Genome analysis and description of Tunturibacter gen. nov. expands the diversity of Terriglobia in tundra soils.

Increased temperatures in Arctic tundra ecosystems are leading to higher microbial respiration rates of soil organic matter, resulting in the release of carbon dioxide and methane. To understand the effects of this microbial activity, it is important to better characterize the diverse microbial communities in Arctic soil. Our goal is to refine our understanding of the phylogenetic diversity of Terriglobia, a common but elusive group within the Acidobacteriota phylum. This will help us link this diversity to variations in carbon and nitrogen usage patterns. We used long-read Oxford Nanopore MinION sequences in combination with metagenomic short-read sequences to assemble complete Acidobacteriota genomes. This allowed us to build multi-locus phylogenies and annotate pangenome markers to distinguish Acidobacteriota strains from several tundra soil isolates. We identified a phylogenetic cluster containing four new species previously associated with Edaphobacter lichenicola. We conclude that this cluster represents a new genus, which we have named Tunturibacter. We describe four new species: Tunturibacter lichenicola comb. nov., Tunturibacter empetritectus sp. nov., Tunturibacter gelidoferens sp. nov., and Tunturibacter psychrotolerans sp. nov. By uncovering new species and strains within the Terriglobia and improving the accuracy of their phylogenetic placements, we hope to enhance our understanding of this complex phylum and shed light on the mechanisms that shape microbial communities in polar soils.

Bacterial and fungal communities in sub-Arctic tundra heaths are shaped by contrasting snow accumulation and nutrient availability.

Climate change is affecting winter snow conditions significantly in northern ecosystems but the effects of the changing conditions for soil microbial communities are not well-understood. We utilized naturally occurring differences in snow accumulation to understand how the wintertime subnivean conditions shape bacterial and fungal communities in dwarf shrub-dominated sub-Arctic Fennoscandian tundra sampled in mid-winter, early, and late growing season. Phospholipid fatty acid (PLFA) and quantitative PCR analyses indicated that fungal abundance was higher in windswept tundra heaths with low snow accumulation and lower nutrient availability. This was associated with clear differences in the microbial community structure throughout the season. Members of Clavaria spp. and Sebacinales were especially dominant in the windswept heaths. Bacterial biomass proxies were higher in the snow-accumulating tundra heaths in the late growing season but there were only minor differences in the biomass or community structure in winter. Bacterial communities were dominated by members of Alphaproteobacteria, Actinomycetota, and Acidobacteriota and were less affected by the snow conditions than the fungal communities. The results suggest that small-scale spatial patterns in snow accumulation leading to a mosaic of differing tundra heath vegetation shapes bacterial and fungal communities as well as soil carbon and nutrient availability.

Reindeer grazing history determines the responses of subarctic soil fungal communities to warming and fertilization.

Composition and functioning of arctic soil fungal communities may alter rapidly due to the ongoing trends of warmer temperatures, shifts in nutrient availability, and shrub encroachment. In addition, the communities may also be intrinsically shaped by heavy grazing, which may locally induce an ecosystem change that couples with increased soil temperature and nutrients and where shrub encroachment is less likely to occur than in lightly grazed conditions. We tested how 4 yr of experimental warming and fertilization affected organic soil fungal communities in sites with decadal history of either heavy or light reindeer grazing using high-throughput sequencing of the internal transcribed spacer 2 ribosomal DNA region. Grazing history largely overrode the impacts of short-term warming and fertilization in determining the composition of fungal communities. The less diverse fungal communities under light grazing showed more pronounced responses to experimental treatments when compared with the communities under heavy grazing. Yet, ordination approaches revealed distinct treatment responses under both grazing intensities. If grazing shifts the fungal communities in Arctic ecosystems to a different and more diverse state, this shift may dictate ecosystem responses to further abiotic changes. This indicates that the intensity of grazing cannot be left out when predicting future changes in fungi-driven processes in the tundra.

Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing.

Arctic soils store vast amounts of carbon and are subject to intense climate change. While the effects of thaw on the composition and activities of Arctic tundra microorganisms has been examined extensively, little is known about the consequences of temperature fluctuations within the subzero range in seasonally frozen or permafrost soils. This study identified tundra soil bacteria active at subzero temperatures using stable isotope probing (SIP). Soils from Kilpisjärvi, Finland, were amended with 13C-cellobiose and incubated at 0, -4 and -16°C for up to 40 weeks. 16S rRNA gene sequence analysis of 13C-labelled DNA revealed distinct subzero-active bacterial taxa. The SIP experiments demonstrated that diverse bacteria, including members of Candidatus Saccharibacteria, Melioribacteraceae, Verrucomicrobiaceae, Burkholderiaceae, Acetobacteraceae, Armatimonadaceae and Planctomycetaceae, were capable of synthesising 13C-DNA at subzero temperatures. Differences in subzero temperature optima were observed, for example, with members of Oxalobacteraceae and Rhizobiaceae found to be more active at 0°C than at -4°C or -16°C, whereas Melioribacteriaceae were active at all subzero temperatures tested. Phylogeny of 13C-labelled 16S rRNA genes from the Melioribacteriaceae, Verrucomicrobiaceae and Candidatus Saccharibacteria suggested that these taxa formed subzero-active clusters closely related to members from other cryo-environments. This study demonstrates that subzero temperatures impact active bacterial community composition and activity, which may influence biogeochemical cycles.

Complete genome sequence of Granulicella tundricola type strain MP5ACTX9(T), an Acidobacteria from tundra soil.

Granulicella tundricola strain MP5ACTX9(T) is a novel species of the genus Granulicella in subdivision 1 Acidobacteria. G. tundricola is a predominant member of soil bacterial communities, active at low temperatures and nutrient limiting conditions in Arctic alpine tundra. The organism is a cold-adapted acidophile and a versatile heterotroph that hydrolyzes a suite of sugars and complex polysaccharides. Genome analysis revealed metabolic versatility with genes involved in metabolism and transport of carbohydrates, including gene modules encoding for the carbohydrate-active enzyme (CAZy) families for the breakdown, utilization and biosynthesis of diverse structural and storage polysaccharides such as plant based carbon polymers. The genome of G. tundricola strain MP5ACTX9(T) consists of 4,309,151 bp of a circular chromosome and five mega plasmids with a total genome content of 5,503,984 bp. The genome comprises 4,705 protein-coding genes and 52 RNA genes.

Moth herbivory enhances resource turnover in subarctic mountain birch forests?

Massive moth outbreaks cause large-scale damage in subarctic mountain birch forests with a concomitant decrease in carbon flux to mycorrhizal fungi and an increased deposition of dissolved carbon and nutrients as moth frass into soil. We investigated impacts of moth herbivory along three replicated gradients with three levels of moth herbivory (undamaged, once damaged, repeatedly damaged) on soil nutrient levels and biological parameters. We found an increase in soil nutrients and in the biomass of enchytraeid worms, which are key faunal decomposers. Fungi bacteria ratio and C:N ratio decreased in humus with increasing severity of herbivory. Our findings suggest enhanced resource turnover in mountain birch forests due to massive moth herbivory. This may provide a shortcut for carbon and nutrient input to subarctic soils, which largely bypasses the main routes of carbon from plants to soil via mycorrhizal and litter-decomposing fungi. Moreover, a temporal shift occurs in carbon allocation to soil, providing decomposers an opportunity to use an early-season peak in resource availability. Our results suggest a hitherto unappreciated role of massive insect herbivore attacks on resource dynamics in subarctic ecosystems.

Acidobacteria dominate the active bacterial communities of Arctic tundra with widely divergent winter-time snow accumulation and soil temperatures.

The timing and extent of snow cover is a major controller of soil temperature and hence winter-time microbial activity and plant diversity in Arctic tundra ecosystems. To understand how snow dynamics shape the bacterial communities, we analyzed the bacterial community composition of windswept and snow-accumulating shrub-dominated tundra heaths of northern Finland using DNA- and RNA-based 16S rRNA gene community fingerprinting (terminal restriction fragment polymorphism) and clone library analysis. Members of the Acidobacteria and Proteobacteria dominated the bacterial communities of both windswept and snow-accumulating habitats with the most abundant phylotypes corresponding to subdivision (SD) 1 and 2 Acidobacteria in both the DNA- and RNA-derived community profiles. However, different phylotypes within Acidobacteria were found to dominate at different sampling dates and in the DNA- vs. RNA-based community profiles. The results suggest that different species within SD1 and SD2 Acidobacteria respond to environmental conditions differently and highlight the wide functional diversity of these organisms even within the SD level. The acidic tundra soils dominated by ericoid shrubs appear to select for diverse stress-tolerant Acidobacteria that are able to compete in the nutrient poor, phenolic-rich soils. Overall, these communities seem stable and relatively insensitive to the predicted changes in the winter-time snow cover.

Complete genome sequence of Granulicella mallensis type strain MP5ACTX8(T), an acidobacterium from tundra soil.

Granulicella mallensis MP5ACTX8(T) is a novel species of the genus Granulicella in subdivision 1of Acidobacteria. G. mallensis is of ecological interest being a member of the dominant soil bacterial community active at low temperatures and nutrient limiting conditions in Arctic alpine tundra. G. mallensis is a cold-adapted acidophile and a versatile heterotroph that hydrolyzes a suite of sugars and complex polysaccharides. Genome analysis revealed metabolic versatility with genes involved in metabolism and transport of carbohydrates. These include gene modules encoding the carbohydrate-active enzyme (CAZyme) family involved in breakdown, utilization and biosynthesis of diverse structural and storage polysaccharides including plant based carbon polymers. The genome of Granulicella mallensis MP5ACTX8(T) consists of a single replicon of 6,237,577 base pairs (bp) with 4,907 protein-coding genes and 53 RNA genes.

Endophytic bacterial communities in three arctic plants from low arctic fell tundra are cold-adapted and host-plant specific.

Endophytic bacteria inhabit internal plant tissues, and have been isolated from a large diversity of plants, where they form nonpathogenic relationships with their hosts. This study combines molecular and culture-dependent approaches to characterize endophytic bacterial communities of three arcto-alpine plant species (Oxyria digyna, Diapensia lapponica and Juncus trifidus) sampled in the low Arctic (69°03'N). Analyses of a 325 bacterial endophyte isolates, as well as seven clone libraries, revealed a high diversity. In particular, members of the Actinobacteria, Bacteroidetes, Firmicutes, Acidobacteria, and Proteobacteria were found. The compositions of the endophytic bacterial communities were dependent on host-plant species as well as on snow cover at sampling sites. Several bacterial genera were found to be associated tightly with specific host-plant species. In particular, Sphingomonas spp. were characteristic for D. lapponica and O. digyna, and their phylogenetic grouping corresponded to the host plant. Most of the endophyte isolates grew well and retained activity at +4 °C, and isolate as well as clone library sequences were often highly similar to sequences from bacteria from cold environments. Taken together, this study shows that arctic plants harbour a diverse community of bacterial endophytes, a portion of which seems to be tightly associated with specific plant species.

Comparative genomic and physiological analysis provides insights into the role of Acidobacteria in organic carbon utilization in Arctic tundra soils.

Acidobacteria are among the most abundant bacterial phyla found in terrestrial ecosystems, but relatively little is known about their diversity, distribution and most critically, their function. Understanding the functional activities encoded in their genomes will provide insights into their ecological roles. Here we describe the genomes of three novel cold-adapted strains of subdivision 1 Acidobacteria. The genomes consist of a circular chromosome of 6.2 Mbp for Granulicella mallensis MP5ACTX8, 4.3 Mbp for Granulicella tundricola MP5ACTX9, and 5.0 Mbp for Terriglobus saanensis SP1PR4. In addition, G. tundricola has five mega plasmids for a total genome size of 5.5 Mbp. The three genomes showed an abundance of genes assigned to metabolism and transport of carbohydrates. In comparison to three mesophilic Acidobacteria, namely Acidobacterium capsulatum ATCC 51196, 'Candidatus Koribacter versatilis' Ellin345, and 'Candidatus Solibacter usitatus' Ellin6076, the genomes of the three tundra soil strains contained an abundance of conserved genes/gene clusters encoding for modules of the carbohydrate-active enzyme (CAZyme) family. Furthermore, a large number of glycoside hydrolases and glycosyl transferases were prevalent. We infer that gene content and biochemical mechanisms encoded in the genomes of three Arctic tundra soil Acidobacteria strains are shaped to allow for breakdown, utilization, and biosynthesis of diverse structural and storage polysaccharides and resilience to fluctuating temperatures and nutrient-deficient conditions in Arctic tundra soils.

Complete genome sequence of Terriglobus saanensis type strain SP1PR4(T), an Acidobacteria from tundra soil.

Terriglobus saanensis SP1PR4(T) is a novel species of the genus Terriglobus. T. saanensis is of ecological interest because it is a representative of the phylum Acidobacteria, which are dominant members of bacterial soil microbiota in Arctic ecosystems. T. saanensis is a cold-adapted acidophile and a versatile heterotroph utilizing a suite of simple sugars and complex polysaccharides. The genome contained an abundance of genes assigned to metabolism and transport of carbohydrates including gene modules encoding for carbohydrate-active enzyme (CAZyme) family involved in breakdown, utilization and biosynthesis of diverse structural and storage polysaccharides. T. saanensis SP1PR4(T) represents the first member of genus Terriglobus with a completed genome sequence, consisting of a single replicon of 5,095,226 base pairs (bp), 54 RNA genes and 4,279 protein-coding genes. We infer that the physiology and metabolic potential of T. saanensis is adapted to allow for resilience to the nutrient-deficient conditions and fluctuating temperatures of Arctic tundra soils.

Granulicella arctica sp. nov., Granulicella mallensis sp. nov., Granulicella tundricola sp. nov. and Granulicella sapmiensis sp. nov., novel acidobacteria from tundra soil.

Four aerobic bacteria, designated MP5ACTX2(T), MP5ACTX8(T), MP5ACTX9(T) and S6CTX5A(T), were isolated from tundra soil of north-western Finland (69° 03' N 20° 50' E). Cells of all isolates were Gram-negative, non-motile rods. Phylogenetic analysis indicated that they belonged to the genus Granulicella of subdivision 1 of the phylum Acidobacteriahttp://dx.doi.org/10.1601/nm.7918. 16S rRNA gene sequence similarity between the new isolates and the type strains of Granulicella aggregans, Granulicella paludicola, Granulicella pectinivorans and Granulicella rosea ranged from 94 to 99 %. Analysis of the RNA polymerase beta subunit (rpoB) gene sequence indicated that the isolates represented novel species of the genus Granulicella (<92 % rpoB sequence similarity between the isolates and members of the genus Granulicella). This was also confirmed by low DNA-DNA relatedness (31 %) between strain S6CTX5A(T) and the type strain of G. pectinivorans, which exhibited 99.1 % 16S rRNA gene sequence similarity and 91.7 % rpoB gene sequence similarity. The isolates grew at pH 3.5-6.5 and at 4-26 °C. Sugars were the preferred growth substrates. The major cellular fatty acids were iso-C(15 : 0), C(16 : 1)ω7c and C(16 : 0) and the major isoprenoid quinone was MK-8. The DNA G+C content was 56-60 mol%. On the basis of phylogenetic analysis and chemotaxonomic and physiological data, the isolates represent four novel species of the genus Granulicella, for which the names Granulicella arctica MP5ACTX2(T) (= ATCC BAA-1858(T) = DSM 23128(T)), Granulicella mallensis MP5ACTX8(T) (= ATCC BAA-1857(T) = DSM 23137(T)), Granulicella tundricola MP5ACTX9(T) (ATCC BAA-1859(T) = DSM 23138(T)) and Granulicella sapmiensis S6CTX5A(T) (= LMG 26174(T) = DSM 23136(T)) are proposed. An emended description of the genus Granulicella is also presented.

Terriglobus saanensis sp. nov., an acidobacterium isolated from tundra soil.

Two aerobic bacterial strains, designated SP1PR4(T) and SP1PR5, were isolated from tundra soil samples collected from Saana fjeld, North-western Finland (69° 03' N 20° 50' E). Cells of both strains were Gram-negative, non-motile rods. Phylogenetic analysis indicated that the strains belong to the genus Terriglobus in subdivision 1 of the phylum Acidobacteria. Strains SP1PR4(T) and SP1PR5 shared identical BOX and ERIC fingerprints and 99.7 % 16S rRNA gene similarity indicating that, together with their identical physiological features, these strains are members of the same species. The 16S rRNA gene sequence similarity of SP1PR4(T) and SP1PR5 with Terriglobus roseus DSM 18391(T) was 97.1 %. A low DNA-DNA hybridization value (<20 %) and rpoB gene sequence similarity (83.6 %) with T. roseus DSM 18391(T) indicated that the tundra soil isolates represent novel members of the genus Terriglobus. Strains SP1PR4(T) and SP1PR5 grew at pH 4.5-7.5 and 4-30 °C. Sugars were the preferred growth substrates. The major cellular fatty acids were iso-C(15 : 0), C(16 : 1)ω7c, iso-C(13 : 0) and C(16 : 0). The DNA G+C content of strain SP1PR4(T) was 57.3 mol%. Based on phylogenetic, chemotaxonomic and physiological analyses, the name Terriglobus saanensis sp. nov. is proposed to accommodate the two strains; the type strain is SP1PR4(T) ( = DSM 23119(T)  = ATCC BAA-1853(T)).

Mucilaginibacter frigoritolerans sp. nov., Mucilaginibacter lappiensis sp. nov. and Mucilaginibacter mallensis sp. nov., isolated from soil and lichen samples.

Five cold-adapted bacteria belonging to the genus Mucilaginibacter were isolated from lichen and soil samples collected from Finnish Lapland and investigated in detail by phenotypic and phylogenetic analyses. Based on 16S rRNA gene phylogeny, the novel strains represent three new branches within the genus Mucilaginibacter. The strains were aerobic, chemo-organotrophic, non-motile rods and formed pigmented, smooth, mucoid colonies on solid media. The strains grew between 0 and 33 °C (optimum growth at 25 °C) and at pH 4.5-8.0 (optimum growth at pH 6.0). The main cellular fatty acids were iso-C(15 : 0), summed feature 3 (C(16 : 1)ω7c/iso-C(15 : 0) 2-OH) and iso-C(17 : 0) 3-OH and the major respiratory quinone was MK-7. The DNA G+C contents were 44.0-46.5 mol%. Based on phylogenetic, phenotypic and chemotaxonomic data, the strains represent three novel species of the genus Mucilaginibacter for which the names Mucilaginibacter frigoritolerans sp. nov. (type strain FT22(T) =ATCC BAA-1854(T) =LMG 25359(T)), Mucilaginibacter lappiensis sp. nov. (type strain ANJLI2(T) =ATCC BAA-1855(T) =LMG 25358(T)) and Mucilaginibacter mallensis sp. nov. (type strain MP1X4(T) =ATCC BAA-1856(T) =LMG 25360(T)) are proposed.

Effect of freeze-thaw cycles on bacterial communities of arctic tundra soil.

The effect of freeze-thaw (FT) cycles on Arctic tundra soil bacterial community was studied in laboratory microcosms. FT-induced changes to the bacterial community were followed over a 60-day period by terminal restriction fragment length polymorphism (T-RFLP) profiles of amplified 16S rRNA genes and reverse transcribed 16S rRNA. The main phylotypes of the active, RNA-derived bacterial community were identified using clone analysis. Non-metric multidimensional scaling ordination of the T-RFLP profiles indicated some shifts in the bacterial communities after three to five FT cycles at -2, -5, and -10 degrees C as analyzed both from the DNA and rRNA. The dominating T-RFLP peaks remained the same, however, and only slight variation was generally detected in the relative abundance of the main T-RF sizes of either DNA or rRNA. T-RFLP analysis coupled to clone analysis of reverse transcribed 16S rRNA indicated that the initial soil was dominated by members of Bacteroidetes, Acidobacteria, Alpha-, Beta-, and Gammaproteobacteria. The most notable change in the rRNA-derived bacterial community was a decrease in the relative abundance of a Betaproteobacteria-related phylotype after the FT cycles. This phylotype decreased, however, also in the control soil incubated at constant +5 degrees C suggesting that the decrease was not directly related to FT sensitivity. The results indicate that FT caused only minor changes in the bacterial community structure.

Bacterial communities in Arctic fjelds of Finnish Lapland are stable but highly pH-dependent.

The seasonal and spatial variations of microbial communities in Arctic fjelds of Finnish Lapland were studied. Phospholipid fatty acid analysis (PLFA) and terminal restriction fragment analysis (T-RFLP) of amplified 16S rRNA genes were used to assess the effect of soil conditions and vegetation on microbial community structures along different altitudes of two fjelds, Saana and Jehkas. Terminal restriction fragments were additionally analysed from c. 160 cloned sequences and isolated bacterial strains and matched with those of soil DNA samples. T-RFLP and PLFA analyses indicated relatively similar microbial communities at various altitudes and under different vegetation of the two fjelds. However, soil pH had a major influence on microbial community composition. Members of the phylum Acidobacteria dominated especially in the low pH soils (pH 4.6-5.2), but above pH 5.5, the relative amount of terminal restriction fragments corresponding to acidobacterial clones was substantially lower. Both T-RFLP and PLFA analysis indicated stable microbial communities as the DNA and fatty acid profiles were similar in spring and late summer samples sampled over 3 years. These results indicate that differences in microbial community composition could be explained primarily by variation in the bedrock materials that cause variation in the soil pH.

Characterization of psychrotolerant heterotrophic bacteria from Finnish Lapland.

A total of 331 aerobic heterotrophic bacterial strains were isolated from various ecosystems of Finnish Lapland (68-69 degrees N) including forest soil, arctic alpine-tundra soil, stream water, lake and mire sediments, lichen and snow algae. Whole cell fatty acid and 16S rRNA gene sequence analysis and microscopy indicated that the isolates were dominated by Gram-negative bacteria, while only 20 Gram-positive strains were isolated. Based on 16S rRNA gene sequences the isolates were members of alpha-, beta-, gamma-Proteobacteria, Gram-positives with low G+C content, Actinobacteria and the Cytophaga/Flexibacter/Bacteroides group. More than one-third of the isolates could be tentatively identified as Pseudomonas spp. which were particularly abundant in the alpine-tundra soils where they represented 60% of all isolates. Other frequently isolated Gram-negative taxa were Burkholderia sp., Collimonas sp., Pedobacter sp., Janthinobacter sp., Duganella sp., Dyella sp. and Sphingomonas sp. Growth temperature ranges and hydrolytic enzyme activities of selected ca.100 strains were screened. The strains were psychrotolerant growing generally at temperatures ranging from 0 to 30 degrees C, as 82% of the isolates grew at 0 degrees C while only 7% grew at 35 degrees C. Protease and lipase activities at 5 degrees C were detected in more than half of the strains while approximately 20% of the strains possessed amylase and/or cellulase activities.

Novosphingobium lentum sp. nov., a psychrotolerant bacterium from a polychlorophenol bioremediation process.

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).

Isolation and characterization of Novosphingobium sp. strain MT1, a dominant polychlorophenol-degrading strain in a groundwater bioremediation system.

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.