Evaluation of Inoculum Addition To Stimulate In Situ Bioremediation of Oily-Sludge-Contaminated Soil

A full-scale study evaluating an inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil was conducted at an oil refinery where the indigenous population of hydrocarbon-degrading bacteria in the soil was very low (10³ to 10⁴ CFU/g of soil). A feasibility study was conducted prior to the full-scale bioremediation study. In this feasibility study, out of six treatments, the application of a bacterial consortium and nutrients resulted in maximum biodegradation of total petroleum hydrocarbon (TPH) in 120 days. Therefore, this treatment was selected for the full-scale study. In the full-scale study, plots A and B were treated with a bacterial consortium and nutrients, which resulted in 92.0 and 89.7% removal of TPH, respectively, in 1 year, compared to 14.0% removal of TPH in the control plot C. In plot A, the alkane fraction of TPH was reduced by 94.2%, the aromatic fraction of TPH was reduced by 91.9%, and NSO (nitrogen-, sulfur-, and oxygen-containing compound) and asphaltene fractions of TPH were reduced by 85.2% in 1 year. Similarly, in plot B the degradation of alkane, aromatic, and NSO plus asphaltene fractions of TPH was 95.1, 94.8, and 63.5%, respectively, in 345 days. However, in plot C, removal of alkane (17.3%), aromatic (12.9%), and NSO plus asphaltene (5.8%) fractions was much less. The population of introduced Acinetobacter baumannii strains in plots A and B was stable even after 1 year. Physical and chemical properties of the soil at the bioremediation site improved significantly in 1 year.     

In Situ Bioremediation Potential of an Oily Sludge-Degrading Bacterial Consortium

A field-scale study was conducted in a 4000 m² plot of land contaminated with an oily sludge by use of a carrier-based hydrocarbon-degrading bacterial consortium for bioremediation. The land belonged to an oil refinery. Prior to this study, a feasibility study was conducted to assess the capacity of the bacterial consortium to degrade oily sludge. The site selected for bioremediation contained approximately 300 tons of oily sludge. The plot was divided into four blocks, based on the extent of contamination. Blocks A, B, and C were treated with the bacterial consortium, whereas Block D was maintained as an untreated control. In Block A, at time zero, i.e., at the beginning of the experiment, the soil contained as much as 99.2 g/kg of total petroleum hydrocarbon (TPH). The application of a bacterial consortium (1 kg carrier-based bacterial consortium/10 m² area) and nutrients degraded 90.2% of the TPH in 120 days, whereas in block D only 16.8% of the TPH was degraded. This study validates the large-scale use of a carrier-based bacterial consortium and nutrients for the treatment of land contaminated with oily sludge, a hazardous hydrocarbon waste generated by petroleum industry. Received: 20 October 2000 / Accepted: 22 March 2001     

Enhancing the biodegradation of total petroleum hydrocarbons in oily sludge by a modified bioaugmentation strategy

The effect of successive inoculation with hydrocarbon-degrading bacteria on the dynamics of petroleum hydrocarbons degradation in soil was investigated in this study. Oily sludge was used as a source of mixed hydrocarbons pollutant. Two bacterial consortia composed of alkanes and polycyclic aromatic hydrocarbon degraders were constructed from bacteria isolated from soil and oily sludge. These consortia were applied to incubated microcosms either in one dose at the onset of the incubation or in two doses at the beginning and at day 62 of the incubation period, which lasted for 198 days. During this period, carbon mineralization was evaluated by respirometry while total petroleum hydrocarbons and its fractions were gravimetrically evaluated by extraction from soil and fractionation. Dosing the bacterial consortia resulted in more than 30% increase in the overall removal of total petroleum hydrocarbons from soil. While alkane removal was only slightly improved, aromatic and asphaltic hydrocarbon fraction removal was significantly enhanced by the addition of the second consortium. Polar compounds (resins) were enriched only as a result of aromatics and asphaltene utilization. Nonetheless, their concentration declined back to the original level by the end of the incubation period.     

Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation

Bioremediation of diesel oil in soil can occur by natural attenuation, or treated by biostimulation or bioaugmentation. In this study we evaluated all three technologies on the degradation of total petroleum hydrocarbons (TPH) in soil. In addition, the number of diesel-degrading microorganisms present and microbial activity as indexed by the dehydrogenase assay were monitored. Soils contaminated with diesel oil in the field were collected from Long Beach, California, USA and Hong Kong, China. After 12 weeks of incubation, all three treatments showed differing effects on the degradation of light (C12-C23) and heavy (C23-C40) fractions of TPH in the soil samples. Bioaugmentation of the Long Beach soil showed the greatest degradation in the light (72.7%) and heavy (75.2%) fractions of TPH. Natural attenuation was more effective than biostimulation (addition of nutrients), most notably in the Hong Kong soil. The greatest microbial activity (dehydrogenase activity) was observed with bioaugmentation of the Long Beach soil (3.3-fold) and upon natural attenuation of the Hong Kong sample (4.0-fold). The number of diesel-degrading microorganisms and heterotrophic population was not influenced by the bioremediation treatments. Soil properties and the indigenous soil microbial population affect the degree of biodegradation; hence detailed site specific characterization studies are needed prior to deciding on the proper bioremediation method.     

Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creosote-contaminated soil

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the alpha-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the gamma-Proteobacteria group (genus Xanthomonas), the alpha-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the gamma-Proteobacteria group (genus Xathomonas), the beta-Proteobacteria group (genera Alcaligenes and Achromobacter), and the alpha-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.     

Restoration of Petroleum-Contaminated Soil Using Phased Bioremediation

A conceptual approach is presented for the restoration of petroleum-contaminated sites by combining bioremediation with revegetation using native plants. Phased bioremediation includes active and passive treatment options for soil containing greater than 1% total petroleum hydrocarbons (TPHs). Phase I is used when initial soil TPH exceeds 1%. Phase I utilizes either active land treatment, with regular soil tillage, or passive bioremediation to attain a treatment endpoint of 1% soil TPH. Passive treatment utilizes static soil and TPH-tolerant plants. Phase II is utilized when soil contains 1% TPH or less. It combines passive bioremediation with revegetation using native plants to complete the site restoration process. The phased approach to bioremediation was developed from results of full-scale field bioremediation and laboratory treatability studies. This approach assumes that the kinetics of TPH biodegradation are initially rapid, followed by a much slower second stage. It provides active initial treatment, followed by lower-cost passive treatment. The selection of either active or passive treatment in Phase I depends on whether total cost or time of treatment is more important. Passive treatment, although less costly than active treatment, generally requires more time. Phased bioremediation may provide a flexible, cost-effective, and technically sound approach for restoration of petroleum-contaminated sites.

Vegetation used with passive bioremediation has several benefits. Plants stabilize soil, preventing erosion and thereby minimizing exposure to soil contaminants. Phytoremediation may also occur within the rhizosphere. The use of native plants has a strong ecological basis. They provide ecological diversity, are aesthetically pleasing and beneficial to wildlife, while requiring little maintenance. Phased bioremediation can provide a flexible, cost-effective, and technically sound approach for the restoration of petroleum-contaminated sites.     

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.     

Assessment of hydrocarbon degradation potentials in a plant–microbe interaction system with oil sludge contamination: A sustainable solution

A pot culture experiment was conducted for 90 days for the evaluation of oil and total petroleum hydrocarbon (TPH) degradation in vegetated and non-vegetated treatments of real-field oil-sludge-contaminated soil. Five different treatments include (T1) control, 2% oil-sludge-contaminated soil; (T2), augmentation of microbial consortium; (T3), Vertiveria zizanioides; (T4), bio-augmentation along with V. zizanioides; and (T5), bio-augmentation with V. zizanioides and bulking agent. During the study, oil reduction, TPH, and degradation of its fractions were determined. Physico-chemical and microbiological parameters of soil were also monitored simultaneously. At the end of the experimental period, oil content (85%) was reduced maximally in bio-augmented rhizospheric treatments (T4 and T5) as compared to control (27%). TPH reduction was observed to be 88 and 89% in bio-augmented rhizospheric soil (T4 and T5 treatments), whereas in non-rhizospheric and control (T2 and T1), TPH reduction was 78 and 37%, respectively. Degradation of aromatic fraction after 90 days in bio-augmented rhizosphere of treatments T4 and T5 was found to 91 and 92%, respectively. In microbial (T2) and Vertiveria treatments (T3), degradation of aromatic fraction was 83 and 68%, respectively. A threefold increase in soil dehydrogenase activity and noticeable changes in organic carbon content and water-holding capacity were also observed which indicated maximum degradation of oil and its fractions in combined treatment of plants and microbes. It is concluded that the plant–microbe soil system helps to restore soil quality and can be used as an effective tool for the remediation of oil-sludge-contaminated sites.     

Protocol for laboratory testing of crude-oil bioremediation products in freshwater conditions

In 1993, the Environmental Protection Agency, National Risk Management Research Laboratory (EPA, NRMRL), with the National Environmental Technology Application Center (NETAC), developed a protocol for evaluation of bioremediation products in marine environments [18]. The marine protocol was adapted for application in freshwater environments by using a chemically defined medium and an oil-degrading consortium as a positive control. Four products were tested using the modified protocol: two with nutrients and an oleophilic component; one with nutrients, sorbent, and organisms; and one microbial stimulant. A separate experiment evaluated the use of HEPES and MOPSO buffers as replacements for phosphate buffer. The oleophilic nutrient products yielded oil degradation similar to the positive control, with an average alkane removal of 97.1±2.3% and an aromatic hydrocarbon removal of 64.8±1.2%. The positive control, which received inoculum plus nutrients, demonstrated alkane degradation of 98.9±0.1% and aromatic degradation of 52.9±0.1%. The sorbent-based product with inoculum failed to demonstrate oil degradation, while the microbial stimulant showed less oil degradation than the positive control. Replacement of phosphate buffer with other buffers had no significant effect on one product's performance. Differences in product performance were easily distinguishable using the protocol, and performance targets for alkane and aromatic hydrocarbon degradation are suggested. Electronic Publication     

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.     

Phenanthrene Biodegradation in Soils Using an Antarctic Bacterial Consortium

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in Antarctic soils is limited by low temperatures, lack of adequate levels of nutrients, low number of PAH-tolerant members in the autochthonous microbiota and low bioavailability of contaminants. In the present work, microcosms systems (performed in 1-L glass flasks containing Antarctic soil supplemented with 1744 ppm of phenanthrene) were used to study (i) the effect of biostimulation with a complex organic source of nutrients (fish meal) combined with a surfactant (Brij 700); (ii) the effect of bioaugmentation with a psychrotolerant PAH-degrading bacterial consortium (M10); (iii) the effect of the combination of both strategies. The authors found that combination of biostimulation and bioaugmentation caused a significant removal (46.6%) of phenanthrene after 56 days under Antarctic environmental conditions. When bioaugmentation or biostimulation were applied separately, nonsignificant reduction in phenanthrene concentration was observed. Microtox test showed a low increase in toxicity only in the most efficient system. Results proved that “in situ” bioremediation process of phenanthrene-contaminated soils is possible in Antarctic stations. In addition, inoculation with a psychrotolerant PAH-degrading bacterial consortium in association with a mix of fish meal and a high-molecular-weight surfactant improved phenanthrene removal and should be the selected strategy when the number of hydrocarbons degrading bacteria in the target soil is low.     

Influence of chemical surfactants on the biodegradation of crude oil by a mixed bacterial culture.

The effects of surfactant physicochemical properties, such as the hydrophile-lipophile balance (HLB) and molecular structure, on the biodegradation of 2% w/v Bow River crude oil by a mixed-bacterial culture were examined. Viable counts increased 4.6-fold and total petroleum hydrocarbon (TPH) biodegradation increased 57% in the presence of Igepal CO-630, a nonylphenol ethoxylate (HLB 13, 0.625 g/L). Only the nonylphenol ethoxylate with an HLB value of 13 substantially enhanced biodegradation. The surfactants from other chemical classes with HLB values of 13 (0.625 g/L) had no effect or were inhibitory. TPH biodegradation enhancement by Igepal CO-630 occurred at concentrations above the critical micelle concentration. When the effect of surfactant on individual oil fractions was examined, the biodegradation enhancement for the saturate and aromatic fractions was the same. In all cases, biodegradation resulted in increased resin and asphaltene concentrations. Optimal surfactant concentrations for TPH biodegradation reduced resin and asphaltene formation. Chemical surfactants have the potential to improve crude oil biodegradation in complex microbial systems, and surfactant selection should consider factors such as molecular structure, HLB, and surfactant concentration.     

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.     

Compositional changes during landfarming of weathered michigan crude oil‐contaminated soii

Laboratory landfarming experiments were conducted to study the bioremediation potential of weathered Michigan crude oil‐contaminated soils. It was found that landfarming was successful in removing up to 90% of the total petroleum hydrocarbons (TPH) in the soil within 22 weeks of treatment. Boiling point analyses of untreated and treated soils indicate a significant removal of TPH compounds independent of molecular weight or carbon number. Up to 85% of heavy petroleum hydrocarbons with carbon numbers above 44 were biode‐graded. In addition, approximately 93% of saturated and 79% of aromatic compounds of the TPH were biodegraded during the 22 week treatment period. The use of polyethylene sheeting as a landfarm cover does not appear to adversely affect biodegradation kinetics under laboratory conditions. Finally, equilibrium leachate concentrations for BTEX and regulated (in Michigan) polynuclear aromatics (PNAs) were below the respective detection limits for each compound. It can be concluded that landfarming of these weathered soils will be highly successful in removing petroleum hydrocarbons while not adversely impacting either ground‐water or surface water quality.     

Biodegradation Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge

This study focuses on the processes influencing hydrocarbon residue persistence in soil, following land treatment of refinery oily sludge. Treating sludge applied to soil resulted in 70% to 90% degradation of total petroleum hydrocarbon (TPH) during 2 months, regardless of their initial concentrations (9 to 60 g/kg soil). Kinetic analyses performed on TPH degradation, in laboratory and field systems, revealed a degradation pattern characterized by two consecutive first-order kinetics reactions in all experimental settings. The first stage lasted about 3 weeks and was characterized by a temperature dependent rate constant of 0.047 day^-1 at 24°C. That value was comparable to the rate constant obtained when combining the individual rate constants of the saturated, aromatic, asphaltene and polar fractions. The subsequent slower stage rate constant was 0.012 day^-1, insensitive to temperature and to hydrocarbon composition. The transition between the two stages (about 21 days) was independent of the experimental temperature and the biodegradation extent during the first stage. It was concluded that the extent of residual accumulation in the soil was determined by the biodegradation efficiency during the first three weeks of treatment when biological processes dominated. During the following period, abiotic processes leading to reduced bioavailability of the TPH were limiting the degradation rate. Practically, as the first few weeks of treatment determine its efficiency, efforts to enhance the biological activity should be directed to that period.     

Monitoring of petroleum hydrocarbon degradative potential of indigenous microorganisms in ozonated soil

This study was performed to investigate the petroleum hydrocarbon (PH) degradative potential of indigenous microorganisms in ozonated soil to better develop combined pre-ozonation/bioremediation technology. Diesel-contaminated soils were ozonated for 0–900min. PH and microbial concentrations in the soils decreased with increased ozonation time. The greatest reduction of total PH (TPH, 47.6%) and aromatics (11.3%) was observed in 900-min ozonated soil. The number of total viable heterotrophic bacteria decreased by three orders of magnitude in the soil. Ozonated soils were incubated for 9weeks for bioremediation. The number of microorganisms in the soils increased during the incubation period, as monitored by culture- and nonculture-based methods. The soils showed additional PH-removal during incubation, supporting the presence of PH-degraders in the soils. The highest removal (25.4%) of TPH was observed during the incubation of 180-min ozonated soil during the incubation while a negligible removal was shown in 900-min ozonated soil. This negligible removal could be explained by the existence of relatively few or undetected PH-degraders in 900-min ozonated soil. After a 9-week incubation of the ozonated soils, 180-min ozonated soil showed the lowest TPH concentration, suggesting that appropriate ozonation and indigenous microorganisms survived ozonation could enhance remediation of PH-contaminated soil. Microbial community composition in 9-week incubated soils revealed a slight difference between 900-min ozonated and unozonated soils, as analyzed by whole cell hybridization. Taken together, this study provided insight into indigenous microbial potential to degrade PH in ozonated soils.     

Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

Enhanced Biodegradation of Petroleum Hydrocarbons in Contaminated Soil

Soil samples taken from a contaminated site in Northern Quebec, Canada, exhibited a low capacity for biodegradation of total petroleum hydrocarbons (TPH), despite a high capacity for the mineralization of aromatic hydrocarbons and a low toxicity of soil leachates as measured by Microtox assay. Toxicity assays directly performed on surface soil, including earthworm mortality and barley seedling emergence, indicated moderate to high levels of toxicity. Soil biostimulation did not improve the removal of petroleum hydrocarbons, while bioaugmentation of soil with a developed enrichment culture increased the efficiency of hydrocarbon removal from 20.4% to 49.2%. A considerable increase in the removal of TPH was obtained in a bioslurry process, enhancing the mass transfer of hydrocarbons from soil to the aqueous phase and increasing the efficiency of hydrocarbon removal to over 70% after 45 days of incubation. The addition of ionic or nonionic surfactants did not have a significant impact on biodegradation of hydrocarbons. The extent of hydrocarbon mineralization during the bioslurry process after 45 days of incubation ranged from 41.3% to 58.9%, indicating that 62.7% to 83.1% of the eliminated TPH were transformed into CO₂ and water.     

Bioremediation of Crude Oil–Contaminated Soil By Immobilized Bacteria on an Agroindustrial Waste—Sunflower Seed Husks

This study reports the immobilization and performance of a hydrocarbon-degrading Rhodococcus sp. strain (designated as QBTo) on sunflower seed husks (SH) for the bioremediation of soils polluted with crude oil. The SH performance as inoculants carrier was compared with peat, which is a vegetal material traditionally used in carrier-based inoculants production. The stability of the immobilized culture under storage conditions was assessed by viability at different times when stored at 25°C and 10°C. The catabolic activity of immobilized and free QTBo cells introduced into sandy loam soil, freshly contaminated with crude oil, was studied in microcosms. A higher number of viable QTBo cells were recovered from the inoculants formulated with SH (QTBo-SH) after prolonged storage at 10°C and 25°C. The microcosms amended with QTBo-SH inoculants showed a removal of about 66% of total petroleum hydrocarbons (TPH), whereas in those inoculated with QTBo-peat inoculants, the decrease was of about 47%. In the control microcosms (noninoculated) and liquid culture–amended soils, the TPH removal was about 28%. SH is a waste of edible oil industry, nontoxic, and biodegradable and has demonstrated to confer to the immobilized cultures greater potential to survive not only during storage but also in the soil environment, improving bioremediation process.     

Microbial Activity and Community Composition during Bioremediation of Diesel-Oil-Contaminated Soil: Effects of Hydrocarbon Concentration,Fertilizers, and Incubation Time

We investigated the influence of three factors—diesel oil concentration [2500, 5000, 10,000, 20,000 mg total petroleum hydrocarbons (TPH) kg⁻¹ soil], biostimulation (unfertilized, inorganic fertilization with NPK nutrients, or oleophilic fertilization with Inipol EAP22), and incubation time—on hydrocarbon removal, enzyme activity (lipase), and microbial community structure [phospholipid fatty acids (PLFA)] in a laboratory soil bioremediation treatment. Fertilization enhanced TPH removal and lipase activity significantly (P ≤ 0.001). The higher the initial contamination, the more marked was the effect of fertilization. Differences between the two fertilizers were not significant (P > 0.05). Microbial communities, as assessed by PLFA patterns, were primarily influenced by the TPH content, followed by fertilization, and the interaction of these two factors, whereas incubation time was of minor importance. This was demonstrated by three-factorial analysis of variance and multidimensional scaling analysis. Low TPH content had no significant effect on soil microbial community, independent of the treatment. High TPH content generally resulted in increased PLFA concentrations, whereby a significant increase in microbial biomass with time was only observed with inorganic fertilization, whereas oleophilic fertilization (Inipol EAP22) tended to inhibit microbial activity and to reduce PLFA contents with time. Among bacteria, PLFA indicative of the Gram-negative population were significantly (P ≤ 0.05) increased in soil samples containing high amounts of diesel oil and fertilized with NPK after 21–38 days of incubation at 20°C. The Gram-positive population was not significantly influenced by TPH content or biostimulation treatment.