Teija Koivula

Means to improve the effect of in situ bioremediation of contaminated soil: an overview of novel approaches

Different aspects of bacterial degradation of organic contaminants in soil, and how to improve the efficiency and reproducibility is discussed in this review. Although bioremediation in principle includes the use of any type of organism in improving the condition of a contaminated site, most commonly bacteria are the degraders and other organisms, such as soil animals or plant roots, play a role in dissemination of bacteria and, indirectly, plasmids between bacteria, and in providing nutrients and co-substrates for the bacteria active in the degradation process. There are a number of different procedures that have been tested more-or-less successfully in attempts to improve reliability, cost efficiency and speed of bioremediation. The methods range from minimal intervention, such as mere monitoring of intrinsic bioremediation, through in situ introduction of nutrients and/or bacterial inocula or improvement of physico-chemical conditions, all the way to excavation followed by on site or ex situ composting in its different varieties. In the past the rule has been that more intervention (leading to higher costs) has been more reliable, but novel ideas are continuously tried out, both as a means to come up with new truly functional applications and also as a line of studies in basic soil microbial ecology. Both approaches generate valuable information needed when predicting outcome of remediation activities, evaluating environmental risks, deciding on cleaning-up approaches, etc. The emphasis of this review is to discuss some of the novel methods for which the value has not been clearly shown, but that in our view merit continued studies and efforts to make them work, separately or in combination.

Hormone-sensitive lipase is closely related to several bacterial proteins, and distantly related to acetylcholinesterase and lipoprotein lipase: Identification of a superfamily of esterases and lipases

We have sequenced a gene from Bacillus acidocaldarius which encodes an open reading frame (ORF3) of 310 amino acids. The ORF3 was found to be related to the mammalian hormone-sensitive lipase (HSL). Searching the protein data base revealed five other bacterial proteins related to the HSL. Upon further sequence comparisons this HSL-group was found to be related to the family of carboxylesterases, and to a family of lipases (lipoprotein, hepatic and pancreatic lipases). The evolutionary relationship of these serine-dependent hydrolytic enzymes has not been studied previously, and it has not been known that these proteins belong to the same superfamily. Finally, the alignment of the HSL with the bacterial proteins allowed us to infer the location of the hormone-sensitive regulatory domain of the HSL-protein.

Tolerance and biodegradation of m‐toluate by Scots pine, a mycorrhizal fungus and fluorescent pseudomonads individually and under associative conditions

The tolerance to, and degradation of m‐toluate by Scots pine (Pinus sylvestris), a symbiotic mycorrhizal fungus (Suillus bovinus) and Pseudomonas fluorescens strains, with or without m‐toluate‐degrading capacity, was determined individually and in all symbiotic/associative plant‐microbe combinations. Fungal survival on medium with m‐toluate was increased in co‐culture with the degradative bacterial strains on agar plates (up to 0·02%, w/v). When fungi were grown in mycorrhizal association with Scots pine seedlings in test‐tube microcosms containing expanded clay pellets and growth media, the fungus was able to withstand m‐toluate concentrations up to 2·0%, w/v in all treatments. The seedling tolerance remained unaltered regardless of the presence or absence of mycorrhizal fungi or biodegradative bacteria. Reduction in m‐toluate levels was only detected in treatments inoculated with bacterial strains harbouring TOL catabolic plasmids. The plant and fungus, alone or in mycorrhizal symbiosis, were unable to cleave m‐toluate. The presence of easily available plant‐derived carbon sources did not impede m‐toluate degradation by the bacteria in the mycorrhizosphere.

EcM fungal community structure, but not diversity, altered in a Pb‐contaminated shooting range in a boreal coniferous forest site in Southern Finland

Boreal forests contain diverse fungal communities that form essential ectomycorrhizal symbioses with trees. To determine the effects of lead (Pb) contamination on ectomycorrhizal fungal communities associated with the dominant pine (Pinus sylvestris L.), we surveyed sporocarps for 3 years, analyzed morphotyped ectomycorrhizal root tips by direct sequencing, and 454-sequenced fungal communities that grew into in-growth bags during a 2-year incubation at a shooting range where sectors vary in the Pb load. We recorded a total of 32 ectomycorrhizal fungi that formed conspicuous sporocarps, 27 ectomycorrhizal fungal phylotypes from 294 root tips, and 116 ectomycorrhizal fungal operation taxonomic unit (OTUs) from a total of 8194 internal transcribed spacer-2 454 sequences. Our ordination analyses by nonparametric multidimensional scaling (NMS) indicated that the Pb enrichment induced a shift in the ectomycorrhizal community composition. This was visible as indicative trends in the sporocarp and root tip data sets, but was explicitly clear in the communities observed in the in-growth bags. The compositional shift in the ectomycorrhizal community was mainly attributable to an increase in the frequencies of OTUs assigned to genus Thelephora and to a decrease in the OTUs assigned to Pseudotomentella, Suillus, and Tylospora in Pb-contaminated areas when compared with the control. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem function remain undetermined.

Cloning and sequencing of a gene encoding acidophilic amylase from Bacillus acidocaldarius

Two starch-degrading enzymes produced by Bacillus acidocaldarius (renamed as Alicyclobacillus acidocaldarius) were identified. According to SDS-PAGE, the apparent molecular masses of the enzymes were 90 and 160 kDa. Eight peptide fragments and the N-terminal end of the 90 kDa polypeptide were sequenced. An oligonucleotide, based on the amino acid sequence of a peptide fragment of the 90 kDa protein, was used to screen a lambda gt10 bank of B. acidocaldarius, and the region encoding the 90 kDa protein was cloned. Unexpectedly, the ORF continued upstream of the N terminus of the 90 kDa protein. The entire ORF was 1301 amino acids (aa) long (calculated molecular mass 140 kDa) and it was preceded by a putative ribosomal binding site and a promoter. Computer analysis showed that the 1301 aa protein was closely related to an alpha-amylase-pullulanase of Clostridium thermohydrosulfuricum. We suggest that the starch-degrading 160 kDa protein of B. acidocaldarius is an alpha-amylase-pullulanase, and the 90 kDa protein is a cleavage product of the 160 kDa protein. Another ORF, apparently in the same transcription unit, was found downstream from the amylase gene. It encoded a protein that was closely related to the maltose-binding protein of Escherichia coli.

Nucleotide sequence of the secY gene from Lactococcus lactis and identification of conserved regions by comparison of four SecY proteins.

SecY is an integral membrane protein which participates in the translocation of proteins through the bacterial cell membrane. We have cloned the secY gene of Lactococcus lactis, and found its deduced protein sequence, 439 amino acids long, to be similar in length to the previously determined SecY proteins of Escherichia coli, Bacillus subtilis and Mycoplasma capricolum. Comparison of the L. lactis SecY to the 3 other SecY proteins revealed 90 conserved amino acid residues (21%). Nearly half of the conserved residues are clustered in 2 of the 10 transmembrane segments, and in 2 of the 6 cytoplasmic regions. Some of the conserved regions are apparently responsible for the interactions of SecY with signal sequences, and the proteins SecE and SecA.

Comparison of partial 16S rRNA gene sequences obtained from activated sludge bacteria

Abstract The cultivated and uncultivated bacterial communities of an activated sludge plant were studied. Two samples were taken and a total of 516 bacterial isolates were classified into groups using their whole-cell protein patterns. The distribution of bacteria into protein-pattern groups differed significantly between the two samples, suggesting variation in culturable bacterial flora. Partial 16S rRNA gene sequences were determined for representatives of the commonest protein-pattern groups. Most of the sequences obtained were previously unknown, but relatively closely related to known sequences of organisms belonging to the α, β or γ subclasses of the proteobacteria, the first two subclasses being predominant. This classification of bacteria isolated on a diluted nutrient-rich medium differed from recent culture-dependent studies using nutrient-rich media. The uncultivated bacterial community was studied by analyzing ten partial 16S rRNA gene sequences cloned directly from activated sludge. None of the cloned sequences was identical to those determined for culturable organisms; or to those in the GenBank database. They were, however, related to the α or β subclasses of the proteobacteria, or to the gram-positive bacteria with a high G+C DNA content.

Deposition of gamma emitters from Chernobyl accident and their transfer in lichen-soil columns.

Lichen-soil column samples were taken from several locations in the Southern Finland between 1986 and 2006. Columns were divided into three parts, upper lichen, lower lichen and underlying soil, and their gamma emitting radionuclides, 134Cs, 137Cs, 103Ru, 95Zr, 106Ru, 110mAg, 125Sb and 144Ce, were measured with gamma spectrometry. Deposition values were calculated as Bq/m2 for each sampling site. Distribution of various radionuclides in the three compartments as a function of time was determined. Both effective and ecological half-lives of all radionuclides were calculated for upper lichen, whole lichen and whole lichen-soil column. A linear relation was derived between the physical half-lives and effective half-lives for whole lichen and for whole lichen-soil column. Reindeer meat activity concentrations of various radionuclides and ensuing radiation doses to reindeer-herding people were also estimated for a hypothetical case where a similar high radioactive pollution, as was taken place in the Southern Finland, would have occurred in the reindeer-herding areas in the Finnish Lapland.

Review ArticlesEx vivo evaluation of N-(3-[18F]fluoropropyl)-2β-carbomethoxy-3β-(4-fluorophenyl)nortropane in rats

INTRODUCTION The dopamine transporter (DAT) ligand N-(3-fluoropropyl)-2 beta-carbomethoxy-3beta-(4-fluorophenyl)nortropane (beta-CFT-FP) was labeled with fluorine-18, and its biodistribution was evaluated in rats ex vivo. METHODS The distribution of 18F radioactivity in the brain and peripheral organs and tissues was determined at several time points 5-120 min after intravenous injection of [18F]beta-CFT-FP. RESULTS The highest brain uptake of [18F]beta-CFT-FP was localized in the striatum; limbic structures also exhibited high uptake. Low uptake was found in the cerebellum. The highest ratio of striatum-to-cerebellum uptake, already reached within 5 min, was 3.1. Pretreatment with the selective DAT inhibitor GBR12909 significantly decreased [18F]beta-CFT-FP uptake in the striatum. In most peripheral tissues, the highest uptake was found at 5 min, indicating fast washout of the radioligand. Some accumulation of (18)F radioactivity was seen in bone as a function of time, reflecting defluorination of the radioligand. CONCLUSION The results indicate that [18F]beta-CFT-FP is a potential radioligand for studying DAT in vivo with positron emission tomography.

Ex vivo evaluation of N-(3-[18F]fluoropropyl)-2β-carbomethoxy-3β-(4-fluorophenyl)nortropane in rats

INTRODUCTION The dopamine transporter (DAT) ligand N-(3-fluoropropyl)-2 beta-carbomethoxy-3beta-(4-fluorophenyl)nortropane (beta-CFT-FP) was labeled with fluorine-18, and its biodistribution was evaluated in rats ex vivo. METHODS The distribution of 18F radioactivity in the brain and peripheral organs and tissues was determined at several time points 5-120 min after intravenous injection of [18F]beta-CFT-FP. RESULTS The highest brain uptake of [18F]beta-CFT-FP was localized in the striatum; limbic structures also exhibited high uptake. Low uptake was found in the cerebellum. The highest ratio of striatum-to-cerebellum uptake, already reached within 5 min, was 3.1. Pretreatment with the selective DAT inhibitor GBR12909 significantly decreased [18F]beta-CFT-FP uptake in the striatum. In most peripheral tissues, the highest uptake was found at 5 min, indicating fast washout of the radioligand. Some accumulation of (18)F radioactivity was seen in bone as a function of time, reflecting defluorination of the radioligand. CONCLUSION The results indicate that [18F]beta-CFT-FP is a potential radioligand for studying DAT in vivo with positron emission tomography.