Bacterial Community Dynamics and Polycyclic Aromatic Hydrocarbon Degradation during Bioremediation of Heavily Creosote-Contaminated Soil

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the α-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the γ-Proteobacteria group (genus Xanthomonas), the α-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the γ-Proteobacteria group (genus Xathomonas), the β-Proteobacteria group (genera Alcaligenes and Achromobacter), and the α-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.     

Identification and quantification of uncultivated Proteobacteria associated with pyrene degradation in a bioreactor treating PAH-contaminated soil

Uncultivated bacteria associated with the degradation of pyrene in a bioreactor treating soil contaminated with polycyclic aromatic hydrocarbons (PAH) were identified by DNA-based stable-isotope probing (SIP) and quantified by real-time quantitative PCR. Most of the 16S rRNA gene sequences recovered from (13)C-enriched DNA fractions clustered phylogenetically within three separate groups of beta- and gamma-Proteobacteria unassociated with described genera and were designated "Pyrene Groups 1, 2 and 3". One recovered sequence was associated with the Sphingomonas genus. Pyrene Groups 1 and 3 were present in very low numbers in the bioreactor but represented 75% and 7%, respectively, of the sequences recovered from 16S rRNA gene clone libraries constructed from (13)C-enriched DNA. In a parallel time-course incubation with unlabelled pyrene, there was between a 2- and 4-order-of-magnitude increase in the abundance of 16S rRNA genes from Pyrene groups 1 and 3 and from targeted Sphingomonas spp. over a 10 day incubation. Sequences from Pyrene Group 2 were 11% of the SIP clone libraries but accounted for 14% of the total bacterial 16S rRNA genes in the bioreactor community. However, the abundance of this group did not increase significantly in response to pyrene disappearance. These data indicate that the primary pyrene degraders in the bioreactor were uncultivated, low-abundance beta- and gamma-Proteobacteria not previously associated with pyrene degradation.     

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.     

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 C(10) to C(32) 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.     

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

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

Robust Hydrocarbon Degradation and Dynamics of Bacterial Communities during Nutrient-Enhanced Oil Spill Bioremediation

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

普通和稀释培养基研究太湖沉积物可培养细菌的多样性

采用普通牛肉汁培养基和 10倍稀释的普通牛肉汁培养基 (以下简称稀释培养基 )研究太湖沉积物中细菌多样性 ,发现在稀释培养基上生长的细菌数量普遍是在普通牛肉汁琼脂培养基上生长的细菌数量的 3～ 5倍。分离得到纯培养物的 16SrDNA部分序列 (5′端约 5 0 0bp)分析表明 ,不同培养基上生长的优势细菌类群存在差别 :普通培养基生长的细菌主要为γ_Proteobacteria(35. 1% ) ,其次为Actinobacteria(2 4 5 % )和Firmicutes(2 2 . 3% )等类群 ,其中大部分细菌与假单胞菌属 (Pseudomoas)、芽孢杆菌属 (Bacillus)和节杆菌属 (Archrobacter)细菌的系统关系密切 ;稀释培养基生长的细菌则主要为Actinobacteria(2 7. 1% )、Firmicutes(2 5 . 7% )、α_Proteobacteria(2 1. 4 % )和γ_Proteobacteria(15. 7% )等类群 ,与芽孢杆菌属 (Bacillus) (2 5. 7% )发育系统关系密切的细菌为优势属。研究结果表明同时采用两种培养基有助于从太湖沉积物中分离到更多种微生物。     

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

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

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.     

Real-Time PCR quantification of PAH-ring hydroxylating dioxygenase (PAH-RHDalpha) genes from Gram positive and Gram negative bacteria in soil and sediment samples

Real-Time PCR based assays were developed to quantify Gram positive (GP) and Gram negative (GN) bacterial populations that are capable of degrading the polycyclic aromatic hydrocarbons (PAH) in soil and sediment samples with contrasting contamination levels. These specific and sensitive Real-Time PCR assays were based on the quantification of the copy number of the gene that encodes the alpha subunit of the PAH-ring hydroxylating dioxygenases (PAH-RHDalpha), involved in the initial step of the aerobic metabolism of PAH. The PAH-RHDalpha-GP primer set was designed against the different allele types present in the data base (narAa, phdA/pdoA2, nidA/pdoA1, nidA3/fadA1) common to the Gram positive PAH degraders such as Rhodococcus, Mycobacterium, Nocardioides and Terrabacter strains. The PAH-RHDalpha-GN primer set was designed against the genes (nahAc, nahA3, nagAc, ndoB, ndoC2, pahAc, pahA3, phnAc, phnA1, bphAc, bphA1, dntAc and arhA1) common to the Gram negative PAH degraders such as Pseudomonas, Ralstonia, Commamonas, Burkholderia, Sphingomonas, Alcaligenes, Polaromonas strains. The PCR clones for DNA extracted from soil and sediment samples using the designed primers showed 100% relatedness to the PAH-RHDalpha genes targeted. Deduced from highly sensitive Real-Time PCR quantification, the ratio of PAH-RHDalpha gene relative to the 16S rRNA gene copy number showed that the PAH-bacterial degraders could represent up to 1% of the total bacterial community in the PAH-contaminated sites. This ratio highlighted a positive correlation between the PAH-bacterial biodegradation potential and the PAH-contamination level in the environmental samples studied.     

Bacterial communities in the initial stage of marine biofilm formation on artificial surfaces

Succession of bacterial communities during the first 36 h of biofilm formation in coastal water was investigated at 3 approximately 15 h intervals. Three kinds of surfaces (i.e., acryl, glass, and steel substratum) were submerged in situ at Sacheon harbor, Korea. Biofilms were harvested by scraping the surfaces, and the compositions of bacterial communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of 16S rRNA genes. While community structure based on T-RFLP analysis showed slight differences by substratum, dramatic changes were commonly observed for all substrata between 9 and 24 h. Identification of major populations by 16S rRNA gene sequences indicated that gamma-Proteobacteria (Pseudomonas, Acinetobacter, Alteromonas, and uncultured gamma-Proteobacteria) were predominant in the community during 0 approximately 9 h, while the ratio of alpha-Proteobacteria (Loktanella, Methylobacterium, Pelagibacter, and uncultured alpha-Proteobacteria) increased 2.6 approximately 4.8 folds during 24 approximately 36 h of the biofilm formation, emerging as the most predominant group. Previously, alpha-Proteobacteria were recognized as the pioneering organisms in marine biofilm formation. However, results of this study, which revealed the bacterial succession with finer temporal resolution, indicated some species of gamma-Proteobacteria were more important as the pioneering population. Measures to control pioneering activities of these species can be useful in prevention of marine biofilm formation.     

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.     

Bacterial Communities Predominant in the Degradation of 13C-Labeled Pyrene in Red Soil

The bacterial community composition using a consortium (CON) with or without methyl-β-cyclodextrin (MCD), used to bioremediate the pyrene-contaminated soil was evaluated through stable-isotope probing (SIP). Microcosms were artificially contaminated with ¹³C pyrene at 15 ppm, and bacterial community composition was determined over a 14-day period. After 14 days, only 29.1% of pyrene was degraded in control, while 90.6% of pyrene was degraded by the bacterial community with MCD, showing the best bioremediation rate of all treatments. After 14 days, PAH degraders became dominated by Proteobacteria. Sphingomonas and Pseudomonas were the predominant genera in all treatments, and Pseudomonas was the main degrader from the bacterial community. The bacterial community was little affected by MCD. The results indicated that the combination of the bacterial community with MCD can be used to bioremediate PAHs polluted soil.     

Natural microbial diversity in superficial sediments of Milazzo Harbor (Sicily) and community successions during microcosm enrichment with various hydrocarbons

Hydrocarbon-contaminated superficial sediments collected from the Harbor of Milazzo (Tirrenean Sea, northern Sicily), a zone strongly affected by anthropogenic activities, were examined for in situ biodegradative capacities. A culture-independent molecular phylogenetic approach was used to study the influence of hydrocarbon and nutrient addition on the activity and diversity of the indigenous microbiota during a microcosm evaluation. The autochthonous microbial community in non-polluted sediments was represented by eubacterial phylotypes grouped within Proteobacteria, CFB and Firmicutes. The archaeal domain was represented by members of Marine Group I of Crenarchaeota. The majority of recovered sequences was affiliated with heterotrophic genera Clostridium and Vibrio, typical members of eutrophic coastal environments. Amendments of hydrocarbons and mineral nutrients to microcosms dramatically changed the initial diversity of the microbial community. Only bacterial phylotypes affiliated with Proteobacteria and CFB division were detected. The decrease in diversity observed in several microcosms could be explained by the strong selection for microorganisms belonging to group of marine hydrocarbonoclastic gamma-Proteobacteria, namely Alcanivorax, Cycloclasticus, Marinobacter, Marinobacterium/Neptunomonas and Thalassolituus. This study demonstrated that nutrient amendment to hydrocarbon-contaminated superficial sediments enhanced the indigenous microbial biodegradation activity and that highly specialized marine hydrocarbonoclastic bacteria, representing a minor fraction in the natural microbial community, play an important role in the biodegradation of petroleum hydrocarbons accidentally entering the coastal environment.     

Biodegrader metabolic expansion during polyaromatic hydrocarbons rhizoremediation

Root-microbe interactions are considered to be the primary process of polyaromatic hydrocarbon (PAH) phytoremediation, since bacterial degradation has been shown to be the dominant pathway for environmental PAH dissipation. However, the precise mechanisms driving PAH rhizostimulation symbiosis remain largely unresolved. In this study, we assessed PAH degrading bacterial abundance in contaminated soils planted with 18 different native Michigan plant species. Phenanthrene metabolism assays suggested that each plant species differentially influenced the relative abundance of PAH biodegraders, though they generally were observed to increase heterotrophic and biodegradative cell numbers relative to unplanted soils. Further study of >1800 phenanthrene degrading isolates indicated that most of the tested plant species stimulated biodegradation of a broader range of PAH compounds relative to the unplanted soil bacterial consortia. These observations suggest that a principal contribution of planted systems for PAH bioremediation may be via expanded metabolic range of the rhizosphere bacterial community.     

Comparison of bioremediation strategies for soil impacted with petrochemical oily sludge

Different bioremediation techniques (natural attenuation, biostimulation and bioaugmentation) in contaminated soils with two oily sludge concentrations (1.5% and 6.0%) in open and closed microcosms systems were assessed during 90 days. The results showed that the highest biodegradation rates were obtained in contaminated soils with 6% in closed microcosms. Addition of microbial consortium and nutrients in different concentrations demonstrated higher biodegradation rate of total petroleum hydrocarbons (TPH) than those of the natural attenuation treatment. Soils treated in closed microcosms showed highest removal rate (84.1 ± 0.9%) when contaminated at 6% and bacterial consortium and nutrients in low amounts were added. In open microcosms, the soil contaminated at 6% using biostimulation with the highest amounts of nutrients (C:N:P of 100:10:1) presented the highest degradation rate (78.7 ± 1.3%). These results demonstrate that the application of microbial consortium and nutrients favored biodegradation of TPH present in oily sludge, indicating their potential applications for treatment of the soils impacted with this important hazardous waste.     

Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation

Bioremediation of diesel oil in soil can occur by natural attenuation, or treated by biostimulation or bioaugmentation. In this study we evaluated all three technologies on the degradation of total petroleum hydrocarbons (TPH) in soil. In addition, the number of diesel-degrading microorganisms present and microbial activity as indexed by the dehydrogenase assay were monitored. Soils contaminated with diesel oil in the field were collected from Long Beach, California, USA and Hong Kong, China. After 12 weeks of incubation, all three treatments showed differing effects on the degradation of light (C12-C23) and heavy (C23-C40) fractions of TPH in the soil samples. Bioaugmentation of the Long Beach soil showed the greatest degradation in the light (72.7%) and heavy (75.2%) fractions of TPH. Natural attenuation was more effective than biostimulation (addition of nutrients), most notably in the Hong Kong soil. The greatest microbial activity (dehydrogenase activity) was observed with bioaugmentation of the Long Beach soil (3.3-fold) and upon natural attenuation of the Hong Kong sample (4.0-fold). The number of diesel-degrading microorganisms and heterotrophic population was not influenced by the bioremediation treatments. Soil properties and the indigenous soil microbial population affect the degree of biodegradation; hence detailed site specific characterization studies are needed prior to deciding on the proper bioremediation method.     

Effects of temperature and biostimulation on oil-degrading microbial communities in temperate estuarine waters

Improved strategies for oil-spill remediation will follow a better understanding of the nature, activities and regulating parameters of petroleum hydrocarbon-degrading microbial communities in temperate marine environments. The addition of crude oil to estuarine water resulted in an immediate change in bacterial community structure, increased abundance of hydrocarbon-degrading microorganisms and a rapid rate of oil degradation, suggesting the presence of a pre-adapted oil-degrading microbial community and sufficient supply of nutrients. Relatively rapid degradation was found at 4°C, the lowest temperature tested; and it was temperature rather than nutrient addition that most influenced the community structure. A detailed phylogenetic analysis of oil-degrading microcosms showed that known hydrocarbonoclastic organisms like Thalassolituus and Cycloclasticus , as well as proposed oil degraders like Roseobacter , were present at both 4°C and 20°C, demonstrating the thermo-versatility of such organisms. Clones related to Oleispira antarctica (98% 16S rRNA similarity), a psychrophilic alkane degrader, were dominant in the 4°C oil-degrading community, whereas other clones constituting a different clade and showing 94% similarity 16S rRNA with O. antarctica were found in situ. These findings demonstrate the potential for intrinsic bioremediation throughout the course of the year in temperate estuarine waters, and highlight the importance of both versatile psychrotolerant and specialized psychrophilic hydrocarbon-degrading microbes in effecting this process at low temperatures.     

Monitoring of microbial diversity and activity during bioremediation of crude oil-contaminated soil with different treatments

The present study compared the microbial diversity and activity during the application of various bioremediation processes to crude oil-contaminated soil. Five different treatments, including natural attenuation (NA), biostimulation (BS), biosurfactant addition (BE), bioaugmentation (BA), and a combined treatment (CT) of biostimulation, biosurfactant addition, and bioaugmentation, were used to analyze the degradation rate and microbial communities. After 120 days, the level of remaining hydrocarbons after all the treatments was similar, however, the highest rate (k) of total petroleum hydrocarbon (TPH) degradatioN was observed with the CT treatment (P < 0.05). The total bacterial counts increased during the first 2 weeks with all the treatments, and then remained stable. The bacterial communities and alkane monooxygenase gene fragment, alkB, were compared by denaturing gradient gel electrophoresis (DGGE). The DGGE analyses of the BA and CT treatments, which included Nocardia sp. H17-1, revealed a simple dominant population structure, compared with the other treatments. The Shannon-Weaver diversity index (H') and Simpson dominance index (D), calculated from the DGGE profiles using 16S rDNA, showed considerable qualitative differences in the community structure before and after the bioremediation treatment as well as between treatment conditions.     

Effects of low temperature and freeze-thaw cycles on hydrocarbon biodegradation in Arctic tundra soil.

Degradation of petroleum hydrocarbons was monitored in microcosms with diesel fuel-contaminated Arctic tundra soil incubated for 48 days at low temperatures (-5, 0, and 7 degrees C). An additional treatment was incubation for alternating 24-h periods at 7 and -5 degrees C. Hydrocarbons were biodegraded at or above 0 degrees C, and freeze-thaw cycles may have actually stimulated hydrocarbon biodegradation. Total petroleum hydrocarbon (TPH) removal over 48 days in the 7, 0, and 7 and -5 degrees C treatments, respectively, was 450, 300, and 600 microg/g of soil. No TPH removal was observed at -5 degrees C. Total carbon dioxide production suggested that TPH removal was due to biological mineralization. Bacterial metabolic activity, indicated by RNA/DNA ratios, was higher in the middle of the experiment (day 21) than at the start, in agreement with measured hydrocarbon removal and carbon dioxide production activities. The total numbers of culturable heterotrophs and of hydrocarbon degraders did not change significantly over the 48 days of incubation in any of the treatments. At the end of the experiment, bacterial community structure, evaluated by ribosomal intergenic spacer length analysis, was very similar in all of the treatments but the alternating 7 and -5 degrees C treatment.