Indigenous Hydrocarbon-degrading Bacteria Research Articles

Interaction between indigenous hydrocarbon-degrading bacteria in reconstituted mixtures for remediation of weathered oil in soil

It has been demonstrated that biostimulation is necessary to investigate the interactions between indigenous bacteria and establish an approach for the bioremediation of soils contaminated with weathered oil. This was achieved by adjusting the carbon (C)/nitrogen (N)/phosphorus (P) ratio to 100/10/1 combined with the application of 0.8 mL/kg Tween-80. In addition, three indigenous bacteria isolated from the same soil were introduced solely or combined concomitantly with stimulation. Removal of n-alkanes and the ratios of n-heptadecane to pristane and n-octadecane to phytane were taken to indicate their biodegradation performance over a period of 16 weeks. One strain of Pseudomonas aeruginosa D7S1 improved the efficiency of the process of stimulation. However, another Pseudomonas aeruginosa, D5D1, inhibited the overall process when combined with other bacteria. One strain of Bacillus licheniformis D1D2 did not affect the process significantly. The Fourier transform infrared analysis of the residual hydrocarbons supported the conclusions pertaining to the biodegradation processes when probing the modifications in densities and stretching. The indigenous bacteria cannot mutually benefit from their metabolisms for bioremediation if augmented artificially. However, the strain Pseudomonas. aeruginosa D7S1 was able to perform better alone than in a consortium of indigenous bacteria.

Microbiome profile of soil and rhizosphere plants growing in traditional oil mining land in Wonocolo, Bojonegoro, Indonesia

Traditional oil mining poses negative effects on the environment through pollution with crude oil. One of the traditional mining sites in Wonocolo, Bojonegoro, Indonesia was reported to contaminate the surrounding area with a high level of crude oil. Therefore, this study aims to examine the microbiome profiles of contaminated soil and the rhizosphere of naturalized plants growing at the sites. It was conducted in Wonocolo, Bojonegoro to obtain an insight into the possible remediation efforts of using indigenous hydrocarbon-degrading bacteria and naturalized plants as in situ remediation agents. The results showed that the soil located close to the oil well-contained a high level of crude oil at 24.8%, and exhibited a distinct microbiome profile compared to those located further which had lower crude oil contamination of 14.15, 10.89, and 4.9%. Soil with the highest level of crude oil contamination had a comparatively higher relative abundance of assA, an anaerobic alkene-degrading gene. Meanwhile, the rhizosphere of the two naturalized plants, Muntingia calabura, and Pennisetum purpureum, exhibited indifferent microbiome profiles compared to the soil. They were found to contain less abundant hydrocarbon-degrading genes, such as C230, PAH-RHD-GP, nahAc, assA, and alkB suggesting that these naturalized plants might not be a suitable tool for in-situ remediation.

Bioaugmentation with zeolite-immobilized bacterial consortium OPK results in a bacterial community shift and enhances the bioremediation of crude oil-polluted marine sandy soil microcosms

A pyrene-degrading consortium OPK containing Mycolicibacterium strains PO1 and PO2, Novosphingobium pentaromativorans PY1 and Bacillus subtilis FW1 effectively biodegraded medium- and long-chain alkanes as well as mixed hydrocarbons in crude oil. The detection of alkB and CYP153 genes in the genome of OPK members supports its phenotypic ability to effectively degrade a broad range of saturated hydrocarbons in crude oil. Zeolite-immobilized OPK was developed as a ready-to-use bioproduct and it exhibited 74% removal of 1000 mg L−1 crude oil within 96 h in sterilized seawater without nutrient supplementation and maintained high crude oil-removal activity under a broad range of pH values (5.0–9.0), temperatures (30–40 °C) and salinities (20–60‰). In addition, the immobilized OPK retained a high crude oil removal efficacy in semicontinuous experiments and showed reusability for at least 5 cycles. Remarkably, bioaugmentation with zeolite-immobilized OPK in sandy soil microcosms significantly increased crude oil (10,000 mg kg−1 soil) removal from 45% to 80.67% within 21 days compared to biostimulation and natural attenuation. Moreover, bioaugmentation with exogenous immobilized OPK stimulated an increase in the relative abundances of Alcanivorax genus, indigenous hydrocarbon-degrading bacteria, which in turn enhanced removal efficiency of crude oil contamination from sandy soil microcosms. The results indicate positive interactions between the bioaugmented immobilized consortium, harboring Mycolicibacterium as a key player, and indigenous Alcanivorax, which exhibited crucial functions for improving crude oil removal efficacy. The knowledge obtained forms an important basis for further synthesis and handling of a promising bio-based product for enhancing the in situ bioremediation of crude oil-polluted marine environments.

Study of bacterial interactions in reconstituted hydrocarbon-degrading bacterial consortia from a local collection, for the bioremediation of weathered oily-soils

To enhance the process of bacterial remediation of weathered hydrocarbons, the area of Dukhan, Qatar, was considered as a model for weathering processes. Self-purification by indigenous hydrocarbon-degrading bacteria showed low performance. Biostimulation/seeding using one or another of the indigenous bacteria improved the performance. Symbiosis between three strains dominating the soil; Bacillus sorensis D11, Bacillus cereus D12, and Pseudomonas stutzeri D13, was highly performant for removal of total petroleum hydrocarbons in the weathered soil. D11, the most sensitive, showed the highest performance when mixed with D12 or D13. D12, less performant than D11, was more active on diesel range organics (DRO: C10-C28), similar to D11. D13 showed a metabolic behavior close to commensal and co-metabolic ones. It was more active on hydrocarbons above C29. Combination of the three strains conducted to the removal of at least 80% of C10-C35 organics in the extract at concentrations of 31.1 mg/g TPH-DRO.

Functional Gene Diversity of Selected Indigenous Hydrocarbon-Degrading Bacteria in Aged Crude Oil.

Crude oil pollution has consistently deteriorated all environmental compartments through the cycle of activities of the oil and gas industries. However, there is a growing need to identify microbes with catabolic potentials to degrade these pollutants. This research was conducted to identify bacteria with functional degradative genes. A crude oil-polluted soil sample was obtained from an aged spill site at Imo River, Ebubu, Komkom community, Nigeria. Bacteria isolates were obtained and screened for hydrocarbon degradation potential by turbidometry assay. Plasmid and chromosomal DNA of the potential degraders were further screened for the presence of selected catabolic genes (C230, Alma, Alkb, nahAC, and PAHRHD(GP)) and identified by molecular typing. Sixteen (16) out of the fifty (50) isolates obtained showed biodegradation activity in a liquid broth medium at varying levels. Bacillus cereus showed highest potential for this assay with an optical density of 2.450 @ 600 nm wavelength. Diverse catabolic genes resident in plasmids and chromosomes of the isolates and, in some cases, both plasmid and chromosomes of the same organism were observed. The C230 gene was resident in >50% of the microbial population tested, while other genes occurred in lower proportions with the least observed in nahAC and PAHRHD. These organisms can serve as potential bioremediation agents.

Isolation, identification and diesel-oil biodegradation capacities of indigenous hydrocarbon-degrading strains of Cellulosimicrobium cellulans and Acinetobacter baumannii from tarball at Terengganu beach, Malaysia

In this study, we isolated two indigenous hydrocarbon-degrading bacteria from tarball found in Rhu Sepuluh beach, Terengganu, Malaysia. These bacteria were identified based on their physiological characteristic and 16S rRNA gene sequence analysis, and they showed 99% similarity with Cellulosimicrobium cellulans DSM 43879 and Acinetobacter baumannii ATCC 19606 respectively. Their hydrocarbon-degrading capabilities were tested using diesel-oil as sole carbon source. Results analysed using GC–MS, showed diesel-oil alkanes were degraded an average 64.4% by C. cellulans and 58.1% by A. baumannii with medium optical density reaching 0.967 (C. cellulans) and 1.515 (A. baumannii) in minimal salt media at 32°C for 10days. Individual diesel-oil alkanes were degraded between 10%–95.4% by C. cellulans and 0.2%–95.9% by A. baumannii. Both strains utilized diesel-oil for growth. The study suggests both strains are part of indigenous hydrocarbon-degrading bacteria in tarball with potential for bioremediation of oil-polluted marine environment.

Selection and characterization of indigenous hydrocarbon-degrading bacteria from tourist ports in the Mediterranean Sea Basin

Pollution by petroleum hydrocarbons is one of the major environmental problems in ports and it is mainly associated with ship/boat traffic and related facilities. Ports are not closed systems and their pollution may impact adjacent coastal areas. Hydrocarbon degraders and particularly the obligate hydrocarbonoclastic bacteria carry out a fundamental and global activity in biological removal of hydrocarbons in marine habitats. This studywas carried out withinMAPMED, amultidisciplinary project aimed to improve the environmental sustainability of tourist ports in the Mediterranean Sea with regard to monitoring and reduction of hydrocarbon pollution. Three tourist ports were selected as case study sites: Cagliari (Italy), El Kantaoui (Tunisia), and Heraklion (Greece). The degradation potential of the autochthonous bacterial communities was evaluated enumerating heterotrophs and hydrocarbon degraders by MPN in the surface seawater. Heterotrophs were significantly more abundant in seawater from Cagliari port as compared to El Kantaoui and Heraklion ports. On the contrary, higher viable titles of dieseland phenanthrene-degraders were found in seawater from El Kantaoui port compared to the other two areas. Hydrocarbon-degrading bacteria were isolated and characterized regarding their phylogenetic position and catabolic abilities. The hydrocarbon degradation activities were evaluated by GC-MS in aerobic batch reactors on diesel as carbon source. The majority of degraders from Cagliari were assigned to Pseudomonas whereas strains from El Kantaoui and Heraklion were assigned to Alcanivorax and Marinobacter. The selection of the most appropriate methodologies for the eco-efficient remediation of petroleum-hydrocarbon contamination of selected sites is currently in progress.

Distribution of Hydrocarbon-Degrading Bacteria in the Soil Environment and Their Contribution to Bioremediation

A real-time PCR quantification method for indigenous hydrocarbon-degrading bacteria (HDB) carrying the alkB gene in the soil environment was developed to investigate their distribution in soil. The detection limit of indigenous HDB by the method was 1 × 10(6) cells/g-soil. The indigenous HDB were widely distributed throughout the soil environment and ranged from 3.7 × 10(7) to 5.0 × 10(8) cells/g-soil, and the ratio to total bacteria was 0.1-4.3%. The dynamics of total bacteria, indigenous HDB, and Rhodococcus erythropolis NDKK6 (carrying alkB R2) during bioremediation were analyzed. During bioremediation with an inorganic nutrient treatment, the numbers of these bacteria were slightly increased. The numbers of HDB (both indigenous bacteria and strain NDKK6) were gradually decreased from the middle stage of bioremediation. Meanwhile, the numbers of these bacteria were highly increased and were maintained during bioremediation with an organic nutrient. The organic treatment led to activation of not only the soil bacteria but also the HDB, so an efficient bioremediation was carried out.

Deep‐sea bacteria enriched by oil and dispersant from the Deepwater Horizon spill

The Deepwater Horizon oil spill resulted in a massive influx of hydrocarbons into the Gulf of Mexico (the Gulf). To better understand the fate of the oil, we enriched and isolated indigenous hydrocarbon-degrading bacteria from deep, uncontaminated waters from the Gulf with oil (Macondo MC252) and dispersant used during the spill (COREXIT 9500). During 20 days of incubation at 5°C, CO(2) evolution, hydrocarbon concentrations and the microbial community composition were determined. Approximately 60% to 25% of the dissolved oil with or without COREXIT, respectively, was degraded, in addition to some hydrocarbons in the COREXIT. FeCl(2) addition initially increased respiration rates, but not the total amount of hydrocarbons degraded. 16S rRNA gene sequencing revealed a succession in the microbial community over time, with an increase in abundance of Colwellia and Oceanospirillales during the incubations. Flocs formed during incubations with oil and/or COREXIT in the absence of FeCl(2) . Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy revealed that the flocs were comprised of oil, carbohydrates and biomass. Colwellia were the dominant bacteria in the flocs. Colwellia sp. strain RC25 was isolated from one of the enrichments and confirmed to rapidly degrade high amounts (approximately 75%) of the MC252 oil at 5°C. Together these data highlight several features that provide Colwellia with the capacity to degrade oil in cold, deep marine habitats, including aggregation together with oil droplets into flocs and hydrocarbon degradation ability.

Dynamics of bacterial community exposed to hydrocarbons and oleophilic fertilizer in high-Arctic intertidal beach

Exposure of pristine microbial environments to hydrocarbon contamination stimulates growth of the initially small fraction of indigenous hydrocarbon-degrading bacteria. Custom-made oleophilic fertilizers have been demonstrated to promote oil bioremediation by boosting this proliferation. In the present study, the temporal dynamics of the bacterial community structure and the individual influences of hydrocarbons and an oleophilic fertilizer in shaping the community structure was explored during a 78 days bioremediation experiment in a high-Arctic intertidal beach environment. A combination of cultivation-independent 16S rRNA gene length-heterogeneity polymerase chain reaction (LH-PCR) profiling and identification of hydrocarbon-degrading isolates based on partial 16S rRNA gene sequences was employed. LH-PCR community profiles in the fertilizer alone and fertilized kerosene plots were largely indistinguishable throughout the experimental period, while kerosene alone plots showed a markedly different composition of dominant groups. This pointed to the fertilizer as the more decisive factor in shaping the community structure. Most prominent LH-PCR fragments which emerged after kerosene or fertilizer addition could be provisionally assigned to bacterial taxa through coinciding LH-PCR fragment lengths with hydrocarbon-degrading isolates obtained from the same type of experimental units. However, a few quantitatively significant LH-PCR groups had no counterparts among the cultivated bacteria. One of these was affiliated to a hitherto unspeciated subgroup within the Alkanindiges/Acinetobacter clade of Moraxellaceae by a 16S rRNA gene cloning approach.

Bioremediation of crude oil-contaminated soil: Comparison of different biostimulation and bioaugmentation treatments

Biostimulation with inorganic fertilizer and bioaugmentation with hydrocarbon utilizing indigenous bacteria were employed as remedial options for 12 weeks in a crude oil-contaminated soil. To promote oil removal, biocarrier for immobilization of indigenous hydrocarbon-degrading bacteria was developed using peanut hull powder. Biodegradation was enhanced with free-living bacterial culture and biocarrier with a total petroleum hydrocarbon removal ranging from 26% to 61% after a 12-week treatment. Oil removal was also enhanced when peanut hull powder was only used as a bulking agent, which accelerated the mass transfer rate of water, oxygen, nutrients and hydrocarbons, and provided nutrition for the microflora. Dehydrogenase activity in soil was remarkably enhanced by the application of carrier material. Metabolites of polycyclic aromatic hydrocarbons were identified by Fourier transform ion cyclotron resonance mass spectrometry.

Biodegradative potential and characterization of bioemulsifiers of marine bacteria isolated from samples of seawater, sediment and fuel extracted at 4000 m of depth (Prestige wreck)

Seawater, sediments and fuel samples extracted from a small area of the Prestige wreck (4000 m deep) were studied to check the microbial activity of the area, the occurrence of indigenous hydrocarbon-degrading bacteria and the presence of bacteria able to produce bioemulsifiers. Twenty-one strains with the capacity to degrade hydrocarbons and/or produce useful bioemulsifiers were selected. Phylogenetic affiliation of these isolates placed them in the genus Bacillus (8 strains), Pseudomonas (3 strains), Halomonas (4 strains), Pseudoalteromonas (1 strain), Brevibacterium (2 strains), and Marinobacter (1 strain) and 2 strains were identified as marine bacterium. Hydrocarbon degradation was established by determining the amount of alkanes and polycyclic aromatic hydrocarbons by GC/MS analyses. In this study, Pseudomonas, Bacillus and Brevibacterium strains were the most efficient hydrocarbon-degrading marine bacteria. In contrast, Halomonas strains produced the highest amount of efficient biopolymers with emulsifying activity. The yield, chemical composition and functional properties of these bioemulsifiers were affected by the incubation time required for production. In addition, supplementation of the hydrocarbon culture medium with bioemulsifiers (S22-BE, S24-BE and AD2-BE) clearly stimulated the growth of strains S25, S28 and S29 and enhanced the ability of these strains to biodegrade alkanes, alkenes and aromatic compounds.

Effects of nutrient and temperature on degradation of petroleum hydrocarbons in sub-Antarctic coastal seawater

In an attempt to evaluate the potential of petroleum bioremediation at high latitudes environments, microcosm studies using Antarctic coastal seawater contaminated with diesel or crude oil were conducted in Kerguelen Archipelago (49°22′S, 70°12′E). Microcosms were incubated at three different temperatures (4, 10 and 20°C). During experiments, changes observed in microbial assemblages (total direct count, heterotrophic cultivable microorganisms and hydrocarbon-degrading microorganisms) were generally similar for all incubation temperatures, but chemical data showed only some slight changes in biodegradation indices [Σ(C12–C20)/Σ(C21–C32) and C17/pristane]. The complete data set provided strong evidence of the presence of indigenous hydrocarbon-degrading bacteria in Antarctic seawater and their high potential for hydrocarbon bioremediation. The rate of oil degradation could be increased by the addition of a commercial fertilizer, but water temperature had little effects on biodegradation efficiency which is in conflict with the typical temperature-related assumption predicting 50% rate reduction when temperature is reduced by 10°C. Global warming of Antarctic seawater should not increase significantly the rate of oil biodegradation in these remote regions.

Porous biocarrier-enhanced biodegradation of crude oil contaminated soil

Soil contamination with crude oil from petrochemicals and oil exploitation is an important worldwide issue. Comparing available remediation techniques, bioremediation is widely considered to be a cost-effective choice; however, slow degradation of crude oil is a common problem due to the low numbers of bacteria capable of degrading petroleum hydrocarbons and the low bioavailability of contaminants in soil. To promote crude oil removal, biocarrier for immobilization of indigenous hydrocarbon-degrading bacteria was developed using porous materials such as activated carbon and zeolite. Microbial biomass reached 10 10 cells g −1 on activated carbon and 10 6 cells g −1 on zeolite. Total microbial and dehydrogenase activities were approximately 12 times and 3 times higher, respectively, in activated carbon than in zeolite. High microbial colonization by spherical and rod shapes were observed for the 5–20 μm thick biofilm on the outer surface of both biocarriers using electronic microscopy. Based on batch-scale experiments containing free-living bacterial cultures and activated carbon biocarrier into crude oil contaminated soil, biocarrier enhanced the biodegradation of crude oil, with 48.89% removal, compared to natural attenuation with 13.0% removal, biostimulation (nutrient supplement only) with 26.3% removal, and bioaugmentation (free-living bacteria) with 37.4% removal. In addition, the biocarrier increased the bacterial population to 10 8 cells g −1 dry soil and total microbial activity to 3.5 A 490. A hypothesis model was proposed to explain the mechanism: the biocarrier improved the oxygen, nutrient mass transfer and water holding capacity of the soil, which were the limiting factors for biodegradation of non-aqueous phase liquid (NAPL) contaminants such as crude oil in soil. Scientific relevance This study explored the role of biocarrier in enhancing biodegradation of hydrophobic contaminants such as crude oil, and discussed the function of biocarrier in improving oxygen mass transfer and soil water holding capacity, etc.

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.

Impact of crude oil on bacteriocenosis of the digestive tract of mollusks.

In this study the effect of crude oil on intestinal bacterial populations of the mollusk Viviparus contactus was investigated. The addition of crude oil into an environment of mollusks induced no clear changes in the saprophytic, amylolytic, and total coliform bacterial counts in the digestive tract of the mollusk. After 10 days of contamination, the saprophytic, amylolytic, and total coliform bacterial numbers were of the same order of magnitude as the initial numbers. Significant numbers of indigenous hydrocarbon-degrading bacteria were observed in the intestinal tracts of mollusks before contamination with crude oil. Introduction of crude oil into the mollusk environment resulted in an increase of 2 orders of magnitude in the number of hydrocarbon-degrading bacteria in the digestive tract. Therefore, measuring the hydrocarbon-degrading bacterial populations in the digestive tracts of hydrobionts can be considered an important component of contaminated-site assessment studies.

Field observations on the variability of crude oil impact on indigenous hydrocarbon-degrading bacteria from sub-Antarctic intertidal sediments

Oil pollution of the oceans has been a problem ever since man began to use fossil fuels. Biodegradation by naturally occurring populations of micro-organisms is a major mechanism for the removal of petroleum from the environment. To examine the effects of crude oil pollution on intertidal bacteria, we repeated the same contamination experiments on nine different sub-Antarctic intertidal beaches using specifically built enclosures (PVC pipe, 15 cm in inner diameter and 30 cm in height). Despite the pristine environmental conditions, significant numbers of indigenous hydrocarbon-degrading bacteria were observed in all the studied beaches. Introduction of oil into these previously oil-free environments resulted in several orders of magnitude of increase in hydrocarbon-degrading micro-organisms within a few days in some of the studied sites but has no obvious effects on two others. The physical environment of the bacterial assemblage seems to play a major role in the biodegradation capacities. After 3 months of contamination, both remaining oil concentrations and biodegradation indexes differ strongly between the different stations. Thus, chemical and biological parameters reveal a strong heterogeneity of biodegradation capacities between the different sites.

Effect of Dispersants on Oil Biodegradation Under Simulated Marine Conditions

ABSTRACT A study was undertaken on the dispersion, microbial colonisation and biodegradation of chemically-dispersed weathered Forties crude oil under simulated marine conditions in laboratory microcosms. The measurements of droplet size, number and microbial colonisation were made using new techniques developed by the project team. Rapid growth of indigenous micro-organisms capable of degrading both crude oil and dispersants was observed in the presence of chemically-dispersed oil. These organisms colonised the dispersed oil and biodegraded the aliphatic and aromatic hydrocarbons. These processes was stimulated by the addition of inorganic nutrients. Some colonised droplets agglomerated into neutrally-buoyant “clusters” (100 µm- 2 mm diameter) consisting of oil, bacteria, protozoa, and nematodes. After substantial hydrocarbon biodegradation these clusters sank to the bottom of the microcosms. No biodegradation or cluster formation was noted in “killed” controls in which biological activity had been inhibited. Different dispersants promoted microbial growth to differing extents. These results suggest that the addition of dispersants can increase the rate of oil biodegradation under natural conditions by promoting the growth of indigenous hydrocarbon-degrading bacteria, as well as increasing the surface area of oil available for microbial colonisation.