Biodegradation of fuel oil hydrocarbons by a mixed bacterial consortium in sandy and loamy soils

The aerobic degradation of light fuel oil in sandy and loamy soils by an environmental bacterial consortium was investigated. Soils were spiked with 1 or 0.1% of oil per dry weight of soil. Acetone extracts of dried soils were analyzed by GC and the overall degradation was calculated by comparison with hydrocarbon recovery from uninoculated soils. In sandy soils, the sum of alkanes n-C(12) to n-C(23) was degraded to about 45% within 6 days at 20 degrees C and to 27-31% within 28 days, provided that moisture and nutrients were replenished. Degradation in loamy soil was about 12% lower. The distribution of recovered alkanes suggested a preferential degradation of shorter chain molecules (n-C(12) to n-C(16)) by the bacterial consortium. Partial 16S rDNA sequences indicated the presence of strains of Pseudomonas aeruginosa, Pseudomonas citronellolis, and Stenotrophomonas maltophilia. Toxicity tests using commercial standard procedures showed a moderate inhibition of bacterial activity. The study showed the applicability of a natural microbial community for the degradation of oil spills into soils at ambient temperatures.     

Sulfur-selective desulfurization of dibenzothiophene and diesel oil by newly isolated Rhodococcus sp. strains

New desulfurizing bacteria able to convert dibenzothiophene into 2-hydroxybiphenyl and sulfate were isolated from contaminated soils collected in Mexican refineries. Random amplified polymorphic DNA analysis showed they were different from previously reported Rhodococcus erythropolis desulfurizing strains. According to 16S rRNA gene sequencing and fatty acid analyses, these new isolates belonged to the genus Rhodococcus. These strains could desulfurize 4,6-dimethyldibenzothiophene which is one of the most difficult dibenzothiophene derivatives to remove by hydrodesulfurization. A deeply hydrodesulfurized diesel oil containing significant amounts of 4,6-dimethyldibenzothiophene was treated with Rhodococcus sp. IMP-S02 cells. Up to 60% of the total sulfur was removed and all the 4,6-dimethyldibenzothiophene disappeared as a result of this treatment.     

Degradation of 1,3-dichloropropene by a soil bacterial consortium and Rhodococcus sp. AS2C isolated from the consortium

A bacterial consortium capable of degrading the fumigant 1,3-D ((Z)- and (E)-1,3-dichloropropene) was enriched from an enhanced soil. This mixedculture degraded (Z)- and (E)-1,3-D only in the presence of a suitable biodegradable organic substrate, such as tryptone, tryptophan, or alanine. After 8 months of subculturing at 2- to 3-week intervals, a strain of Rhodococcus sp. (AS2C) that was capable of degrading 1,3-D cometabolically in the presenceof a suitable second substrate was isolated. (Z)-3-chloroallyl alcohol (3-CAA) and (Z)-3-chloroacrylic acid (3-CAAC), and (E)-3-CAA and (E)-3-CAAC were the metabolites of (Z)- and (E)-1,3-D, respectively. (E)-1,3-D was degraded faster than (Z)-1,3-D by the strain AS2C and the consortium. AS2C also degraded (E)-3-CAA faster than (Z)-3-CAA. Isomerization of (E)-1,3-D to (Z)-1,3-D orthe (Z) form to the (E) form did not occur.     

Studies on composition and stability of a large membered bacterial consortium degrading phenol

A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l⁻¹ phenol for four years at 28 ± 3 °C. The consortium could degrade 99% of 500 mg l⁻¹ phenol after 24 hours incubation with a biomass increase of 2.6 × 10⁷ to 4 × 10¹² CFU ml⁻¹. Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium. Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l⁻¹) and by mixing with either 160 mg l⁻¹ glucose or 50 mg l⁻¹ of 2,4-dichlorophenol in the medium. Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms. A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway. The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications.     

芘降解菌群驯化过程中的演变

【目的】筛选、驯化获得芘的高效降解菌群,为利用其对多环芳烃污染土壤进行生物修复奠定理论基础。【方法】利用芘作为底物在矿物盐培养基(MSM)中富集驯化,得到了一个混合菌群PYR,利用分光光度计和HPLC测定混合菌群的生长与降解的关系,并对混合菌群降解多环芳烃底物的广谱性和降解芘性能的稳定性进行了测定;通过冷冻干燥的方法对菌群进行了保藏,通过HPLC的方法对保藏后复壮的菌群的降解性能进行了测定;通过培养和非培养方法对菌群的多样性进行了调查,通过构建16S rRNA基因文库的方法,分析焦化厂原始土壤中菌群组成和混合菌群转接3次(PYR-3)、6次(PYR-6)和9次(PYR-9)后的组成变化。【结果】该混合菌群可以利用芘作为唯一碳源和能源生长,12 d对芘的降解率为89%,对于菲(86%)及荧蒽(49%)也具有较高的降解率,但不能降解萘和茚并芘,并且该菌群的降解活性经过多次转接和冷冻干燥保藏保持稳定,从混合菌群中分离得到了9株菌,这9株菌分布在无色杆菌属(Achromobacter),芽胞杆菌属(Bacillus),节杆菌属(Arthrobacter),微小杆菌属(Exiguobacterium)和类土地杆菌属(Parapedobacter)。系统进化分析表明变形菌门是土壤原样(100%)及以芘为底物富集的混合菌群中的主要类群(PYR-3,83%),与原土壤样品相似,PYR-3中γ-Proteobacteria(占变形菌门的77%)中假单胞菌属的菌依然占主导地位,但由于菌群的多样性增加,假单胞菌属所占比例减少。随着富集代数的增加,菌群的多样性进一步增加,γ-Proteobacteria的比例在混合菌群中的比例下降(PYR-6中占变形菌门的33%,PYR-9中占变形菌门的18%),而β-Proteobacteria在混合菌群中的比例上升(PYR-3中占变形菌门的13%,PYR-6中占变形菌门的36%,PYR-9中占变形菌门的55%)。【结论】混合菌群具有很强的芘的降解性能,并且随着传代次数的增加,菌群的组成趋于稳定。     

Study of a hexane-degrading consortium in a biofilter and in liquid culture: biodiversity, kinetics and characterization of degrading strains

A gasoline-degrading consortium, originating from a Mexican soil, was used to study its hexane-degradation kinetics in liquid culture and in a biofilter with mineral support. The biodiversity of the consortium depending on the culture conditions and electron and energy source (gasoline, hexane in liquid or hexane in the biofilter) was analyzed using a 16S rRNA-based approach. Significant differences between the populations were observed, indicating a probable adaptation to the substrate. Two strains, named SP2B and SP72-3, isolated from the consortium, belonged to Actinomycetes and demonstrated a high metabolic potential in hexane degradation. Even though the SP2B strain was related to Rhodococcus ruber DSM 43338(T) by phylogenetic studies, it displayed enlarged metabolic properties in hexane and other short-alkane degradation compared with the collection strain.     

Deep desulfurization of diesel oil and crude oils by a newly isolated Rhodococcus erythropolis strain

The soil-isolated strain XP was identified as Rhodococcus erythropolis. R. erythropolis XP could efficiently desulfurize benzonaphthothiophene, a complicated model sulfur compound that exists in crude oil. The desulfurization product of benzonaphthothiophene was identified as alpha-hydroxy-beta-phenyl-naphthalene. Resting cells could desulfurize diesel oil (total organic sulfur, 259 ppm) after hydrodesulfurization. The sulfur content of diesel oil was reduced by 94.5% by using the resting cell biocatalyst for 24 h at 30 degrees C. Biodesulfurization of crude oils was also investigated. After 72 h of treatment at 30 degrees C, 62.3% of the total sulfur content in Fushun crude oil (initial total sulfur content, 3,210 ppm) and 47.2% of that in Sudanese crude oil (initial total sulfur, 1,237 ppm) were removed. Gas chromatography with pulsed-flame photometric detector analysis was used to evaluate the effect of R. erythropolis XP treatment on the sulfur content in Fushun crude oil, and it was shown that most organic sulfur compounds were eliminated after biodesulfurization.     

Adaptation of coimmobilized Rhodococcus cells to oil hydrocarbons in a column bioreactor

A possible adaptation of the association of Rhodococcus ruber and Rhodococcus opacus strains immobilized on modified sawdust to oil hydrocarbons in a column bioreactor was investigated. In the bioreactor, the bacterial population showed higher hydrocarbon and antibiotic resistance accompanied by the changes in cell surface properties (hydrophobicity, electrokinetic potential) and in the content of cellular lipids and biosurfactants. The possibility of using adapted Rhodococcus strains for the purification of oil-polluted water in the bioreactor was demonstrated.     

Bioremediation of gasoline contaminated soil by a bacterial consortium amended with poultry litter, coir pith and rhamnolipid biosurfactant.

The aim of the present study was to find methods for enhancing rates of hydrocarbon biodegradation in gasoline contaminated soil by ex situ bioremediation. Red soil (RS) was treated with gasoline-spilled soil (GS) from a gasoline station and different combinations of amendments were prepared using (i) mixed bacterial consortium (MC), (ii) poultry litter (PL), (iii) coir pith (CP) and (iv) rhamnolipid biosurfactant (BS) produced by Pseudomonas sp. DS10-129. The study was conducted for a period of 90 days during which bacterial growth, hydrocarbon degradation and growth parameters of Phaseolus aureus RoxB including seed germination, chlorophyll content, shoot and root length were measured. Approximately 67% and 78% of the hydrocarbons were effectively degraded within 60 days in soil samples amended with RS + GS + MC + PL + CP + BS at 0.1% and 1%. Maximum percentage of seed germination, shoot length, root length and chlorophyll content in P. aureus were recorded after 60 days in the above amendments. Further incubation to 90 days did not exhibit significant improvements. Statistical analysis using analysis of variance (ANOVA) and Duncan's multiple range test (DMRT) revealed that the level of amendments, incubation time and combination of amendments significantly influenced bacterial growth, hydrocarbon degradation, seed germination and chlorophyll content at a 1% probability level. All tested additives MC, PL, CP and rhamnolipid BS had significant positive effects on the bioremediation of GS.     

Phylogenetic analysis of bacterial populations in an anaerobic microbial consortium capable of degrading saturation concentrations of tetrachloroethylene

An anaerobic microbial consortium able to biodegrade saturation levels of perchloroethylene (PCE) in a column containing a source zone of PCE was examined phylogenetically to determine microbial community structure and spatial variation in relation to the PCE source. The consortium was comprised of at least 34 members with 7 organisms sharing affiliations with known respiratory or cometabolic dechlorinators. Seven other organisms had their closest phylogenetic relative detected in other environments containing chlorinated compounds. Based on denaturing gradient gel electrophoresis, significant Bacteria were Dehalococcoides ethenogenes, Shewanella putrefaciens, and an Acetobacterium species. Spatial variations in community structure of the consortium relative to the PCE source zone were observed. A Pseudomonas species was predominant in a zone 30 cm from the PCE source. A Methanothrix species was predominant at points over 85 cm from the source zone. A Trichlorobacter species was detected where PCE concentrations were highest, up to 85 cm from the PCE source, whereas D. ethenogenes was ubiquitous to over 128 cm from the PCE source.     

Survival of a bacterial/archael consortium on building materials as revealed by molecular methods

The analysis of microbial communities using molecular techniques has become a common method to describe their components. In this study some building materials were inoculated with a microbial consortium of 14 microorganisms, subjected to alternate wetting/drying cycles for six months, and left to rest for eight years. At the end of the resting period, most members of the consortium were detected by molecular methods, which indicate a remarkable preservation of the DNA, although only one bacterium was able to grow in a culture medium. The experimental approach we carried out indicates that the list of microorganisms obtained from DNA analyses reflects not only the actual composition of the microbial communities, but is also a reflection of the microorganisms that were once active on the building materials.     

Effect of yeast extract on fluoranthene degradation and aromatic ring dioxygenase expressing bacterial community structure of a fluoranthene degrading bacterial consortium

Fluoranthene (Fla) is a high molecular weight polycyclic aromatic hydrocarbon that exerts hazardous effects on living organisms. An efficient Fla degrading bacterial consortium LP was enriched from an oil contaminated soil sample, with and without yeast extract as a supplement. Objective of the present study was to see if there was any differential effect of yeast extract addition on Fla degradation potential and aromatic ring dioxygenase expressing bacteria (ARDB) of the enrichments. Primary enrichment of the soil sample was carried out in minimal salt medium (MSM) added with 500 mg l⁻¹ Fla and 0.05% yeast extract (YMSM). Secondary, tertiary and subsequent enrichments were prepared in YMSM and MSM after every sixteen days of incubation. Fla was efficiently degraded by YMSM enriched culture than MSM enriched culture. However, when MSM enrichment was incubated longer instead of further subculturings, it also degraded Fla efficiently. All three enrichments exhibited growth of bacterial colonies on Fla sprayed minimal agar plates however only YMSM enrichment showed clear zone forming bacterial colonies. A positive effect was observed of yeast extract on ARDB population of LP consortium. To our limited knowledge this is first time that effect of yeast extract on ARDB population was studied.     

Enhanced biodegradation of diesel oil by a newly identified Rhodococcus baikonurensis EN3 in the presence of mycolic acid

AIMS: The aim of the present study was to isolate and characterize a bacterium, strain EN3, capable of using diesel oil as a major carbon and energy source, and to analyse the enhancement of diesel oil degradation by this organism using synthetic mycolic acid (2-hexyl-3-hydroxyldecanoic acid). METHOD AND RESULTS: An actinomycete with the ability to degrade diesel oil was isolated from oil contaminated soil and characterized. The strain had phenotypic properties consistent with its classification in the genus Rhodococcus showing a 16S rRNA gene similarity of 99.7% with Rhodococcus baikonurensis DSM 44587(T). The ability of the characterized strain to degrade diesel oil at various concentrations (1000, 5000, 10 000 and 20 000 mg l(-1)) was determined. The effect of synthetic mycolic acid on the biodegradation of diesel oil was investigated at the 20 000 mg l(-1) concentration; the surfactant was added to the flask cultures at three different concentrations (10, 50 and 100 mg l(-1)) and degradation followed over 7 days. Enhanced degradation was found at all three concentrations of the surfactant. In addition, the enhancement of diesel oil degradation by other surfactants was observed. CONCLUSIONS: The synthetic mycolic acid has potential for the remediation of petroleum-contaminated sites from both an economic and applied perspective as it can stimulate biodegradation at low concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that the synthesized mycolic acid can be used for potential applications in the bioremediation industries, for example, in oil spill clean-up, diesel fuel remediation and biostimulation.     

Effect of rapeseed oil on the degradation of polycyclic aromatic hydrocarbons in soil by Rhodococcus wratislaviensis

The effect of rapeseed oil (0, 0.1 and 1% w/w) on the degradation of polycyclic aromatic hydrocarbons (PAH) by Rhodococcus wratislaviensis was studied in soils artificially contaminated with phenanthrene, anthracene, pyrene and benzo(a)pyrene (50 mg kg⁻¹ each), during 49 days at 30 °C. Without or with 0.1% of rapeseed oil, R. wratislaviensis degraded >90% of phenanthrene and anthracene in 14 days and mineralised approx. 23% of ¹⁴C-phenanthrene. The native microflora degraded pyrene (90% degradation; 75% mineralisation) and benzo(a)pyrene (30% degradation, no mineralisation). With 1% rapeseed oil, R. wratislaviensis degraded only 66% of the phenanthrene and mineralised 12.4%, and had no effect on other PAH, while degradation by the native microflora was inhibited. On the other hand, the addition of 1% oil promoted degradation of benzo(a)pyrene (75%) and anthracene (90%) and anthraquinone was produced at high concentrations and accumulated. Two distinct processes gave degradation of PAH, one biological and one abiotic. Biological processes mainly degraded phenanthrene and pyrene, either by R. wratislaviensis or by the indigenous microflora. Benzo(a)pyrene was degraded mainly by an abiotic process in the presence of 1% rapeseed oil. Anthracene was degraded by a combination of both processes.PAH are often found in contaminated soils and there is the need of developing techniques that can be applied in the remediation of these sites, where PAH, specially those with high molecular weight, pose health and environmental risks. There is a continuous search for efficient microorganisms able to degrade these pollutants and for methods to enhance their degradation and bioavailability, e.g. by the use of vegetable oils. This paper presents a novel process for the degradation of PAH by a combined biological/abiotic system.     

Determination of gasoline range,diesel range,and mineral oil range organics in soils and water by flame lonization gas chromatography

This article describes a method for the determination of gasoline range, diesel range, and mineral oil range organics in soils and water. It represents the culmination of a series of efforts to go beyond typical GRO and DRO methodology currently available in the literature to include a quantitative determination of mineral oil organics having a boiling range up to C₄₄. It also is the result of an attempt to develop a cost‐effective method that enables the analyst to quantify three different types of hydrocarbon components in one GC run under conditions without a concentration step. Method performance is comparable to that of current protocols for GRO and DRO determinations and validated further by comparisons to certified standards and in‐house standards. Accuracy as percentage recovery for GRO in water is 82 to 84 and 91 to 92% for soils. Accuracy as percentage recovery for MRO in water is 84 to 102 and 75 to 80% for soils. Accuracy as percentage recovery for DRO in water is 78 to 100 and 71 to 90% for soils. Results from the analysis of in‐house standards and certified standards for DRO and MRO gave higher recoveries than was demonstrated in the MDL studies. The MDL for DRO in water is 31 mg/l and 14 mg/kg for soils. The MDL for GRO in water is 8 mg/l and 4 mg/kg for soils. For MRO in water, the MDL is 7 mg/l and for soils 10 mg/kg. Future proposed improvements to this method will involve updated software that will allow automatic blank subtraction, automatic calculation of surrogate recoveries and the automatic incorporation of dry weight factors in the final calculations for soils. In addition, a GRO method with a run time of only 24 min will be used routinely when only GRO analyses have to be performed.