Phylogenetic diversity of bacterial communities associated with bioremediation of crude oil in microcosms

Bioremediation, mainly by indigenous bacteria, has been regarded as an effective way to clean up oil pollution after an oil spill. In order to obtain a systematic understanding of the succession of bacterial communities associated with oil bioremediation, sediments collected from the Penglai 19-3 oil platform were co-incubated with crude oil. Oil biodegradation was assessed on the basis of changes in oil composition monitored by GC–MS. Changes in the bacterial community structure were detected by two 16S rRNA gene based culture-independent methods, denaturing gradient gel electrophoresis (DGGE) and clone library. The results suggested that crude oil was rapidly degraded during the 30-day bioremediation period. Bacteria affiliated with the genus Pseudomonas dominated all three clone libraries. But dramatic changes were also detected in the process of biodegradation of crude oil. The “professional hydrocarbonocastic bacteria” (e.g., Alcanivorax) became abundant in the two samples during the bioremediation period. Meanwhile, δ-proteobacteria was only detected in the two samples. Information on the bacterial community revealed in this study will be useful in developing strategies for bioremediation of crude oil dispersed in the marine ecosystem.     

Use of hopane as a conservative biomarker for monitoring the bioremediation effectiveness of crude oil contaminating a sandy beach

Much of the variability inherent in crude oil bioremediation field studies can be eliminated by normalizing analyte concentrations to the concentration of a nonbiodegradable biomarker such as hopane. This was demonstrated with data from a field study in which crude oil was intentionally released onto experimental plots on the Delaware shoreline. Five independent replicates of three treatments were examined: no nutrient addition, addition of inorganic mineral nutrients alone, and nutrient addition plus indigenous oil-degrading microorganisms from the site. Samples collected biweekly were analyzed for the Most Probable Numbers (MPNs) of alkane and aromatic degraders and oil component analysis by GC/MS. The data were normalized to either the mass of sand that was extracted or to the concentration of hopane that was measured. Hopane normalization enabled detection of significant treatment differences in hydrocarbon biodegradation that were not detected when the data were normalized to sand mass. First-order loss rates for the hopane-normalized data were lower than those for the sand-normalized data because hopane normalization accounts only for loss due to biodegradation whereas sand normalization includes all loss mechanisms. Plots amended with nutrients alone and nutrients plus the inoculum showed enhanced removal of hydrocarbons compared to unamended control plots. However, no differences were detected between the nutrient-amended plots and the nutrient/inoculum-amended plots. Received 06 November 1995/ Accepted in revised form 26 June 1996     

Hydrocarbon bioremediation of a mineral-base contaminated waste from crude oil extraction by indigenous bacteria

The susceptibility to bioremediation of the hydrocarbons contained in a waste from crude oil extraction was examined. Laboratory scale batch reactors were inoculated with indigenous bacteria and biodegradation was followed for 45 days. The total hydrocarbon content was reduced to 70% of its initial value at the end of the experiments. Saturated and aromatic hydrocarbons were the most readily degraded fractions with, respectively, 70% and 60% of the fraction remaining at the end of the experiment. A minor degradation was observed in the resins fraction (20%), whereas the asphaltenes fraction remained almost constant.The substrate preferences of the natural population towards various fractions of the crude oil were determined by both the length of the lag phase and the slope of the exponential growth in a mineral salt-base medium containing either of the different hydrocarbon fraction as the sole source of carbon. The highest consumption rate for every fraction during the time course experiments was in agreement with the shortest lag phase and the greatest exponential growth slope in the corresponding selective media, indicating changes in the population composition.     

Towards efficient crude oil degradation by a mixed bacterial consortium

A laboratory study was undertaken to assess the optimal conditions for biodegradation of Bombay High (BH) crude oil. Among 130 oil degrading bacterial cultures isolated from oil contaminated soil samples, Micrococcus sp. GS2-22, Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73, Bacillus sp. DS6-86 and Pseudomonas sp. DS10-129 were selected for the study based on the efficiency of crude oil utilisation. A mixed bacterial consortium prepared using the above strains was also used. Individual bacterial cultures showed less growth and degradation than did the mixed bacterial consortium. At 1% crude oil concentration, the mixed bacterial consortium degraded a maximum of 78% of BH crude oil. This was followed by 66% by Pseudomonas sp. DS10-129, 59% by Bacillus sp. DS6-86, 49% by Micrococcus sp. GS2-22, 43% by Corynebacterium sp. GS5-66 and 41% by Flavobacterium sp. DS5-73. The percentage of degradation by the mixed bacterial consortium decreased from 78% to 52% as the concentration of crude oil was increased from 1% to 10%. Temperature of 30 degrees C and pH 7.5 were found to be optima for maximum biodegradation.     

Effectiveness of bioremediation of crude oil contaminated subantarctic intertidal sediment: the microbial response

A field study was initiated in February 1996 in a remote sandy beach of The Grande Terre (Kerguelen Archipelago, 69° 42° E, 49° 19° S) with the objective of determining the long-term effects of some bioremediation agents on the biodegradation rate and the toxicity of oil residues under severe subantarctic conditions. A series of 10 experimental plots were settled firmly into sediment. Each plot received 2L of Arabian light crude oil and some of them were treated with bioremediation agents: slow release fertilizer Inipol EAP-22 (Elf Atochem) or fish composts. Plots were sampled on a regular basis over a 3-year period. A two-order of magnitude increase of saprophytic and hydrocarbon-utilizing microorganisms occurred during the first month of the experiment in all treated enclosures, but no clear differences appeared between the plots. Very high microbial populations were present during the experiment. Biodegradation within treated spots was faster than within the untreated ones and appeared almost complete after 6 months as indicated by the degradation index of aliphatic hydrocarbons within all plots. The analysis of interstitial water collected below the oily residues presented no toxicity. However, a high toxicity signal, using Microtox solid phase, appeared for all oiled sand samples with a noticeable reduction with time even if the toxicity signal remained present and strong after 311 days of oil exposition. As a conclusion, it is clear that the microbial response was rapid and efficient in spite of the severe weather conditions, and the rate of degradation was improved in presence of bioremediation agents. However, the remaining residues had a relatively high toxicity.     

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     

A folding artificial substratum sampler for use in standing water

A new easily retrieved folding artificial substratum sampler is described and its performance compared with that of trays in Cow Green Reservoir at a depth of 15–18 m.     

Assessment of crude oil degradation efficiency of newly isolated actinobacteria reveals untapped bioremediation potentials

Bioremediation is gaining favorable attention as a more economical and environmentally friendly technique for the remediation of crude oil hydrocarbons. This makes the search for crude oil–degrading microbes very crucial. In this study, the isolation and identification of actinobacteria in soil samples from a selected crude oil spill site were carried out. Eighteen isolates from different soil depths (20–120 cm) were screened for their ability to grow on crude oil–based medium (COBM). Actinomyces naeslundii, Actinomyces viscosus, Actinomyces israelii, Actinomyces meyeri, and Nocardia formicae from a 20 cm soil depth exhibited higher growth profiles on COBM than on glucose-based medium (GBM). A. viscosus and A. isrealii exhibited 5- and 3-fold increase in growth over GBM and were selected for biodegradation studies. Growth kinetics and residual crude oil were used to measure the degradation efficiency of A. viscosus and A. israeli over varying crude oil concentrations. Surprisingly, A. viscosus and A. isrealii achieved 98% degradation of 10 g/L crude oil in 12 days and 97% degradation of 30, 50, and 75 g/L in 16 and 18 days, respectively. Specific activity of total peroxidase was assayed over the biodegradation period. Peroxidase activity increased with degradation efficiency of A. viscosus and A. isrealii, suggesting that peroxidases play a key role in the crude oil biodegradation process. The unique tolerance exhibited by A. viscosus and A. israelii to crude oil and high crude oil degradation efficiencies indicate their promising potential for bioremediation applications.     

Efficacy of intervention strategies for bioremediation of crude oil in marine systems and effects on indigenous hydrocarbonoclastic bacteria

There is little information on how different strategies for the bioremediation of marine oil spills influence the key indigenous hydrocarbon-degrading bacteria (hydrocarbonoclastic bacteria, HCB), and hence their remediation efficacy. Therefore, we have used quantitative polymerase chain reaction to analyse changes in concentrations of HCB in response to intervention strategies applied to experimental microcosms. Biostimulation with nutrients (N and P) produced no measurable increase in either biodegradation or concentration of HCB within the first 5 days, but after 15 days there was a significant increase (29%; P < 0.05) in degradation of n-alkanes, and an increase of one order of magnitude in concentration of Thalassolituus (to 10(7) cells ml(-1)). Rhamnolipid bioemulsifier additions alone had little effect on biodegradation, but, in combination with nutrient additions, provoked a significant increase: 59% (P < 0.05) more n-alkane degradation by 5 days than was achieved with nutrient additions alone. The very low Alcanivorax cell concentrations in the microcosms were hardly influenced by addition of nutrients or bioemulsifier, but strongly increased after their combined addition, reflecting the synergistic action of the two types of biostimulatory agents. Bioaugmentation with Thalassolituus positively influenced hydrocarbon degradation only during the initial 5 days and only of the n-alkane fraction. Bioaugmentation with Alcanivorax was clearly much more effective, resulting in 73% greater degradation of n-alkanes, 59% of branched alkanes, and 28% of polynuclear aromatic hydrocarbons, in the first 5 days than that obtained through nutrient addition alone (P < 0.01). Enhanced degradation due to augmentation with Alcanivorax continued throughout the 30-day period of the experiment. In addition to providing insight into the factors limiting oil biodegradation over time, and the competition and synergism between HCB, these results add weight to the use of bioaugmentation in oil pollution mitigation strategies.     

一种净化水质的复合生物修复系统

在实验室内利用人工模拟方法,选择水蕹菜(Ipomoea aquatica)、泥鳅(Misgurus anguillicaudatus)、沼泽红假单胞菌(Rhodopseudomonas palustris)为工程物种,构建一套水生经济植物-水生动物-微生物复合生物修复系统进行污水修复,研究该系统中动植物生物量及水质指标的变化。结果表明,在23d的实验周期中,水体铵态氮(NH4+-N)下降96.5%,硝态氮(NO3--N)下降82.2%,总磷(TP)下降53.2%,化学需氧量(CODMn)下降24.5%。水蕹菜平均增重31.2%,泥鳅平均增重6.1%。这种复合的生物修复模式具有较好的经济效益与环境效益。     

Influence of crude oil on changes of bacterial communities in Arctic sea-ice

The danger of a petroleum hydrocarbon spillage in the polar, ice-covered regions is increasing due to oil exploration in Arctic offshore areas and a growing interest in using the Northern Sea Route (NSR) as an alternative transportation route for Arctic oil and gas. However, little is known about the potential impact of accidental oil spills on this environment. We investigated the impact of crude oil on microbial community composition in six different Arctic sea-ice samples incubated with crude oil at 1 degrees C in microcosms for one year. Alterations in the composition of bacterial communities were analyzed with the culture-independent molecular methods DGGE (denaturing gradient gel electrophoresis) and FISH (fluorescence in situ hybridization). DGGE, FISH and cultivation methods revealed a strong shift in community composition toward the gamma-proteobacteria in sea-ice and melt pool samples incubated with crude oil. Marinobacter spp., Shewanella spp. and Pseudomonas spp. were the predominant phylotypes in the oil-treated microcosms. The ability of indigenous sea-ice bacteria to degrade hydrocarbons at low temperature (1 degrees C) was tested using four representative strains cultivated from sea-ice enriched with crude oil. [14C]Hexadecane was degraded by the sea-ice isolates at 20-50% capacity of the mesophilic type strain Marinobacter hydrocarbonoclasticus, a known hydrocarbon degrader, incubated at 22 degrees C.     

Robust hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced oil spill bioremediation

Degradation of oil on beaches is, in general, limited by the supply of inorganic nutrients. In order to obtain a more systematic understanding of the effects of nutrient addition on oil spill bioremediation, beach sediment microcosms contaminated with oil were treated with different levels of inorganic nutrients. Oil biodegradation was assessed respirometrically and on the basis of changes in oil composition. Bacterial communities were compared by numerical analysis of denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Nutrient amendment over a wide range of concentrations significantly improved oil degradation, confirming that N and P limited degradation over the concentration range tested. However, the extent and rate of oil degradation were similar for all microcosms, indicating that, in this experiment, it was the addition of inorganic nutrients rather than the precise amount that was most important operationally. Very different microbial communities were selected in all of the microcosms. Similarities between DGGE profiles of replicate samples from a single microcosm were high (95% +/- 5%), but similarities between DGGE profiles from replicate microcosms receiving the same level of inorganic nutrients (68% +/- 5%) were not significantly higher than those between microcosms subjected to different nutrient amendments (63% +/- 7%). Therefore, it is apparent that the different communities selected cannot be attributed to the level of inorganic nutrients present in different microcosms. Bioremediation treatments dramatically reduced the diversity of the bacterial community. The decrease in diversity could be accounted for by a strong selection for bacteria belonging to the alkane-degrading Alcanivorax/Fundibacter group. On the basis of Shannon-Weaver indices, rapid recovery of the bacterial community diversity to preoiling levels of diversity occurred. However, although the overall diversity was similar, there were considerable qualitative differences in the community structure before and after the bioremediation treatments.     

Use of an internal standard in monitoring the bacterial degradation of crude oil.

Hexachloroethane is nonvolatile, insoluble in water, and apparently not toxic to or metabolized by bacteria. Its addition to cultures growing at the expense of crude oil thus provides an internal standard against which the rate of degradation of individual crude oil components can be conveniently and reproducibly measured.     

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.     

Selection of non-conventional yeasts and their use in immobilized form for the bioremediation of olive oil mill wastewaters

The yeast population dynamics in olive wastewaters (OMW), sampled in five mills from Salento (Apulia, Southern Italy), were investigated. Three hundred yeasts were isolated in five industrial mills and identified by molecular analysis. Strains belonging to Geotrichum, Saccharomyces, Pichia, Rhodotorula and Candida were detected. Five G. candidum strains were able to grow in OMW as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One G. candidum isolate was selected for whole-cell immobilization in calcium alginate gel. The COD and phenolic reduction obtained with immobilized cells showed a 2.2- and 2-fold increase compared to the removal obtained with free cells, respectively. The immobilization system enhanced yeast oxidative activity by avoiding the presence of microbial protease in treated OMW. To our knowledge, this is the first report on G. candidum whole-cell immobilization for OMW bioremediation.     

Enhanced bioremediation of oil spills in the sea

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A bioengineering system for in situ bioremediation of contaminated groundwater

Much of the past and current focus of bioremediation has been on laboratory studies of microbial processes. By necessity, early studies have ignored important field properties, parameters, and processes that control the ultimate success of in situ bioremediation of contaminated groundwater. This paper presents a bioengineering systems approach that examines the impact of some of these field variables on common bioremediation practices. Using simple systems, the niche of biostimulation is shown to be aquifers with high contaminant sorption. A novel gas-phase biostimulation filter and a novel resting-state bioaugmentation/biofilter approach which show promise for effective field implementation are discussed. Received 08 December 1995/ Accepted in revised form 30 July 1996