolites of the former form an ecological niche where they thrive. The accumulation of water at the bottom of storage tanks and in oil pipelines is a primary prerequisite for development of microorganisms in fuels and oil and their subsequent biological fouling. Ability of microorganisms to grow both in a water phase and on inter-phase of water/hydrocarbon as well as the generation of products of their metabolism worsen the physical and chemical properties of oils and fuels. This activity also increases the amount of suspended solids, leads to the formation of slimes and creates a variety of operational problems. Nowadays various test-systems are utilized for microbial monitoring in crude oils and fuels; thus allowing an express determination of both the species and the quantities of microorganisms present. To suppress microbial growth in oils and fuels, both physico-mechanical and chemical methods are applied. Among chemical methods, the preference is given to substances such as biocides, additives, the anti-freezing agents etc that do not deteriorate the quality of oil and fuels and are environmentally friendly. This review is devoted to the analysis of the present knowledge in the field of microbial fouling of crude oils and oil products. The methods utilized for monitoring of microbial contamination and prevention of their undesirable activities are also evaluated. The special focus is given to Russian scientific literature devoted to crude oil and oil products biodeterioration.     

Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil,shale oil or diesel fuel

The coastal waters of the Baltic Sea are constantly threatened by oil spills, due to the extensive transportation of oil products across the sea. To characterise the hydrocarbon-degrading bacterial community of this marine area, microcosm experiments on diesel fuel, crude oil and shale oil were performed. Analysis of these microcosms, using alkane monooxygenase (alkB) and 16S rRNA marker genes in PCR-DGGE experiments, demonstrated that substrate type and concentration strongly influence species composition and the occurrence of alkB genes in respective oil degrading bacterial communities. Gammaproteobacteria (particularly the genus Pseudomonas) and Alphaproteobacteria were dominant in all microcosms treated with oils. All alkB genes carried by bacterial isolates (40 strains), and 8 of the 11 major DGGE bands from the microcosms, had more than 95% sequence identity with the alkB genes of Pseudomonas fluorescens. However, the closest relatives of the majority of sequences (54 sequences from 79) of the alkB gene library from initially collected seawater DNA were Actinobacteria. alkB gene expression, induced by hexadecane, was recorded in isolated bacterial strains. Thus, complementary culture dependent and independent methods provided a more accurate picture about the complex seawater microbial communities of the Baltic Sea.     

Fermentation of a wheat straw acid hydrolysate by Bacillus stearothermophilus T-13 in continuous culture with partial cell recycle

The effects of pretreatment by overliming and addition of nutrients (yeast extract, tryptone and ammonium chloride) on fermentation of mixed sugars derived by acid hydrolysis of the hemicellulose fraction of wheat straw with Bacilllus stearothermophilus strain T-13, an l-lactate dehydrogenase deficient mutant was investigated in continuous culture with partial cell recycle. Pretreatment and addition of nutrients to the hydrolysate improved the fermentation considerably. Sugar utilization, ethanol yields and productivities obtained in the treated hydrolysate with added nutrients were comparable to those obtained in a synthetic medium. Sugar utilization in the synthetic medium and treated and crude hydrolysates with added nutrients were 86%, 89% and 56%, respectively, compared with 45% in the treated hydrolysate without extra nutrients. Ethanol yields obtained were 0.32 g g⁻¹ sugars and 0.38 g g⁻¹ sugars in the treated hydrolysate with and without extra nutrients, respectively, compared with 0.24 g g⁻¹ sugars in the crude hydrolysate with added nutrients. Continuous culture with partial cell recycle significantly increased the rate of ethanol production (0.60–1.02 g litre⁻¹ h⁻¹) in the various media and the stability of the mutant strain compared with conventional continuous culture.     

Effect of soybean oil and glucose on sophorose lipid fermentation by Torulopsis bombicola in continuous culture

The effect of soybean oil and glucose on the growth of Torulopsis bombicola and sophorose lipid production in continuous culture was investigated. As the dilution rate in 100 g/l glucose and 100 g/l soybean oil medium was increased, the dry cell weight and sophorose lipid concentration decreased. Sophorose lipid productivity, however, was maximum at a dilution rate of 0.03 h⁻¹. The cell yield from glucose and the sophorose lipid production from soybean oil were approximately constant regardless of the dilution rate. The specific consumption rate of soybean oil was closely related to the specific production rate of sophorose lipid. These results suggest that soybean oil was used only for sophorose lipid production whereas glucose was used only for cell mass and maintenance. When the soybean oil concentration was varied at fixed dilution rate in 100 g/l glucose medium, a high concentration of soybean oil was found to inhibit sophorose lipid production. Received: 9 January 1997 / Received revision: 5 March 1997 / Accepted: 13 April 1997     

Growth kinetics of a bacteriophage in continuous culture

Lytic coliphage Qbeta was grown in continuously cultured host bacteria using a cascade of stirred flow reactors. The apparatus was constructed so that the steady stream of exponentially growing bacterial cells passing through the stirred flow reactors served to prevent coevolution brought about by host-parasite interactions. Wall growth was the primary cause for deviation from ideal continuous culture conditions and is largely dependent on the surface structure of the host bacteria. Using an Escherichia coli strain deficient in adhesive type I pili expression, the desynchronization of single burst events could easily be followed over the course of four infection latency periods. Computer simulations based on a two-stage model for the Qbeta infection cycle were in perfect agreement with the experimental data. Applications of the optimized system to strategies of molecular evolution are discussed. (c) 1996 John Wiley & Sons, Inc.     

Enrichment of DNRA bacteria in a continuous culture

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are competing microbial nitrate-reduction processes. The occurrence of DNRA has been shown to be effected qualitatively by various parameters in the environment. A more quantitative understanding can be obtained using enrichment cultures in a laboratory reactor, yet no successful DNRA enrichment culture has been described. We showed that a stable DNRA-dominated enrichment culture can be obtained in a chemostat system. The enrichment was based on the hypothesis that nitrate limitation is the dominant factor in selecting for DNRA. First, a conventional denitrifying culture was enriched from activated sludge, with acetate and nitrate as substrates. Next, the acetate concentration in the medium was increased to obtain nitrate-limiting conditions. As a result, conversions shifted from denitrification to DNRA. In this selection of a DNRA culture, two important factors were the nitrate limitation and a relatively low dilution rate (0.026 h⁻¹). The culture was a highly enriched population of Deltaproteobacteria most closely related to Geobacter lovleyi, based on 16S rRNA gene sequencing (97% similarity). We established a stable and reproducible cultivation method for the enrichment of DNRA bacteria in a continuously operated reactor system. This enrichment method allows to further investigate the DNRA process and address the factors for competition between DNRA and denitrification, or other N-conversion pathways.