Verification of Degradation of <Emphasis Type="Italic">n</Emphasis>-Alkanes in Diesel Oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Strain WatG in Soil Microcosms

Degradation of n-alkanes in diesel oil by Pseudomonas aeruginosa strain WatG (WatG) was verified in soil microcosms. The total petroleum hydrocarbon (TPH) degradation level in two bioaugmentation samples was 51% and 46% for 1 week in unsterilized and sterilized soil microcosms, respectively. The TPH degradation in the biostimulation was of control level (15%). The TPH degradation in aeration-limited samples was clearly reduced when compared with that in aeration-unlimited ones under both sterilized and unsterilized conditions. Addition of WatG into soil microcosms was accompanied by dirhamnolipid production only in the presence of diesel oil. These findings suggest that degradation of n-alkanes in diesel oil in soil microcosms would be facilitated by bioaugmentation of WatG, with production of dirhamnolipid, and also by participation of biostimulated indigenous soil bacteria.     

Degradation of long-chain n-alkanes (C36 and C40) by Pseudomonas aeruginosa strain WatG

Pseudomonas aeruginosa strain WatG was unable to utilize either n-hexatriacontane (C₃₆) or n-tetracontane (C₄₀), which are both insoluble in a mineral salts medium (MSM), as a sole carbon source. However, when C₃₆ and C₄₀ were added to MSM containing crude oil, more than 25% of each of the compounds was degraded by this strain after 2 weeks at 30 °C. These results demonstrate that P. aeruginosa strain WatG has the ability to degrade long-chain alkanes up to C₄₀, when they are solubilized by crude oil components.     

Selection of functional consortium for crude oil-contaminated soil remediation

Microorganisms with high oil-degrading performance are essential for bioremediation of soil contaminated with crude oil. A positive end dilution method was employed for the selection of crude oil-degrading functional consortium from contaminated soil. The selected consortium was consisted of Rhizobiales sp., Pseudomonas sp., Brucella sp., Bacillus sp., Rhodococcus sp., Microbacterium sp. and Roseomonas sp. and removed nearly 52.1% of crude oil at initial concentration of 10,000 mg l⁻¹ at 30 °C within 7 days, with removal of aliphatic hydrocarbons by 71.4% and aromatic hydrocarbons by 36.0%, respectively. The effectiveness of the consortium for bioaugmentation was confirmed with microcosm test by contaminated soil (1.0 kg) from Karemary Oilfield, China. The removal efficiency of crude oil was enhanced to >50% in microcosms with the consortium compared with 8-13% or lower in controls over a 60 day period. The crude oil removal reaction was probably first order reaction and the rate was greatly enhanced by bioaugmentation. Supplementation of nitrogen and phosphate sources had limited effect on the oil removal in the tested soil.     

Molinate biodegradation in soils: natural attenuation versus bioaugmentation

The aims of the present study were to assess the potential of natural attenuation or bioaugmentation to reduce soil molinate contamination in paddy field soils and the impact of these bioremediation strategies on the composition of soil indigenous microbiota. A molinate mineralizing culture (mixed culture DC) was used as inoculum in the bioaugmentation assays. Significantly higher removal of molinate was observed in bioaugmentation than in natural attenuation microcosms (63 and 39 %, respectively) after 42 days of incubation at 22 °C. In the bioaugmentation assays, the impact of Gulosibacter molinativorax ON4^T on molinate depletion was observed since the gene encoding the enzyme responsible for the initial molinate breakdown (harboured by that actinobacterium) was only detected in inoculated microcosms. Nevertheless, the exogenous mixed culture DC did not overgrow as the heterotrophic counts of the bioaugmentation microcosms were not significantly different from those of natural attenuation and controls. Moreover, the actinobacterial clone libraries generated from the bioaugmentation microcosms did not include any 16S rRNA gene sequences with significant similarity to that of G. molinativorax ON4^T. The multivariate analysis of the 16S rRNA DGGE patterns of the soil microcosm suggested that the activity of mixed culture DC did not affect the soil bacterial community structure since the DGGE patterns of the bioaugmentation microcosms clustered with those of natural attenuation and controls. Although both bioremediation approaches removed molinate without indigenous microbiota perturbation, the results suggested that bioaugmentation with mixed culture DC was more effective to treat soils contaminated with molinate.     

Characterization of a Novel Cr6+ Reducing Pseudomonas sp. with Plant Growth–Promoting Potential

The isolate RNP4 obtained from a long-term tannery waste contaminated soil was characterized and presumptively identified as Pseudomonas sp. The strain RNP4 tolerated concentrations up to 450 mg Cr⁶⁺/L on a Luria-Bartani (LB) agar medium and reduced a substantial amount of Cr⁶⁺ to Cr³⁺ in the LB liquid medium. The ability of performing multifarious activities in tandem suggested the uniqueness of isolate RNP4. The strain produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. The strain also exhibited the production of siderophore and solubilization of phosphorus in mineral salt medium and SRS1 medium, respectively. Concurrent production of IAA and siderophore and the solubilization of phosphorus revealed its plant growth promotion potential. Furthermore, the strain was able to promote the growth of black gram, Indian mustard, and pearl millet in the presence of Cr⁶⁺. Thus, the innate capability of this novel isolate for parallel bioremediation and plant growth promotion has significance in the management of environmental and agricultural problems.     

Application of the knowledge-based approach to strain selection for a bioaugmentation process of phenanthrene- and Cr(VI)-contaminated soil

Aims: The objective of this study was to apply the knowledge‐based approach to the selection of an inoculum to be used in bioaugmentation processes to facilitate phenanthrene degradation in phenanthrene‐ and Cr(VI)‐co‐contaminated soils. Methods and Results: The bacterial community composition of phenanthrene and phenanthrene‐ and Cr(VI)‐co‐contaminated microcosms, determined by denaturing gradient gel electrophoresis analysis, showed that members of the Sphingomonadaceae family were the predominant micro‐organisms. However, the Cr(VI) contamination produced a selective change of predominant Sphingomonas species, and in co‐contaminated soil microcosms, a population closely related to Sphingomonas paucimobilis was naturally selected. The bioaugmentation process was carried out using the phenanthrene‐degrading strain S. paucimobilis 20006FA, isolated and characterized in our laboratory. Although the strain showed a low Cr(VI) resistance (0·250 mmol l^?1); in liquid culture, it was capable of reducing chromate and degrading phenanthrene simultaneously. Conclusion: The inoculation of this strain managed to moderate the effect of the presence of Cr(VI), increasing the biological activity and phenanthrene degradation rate in co‐contaminated microcosm. Significance and Impact of the Study: In this study, we have applied a novel approach to the selection of the adequate inoculum to enhance the phenanthrene degradation in phenanthrene‐ and Cr(VI)‐co‐contaminated soils.     

Kinetic Modelling and Half-Life Study on Bioremediation of Soil Co-Contaminated with Lubricating Motor Oil and Lead Using Different Bioremediation Strategies

The focus of this study was to investigate the effect of nutrient supplement (urea fertilizer) and microbial species augmentation (mixed culture of Aeromonas, Micrococcus, and Serratia sp.) on biodegradation of lubricating motor oil (LMO) and lead uptake by the autochthonous microorganism in LMO and lead-impacted soil were investigated. The potential inhibitory effects of lead on hydrocarbon utilization were investigated over a wide range of lead concentrations (25–200 mg/kg) owing to the complex co-contamination problem frequently encountered in most sites. Under aerobic conditions, total petroleum hydrocarbons (TPH) removal was 45.3% in the natural attenuation microcosm while a maximum of 72% and 68.2% TPH removal was obtained in biostimulation and bioaugmentation microcosms, respectively. Lead addition, as lead nitrate, to soil samples reduced the number of hydrocarbon degraders in all samples by a wide range (11–52%) depending on concentration and similarly, the metabolic activities were affected as observed in mineralization of LMO (3–60%) in soils amended with various lead concentrations. Moreover, the uptake of lead by the autochthonous microorganisms in the soil reduced with increase in the initial lead concentration. First-order kinetics described the biodegradation of LMO very well. The biodegradation rate constants were 0.015, 0.033, and 0.030 day^?1 for LMO degradation in natural attenuation, biostimulation and bioaugmentation treatment microcosms, respectively. The presence of varying initial lead concentration reduced the biodegradation rate constant of LMO degradation in the biostimulation treatment microcosm. Half-life times were 46.2, 21, and 23 days for LMO degradation in natural attenuation, biostimulation and bioaugmentation treatment microcosms, respectively. The half-life time in the biostimulation treatment microcosm was increased with a range between 10.7 and 39.2 days by the presence of different initial lead concentration. The results have promising potential for effective remediation of soils co-contaminated with hydrocarbons and heavy metals.     

Bioaugmentation of Atrazine and Fenamiphos Impacted Groundwater: Laboratory Evaluation

After the failure of a three-month pump-and-treat exercise to clean up an aquifer contaminated with the pesticides atrazine and fenamiphos, microcosm experiments using ¹⁴C-labeled compounds were undertaken to determine under what conditions bioremediation would be most effective, and to investigate the prospects for the use of bioaugmentation. The calculated half-lives for atrazine and fenamiphos mineralization to carbon dioxide in unamended, anaerobic aquifer material were 730 and 1,000 years, respectively. Oxygenation, coupled with bioaugmentation with enrichments of atrazine-mineralizing bacteria obtained from the contaminated site or an imported, atrazine-mineralizing pure strain, Pseudomonas sp. strain ADP, decreased the half-life of atrazine mineralization, to >20 days. Although strain ADP does not use atrazine as a source of carbon and energy, amendment of the aquifer material with citrate, which strain ADP uses as a source of carbon and energy, did not appreciably stimulate the mineralization rate of atrazine in the microcosms, suggesting that the aquifer contains enough natural organic carbon for atrazine mineralization. Aerobic enrichments of fenamiphos-degrading bacteria were prepared; however, oxygenation and bioaugmentation of aquifer material with these strains did not enhance mineralization of fenamiphos within the time constraints of the experiments. The shortest calculated half-life of fenamiphos mineralization in the microcosms was 6.8 years, which is exceedingly long compared with the half-life of fenamiphos in most surface soils.     

Occurrence and microbial degradation of fipronil residues in tropical highland rhizosphere soils of Kerala,India

The aim of the current study was to analyze the abundance and activity of soil microflora in response to fipronil residues, as well as conjointly to isolate and identify bacteria for the bioremediation of fipronil contaminated soils in the cardamom plantations of Idukki district, Kerala. Soil samples collected from rhizosphere areas of six completely different cardamom plantations were analyzed for fipronil residues, physicochemical properties, biochemical properties, and microbial abundance. Biodegradation studies using isolated bacteria were done both in liquid medium and in soil microcosm fortified with fipronil. Fipronil residues were detected in all sampling sites. Canonical correlation analysis revealed that the influence of fipronil on soil physicochemical properties was more pronounced than that on soil microbial properties. The presence of fipronil residues in the soil did not adversely affect bacterial abundance and activity. Two bacterial strains Staphylococcus arlettae and Bacillus thuringiensis could degrade fipronil in both liquid culture and soil. Paired sample T-test and degradation kinetic study recorded that the bacterial strain S. arlettae was more efficient (81.94%) in fipronil degradation than B. thuringiensis (65.98%). The results revealed the potential for in situ bioremediation of fipronil contaminated soil by bioaugmentation using efficient bacterial isolates.     

Biodegradability of bacterial surfactants

This work aimed at evaluating the biodegradability of different bacterial surfactants in liquid medium and in soil microcosms. The biodegradability of biosurfactants by pure and mixed bacterial cultures was evaluated through CO₂ evolution. Three bacterial strains, Acinetobacter baumanni LBBMA ES11, Acinetobacter haemolyticus LBBMA 53 and Pseudomonas sp. LBBMA 101B, used the biosurfactants produced by Bacillus sp. LBBMA 111A (mixed lipopeptide), Bacillus subtilis LBBMA 155 (lipopeptide), Flavobacterium sp. LBBMA 168 (mixture of flavolipids), Dietzia Maris LBBMA 191(glycolipid) and Arthrobacter oxydans LBBMA 201(lipopeptide) as carbon sources in minimal medium. The synthetic surfactant sodium dodecyl sulfate (SDS) was also mineralized by these microorganisms, but at a lower rate. CO₂ emitted by a mixed bacterial culture in soil microcosms with biosurfactants was higher than in the microcosm containing SDS. Biosurfactant mineralization in soil was confirmed by the increase in surface tension of the soil aqueous extracts after incubation with the mixed bacterial culture. It can be concluded that, in terms of biodegradability and environmental security, these compounds are more suitable for applications in remediation technologies in comparison to synthetic surfactants. However, more information is needed on structure of biosurfactants, their interaction with soil and contaminants and scale up and cost for biosurfactant production.     

Rhamnolipid production using Shewanella seohaensis BS18 and evaluation of its efficiency along with phytoremediation and bioaugmentation for bioremediation of hydrocarbon contaminated soils

Abstract

This study reports the combined use of a rhamnolipid type biosurfactant (BS) along with phytoremediation and bioaugmentation (BA) for bioremediation of hydrocarbon-contaminated soils. Bacterial isolates obtained from hydrocarbon contaminated soil were screened for rhamnolipid production and isolate BS18, identified as Shewanella seohaensis, was selected for bioremediation experiments. Growth of BS18 in mineral salt medium (MSM) with diesel oil as the carbon source showed a maximum biomass of 8.2?g L^?1, rhamnolipid production of 2.2?mg g^?1 cell dry weight, surface tension reduction of 28.6?mN/m and emulsification potential (EI₂₄%) of 65.6. Characterization of rhamnolipid based on Fourier transmittance infrared (FTIR) analysis confirmed the presence of OH, CH₂/CH₃, C=O, and COO stretching vibrations, respectively, which are distinctive features of rhamnolipid type BSs. In bioremediation experiments, the lowest hydrocarbon concentration of 2.1?mg g^?1 of soil for non-sterilized soil and 4.3?mg g^?1 of soil for sterilized soil was recorded in the combined application of rhamnolipid, phytoremediation, and BA. This treatment also yielded the highest hydrocarbon degrading bacterial population (6.4 Log Cfu g^?1 of soil), highest plant biomass (8.3?g dry weight plant^?1), and the highest hydrocarbon uptake (512.3?mg Kg^?1 of plant).     

Isolation and Characterization of A Metal-resistant Pseudomonas Aeruginosa Strain

Summary The use of microorganisms to remove heavy metals from industrial effluent is an area of extensive research and development. Attempts have been made to isolate and characterize metal-resistant microorganisms from treated oil mill industry effluent wastewater samples. The metal-resistant organisms that showed values of minimum inhibitory concentration towards metals (Cd, Cr, Ni and Pb) ranging from 100 to 800 ppm level were screened. A potent metal-resistant organism, isolate BC15 from the wastewater samples was tentatively identified as Pseudomonas sp. Detailed analysis of morphological, biochemical and 16S rDNA sequence of the isolate revealed that it is closely related to Pseudomonas aeruginosa (94%). Pseudomonas BC15 was capable of absorbing 93% Ni, 65% Pb, 50% Cd and 30% Cr within 48 h from the medium containing 100 mg of each heavy metal per liter. The multiple metal tolerance of this strain was also associated with resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, erythromycin, kanamycin and streptomycin.     

Biodegradation of α and β endosulfan in broth medium and soil microcosm by bacterial strain Bordetella sp. B9

Bacterial strains were isolated from endosulfan treated soil to study the microbial degradation of this pesticide in broth medium and soil microcosm. The isolates were grown in minimal medium and screened for endosulfan degradation. The strain, which utilized endosulfan and showed maximum growth, was selected for detail studies. Maximum degrading capability in shake flask culture was shown by Bordetella sp. B9 which degraded 80% of α endosulfan and 86% of β endosulfan in 18 days. Soil microcosm study was also carried out using this strain in six different treatments. Endosulfan ether and endosulfan lactone were the main metabolites in broth culture, while in soil microcosm endosulfan sulfate was also found along with endosulfan ether and endosulfan lactone. This bacterial strain has a potential to be used for bioremediation of the contaminated sites.     

不同来源溶藻菌的分离、鉴定及溶藻效果比较

为了探究从何种类型的自然生境中更易分离得到溶藻微生物,采用高氏1号培养基分别从水库底泥、湖泊底泥、农田土壤、林地土壤等四种来源共36份样品中分离了7 600株菌,并最终从中筛选得到了5株溶铜绿微囊藻(Microcystis aeruginosa)的溶藻菌,其中4株为假单胞菌(Pseudomonas sp.),1株为黄杆菌(Flavobacterium sp.),5株菌溶藻效率的变化范围为62%~95%。结果表明,当采用高氏1号培养基作为分离培养基时,湖泊底泥和水库底泥中的成功筛选概率最高,农田土壤次之,而林地土壤中则难以筛选得到,假单胞菌是较容易筛选得到的溶藻菌。     

Degradation of Aroclor 1221 and survival of strains in soil microcosms

Three bacterial strains able to use different aromatic compounds as the sole carbon and energy source were tested for their potential to degrade Aroclor 1221 in soil microcosms when present in mixed culture. Disappearance of polychlorinated biphenyls (PCBs), occurrence of metabolites, release of chloride, and survival of the laboratory-selected strains were investigated under different conditions. In principle, complete mineralization of various congeners of Aroclor 1221, a technical mixture of PCBs, by the mixed culture was possible. The autochthonous microflora negatively affected the degradation due to formation of a toxic compound from 4-chlorobenzoate. 4-Chlorobenzoate was produced by one of the added strains, Pseudomonas sp. JHK, during degradation of 4-chlorobiphenyl. The unknown metabolite of 4-chlorobenzoate led to a rapid decrease in viable counts of the laboratory-selected strains in the soil microcosm.Correspondence to: J. Havel     

Isolation and application of Gordonia sp. JC11 for removal of boat lubricants

Boat lubricants are continuously released into the marine environment and thereby cause chronic oil pollution. This study aims to isolate lubricant-degrading microorganisms from Thai coastal areas as well as to apply a selected strain for removal of boat lubricants. Ten microorganisms in the genera of Gordonia, Microbacterium, Acinetobacter, Pseudomonas, Brucella, Enterococcus and Candida were initially isolated by crude oil enrichment culture techniques. The lubricant-removal activity of these isolates was investigated with mineral-based lubricants that had been manufactured for the 4-stroke diesel engines of fishing boats. Gordonia sp. JC11, the most effective strain was able to degrade 25-55% of 1,000 mg L(-1) total hydrocarbons in six tested lubricants, while only 0-15% of the lubricants was abiotically removed. The bacterium had many characteristics that promoted lubricant degradation such as hydrocarbon utilization ability, emulsification activity and cell surface hydrophobicity. For bioaugmentation treatment of lubricant contaminated seawater, the inoculum of Gordonia sp. JC11 was prepared by immobilizing the bacterium on polyurethane foam (PUF). PUF-immobilized Gordonia sp. JC11 was able to remove 42-56% of 100-1,000 mg L(-1) waste lubricant No. 2 within 5 days. This lubricant removal efficiency was higher than those of free cells and PUF without bacterial cells. The bioaugmentation treatment significantly increased the number of lubricant-degrading microorganisms in the fishery port seawater microcosm and resulted in rapid removal of waste lubricant No. 2.     

Low temperature bioremediation of oil-contaminated soil using biostimulation and bioaugmentation with a Pseudomonas sp. from maritime Antarctica

AIMS: To identify native Antarctic bacteria capable of oil degradation at low temperatures. METHODS AND RESULTS: Oil contaminated and pristine soils from Signy Island (South Orkney Islands, Antarctica) were examined for bacteria capable of oil degradation at low temperatures. Of the 300 isolates cultured, Pseudomonas strain ST41 grew on the widest range of hydrocarbons at 4 degrees C. ST41 was used in microcosm studies of low temperature bioremediation of oil-contaminated soils. Microcosm experiments showed that at 4 degrees C the levels of oil degradation increased, relative to the controls, with (i) the addition of ST41 to the existing soil microbial population (bioaugmentation), (ii) the addition of nutrients (biostimulation) and to the greatest extent with (iii) a combination of both treatments (bioaugmentation and biostimulation). Addition of water to oil contaminated soil (hydration) also enhanced oil degradation, although less than the other treatments. Analysis of the dominant species in the microcosms after 12 weeks, using temporal temperature gradient gel electrophoresis, showed Pseudomonas species to be the dominant soil bacteria in both bioaugmented and biostimulated microcosms. CONCLUSIONS: Addition of water and nutrients may enhance oil degradation through the biostimulation of indigenous oil-degrading microbial populations within the soil. However, bioaugmentation with Antarctic bacteria capable of efficient low temperature hydrocarbon degradation may enhance the rate of bioremediation if applied soon after the spill. SIGNIFICANCE AND IMPACT OF THE STUDY: In the future, native soil bacteria could be of use in bioremediation technologies in Antarctica.     

Bioremediation of soil polluted with fuels by sequential multiple injection of native microorganisms: Field-scale processes in Poland

Research was conducted to estimate impact of the multiple bioaugmentation on the treatment of soil contaminated by fuels - diesel oil and aircraft fuel. The bacteria used to inoculate the remediation plots were isolated from the polluted soil and proliferated in field conditions. The amount of biomass applied to the polluted soil was set to ensure the total number of bacteria in soil 10⁷-10⁸ cfu/g d.w. The multiple inoculation of soil with indigenous bacteria active in diesel oil and engine oil (plot A) degradation increased bioremediation effectiveness by 50% in comparison to the non-inoculated control soil and by 30% in comparison to the soil that was inoculated only once. The multiple inoculation of soil with indigenous microorganisms was then applied in bioremediation of the soil polluted with double high concentration of diesel oil (soil B) and in bioremediation of the soil polluted with aircraft fuel (soil C). The process efficiency was 80% and 98% removal of TPH for soil B and C, respectively.     

Method for measuring substrate preferences by individual members of microbial consortia proposed for bioaugmentation

In this study we used the assimilation of isotope labeled CO(2) to measure the substrate preferences by two different bioaugmentation mixtures proposed for bioremediation of diesel oil contamination. All active microorganisms assimilate CO(2) in various carboxylation processes involved in growth. The CO(2) assimilation by the two mixtures was measured upon addition of glucose, diesel oil or specific compounds present in diesel oil (naphthalene, toluene, hexadecane, and octane). It was shown that within short term incubations with diesel oil (<5 h), one bioaugmentation mixture was superior to the other regarding the assimilation of CO(2). This observation was confirmed in a labor-intensive long term microcosm study (60 days). The applied method open various possibilities for fast pre-testing of substrate-preferences by microbial-bioaugmentation mixtures without microcosm experiments, onsite tests, and complicated chemical analysis. This study also demonstrates the possibility to obtain further information on the substrate preferences at a single cell level of phylogenetically defined microbial subgroups in bioaugmentation mixtures, based on combined analyses of microautoradiography and fluorescence in situ hybridization.