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.     

Enhanced crude oil biodegradation in soil via biostimulation

Research on feasible methods for the enhancement of bioremediation in soil contaminated by crude oil is vital in oil-exporting countries such as Kuwait, where crude oil is a major pollutant and the environment is hostile to biodegradation. This study investigated the possibility of enhancing crude oil bioremediation by supplementing soil with cost-effective organic materials derived from two widespread locally grown trees, Conocarpus and Tamarix. Amendments in soils increased the counts of soil microbiota by up to 98% and enhanced their activity by up to 95.5%. The increase in the biodegradation of crude oil (75%) and high levels of alkB expression substantiated the efficiency of the proposed amendment technology for the bioremediation of hydrocarbon-contaminated sites. The identification of crude-oil-degrading bacteria revealed the dominance of the genus Microbacterium (39.6%), Sphingopyxis soli (19.3%), and Bordetella petrii (19.6%) in unamended, Conocarpus-amended, and Tamarix-amended contaminated soils, respectively. Although soil amendments favored the growth of Gram-negative bacteria and reduced bacterial diversity, the structures of bacterial communities were not significantly altered.     

Characterisation of biodegradation capacities of environmental microflorae for diesel oil by comprehensive two-dimensional gas chromatography

In contaminated soils, efficiency of natural attenuation or engineered bioremediation largely depends on biodegradation capacities of the local microflorae. In the present study, the biodegradation capacities of various microflorae towards diesel oil were determined in laboratory conditions. Microflorae were collected from 9 contaminated and 10 uncontaminated soil samples and were compared to urban wastewater activated sludge. The recalcitrance of hydrocarbons in tests was characterised using both gas chromatography (GC) and comprehensive two-dimensional gas chromatography (GC×GC). The microflorae from contaminated soils were found to exhibit higher degradation capacities than those from uncontaminated soil and activated sludge. In cultures inoculated by contaminated-soil microflorae, 80% of diesel oil on an average was consumed over 4-week incubation compared to only 64% in uncontaminated soil and 60% in activated sludge cultures. As shown by GC, n-alkanes of diesel oil were totally utilised by each microflora but differentiated degradation extents were observed for cyclic and branched hydrocarbons. The enhanced degradation capacities of impacted-soil microflorae resulted probably from an adaptation to the hydrocarbon contaminants but a similar adaptation was noted in uncontaminated soils when conifer trees might have released natural hydrocarbons. GC×GC showed that a contaminated-soil microflora removed all aromatics and all branched alkanes containing less than C₁₅. The most recalcitrant compounds were the branched and cyclic alkanes with 15–23 atoms of carbon.     

Biodegradation potential of oily sludge by pure and mixed bacterial cultures

The biodegradation capacity of aliphatic and aromatic hydrocarbons of petrochemical oily sludge in liquid medium by a bacterial consortium and five pure bacterial cultures was analyzed. Three bacteria isolated from petrochemical oily sludge, identified as Stenotrophomonas acidaminiphila, Bacillus megaterium and Bacillus cibi, and two bacteria isolated from a soil contaminated by petrochemical waste, identified as Pseudomonas aeruginosa and Bacillus cereus demonstrated efficiency in oily sludge degradation when cultivated during 40 days. The bacterial consortium demonstrated an excellent oily sludge degradation capacity, reducing 90.7% of the aliphatic fraction and 51.8% of the aromatic fraction, as well as biosurfactant production capacity, achieving 39.4% reduction of surface tension of the culture medium and an emulsifying activity of 55.1%. The results indicated that the bacterial consortium has potential to be applied in bioremediation of petrochemical oily sludge contaminated environments, favoring the reduction of environmental passives and increasing industrial productivity.     

Soil initial bacterial diversity and nutrient availability determine the rate of xenobiotic biodegradation

Understanding the relative importance of soil microbial diversity, plants and nutrient management is crucial to implement an effective bioremediation approach to xenobiotics-contaminated soils. To date, knowledge on the interactive effects of soil microbiome, plant and nutrient supply on influencing biodegradation potential of soils remains limited. In this study, we evaluated the individual and interactive effects of soil initial bacterial diversity, nutrient amendments (organic and inorganic) and plant presence on the biodegradation rate of pyrene, a polycyclic aromatic hydrocarbon. Initial bacterial diversity had a strong positive impact on soil biodegradation potential, with soil harbouring higher bacterial diversity showing ~ 2 times higher degradation rates than soils with lower bacterial diversity. Both organic and inorganic nutrient amendments consistently improved the degradation rate in lower diversity soils and had negative (inorganic) to neutral (organic) effect in higher diversity soils. Interestingly, plant presence/type did not show any significant effect on the degradation rate in most of the treatments. Structural equation modelling demonstrated that initial bacterial diversity had a prominent role in driving pyrene biodegradation rates. We provide novel evidence that suggests that soil initial microbial diversity, and nutrient amendments should be explicitly considered in the design and employment of bioremediation management strategies for restoring natural habitats disturbed by organic pollutants.     

Nutrient Amendments of Inorganic Fertiliser and Oil Palm Empty Fruit Bunch and Their Influence on Bacterial Species Dominance and Degradation of the Associated Crude Oil Constituents

The effects of inorganic commercial fertiliser (N:P:K = 8:8:1) and oil palm empty fruit bunch (EFB) as nutrient amendments for crude oil degradation and microbial population shift by a microbial consortium [Pseudomonas sp. (UKMP-14T), Acinetobacter sp. (UKMP-12T), Trichoderma sp. (TriUKMP-1M and TriUKMP-2M)] were assessed. The bacterial populations present during crude oil degradation were analysed by spread plate method and 16S rRNA sequences, whereas the presence of fungi was assessed by growth on potato dextrose agar. Crude oil degradation analysed using gas chromatography-flame ionisation detection showed total petroleum hydrocarbon reduced between 70 and 100%, depending on the type of amendments compared to control (≈55%) after 30 days of incubation. Nutrient amendments using NPK fertiliser or EFB were found to influence the domination of different bacterial species, which in turn preferentially utilised different hydrocarbons. This study suggested different nutrient amendments could be used to preferentially select bacteria to degrade different components of crude oil, particularly pertaining to the recalcitrant phytane. This information is very useful for application of in situ bioremediation of soil hydrocarbon contamination.     

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.     

Studies on Biodegradation of Kerosene in Soil under Different Bioremediation Strategies

The effectiveness of bioremediation is often a function of the microbial population and how they can be enriched and maintained in an environment. Strategies for inexpensive in situ bioremediation of soil contaminated with petroleum hydrocarbons include stimulation of the indigenous microorganisms by introduction of nutrients (biostimulation) and/or through inoculation of an enriched mixed microbial culture into soil (bioaugmentation). To demonstrate the potential use of bioremediation in soil contaminated with kerosene, a laboratory study with the objective of evaluating and comparing the effects of bioattenuation, biostimulation, bioaugmentation, and combined biostimulation and bioaugmentation was performed. The present study dealt with the biodegradation of kerosene in soil under different bioremediation treatment strategies: bioattenuation, biostimulation, bioaugmentation, and combined biostimulation and bioaugmentation, respectively. Each treatment strategy contained 10% (w/w) kerosene in soil as a sole source of carbon and energy. After 5 weeks of remediation, the results revealed that bioattenuation, bioaugmentation, biostimulation, and combined biostimulation and bioaugmentation exhibited 44.1%, 67.8%, 83.1%, and 87.3% kerosene degradation, respectively. Also, the total hydrocarbon-degrading bacteria (THDB) count in all the treatments increased with time up till the second week after which it decreased. The highest bacterial growth was observed for combined biostimulation and bioaugmentation treatment strategy. A first-order kinetic model equation was fitted to the biodegradation data to further evaluate the rate of biodegradation and the results showed that the specific degradation rate constant (k) value was comparatively higher for combined biostimulation and bioaugmentation treatment strategy than the values for other treatments. Therefore, value of the kinetic parameter showed that the degree of effectiveness of these bioremediation strategies in the clean up of soil contaminated with kerosene is in the following order: bioattenuation < bioaugmentation < biostimulation < combined biostimulation and bioaugmentation. Conclusively, the present work has defined combined biostimulation and bioaugmentation treatment strategy requirements for kerosene oil degradation and thus opened an avenue for its remediation from contaminated soil.     

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.     

Towards efficient crude oil degradation by a mixed bacterial consortium

A laboratory study was undertaken to assess the optimal conditions for biodegradation of Bombay High (BH) crude oil. Among 130 oil degrading bacterial cultures isolated from oil contaminated soil samples, Micrococcus sp. GS2-22, Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73, Bacillus sp. DS6-86 and Pseudomonas sp. DS10-129 were selected for the study based on the efficiency of crude oil utilisation. A mixed bacterial consortium prepared using the above strains was also used. Individual bacterial cultures showed less growth and degradation than did the mixed bacterial consortium. At 1% crude oil concentration, the mixed bacterial consortium degraded a maximum of 78% of BH crude oil. This was followed by 66% by Pseudomonas sp. DS10-129, 59% by Bacillus sp. DS6-86, 49% by Micrococcus sp. GS2-22, 43% by Corynebacterium sp. GS5-66 and 41% by Flavobacterium sp. DS5-73. The percentage of degradation by the mixed bacterial consortium decreased from 78% to 52% as the concentration of crude oil was increased from 1% to 10%. Temperature of 30 degrees C and pH 7.5 were found to be optima for maximum biodegradation.     

Effect of various amendments on heavy mineral oil bioremediation and soil microbial activity

To examine the effects of amendments on the degradation of heavy mineral oil, we conducted a pilot-scale experiment in the field for 105 days. During the experiment, soil samples were collected and analyzed periodically to determine the amount of residual hydrocarbons and evaluate the effects of the amendments on microbial activity. After 105 days, the initial level of contamination (7490+/-480 mg hydrocarbon kg(-1) soil) was reduced by 18-40% in amended soils, whereas it was only reduced by 9% in nonamended soil. Heavy mineral oil degradation was much faster and more complete in compost-amended soil than in hay-, sawdust-, and mineral nutrient-amended soils. The enhanced degradation of heavy mineral oil in compost-amended soil may be a result of the significantly higher microbial activity in this soil. Among the studied microbial parameters, soil dehydrogenase, lipase, and urease activities were strongly and negatively correlated with heavy mineral oil biodegradation (P<0.01) in compost-amended soil.     

A refinery sludge deposition site: presence of nahH and alkJ genes and crude oil biodegradation ability of bacterial isolates

204 bacterial isolates from four Greek refinery sludge deposition sites were investigated for the presence of nahH and alkJ genes encoding key enzymes of both aromatic and aliphatic hydrocarbon degradation pathways by PCR and DNA hybridisation. Members of Pseudomonas, Acinetobacter, Bacillus, Rhodococcus and Arthrobacter play important role in bioremediation processes in sandy/loam soil contaminated with oil and nahH and alkJ genes were present in the 73% of the isolates. Consortia of bacterial isolates that were used for biodegradation of aliphatic and aromatic hydrocarbons in crude oil using liquid cultures exhibited rates from 35% to 48% within 10 days of incubation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhancing bioremediation of diesel-fuel-contaminated soil in a boreal climate: Comparison of biostimulation and bioaugmentation

Cold conditions delay bioremediation of oil hydrocarbons, but other bottlenecks also affect the outcome. Means to stimulate biodegradation of diesel oil hydrocarbons in contaminated soil were compared. Different combinations of nutrients, bulking agent, aeration, and microbial inocula were examined in lab simulations, and effective combinations were tested in field conditions. Bacterial communities were investigated by cloning and sequencing 16S-rRNA genes. Efficient degradation was attained when slow-release nutrients and aeration were used simultaneously. Bacterial inocula did not advance soil remediation, nor did they have any lasting effect on bacterial densities. Bacteria belonging to Proteobacteria were dominant in all cases. In the field test, a bulking agent promoting air passage through the soil ensured sufficient aeration, while forced air decreased the soil moisture excessively. We concluded that biostimulation via optimization of nitrogen and oxygen supply significantly improved bioremediation of oil-contaminated soil, while bioaugmentation had no additional effect.     

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.     

Biodegradation of trinitrotoluene (TNT) by indigenous microorganisms from TNT-contaminated soil,and their application in TNT bioremediation

Soil and groundwater contaminated by munitions compounds is a crucial issue in environmental protection. Trinitrotoluene (TNT) is highly toxic and carcinogenic; therefore, the control and remediation of TNT contamination is a critical environmental issue. In this study, the authors characterized the indigenous microbial isolates from a TNT-contaminated site and evaluated their activity in TNT biodegradation. The bacteria Achromobacter sp. BC09 and Citrobacter sp. YC4 isolated from TNT-contaminated soil by enrichment culture with TNT as the sole carbon and nitrogen source (strain BC09) and as the sole nitrogen but not carbon source (strain YC4) were studied for their use in TNT bioremediation. The efficacy of degradation of TNT by indigenous microorganisms in contaminated soil without any modification was insufficient in the laboratory-scale pilot experiments. The addition of strains BC09 and YC4 to the contaminated soil did not significantly accelerate the degradation rate. However, the addition of an additional carbon source (e.g., 0.25% sucrose) could significantly increase the bioremediation efficiency (ca. decrease of 200 ppm for 10 days). Overall, the results suggested that biostimulation was more efficient as compared with bioaugmentation. Nevertheless, the combination of biostimulation and bioaugmentation using these indigenous isolates is still a feasible approach for the development of bioremediation of TNT pollution.     

Influence of biochar and compost on phytoremediation of oil-contaminated soil

The use of pyrolyzed carbon, biochar, as a soil amendment is of potential interest for improving phytoremediation of soil that has been contaminated by petroleum hydrocarbons. To examine this question, the research reported here compared the effects of biochar, plants (mesquite tree seedlings), compost and combinations of these treatments on the rate of biodegradation of oil in a contaminated soil and the population size of oil-degrading bacteria. The presence of mesquite plants significantly enhanced oil degradation in all treatments except when biochar was used as the sole amendment without compost. The greatest extent of oil degradation was achieved in soil planted with mesquite and amended with compost (44% of the light hydrocarbon fraction). Most probable number assays showed that biochar generally reduced the population size of the oil-degrading community. The results of this study suggest that biochar addition to petroleum-contaminated soils does not improve the rate of bioremediation. In contrast, the use of plants and compost additions to soil are confirmed as important bioremediation technologies.     

Laboratory study on the bioremediation of petrochemical sludge-contaminated soil

This study evaluated by biological and chemical analyses the effectiveness of bioremediation of sludge from the petrochemical industry in systems containing artificially contaminated soil. The sludge–soil systems were prepared with three different initial concentrations of sludge, and during bioremediation 86–95% of the hydrocarbons was eliminated. Simultaneously, soil bacterial populations and inhibition of seed germination by aqueous extracts increased in all sludge–soil systems during the first 180 days of treatment. After 1 year of bioremediation, a loss in the catabolic capacity of the Gram-negative bacterial population was observed, but was not dependent on the initial sludge concentration. Furthermore, residual levels of hydrocarbons and seed germination inhibitory effect decreased sharply, but some level of toxicity remained in the systems containing the highest initial sludge concentration. Independent of the initial sludge concentration, the contaminated soils did not re-establish their original features even when residual hydrocarbon concentrations suggested the end of the process.     

Application of biosurfactant produced from peanut oil cake by Lactobacillus delbrueckii in biodegradation of crude oil

Lactobacillus delbrueckii cultured with peanut oil cake as the carbon source yielded 5.35 mg ml(-1) of biosurfactant production. Five sets of microcosm biodegradation experiments were carried out with crude oil as follows: set 1 - bacterial cells+crude oil, set 2 - bacterial cells+crude oil+fertilizer, set 3 - bacterial cells+crude oil+biosurfactant, set 4 - bacterial cells+crude oil+biosurfactant+fertilizer, set 5 - with no bacterial cells, fertilizer and biosurfactant (control). Maximum degradation of crude oil was observed in set 4 (75%). Interestingly, when biosurfactant and bacterial cells were used (set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in set 4 was 7% higher degradation level in microcosm experiments. It is evident from the results that biosurfactants alone is capable of promoting biodegradation to a large extent without added fertilizers.     

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

Comparison of bio-augmentation and composting for remediation of oily sludge: A field-scale study in China

Two bioremediation technologies were performed in order to explore a better treatment process for an oily sludge restoration in China during 2004. The bioremediation by augmentation of biopreparation was compared with a conventional composting. The oily sludge and oil-polluted soil were received from an oil production plant. The total hydrocarbon content (THC) varied from 327.7 to 371.2 g kg⁻¹ of dry sludge and the THC in contaminated soil was 151.0 g kg⁻¹. Before application of preparation, straw, sawdust, top sand and pure soil were added in different proportions to the sludge and soil and mixed thoroughly. Such sludge and soil composites were used for negative controls and for activation of indigenous oil degrading microorganisms with addition of fertilizer (positive controls). For composting, crude manure and straw were added to the oily sludge and the THC was 101.4 g kg⁻¹. The biopreparation was applied every 2 weeks and experiment lasted 56 days under the ambient temperature. The sludge was mixed and watered every 3 days. After three times of biopreparation application, the THC decreased by 46–53% in the oily sludge and soil, while in the positive controls (activation of indigenous microorganisms) the THC decreased by 13–23%, and there was no oil degradation in negative controls After composting, the THC decreased by 31% in the oily sludge. The planting of Tall Fescue (Festuca arundinace) revealed a decrease of sludge toxicity after application of both bioremediation technologies and additionally decreased the THC by 5–7%.