Spatial patterns of microbial diversity and activity in an aged creosote-contaminated site.

Restoration of polluted sites via in situ bioremediation relies heavily on the indigenous microbes and their activities. Spatial heterogeneity of microbial populations, contaminants and soil chemical parameters on such sites is a major hurdle in optimizing and implementing an appropriate bioremediation regime. We performed a grid-based sampling of an aged creosote-contaminated site followed by geostatistical modelling to illustrate the spatial patterns of microbial diversity and activity and to relate these patterns to the distribution of pollutants. Spatial distribution of bacterial groups unveiled patterns of niche differentiation regulated by patchy distribution of pollutants and an east-to-west pH gradient at the studied site. Proteobacteria clearly dominated in the hot spots of creosote pollution, whereas the abundance of Actinobacteria, TM7 and Planctomycetes was considerably reduced from the hot spots. The pH preferences of proteobacterial groups dominating in pollution could be recognized by examining the order and family-level responses. Acidobacterial classes came across as generalists in hydrocarbon pollution whose spatial distribution seemed to be regulated solely by the pH gradient. Although the community evenness decreased in the heavily polluted zones, basal respiration and fluorescein diacetate hydrolysis rates were higher, indicating the adaptation of specific indigenous microbial populations to hydrocarbon pollution. Combining the information from the kriged maps of microbial and soil chemistry data provided a comprehensive understanding of the long-term impacts of creosote pollution on the subsurface microbial communities. This study also highlighted the prospect of interpreting taxa-specific spatial patterns and applying them as indicators or proxies for monitoring polluted sites.

High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms.

Cyanobacterial mass occurrences (water blooms) cause ecological, economic and health problems worldwide. Still, little is known about heterotrophic bacteria associated with cyanobacteria and the interactions between those organisms. We isolated 460 bacterial strains from more than 40 lakes and rivers (151 samples), Baltic Sea (32 samples) and treated drinking water of seven treatment plants (29 samples). The water bodies and the raw water of the treatment plants were frequently dominated by high numbers of cyanobacteria. Various growth media were used to isolate the strains. Analysis of partial 16S rRNA gene fragments (701-905 bp for 358 strains and 413-497 bp for 102 strains) classified the isolated bacteria as Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes and Deinococcus-Thermus. Some of these isolates represented possible new bacterial orders, families, genera or species. We isolated various potentially pathogenic bacteria, such as Aeromonas, Vibrio, Acinetobacter and Pseudomonas, that may cause adverse health effects in humans and animals and should be taken into consideration when assessing the risks caused by cyanobacterial blooms. Several strains also inhibited or enhanced the growth of cyanobacteria. Most of such strains had an enhancing effect on the cyanobacterial growth. Other isolates were affiliated with genera such as Sphingomonas or Flavobacterium, which include strains that are capable of degrading cyanobacterial toxins or other recalcitrant and problematic organic compounds. The isolated strains provide a large group of bacteria that could be used in assessing and controlling the harmful effects of cyanobacteria.

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.

First report of saxitoxin in Finnish lakes and possible associated effects on human health.

This study is the first report of saxitoxin in cyanobacterial blooms in Finland. Bloom samples (n = 50) were collected from Finnish freshwater sites during summer months of 2002 and 2003. These samples were screened for the presence of paralytic shellfish toxins (PSTs) using the Jellett rapid PSP screening test. Samples testing positive for PSTs (n = 7) were further analyzed with saxiphilin- and voltage-gated sodium channel [(3)H]-STX-binding radioreceptor assays and liquid chromatography using fluorescence and mass spectrometric analysis. The results indicated that saxitoxin (STX) was the only PST analogue in the samples and that it was present in high concentrations, as much as 1 mg L(-1). Microscopic analysis revealed that 95%-100% of the phytoplankton in the positive samples consisted of Anabaena lemmermannii. The trophic status of lakes in which STX-containing blooms were found varied from oligotrophic to hypertrophic. All the lakes had high nitrogen-to-phosphorus ratios. In some instances, samples had been collected from sites where swimmers had reported adverse health effects, and in three such cases, reported adverse health effects were associated with sites from which samples testing positive for STX had been received. Symptoms of fever, eye irritation, abdominal pains, and skin rash were reported in children aged 2-10 years after exposure to the water. These were not the adverse human symptoms typical of STX poisoning; rather, they represented acute effects often reported following recreational exposure to cyanobacterial blooms.

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.