Bacterial Succession in a Petroleum Land Treatment Unit

Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C₁₀ to C₃₂ range. The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts. Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation. TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project. A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation. Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study. The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased. For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones. Two phylotypes associated with Flavobacterium and Pseudomonas were dominant in TRF patterns from samples during rapid TPH degradation. After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while Flavobacterium and Pseudomonas phylotypes decreased in abundance. These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.     

Do plants modulate biomass allocation in response to petroleum pollution?

Biomass allocation is an important plant trait that responds plastically to environmental heterogeneities. However, the effects on this trait of pollutants owing to human activities remain largely unknown. In this study, we investigated the response of biomass allocation of Phragmites australis to petroleum pollution by a ¹³CO₂ pulse-labelling technique. Our data show that plant biomass significantly decreased under petroleum pollution, but the root–shoot ratio for both plant biomass and ¹³C increased with increasing petroleum concentration, suggesting that plants could increase biomass allocation to roots in petroleum-polluted soil. Furthermore, assimilated ¹³C was found to be significantly higher in soil, microbial biomass and soil respiration after soils were polluted by petroleum. These results suggested that the carbon released from roots is rapidly turned over by soil microbes under petroleum pollution. This study found that plants can modulate biomass allocation in response to petroleum pollution.     

Phytoremediation of Light Crude Oil by Maize (Zea mays L.) Bio-Augmented with Plant Growth Promoting Bacteria

The aim of this study was to evaluate the converged effect of maize and plant growth promoting bacteria on degradation of petroleum hydrocarbons under axenic conditions. Artificially spiked sand with 10 g kg^?1 light crude oil was planted with maize alone and in combination with eight bacterial isolates having plant growth promotion and bioremediation potential to observe the dissipation of petroleum hydrocarbons. Results showed remarkable suppression of maize growth and biomass production due to phytotoxicity of the crude oil contamination. However, bio-augmentation of plants with bacteria having ACC-deaminase activity significantly compensated the reduction in plant growth compared to uninoculated plants. The results revealed that plants bio-augmented with PM32Y exhibited significant increase in root length (75%), plant height (74%), and biomass (67%) as compared to uninoculated plants after 60 days of planting. The same bacterium in convergence with maize caused 43% degradation of petroleum hydrocarbons as compared to the unplanted and uninoculated control. Amplification, sequencing and phylogenetic analysis of 16S rRNA gene sequence identified PM32Y bacterium as Bacillus subtilis strain. It is concluded that bio-augmentation of plants with plant growth promoting bacteria having bioremediation potential and ACC-deaminase activity can successfully be used in phytoremediation of petroleum hydrocarbons.     

Bacteria Belonging to the Genus Cycloclasticus Play a Primary Role in the Degradation of Aromatic Hydrocarbons Released in a Marine Environment

To identify the bacteria that play a major role in the aerobic degradation of petroleum polynuclear aromatic hydrocarbons (PAHs) in a marine environment, bacteria were enriched from seawater by using 2-methylnaphthalene, phenanthrene, or anthracene as a carbon and energy source. We found that members of the genus Cycloclasticus became predominant in the enrichment cultures. The Cycloclasticus strains isolated in this study could grow on crude oil and degraded PAH components of crude oil, including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. To deduce the role of Cycloclasticus strains in a coastal zone oil spill, propagation of this bacterial group on oil-coated grains of gravel immersed in seawater was investigated in beach-simulating tanks that were 1 m wide by 1.5 m long by 1 m high. The tanks were two-thirds filled with gravel, and seawater was continuously introduced into the tanks; the water level was varied between 30 cm above and 30 cm below the surface of the gravel layer to simulate a 12-h tidal cycle. The number of Cycloclasticus cells associated with the grains was on the order of 10³ cells/g of grains before crude oil was added to the tanks and increased to 3 × 10⁶ cells/g of grains after crude oil was added. The number increased further after 14 days to 10⁸ cells/g of grains when nitrogen and phosphorus fertilizers were added, while the number remained 3 × 10⁶ cells/g of grains when no fertilizers were added. PAH degradation proceeded parallel with the growth of Cycloclasticus cells on the surfaces of the oil-polluted grains of gravel. These observations suggest that bacteria belonging to the genus Cycloclasticus play an important role in the degradation of petroleum PAHs in a marine environment.     

Microbial community dynamics during assays of harbour oil spill bioremediation: a microscale simulation study

AIMS: Microcosm experiments simulating an oil spill event were performed to evaluate the response of the natural microbial community structure of Messina harbour seawater following the accidental load of petroleum. METHODS AND RESULTS: An experimental harbour seawater microcosm, supplemented with nutrients and crude oil, was monitored above 15 days in comparison with unpolluted ones (control microcosms). Bacterial cells were counted with a Live/Dead BacLight viability kit; leucine aminopeptidase, beta-glucosidase, alkaline phosphatase, lipase and esterase enzymes were measured using fluorogenic substrates. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil addition stimulated an increase of the total bacterial abundance, leucine aminopeptidase and phosphatase activity rates, as well as a change in the community structure. This suggested a prompt response of micro-organisms to the load of petroleum hydrocarbons. CONCLUSIONS: The present study on the viability, specific composition and metabolic characteristics of the microbial community allows a more precise assessment of oil pollution. Both structural and functional parameters offer interesting perspectives as indicators to monitor changes caused by petroleum hydrocarbons. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of microbial structural successions at oil-polluted sites is essential for environmental bioremediation. Data obtained in microcosm studies improve our understanding of natural processes occurring during oil spills.     

Persistence and Biodegradation of Spilled Residual Fuel on an Estuarine Beach

The enrichment of hydrocarbon-degrading bacteria and the persistence of petroleum hydrocarbons on an estuarine beach after a spill of residual fuel oil on 11 April 1973 in Upper Narragansett Bay, R.I. was investigated. A rapid enrichment occurred during days 4 to 16 after the oil spill and a significant population of hydrocarbon-degrading bacteria was maintained in the beach sand for at least a year. The concentration of petroleum hydrocarbons in the mid-tide area declined rapidly during the bacterial enrichment period, remained fairly constant throughout the summer, and then declined to a low concentration after 1 year. An increased concentration of branched and cyclic aliphatic hydrocarbons in the low-tide sediment 128 days after the spill suggested a migration of hydrocarbons during the summer. Hydrocarbon biodegradation was apparent during the winter months at a rate of less than 1 mug of hydrocarbon per g of dry sediment per day.     

Biodegradation of petroleum hydrocarbons by filamentous fungi (Aspergillus ustus and Purpureocillium lilacinum) isolated from used engine oil contaminated soil

The use of microorganisms for remediation and restoration of hydrocarbons contaminated soils is an effective and economic solution. The current study aims to find out efficient telluric filamentous fungi to degrade petroleum hydrocarbons pollutants. Six fungal strains were isolated from used engine (UE) oil contaminated soil. Fungi were screened for their ability to degrade crude oil, diesel and UE oil using 2.6-dichlorophenol indophenol (DCPIP). Two isolates were selected, identified and registered at NCBI as Aspergillus ustus HM3.aaa and Purpureocillium lilacinum HM4.aaa. Fungi were tested for their tolerance to different concentration of petroleum oils using radial growth diameter assay. Hydrocarbons removal percentage was evaluated gravimetrically. The degradation kinetic of crude oil was studied at a time interval of 10 days. A.ustus was the most tolerant fungi to high concentration of petroleum oils in solid medium. Quantitative analysis showed that crude oil was the most degraded oil by both isolate; P. lilacinium and A. ustus removed 44.55% and 30.43% of crude oil, respectively. The two fungi were able to degrade, respectively, 27.66 and 21.27% of diesel and 14.39 and 16.00% of UE oil. As compared to the controls, these fungi accumulated high biomass in liquid medium with all petroleum oils. Likewise, crude oil removal rate constant (K) and half-lives (t_1/2) were 0.02 day⁻¹, 34.66 day and 0.015 day⁻¹, 46.21 day for P. lilacinium and A. ustus, respectively. The selected fungi appear interesting for petroleum oils biodegradation and their application for soil bioremediation require scale-up studies.     

Role of Bacterial Exopolysaccharides (EPS) in the Fate of the Oil Released during the Deepwater Horizon Oil Spill

Halomonas species are recognized for producing exopolysaccharides (EPS) exhibiting amphiphilic properties that allow these macromolecules to interface with hydrophobic substrates, such as hydrocarbons. There remains a paucity of knowledge, however, on the potential of Halomonas EPS to influence the biodegradation of hydrocarbons. In this study, the well-characterized amphiphilic EPS produced by Halomonas species strain TG39 was shown to effectively increase the solubilization of aromatic hydrocarbons and enhance their biodegradation by an indigenous microbial community from oil-contaminated surface waters collected during the active phase of the Deepwater Horizon oil spill. Three Halomonas strains were isolated from the Deepwater Horizon site, all of which produced EPS with excellent emulsifying qualities and shared high (97-100%) 16S rRNA sequence identity with strain TG39 and other EPS-producing Halomonas strains. Analysis of pyrosequence data from surface water samples collected during the spill revealed several distinct Halomonas phylotypes, of which some shared a high sequence identity (≥97%) to strain TG39 and the Gulf spill isolates. Other bacterial groups comprising members with well-characterized EPS-producing qualities, such as Alteromonas , Colwellia and Pseudoalteromonas , were also found enriched in surface waters, suggesting that the total pool of EPS in the Gulf during the spill may have been supplemented by these organisms. Roller bottle incubations with one of the Halomonas isolates from the Deepwater Horizon spill site demonstrated its ability to effectively produce oil aggregates and emulsify the oil. The enrichment of EPS-producing bacteria during the spill coupled with their capacity to produce amphiphilic EPS is likely to have contributed to the ultimate removal of the oil and to the formation of oil aggregates, which were a dominant feature observed in contaminated surface waters.     

Hydrocarbon-degrading bacteria and the bacterial community response in gulf of Mexico beach sands impacted by the deepwater horizon oil spill

A significant portion of oil from the recent Deepwater Horizon (DH) oil spill in the Gulf of Mexico was transported to the shoreline, where it may have severe ecological and economic consequences. The objectives of this study were (i) to identify and characterize predominant oil-degrading taxa that may be used as model hydrocarbon degraders or as microbial indicators of contamination and (ii) to characterize the in situ response of indigenous bacterial communities to oil contamination in beach ecosystems. This study was conducted at municipal Pensacola Beach, FL, where chemical analysis revealed weathered oil petroleum hydrocarbon (C₈ to C₄₀) concentrations ranging from 3.1 to 4,500 mg kg⁻¹ in beach sands. A total of 24 bacterial strains from 14 genera were isolated from oiled beach sands and confirmed as oil-degrading microorganisms. Isolated bacterial strains were primarily Gammaproteobacteria, including representatives of genera with known oil degraders (Alcanivorax, Marinobacter, Pseudomonas, and Acinetobacter). Sequence libraries generated from oiled sands revealed phylotypes that showed high sequence identity (up to 99%) to rRNA gene sequences from the oil-degrading bacterial isolates. The abundance of bacterial SSU rRNA gene sequences was ∼10-fold higher in oiled (0.44 × 10⁷ to 10.2 × 10⁷ copies g⁻¹) versus clean (0.024 × 10⁷ to 1.4 × 10⁷ copies g⁻¹) sand. Community analysis revealed a distinct response to oil contamination, and SSU rRNA gene abundance derived from the genus Alcanivorax showed the largest increase in relative abundance in contaminated samples. We conclude that oil contamination from the DH spill had a profound impact on the abundance and community composition of indigenous bacteria in Gulf beach sands, and our evidence points to members of the Gammaproteobacteria (Alcanivorax, Marinobacter) and Alphaproteobacteria (Rhodobacteraceae) as key players in oil degradation there.     

Efficiency of Opuntia ficus in the phytoremediation of a soil contaminated with used motor oil and lead,compared to that of Lolium perenne and Aloe barbadensis

Industrial pollutants such as heavy metals and hydrocarbons in soils represent a serious concern due to their persistence and negative effects on the environment, affecting cellular processes in living organisms and even causing mutations and cancer. The main objectives of this work were to evaluate the efficiency of Opuntia ficus in the phytoremediation of a soil polluted with used motor oil. Two other species, one with different and one with similar characteristics, relatively, were used for comparison purposes: Lolium perenne and Aloe barbadensis. The effect of the plants on lead solubility and bioaccumulation, the biomass production of each specie and the microbial counts and bacterial identification for each experiment was studied. Total petroleum hydrocarbons (TPH) were measured every 5 weeks throughout the 20-week phytoremediation experiment. At the end of the experiment soluble Pb, Pb extracted by the plant species, microbiological counts, total biomass and bacterial species in soil were analyzed. Even though Lolium perenne showed the highest TPH removal (47%), Opuntia ficus produced the highest biomass and similar removal (46%). Since Opuntia ficus requires low amounts of water and grows fast, it would be a suitable option in the remediation of soils polluted with hydrocarbons and/or heavy metals.     

Stimulation of Diesel Fuel Biodegradation by Indigenous Nitrogen Fixing Bacterial Consortia

Abstract Successful stimulation of N₂ fixation and petroleum hydrocarbon degradation in indigenous microbial consortia may decrease exogenous N requirements and reduce environmental impacts of bioremediation following petroleum pollution. This study explored the biodegradation of petroleum pollution by indigenous N₂ fixing marine microbial consortia. Particulate organic carbon (POC) in the form of ground, sterile corn-slash (post-harvest leaves and stems) was added to diesel fuel amended coastal water samples to stimulate biodegradation of petroleum hydrocarbons by native microorganisms capable of supplying a portion of their own N. It was hypothesized that addition of POC to petroleum amended water samples from N-limited coastal waters would promote the growth of N₂ fixing consortia and enhance biodegradation of petroleum. Manipulative experiments were conducted using samples from coastal waters (marinas and less polluted control site) to determine the effects of POC amendment on biodegradation of petroleum pollution by native microbial consortia. Structure and function of the microbial consortia were determined by measurement of N₂ fixation (acetylene reduction), hydrocarbon biodegradation (¹⁴C hexadecane mineralization), bacterial biomass (AODC), number of hydrocarbon degrading bacteria (MPN), and bacterial productivity (³H-thymidine incorporation). Throughout this study there was a consistent enhancement of petroleum hydrocarbon degradation in response to the addition of POC. Stimulation of diesel fuel biodegradation following the addition of POC was likely attributable to increases in bacterial N₂ fixation, diesel fuel bioavailability, bacterial biomass, and metabolic activity. Toxicity of the bulk phase water did not appear to be a factor affecting biodegradation of diesel fuel following POC addition. These results indicate that the addition of POC to diesel-fuel-polluted systems stimulated indigenous N₂ fixing microbial consortia to degrade petroleum hydrocarbons. Received: 29 December 1998; Accepted: 6 April 1999     

Bioremediation of oil-polluted soils: Using the [13C]/[12C] ratio to characterize microbial products of oil hydrocarbon biodegradation

We compared data on the extent of bioremediation in soils polluted with oil. The data were obtained using conventional methods of hydrocarbon determination: extraction gas chromatography-mass spectrometry, extraction IR spectroscopy, and extraction gravimetry. Due to differences in the relative abundances of the stable carbon isotopes (¹³C/¹²C) in oil and in soil organic matter, these ratios could be used as natural isotopic labels of either substance. Extraction gravimetry in combination with characteristics of the carbon isotope composition of organic products in the soil before and after bioremediation was shown to be the most informative approach to an evaluation of soil bioremediation. At present, it is the only method enabling quantification of the total petroleum hydrocarbons in oil-polluted soil, as well as of the amounts of hydrocarbons remaining after bioremediation and those microbially transformed into organic products and biomass.     

Optimization of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> (KU923381) for diesel oil degradation using response surface methodology (RSM)

Efficiency of Enterobacter cloacae KU923381 isolated from petroleum hydrocarbon contaminated soil was evaluated in batch culture and bioreactor mode. The isolate were screened for biofilm formation using qualitative and quantitative assays. Response surface methodology (RSM) was used to study the effect of pH, temperature, glucose concentration, and sodium chloride on diesel degradation. The predicted values for diesel oil degradation efficiency by the statistical designs are in a close agreement with experimental data (R ² = 99.66%). Degradation efficiency is increased by 36.78% at pH = 7, temperature = 35°C, glucose = 5%, and sodium chloride concentration = 5%. Under the optimized conditions, the experiments were performed for diesel oil degradation by gas chromatographic mass spectrometric analysis (GC-MS). GC-MS analysis confirmed that E. cloacae had highly degrade hexadecane, heptadecane, tridecane, and docosane by 99.71%, 99.23%, 99.66%, and 98.34% respectively. This study shows that rapid bioremoval of hydrocarbons in diesel oil is acheived by E. cloacae with abet of biofilm formation. The potential use of the biofilms for preparing trickling filters (gravel particles) for the degradation of hydrocarbons from petroleum wastes before their disposal in the open environment is highly suggested. This is the first successful attempt for artificially establishing petroleum hydrocarbon degrading bacterial biofilm on solid substrates in bioreactor.     

The impact of petroleum hydrocarbons (diesel) on periphyton in an impacted tropical estuary based on in situ microcosms

The distribution of petroleum hydrocarbons and their effects on the periphytic algal biomass using in situ microcosms were investigated in Ponggol estuary located on the northeastern coast of Singapore. Dissolved or dispersed petroleum hydrocarbon (DDPH) concentrations in the surface and bottom waters and absorbed or adsorbed petroleum hydrocarbon (AAPH) concentrations in sediments were monitored from July 1999 to June 2000. Results showed concentrations ranging from 4.42 to 248.94 μg l⁻¹, from 0.35 to 1099.65 μg l⁻¹, and from 20.55 to 541.01 mg kg⁻¹ for DDPH in surface and bottom waters and AAPH in sediments, respectively. Accidental spillages of fuel from dredgers operating in the estuary, fuel and engine oil from recreational boats, shipping operations in the adjacent strait, and runoff monsoon drains in the vicinity were some of the possible sources of petroleum hydrocarbons in the estuary. An assessment of environmentally realistic concentrations of petroleum hydrocarbons on periphytic algal biomass using in situ microcosms revealed signs of acute toxicity. A reduction in periphytic algal biomass (with respect to controls) of 68-93% was observed for various treatments exposed to diesel.     

Impact of Oil on Bacterial Community Structure in Bioturbated Sediments

Oil spills threaten coastlines where biological processes supply essential ecosystem services. Therefore, it is crucial to understand how oil influences the microbial communities in sediments that play key roles in ecosystem functioning. Ecosystems such as sediments are characterized by intensive bioturbation due to burrowing macrofauna that may modify the microbial metabolisms. It is thus essential to consider the bioturbation when determining the impact of oil on microbial communities. In this study, an experimental laboratory device maintaining pristine collected mudflat sediments in microcosms closer to true environmental conditions – with tidal cycles and natural seawater – was used to simulate an oil spill under bioturbation conditions. Different conditions were applied to the microcosms including an addition of: standardized oil (Blend Arabian Light crude oil, 25.6 mg.g⁻¹ wet sediment), the common burrowing organism Hediste (Nereis) diversicolor and both the oil and H. diversicolor. The addition of H. diversicolor and its associated bioturbation did not affect the removal of petroleum hydrocarbons. After 270 days, 60% of hydrocarbons had been removed in all microcosms irrespective of the H. diversicolor addition. However, 16S-rRNA gene and 16S-cDNA T-RFLP and RT-PCR-amplicon libraries analysis showed an effect of the condition on the bacterial community structure, composition, and dynamics, supported by PerMANOVA analysis. The 16S-cDNA libraries from microcosms where H. diversicolor was added (oiled and un-oiled) showed a marked dominance of sequences related to Gammaproteobacteria. However, in the oiled-library sequences associated to Deltaproteobacteria and Bacteroidetes were also highly represented. The 16S-cDNA libraries from oiled-microcosms (with and without H. diversicolor addition) revealed two distinct microbial communities characterized by different phylotypes associated to known hydrocarbonoclastic bacteria and dominated by Gammaproteobacteria and Deltaproteobacteria. In the oiled-microcosms, the addition of H. diversicolor reduced the phylotype-richness, sequences associated to Actinobacteria, Firmicutes and Plantomycetes were not detected. These observations highlight the influence of the bioturbation on the bacterial community structure without affecting the biodegradation capacities.     

Hydrocarbons and fatty acid methyl esters in bacterial biomass before and after physicochemical treatment

Hydrocarbons and fatty acid methyl esters were identified by chromatography-mass spectrometry in extracts of the native biomass of bacteria: chemoorganoheterotrophic Arthrobacter sp. and Pseudomonas aeruginosa and chemolithoautotrophic Carboxydothermus sp. Ultrasound treatment of bacterial biomass and mild thermolysis were shown to promote formation of a broad spectrum of hydrocarbons from bacterial biomass. The biomarker stigmastane belonging to the sterane group was found in P. aeruginosa biomass after thermolysis at 110°C in an open vial. Alkane composition in P. aeruginosa biomass before and after thermolysis at 300°C in a sealed container remained unchanged, indicating the possibility of preservation of hydrocarbons of bacterial origin in sealed layers under high temperature and elevated pressure.     

Potential of restoration and phytoremediation with Juncus roemerianus for diesel-contaminated coastal wetlands

Oil spills may considerably damage sensitive coastal wetlands. In this study, the tolerance limits of a dominant coastal salt marsh plant, Juncus roemerianus, to diesel oil and its phytoremediation effectiveness in wetland environments were investigated in the greenhouse. J. roemerianus was transplanted into salt marsh sediment contaminated with diesel fuel at concentrations of 0, 20, 40, 80, 160, 320, and 640 mg diesel g^?1 dry sediment. Plant stem density, shoot height, aboveground biomass and belowground biomass were detrimentally impacted at high oil dosages even 1 year after transplantation. Tolerance limits were estimated between 160 and 320 mg g^?1 based on various plant variables. Importantly, J. roemerianus enhanced oil degradation; at the 40 mg/g diesel dosage, concentrations of residual total petroleum hydrocarbons (TPH) in the sediment planted with J. roemerianus were significantly lower than those of unplanted sediments 1 year after treatment initiation. Furthermore, concentrations of total targeted polycyclic aromatic hydrocarbons (PAHs) and n-alkanes in the J. roemerianus planted treatment were, respectively, about 3% and 15% of the unplanted treatment. Concentration reduction in all categories of hydrocarbons suggested that J. roemerianus effectively phytoremediated the diesel-contaminated wetlands.     

Biodegradation of Petroleum Hydrocarbons in a Soil Polluted Sample by Oil-Based Drilling Cuttings

Microbial degradation of hydrocarbons in soils polluted by oil-based drilling mud and cuttings has been investigated by static methods such as composting or biopiling. Bioremediation of polluted soils by oil-based drilling cuttings through a slurry bioreactor has not previously been reported. The main aim of this work is to monitor hydrocarbon biodegradation in slurry of drilling cuttings and unpolluted soils and the effects of nutrients on it. Indigenous, bacterial-mixed culture isolated from a polluted soil by drilling cuttings adapted to drilling mud concentrations up to 15% (v/v) was done during a 15-month program. The total petroleum hydrocarbons’ (TPHs) removal efficiency in C/N/P 100/5/1 ratio was 90.5 and 79.85% under experimental and control conditions, respectively. The microbial count on the first day, 15 × 10⁷ CFUg^?1, reached 20 × 10⁹ CFUg^?1on the twenty-first day at experimental conditions. The TPH removal efficiency in C/N/P 100/10/2 was 92.5 and 82.25% at experiment and control, respectively. Increasing nitrogen and phosphorous amount couldn't increase microbial count in comparison with C/N/P ratio 100/5/1. The measured biomass contents and microbial counts in experiments were significantly higher than the control and confirmed hydrocarbons’ biodegradation during the time. Results showed that slurry bioreactors could accelerate the biodegradation of TPHs and reduce remediation time in soil polluted by oil-based drilling cuttings.     

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly ¹³C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.     

Bacterial Community Response to Petroleum Hydrocarbon Amendments in Freshwater,Marine, and Hypersaline Water-Containing Microcosms

Hydrocarbon-degrading bacterial communities from freshwater, marine, and hypersaline Brazilian aquatic ecosystems (with water salinities corresponding to 0.2%, 4%, and 5%, respectively) were enriched with different hydrocarbons (heptadecane, naphthalene, or crude oil). Changes within the different microcosms of bacterial communities were analyzed using cultivation approaches and molecular methods (DNA and RNA extraction, followed by genetic fingerprinting and analyses of clone libraries based on the 16S rRNA-coding gene). A redundancy analysis (RDA) of the genetic fingerprint data and a principal component analysis (PCA) of the clone libraries revealed hydrocarbon-enriched bacterial communities specific for each ecosystem studied. However, within the same ecosystem, different bacterial communities were selected according to the petroleum hydrocarbon used. In general, the results demonstrated that Acinetobacter and Cloacibacterium were the dominant genera in freshwater microcosms; the Oceanospirillales order and the Marinobacter, Pseudomonas, and Cycloclasticus genera predominated in marine microcosms; and the Oceanospirillales order and the Marinobacter genus were selected in the different hydrocarbon-containing microcosms in hypersaline water. Determination of total petroleum hydrocarbons (TPHs) in all microcosms after 32 days of incubation showed a decrease in the hydrocarbon concentration compared to that for the controls. A total of 50 (41.3%) isolates from the different hydrocarbon-contaminated microcosms were associated with the dominant operational taxonomic units (OTUs) obtained from the clone libraries, and their growth in the hydrocarbon contaminating the microcosm from which they were isolated as the sole carbon source was observed. These data provide insight into the general response of bacterial communities from freshwater, marine, and hypersaline aquatic ecosystems to petroleum hydrocarbon contamination.