Effects of Dispersant on the Petroleum Hydrocarbon Biodegradation and Microbial Communities in Seawater from the Baltic Sea and Norwegian Sea.

Dispersants have been used in several oil spill accidents, but little information is available on their effectiveness in Baltic Sea conditions with low salinity and cold seawater. This study investigated the effects of dispersant use on petroleum hydrocarbon biodegradation rates and bacterial community structures. Microcosm experiments were conducted at 5 °C for 12 days with North Sea crude oil and dispersant Finasol 51 with open sea Gulf of Bothnia and coastal Gulf of Finland and Norwegian Sea seawater. Petroleum hydrocarbon concentrations were analysed with GC-FID. Bacterial community structures were studied using 16S rDNA gene amplicon sequencing, and the abundance of genes involved in hydrocarbon degradation with quantitative PCR. The highest oil degradation gene abundances and oil removal were observed in microcosms with coastal seawater from the Gulf of Bothnia and Gulf of Finland, respectively, and the lowest in the seawater from the Norwegian Sea. Dispersant usage caused apparent effects on bacterial communities in all treatments; however, the dispersant's effect on the biodegradation rate was unclear due to uncertainties with chemical analysis and variation in oil concentrations used in the experiments.

Oil degradation potential of microbial communities in water and sediment of Baltic Sea coastal area.

Two long-term potentially oil exposed Baltic Sea coastal sites near old oil refineries and harbours were compared to nearby less exposed sites in terms of bacterial, archaeal and fungal microbiomes and oil degradation potential. The bacterial, archaeal and fungal diversities were similar in oil exposed and less exposed sampling sites based on bacterial and archaeal 16S rRNA gene and fungal 5.8S rRNA gene amplicon sequencing from both DNA and RNA fractions. The number of genes participating in alkane degradation (alkB) or PAH-ring hydroxylation (PAH-RHDα) were detected by qPCR in all water and sediment samples. These numbers correlated with the number of bacterial 16S rRNA gene copies in sediment samples but not with the concentration of petroleum hydrocarbons or PAHs. This indicates that both the clean and the more polluted sites at the Baltic Sea coastal areas have a potential for petroleum hydrocarbon degradation. The active community (based on RNA) of the coastal Baltic Sea water differed largely from the total community (based on DNA). The most noticeable difference was seen in the bacterial community in the water samples were the active community was dominated by Cyanobacteria and Proteobacteria whereas in total bacterial community Actinobacteria was the most abundant phylum. The abundance, richness and diversity of Fungi present in water and sediment samples was in general lower than that of Bacteria and Archaea. Furthermore, the sampling location influenced the fungal community composition, whereas the bacterial and archaeal communities were not influenced. This may indicate that the fungal species that are adapted to the Baltic Sea environments are few and that Fungi are potentially more vulnerable to or affected by the Baltic Sea conditions than Bacteria and Archaea.

Bioremediation strategies for removal of residual atrazine in the boreal groundwater zone.

Strategies for bioremediation of atrazine, a pesticide commonly polluting groundwater in low concentrations, were studied in two boreal nonagricultural soils. Atrazine was not mineralized in soil without bioremediation treatments. In biostimulation treatment with molasses, up to 52% of atrazine was mineralized at 10 °C, even though the degradation gene copy numbers did not increase. Incubations with radioactively labeled atrazine followed by microautoradiographic analysis revealed that bioremediation strategies increased the relative proportion of active degraders from 0.3 up to 1.9% of the total bacterial count. These results indicate that atrazine degradation might not solely be facilitated by atzA/trzN-atzB genes. In combined biostimulation treatment using citrate or molasses and augmentation with Pseudomonas citronellolis ADP or Arthrobacter aurescens strain TC1, up to 76% of atrazine was mineralized at 30 °C, and the atrazine degradation gene numbers increased up to 10(7) copies g(-1) soil. Clone libraries from passive samplers in groundwater monitoring wells revealed the presence of phylogenetic groups formerly shown to include atrazine degraders, and the presence of atrazine degradation genes atzA and atzB. These results show that the mineralization of low concentrations of atrazine in the groundwater zone at low temperatures is possible by bioremediation treatments.

Soil mesocosm studies on atrazine bioremediation.

Accumulation of pesticides in the environment causes serious issues of contamination and toxicity. Bioremediation is an ecologically sound method to manage soil pollution, but the bottleneck here, is the successful scale-up of lab-scale experiments to field applications. This study demonstrates pilot-scale bioremediation in tropical soil using atrazine as model pollutant. Mimicking field conditions, three different bioremediation strategies for atrazine degradation were explored. 100 kg soil mesocosms were set-up, with or without atrazine application history. Natural attenuation and enhanced bioremediation were tested, where augmentation with an atrazine degrading consortium demonstrated best pollutant removal. 90% atrazine degradation was observed in six days in soil previously exposed to atrazine, while soil without history of atrazine use, needed 15 days to remove the same amount of amended atrazine. The bacterial consortium comprised of 3 novel bacterial strains with different genetic atrazine degrading potential. The progress of bioremediation was monitored by measuring the levels of atrazine and its intermediate, cyanuric acid. Genes from the atrazine degradation pathway, namely, atzA, atzB, atzD, trzN and trzD were quantified in all mesocosms for 60 days. The highest abundance of all target genes was observed on the 6th day of treatment. trzD was observed in the bioaugmented mesocosms only. The bacterial community profile in all mesocosms was monitored by LH-PCR over a period of two months. Results indicate that the communities changed rapidly after inoculation, but there was no drastic change in microbial community profile after 1 month. Results indicated that efficient bioremediation of atrazine using a microbial consortium could be successfully up-scaled to pilot scale.

Dominant petroleum hydrocarbon-degrading bacteria in the Archipelago Sea in South-West Finland (Baltic Sea) belong to different taxonomic groups than hydrocarbon degraders in the oceans.

The natural petroleum hydrocarbon degrading capacity of the Archipelago Sea water in S-W Finland was studied in a microcosm experiment. Pristine and previously oil exposed sites were examined. Bacterial community fingerprinting was performed using terminal restriction fragment length polymorphism (T-RFLP) and samples from selected microcosms were sequenced. The abundance of PAH degradation genes was measured by quantitative PCR. Bacterial communities in diesel exposed microcosms diverged from control microcosms during the experiment. Gram positive PAH degradation genes dominated at both sites in situ, whereas gram negative PAH degrading genes became enriched in diesel microcosms. The dominant bacterial groups after a 14 days of diesel exposure were different depending on the sampling site, belonging to the class Actinobacteria (32%) at a pristine site and Betaproteobacteria (52%) at a previously oil exposed site. The hydrocarbon degrading bacteria in the Baltic Sea differ from those in the oceans, where most hydrocarbon degraders belong to Gammaproteobacteria.

Monitoring bioremediation of atrazine in soil microcosms using molecular tools.

Molecular tools in microbial community analysis give access to information on catabolic potential and diversity of microbes. Applied in bioremediation, they could provide a new dimension to improve pollution control. This concept has been demonstrated in the study using atrazine as model pollutant. Bioremediation of the herbicide, atrazine, was analyzed in microcosm studies by bioaugmentation, biostimulation and natural attenuation. Genes from the atrazine degrading pathway atzA/B/C/D/E/F, trzN, and trzD were monitored during the course of treatment and results demonstrated variation in atzC, trzD and trzN genes with time. Change in copy number of trzN gene under different treatment processes was demonstrated by real-time PCR. The amplified trzN gene was cloned and sequence data showed homology to genes reported in Arthrobacter and Nocardioides. Results demonstrate that specific target genes can be monitored, quantified and correlated to degradation analysis which would help in predicting the outcome of any bioremediation strategy.

Monitored natural attenuation.

Monitored natural attenuation (MNA) is an in situ remediation technology that relies on naturally occurring and demonstrable processes in soil and groundwater which reduce the mass and concentration of the contaminants. Natural attenuation (NA) involves both aerobic and anaerobic degradation of the contaminants due to the fact that oxygen is used up near the core of the contaminant plume. The aerobic and anaerobic microbial processes can be assessed by microbial activity measurements and molecular biology methods in combination with chemical analyses. The sampling and knowledge on the site conditions are of major importance for the linkage of the results obtained to the conditions in situ. Rates obtained from activity measurements can, with certain limitations, be used in modeling of the fate of contaminants whereas most molecular methods mainly give qualitative information on the microbial community and gene abundances. However, molecular biology methods are fast and describe the in situ communities and avoid the biases inherent to activity assays requiring laboratory incubations.

[Not Available].

Relatively little is known about the microbial communities adapted to soil environments contaminated with aged complex hydrocarbon mixtures, especially in the subsurface soil layers. In this work we studied the microbial communities in two different soil profiles down to the depth of 7 m which originated from a 30-year-old site contaminated with petroleum hydrocarbons (PHCs) and from a clean site next to the contaminated site. The concentration of oxygen in the contaminated soil profile was strongly reduced in soil layers below 1 m depth but not in the clean soil profile. Total microbial biomass and community composition was analyzed by phospholipid fatty acid (PLFA) measurements. The diversity of fungi and actinobacteria was investigated more in detail by construction of rDNA-based clone libraries. The results revealed that there was a significant and diverse microbial community in subsoils at depth below 2 m, also in conditions where oxygen was limiting. The diversity of actinobacteria was different in the two soil profiles; the contaminated soil profile was dominated by Mycobacterium -related sequences whereas sequences from the clean soil samples were related to other, generally uncultured organisms, some of which may represent two new subclasses of actinobacteria. One dominating fungal sequence which matched with the ascomycotes Acremonium sp. and Paecilomyces sp. was identified both in clean and in contaminated soil profiles. Thus, although the relative amounts of fungi and actinobacteria in these microbial communities were highest in the upper soil layers, many representatives from these groups were found in hydrocarbon contaminated subsoils even under oxygen limited conditions.

Functional gene abundances (nahAc, alkB, xylE) in the assessment of the efficacy of bioremediation.

In this study, we compared the mineralization rates of three selected (14)C-labeled hydrocarbon compounds, octacosane, toluene, and naphthalene, with the presence of the corresponding functional genes (alkB, xylE, nahAc) in a large number of soil samples representing different types of soil and petroleum hydrocarbon contamination. Functional genes were enumerated by the replicate limited dilution (RLD) polymerase chain reaction (PCR) technique. RLD-PCR was further compared to real-time PCR measurements for nahAc and xylE for some samples. At a heating oil-contaminated site, octacosane mineralization rates were higher (on average 0.0015 day(-1)) when compared to aerobic naphthalene and toluene mineralization (on average 0.00003 and 0.0007 day(-1)). The corresponding gene abundances measured by RLD-PCR were on average 0.95, 0.3, and 0.13 x 10(3) gene copies g(-1) soil for alkB, nahAc, and xylE, respectively. At a site contaminated with gasoline, the situation was the opposite: Toluene mineralization was the highest (on average 0.0031 day(-1)), and only xylE genes could be detected (on average 0.13 x 10(3) gene copies g(-1) soil by RLD-PCR). XylE and nahAc gene abundances were correlated with the (14)C-toluene and naphthalene mineralization activities, respectively, in samples from aerobic layers. AlkB gene abundances were not correlated with the octacosane mineralization. Real-time PCR was a more sensitive method than RLD-PCR by a factor of 1,200 for nahAc and 300 for xylE. In conclusion, functional gene abundances seemed to reflect the type of the contamination. With optimized assays, the gene abundances can be used to assess bioremediation efficacy.

Advances in monitoring of catabolic genes during bioremediation.

Biodegradation of xenobiotic compounds by microbes is exploited in the clean up of contaminated environments by bioremediation. Catabolic (or functional) genes encode for specific enzymes in catabolic pathways such as key enzymes in xenobiotic degradation pathways. By assessing the abundance or the expression of key genes in environmental samples one can get a potential measure of the degradation activity. One way of assessing the abundance and expression of specific catabolic genes is by analyzing the metagenomic DNA and RNA from environmental samples. Three major challenges in the detection and quantification of catabolic genes in bioremediation studies are 1) the accurate and sensitive quantification from environmental samples 2) the coverage of the enzymatic potential by the targeted genes 3) the validation of the correlation with actual observed degradation activities in field cases. New advances in realtime PCR, functional gene arrays and meta-transcriptomics have improved the applicability of catabolic gene assessment during bioremediation.

Occurrence and rates of terminal electron-accepting processes and recharge processes in petroleum hydrocarbon-contaminated subsurface.

The occurrence and rates of terminal electron acceptor processes, and recharge processes in the unsaturated zone of a boreal site contaminated with petroleum hydrocarbons in the range C(10) to C(40) were examined. Soil microcosms were used to determine the rates of denitrification, iron (Fe) reduction, sulfate (SO(4)) reduction, and methanogenesis in two vertical soil profiles contaminated with oil, and in a noncontaminated reference sample. Furthermore, the abundances of the 16S rRNA genes belonging to Geobacteracaea in the samples were determined by real-time quantitative polymerase chain reaction (PCR). Analyses of ground water chemistry and soil gas composition were also performed together with continuous in situ monitoring of soil water and ground water chemistry. Several lines of evidence were obtained to demonstrate that both Fe reduction and methanogenesis played significant roles in the vertical profiles: Fe reduction rates up to 3.7 nmol h(-1) g(-1) were recorded and they correlated with the abundances of the Geobacteracaea 16S rRNA genes (range: 2.3 x 10(5) to 4.9 x 10(7) copies g(-1)). In the ground water, ferrous iron (Fe(2+)) concentration up to 55 mg L(-1) was measured. Methane production rates up to 2.5 nmol h(-1) g(-1) were obtained together with methane content up to 15% (vol/vol) in the soil gas. The continuous monitoring of soil water and ground water chemistry, microcosm experiments, and soil gas monitoring together demonstrated that the high microbial activity in the unsaturated zone resulted in rapid removal of oxygen from the infiltrating recharge thus leaving the anaerobic microbial processes dominant below 1.5 m depth both in the unsaturated and the saturated zones of the subsurface.

Presence of unintended Agrobacterium tumefaciens cloning vector sequences in genetically modified plants.

Agrobacterium transformation was used in the production of genetically modified plants from oilseed rape (Brassica napus) and tobacco (Nicotiana tabacum). After inoculation stop with the antibiotic timentin, a subsequent one-week treatment eliminated the vector bacterium from the oilseed rape plate explant cultures. From the tobacco, however, we recorded vector-derived signals one week after potting the regenerants in the greenhouse and still 10 weeks later. Genetically modified plants produced through Agrobacterium-transformation therefore cannot be guaranteed to be completely free of unintended vector sequences after antibiotic treatment.

Use of potassium formate in road winter deicing can reduce groundwater deterioration.

We present here an aquifer scale study on the fate of potassium formate, an alternative, weakly corrosive deicing agent in soil and subsurfaces. Potassium formate was used to deice a stretch of a highway in Finland. The fate of the formate was examined by monitoring the groundwater chemistry in the underlying aquifer of which a conceptual model was constructed. In addition, we determined aerobic and anaerobic biodegradation rates of formate at low temperatures (-2 to +6 degrees C) in soil microcosms. Our results show that the formate did not enter the saturated zone through the thin vadose zone; thus, no undesirable changes in the groundwater chemistry were observed. Furthermore, the conceptual model explained the distribution of chloride in the aquifer used in deicing for the past 30 years. We recorded mineralization potential up to 97% and up to 17% within 24 h under aerobic and anaerobic conditions, respectively, in the soil and subsurface samples obtained from the site. This demonstrates that biodegradation in the topsoil layers was responsible for the removal of the formate. We conclude that the use of potassium formate can potentially help diminish the negative impacts of road winter deicing on groundwater without jeopardizing traffic safety.

Potential for aerobic and anaerobic biodegradation of petroleum hydrocarbons in boreal subsurface.

We studied the role of aerobic and anaerobic petroleum hydrocarbon degradation at a boreal, light-weight fuel and lubrication oil contaminated site undergoing natural attenuation. At the site, anoxic conditions prevailed with high concentrations of CH4 (up to 25% v/v) and CO2 (up to 18% v/v) in the soil gas throughout the year. Subsurface samples were obtained mainly from the anoxic parts of the site and they represented both the unsaturated and saturated zone. The samples were incubated in microcosms at near in situ conditions (i.e. in situ temperature 8 degrees C, aerobic and anaerobic conditions, no nutrient amendments) resulting in the removal of mineral oil (as determined by gas chromatography) aerobically as well as anaerobically. In the aerobic microcosms on average 31% and 27% of the initial mineral oil was removed during a 3- and 4-month incubation, respectively. In the anaerobic microcosms, on average 44% and 15% of the initial mineral oil was removed during a 12- and 10-month anaerobic incubation, respectively, and e.g. n-alkanes from C11 to C15 were removed. A methane production rate of up to 2.5 microg CH4 h(-1) g(-1) dwt was recorded in these microcosms. In the aerobic as well as anaerobic microcosms, typically 90% of the mineral oil degraded belonged to the mineral oil fraction that eluted from the gas chromatograph after C10 and before C15, while 10% belonged to the fraction that eluted after C15 and before C40. Our results suggest that anaerobic petroleum hydrocarbon degradation, including n-alkane degradation, under methanogenic conditions plays a significant role in the natural attenuation in boreal conditions.

The abundance of nahAc genes correlates with the 14C-naphthalene mineralization potential in petroleum hydrocarbon-contaminated oxic soil layers.

In this study, we evaluated whether the abundance of the functional gene nahAc reflects aerobic naphthalene degradation potential in subsurface and surface samples taken from three petroleum hydrocarbon contaminated sites in southern Finland. The type of the contamination at the sites varied from lightweight diesel oil to high molecular weight residuals of crude oil. Samples were collected from both oxic and anoxic soil layers. The naphthalene dioxygenase gene nahAc was quantified using a replicate limiting dilution-polymerase chain reaction (RLD-PCR) method with a degenerate primer pair. In the non-contaminated samples nahAc genes were not detected. In the petroleum hydrocarbon-contaminated oxic soil samples nahAc gene abundance [range 3 x 10(1)-9 x 10(4) copies (g dry wt soil)(-1)] was correlated (Kendall non-parametric correlation r2=0.459, p<0.01) with the aerobic 14C-naphthalene mineralization potential (range 1 x 10(-5)-0.1 d(-1)) measured in microcosms at in situ temperatures (8 degrees C for subsurface and 20 degrees C for surface soil samples). In these samples nahAc gene abundance was also correlated with total microbial cell counts (r2=0.471, p<0.01), respiration rate (r2=0.401, p<0.01) and organic matter content (r2=0.341, p<0.05). NahAc genes were amplified from anoxic soil layers indicating that, although involved in aerobic biodegradation of naphthalene, these genes or related sequences were also present in the anoxic subsurface. In the samples taken from the anoxic layers, the aerobic 14C-naphthalene mineralization rates were not correlated with nahAc gene abundance. In conclusion, current sequence information provides the basis for a robust tool to estimate the naphthalene degradation potential at oxic zones of different petroleum hydrocarbon-contaminated sites undergoing in situ bioremediation.