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 α-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the γ-Proteobacteria group (genus Xanthomonas), the α-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the γ-Proteobacteria group (genus Xathomonas), the β-Proteobacteria group (genera Alcaligenes and Achromobacter), and the α-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.     

Bacterial Community Dynamics during Bioremediation of Diesel Oil-Contaminated Antarctic Soil

The effect of nutrient and inocula amendment in a bioremediation field trial using a nutrient-poor Antarctic soil chronically contaminated with hydrocarbons was tested. The analysis of the effects that the treatments caused in bacterial numbers and hydrocarbon removal was combined with the elucidation of the changes occurring on the bacterial community, by 16S rDNA-based terminal restriction fragment length polymorphism (T-RFLP) typing, and the detection of some of the genes involved in the catabolism of hydrocarbons. All treatments caused a significant increase in the number of bacteria able to grow on hydrocarbons and a significant decrease in the soil hydrocarbon content, as compared to the control. However, there were no significant differences between treatments. Comparison of the soil T-RFLP profiles indicated that there were changes in the structure and composition of bacterial communities during the bioremediation trial, although the communities in treated plots were highly similar irrespective of the treatment applied, and they had a similar temporal dynamics. These results showed that nutrient addition was the main factor contributing to the outcome of the bioremediation experiment. This was supported by the lack of evidence of the establishment of inoculated consortia in soils, since their characteristic electrophoretic peaks were only detectable in soil profiles at the beginning of the experiment. Genetic potential for naphthalene degradation, evidenced by detection of nahAc gene, was observed in all soil plots including the control. In treated plots, an increase in the detection of catechol degradation genes (nahH and catA) and in a key gene of denitrification (nosZ) was observed as well. These results indicate that treatments favored the degradation of aromatic hydrocarbons and probably stimulated denitrification, at least transiently. This mesocosm study shows that recovery of chronically contaminated Antarctic soils can be successfully accelerated using biostimulation with nutrients, and that this causes a change in the indigenous bacterial communities and in the genetic potential for hydrocarbon degradation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Robust Hydrocarbon Degradation and Dynamics of Bacterial Communities during Nutrient-Enhanced Oil Spill Bioremediation

Degradation of oil on beaches is, in general, limited by the supply of inorganic nutrients. In order to obtain a more systematic understanding of the effects of nutrient addition on oil spill bioremediation, beach sediment microcosms contaminated with oil were treated with different levels of inorganic nutrients. Oil biodegradation was assessed respirometrically and on the basis of changes in oil composition. Bacterial communities were compared by numerical analysis of denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Nutrient amendment over a wide range of concentrations significantly improved oil degradation, confirming that N and P limited degradation over the concentration range tested. However, the extent and rate of oil degradation were similar for all microcosms, indicating that, in this experiment, it was the addition of inorganic nutrients rather than the precise amount that was most important operationally. Very different microbial communities were selected in all of the microcosms. Similarities between DGGE profiles of replicate samples from a single microcosm were high (95% ± 5%), but similarities between DGGE profiles from replicate microcosms receiving the same level of inorganic nutrients (68% ± 5%) were not significantly higher than those between microcosms subjected to different nutrient amendments (63% ± 7%). Therefore, it is apparent that the different communities selected cannot be attributed to the level of inorganic nutrients present in different microcosms. Bioremediation treatments dramatically reduced the diversity of the bacterial community. The decrease in diversity could be accounted for by a strong selection for bacteria belonging to the alkane-degrading Alcanivorax/Fundibacter group. On the basis of Shannon-Weaver indices, rapid recovery of the bacterial community diversity to preoiling levels of diversity occurred. However, although the overall diversity was similar, there were considerable qualitative differences in the community structure before and after the bioremediation treatments.     

Bioremediation of Soil Artificially Contaminated with Petroleum Hydrocarbon Oil Mixtures: Evaluation of the Use of Animal Manure and Chemical Fertilizer

The search for cheaper and environmentally friendly options of enhancing petroleum hydrocarbon degradation has continued to elicit research interest. One of such options is the use of animal manure as biostimulating agents. A combination of treatments consisting of the application of poultry manure, piggery manure, goat manure, and chemical fertilizer was evaluated in situ during a period of 4 weeks of remediation. Each treatment contained petroleum hydrocarbon mixture (kerosene, diesel oil, and gasoline mixtures) (10% w/w) in soil as a sole source of carbon and energy. After 4 weeks of remediation, the results showed that poultry manure, piggery manure, goat manure, and NPK (nitrogen, phosphorous, and potash [potassium]) fertilizer exhibited 73%, 63%, 50%, and 39% total petroleum hydrocarbon degradation, respectively. Thus, all the biostimulating treatment strategies showed the ability to enhance petroleum hydrocarbon microbial degradation. However, poultry manure, piggery manure, and goat manure treatments showed greater petroleum hydrocarbon reductions than NPK fertilizer treatment. A first-order kinetic equation was fitted to the biodegradation data and the specific degradation rate constant (k) values obtained showed that the order of effectiveness of these biostimulating strategies in the cleanup of soil contaminated with petroleum hydrocarbon mixtures (mixture of kerosene, diesel oil, and gasoline) is NPK fertilizer < goat manure < piggery manure < poultry manure. Therefore, this present work has indicated that the application of poultry manure, piggery manure, goat manure, and chemical fertilizer could enhance petroleum hydrocarbon degradation with poultry manure, showing a greater effectiveness and thus could be one of the severally sought environmentally friendly ways of remediating natural ecosystem contaminated with crude oil.     

Integrated Approach for Polycyclic Aromatic Hydrocarbon Solubilization from the Soil Matrix to Enhance Bioremediation

Polycyclic aromatic hydrocarbons (PAHs) are known to be toxic to living organisms and have been identified as carcinogenic. In this study, a pathway of surfactant flushing, chemical oxidation, and biological treatment is proposed to remediate the soils polluted with the hydrophobic PAHs. Different surfactants such as Tween 80, Brij 35, sodium dodecyl sulfate (SDS), and polyethylene glycol (PEG) 6000 were tested in order to increase the PAH solubilization from the soil matrix. The maximum desorption efficiency of naphthalene and anthracene were found to be 56.5% and 59%, respectively, when Brij and SDS were used. The soluble PAH in the aqueous phase was amended with sodium thiosulfate (3%) to oxidize the PAH into a more bioavailable form. The chemical oxidation with subsequent biodegradation by Pseudomonas aeruginosa exhibited the relatively high PAH degradation rate (1.24 times higher) when compared with chemical oxidation alone. These results display the efficiency of chemical pretreatment of PAH-contaminated soil for improved bioremediation.     

An Investigation of the Ability of a 14C-Labeled Hydrocarbon Mineralization Test to Predict Bioremediation of Soils Contaminated with Petroleum Hydrocarbons

Bioremediation is a widely accepted technology for the remediation of hydrocarbon-contaminated soil. Treatability studies are usually carried out to assess the biodegradation potential of the contaminants and to design optimal treatments. Laboratory studies measuring soil respiration are often used. One method consists of monitoring the mineralization of a ¹⁴C-labeled hydrocarbon surrogate added to the contaminated soil. This study investigates the ability of this method to properly predict the removal of the hydrocarbon contaminants initially found in soils. Mineralization of ¹⁴C-labeled hexadecane was monitored in seven soils contaminated with various hydrocarbon mixtures, both fresh and weathered, in microcosm experiments. Reduction of total petroleum hydrocarbon (TPH) concentrations was measured simultaneously in separate microcosms. Both types of microcosms were subjected to the same amendment regimes. For all soils, poor correlation was observed between the mineralization and TPH reduction data sets. Mineralization data supported contaminants removal data in only one soil. Findings indicate that the radioactive surrogate method does not reliably predict the extent of, and the effect of amendments on, the removal of the hydrocarbons initially present in soil, and may therefore predict suboptimal treatment regimes. Recommendations for soil treatability protocols are provided.     

Dynamics of Bacterial Community Composition and Activity during a Mesocosm Diatom Bloom

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment (≈24 μg of chlorophyll a liter⁻¹). At this time bacterial abundance abruptly decreased from 2.8 × 10⁶ to 0.75 × 10⁶ ml⁻¹, and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-μm size fraction towards the >1.0-μm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized α-Proteobacteria- and Cytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, β-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.     

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.     

Bacterial Community Response to Petroleum Hydrocarbon Amendments in Freshwater,Marine, and Hypersaline Water-Containing Microcosms

Hydrocarbon-degrading bacterial communities from freshwater, marine, and hypersaline Brazilian aquatic ecosystems (with water salinities corresponding to 0.2%, 4%, and 5%, respectively) were enriched with different hydrocarbons (heptadecane, naphthalene, or crude oil). Changes within the different microcosms of bacterial communities were analyzed using cultivation approaches and molecular methods (DNA and RNA extraction, followed by genetic fingerprinting and analyses of clone libraries based on the 16S rRNA-coding gene). A redundancy analysis (RDA) of the genetic fingerprint data and a principal component analysis (PCA) of the clone libraries revealed hydrocarbon-enriched bacterial communities specific for each ecosystem studied. However, within the same ecosystem, different bacterial communities were selected according to the petroleum hydrocarbon used. In general, the results demonstrated that Acinetobacter and Cloacibacterium were the dominant genera in freshwater microcosms; the Oceanospirillales order and the Marinobacter, Pseudomonas, and Cycloclasticus genera predominated in marine microcosms; and the Oceanospirillales order and the Marinobacter genus were selected in the different hydrocarbon-containing microcosms in hypersaline water. Determination of total petroleum hydrocarbons (TPHs) in all microcosms after 32 days of incubation showed a decrease in the hydrocarbon concentration compared to that for the controls. A total of 50 (41.3%) isolates from the different hydrocarbon-contaminated microcosms were associated with the dominant operational taxonomic units (OTUs) obtained from the clone libraries, and their growth in the hydrocarbon contaminating the microcosm from which they were isolated as the sole carbon source was observed. These data provide insight into the general response of bacterial communities from freshwater, marine, and hypersaline aquatic ecosystems to petroleum hydrocarbon contamination.     

Community Dynamics of Free-living and Particle-associated Bacterial Assemblages during a Freshwater Phytoplankton Bloom

Bacterial community dynamics were followed in a 19-day period during an induced diatom bloom in two freshwater mesocosms. The main goal was to compare diversity and succession among free-living (<10 MM) AND PARTICLE-ASSOCIATED (>10 mm) bacteria. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rDNA showed the highest number of bands among free-living bacteria, but with a significant phylogenetic overlap in the two size fractions indicating that free-living bacteria were also important members of the particle-associated bacterial assemblage. Whereas the number of bands in the free-living fraction decreased during the course of the bloom, several phylotypes unique to particles appeared towards the end of the experiment. Besides the primer set targeting Bacteria, a primer set targeting most members of the Cytophaga-Flavobacterium (CF)-cluster of the Cytophaga-Flavobacterium-Bacteroides group and a primer set mainly targeting a-Proteobacteria were applied. PCR-DGGE analyses revealed that a number of phylotypes targeted by those primer sets were found solely on particles. Almost all sequenced bands from the bacterial DGGE gel were related to phylogenetic groups commonly found in freshwater: a-Proteobacteria, CF, and Firmicutes. Despite the use of primers intended to be specific mainly for a-Proteobacteria most bands sequenced from the a-proteobacterial DGGE gel formed a cluster within the Verrucomicrobiales subdivision of the Verrucomicrobia division and were not related to a-Proteobacteria. Bands sequenced from the CF DGGE gel were related to members of the CF cluster. From the present study, we suggest that free-living and particle-associated bacterial communities should not be perceived as separate entities, but rather as interacting assemblages, where the extent of phylogenetic overlap is dependent on the nature of the particulate matter.     

Use of Experimental Factorial Design for Optimization of Hexavalent Chromium Removal by a Bacterial Consortium: Soil Microcosm Bioremediation

Hexavalent chromium Cr(VI) is regularly introduced into the environment through diverse anthropogenic activities. It is highly toxic, mutagenic and carcinogenic, and because of its solubility in water, chromate contamination can be difficult to contain. Bacteria can reduce chromate to insoluble and less toxic trivalent chromium Cr(III), and thus increasing attention is paid to chromate bioremediation to reduce its ecotoxicological impacts. In this study, the factorial design 2³ was employed to optimize critical parameters responsible for higher Cr(VI) removal by a bacterial consortium. The factors considered were pH, temperature, and inoculum size at two markedly different levels. All three dependent variables have significant effect on Cr(VI) reduction. Optimal Cr(VI) removal by the bacterial consortium occurred at pH 9, temperature 37°C, and inoculum size OD = 3. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.984, thus ensuring a satisfactory adjustment of the second-order regression model with the experimental data. In addition, the effect of bioaugmentation of Cr(VI)-polluted soil microcosms with the bacterial consortium was investigated using the best factor levels. Contaminated soil by 20 and 60 mg/Kg of Cr(VI) showed reductions of 83% and 65% of initial Cr(VI) by the bacterial consortium, suggesting that this bacterial consortium might diminish phytoavailable Cr(VI) in soil and be useful for cleaning up chromium-contaminated sites.     

On-Site Treatment of Contaminated Soils: An Approach to Bioremediation of Weathered Petroleum Compounds

A bench-scale investigation was conducted prior to on-site bioremediation of 52,000 cubic yards of contaminated soil containing weathered, structurally complex petroleum compounds from an inactive oil refinery. Addition of bulking agents was required to improve soil physical properties. A supplemental study was also conducted to evaluate the effectiveness of bio-enhancement products. Loss of n-alkanes was rapid in soil mixtures containing a high nitrogen sludge compost, but very slow in mixtures containing wood products as bulking agents. By completion of the study at day 110, the isoprenoids pristane and phytane had nearly disappeared from mixtures containing sludge compost. Clearly, pristane and phytane are inadequate biomarkers when conditions favor an advanced stage of biodegradation. Nearly half the complex branched and cyclic alkanes in the unresolved complex mixture also degraded. After 70 days, depletion of dibenzo-thiophenes and phenan-threnes was 75 and 90%, respectively. The most stable PAHs within each group were the highly methylated homologues. Because of their complex structures, both steranes and hopanes were stable in all soil mixtures. Data were normalized to hopanes as a conserved internal standard or biomarker. Use of hopane-normalized data successfully eliminated much of the data variability and permitted a more accurate assessment of biodegradation. A relatively slow decline in total hydrocarbons occurred later in the study. This slowing tendency of microbial utilization is caused not only by substrate depletion, but also because remaining hydrocarbons are structurally more complex and persistent. Because of this, it is important to avoid using kinetic data from early stages of bioremediation to predict later hydrocarbon losses, such as the time required to attain a cleanup standard. In the supplemental study, an oleophilic fertilizer product accelerated hydrocarbon degradation when compared with a conventional fertilizer. This product will be tested in combination with organic bulking agents under field conditions to determine its cost effectiveness.     

Bacterial adhesion: From mechanism to control

Bacterial adhesion is the initial step in colonization and biofilm formation. Biofilms can, on the one hand, be detrimental to both human life and industrial processes, for example, causing infection, pathogen contamination, and slime formation, while on the other hand, be beneficial in environmental technologies and bioprocesses. For control and utilization of bacterial adhesion and biofilms, adhesion mechanisms must be elucidated. Conventional physicochemical approaches based on Lifshitz-van der Waals, electrostatic and acid–base interactions provide important models of bacterial adhesion but have a limited capacity to provide a complete understanding of the complex adhesion process of real bacterial cells. In conventional approaches, bacterial cells, whose surfaces are structurally and chemically heterogeneous, are often described from the viewpoint of their overall cellular properties. Cell appendages such as polysaccharide chains and proteinous nanofibers have an important function bridging between cells and the substratum in conventional adhesion models, but sometimes cause deviation from the models of cell adhesion. In reality, cell appendages are responsible for specific and nonspecific cell adhesion to biotic and abiotic surfaces. This paper reviews conventional physicochemical models and cell appendage-mediated cell adhesion. State-of-the-art technologies for controlling microbial adhesion and biofilm formation are also described. These technologies are based on the adhesion mechanisms.     

Characterization of Novel Polycyclic Aromatic Hydrocarbon Dioxygenases from the Bacterial Metagenomic DNA of a Contaminated Soil

Ring-hydroxylating dioxygenases (RHDs) play a crucial role in the biodegradation of a range of aromatic hydrocarbons found on polluted sites, including polycyclic aromatic hydrocarbons (PAHs). Current knowledge on RHDs comes essentially from studies on culturable bacterial strains, while compelling evidence indicates that pollutant removal is mostly achieved by uncultured species. In this study, a combination of DNA-SIP labeling and metagenomic sequence analysis was implemented to investigate the metabolic potential of main PAH degraders on a polluted site. Following in situ labeling using [¹³C]phenanthrene, the labeled metagenomic DNA was isolated from soil and subjected to shotgun sequencing. Most annotated sequences were predicted to belong to Betaproteobacteria, especially Rhodocyclaceae and Burkholderiales, which is consistent with previous findings showing that main PAH degraders on this site were affiliated to these taxa. Based on metagenomic data, four RHD gene sets were amplified and cloned from soil DNA. For each set, PCR yielded multiple amplicons with sequences differing by up to 321 nucleotides (17%), reflecting the great genetic diversity prevailing in soil. RHDs were successfully overexpressed in Escherichia coli, but full activity required the coexpression of two electron carrier genes, also cloned from soil DNA. Remarkably, two RHDs exhibited much higher activity when associated with electron carriers from a sphingomonad. The four RHDs showed markedly different preferences for two- and three-ring PAHs but were poorly active on four-ring PAHs. Three RHDs preferentially hydroxylated phenanthrene on the C-1 and C-2 positions rather than on the C-3 and C-4 positions, suggesting that degradation occurred through an alternate pathway.     

The Bioremediation Potential of Hydrocarbonoclastic Bacteria Isolated From a Mangrove Contaminated by Petroleum Hydrocarbons on the Cilacap Coast,Indonesia

The main purpose of the study was to isolate strains of bacteria capable of degrading hydrocarbons from contaminated mangroves and to investigate the ability of the isolated bacteria to degrade total petroleum hydrocarbons (TPH) in a microcosm model of an oily sludge. The potential use of these bacteria strains as environmental clean-up agents was tested by culturing them with six different polyaromatic hydrocarbon (PAH) compounds (phenothiazine, fluorene, fluoranthene, dibenzothiophene, phenanthrene, and pyrene). Six viable and culturable bacteria were isolated, and the 16S rDNA sequence for each was amplified using the primers 9F and 1510R. Sequence results were compared using the National Center for Biotechnology Information (NCBI) BLAST program and, combined with phenotypic and phylogenetic data, were used to identify three strains that belonged to the Bacillus genus and were most closely related (98–99%) to Bacillus aquimaris, Bacillus megaterium, and Bacillus pumilus. The other three strains were closely related (98–100%) to Flexibacteraceae bacterium, Halobacilus trueperi, and Rhodobacteraceae bacterium. Two isolates, BA-PZN and BM-PFFP, which were related to Bacillus aquimaris and Bacillus megaterium, respectively, were further characterized and showed great potential for the removal of more complex hydrocarbon compounds in the oily microcosm model.     

Purple Bacterial Light-harvesting Complexes: From Dreams to Structures

This paper describes the main stages involved in the research efforts designed to try and understand the structure and function of purple bacterial antenna complexes. Wherever possible the work has been illustrated by pictures of the major people who carried it out.