The effects of pesticides on the diversity of culturable soil bacteria

The numbers of culturable soil bacteria in plots that had received either no pesticides or the full combination (aldicarb, chlorfenvinphos, benomyl, glyphosate, plus chlorotoluron or triadimefon) over a 20 year period were compared. Differences were very small although there were consistently higher numbers on the treated plot, possibly reflecting the greater crop yields which had been reported previously. There was no significant difference in numbers of bacterial colonies with homology to a nif gene probe in soils from the two plots. Genetic fingerprinting of Pseudomonas fluorescens isolates from the plots, using ERIC-PCR, showed that the dominant strains in the two populations were not the same although there was no obvious difference in the degree of diversity. Substrate utilization by microbial populations from the two plots was compared using Biolog plates. The population from the pesticide-treated plot showed a higher rate of substrate utilization which could reflect a slightly higher inoculum of heterotrophic bacteria, but could also indicate greater metabolic diversity in the population.     

Effect of Crude Oil and Chemical Additives on Metabolic Activity of Mixed Microbial Populations in Fresh Marsh Soils

Abstract Hydrocarbons increase abundance of hydrocarbon-degrading microorganisms, but also decrease microbial diversity. This could disrupt ecosystem dynamics by altering soil organic matter mineralization and resultant nutrient remineralization rates. Crude oil, which is known to contain toxins and reduce microbial diversity, was hypothesized to reduce gross metabolic activity of mixed microbial populations in wetland soils. Soil respiration and Eh were compared, for 6 months, among microcosms containing marsh soils that differed in soil organic matter (Panicum hemitomon Shult. or Sagittaria lancifolia L. dominated marshes), crude oil (Arabian crude, Louisiana crude, or no oil), and additives (a cleaner, a dispersant, fertilizer, or no additive). No treatment slowed activity; instead, Louisiana plus fertilizer and all Arabian treatments temporarily accelerated activity. Additional C respired from oiled microcosms exceeded C added as crude oil by 1.4 to 3.5 times. Thus, much additional C originated from soil organic matter rather than crude oil. Crude oils temporarily lowered soil Eh, which is consistent with accelerated metabolism and demand for electron acceptors. The lack of inhibition observed at the community level does not necessarily indicate an absence of toxicity. Instead, tolerant species with metabolic versatility probably maintained activity. Stimulation probably resulted from removal of micronutrient limitation, rather than removal of grazing pressure or macronutrient limitation. Regardless, accelerated soil organic matter mineralization surely accelerated nutrient remineralization. This might explain some reports of crude oil stimulating plant growth. These results are not inconsistent with theoretical and experimental conclusions regarding effects of biodiversity on ecosystem stability and productivity, nor are they inconsistent with conclusions that crude oils contain components that are toxic to microbes, vegetation, and fauna. However, these data do indicate that crude oils also contain components that temporarily stimulate metabolic activity of surviving microbes. Received: 27 April 1998; Accepted: 15 July 1998     

Evaluation of inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil

A full-scale study evaluating an inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil was conducted at an oil refinery where the indigenous population of hydrocarbon-degrading bacteria in the soil was very low (10(3) to 10(4) CFU/g of soil). A feasibility study was conducted prior to the full-scale bioremediation study. In this feasibility study, out of six treatments, the application of a bacterial consortium and nutrients resulted in maximum biodegradation of total petroleum hydrocarbon (TPH) in 120 days. Therefore, this treatment was selected for the full-scale study. In the full-scale study, plots A and B were treated with a bacterial consortium and nutrients, which resulted in 92.0 and 89.7% removal of TPH, respectively, in 1 year, compared to 14.0% removal of TPH in the control plot C. In plot A, the alkane fraction of TPH was reduced by 94.2%, the aromatic fraction of TPH was reduced by 91.9%, and NSO (nitrogen-, sulfur-, and oxygen-containing compound) and asphaltene fractions of TPH were reduced by 85.2% in 1 year. Similarly, in plot B the degradation of alkane, aromatic, and NSO plus asphaltene fractions of TPH was 95.1, 94.8, and 63.5%, respectively, in 345 days. However, in plot C, removal of alkane (17.3%), aromatic (12.9%), and NSO plus asphaltene (5.8%) fractions was much less. The population of introduced Acinetobacter baumannii strains in plots A and B was stable even after 1 year. Physical and chemical properties of the soil at the bioremediation site improved significantly in 1 year.     

Effect of Four Dispersants on Biodegradation and Growth of Bacteria on Crude Oil

Four chemical dispersants, Corexit 8666, Gamlen Sea Clean, G. H. Woods Degreaser-Formula 11470, and Sugee 2 were examined singly and in individual combinations with Arabian Crude Oil (1:1 ratio) at 10 and 25 C for their effects on the growth of bacteria indigenous to local marine waters, the bacterial population composition, and biodegradation of crude oil; in addition, their emulsifying capacities, at approximately 24 C, were determined. None of the dispersants used alone were toxic even at relatively high concentrations (1.25%), although Gamlen Sea Clean and G. H. Woods Degreaser-Formula 11470 did cause an increase in the lag phase which was more pronounced at 10 than at 25 C; addition of the crude oil reduced the lag phase increase. All of the dispersants used alone supported good growth of microorganisms, but qualitative population shifts were caused by the dispersant-oil combinations. The degrees of degradation of the n-alkane fraction of the crude oil varied depending upon the dispersant used. Under these test conditions, only Sugee 2, which had the poorest emulsifying capacity, promoted n-alkane degradation compared with the values obtained by using the crude oil alone.     

Distinct responses in ammonia-oxidizing archaea and bacteria after addition of biosolids to an agricultural soil

The recently discovered ammonia-oxidizing archaea (AOA) have been suggested as contributors to the first step of nitrification in terrestrial ecosystems, a role that was previously assigned exclusively to ammonia-oxidizing bacteria (AOB). The current study assessed the effects of agricultural management, specifically amendment of soil with biosolids or synthetic fertilizer, on nitrification rates and copy numbers of archaeal and bacterial ammonia monooxygenase (amoA) genes. Anaerobically digested biosolids or synthetic fertilizer was applied annually for three consecutive years to field plots used for corn production. Biosolids were applied at two loading rates, a typical agronomic rate (27 Mg hectare(-1) year(-1)) and double the agronomic rate (54 Mg hectare(-1) year(-1)), while synthetic fertilizer was applied at an agronomic rate typical for the region (291 kg N hectare(-1) year(-1)). Both biosolids amendments and synthetic fertilizer increased soil N and corn yield, but only the biosolids amendments resulted in significant increases in nitrification rates and increases in the copy numbers of archaeal and bacterial amoA genes. In addition, only archaeal amoA gene copy numbers increased in response to biosolids applied at the typical agronomic rate and showed a significant correlation with nitrification rates. Finally, copy numbers of archaeal amoA genes were significantly higher than copy numbers of bacterial amoA genes for all treatments. These results implicate AOA as being primarily responsible for the increased nitrification observed in an agricultural soil amended with biosolids. These results also support the hypothesis that physiological differences between AOA and AOB may enable them to occupy distinct ecological niches.     

Effect of Environmental Parameters on Bacterial Degradation of Bunker C Oil, Crude Oils, and Hydrocarbons

Mixed microbial cultures, previously enriched on Bunker C fuel oil, grew on and degraded Bunker C fuel oil at temperatures ranging from 5 to 28 C. At 15 C, 41 to 85% of the benzene-soluble components of Bunker C disappeared after incubation for 7 days; at 5 C the values ranged from 21 to 52% after 14 days of incubation. A Nocardia sp. isolated from a culture enriched on Bunker C oil grew on Venezuelan crude oil, Bunker C, hexadecane, and a hydrocarbon mixture at temperatures of 5 and 15 C. The 10-C decrease in temperature resulted in an average 2.2-fold decrease in generation time of the bacteria. Gas-liquid chromatographic measurements of Venezuelan and Arabian crude oils which had been incubated with the Nocardia sp. showed significant degradation of the n-alkane portion and the chromatographically unresolved components of the oils. The concentration of elemental nitrogen required to bring about the disappearance of 1 mg of hexadecane by the Nocardia sp. was 0.5 mg. The results confirm suggestions that the rate of natural biodegradation of oil in marine temperate-to-polar zones is probably limited by low temperatures and phosphorus concentrations, but suggest that the concentrations of nitrogen occurring naturally are probably not rate-limiting factors.     

Indigenous Sediment Microbial Activity in Response to Nutrient Enrichment and Plant Growth Following a Controlled Oil Spill on a Freshwater Wetland

The population density and activity of a microbial community associated with the sediment and rhizosphere of an intertidal freshwater wetland dominated by Scirpus pungens was monitored before and following the application of weathered Mesa light crude oil and fertilizers. The influence of nutrient enrichment (fertilizers) and plant growth on oil degradation rates was determined from the resulting data. The study plots (four blocks of replicates) were subjected to five treatments: oil only (natural attenuation); oil plus ammonium nitrate and phosphate, with regular cropping of the plants; oil plus ammonium nitrate and phosphate; oil plus sodium nitrate and phosphate; no oil, ammonium nitrate and phosphate. The plots were regularly monitored in the field for gas production (carbon dioxide and nitrous oxide), and samples were collected for laboratory analysis of denitrification activity, aliphatic and aromatic hydrocarbon degradation activity, and total heteroptrophic bacteria.

The viable bacterial population density increased during the first 4 weeks in oiled and unoiled experimental plots that were fertilized. In contrast, population densities in untreated areas remained relatively unchanged throughout the monitoring period. The microbial population demonstrated a rapid and sustained increase in naphthalene mineralization activity in plots that were both fertilized and oiled. Hexadecane mineralization activity increased in response to fertilizer application, with ammonium nitrate causing a larger increase than sodium nitrate. A very significant difference observed in the mineralization of hexadecane was that the surface sediments were much more active than the subsurface sediments. This difference became even more pronounced in the second year of monitoring, even though the treatment regime had been discontinued. This compartmentalization of mineralization activity was not observed for naphthalene. Following fertilizer application, field and laboratory evaluation of nitrogen metabolism in the sediments indicated significant denitrification activity that was not adversely affected by oiling. The results demonstrated that the application of fertilizers stimulated the activities of indigenous hydrocarbon-degrading and denitrifying bacteria, and the presence of oil either enhanced or had no detrimental effect on these activities. As a remediation strategy, the application of fertilizers to a wetland shoreline following an oil spill would promote the growth of indigenous plants and their associated microbial flora, resulting in increased metabolic activity and the potential for increased oil degradation activity.     

Indigenous Sediment Microbial Activity in Response to Nutrient Enrichment and Plant Growth Following a Controlled Oil Spill on a Freshwater Wetland

The population density and activity of a microbial community associated with the sediment and rhizosphere of an intertidal freshwater wetland dominated by Scirpus pungens was monitored before and following the application of weathered Mesa light crude oil and fertilizers. The influence of nutrient enrichment (fertilizers) and plant growth on oil degradation rates was determined from the resulting data. The study plots (four blocks of replicates) were subjected to five treatments: oil only (natural attenuation); oil plus ammonium nitrate and phosphate, with regular cropping of the plants; oil plus ammonium nitrate and phosphate; oil plus sodium nitrate and phosphate; no oil, ammonium nitrate and phosphate. The plots were regularly monitored in the field for gas production (carbon dioxide and nitrous oxide), and samples were collected for laboratory analysis of denitrification activity, aliphatic and aromatic hydrocarbon degradation activity, and total heteroptrophic bacteria. The viable bacterial population density increased during the first 4 weeks in oiled and unoiled experimental plots that were fertilized. In contrast, population densities in untreated areas remained relatively unchanged throughout the monitoring period. The microbial population demonstrated a rapid and sustained increase in naphthalene mineralization activity in plots that were both fertilized and oiled. Hexadecane mineralization activity increased in response to fertilizer application, with ammonium nitrate causing a larger increase than sodium nitrate. A very significant difference observed in the mineralization of hexadecane was that the surface sediments were much more active than the subsurface sediments. This difference became even more pronounced in the second year of monitoring, even though the treatment regime had been discontinued. This compartmentalization of mineralization activity was not observed for naphthalene. Following fertilizer application, field and laboratory evaluation of nitrogen metabolism in the sediments indicated significant denitrification activity that was not adversely affected by oiling. The results demonstrated that the application of fertilizers stimulated the activities of indigenous hydrocarbon-degrading and denitrifying bacteria, and the presence of oil either enhanced or had no detrimental effect on these activities. As a remediation strategy, the application of fertilizers to a wetland shoreline following an oil spill would promote the growth of indigenous plants and their associated microbial flora, resulting in increased metabolic activity and the potential for increased oil degradation activity.     

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.     

Evaluation of Inoculum Addition To Stimulate In Situ Bioremediation of Oily-Sludge-Contaminated Soil

A full-scale study evaluating an inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil was conducted at an oil refinery where the indigenous population of hydrocarbon-degrading bacteria in the soil was very low (10³ to 10⁴ CFU/g of soil). A feasibility study was conducted prior to the full-scale bioremediation study. In this feasibility study, out of six treatments, the application of a bacterial consortium and nutrients resulted in maximum biodegradation of total petroleum hydrocarbon (TPH) in 120 days. Therefore, this treatment was selected for the full-scale study. In the full-scale study, plots A and B were treated with a bacterial consortium and nutrients, which resulted in 92.0 and 89.7% removal of TPH, respectively, in 1 year, compared to 14.0% removal of TPH in the control plot C. In plot A, the alkane fraction of TPH was reduced by 94.2%, the aromatic fraction of TPH was reduced by 91.9%, and NSO (nitrogen-, sulfur-, and oxygen-containing compound) and asphaltene fractions of TPH were reduced by 85.2% in 1 year. Similarly, in plot B the degradation of alkane, aromatic, and NSO plus asphaltene fractions of TPH was 95.1, 94.8, and 63.5%, respectively, in 345 days. However, in plot C, removal of alkane (17.3%), aromatic (12.9%), and NSO plus asphaltene (5.8%) fractions was much less. The population of introduced Acinetobacter baumannii strains in plots A and B was stable even after 1 year. Physical and chemical properties of the soil at the bioremediation site improved significantly in 1 year.     

Comparison of autochthonous bacteria and commercially available cultures with respect to their effectiveness in fuel oil degradation

Summary This study examined the microbial degradation of fuel oil by nine highly adapted different commercially available mixed bacterial cultures (DBC-plus, Flow Laboratories, Meckenheim, F.R.G.) and a bacterial community from a domestic sewage sludge sample. All mixed cultures were cultivated under aerobic batch conditions shaking (110 rpm) at 20°C in a mineral base medium containing 1 or 5% (v/v) fuel oil as the sole carbon source. Percent degradation of fuel oil and the n-alkane fraction was recorded for the nine DBC-plus cultures and the mixed population of the activated sludge sample. The increase in colony counts, protein, and optical density was studied during a 31-day incubation period for DBC-plus culture A, DBC-plus culture A2 and the activated sludge sample. The activated sludge mixed culture was most effective in degrading fuel oil, but various isolated bacterial strains from this bacterial community were not able to grow on fuel oil as the sole carbon source. In contrast, the n-alkane degradation rates of the DBC-cultures were lower, but single strains from the commercially available mixed cultures were able to mineralize fuel oil hydrocarbons. Strains ofPseudomonas aeruginosa were isolated most frequently and these organisms were able to grow very rapidly on fuel oil as a complex sole carbon source. The results indicate that fuel oil degradation in domestic sewage sludge is performed by mixed populations of naturally occurring bacteria and does not depend on the application of highly adapted commercially available cultures.     

A comparative immunological investigation of the alkane hydroxylating cytochrome P-450 from the yeast Candida maltosa

The immunological relations of the cytochrome P-450 from the n-alkane utilizing yeast Candida maltosa to cytochrome P-450 forms of other organisms - yeasts, bacteria and mammalia - were investigated using a solid-phase double-antibody radioimmunoassay. Only the microsomal fraction of other n-alkane utilizing yeasts shows a distinct cross-reaction with an antiserum against cytochrome P-450 from Candida maltosa. Neither the tested bacterial nor the mammalian cytochromes P-450 cross-react with the antiserum.     

Microbial populations and hydrocarbon biodegradation potentials in fertilized shoreline sediments affected by the T/V Exxon Valdez oil spill.

The effort of clean up the T/V Exxon Valdez oil spill in Prince William Sound, Alaska, included the use of fertilizers to accelerate natural microbial degradation of stranded oil. A program to monitor various environmental parameters associated with this technique took place during the summer of 1990. Microbiological assays for numbers of heterotrophic and oil-degrading microbes and their hydrocarbon mineralization potentials were performed in support of this program. Fertilizer addition resulted in higher hexadecane and phenanthrene mineralization potentials on treated plots than on untreated reference plots. Microbial numbers in treated and reference surface sediments were not significantly different immediately after the first nutrient application in May 1990. However, subsurface sediments from treated plots had higher numbers of hydrocarbon degraders than did reference sediments shortly after treatment. The second application of fertilizer, later in summer, resulted in surface and subsurface increases in numbers of hydrocarbon degraders with respect to reference sediments at two of the three study sites. Elevated mineralization potentials, coupled with increased numbers of hydrocarbon degraders, indicated that natural hydrocarbon biodegradation was enhanced. However, these microbiological measurements alone are not sufficient to determine in situ rates of crude oil biodegradation.     

Microbial populations and hydrocarbon biodegradation potentials in fertilized shoreline sediments affected by the T/V Exxon Valdez oil spill.

The effort of clean up the T/V Exxon Valdez oil spill in Prince William Sound, Alaska, included the use of fertilizers to accelerate natural microbial degradation of stranded oil. A program to monitor various environmental parameters associated with this technique took place during the summer of 1990. Microbiological assays for numbers of heterotrophic and oil-degrading microbes and their hydrocarbon mineralization potentials were performed in support of this program. Fertilizer addition resulted in higher hexadecane and phenanthrene mineralization potentials on treated plots than on untreated reference plots. Microbial numbers in treated and reference surface sediments were not significantly different immediately after the first nutrient application in May 1990. However, subsurface sediments from treated plots had higher numbers of hydrocarbon degraders than did reference sediments shortly after treatment. The second application of fertilizer, later in summer, resulted in surface and subsurface increases in numbers of hydrocarbon degraders with respect to reference sediments at two of the three study sites. Elevated mineralization potentials, coupled with increased numbers of hydrocarbon degraders, indicated that natural hydrocarbon biodegradation was enhanced. However, these microbiological measurements alone are not sufficient to determine in situ rates of crude oil biodegradation.     

Bioremediation Assessment of Hydrocarbon-Contaminated Soils from the High Arctic

The bioremediation potential of hydrocarbon-contaminated soils from the most northerly inhabited station in the world, Canadian Forces Station - Alert, was assessed. Microbial enumeration, by both viable plate counts and direct counts, combined with molecular analysis (polymerase chain reaction and colony hybridization) for hydrocarbon catabolic genes (alkB, ndoB, xylE), demonstrated the presence of significant numbers of cold-adapted hydrocarbon-degrading microorganisms. The degradative activity of these populations was assessed by mineralization of ¹⁴Clabeled hexadecane (C16) at 5°C in untreated and treated soils. Although very low rates of C16 mineralization were observed in the untreated soils, nutrient supplementation with a fertilizer markedly increased C16 mineralization. Highly active cold-adapted hydrocarbon-degrading consortia were prepared from soil slurries, and their degradative potentials were monitored by biomass measurements and mineralization activity. Bio augmentation of the contaminated soils with consortia containing the greatest percentages of degradative bacteria resulted in the shortest C16 mineralization acclimation period. However, treatment with the consortia plus fertilizer did not appreciably increase C16 mineralization or reduce total petroleum hydrocarbon concentrations to a greater extent than did the fertilizer treatment alone. These results indicate that the soils possessed sufficient numbers of cold-adapted degradative bacteria, and that fertilizer application alone was sufficient to obtain elevated levels of degradative activity at low ambient summer temperatures.     

Monitoring Soil Ecosystem Recovery Following Bioremediation of a Terrestrial Crude Oil Spill With and Without a Fertilizer Amendment

The effect of fertilizer as an amendment in the bioremediation of a terrestrial crude oil spill has been investigated in terms of the subsequent recovery of the soil ecosystem following bioremediation. Two different spills in the same area with different initial hydrocarbon concentrations (33,500 mg kg^-1 and 4,800 mg kg^-1) were compared. At the higher initial hydrocarbon concentration fertilizer addition increased the rate of bioremediation (first-order rate constant of 0.0033 days-1 with fertilizer amendment vs. 0.0020 days-1 without) and resulted in more rapid recovery of soil bacteria (numbers, community structure, diversity) and nematodes (trophic diversity and community structure). The effect of the fertilizer amendment was more significant at the higher initial concentration of crude oil hydrocarbons, presumably due to greater depletion of soil nutrient pools in the absence of the amendment. A second objective of this work was to identify sensitive and cost-effective ecological indicators useful for monitoring the recovery of soil ecosystems impacted by crude oil. Ecological indicators used included: microbial numbers, community structure, and activity as revealed by biomarker analysis (phospholipid fatty acids); nitrogen availability; nematode numbers and community structure (trophic groups and colonizer-persister classes); and ultimately, plant cover and diversity. All ecological indicators investigated were sensitive to disturbances in the soil food web in a hydrocarbon-impacted site. However, nematode community structure analysis offered the greatest sensitivity coupled with low cost and readily available sources for the analysis.     

Variability in Pseudomonas aeruginosa lipopolysaccharide expression during crude oil degradation

Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.     

Microbial Population Changes during Bioremediation of an Experimental Oil Spill

Three crude oil bioremediation techniques were applied in a randomized block field experiment simulating a coastal oil spill. Four treatments (no oil control, oil alone, oil plus nutrients, and oil plus nutrients plus an indigenous inoculum) were applied. In situ microbial community structures were monitored by phospholipid fatty acid (PLFA) analysis and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) to (i) identify the bacterial community members responsible for the decontamination of the site and (ii) define an end point for the removal of the hydrocarbon substrate. The results of PLFA analysis demonstrated a community shift in all plots from primarily eukaryotic biomass to gram-negative bacterial biomass with time. PLFA profiles from the oiled plots suggested increased gram-negative biomass and adaptation to metabolic stress compared to unoiled controls. DGGE analysis of untreated control plots revealed a simple, dynamic dominant population structure throughout the experiment. This banding pattern disappeared in all oiled plots, indicating that the structure and diversity of the dominant bacterial community changed substantially. No consistent differences were detected between nutrient-amended and indigenous inoculum-treated plots, but both differed from the oil-only plots. Prominent bands were excised for sequence analysis and indicated that oil treatment encouraged the growth of gram-negative microorganisms within the α-proteobacteria and Flexibacter-Cytophaga-Bacteroides phylum. α-Proteobacteria were never detected in unoiled controls. PLFA analysis indicated that by week 14 the microbial community structures of the oiled plots were becoming similar to those of the unoiled controls from the same time point, but DGGE analysis suggested that major differences in the bacterial communities remained.     

Microbial population changes during bioremediation of an experimental oil spill.

Three crude oil bioremediation techniques were applied in a randomized block field experiment simulating a coastal oil spill. Four treatments (no oil control, oil alone, oil plus nutrients, and oil plus nutrients plus an indigenous inoculum) were applied. In situ microbial community structures were monitored by phospholipid fatty acid (PLFA) analysis and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) to (i) identify the bacterial community members responsible for the decontamination of the site and (ii) define an end point for the removal of the hydrocarbon substrate. The results of PLFA analysis demonstrated a community shift in all plots from primarily eukaryotic biomass to gram-negative bacterial biomass with time. PLFA profiles from the oiled plots suggested increased gram-negative biomass and adaptation to metabolic stress compared to unoiled controls. DGGE analysis of untreated control plots revealed a simple, dynamic dominant population structure throughout the experiment. This banding pattern disappeared in all oiled plots, indicating that the structure and diversity of the dominant bacterial community changed substantially. No consistent differences were detected between nutrient-amended and indigenous inoculum-treated plots, but both differed from the oil-only plots. Prominent bands were excised for sequence analysis and indicated that oil treatment encouraged the growth of gram-negative microorganisms within the alpha-proteobacteria and Flexibacter-Cytophaga-Bacteroides phylum. alpha-Proteobacteria were never detected in unoiled controls. PLFA analysis indicated that by week 14 the microbial community structures of the oiled plots were becoming similar to those of the unoiled controls from the same time point, but DGGE analysis suggested that major differences in the bacterial communities remained.