Native fungal community remains resilient during bioremediation of DBP pollution by exogenous Gordonia phthalatica QH-11T.

Microbial bioremediation is a highly effective method to degrade phthalates in the environment. However, the response of native microbial communities to the exogenously introduced microorganism remains unknown. In this study, the native fungal community was monitored by amplicon sequencing of the fungal ITS region during the restoration process of the di-n-butyl phthalate (DBP)-contaminated soils with Gordonia phthalatica QH-11T. Our results showed that the diversity, composition, and structure of the fungal community in the bioremediation treatment did not differ from the control, and no significant correlations were found between number of Gordonia and variation of fungal community. It was also observed that DBP pollution initially increased the relative abundance of plant pathogens and soil saprotrophs first, but their proportions returned to the initial level. Molecular ecological network analysis showed that DBP pollution increased the network complexity, while the network was not significantly altered by bioremediation. Overall, the introduction of Gordonia was shown to not have a long-term impact on the native soil fungal community. Therefore, this restoration method can be considered safe in terms of soil ecosystem stability. The present study provides a deeper insight into the effect of bioremediation on fungal communities and provides an extended basis to further explore the ecological risks of introducing exogenous microorganisms.

Whole genome sequencing and transcriptomics-based characterization of a novel ß-cypermethrin-degrading Gordonia alkanivorans GH-1 isolated from fermented foods.

Beta-cypermethrin (ß-CY) is an organic compound that is widely used as a synthetic pesticide in agriculture and family. Excessive accumulation of ß-CY inevitably causes environmental pollution, which has led to food safety and human health concerns. Identification of microorganisms from food sources that are capable of ß-CY biodegradation may help prevent pollution due to ß-CY accumulation. Here, Gordonia alkanivorans GH-1, which was isolated from the traditional Sichuan fermented food, Pixian Doubanjiang, could not only degrade 82.76% of 50 mg/L ß-CY at 96 h, but also degraded the intermediate degradation products including dibutyl phthalate (DBP), benzoic acid (BA) and phenol (Ph). This bacterial strain, thus, effectively improved the efficiency of removal of ß-CY and its related metabolites, without being limited by toxic intermediates. Whole genome sequencing and transcriptomics analyses have demonstrated that the bacteria affected the transcription of genes related to cell response and material transport under the stress induced by ß-CY, and thereby promoted degradation and transformation of ß-CY. Moreover, a complete pathway of ß-CY degradation is proposed based on the key genes involved in degradation. This study provides important theoretical significance and reference value for eliminating pesticide residues in agricultural products and food to ensure food safety.

Bioinformatic characterization of endolysins and holin-like membrane proteins in the lysis cassette of phages that infect Gordonia rubripertincta.

Holins are bacteriophage-encoded transmembrane proteins that function to control the timing of bacterial lysis event, assist with the destabilization of the membrane proton motive force and in some models, generate large "pores" in the cell membrane to allow the exit of the phage-encoded endolysin so they can access the peptidoglycan components of the cell wall. The lysis mechanism has been rigorously evaluated through biochemical and genetic studies in very few phages, and the results indicate that phages utilize endolysins, holins and accessory proteins to the outer membrane to achieve cell lysis through several distinct operational models. This observation suggests the possibility that phages may evolve novel variations of how the lysis proteins functionally interact in an effort to improve fitness or evade host defenses. To begin to address this hypothesis, the current study utilized a comprehensive bioinformatic approach to systematically identify the proteins encoded by the genes within the lysis cassettes in 16 genetically diverse phages that infect the Gram-positive Gordonia rubripertincta NRLL B-16540 strain. The results show that there is a high level of diversity of the various lysis genes and 16 different genome organizations of the putative lysis cassette, many which have never been described. Thirty-four different genes encoding holin-like proteins were identified as well as a potential holin-major capsid fusion protein. The holin-like proteins contained between 1-4 transmembrane helices, were not shared to a high degree amongst the different phages and are present in the lysis cassette in a wide range of combinations of up to 4 genes in which none are duplicated. Detailed evaluation of the transmembrane domains and predicted membrane topologies of the holin-like proteins show that many have novel structures that have not been previously characterized. These results provide compelling support that there are novel operational lysis models yet to be discovered.

Biodegradation of di-n-octyl phthalate by Gordonia sp. Lff and its application in soil.

A previous isolated Gordonia sp. (Lff) was used to degrade di-n-octyl phthalate (DOP) contamination in both aqueous solution and soil. The influence of temperature, pH, inoculum size, salt content and initial concentration of DOP on DOP degradation by Lff were analysed. The response of soil bacterial community to DOP and Lff was also analysed by Illumina MiSeq sequence method. Results showed that the optimal temperature, pH, inoculum size and salt content were 35oC, 8.0, 5% and <5%, respectively. Under the optimal condition, more than 91.25% of DOP with different initial concentrations (100-2000 mg/L) could be degraded by Lff. Kinetics analysis indicated that biodegradation of DOP by Lff could be described by first-order kinetics (R2 > 0.917) with the half-life (t1/2) changing irregularly between 0.58 and 0.83 d. In addition, Lff enhanced the removal of DOP in soil and alleviated the toxicity of DOP on soil microorganisms. Furthermore, its influence on soil bacterial community is not obvious. These results suggested that Lff was effective in remediating DOP contamination in different environments.

Functional characterization of the transcription regulator WhiB1 from Gordonia sp. IITR100.

WhiB is a transcription regulator which has been reported to be involved in the regulation of cell morphogenesis, cell division, antibiotic resistance, stress, etc., in several members of the family Actinomycetes. The present study describes functional characterization of a WhiB family protein, WhiB1 (protein ID: WP_065632651.1), from Gordonia sp. IITR100. We demonstrate that WhiB1 affects chromosome segregation and cell morphology in recombinant Escherichia coli, Gordonia sp. IITR100 as well as in Rhodococcus erythropolis. Multiple sequence alignment suggests that WhiB1 is a conserved protein among members of the family Actinomycetes. It has been reported that overexpression of WhiB1 leads to repression of the biodesulfurization operon in recombinant E. coli, Gordonia sp. IITR100 and R. erythropolis. A WhiB1-mut containing a point mutation Q116A in the DNA binding domain of WhiB1 led to partial alleviation of repression of the biodesulfurization operon. We show for the first time that the WhiB family protein WhiB1 is also involved in repression of the biodesulfurization operon by directly binding to the dsz promoter DNA.

Application of a multiple lines of evidence approach to document natural attenuation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in groundwater.

This study utilized innovative analyses to develop multiple lines of evidence for natural attenuation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in groundwater at the U.S. Department of Energy's Pantex Plant. RDX, as well as the degradation product 4-nitro-2,4-diazabutanal (NDAB; produced by aerobic biodegradation or alkaline hydrolysis) were detected in a large portion of the plume, with lower concentrations of the nitroso-containing metabolites produced during anaerobic biodegradation. 16S metagenomic sequencing detected the presence of bacteria known to aerobically degrade RDX (e.g., Gordonia, Rhodococcus) and NDAB (Methylobacterium), as well as the known anoxic RDX degrader Pseudomonas fluorescens I-C. Proteomic analysis detected both the aerobic RDX degradative enzyme XplA, and the anoxic RDX degradative enzyme XenB. Groundwater enrichment cultures supplied with low concentrations of labile carbon confirmed the potential of the extant groundwater community to aerobically degrade RDX and produce NDAB. Compound-specific isotope analysis (CSIA) of RDX collected at the site showed fractionation of nitrogen isotopes with δ15N values ranging from approximately -5‰ to +9‰, providing additional evidence of RDX degradation. Taken together, these results provide evidence of in situ RDX degradation in the Pantex Plant groundwater. Furthermore, they demonstrate the benefit of multiple lines of evidence in supporting natural attenuation assessments, especially with the application of innovative isotopic and -omic technologies.

Effect of soil organic matter on petroleum hydrocarbon degradation in diesel/fuel oil-contaminated soil.

The purpose of this study is to investigate the effect of soil organic matter (SOM) content levels on the biodegradation of total petroleum hydrocarbons (TPH). Batch experiments were conducted with soils with 2% or 10% organic matter that had been contaminated by diesel or fuel oil. In addition to the TPH (diesel or fuel oil) degradation efficiency, a comprehensive investigation was conducted on the TPH-degrading microbial community using molecular tools including oligonucleotide microarray technique and terminal restriction fragment length polymorphism analysis (T-RFLP). TPH was reduced from 10,000 mg/kg to 1849-4352 mg/kg dry weight soil. Higher biodegradation efficiencies and kinetic rate constants were observed in higher SOM contents. Hydrocarbon fractional analyses were conducted to explain the optimal operation with relatively low resin and aromatic fractions detected at the end of the remediation. The bacterial and fungal counts in the 10% SOM were approximately 10 CFU/g to 102 CFU/g above those in the 2% SOM, and the lowest fungal level was found when the least TPH degradability was measured. The internal transcribed spacer microarray identified the microorganisms that were introduced and proved their survival. The associated growth pattern confirmed that different kinds of contamination oils affected the microbial community diversity over time. Both the microarray and T-RFLP profiles indicated that Gordonia alkanivorans, G. desulfuricans, and Rhodococcus erythoropolis were the dominant bacteria, while Fusarium oxysporum and Aspergillus versicolor were the dominant fungi. The T-RFLP-derived nonmetric multidimensional scaling concluded that the dynamics of the microbial communities were impacted by the TPH degradation stages.

Spatially-distinct redox conditions and degradation rates following field-scale bioaugmentation for RDX-contaminated groundwater remediation.

In situ bioaugmentation for cleanup of an hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-contaminated groundwater plume was recently demonstrated. Results of a forced-gradient, field-scale cell transport test with Gordonia sp. KTR9 and Pseudomonas fluorescens strain I-C cells (henceforth "KTR9" and "Strain I-C") showed these strains were transported 13 m downgradient over 1 month. Abundances of xplA and xenB genes, respective indicators of KTR9 and Strain I-C, approached injection well cell densities at 6 m downgradient, whereas gene abundances (and conservative tracer) had begun to increase at 13 m downgradient at test conclusion. In situ push-pull tests were subsequently completed to measure RDX degradation rates in the bioaugmented wells under ambient gradient conditions. Time-series monitoring of RDX, RDX end-products, conservative tracer, xplA and xenB gene copy numbers and XplA and XenB protein abundance were used to assess the efficacy of bioaugmentation and to estimate the apparent first-order RDX degradation rates during each test. A collective evaluation of redox conditions, RDX end-products, varied RDX degradation kinetics, and biomarkers indicated that Strain I-C and KTR9 rapidly degraded RDX. Results showed bioaugmentation is a viable technology for accelerating RDX cleanup in the demonstration site aquifer and may be applicable to other sites. Full-scale implementation considerations are discussed.

Analysis of genome sequence and trehalose lipid production peculiarities of the thermotolerant Gordonia strain.

Gordoniae are one of the most promising hydrocarbon-oxidizing actinobacteria. Here we present the genome sequence analysis of thermotolerant strain Gordonia sp. 1D isolated from oil-refinery soil. It is capable of alkane consumption and biosurfactant production at temperatures of up to 50°C. Gordonia sp. 1D demonstrates maximum biosurfactant production when grown on hexadecane, and at 40°C it was slightly higher than at 27°C: 35 and 39 mN/m, respectively. For the first time, it was experimentally confirmed that the carbohydrate component of extracellular biosurfactants produced by strain 1D is trehalose. In addition, genes for the production of trehalose lipid biosurfactants were identified. The genetic determinants for two different pathways for trehalose synthesis were found. The strain carries genes otsA and otsB involved in de novo trehalose biosynthesis. Moreover, the genes treY and treZ responsible for trehalose biosynthesis from maltooligosaccharides and starch or glycogen were identified.

Characterization of DNA binding and ligand binding properties of the TetR family protein involved in regulation of dsz operon in Gordonia sp. IITR100.

Gordonia sp. IITR100 is a biodesulfurizing bacterium which can metabolize dibenzothiophene (DBT) to 2 hydroxybiphenyl in four steps via the 4S pathway. The genes involved in the metabolism are present in the form of an operon, dszABC, which gets activated by a TetR family protein. Here, we report the detailed characterization of the DNA binding and ligand binding property of the TetR family protein. The protein was found to be conserved across other desulfurizing organisms. The protein was purified and was found to exist as dimer. The presence of ligand binding site was identified by docking studies and the structural changes in the protein upon ligand binding were determined by CD spectroscopy and tryptophan fluorescence. Further, it was determined that this protein binds to an imperfect palindromic DNA sequence present in the dsz promoter DNA. Binding to the DNA also changes conformation of the protein.

Methylation, sugar puckering and Z-form status of DNA from a heavy metal-acclimated freshwater Gordonia sp.

Heavy metal acclimation of bacteria is of particular interest in many aspects. It could add to our understanding of adaptation strategies applied by bacteria, as well as help us in devising ways to use such adaptive bacteria for bioremediation. In this study, we have explored the changes in the DNA of an aquatic Gordonia sp. acclimated to silver, cadmium, and lead. We have measured the changes in the DNA extracted from the acclimated bacteria by using ATR-FTIR coupled with unsupervised and supervised pattern recognition algorithms. Although whole-cell FTIR studies do reveal nucleic acid changes, the special care should be taken when considering marker nucleic acid bands in such spectra, as various other cell or tissue constituents also yield IR bands in the same region. An FTIR study on isolated DNA can be used to avoid this problem. The IR spectral profiles of the DNA molecules revealed significant changes in the backbone and sugar conformations of upon acclimation. We then further analyzed the DNA's global cytosine-methylation patterns of the heavy metal-acclimated bacteria. We aimed to find out whether epigenetic mechanisms operate in bacteria for survival and growth in inhibitory heavy metal concentrations or not. We found hypermethylation in Cd acclimation but hypomethylation for both Pb and Ag in Gordonia sp. Our results imply that changes in the conformational and methylation states of DNA seem to let bacteria to thrive in otherwise inhibitory conditions and mark the involvement of epigenetic modulation in acclimation processes.

Effects of natural organic matters on bioavailability of petroleum hydrocarbons in soil-water environments.

The bioremediation efficiency of petroleum hydrocarbons in natural soil-water systems is regulated by active microbial populations and other system parameters. Relevant factors include the transfer rate of petroleum contaminants from a medium into microorganisms, the partitioning behavior of contaminants from water into the soil organic matter (SOM), and the influence of the dissolved organic matter (DOM) on the contaminant level in water. The objectives of this study was aimed to determine the correlation among bioavailability of petroleum hydrocarbons, SOM content, and DOM level in soil-water systems. Heptadecane, pristane, and decylcyclohexane were selected as model hydrocarbon contaminants. The bioavailability of target contaminants in soil was examined using soils of different SOM contents (2% and 20%) in slurry bioreactors. In addition, the contaminant bioavailability as affected by various DOM levels (0-100 mgC/L) was also examined. The results showed that the SOM content affected the degrading rate of hydrocarbons significantly, where the rate constant was 4 times higher in 2% SOM microcosm than in the 20% SOM bioreactor for heptadecane degradation. Similarly, the pristane degrading efficiency after 240 h operation was 95% for the 2% SOM microcosm and only 38% for the 20% SOM microcosm. The hydrocarbon degradation rates in water phase were found to be enhanced by the added DOM level. A positive correlation existed between the contaminant bioavailability and the contaminant level in water as impacted by the SOM content in soil and the DOM level in water.

Biodegradation of Structurally Diverse Phthalate Esters by a Newly Identified Esterase with Catalytic Activity toward Di(2-ethylhexyl) Phthalate.

Herein, we report a double enzyme system to degrade 12 phthalate esters (PAEs), particularly bulky PAEs, such as the widely used bis(2-ethylhexyl) phthalate (DEHP), in a one-pot cascade process. A PAE-degrading bacterium, Gordonia sp. strain 5F, was isolated from soil polluted with plastic waste. From this strain, a novel esterase (GoEst15) and a mono(2-ethylhexyl) phthalate hydrolase (GoEstM1) were identified by homology-based cloning. GoEst15 showed broad substrate specificity, hydrolyzing DEHP and 10 other PAEs to monoalkyl phthalates, which were further degraded by GoEstM1 to phthalic acid. GoEst15 and GoEstM1 were heterologously coexpressed in Escherichia coli BL21 (DE3), which could then completely degrade 12 PAEs (5 mM), within 1 and 24 h for small and bulky substrates, respectively. To our knowledge, GoEst15 is the first DEHP hydrolase with a known protein sequence, which will enable protein engineering to enhance its catalytic performance in the future.

Biodegradation of di-(2-ethylhexyl) phthalate by a newly isolated Gordonia sp. and its application in the remediation of contaminated soils.

A bacterial strain (Gordonia sp. Lff) capable of efficiently degrading di-(2-ethylhexyl) phthalate (DEHP) was isolated from river sludge. The optimal pH and temperature for the degradation of DEHP by Lff were 7.0 and 35 °C, respectively. Lff could degrade high concentrations of DEHP (100-2000 mg/L) with a degradation efficiency of over 91.43%. The DEHP degradation curves fit well with first-order kinetics, with a half-life ranging from 0.598 to 0.746 d. Substrate inhibition analyses showed that the maximum specific degradation rate, half-saturation constant and inhibition constant were 0.8 d-1, 45.8 mg/L and 462.18 mg/L, respectively. A detailed biodegradation pathway of DEHP was proposed based on GC-MS analysis. Furthermore, Lff could also efficiently degrade DEHP in soils. DEHP or DEHP plus Lff changed the bacterial community in soils, and Lff accelerated the shaping of the bacterial community. To the best of our knowledge, this study is the first to perform a detailed investigation into the biodegradation of DEHP in soil by Gordonia sp. and its effect on the soil bacterial community. These results suggest that Lff is an ideal candidate for the bioremediation of DEHP-contaminated environments.

Enhanced plasmid-mediated bioaugmentation of RDX-contaminated matrices in column studies using donor strain Gordonia sp. KTR9.

Horizontal gene transfer (HGT) is the lateral movement of genetic material between organisms. The RDX explosive-degrading bacterium Gordonia sp. KTR9 has been shown previously to transfer the pGKT2 plasmid containing the RDX degradative genes (xplAB) by HGT. Overall, fitness costs to the transconjugants to maintain pGKT2 was determined through growth and survivability assessments. Rhodococcus jostii RHA1 transconjugants demonstrated a fitness cost while other strains showed minimal cost. Biogeochemical parameters that stimulate HGT of pGKT2 were evaluated in soil slurry mating experiments and the absence of nitrogen was found to increase HGT events three orders of magnitude. Experiments evaluating RDX degradation in flow-through soil columns containing mating pairs showed 20% greater degradation than columns with only the donor KTR9 strain. Understanding the factors governing HGT will benefit bioaugmentation efforts where beneficial bacteria with transferrable traits could be used to more efficiently degrade contaminants through gene transfer to native populations.

Bioremediation of dibutyl phthalate in a simulated agricultural ecosystem by Gordonia sp. strain QH-11 and the microbial ecological effects in soil.

Bioremediation of organic pollutants has been identified as an economically efficient and environmentally friendly method. Here, a pot experiment was conducted to evaluate the bioremediation efficiency of dibutyl phthalate (DBP) by Gordonia phthalatica sp. nov. QH-11 in agricultural soils, along with the effect of this exogenous organism on the native microbial community and ecosystem functions during the bioremediation process. The results showed that inoculation with strain QH-11 accelerated DBP degradation in the soil and decreased DBP accumulation in plants, thereby reducing the health risks associated with vegetables grown in those soils. High-throughput sequencing demonstrated that both DBP contamination and the bioremediation process significantly altered prokaryotic community composition, structure, and network interactions; however, these effects were greatly reduced after 30 d. Dibutyl phthalate affected the prokaryotic community by influencing soil properties rather than directly impacting on microorganisms. In addition, ecosystem functions, like the nitrogen cycle, were significantly altered. Contamination with DBP promoted nitrogen fixation and the denitrification processes while inhibiting nitrification. Bioremediation may mitigate some of the changes to nitrogen cycling, helping to maintain the balance of prokaryotic community function. According to this study, bioremediation through highly efficient degradation bacteria may be a safe and promising method for reducing PAEs contamination in soil-vegetable systems.

Biodesulfurization of diesel oil in oil-water two phase reaction system by Gordonia sp. SC-10.

OBJECTIVES: Different sulfur contents of diesel oils were used for biodesulfurization to study the desulfurization capacity of Gordonia sp. SC-10 in oil-water two-phase reaction system. RESULTS: Gordonia sp. SC-10 showed great properties in desulfurizing diesel oil with different sulfur contents. This bacterium could decrease sulfur contents in different diesel oils from 194.7 ± 3.7 to 30.4 ± 0.5 mg/l and from 3035.3 ± 23.8 to 1792.8 ± 48.9 mg/l, respectively. Furthermore, this bacterium could desulfurize broad range of organosulfur compounds and had strong desulfurization activity against alkylated DBTs. For low-sulfur diesel oil, sulfur could be removed from 10.2 ± 0.1 to 5.0 ± 0.1 mg/l. CONCLUSIONS: The newly isolated bacteria Gordonia sp. SC-10 showed a good performance in desulfurizing diesel oils, and it might be a useful desulfurizing biocatalyst to enable the industrialized application of biodesulfurization process.

Harnessing the catabolic versatility of Gordonia species for detoxifying pollutants.

The genus Gordonia includes variedly pigmented aerobic, non-motile, non-sporulating Gram positive (sometimes variable) coccoid forms and rods. Different isolates display distinguishing physiological traits and biochemical properties that are significant in remediation applications. Strains inherently prevalent in soils, seawater, sediments and wastewaters can degrade hydrocarbons. Immobilized cells and microbial consortia containing Gordonia species have been used for in situ applications. Hydrocarbon uptake in this Actinomycete is mediated by attachment to large droplets or by pseudosolubilization of substrates. Hydrocarbons so internalized are degraded by relevant enzymes that are innately present in this microorganism. Wild-type and recombinant strains also mediate desulfurization of polyaromatic sulfur heterocyclic compounds. This organism is metabolically capable of bringing about detoxification of phthalate esters. Two species namely, Gordonia polyisoprenivorans and Gordonia westfalica mediate degradation of rubber and the metabolic pathways involved in the process have been well-understood. Some members are able to transform nitriles into commercially valuable products and others degrade the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine. Cholesterol, pyridine derivatives, fuel oxygenates, thiodiglycol, bis-(3-pentafluorophenylpropyl)-sulfide and 6:2 fluorotelomersulfonate are also biotransformed or degraded by Gordonia species. Some members of this genus are significant in the treatment of wastewaters including those that are rich in steroids and lignin. There are also several patents highlighting the commercial relevance of this genus. On account of its diverse catabolic properties, this Actinomycete has become important in bioremediation of polluted environments.

LcpRVH2 - regulating the expression of latex-clearing proteins in Gordonia polyisoprenivorans VH2.

Gordonia polyisoprenivorans VH2 harbours two latex clearing proteins, which are responsible for the cleavage of poly(cis-1,4-isoprene) into oligoisoprenes, thereby allowing growth in presence of, e.g. natural rubber. A gene coding for a putative regulator of the TetR-family (lcpRVH2) is located 131 bp upstream of lcp1VH2. We heterologously expressed lcpRVH2 in Escherichia coli, and purified and characterized the protein with respect to its ability to bind to the operator region of lcp1VH2. LcpRVH2 forms a dimer in its native state. The size of the dimer was determined to be 52.7 kDa by size exclusion chromatography, whereas the calculated size of a monomer was 24.1 kDa. Electrophoretic mobility shift assays (EMSAs) with the purified protein revealed a shift upon binding to the intergenic region between lcpRVH2 and lcp1VH2. Within this region, an inverted repeat was identified in silico, probably being the binding site of LcpRVH2. This binding sequence was confirmed by a DNase I footprinting assay. A shift also occurred in EMSAs with this 44 bp sequence only. Interestingly, no regulator was detected upstream of the second lcp (lcp2VH2). Therefore, we performed EMSA studies with LcpRVH2 and the putative operator region upstream of lcp2VH2, and discovered by DNase I footprinting another binding sequence upstream of lcp2VH2. Hence, we concluded that LcpRVH2 binds the operator region of both lcps and, most likely, regulates their expression in G. polyisoprenivorans VH2.

Biodegradation of A-fuel oil in soil samples with bacterial mixtures of Rhodococcus and Gordonia strains under low temperature conditions.

Bioaugmentation is an effective treatment to clean up polluted sites using contaminant-degrading bacteria. However, this treatment is influenced by various environmental conditions, including temperature. In this study, an effective bioaugmentation system under low temperature condition was developed with three Rhodococcus (strains A, C, and D) and one Gordonia (strain B) oil-degraders, which are officially permitted for bioaugmentation applications in Japan. The oil-degrading ability of each strain and mixture was assessed in liquid culture and in model soils supplemented with A-fuel oil. In liquid culture, Rhodococcus strains A and C degraded the A-fuel oil in cold temperature conditions (15°C and 10°C) as well as in mesophilic condition (30°C). In the model soil samples, the mixture of four degraders was the most effective at removing the A-fuel oil under mesophilic condition (>90%), suggesting that strains B and/or D might have factors that promote degradation. In contrast, A-fuel oil was efficiently removed (>80%) in the soil samples inoculated with A or C as well as that with mixture in cold temperature condition, suggesting that strains A and C were the major degraders under cold condition. Our results indicate that the four degraders could be applied to the bioaugmentation in cold areas.