Development of a standard bacterial consortium for laboratory efficacy testing of commercial freshwater oil spill bioremediation agents

Six crude oil-degrading bacterial strains isolated from different soil and water environments were combined to create a defined consortium for use in standardized efficacy testing of commercial oil spill bioremediation agents (OSBA). The isolates were cryopreserved in individual aliquots at pre-determined cell densities, stored at −70°C, and thawed for use as standardized inocula as needed. Aliquots were prepared with precision (typically within 10% of the mean) ensuring reproducible inoculation. Five of the six strains displayed no appreciable loss of viability during cryopreservation exceeding 2.5 years, and five isolates demonstrated stable hydrocarbon-degrading phenotypes during inoculum preparation and storage. When resuscitated, the defined consortium reproducibly biodegraded Alberta Sweet Mixed Blend crude oil (typically ± 7% of the mean of triplicate cultures), as determined by quantitative gas chromatography–mass spectrometry of various analyte classes. Reproducible biodegradation was observed within a batch of inoculum in trials spanning 2.5 years, and among three batches of inoculum prepared more than 2 years apart. Biodegradation was comparable after incubation for 28 days at 10°C or 14 days at 22°C, illustrating the temperature tolerance of the bacterial consortium. The results support the use of the synthetic consortium as a reproducible, predictable inoculum to achieve standardized efficacy tests for evaluating commercial OSBA. Received 31 August 1998/ Accepted in revised form 30 November 1998     

Development of a method for the application of solid-phase microextraction to monitor biodegradation of volatile hydrocarbons during bacterial growth on crude oil

A quantitative solid-phase microextraction, gas chromatography, flame ionization detector (SPME-GC-FID) method for low-molecular-weight hydrocarbons from crude oil was developed and applied to live biodegradation samples. Repeated sampling was achieved through headspace extractions at 30°C for 45 min from flasks sealed with Teflon Mininert. Quantification without detailed knowledge of oil–water–air partition coefficients required the preparation of standard curves. An inverse relationship between retention time and mass accumulated on the SPME fibre was noted. Hydrocarbons from C₅ to C₁₆ were dated and those up to C₁₁ were quantified. Total volatiles were quantified using six calibration curves. Biodegradation of volatile hydrocarbons during growth on crude oil was faster and more complete with a mixed culture than pure isolates derived therefrom. The mixed culture degraded 55% of the compounds by weight in 4 days versus 30–35% by pure cultures of Pseudomonas aeruginosa, Rhodococcus globerulus or a co-culture of the two. The initial degradation rate was threefold higher for the mixed culture, reaching 45% degradation after 48 h. For the mixed culture, the degradation rate of individual alkanes was proportional to the initial concentration, decreasing from hexane to undecane. P. fluorescens was unable to degrade any of the low-molecular-weight hydrocarbons and methylcyclohexane was recalcitrant in all cases. Overall, the method was found to be reliable and cost-effective. Journal of Industrial Microbiology & Biotechnology (2000) 25, 155–162. Received 04 March 2000/ Accepted in revised form 25 June 2000     

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     

The role of indigenous bacterial and fungal soil populations in the biodegradation of crude oil in a desert soil

The biodegradation capacity of indigenous microbial populations was examined in a desert soil contaminated with crude oil. To evaluate biodegradation, soil samples supplemented with 5, 10 or 20% (w/w) of crude oil were incubated for 90 days at 30 °C. The effect of augmentation of the soil with vermiculite (50% v/v) as a bulking agent providing increased surface/volume ratio and improved soil aeration was also tested. Maximal biodegradation (91%) was obtained in soil containing the highest concentration of crude oil (20%) and supplemented with vermiculite; only 74% of the oil was degraded in samples containing the same level of crude oil but lacking vermiculite. Gas chromatograms of distilled fractions of crude oil extracted from the soil before and after incubation demonstrated that most of the light and part of the intermediate weight fractions initially present in the oil extracts could not be detected after incubation. Monitoring of microbial population densities revealed an initial decline in bacterial viable counts after exposure to oil, presumably as a result of the crude oil’s toxicity. This decline was followed by a steep recovery in microbial population density, then by a moderate increase that persisted until the end of incubation. By contrast, the inhibitory effect of crude oil on the fungal population was minimal. Furthermore, the overall increased growth response of the fungal population, at all three levels of contamination, was about one order of magnitude higher than that of the bacterial population.     

Cellulose degradation by a mixed bacterial culture

Summary An integrated mixed bacterial culture consisting of four strains has been isolated by a batch enrichment technique. The cellulolytic member (strain D) is aCellulomonas sp. and the others are non-cellulolytic. The interaction between strains D and C is pronounced and appears to involve an exchange of reducing sugars and growth factors. The symbiotic relationship of this naturally occurring mixed culture is therefore one of mutualism. The filter paper cellulase and carboxymethyl cellulase activities in extracellular fluid are high, while -glucosidase activity is low. The mixed culture digests a variety of lignocellulosics efficiently and is of fundamental interest in the study of microbial interrelationships.     

Biodegradation of fuel oil hydrocarbons by a mixed bacterial consortium in sandy and loamy soils

The aerobic degradation of light fuel oil in sandy and loamy soils by an environmental bacterial consortium was investigated. Soils were spiked with 1 or 0.1% of oil per dry weight of soil. Acetone extracts of dried soils were analyzed by GC and the overall degradation was calculated by comparison with hydrocarbon recovery from uninoculated soils. In sandy soils, the sum of alkanes n-C(12) to n-C(23) was degraded to about 45% within 6 days at 20 degrees C and to 27-31% within 28 days, provided that moisture and nutrients were replenished. Degradation in loamy soil was about 12% lower. The distribution of recovered alkanes suggested a preferential degradation of shorter chain molecules (n-C(12) to n-C(16)) by the bacterial consortium. Partial 16S rDNA sequences indicated the presence of strains of Pseudomonas aeruginosa, Pseudomonas citronellolis, and Stenotrophomonas maltophilia. Toxicity tests using commercial standard procedures showed a moderate inhibition of bacterial activity. The study showed the applicability of a natural microbial community for the degradation of oil spills into soils at ambient temperatures.     

From water‐in‐oil to oil‐in‐water emulsions to optimize the production of fatty acids using ionic liquids in micellar systems

Biocatalysis is nowadays considered as one of the most important tools in green chemistry. The elimination of multiple steps involved in some of the most complex chemical synthesis, reducing the amounts of wastes and hazards, thus increasing the reaction yields and decreasing the intrinsic costs, are the major advantages of biocatalysis. This work aims at improving the enzymatic hydrolysis of olive oil to produce valuable fatty acids through emulsion systems formed by long alkyl chain ionic liquids (ILs). The optimization of the emulsion and the best conditions to maximize the production of fatty acids were investigated. The stability of the emulsion was characterized considering the effect of several parameters, namely, the IL and its concentration and different water/olive oil volumetric ratios. ILs from the imidazolium and phosphonium families were evaluated. The results suggest that the ILs effect on the hydrolysis performance varies with the water concentration and the emulsion system formed, that is, water‐in‐oil or oil‐in‐water emulsion. Although at low water concentrations, the presence of ILs does not present any advantages for the hydrolysis reaction, at high water contents (in oil‐in‐water emulsions), the imidazolium‐based IL acts as an enhancer of the lipase catalytic capacity, super‐activating 1.8 times the enzyme, and consequently promoting the complete hydrolysis of the olive oil for the highest water contents [85% (v/v)]. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1473–1480, 2015     

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.     

Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions

The effect of interfacial surfactant molecules on oxygen transfer through oil/water phase boundary has been studied in FlurO(2) (TM) emulsions, i.e., perfluorocarbon (PFC) emulsions developed as oxygen carriers in cell culture. Measurements of oxygen permeability were made with a polarographic oxygen electrode in pure PFCs and in emulsions with various PFC volume fractions. Comparison of the experimental results with the theoretically derived values of relative oxygen permeability clearly indicates that the mass transfer resistance caused by the interfacial surfactant layer in PFC emulsions is insignificant. Therefore, oxygen dissolved in the enclosed PFC phase is readily available to cells growing in the aqueous media and FlurO(2) emulsions with very fine emulsion particles (< 0.2 mum) can be used to effectively enhance gas/liquid interfacial oxygen transfer in bioreactors. The inadequacy in describing mass transfer in heterogeneous systems, such as the PFC emulsions, by conventional concentration-based oxygen diffusion coefficients has also been discussed.     

Proteomic tracking and analysis of a bacterial mixed culture

To improve the understanding of microbial behaviors in communities, proteomic tracking, an approach for relative quantification of species-specific population dynamics of mixed cultures, was developed. Therefore, a bacterial mixed culture was analyzed during batch cultivations with and without addition of the antibiotic Ceftazidime. The community was composed of Burkholderia cepacia, Pseudomonas aeruginosa, and Staphylococcus aureus, pathogens causing infections in cystic fibrosis patients. Gel-based proteomics and mass spectrometry were used to obtain qualitative and quantitative proteomic data. During cultivation, P. aeruginosa became dominant within the mixed culture while S. aureus was inhibited in growth. Analysis of samples - taken along cultivation - revealed about 270 differentially expressed proteins. Some of those proteins are related to bacterial interactions, response to antibiotic treatment or metabolic shifts. For instance, the enzymes PhzS(flavin-containing monooxygenase), PhzD (phenazine biosynthesis protein), and PhzG2 (pyridoxamine 5'-phosphate oxidase) indicated the production of the antibiotic pigment pyocyanine by P. aeruginosa that is related to oxidative stress and therefore, might inhibit growth of S. aureus. Overall, the strategy applied not only allows species-specific tracking of the community composition but also provides valuable insights into the behavior of mixed cultures.     

Biodegradation of a dilute waste oil emulsion applied to soil

Summary The use of land treatment for disposal of a dilute waste oil emulsion generated by an aluminum rolling industry was investigated. Major components of the waste, identified by gas chromatography and mass spectrometry, were linear and branched (C₁₂–C₂₅) and fatty acid emulsifiers (primarily, isomers of oleic acid). Hexadecane and pristane were readily biodegraded in vitro when added to soil collected from the waste disposal site. Hydrocarbons and fatty acids extracted from the waste were similarly, biodegraded, however, the rate of decomposition may have depended on the history of waste applications to soil collected from the land treatment site. The apparent half-life of resolvable waste hydrocarbons and fatty acids was 9.5 days in soil which had received waste applications averaging 25.4l m^–2 wk^–1. In contrast, soil receiving either 50.8l m^–2 wk^–1 or no waste application during summer 1987 apparent exhibited half-lives of 28.1 and 60.3 days, respectively. Waste components were restricted to the upper 48 cm of the soil cores collected from the disposal site. Core samples also provided evidence for biodegradation of hydrocarbons and fatty acids as well as an accumulation of other compounds not readily resolvable by gas chromatographyPublished with the approval of the Director of the West Virginia University Agriculture and Forestry Experiment Station as Scientific Article # 2122.     

Isolation and characterization of a symbiotic cellulolytic mixed bacterial culture

Summary By using batch-culture enrichment techniques a mixed culture of two bacterial spe cies identified as Cellulomonas flavigena and Xanthomonas sp was isolated. The capacity of both bacteria to grow as pure cultures in a min eral medium with alkaline pretreated sugar cane bagasse or cellobiose was tested. C. flavigena as pure culture was able to grow on both substrates only when yeast extract or biotin and thiamine were added to the culture medium, while Xanthomonas sp. could not grow on sugar cane ba gasse, but assimilated cellobiose if yeast extract was supplied. However, both bacteria in mixed culture grew very well on both substrates and did not require any growth factor. It was concluded that the interaction was favourable to both species. The mixed culture had the capacity to degrade a number of different agricul tural wastes and to use them as the sole carbon and energy source for the production mainly of biomass. More than 80% of pineapple bagasse, without chemical pretreatment, was used up by the microbial system.     

Molecular diversity analysis and bacterial population dynamics of an adapted seawater microbiota during the degradation of Tunisian zarzatine oil

The indigenous microbiota of polluted coastal seawater in Tunisia was enriched by increasing the concentration of zarzatine crude oil. The resulting adapted microbiota was incubated with zarzatine crude oil as the only carbon and energy source. Crude oil biodegradation capacity and bacterial population dynamics of the microbiota were evaluated every week for 28 days (day 7, day 14, day 21, and day 28). Results show that the percentage of petroleum degradation was 23.9, 32.1, 65.3, and 77.8%, respectively. At day 28, non-aromatic and aromatic hydrocarbon degradation rates reached 92.6 and 68.7%, respectively. Bacterial composition of the adapted microflora was analysed by 16S rRNA gene cloning and sequencing, using total genomic DNA extracted from the adapted microflora at days 0, 7, 14, 21, and 28. Five clone libraries were constructed and a total of 430 sequences were generated and grouped into OTUs using the ARB software package. Phylogenetic analysis of the adapted microbiota shows the presence of four phylogenetic groups: Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Diversity indices show a clear decrease in bacterial diversity of the adapted microflora according to the incubation time. The Proteobacteria are the most predominant (>80%) at day 7, day 14 and day 21 but not at day 28 for which the microbiota was reduced to only one OTU affiliated with the genus Kocuria of the Actinobacteria. This study shows that the degradation of zarzatine crude oil components depends on the activity of a specialized and dynamic seawater consortium composed of different phylogenetic taxa depending on the substrate complexity.     

Integration of biosorption and biodegradation in a fed-batch mode for the enhanced crude oil remediation

Microbial bioremediation of oil-contaminated sites is still a challenge due to the slower rate and susceptibility of microbes to a higher concentration of oil. The poor bioavailability, hydrophobicity, and non-polar nature of oil slow down microbial biodegradation. In this study, biodegradation of crude oil is performed in fed-batch mode using an oil-degrader Pseudomonas aeruginosa to address the issue of substrate toxicity. The slower biodegradation was integrated with faster biosorption for effective oil remediation. Highly fibrous and porous sugarcane bagasse was surface modified with hydrophobic octyl groups to improve the surface-oil interactions. The microbe showed 2 folds enhanced oil degradation in the fed-batch study, which was further increased by 1·5 folds in the integrated biosorption coupled biodegradation approach. The biosorption-assisted biodegradation approach supported the microbial growth to 2 folds higher than the fed-batch study without biosorbent. The analysis of biosurfactant production indicated the 3 folds higher concentration in fed-batch modes as compared to batch study. In the integrated strategy, the concentration of contaminant (oil) reduces to quite a tolerable level to microbes, which improved effective metabolism and thus overall biodegradation. This study puts forward a promising strategy for improved degradation of hazardous hydrophobic contaminants in a sustainable, economic and eco-friendly manner.     

Lipase applications in oil hydrolysis with a case study on castor oil: a review

Lipase (triacylglycerol acylhydrolase) is a unique enzyme which can catalyze various types of reactions such as hydrolysis, esterification, alcoholysis etc. In particular, hydrolysis of vegetable oil with lipase as a catalyst is widely studied. Free lipase, lipase immobilized on suitable support, lipase encapsulated in a reverse micelle and lipase immobilized on a suitable membrane to be used in membrane reactor are the most common ways of employing lipase in oil hydrolysis. Castor oil is a unique vegetable oil as it contains high amounts (90%) of a hydroxy monounsaturated fatty acid named ricinoleic acid. This industrially important acid can be obtained by hydrolysis of castor oil. Different conventional hydrolysis processes have certain disadvantages which can be avoided by a lipase-catalyzed process. The degree of hydrolysis varies widely for different lipases depending on the operating range of process variables such as temperature, pH and enzyme loading. Immobilization of lipase on a suitable support can enhance hydrolysis by suppressing thermal inactivation and estolide formation. The presence of metal ions also affects lipase-catalyzed hydrolysis of castor oil. Even a particular ion has different effects on the activity of different lipases. Hydrophobic organic solvents perform better than hydrophilic solvents during the reaction. Sonication considerably increases hydrolysis in case of lipolase. The effects of additives on the same lipase vary with their types. Nonionic surfactants enhance hydrolysis whereas cationic and anionic surfactants decrease it. A single variable optimization method is used to obtain optimum conditions. In order to eliminate its disadvantages, a statistical optimization method is used in recent studies. Statistical optimization shows that interactions between any two of the following pH, enzyme concentration and buffer concentration become significant in presence of a nonionic surfactant named Span 80.     

灌草凋落物混合添加对原油污染土壤修复效果的影响

以陕北地区常见的10种灌草植物凋落物组成9种混合物,分别将单种或混合凋落物以2%的比例（质量分数）混入15 g/kg原油污染土壤,在室温（20-25℃）恒湿条件下进行150 d的室内模拟修复试验,分析凋落物混合添加对其修复油污土壤能力的影响。结果表明：（1）较之自然衰减,单种凋落物处理普遍显著提高了污染物降解率（原油降解率提高35%-85%）以及土壤硝态氮、有效磷和速效钾含量（提高0.36-56倍）,且多数处理显著提高了污染土壤中蔗糖酶、脲酶、碱性磷酸酶和脱氢酶的活性（提高0.4-6.8倍）。（2）白羊草+杠柳+狼牙刺、胡枝子+铁杆蒿、胡枝子+狗娃花+黄蒿或铁杆蒿+杠柳凋落物混合在促进原油及其组分降解时呈协同作用,使其降解率较单种处理再提高5%-28%,或较基于单种处理结果的预测值提高5%-17%,但其同时拮抗削弱凋落物对土壤速效氮（特别是硝态氮）的补充作用（较预测值降低6%-78%）及（或）酶活性（特别是蔗糖酶和脱氢酶）的刺激作用（较预测值降低14%-67%）。在实际使用中可通过上述混合强化凋落物对污染物的降解能力,但需配合其他修复手段改善污染土壤生化性质。白羊草+狼牙刺、铁杆蒿+狼牙刺、杠柳+狼牙刺凋落物混合使各种污染物降解率较单种处理或预测值显著降低,在使用时应对不同凋落物进行分离,以避免削弱其修复效果。（3）总体而言,添加多酚、黄酮、有机酸和磷含量高,碳含量和碳磷比低且化学多样性更高的混合凋落物更有利于降解原油污染物,但同时其含有的上述次生代谢物可能不利于土壤养分状况和酶活性的恢复。     

A Model for diffusion controlled bioavailability of crude oil components

Crude oil is a complex mixture ofseveral different structural classes of compoundsincluding alkanes, aromatics, heterocyclic polarcompounds, and asphaltenes. The rate and extent ofmicrobial degradation of crude oil depends on theinteraction between the physical and biochemicalproperties of the biodegradable compounds and theirinteractions with the non-biodegradable fraction. Inthis study we have systematically altered theconcentration of non-biodegradable material in thecrude oil and analyzed its impact on transport of thebiodegradable components of crude oil to themicroorganisms. We have also developed a mathematicalmodel that explains and accounts for the dependence ofbiodegradation of crude oil through a putativebioavailability parameter. Experimental resultsindicate that as the asphaltene concentration in oilincreases, the maximum oxygen uptake in respirometersdecreases. The mathematically fitted bioavailabilityparameter of degradable components of oil alsodecreases as the asphaltene concentration increases.     

微生物提高原油采出率的室内研究

微生物采油技术是一种提高原油采出率的卓有成效的方法。本文通过在实验室条件下模拟江苏油田韦4区块的储层情况,进行岩芯的驱替实验,对微生物采油技术在韦4区块的运用作了一些研究和评价,并得出应用结论,为该技术在现场的实验奠定了理论基础。     

Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol

Summary A continuous culture technique was used to optimize the medium composition and growth conditions of a mixed bacterial culture utilizing methanol. The improved medium resulted in satisfactory growth, high-yield coefficients and gave a product containing reduced polysaccharide concentrations. Optimal growth and biomass yields occurred at pH 6.8 a temperature of 37° C and dissolved oxygen at >20% saturation. The maximum growth rate was 0.58 h^–1 and maximum biomass yield 0.48 g g^–1. The protein content of the product ranged between 81%–83%, and nucleic acid content between 10%–12%, increasing with growth rate. The amino acid profile of the mixed culture product met and, in some cases, exceeded the UN Food and Agricultural Organization standard, indicating a good source of feed protein.Offprint requests to: A. S. Abu-Ruwaida