Characterization of hydrocarbon-degrading microbial populations in contaminated and pristine Alpine soils

Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).     

Temporal hydrochemical and microbial variations in microcosm experiments from sites contaminated with chloromethanes under biostimulation with lactic acid

The aim of this research was to identify the sequence of degradation processes that leads to the selective enrichment of microorganisms involved in the degradation of carbon tetrachloride and chloroform under conditions of natural attenuation and lactic acid biostimulation. To this end, a comparative study using microcosm experiments was conducted to analyze these two scenarios. The authors used groundwater and sediment collected from a field site located at a petrochemical complex to create the microcosms. Chemical, compound-specific isotope and microbial analyses were performed. A significant finding of this work was the abiotic degradation of carbon tetrachloride. Another result was the identification of biotic reductive dechlorination of chloroform by a bacterium of the Clostridiales order. This study showed that biostimulation with lactic acid produced faster degradation rates of carbon tetrachloride and chloroform. Lactic acid acted as an electron donor and promoted a decrease in the concentration of other electron acceptors such as nitrate and sulfate, which competed with chloromethanes. Thus, biostimulation could be an efficient remediation strategy for sites contaminated with chloromethanes, especially when a site's complex pollution history results in chemical background concentrations that are high in compounds that could potentially reduce natural attenuation.     

Effects of salinity on bacterioplankton: field and microcosm experiments

Bacterial populations in a tidal estuary were monitored with the aim of investigating (i) the relationship between planktonic bacteria and salinity and (ii) the survival of allochthonous bacteria in microcosm experiments at different salinities. With the increase in salinity, bacterial numbers decreased in a curvilinear fashion, rather than monotonically. Maximal abundance for different bacterial groups was found between 7 and 22%₀ S. The salinity alone explained between 19 and 58% of bacterial variability. In microcosm experiments the adverse effect of salinity on the survival of freshwater bacteria and indicators of faecal contamination was particularly important. In freshwater and low salinity experiments grazing by protozoans had similar effects on the survival of allochthonous bacteria.     

Molecular community analysis of microbial diversity

New technologies that avoid the need for either gene amplification (e.g. microarrays) or nucleic acid extraction (e.g. in situ PCR) have recently been implemented in microbial ecology. Together with new approaches for culturing microorganisms and an increased understanding of the biases of molecular methods, these techniques form the most exciting advances in this field during the past year.     

Effects of a bacterial algicide,IRI-160AA,on dinoflagellates and the microbial community in microcosm experiments

Filtrates from the bacterium Shewanella sp. IRI-160 (termed IRI-160AA) have been shown to inhibit population growth and kill a variety of dinoflagellates grown in culture. Here we test the immediate efficacy of IRI-160AA in laboratory microcosms initiated from three natural dinoflagellate blooms (Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum). We measured target dinoflagellate abundance, total chlorophyll-a, photosystem II (PSII) photochemistry, and changes to the prokaryotic and eukaryotic community composition over 2–3 days of IRI-160AA incubation. Naked dinoflagellates were impacted more, while abundance of the thecate P. minimum was not affected. However, dinoflagellate growth inhibition was generally lower than that observed in uni-algal cultures, and took longer to occur. Eukaryotic community composition in IRI-160AA treated microcosms was significantly different from control incubations, and was driven predominantly by increases in heterotrophic protists (e.g. Euplotes sp. and Paraphysomonas sp.). Similarly, significant changes to the prokaryotic community structure were evident. Microcosms of G. instriatum with higher algicide concentrations indicated that algicidal activity was enhanced in a dose dependent manner. Furthermore, total ciliate abundance as well as a bactivorous chyrsophyte (Paraphysomonas sp.) increased in a dose dependent manner. Total diatom abundance increased at lower IRI-160AA concentrations, but increased less with increasing dose. Overall, the bio-activity of IRI-160AA on naturally occurring dinoflagellates in mixed natural microbial communities is encouraging from the applied perspective of using the active compound(s) in IRI-160AA as natural agent(s) to manage harmful dinoflagellate blooms.     

Bacterial community changes in TCE biodegradation detected in microcosm experiments

Laboratory microcosm experiments were set up to model biodegradation of trichloroethylene (TCE). Groundwater samples from two contaminated sites were taken, one of them with low (70 mg L⁻¹), the other with high sulfate (685 mg L⁻¹) concentration. In order to assess the biodegradative potential of natural microbiota, supplementary substrates (whey or molasses) were added to the bottles. At day 54, 98, 155, and 318, chemical and bacteriological parameters (i.e., Dehalococcoides test) were investigated. Terminal restriction fragment length polymorphism (T-RFLP) based diversity assessments were carried out to observe the bacterial community changes. Whey and molasses enhanced degradation at different rates. In the case of samples with high sulfate content and amended with whey, no ethylene, ethane, or methane was generated. Both ethylene and methane production was detected in samples of low sulfate content with added whey. The results of Dehalococcoides tests were positive for all control and amended samples. Based on T-RFLP analysis, the bacterial communities of high sulfate concentration groundwater microcosms amended with molasses or whey were very similar, while the communities of groundwater samples with low sulfate concentration were different when supplemented with whey or molasses. The rRNA and rDNA based investigations suggest that the proportions of the active microbes and the microbes present in the microcosms differ.     

Bacterial community changes in TCE biodegradation detected in microcosm experiments

Laboratory microcosm experiments were set up to model biodegradation of trichloroethylene (TCE). Groundwater samples from two contaminated sites were taken, one of them with low (70 mg L⁻¹), the other with high sulfate (685 mg L⁻¹) concentration. In order to assess the biodegradative potential of natural microbiota, supplementary substrates (whey or molasses) were added to the bottles. At day 54, 98, 155, and 318, chemical and bacteriological parameters (i.e., Dehalococcoides test) were investigated. Terminal restriction fragment length polymorphism (T-RFLP) based diversity assessments were carried out to observe the bacterial community changes. Whey and molasses enhanced degradation at different rates. In the case of samples with high sulfate content and amended with whey, no ethylene, ethane, or methane was generated. Both ethylene and methane production was detected in samples of low sulfate content with added whey. The results of Dehalococcoides tests were positive for all control and amended samples. Based on T-RFLP analysis, the bacterial communities of high sulfate concentration groundwater microcosms amended with molasses or whey were very similar, while the communities of groundwater samples with low sulfate concentration were different when supplemented with whey or molasses. The rRNA and rDNA based investigations suggest that the proportions of the active microbes and the microbes present in the microcosms differ.     

Dynamics of seagrass bed microbial communities in artificial Chattonella blooms: A laboratory microcosm study

The influence of algicidal and growth-inhibiting bacteria in a seagrass (Zostera marina) bed, and their capability of controlling blooms of the fish-killing raphidophyte flagellate, Chattonella antiqua, were examined in laboratory microcosm experiments. Bacterial communities in seawater collected from the seagrass bed and Z. marina biofilm suppressed artificial Chattonella blooms in the presence of their natural competitors and predators. Phylogenetic analysis suggest that considerable numbers of bacteria that suppress Chattonella, including algicidal or growth-inhibiting bacteria isolated from seagrass biofilm and seawater from the seagrass bed, are members of Proteobacteria that can decompose lignocellulosic compounds. A direct comparison of partial 16S rRNA gene sequences (500 bp) revealed that the growth-limiting bacterium (strain ZM101) isolated from Z. marina biofilm belonged to the genus Phaeobacter (Alphaproteobacteria) showed 100% similarity with strains of growth-limiting bacteria isolated from seawater of both the seagrass bed and nearshore region, suggesting that the origin of these growth-limiting bacteria are the seagrass biofilm or seawater surrounding the seagrass bed. This study demonstrates that Chattonella growth-limiting bacteria living on seagrass biofilm and in the adjacent seawater can suppress Chattonella blooms, suggesting the possibility of Chattonella bloom prevention through restoration, protection, or introduction of seagrass in coastal areas.     

人工微宇宙下粘细菌捕食对微生物群落结构的影响

【目的】人工微宇宙条件下测试粘细菌捕食对微生物群落结构的影响,模拟粘细菌捕食对微生态系统的调控作用。【方法】采用Lawn predation法,测定粘细菌EGB对9种猎物菌的捕食直径,以确定其对猎物菌的捕食能力。通过高通量测序技术分析粘细菌捕食引起的微生物群落结构变化。【结果】粘细菌EGB对9种不同猎物菌的捕食能力差异显著,粘细菌对热带芽孢杆菌的捕食能力显著高于其他细菌(P<0.05)。在含有9种猎物细菌的人工微宇宙系统中添加不等量粘细菌,均可显著降低细菌群落的多样性指数(Shannon)。PCoA结果表明粘细菌捕食可影响微宇宙微生物群落结构。人工微宇宙培养24 h后,7种猎物菌相对丰度显著降低(LefSe,P<0.05),但洋葱伯克霍尔德菌的相对丰度显著升高(P<0.05)。人工微宇宙实验的结果表明,粘细菌添加是造成其微生物群落结构改变的主要影响因素,且添加最小剂量的粘细菌(1 mL)也有显著的影响效果;随着培养时间的增加,洋葱伯克霍尔德菌是唯一能抵抗粘细菌捕食并具有较高丰度的猎物细菌。【结论】粘细菌捕食能够调控微宇宙中微生物的群落结构,为其对土壤生态的调控研究奠定了理论基础。     

Comparative phylogenetic analysis of microbial communities in pristine and hydrocarbon-contaminated Alpine soils

A molecular characterization of pristine and petroleum hydrocarbon-contaminated Alpine soils sampled in Tyrol (Austria) was performed. To identify predominant bacteria, PCR-amplified 16S rRNA gene fragments from five pristine and nine contaminated soils were analysed using denaturing gradient gel electrophoresis (DGGE). Sequencing and phylogenetic analyses demonstrated that the majority of the DGGE bands represented bacteria in the Actinobacteria and Proteobacteria phyla: 18 and 73%, respectively, in pristine soils, compared with 20 and 76%, respectively, in contaminated soils. A different distribution pattern of bacterial classes in the Proteobacteria was observed between pristine and contaminated soils. The relative proportion of microorganisms belonging to the Alphaproteobacteria was larger in pristine (46%) than in contaminated (24%) soils, while Betaproteobacteria and Gammaproteobacteria were detected only in the hydrocarbon-contaminated soils. This result compared favourably with earlier work in which hydrocarbon-degradation genotypes, largely pseudomonads and Acinetobacter, belonging to the Gammaproteobacteria, were enriched following oil hydrocarbon contamination. In contrast, members of the Actinobacteria phylum, represented by Rhodococcus and Mycobacterium, were found in pristine soils where contamination events had not occurred. The results demonstrate a significant shift in the microbial community structure in Alpine soils following contamination. Furthermore, more potentially novel phylotypes were found in the pristine soils than in the contaminated soils.     

Molecular analysis of microbial community structure in an arsenite-oxidizing acidic thermal spring

Electron microscopy (EM), denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing were used to examine the structure and diversity of microbial mats present in an acid-sulphate–chloride (pH 3.1) thermal (58–62°C) spring in Norris Basin, Yellowstone National Park, WY, USA, exhibiting rapid rates of arsenite oxidation. Initial visual assessments, scanning EM and geochemical measurements revealed the presence of three distinct mat types. Analysis of 16S rDNA fragments with DGGE confirmed the presence of different bacterial and archaeal communities within these zones. Changes in the microbial community appeared to coincide with arsenite oxidation activity. Phylogenetic analysis of 1400 bp 16S rDNA sequences revealed that clone libraries prepared from both arsenic redox active and inactive bacterial communities were dominated by sequences phylogenetically related to Hydrogenobacter acidophilus and Desulphurella sp. The appearance of archaeal 16S rDNA sequences coincided with the start of arsenite oxidation, and sequences were obtained showing affiliation with both Crenarchaeota and Euryarchaeota . The majority of archaeal sequences were most similar to sequences obtained from marine hydrothermal vents and other acidic hot springs, although the level of similarity was typically just 90%. Arsenite oxidation in this system may result from the activities of these unknown archaeal taxa and/or the previously unreported arsenic redox activity of H. acidophilus - or Desulphurella -like organisms. If the latter, arsenite oxidation must be inhibited in the initial high-sulphide zone of the spring, where no change in the distribution of arsenite versus arsenate was observed.     

Comparing field and microcosm experiments: a case study on methano- and methylo-trophic bacteria in paddy soil

Methane-oxidising bacteria (MOB) play an important role in the reduction of methane emissions from rice agriculture. In rice fields, they are subjected to many environmental and field management parameters, which may have a significant impact on their community composition. To study this in greater detail, the community structure of methano- and methylo-trophic bacteria was investigated in a rice field in northern Italy during the summer 1999 and compared to a microcosm study described previously. We used PCR-based denaturing gradient gel electrophoresis applying 16S rDNA (9alpha and 10gamma) and mxaF (methanol-dehydrogenase) primer sets. In parallel, population size and activity of MOB were determined. This study provides the first comprehensive investigation of different compartments (bulk soil, rhizosphere, rhizoplane, and homogenate) throughout an entire rice-growing season in the field. Lower cell numbers of MOB were detected in the field compared to the microcosms, possibly due to lower CH4 concentrations in the soil pore water. In both studies, growth of MOB occurred predominantly at the root surface (rhizoplane) and in the root (homogenate), whereas cell numbers in bulk soil showed only minor changes throughout the season. Molecular analysis detected only few changes in alpha-proteobacterial methylotrophs during the season, whereas a higher variability was detected in gamma-proteobacteria. Nevertheless, the sequences of electrophoretic bands showed that the diversity in the field study and in the microcosms was comparable. Activity patterns of MOB and the population structure of methylotrophic bacteria agreed well between both studies, even though the detected quantities differed. Extrapolations of microcosm data to the field scale are thus possible, but should be used carefully when concerning quantitative changes.     

Testing the differential predation hypothesis for the invasion of rusty crayfish in a stream community: laboratory and field experiments

1. We tested the hypothesis that the non‐native rusty crayfish (Orconectes rusticus) is less vulnerable to predators than two native species (O. propinquus and O. obscurus) it is replacing in streams of the upper Susquehanna River catchment (New York, U.S.A.). 2. We used laboratory experiments to compare species‐specific predation rates by smallmouth bass (Micropterus dolomieu) on crayfish of equal size and field tethering experiments to compare relative predation rates between native O. propinquus and non‐native O. rusticus by the suite of crayfish predators in our system. We predicted that crayfish size would affect predation rate but that predation rates would be equal among species when size was controlled. 3. We also tested for two potential artefacts of tethering. We tethered crayfish in cages to test whether the ability to escape from tethers is size specific, and we tested whether tethering alters differential predation among crayfish species by the smallmouth bass. 4. In the laboratory, smallmouth bass predation on rusty crayfish was lower than on either of the native species. In the field, predation risk for tethered crayfish was inversely related to size but did not differ among species when size was taken into account. Because rusty crayfish in the field experiment were slightly larger than the native species, as in nature, mortality was overall lower for the rusty crayfish. 5. In cages, smaller crayfish were more probably to escape from tethers than larger ones, an artefact that may partially confound results from our tethering experiments. Unexpectedly, tethering nearly eliminated predation by smallmouth bass. This artefact prevented us from testing for an interaction of tethering with differential predation and means that the results of field tethering experiments do not include any contribution from smallmouth bass predation. 6. Our experiments highlight the importance of explicitly considering potential artefacts that could confound results. 7. Our results indicate that differential predation contributes to the rusty crayfish's invasion of a stream community. In our study system, predation rates on rusty crayfish are lower than for native species mostly because of selection by predators for smaller crayfish; species‐specific characteristics such as behaviour that further reduce predation may also contribute, especially where smallmouth bass predation is important.     

Phylogenetic characterization of a polychlorinated-dioxin- dechlorinating microbial community by use of microcosm studies

Microcosms capable of reductive dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed in glass bottles by seeding them with a polluted river sediment and incubating them anaerobically with an organic medium. All of the PCDD/F congeners detected were equally reduced without the accumulation of significant amounts of less-chlorinated congeners as the intermediate or end products. Alternatively, large amounts of catechol and salicylic acid were produced in the upper aqueous phase. Thus, the dechlorination of PCDD/Fs and the oxidative degradation of the dechlorinated products seemed to take place simultaneously in the microcosm. Denaturing gel gradient electrophoresis and clone library analyses of PCR-amplified 16S rRNA genes from the microcosm showed that members of the phyla Firmicutes, Proteobacteria, and Bacteroidetes predominated. A significant number of Chloroflexi clones were also detected. Quantitative real-time PCR with specific primer sets showed that the 16S rRNA genes of a putative dechlorinator, "Dehalococcoides," and its relatives accounted for 0.1% of the total rRNA gene copies of the microcosm. Most of the clones thus obtained formed a cluster distinct from the typical "Dehalococcoides" group. Quinone profiling indicated that ubiquinones accounted for 18 to 25% of the total quinone content, suggesting the coexistence and activity of ubiquinone-containing aerobic bacteria. These results suggest that the apparent complete dechlorination of PCDD/Fs found in the microcosm was due to a combination of the dechlorinating activity of the "Dehalococcoides"-like organisms and the oxidative degradation of the dechlorinated products by aerobic bacteria with aromatic hydrocarbon dioxygenases.     

Molecular and ecological analyses of microbial community structures in biofilms of a full-scale Aerated Up-Flow Biobead process

Molecular and cultivation techniques were used to characterize the bacterial communities of biobead reactor biofilms in a sewage treatment plant to which an Aerated Up-Flow Biobead process was applied. With this biobead process, the monthly average values of various chemical parameters in the effluent were generally kept under the regulation limits of the effluent quality of the sewage treatment plant during the operation period. Most probable number (MPN) analysis revealed that the population of denitrifying bacteria was abundant in the biobead #1 reactor, denitrifying and nitrifying bacteria coexisted in the biobead #2 reactor, and nitrifying bacteria prevailed over denitrifying bacteria in the biobead #3 reactor. The results of the MPN test suggested that the biobead #2 reactor was a transition zone leading to acclimated nitrifying biofilms in the biobead #3 reactor. Phylogenetic analysis of 16S rDNA sequences cloned from biofilms showed that the biobead #1 reactor, which received a high organic loading rate, had much diverse microorganisms, whereas the biobead #2 and #3 reactors were dominated by the members of Proteobacteria. DGGE analysis with the ammonia monooxygenase (amoA) gene supported the observation from the MPN test that the biofilms of September were fully developed and specialized for nitrification in the biobead reactor #3. All of the DNA sequences of the amoA DGGE bands were very similar to the sequence of the amoA gene of Nitrosomonas species, the presence of which is typical in the biological aerated filters. The results of this study showed that organic and inorganic nutrients were efficiently removed by both denitrifying microbial populations in the anaerobic tank and heterotrophic and nitrifying bacterial biofilms well-formed in the three functional biobead reactors in the Aerated Up-Flow Biobead process.     

Ecological effects of Irgarol 1051 and Diuron on a coastal meiobenthic community: A laboratory microcosm experiment

After the Tributyltin world ban, Irgarol 1051 and Diuron have been the most commonly used biocides in antifouling paints. When adsorbed to suspended particulate matter or introduced as paint particles, these compounds accumulate in marine sediments and potentially cause ecological damage to benthic organisms. Therefore, a microcosm experiment was designed to evaluate the effects of Irgarol 1051 and Diuron, individually, on a meiofaunal community with emphasis on the dominant nematode assemblages. The experiment tested two factors: “Treatment” (two types of controls and three environmentally relevant concentrations of each contaminant) and “Exposure time” (5, 15 and 30 days). Significant declines in meiofauna density, nematode species richness and diversity, and changes in multivariate community structure were observed for both biocides at all exposure levels when compared to controls. Decreases occurred early on, within five days of exposure, which suggests that mortality, and not sub-lethal effects, has befallen upon the organisms. Sediment chlorophyll a and pheopigment concentrations, and redox potential were monitored to verify any indirect effects to the fauna through changes in the environment and results gave no indications of such mediated effects pointing to a direct toxic effect of both Irgarol and Diuron on the meiofauna. Although contaminated treatments showed a significant decline in the relative abundances of a particular functional group represented by the larger, longer-lived species, we did not observe the typical expected switch to smaller, more opportunistic taxa. Indeed, differences between controls and contaminated treatments were mainly due to an overall reduction in densities of the most abundant species in contaminated treatments. The high mortality (ca. 50% decline in total abundances), changes in community structure and species loss observed at biocide levels frequently encountered in the field suggest Irgarol and Diuron as a threat to benthic communities. Such severe effects contrast to other studies that have detected lower impacts, suggesting the free-living nematodes as potential indicators of marine pollution and the microcosm approach using natural communities as an impending tool for ecotoxicological studies.     

Soil microbial community analysis of between no-till and tillage in a controlled horticultural field

The present study evaluated the changes of soil microbial communities that were subjected to no-till and compared the results to those subject to tillage for organic farming in a controlled horticultural field by fatty acid methyl ester. Fungi (P < 0.001), gram-positive bacteria (P < 0.001), arbuscular mycorrhizal fungi (P < 0.01), and actinomycetes (P < 0.01) in the no-till soils were significantly larger than those in the tillage soils. The no-till in the subsoil had a significantly lower ratio of cy17:0 to 16:1ω7c compared to that of tillage, indicating that microbial stress decreased because the soils were not disturbed (P < 0.05). Fungi should be considered as a potential factor responsible for the obvious microbial community differentiation that was observed between the no-till and tillage areas in a controlled horticultural field.     

Bioremediation of petroleum hydrocarbon contaminated soil: Effects of strategies and microbial community shift

Biodegradation of petroleum hydrocarbon oil (14,000 mg kg⁻¹) were investigated in six biopiles batches, differing in the remediation strategy: bioaugmentation (selected consortium and kitchen waste were introduced), biostimulation (added with rhamnolipid, high-level, or low-level nutrient), and bioaugmentation plus biostimulation (added both with rhamnolipid and bacterial consortia). After the 140-day operation, the kitchen waste (KW) and the low-level nutrient (NEL) batches achieved the highest total petroleum hydrocarbon degradation efficiency (>80%). The result of the hydrocarbon analysis revealed that the bioaugmentation approaches were the most effective ones in removing aromatic component (64% and 68%), and KW and NEL were the only two approaches that can remove the polar component with positive efficiency, 11% and 21%, respectively. The terminal-restriction fragment length polymorphism percentage (T-RFLP) abundance applied with nonmetric multidimensional scaling indicated a similarity of the bacterial communities during the early fastest remediation stage. The results of the oligonucleotide array targeting the ribosomal internal transcribed spacer (ITS) region, along with the hydrocarbon fractional analysis, indicated a successive degradation completed by the bacterial-fungi consortia. Before Day 70, the bacterial community was dominant in decomposing the saturated and partially aromatic hydrocarbons. After Day 70, the fungal community found to be dynamic and responsible for degradation of the polar hydrocarbons composing of recalcitrant metabolites.