Kirsten S. Jørgensen

Bioremediation of petroleum hydrocarbon-contaminated soil by composting in biopiles.

Composting of contaminated soil in biopiles is an ex situ technology, where organic matter such as bark chips are added to contaminated soil as a bulking agent. Composting of lubricating oil-contaminated soil was performed in field scale ( [Formula: see text] m(3)) using bark chips as the bulking agent, and two commercially available mixed microbial inocula as well as the effect of the level of added nutrients (N,P,K) were tested. Composting of diesel oil-contaminated soil was also performed at one level of nutrient addition and with no inoculum. The mineral oil degradation rate was most rapid during the first months, and it followed a typical first order degradation curve. During 5 months, composting of the mineral oil decreased in all piles with lubrication oil from approximately 2400 to 700 mg (kg dry w)(-1), which was about 70% of the mineral oil content. Correspondingly, the mineral oil content in the pile with diesel oil-contaminated soil decreased with 71% from 700 to 200 mg (kg dry w)(-1). In this type of treatment with addition of a large amount of organic matter, the general microbial activity as measured by soil respiration was enhanced and no particular effect of added inocula was observed.

In Situ Bioremediation

In situ bioremediation technologies are sustainable because they neither require transport and deposition of contaminated soil, nor groundwater pumping, treatment, and discharge to recipients. Successful full-scale cases have benefited from the understanding of the prevailing environmental conditions and microbial communities present in both the unsaturated and saturated zones. In particular, the expanding knowledge on anaerobic degradation pathways and degrader communities have encouraged the use of monitored natural attenuation (MNA) that relies on the monitoring of indicators of aerobic or anaerobic degradation processes and naturally develops in the subsurface upon contamination. The treatment of the unsaturated zone can be performed by MNA, by enhancing the natural attenuation by addition of air in bioventing, or by the infiltration with nutrients. The saturated zone can be treated by MNA, by enhancing the aerobic degradation by air sparging, or by addition of oxygen releasing compounds. More recently, the enhancement of anaerobic processes responsible for the biodegradation of contaminants has been used by adding alternative electron acceptors or electron donors. The use of inocula has proven to be successful in full scale for the remediation of methyl tert -butyl ether (MTBE) and chlorinated solvents.

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

We studied the role of aerobic and anaerobic petroleum hydrocarbon degradation ata boreal, light-weight fuel and lubrication oil contaminated site undergoing naturalattenuation. At the site, anoxic conditions prevailed with high concentrations ofCH4 (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 wereincubated in microcosms at near in situ conditions (i.e. in situ temperature 8 °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 μg CH4 h-1 g-1 dwt was recorded in thesemicrocosms. In the aerobic as well as anaerobic microcosms, typically 90% of themineral oil degraded belonged to the mineral oil fraction that eluted from the gaschromatograph 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.

Microbial functional activity during composting of chlorophenol-contaminated sawmill soil

Microbial status during a successful full-scale bioremediation by composting chlorophenol-contaminated soil was studied in three different ways: conventional enumeration of microbes on selective and general media, microbial activity assessed by soil respiration, and community structure studied by the utilization pattern of a large range of substrates using Biolog® microtitre plates. Utilization of ammonium, nitrate and soluble P was also followed. Chlorophenols were well removed in all compost piles which were mixtures of contaminated soil and bark chips or straw compost. The best indicator of the actual chlorophenol degradation efficiency was the number of microbes growing on plates with 2 mM pentachlorophenol (PCP) as the sole carbon source. Nutrient analyses showed that ammonium was rapidly used, and nitrification took place in some of the compost piles. The data from Biolog® microtitre plates was analyzed using the toolbox of Matlab® mathematical software. The areas under the substrate utilization curve were integrated for each substrate used, and they were used for principal component analysis. We were able to see pile-specific substrate usage for piles containing straw compost, but not for pile containing bark chips. All these characteristic substrates were either amino acids or amines. The results suggested that fast-growing microbes responsible for utilization of easily available substrates, measured by respiratory activity and substrate utilization patterns in Biolog, originated mainly from the added bulking agents, straw compost and bark chips. The chlorophenol-degraders originating from contaminated soil seemed not directly to contribute to the Biolog utilization pattern, but probably had benefited from the enhanced general microbial activity in the composts by cometabolism or synergism.

Biomarkers for monitoring efficacy of bioremediation by microbial inoculants.

Bioaugmentation of contaminated sites with microbes that are adapted or genetically engineered for degradation of specific toxic compounds is an area that is currently being explored as a clean-up option. Biomarkers have been developed to track the survival and efficacy of specific bacteria that are used as inocula for bioremediation of contaminated soil. Examples of biomarkers include the luc gene, encoding firefly luciferase and the gfp gene, encoding the green fluorescent protein (GFP). The luc gene was used to tag different bacteria used for bioremediation of gasoline or chlorophenols. The bacteria were monitored on the basis of luciferase activity in cell extracts from soil. The gfp gene was also used to monitor bacteria during degradation of chlorophenol in soil, based on fluorescence of the GFP protein. Other biomarkers can also be used for monitoring of microbial inocula used for bioaugmentation of contaminated sites. The choice of biomarker and monitoring system depends on the particular site, bacterial strain and sensitivity and specificity of detection required.

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.

Maintenance and Impacts of an Inoculated mer/luc-Tagged Pseudomonas fluorescens on Microbial Communities in Birch Rhizospheres Developed on Humus and Peat

Antagonistic bacteria represent promising biocontrol agents for improving forest production in seedling nurseries or forest soils. The fate of an introduced mer/luc-tagged antagonistic Pseudomonas fluorescens 31K3 was monitored in the rhizosphere of silver birch (Betula pendula) seedlings grown in microcosms containing forest humus or nursery peat. The inoculated strain (108 cfu g?1 soil) was unable to establish in significant numbers in either soil type and turned nonculturable in humus. Detection in both soils was possible only via luminescence of enrichment cultures 80 days post-inoculation. Despite low P. fluorescens survival, inoculation had a positive effect on seedling growth. Limited impact of inoculation on the indigenous microbial communities was identified following analyses of respiration and denitrification potential, community-level physiological profiles and molecular fingerprinting of fungi and eubacteria, and Pseudomonas community structures. The minor changes observed in the indigenous microbial communities, including mycorrhiza development, were not consistent between humus and peat growth substrates. It was concluded that the rhizosphere-related microbial communities developed in both of these highly organic soil systems are highly buffered against introduction of foreign bacteria.

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.

Iron–Manganese Concretions Sustaining Microbial Life in the Baltic Sea: The Structure of the Bacterial Community and Enrichments in Metal-Oxidizing Conditions

The abundant deposits of spherical iron-manganese concretions in the Gulf of Finland are colonized by bacteria in vast numbers. Communities on the surface and in the porous interior have formed two separate clusters, in accordance with their genetic differences. The overall bacterial community in the concretions was highly diverse, representing 12 phyla. Half of the bacteria were affiliated with the most common classes of Proteobacteria, while a third of the bacteria were unclassified. Cloned 16S rRNA-gene sequences of the concretion bacteria showed high scores for similarity to the sequences obtained from sea sediments, metal-rich environments, and ocean crust. The clone library of native concretions was not dominated by known Fe- and Mn-oxidizing species. Known Mn-oxidizing bacteria Sphingomonas, Pseudomonas, and Bacillus were enriched in experiments with Mn2+-containing liquid media, whereas Prosthecobacter (Verrucomicrobia) and Rheinheimera were enriched in semisolid media possibly better simulating the natural conditions in the concretions. In a corresponding experiment, the Fe2+-oxygen gradient favored the enrichment of Shewanella baltica and Thalassolituus oleivorans, which are known to reduce Fe and to degrade petroleum hydrocarbons, respectively. An individual spherical concretion forms a microcosm for a diverse microbial community having potential to oxidize Fe and Mn as shown in cultivation experiments. Therefore, bacteria may significantly affect the formation of the concretions in the Gulf of Finland.

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.