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Denitrification is an important microbial process in aquatic ecosystems that can reduce the effects of eutrophication. Here, quantification and pyrosequencing of nirS, nirK, and nosZ genes encoding for nitrite and nitrous oxide reductases was performed in sediment samples from four boreal lakes to determine the structure and seasonal stability of denitrifying microbial populations. Sediment quality and nitrate concentrations were linked to the quantity and diversity of denitrification genes, the abundance of denitrifying populations (nirS and nosZ genes) correlated with coupled nitrification-denitrification (Dn), and the denitrification of the overlying water NO3 (-) (Dw) correlated with the nirS/nirK ratio. The number of core nirS, nirK, and nosZ operational taxonomical units (OTUs) was low (6, 7, and 3, respectively), and most of these core OTUs were shared among the lakes. Dominant nirK sequences matched best with those of the order Rhizobiales, which was one of the main bacterial orders present in the sediment microbiomes, whereas the dominant nirS sequences were affiliated with the order Burkholderiales. Over half of the nosZ sequences belonged to a single OTU of the order Burkholderiales, but coupled nitrification-denitrification rate correlated with another dominant nosZ OTU assigned to the order Rhodospirillales. The study indicates that a few core proteobacterial clusters may drive denitrification in boreal lake sediments, as the same Alpha- and Betaproteobacteria denitrifier clusters were present in different lakes and seasons.

Mid-summer N(2) profiles were analyzed from nine oxygen-stratified, humic-acid-rich lakes using a continuous flow isotope ratio mass spectrometer and a Gasbench II device. Sample preparation steps were performed under water to avoid air contamination. The instrument precision for the δ(15)N measurement was high (0.03‰), but for the whole sampling and analysis procedure the mean deviation between replicate samples was 0.13‰ for the δ(15)N measurements and 5.5% for the N(2) gas concentration analysis. The results show that the Gasbench peripheral was suitable for measurement of the (15)N natural abundance of dissolved nitrogen gas, with denitrification indicated by the oversaturation and slightly (<1‰) depleted δ(15)N values of the dissolved N(2) gas in the suboxic zones of some of the study lakes. Calculated values for the denitrified (excess) N(2) varied between -5.3 and 0.7‰. The denitrification potential was determined using the (15)N tracer method, with results showing nitrate-inducible denitrification and no signs of anaerobic ammonium oxidation (anammox)., (Copyright © 2011 John Wiley & Sons, Ltd.)

The influence of rearing conditions on Flavobacterium columnare infection of rainbow trout, Oncorhynchus mykiss (Walbaum), was studied experimentally in the laboratory and at a fish farm. In experiment I, the effect of parasitic infection on columnaris disease was studied using F. columnare carrier fish. The fish were exposed to Diplostomum spathaceum cercariae and a set of other stressors in order to induce clinical columnaris infection. Parasitic infection and other stressors failed to induce the disease. Disease occurred when the fish were challenged with F. columnare, but D. spathaceum infection did not enhance the severity of the infection. In experiment II, the influence of rearing density and water temperature was studied. Overall mortality was highest in fish at normal rearing density with high temperature (+23 degrees C). At low temperature (+18 degrees C) mortality was not affected by rearing density, but the transmission of columnaris disease was faster at normal rearing density at both temperatures. This supports the view that reduction of fish density could be used in prevention of columnaris disease especially if water temperature is high. Because the lower rearing density can also decrease the transmission of ectoparasites and penetrating endoparasites, it could be an efficient tool in ecological disease management.

A polychlorophenol-degrading strain, designated MT1(T), and three MT1-like strains, MT101, MT103 and MT104, were isolated from a cold (4-8 degrees C) fluidized-bed process treating chlorophenol-contaminated groundwater in southern Finland. The organisms were Gram-negative, rod-shaped, catalase-positive, non-spore-forming and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strains belonged to the alpha-4 subclass of the Proteobacteria and were members of the genus Novosphingobium. The highest 16S rRNA gene sequence similarity observed for these strains was 96.5 % with the type strains of Novosphingobium hassiacum, Novosphingobium aromaticivorans and Novosphingobium subterraneum. Chemotaxonomic data (major ubiquinone: Q-10; major polyamine: spermidine; major polar lipids: phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and sphingoglycolipid; major fatty acids: 18 : 1omega7c, 16 : 1omega7c and 2-OH 14 : 0) as well as the ability to reduce nitrate supported the affiliation of the strains to the genus Novosphingobium. Based on the phylogenetic analysis, whole-cell fatty acid composition as well as biochemical and physiological characteristics, the MT1-like strains were highly similar and could be separated from all recognized Novosphingobium species. The novel species Novosphingobium lentum sp. nov. is proposed to accommodate strains MT1(T) (=DSM 13663(T)=CCUG 45847(T)), MT101 (=CCUG 45849), MT103 (=CCUG 45850) and MT104 (=CCUG 45851).

A two-stage pilot-scale thermophilic aerobic suspended carrier biofilm process (SCBP) was set up for the on-site treatment of pulp and paper mill whitewater lining. The microbial diversity in this process was analyzed by length heterogeneity analysis of PCR-amplified 16S ribosomal DNA. The primer pair selected for PCR amplification was first evaluated by a computational analysis of fragment lengths in ten main phylogenetical eubacterial groups. The fragment contained the first third of the 16S rRNA gene, which was shown to vary naturally between 465 and 563 bp in length. The length heterogeneity analysis of polymerase chain reaction (LH-PCR) profile of the biomass attached to carrier elements was found to be diverse in both stages of the SCBP. During normal operating conditions, sequences belonging to beta-Proteobacteria, Cytophaga/Flexibacter/Bacteroides group and gamma-Proteobacteria were assigned to the most prominent LH-PCR peak. Samples from the suspended biomass consisted of completely different bacterial populations, which were, however, similar in the serial reactors. The pilot process experienced alkaline shocks, after which Bacillus-like sequences were detected in both the biofilm and suspended biomass. However, when the conditions were reversed, the normal microbial population in the biofilm recovered rapidly without further biomass inoculations. This study shows that LH-PCR is a valuable method for profiling microbial diversity and dynamics in industrial wastewater processes.

The chlorophenol degradation pathway in Sphingobium chlorophenolicum is initiated by the pcpB gene product, pentachlorophenol-4-monooxygenase. The distribution of the gene was studied in a phylogenetically diverse group of polychlorophenol-degrading bacteria isolated from contaminated groundwater in Kärkölä, Finland. All the sphingomonads isolated were shown to share pcpB gene homologs with 98.9 to 100% sequence identity. The gene product was expressed when the strains were induced by 2,3,4,6-tetrachlorophenol. A comparative analysis of the 16S rDNA and pcpB gene trees suggested that a recent horizontal transfer of the pcpB gene was involved in the evolution of the catabolic pathway in the Kärkölä sphingomonads. The full-length Kärkölä pcpB gene allele had approximately 70% identity with the three pcpB genes previously sequenced from sphingomonads. It was very closely related to the environmental clones obtained from chlorophenol-enriched soil samples (M. Beaulieu, V. Becaert, L. Deschenes, and R. Villemur, Microbiol. Ecol. 40:345-355, 2000). The gene was not present in polychlorophenol-degrading nonsphingomonads isolated from the Kärkölä source.

A high-rate fluidized-bed bioreactor has been treating polychlorophenol-contaminated groundwater in southern Finland at 5 to 8 degrees C for over 6 years. We examined the microbial diversity of the bioreactor using three 16S ribosomal DNA (rDNA)-based methods: denaturing gradient gel electrophoresis, length heterogeneity-PCR analysis, and restriction fragment length polymorphism analysis. The molecular study revealed that the process was dependent on a stable bacterial community with low species diversity. The dominant organism, Novosphingobium sp. strain MT1, was isolated and characterized. Novosphingobium sp. strain MT1 degraded the main contaminants of the groundwater, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, at 8 degrees C. The strain carried a homolog of the pcpB gene, coding for the pentachlorophenol-4-monooxygenase in Sphingobium chlorophenolicum. Spontaneous deletion of the pcpB gene homolog resulted in the loss of degradation ability. Phenotypic dimorphism (planktonic and sessile phenotypes), low growth rate (0.14 to 0.15 h(-1)), and low-copy-number 16S rDNA genes (single copy) were characteristic of strain MT1 and other MT1-like organisms isolated from the bioreactor.

Pentachlorophenol 4-monooxygenase (PCP4MO) from Sphingomonas chlorophenolica is a flavoprotein that hydroxylates PCP in the presence of NADPH and oxygen. In order to investigate the structure and function of active site, recombinant PCP4MO (rePCP4MO) was produced in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Moreover, a tobacco etch virus (TEV) protease cleavage site (EKLYFQG) was introduced into GST-PCP4MO and a his-tagged TEV protease was employed. Hence, a two-step purification protocol was developed which allowed obtaining 15-20 mg of rePCP4MO from 1 L culture. The rePCP4MO revealed identity with native enzyme by SDS-PAGE and N-terminal sequence analyses. Furthermore, a polyclonal PCP4MO antibody was produced with GST-PCP4MO and purified by immunoaffinity chromatography, where both the native and recombinant forms of PCP4MO showed interaction. However, rePCP4MO was identified as apoprotein with no evidence for a typical flavoprotein spectrum. The catalytic activity could be detected in the presence of FAD. The K(m) and V(max) values for PCP were 50 microM and 30 nmol/min/mg, respectively., (Copyright 2001 Academic Press.)

Effects of low temperature and low oxygen partial pressure on the occurrence and activity of 2,3,4,6-tetrachlorophenol degrading bacteria in a boreal chlorophenol contaminated groundwater and a full-scale fluidized-bed bioreactor were studied using four polychlorophenol degrading bacterial isolates of different phylogenetic backgrounds. These included an alpha-proteobacterial Sphingomonas sp. strain MT1 isolated from the full-scale bioreactor and three isolates from the contaminated groundwater which were identified as beta-proteobacterial Herbaspirillum sp. K1, a Gram-positive bacterium with high G + C content Nocardioides sp. K44 and an alpha-proteobacterial Sphingomonas sp. K74. The Sphingomonas strains K74 and MT1 and Nocardioides sp. K44 degraded 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol as the sole carbon and energy sources. Close to stoichiometric inorganic chloride release with the 2,3,4,6-tetrachlorophenol removal and the absence of methylation products indicated mineralization. Tetrachlorophenol degradation by the Herbaspirillum sp. K1 was enhanced by yeast extract, malate, glutamate, pyruvate, peptone and casitone. At 8 degrees C, Sphingomonas sp. K74 had the highest specific degradation rate (mu(max) = 4.9 x 10(-2) mg h(-1) cell(-1)) for 2,3,4,6-tetrachlorophenol. The Nocardioides strain K44 had the highest affinity (K(s) = 0.46 mg l(-1)) fortetrachlorophenol. K1 and MT1 grew microaerophilically in semisolid glucose medium. Furthermore, the growth of MT1 was inhibited in liquid glucose medium at high oxygen partial pressure indicating sensitivity to accumulating toxic oxygen species. On the other hand, trichlorophenol degradation was not affected by oxygen concentration (2-21%). The isolates K44, K74 and MT1, with optimum growth temperatures between 23 and 25 degrees C, degraded tetrachlorophenol faster at 8 degrees C than at room temperature indicating distinctly different temperature optima for chlorophenol degradation and growth on complex media. These results show efficient polychlorophenol degradation by the isolates at the boreal groundwater conditions, i.e., at low temperature and low oxygen concentrations. Differences in chlorophenol degradation and sensitivities to chlorophenols and oxygen among the isolates indicate that the phylogenetically different chlorophenol degraders have found different niches in the contaminated groundwater and thus potential for contaminant degradation under a variety of saturated subsurface conditions.

Psychrophilic actinobacterial isolates from permanently cold groundwater in Finland were characterized using a polyphasic approach. Growth on agar plates was observed at temperatures down to -2 degrees C, with an optimum at 15-17 degrees C, but no growth was observed at 30 degrees C. The peptidoglycan type was B2y and the characteristic diamino acid was diaminobutyric acid. The cell wall sugars of strain K265T were rhamnose, ribose, xylose and mannose and those of strain K300T were glucose, rhamnose and xylose. The polar lipids included phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid and two glycolipids. The main whole-cell fatty acids were 12-methyltetradecanoic acid, 14-methylpentadecanoic acid and 14-methylhexadecanoic acid. Large amounts of anteiso-1,1-dimethoxy-pentadecane and also iso-1,1-dimethoxyhexadecane were present as diagnostic markers. The predominant menaquinones were MK-9 and MK-10. The G+C content of the DNA of strains K265T and K300T was 64.4 and 67.8 mol%, respectively. Comparison of 16S rRNA gene sequences revealed that strains K265T and K300T represent a new lineage among the type-B-peptidoglycan actinomycetes. The closest relatives were Clavibacter michiganensis, Frigoribacterium faeni and Rathayibacter rathayi. On the basis of 16S rDNA sequence, G+C content and chemotaxonomical and physiological characteristics, K265T and K300T clearly represent a new genus. The genus Subtercola gen. nov. is described, together with two species, namely Subtercola boreus sp. nov. (type strain K300T = DSM 13056T = CCUG 43135T) and Subtercola frigoramans sp. nov (type strain K265T = DSM 13057T = CCUG 43136T).

Chlorophenol-degrading bacteria from a long-term polluted groundwater aquifer were characterized. All isolates degraded 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol at concentrations detected in the contaminated groundwater (< 10 mg 1(-1)). Pentachlorophenol was degraded by three isolates when present alone. In two gram-positive isolates, 2,3,4,6-tetrachlorophenol was required as an inducer for the degradation of pentachlorophenol. The gram-positive isolates were sensitive to pentachlorophenol, with an IC50 value of 5 mg/l. Isolates belonging to the Cytophaga/Flexibacter/Bacteroides phylum had IC50 values of 25 and 63 mg/l. Isolates belonging to alpha-, beta- and gamma-Proteobacteria generally tolerated the highest pentachlorophenol concentrations (> 100 mg/l). Polychlorophenol-degrading capacity was found in strains of Nocardioides, Pseudomonas, Ralstonia, Flavobacterium, and Caulobacter previously not known to degrade polychlorophenols. In addition, six polychlorophenol-degrading sphingomonads were found.