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1.  Effects of different compost amendments on the abundance and composition of alkB harboring bacterial communities in a soil under industrial use contaminated with hydrocarbons 
Alkane degrading microorganisms play an important role for the bioremediation of petrogenic contaminated environments. In this study, we investigated the effects of compost addition on the abundance and diversity of bacteria harboring the alkane monooxygenase gene (alkB) in an oil-contaminated soil originated from an industrial zone in Celje, Slovenia (Technosol). Soil without any amendments (control soil) and soil amended with two composts differing in their maturation stage and nutrient availability, were incubated under controlled conditions in a microcosm experiment and sampled after 0, 6, 12, and 36 weeks of incubation. As expected the addition of compost stimulated the degradation of alkanes in the investigated soil shortly after the addition. By using quantitative real-time PCR higher number of alkB genes were detected in soil samples amended with compost compared to the control soils. To get an insight into the composition of alkB harboring microbial communities, we performed next generation sequencing of amplicons of alkB gene fragment. Richness and diversity of alkB gene harboring prokaryotes was higher in soil mixed with compost compared to control soils with stronger effects of the less maturated, nutrient poor compost. The phylogenetic analysis of communities suggested that the addition of compost stimulated the abundance of alkB harboring Actinobacteria during the experiment independent from the maturation stage of the compost. AlkB harboring γ-proteobacteria like Shewanella or Hydrocarboniphaga as well as α-proteobacteria of the genus Agrobacterium responded also positively to the addition of compost to soil. The amendment of the less maturated, nutrient poor compost resulted in addition in a large increase of alkB harboring bacteria of the Cytophaga group (Microscilla) mainly at the early sampling time points. Our data indicates that compost amendments significantly change abundance and diversity pattern of alkB harboring microbes in Technosol and might be a useful agent to stimulate bioremediation of hydrocarbons in contaminated soils.
doi:10.3389/fmicb.2014.00096
PMCID: PMC3952045  PMID: 24659987
alkane monooxygenase alkB; compost; contaminated soils; bioremediation; next generation sequencing
2.  Evaluation of the biodegradation of Alaska North Slope oil in microcosms using the biodegradation model BIOB 
We present the details of a numerical model, BIOB that is capable of simulating the biodegradation of oil entrapped in the sediment. The model uses Monod kinetics to simulate the growth of bacteria in the presence of nutrients and the subsequent consumption of hydrocarbons. The model was used to simulate experimental results of Exxon Valdez oil biodegradation in laboratory columns (Venosa et al., 2010). In that study, samples were collected from three different islands: Eleanor Island (EL107), Knight Island (KN114A), and Smith Island (SM006B), and placed in laboratory microcosms for a duration of 168 days to investigate oil bioremediation through natural attenuation and nutrient amendment. The kinetic parameters of the BIOB model were estimated by fitting to the experimental data using a parameter estimation tool based on Genetic Algorithms (GA). The parameter values of EL107 and KN114A were similar whereas those of SM006B were different from the two other sites; in particular biomass growth at SM006B was four times slower than at the other two islands. Grain size analysis from each site revealed that the specific surface area per unit mass of sediment was considerably lower at SM006B, which suggest that the surface area of sediments is a key control parameter for microbial growth in sediments. Comparison of the BIOB results with exponential decay curves fitted to the data indicated that BIOB provided better fit for KN114A and SM006B in nutrient amended treatments, and for EL107 and KN114A in natural attenuation. In particular, BIOB was able to capture the initial slow biodegradation due to the lag phase in microbial growth. Sensitivity analyses revealed that oil biodegradation at all three locations were sensitive to nutrient concentration whereas SM006B was sensitive to initial biomass concentration due to its slow growth rate. Analyses were also performed to compare the half-lives of individual compounds with that of the overall polycyclic aromatic hydrocarbons (PAHs).
doi:10.3389/fmicb.2014.00212
PMCID: PMC4030209  PMID: 24860560
oil spill; biodegradation; numerical modeling
3.  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.
doi:10.1128/AEM.67.4.1675-1681.2001
PMCID: PMC92784  PMID: 11282620
4.  Bioremediation Potential of Terrestrial Fuel Spills † 
A bioremediation treatment that consisted of liming, fertilization, and tilling was evaluated on the laboratory scale for its effectiveness in cleaning up a sand, a loam, and a clay loam contaminated at 50 to 135 mg g of soil−1 by gasoline, jet fuel, heating oil, diesel oil, or bunker C. Experimental variables included incubation temperatures of 17, 27, and 37°C; no treatment; bioremediation treatment; and poisoned evaporation controls. Hydrocarbon residues were determined by quantitative gas chromatography or, in the case of bunker C, by residual weight determination. Four-point depletion curves were obtained for the described experimental variables. In all cases, the disappearance of hydrocarbons was maximal at 27°C and in response to bioremediation treatment. Poisoned evaporation controls underestimated the true biodegradation contribution, but nevertheless, they showed that biodegradation makes only a modest contribution to gasoline disappearance from soil. Bunker C was found to be structurally recalcitrant, with close to 80% persisting after 1 year of incubation. The three medium distillates, jet fuel, heating oil, and diesel oil, increased in persistence in the listed order but responded well to bioremediation treatment under all test conditions. With bioremediation treatment, it should be possible to reduce hydrocarbons to insignificant levels in contaminated soils within one growing season.
PMCID: PMC183400  PMID: 16348139
5.  Changes in bacterial community of anthracene bioremediation in municipal solid waste composting soil*  
Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in a municipal solid waste (MSW) composting site. Knowledge of changes in microbial structure is useful to identify particular PAH degraders. However, the microbial community in the MSW composting soil and its change associated with prolonged exposure to PAHs and subsequent biodegradation remain largely unknown. In this study, anthracene was selected as a model compound. The bacterial community structure was investigated using terminal restriction fragment length polymorphism (TRFLP) and 16S rRNA gene clone library analysis. The two bimolecular tools revealed a large shift of bacterial community structure after anthracene amendment and subsequent biodegradation. Genera Methylophilus, Mesorhizobium, and Terrimonas had potential links to anthracene biodegradation, suggesting a consortium playing an active role.
doi:10.1631/jzus.B1000440
PMCID: PMC3167910  PMID: 21887852
Anthracene; Microbial community; Biodegradation; Bioremediation
6.  Isolation and characterisation of polychlorinated biphenyl (PCB) degrading fungi from a historically contaminated soil 
Background
Polychlorinated biphenyls (PCBs) are widespread toxic pollutants. Bioremediation might be an effective, cost competitive and environment-friendly solution for remediating environmental matrices contaminated by PCBs but it is still unsatisfactory, mostly for the limited biodegradation potential of bacteria involved in the processes. Very little is known about mitosporic fungi potential in PCB bioremediation and their occurrence in actual site historically contaminated soils. In the present study, we characterised the native mycoflora of an aged dump site soil contaminated by about 0.9 g kg-1 of Aroclor 1260 PCBs and its changing after aerobic biotreatment with a commercial complex source of bacteria and fungi. Fungi isolated from the soil resulting from 120 days of treatment were screened for their ability to adsorb or metabolise 3 target PCBs.
Results
The original contaminated soil contained low loads of few fungal species mostly belonging to the Scedosporium, Penicillium and Aspergillus genera. The fungal load and biodiversity generally decreased throughout the aerobic treatment. None of the 21 strains isolated from the treated soil were able to grow on biphenyl (200 mg L-1) or a mixture of 2-chlorobiphenyl, 4,4'-dichlorobiphenyl and 2,2',5,5'-tetrachlorobiphenyl (20 mg L-1 each) as sole carbon sources. However, 16 of them grew in a mineral medium containing the same PCBs mixture and glucose (10 g L-1). Five of the 6 isolates, which displayed the faster and more extensive growth under the latter conditions, were found to degrade the 3 PCBs apparently without the involvement of ligninolytic enzymes; they were identified as Penicillium chrysogenum, Scedosporium apiospermum, Penicillium digitatum and Fusarium solani. They are the first PCB degrading strains of such species reported so far in the literature.
Conclusion
The native mycoflora of the actual site aged heavily contaminated soil was mainly constituted by genera often reported as able to biodegrade organopollutants. It was generally remarkably reduced after the biotreatment, which however resulted in the selection of few mitosporic fungal species able to biodegrade PCBs. This is the first study in which an extensive characterisation of the cultivable indigenous mycoflora of an actual site aged PCB contaminated soil, as well as its changes upon soil bioremediation treatment, was conducted. Moreover, this is the first paper in which 5 strains ascribable to 4 mitosporic species able to biodegrade PCB are reported in the literature.
doi:10.1186/1475-2859-8-5
PMCID: PMC2636759  PMID: 19138404
7.  Efficiency of Indigenous and Inoculated Cold-Adapted Soil Microorganisms for Biodegradation of Diesel Oil in Alpine Soils 
Biodegradation of diesel oil (5 g(middot)kg [soil dry weight](sup-1)) was investigated in five alpine subsoils, differing in soil type and bedrock, in laboratory experiments during 20 days at 10(deg)C. The biodegradation activities of the indigenous soil microorganisms and of a psychrotrophic diesel oil-degrading inoculum and the effect of biostimulation by inorganic fertilization (C/N/P ratio = 100:10:2) were determined. Fertilization significantly enhanced diesel oil biodegradation activity of the indigenous soil microorganisms. Biostimulation by fertilization enhanced diesel oil biodegradation to a significantly greater degree than bioaugmentation with the psychrotrophic inoculum. In none of the five soils did fertilization plus inoculation result in a higher decontamination than fertilization alone. A total of 16 to 23% of the added diesel oil contamination was lost by abiotic processes. Total decontamination without and with fertilization was in the range of 16 to 31 and 27 to 53%, respectively.
PMCID: PMC1389197  PMID: 16535642
8.  Crude Oil Treatment Leads to Shift of Bacterial Communities in Soils from the Deep Active Layer and Upper Permafrost along the China-Russia Crude Oil Pipeline Route 
PLoS ONE  2014;9(5):e96552.
The buried China-Russia Crude Oil Pipeline (CRCOP) across the permafrost-associated cold ecosystem in northeastern China carries a risk of contamination to the deep active layers and upper permafrost in case of accidental rupture of the embedded pipeline or migration of oil spills. As many soil microbes are capable of degrading petroleum, knowledge about the intrinsic degraders and the microbial dynamics in the deep subsurface could extend our understanding of the application of in-situ bioremediation. In this study, an experiment was conducted to investigate the bacterial communities in response to simulated contamination to deep soil samples by using 454 pyrosequencing amplicons. The result showed that bacterial diversity was reduced after 8-weeks contamination. A shift in bacterial community composition was apparent in crude oil-amended soils with Proteobacteria (esp. α-subdivision) being the dominant phylum, together with Actinobacteria and Firmicutes. The contamination led to enrichment of indigenous bacterial taxa like Novosphingobium, Sphingobium, Caulobacter, Phenylobacterium, Alicylobacillus and Arthrobacter, which are generally capable of degrading polycyclic aromatic hydrocarbons (PAHs). The community shift highlighted the resilience of PAH degraders and their potential for in-situ degradation of crude oil under favorable conditions in the deep soils.
doi:10.1371/journal.pone.0096552
PMCID: PMC4008593  PMID: 24794099
9.  Robust Hydrocarbon Degradation and Dynamics of Bacterial Communities during Nutrient-Enhanced Oil Spill Bioremediation 
Applied and Environmental Microbiology  2002;68(11):5537-5548.
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.
doi:10.1128/AEM.68.11.5537-5548.2002
PMCID: PMC129918  PMID: 12406747
10.  Bioremediation (Natural Attenuation and Biostimulation) of Diesel-Oil-Contaminated Soil in an Alpine Glacier Skiing Area 
We investigated the feasibility of bioremediation as a treatment option for a chronically diesel-oil-polluted soil in an alpine glacier area at an altitude of 2,875 m above sea level. To examine the efficiencies of natural attenuation and biostimulation, we used field-incubated lysimeters (mesocosms) with unfertilized and fertilized (N-P-K) soil. For three summer seasons (July 1997 to September 1999), we monitored changes in hydrocarbon concentrations in soil and soil leachate and the accompanying changes in soil microbial counts and activity. A significant reduction in the diesel oil level could be achieved. At the end of the third summer season (after 780 days), the initial level of contamination (2,612 ± 70 μg of hydrocarbons g [dry weight] of soil−1) was reduced by (50 ± 4)% and (70 ± 2)% in the unfertilized and fertilized soil, respectively. Nonetheless, the residual levels of contamination (1,296 ± 110 and 774 ± 52 μg of hydrocarbons g [dry weight] of soil−1 in the unfertilized and fertilized soil, respectively) were still high. Most of the hydrocarbon loss occurred during the first summer season ([42 ± 6]% loss) in the fertilized soil and during the second summer season ([41 ± 4]% loss) in the unfertilized soil. In the fertilized soil, all biological parameters (microbial numbers, soil respiration, catalase and lipase activities) were significantly enhanced and correlated significantly with each other, as well as with the residual hydrocarbon concentration, pointing to the importance of biodegradation. The effect of biostimulation of the indigenous soil microorganisms declined with time. The microbial activities in the unfertilized soil fluctuated around background levels during the whole study.
doi:10.1128/AEM.67.7.3127-3133.2001
PMCID: PMC92991  PMID: 11425732
11.  Effect of 2-hydroxybenzoate on the maintenance of naphthalene-degrading pseudomonads in seeded and unseeded soil. 
Applied and Environmental Microbiology  1991;57(10):2873-2879.
The addition of specific nontoxic inducers of catabolic operons to contaminated sites is an approach that may enhance the efficiency of in situ biodegradation. We determined the genetic response of six pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of available carbon, and their DNA sequences show significant homology to the nahAB genes of the degradative plasmid NAH7. Duplicate nonsterile soil cultures were incubated for up to 30 days. Experimental soil cultures were seeded with naphthalene-degrading strains (10(8) CFU g-1) originally isolated from the soil and amended with salicylate (16 or 160 micrograms g-1). Soil samples were analyzed periodically for the population density of heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype in the bacterial community. At 160 micrograms g-1, salicylate sustained the density of naphthalene degraders at the introduced density for 30 days in addition to producing a two- to sixfold increase in the occurrence in the bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 micrograms of salicylate g-1, but this concentration of salicylate induced a significant increase in the level of nah-related genes in the population.
Images
PMCID: PMC183889  PMID: 1746947
12.  Investigation of potential rhizospheric isolate for cypermethrin degradation 
3 Biotech  2012;3(1):33-43.
Rhizoremediation is the use of plant–microbe interaction for the enhanced degradation of contaminants. Rhizosphere bioremediation of pyrethroid pesticides will offer an attractive and potentially inexpensive approach for remediation of contaminated soil. The present study was done with the aim of establishment of highly effective remediation method using plant with degradative rhizosphere and isolation of naturally occurring rhizosphere associated potential degrader providing the possibility of both environmental and insitu detoxification of cypermethrin contamination. The remediation efficacy of Pennisetum pedicellatum was investigated using green house pot culture experiments in cypermethrin amended potting soil mix (10, 25, 50, 75 and 100 mg/kg) for periodic evaluation of changes in concentration. Total proportion of cypermethrin degraders was found to be higher in rhizosphere soil compared to bulk soil. The cypermethrin degrading strain associated with rhizosphere capable of surviving at higher concentrations of cypermethrin was designated as potential degrader. On the basis of morphological characteristics, biochemical tests and 16S rDNA analysis, isolate was identified as Stenotrophomonas maltophilia MHF ENV 22. Bioremediation data of cypermethrin by strain MHF ENV22 examined by HPLC and mass spectroscopy, indicated 100, 50 and 58 % degradation within the time period of 72, 24 and 192 h at concentrations 25, 50 and 100 mg/kg, respectively. This is the first report of effective degradation of cypermethrin by Stenotrophomonas spp. isolated from rhizosphere of Pennisetum pedicellatum. Rhizoremediation strategy will be of immense importance in remediation of cypermethrin residues to a level permissible for technogenic and natural environment.
doi:10.1007/s13205-012-0067-3
PMCID: PMC3563741
Rhizoremediation; Soil; Cypermethrin; Stenotrophomonas maltophilia MHF ENV 22
13.  Response of Archaeal Communities in Beach Sediments to Spilled Oil and Bioremediation 
While the contribution of Bacteria to bioremediation of oil-contaminated shorelines is well established, the response of Archaea to spilled oil and bioremediation treatments is unknown. The relationship between archaeal community structure and oil spill bioremediation was examined in laboratory microcosms and in a bioremediation field trial. 16S rRNA gene-based PCR and denaturing gradient gel analysis revealed that the archaeal community in oil-free laboratory microcosms was stable for 26 days. In contrast, in oil-polluted microcosms a dramatic decrease in the ability to detect Archaea was observed, and it was not possible to amplify fragments of archaeal 16S rRNA genes from samples taken from microcosms treated with oil. This was the case irrespective of whether a bioremediation treatment (addition of inorganic nutrients) was applied. Since rapid oil biodegradation occurred in nutrient-treated microcosms, we concluded that Archaea are unlikely to play a role in oil degradation in beach ecosystems. A clear-cut relationship between the presence of oil and the absence of Archaea was not apparent in the field experiment. This may have been related to continuous inoculation of beach sediments in the field with Archaea from seawater or invertebrates and shows that the reestablishment of Archaea following bioremediation cannot be used as a determinant of ecosystem recovery following bioremediation. Comparative 16S rRNA sequence analysis showed that the majority of the Archaea detected (94%) belonged to a novel, distinct cluster of group II uncultured Euryarchaeota, which exhibited less than 87% identity to previously described sequences. A minor contribution of group I uncultured Crenarchaeota was observed.
doi:10.1128/AEM.70.5.2614-2620.2004
PMCID: PMC404411  PMID: 15128510
14.  Isolation and Identification of hydrocarbon degrading bacteria from Ennore creek 
Bioinformation  2013;9(3):150-157.
The widespread problem caused due to petroleum products, is their discharge and accidental spillage in marine environment proving to be hazardous to the surroundings as well as life forms. Thus remediation of these hydrocarbons by natural decontamination process is of utmost importance. Bioremediation is a non-invasive and cost effective technique for the clean-up of these petroleum hydrocarbons. In this study we have investigated the ability of microorganisms present in the sediment sample to degrade these hydrocarbons, crude oil in particular, so that contaminated soils and water can be treated using microbes. Sediments samples were collected once in a month for a period of twelve months from area surrounding Ennore creek and screened for hydrocarbon degrading bacteria. Of the 113 crude oil degrading isolates 15 isolates were selected and cultivated in BH media with 1% crude oil as a sole carbon and energy source. 3 efficient crude oil bacterial isolates Bacillus subtilis I1, Pseudomonas aeruginosa I5 and Pseudomonas putida I8 were identified both biochemically and phylogenetically. The quantitative analysis of biodegradation is carried out gravimetrically and highest degradation rate, 55% was recorded by Pseudomonas aeruginosa I5 isolate.
doi:10.6026/97320630009150
PMCID: PMC3569603  PMID: 23424279
15.  Kinetic parameters for nutrient enhanced crude oil biodegradation in intertidal marine sediments 
Availability of inorganic nutrients, particularly nitrogen and phosphorous, is often a primary control on crude oil hydrocarbon degradation in marine systems. Many studies have empirically determined optimum levels of inorganic N and P for stimulation of hydrocarbon degradation. Nevertheless, there is a paucity of information on fundamental kinetic parameters for nutrient enhanced crude oil biodegradation that can be used to model the fate of crude oil in bioremediation programmes that use inorganic nutrient addition to stimulate oil biodegradation. Here we report fundamental kinetic parameters (Ks and qmax) for nitrate- and phosphate-stimulated crude oil biodegradation under nutrient limited conditions and with respect to crude oil, under conditions where N and P are not limiting. In the marine sediments studied, crude oil degradation was limited by both N and P availability. In sediments treated with 12.5 mg/g of oil but with no addition of N and P, hydrocarbon degradation rates, assessed on the basis of CO2 production, were 1.10 ± 0.03 μmol CO2/g wet sediment/day which were comparable to rates of CO2 production in sediments to which no oil was added (1.05 ± 0.27 μmol CO2/g wet sediment/day). When inorganic nitrogen was added alone maximum rates of CO2 production measured were 4.25 ± 0.91 μmol CO2/g wet sediment/day. However, when the same levels of inorganic nitrogen were added in the presence of 0.5% P w/w of oil (1.6 μmol P/g wet sediment) maximum rates of measured CO2 production increased more than four-fold to 18.40 ± 1.04 μmol CO2/g wet sediment/day. Ks and qmax estimates for inorganic N (in the form of sodium nitrate) when P was not limiting were 1.99 ± 0.86 μmol/g wet sediment and 16.16 ± 1.28 μmol CO2/g wet sediment/day respectively. The corresponding values for P were 63 ± 95 nmol/g wet sediment and 12.05 ± 1.31 μmol CO2/g wet sediment/day. The qmax values with respect to N and P were not significantly different (P < 0.05). When N and P were not limiting Ks and qmax for crude oil were 4.52 ± 1.51 mg oil/g wet sediment and 16.89 ± 1.25 μmol CO2/g wet sediment/day. At concentrations of inorganic N above 45 μmol/g wet sediment inhibition of CO2 production from hydrocarbon degradation was evident. Analysis of bacterial 16S rRNA genes indicated that Alcanivorax spp. were selected in these marine sediments with increasing inorganic nutrient concentration, whereas Cycloclasticus spp. were more prevalent at lower inorganic nutrient concentrations. These data suggest that simple empirical estimates of the proportion of nutrients added relative to crude oil concentrations may not be sufficient to guarantee successful crude oil bioremediation in oxic beach sediments. The data we present also help define the maximum rates and hence timescales required for bioremediation of beach sediments.
doi:10.3389/fmicb.2014.00160
PMCID: PMC3990054  PMID: 24782848
oil spill; bioremediation; kinetics; Ks; half saturation constant; maximal rates; Alcanivorax; Cycloclasticus
16.  Rhamnolipids Increase the Phytotoxicity of Diesel Oil Towards Four Common Plant Species in a Terrestrial Environment 
Water, Air, and Soil Pollution  2012;223(7):4275-4282.
The study focused on assessing the influence of rhamnolipids on the phytotoxicity of diesel oil-contaminated soil samples. Tests evaluating the seed germination and growth inhibition of four terrestrial plant species (alfalfa, sorghum, mustard and cuckooflower) were carried out at different rhamnolipid concentrations (ranging from 0 to 1.200 mg/kg of wet soil). The experiments were performed in soil samples with a different diesel oil content (ranging from 0 to 25 ml/kg of wet soil). It was observed that the sole presence of rhamnolipids may be phytotoxic at various levels, which is especially notable for sorghum (the germination index decreased to 41 %). The addition of rhamnolipids to diesel oil-contaminated soil samples contributed to a significant increase of their phytotoxicity. The most toxic effect was observed after a rhamnolipid-supplemented diesel oil biodegradation, carried out with the use of a hydrocarbon-degrading bacteria consortium. The supplemention of rhamnolipids (600 mg/kg of wet soil) resulted in a decrease of seed germination of all studied plant species and an inhibition of microbial activity, which was measured by the 2,3,5-triphenyltetrazolium chloride tests. These findings indicate that the presence of rhamnolipids may considerably increase the phytotoxicity of diesel oil. Therefore, their use at high concentrations, during in situ bioremediation processes, should be avoided in a terrestrial environment.
doi:10.1007/s11270-012-1190-9
PMCID: PMC3409371  PMID: 22865941
Bacterial consortium; Diesel oil; Rhamnolipids; Seed germination; Phytotoxicity
17.  Biodegradation of [(sup14)C]Benzo[a]pyrene Added in Crude Oil to Uncontaminated Soil 
Applied and Environmental Microbiology  1997;63(11):4511-4515.
To investigate the possible cometabolic biodegradation of benzo[a]pyrene (BaP), crude oil spiked with [7-(sup14)C]BaP and unlabeled BaP was added to soil with no known pollution history, to give 34 g of oil and 67 mg of BaP/kg of dry soil. The oil-soil mixture was amended with mineral nutrients and incubated in an airtight container with continuous forced aeration. Total CO(inf2) and (sup14)CO(inf2) in the off-gas were trapped and quantified. Soil samples were Soxhlet extracted with dichloromethane at seven time points during the 150-day incubation period, and the extracted soil was subjected to further fractionation in order to recover reversibly and irreversibly bound radiocarbon. Radiocarbon recovery was 100% (plusmn) 3% for each time point. During the first 50 days of incubation, no (sup14)CO(inf2) was evolved, but over the next 100 days, 50% of the BaP radiocarbon was evolved as (sup14)CO(inf2). At 150 days, only 5% of the intact BaP and 23% of the crude oil remained. Of the remaining radiolabel, 20% was found in solvent-extractable metabolites and 25% was incorporated into soil organic matter. Only 1/10 of this could be solubilized by chemical hydrolysis. An abiotic control experiment exhibited binding of only 2% of the BaP, indicating the microbial nature of the BaP transformations. We report that in soil containing suitable cosubstrates, BaP can be completely degraded.
PMCID: PMC1389291  PMID: 16535735
18.  Bioavailability of Heavy Metals in Soil: Impact on Microbial Biodegradation of Organic Compounds and Possible Improvement Strategies 
Co-contamination of the environment with toxic chlorinated organic and heavy metal pollutants is one of the major problems facing industrialized nations today. Heavy metals may inhibit biodegradation of chlorinated organics by interacting with enzymes directly involved in biodegradation or those involved in general metabolism. Predictions of metal toxicity effects on organic pollutant biodegradation in co-contaminated soil and water environments is difficult since heavy metals may be present in a variety of chemical and physical forms. Recent advances in bioremediation of co-contaminated environments have focussed on the use of metal-resistant bacteria (cell and gene bioaugmentation), treatment amendments, clay minerals and chelating agents to reduce bioavailable heavy metal concentrations. Phytoremediation has also shown promise as an emerging alternative clean-up technology for co-contaminated environments. However, despite various investigations, in both aerobic and anaerobic systems, demonstrating that metal toxicity hampers the biodegradation of the organic component, a paucity of information exists in this area of research. Therefore, in this review, we discuss the problems associated with the degradation of chlorinated organics in co-contaminated environments, owing to metal toxicity and shed light on possible improvement strategies for effective bioremediation of sites co-contaminated with chlorinated organic compounds and heavy metals.
doi:10.3390/ijms140510197
PMCID: PMC3676836  PMID: 23676353
biodegradation; biosorption; chlorinated compounds; co-contamination; heavy metals
19.  Application of Organic Amendments to a Coastal Saline Soil in North China: Effects on Soil Physical and Chemical Properties and Tree Growth 
PLoS ONE  2014;9(2):e89185.
The ability of the following four organic amendments to ameliorate saline soil in coastal northern China was investigated from April 2010 to October 2012 in a field experiment: green waste compost (GWC), sedge peat (SP), furfural residue (FR), and a mixture of GWC, SP and FR (1∶1∶1 by volume) (GSF). Compared to a non-amended control (CK), the amendments, which were applied at 4.5 kg organic matter m−3, dramatically promoted plant growth; improved soil structure; increased the cation exchange capacity (CEC), organic carbon, and available nutrients; and reduced the salt content, electrical conductivity (EC), and exchangeable sodium percentage (ESP). At the end of the experiment in soil amended with GSF, bulk density, EC, and ESP had decreased by 11, 87, and 71%, respectively, and total porosity and organic carbon had increased by 25 and 96% respectively, relative to the CK. The GSF treatment resulted in a significantly lower Na++K+ content than the other treatments. CEC and the contents of available N, P, and K were significantly higher in the GSF-treated soil than in the CK and were the highest in all treatments. The FR treatment resulted in the lowest pH value and Ca2+ concentration, which decreased by 8% and 39%, respectively, relative to the CK. Overall, the results indicate that a combination of green waste compost, sedge peat and furfural residue (GSF treatment) has substantial potential for ameliorating saline soils in the coastal areas of northern China, and it works better than each amendment alone. Utilization of GWC and FR can be an alternative organic amendment to substitute the nonrenewable SP in saline soil amelioration.
doi:10.1371/journal.pone.0089185
PMCID: PMC3928440  PMID: 24558486
20.  Endophytic microorganisms—promising applications in bioremediation of greenhouse gases 
Bioremediation is a technique that uses microbial metabolism to remove pollutants. Various techniques and strategies of bioremediation (e.g., phytoremediation enhanced by endophytic microorganisms, rhizoremediation) can mainly be used to remove hazardous waste from the biosphere. During the last decade, this specific technique has emerged as a potential cleanup tool only for metal pollutants. This situation has changed recently as a possibility has appeared for bioremediation of other pollutants, for instance, volatile organic compounds, crude oils, and radionuclides. The mechanisms of bioremediation depend on the mobility, solubility, degradability, and bioavailability of contaminants. Biodegradation of pollutions is associated with microbial growth and metabolism, i.e., factors that have an impact on the process. Moreover, these factors have a great influence on degradation. As a result, recognition of natural microbial processes is indispensable for understanding the mechanisms of effective bioremediation. In this review, we have emphasized the occurrence of endophytic microorganisms and colonization of plants by endophytes. In addition, the role of enhanced bioremediation by endophytic bacteria and especially of phytoremediation is presented.
doi:10.1007/s00253-013-5235-9
PMCID: PMC3825493  PMID: 24048641
Bioremediation; Greenhouse gases; Plant; Endophytes
21.  Biodegradability of commercial and weathered diesel oils 
Brazilian Journal of Microbiology  2008;39(1):133-142.
This work aimed to evaluate the capability of different microorganisms to degrade commercial diesel oil in comparison to a weathered diesel oil collected from the groundwater at a petrol station. Two microbiological methods were used for the biodegradability assessment: the technique based on the redox indicator 2,6 -dichlorophenol indophenol (DCPIP) and soil respirometric experiments using biometer flasks. In the former we tested the bacterial cultures Staphylococcus hominis, Kocuria palustris, Pseudomonas aeruginosa LBI, Ochrobactrum anthropi and Bacillus cereus, a commercial inoculum, consortia obtained from soil and groundwater contaminated with hydrocarbons and a consortium from an uncontaminated area. In the respirometric experiments it was evaluated the capability of the native microorganisms present in the soil from a petrol station to biodegrade the diesel oils. The redox indicator experiments showed that only the consortia, even that from an uncontaminated area, were able to biodegrade the weathered diesel. In 48 days, the removal of the total petroleum hydrocarbons (TPH) in the respirometric experiments was approximately 2.5 times greater when the commercial diesel oil was used. This difference was caused by the consumption of labile hydrocarbons, present in greater quantities in the commercial diesel oil, as demonstrated by gas chromatographic analyses. Thus, results indicate that biodegradability studies that do not consider the weathering effect of the pollutants may over estimate biodegradation rates and when the bioaugmentation is necessary, the best strategy would be that one based on injection of consortia, because even cultures with recognised capability of biodegrading hydrocarbons may fail when applied isolated.
doi:10.1590/S1517-838220080001000028
PMCID: PMC3768377  PMID: 24031193
biodegradability; bioremediation; commercial oil diesel; weathered diesel oil
22.  Utilization of oleo-chemical industry by-products for biosurfactant production 
AMB Express  2013;3:68.
Biosurfactants are the surface active compounds produced by micro-organisms. The eco-friendly and biodegradable nature of biosurfactants makes their usage more advantageous over chemical surfactants. Biosurfactants encompass the properties of dropping surface tension, stabilizing emulsions, promoting foaming and are usually non- toxic and biodegradable. Biosurfactants offer advantages over their synthetic counterparts in many applications ranging from environmental, food, and biomedical, cosmetic and pharmaceutical industries. The important environmental applications of biosurfactants include bioremediation and dispersion of oil spills, enhanced oil recovery and transfer of crude oil. The emphasis of present review shall be with reference to the commercial production, current developments and future perspectives of a variety of approaches of biosurfactant production from the micro-organisms isolated from various oil- contaminated sites and from the by-products of oleo-chemical industry wastes/ by-products viz. used edible oil, industrial residues, acid oil, deodorizer distillate, soap-stock etc.
doi:10.1186/2191-0855-3-68
PMCID: PMC3923564  PMID: 24262384
Biosurfactants; Agro- chemical waste; Rhamnolipid; Oil industry
23.  In situ detection of anaerobic alkane metabolites in subsurface environments 
Alkanes comprise a substantial fraction of crude oil and refined fuels. As such, they are prevalent within deep subsurface fossil fuel deposits and in shallow subsurface environments such as aquifers that are contaminated with hydrocarbons. These environments are typically anaerobic, and host diverse microbial communities that can potentially use alkanes as substrates. Anaerobic alkane biodegradation has been reported to occur under nitrate-reducing, sulfate-reducing, and methanogenic conditions. Elucidating the pathways of anaerobic alkane metabolism has been of interest in order to understand how microbes can be used to remediate contaminated sites. Alkane activation primarily occurs by addition to fumarate, yielding alkylsuccinates, unique anaerobic metabolites that can be used to indicate in situ anaerobic alkane metabolism. These metabolites have been detected in hydrocarbon-contaminated shallow aquifers, offering strong evidence for intrinsic anaerobic bioremediation. Recently, studies have also revealed that alkylsuccinates are present in oil and coal seam production waters, indicating that anaerobic microbial communities can utilize alkanes in these deeper subsurface environments. In many crude oil reservoirs, the in situ anaerobic metabolism of hydrocarbons such as alkanes may be contributing to modern-day detrimental effects such as oilfield souring, or may lead to more beneficial technologies such as enhanced energy recovery from mature oilfields. In this review, we briefly describe the key metabolic pathways for anaerobic alkane (including n-alkanes, isoalkanes, and cyclic alkanes) metabolism and highlight several field reports wherein alkylsuccinates have provided evidence for anaerobic in situ alkane metabolism in shallow and deep subsurface environments.
doi:10.3389/fmicb.2013.00140
PMCID: PMC3671572  PMID: 23761789
anaerobic; metabolites; alkylsuccinates; alkanes; subsurface
24.  Toxicological evaluation of vegetable oils and biodiesel in soil during the biodegradation process 
Brazilian Journal of Microbiology  2012;43(4):1576-1581.
Vegetable oils and their derivatives, like biodiesel, are used extensively throughout the world, thus posing an environmental risk when disposed. Toxicity testing using test organisms shows how these residues affect ecosystems. Toxicity tests using earthworms (Eisenia foetida) are widespread because they are a practical resource for analyzing terrestrial organisms. For phytotoxicological analysis, we used seeds of arugula (Eruca sativa) and lettuce (Lactuca sativa) to analyze the germination of seeds in contaminated soil samples. The toxicological experiment was conducted with four different periods of biodegradation in soil: zero days, 60 days, 120 days and 180 days. The studied contaminants were soybean oil (new and used) and biodiesel (B100). An evaluation of the germination of both seeds showed an increased toxicity for all contaminants as the biodegradation occurred, biodiesel being the most toxic among the contaminants. On the other hand, for the tests using earthworms, the biodiesel was the only contaminant that proved to be toxic. Therefore, the higher toxicity of the sample containing these hydrocarbons over time can be attributed to the secondary compounds formed by microbial action. Thus, we conclude that the biodegradation in soil of the studied compounds requires longer periods for the sample toxicity to be decreased with the action of microorganisms.
doi:10.1590/S1517-838220120004000042
PMCID: PMC3769042  PMID: 24031989
bioremediation; toxicity; soybean oil; soil.
25.  Biodegradation of Toluene Under Seasonal and Diurnal Fluctuations of Soil-Water Temperature 
Water, Air, and Soil Pollution  2012;223(7):3579-3588.
An increasing interest in bioremediation of hydrocarbon polluted sites raises the question of the influence of seasonal and diurnal changes on soil-water temperature on biodegradation of BTEX, a widespread group of (sub)-surface contaminants. Therefore, we investigated the impact of a wide range of varying soil-water temperature on biodegradation of toluene under aerobic conditions. To see the seasonal impact of temperature, three sets of batch experiments were conducted at three different constant temperatures: 10°C, 21°C, and 30°C. These conditions were considered to represent (1) winter, (2) spring and/or autumn, and (3) summer seasons, respectively, at many polluted sites. Three additional sets of batch experiments were performed under fluctuating soil-water temperature cases (21<>10°C, 30<>21°C, and 10<>30°C) to mimic the day–night temperature patterns expected during the year. The batches were put at two different temperatures alternatively to represent the day (high-temperature) and night (low-temperature) times. The results of constant- and fluctuating-temperature experiments show that toluene degradation is strongly dependent on soil-water temperature level. An almost two-fold increase in toluene degradation time was observed for every 10°C decrease in temperature for constant-temperature cases. Under fluctuating-temperature conditions, toluene degraders were able to overcome the temperature stress and continued thriving during all considered weather scenarios. However, a slightly longer time was taken compared to the corresponding time at daily mean temperature conditions. The findings of this study are directly useful for bioremediation of hydrocarbon-polluted sites having significant diurnal and seasonal variations of soil-water temperature.
doi:10.1007/s11270-011-1052-x
PMCID: PMC3409364  PMID: 22865939
Biodegradation; Toluene; BTEX; Varying temperature; Soil-water pollution

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