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1 Department of Ecological Microbiology, University of Bayreuth, 95440 Bayreuth, Germany
2 Electron Microscopy Laboratory, University of Bayreuth, 95440 Bayreuth, Germany
Correspondence
Harold L. Drake
hld{at}uni-bayreuth.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Dechloromonas denitrificans ED1T, Flavobacterium denitrificans ED5T and Paenibacillus terrae MH72 are AJ318917, AJ318907 and AJ318910, respectively.
A table of the substrate utilization profiles of earthworm-gut isolates and transmission electron micrographs of strains ED5T and MH72 are available as supplementary material in IJSEM Online.
Present address: Labor L+S AG, Mangelsfeld 4, 97708 Bad Bocklet, Germany. 
Present address: Department of Microbiology, University of Aarhus, 8000 Aarhus, Denmark.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Matthies et al., 1999; Borken et al., 2000). The annual global potential of earthworms to produce N2O has been estimated at 3x108 kg N2O (Drake et al., 2005). The earthworm gut is where N2O originates in the earthworm (Ihssen et al., 2003; Horn et al., 2003), and the emission of this gas is possibly due to the activation of ingested soil micro-organisms in the special microenvironment of the earthworm gut (Horn et al., 2003). The emission of N2O by earthworms is stimulated by nitrate and nitrite, and denitrifying bacteria appear to be involved in this emission (Karsten & Drake, 1997; Matthies et al., 1999). The dissimilative reduction of nitrate to nitrite or ammonium can also result in a significant production of N2O (Anderson & Levine, 1986). Organisms isolated from earthworm casts have a higher probability of reducing nitrate than do soil isolates (Furlong et al., 2002), thus verifying the presence of nitrate-reducers in gut contents of the earthworm and also supporting the probability that soil nitrate-reducers capable of producing N2O are activated during gut passage (Horn et al., 2003).
Twenty five N2O-producing isolates were recently obtained from dilution series of earthworm-gut homogenates (Ihssen et al., 2003). At the time of their isolation, four of these isolates, ED1T, ED5T, MH21T and MH72, shared 
97 % 16S rRNA gene sequence similarity with their closest cultured and characterized relatives, and presumably represented novel species in the genera Dechloromonas (ED1T), Flavobacterium (ED5T) and Paenibacillus (MH21T and MH72) (Ihssen et al., 2003). These N2O-producing isolates were characterized and assessed for their ability to produce N2O under conditions simulating that of the earthworm gut.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Gas phases were 100 % argon for anoxic media and air for oxic media; the pH was adjusted to 7·0. Solidified media contained 15 g agar per litre. The following media were utilized (unless otherwise indicated, amounts are grams per litre). Medium A: tryptic soy broth (TSB) without dextrose (Difco), 2·75. Medium B: Medium A plus 5 mM KNO3 (Karsten & Drake, 1997). Medium C: Medium B plus 50 mM sodium phosphate buffer, pH 7·0. Medium D: tryptone, 2; yeast extract, 2; KNO2, 1 mM; N-acetylglucosamine, 2 mM. Medium E: KH2PO4, 2·68; K2HPO4.3H2O, 0·073; MgCl2.6H2O, 0·05; NaCl, 0·4; NH4Cl, 0·125; CaCl2.2H2O, 0·01; trace element solution (Karsten & Drake, 1995), 5 ml; B-vitamin solution (Karsten & Drake, 1995), 5 ml. Medium F: TSB, 27·5; glucose, 10 mM. Medium G: NB, nutrient broth (Difco), 0·8; KNO2, 1 mM; N-acetylglucosamine, 2 mM. Nitrite, glucose, N-acetylglucosamine and other substrates were added from sterile, anoxic stock solutions after the media were cooled to 55 °C. Media C, A, F and E were used for determining the substrate range of ED1T, ED5T, MH21T and MH72, respectively. The concentrations used for determining the substrate range of ED1T under denitrifying conditions were: sugars, 2 mM; organic acids, 5–20 mM; alcohols, 10–20 mM; aromatic compounds, 5 mM. The concentrations used for determining the substrate range of ED5T, MH21T and MH72 under oxic conditions were 5 mM. Cultures were incubated at 15 °C in the dark.
Electron microscopy.
Strains were grown under oxic conditions on liquid or solidified Medium F. For negative staining, cells were harvested from liquid media by centrifugation (2000 g, 10 min), suspended in distilled water, and adsorbed to carbon film (Valentine et al., 1968). Cells were stained with an aqueous uranyl acetate solution (2 %, w/v, pH 4·8). For thin section preparations, cells were fixed in glutaraldehyde/OsO4 (Traub et al., 1976; Küsel et al., 2000). The fixed specimens were treated with tannic acid (1 %, w/v, 0·1 M cacodylate buffer, pH 7·2) (Bayer & Easterbrook, 1991). Specimens were embedded in Spurr's resin after dehydration in ethyl alcohol (Spurr, 1969; Traub et al., 1976).
Membrane preparation and redox difference spectra.
Cell-free extracts, cytoplasmic fractions and membranes were prepared from cells grown under oxic conditions or anoxic conditions with nitrate (10 mM) as terminal electron acceptor. Oxic preparations (Fröstl et al., 1996) of cellular fractions were reduced with sodium dithionite, and reduced-minus-oxidized spectra were measured at room temperature with an Uvikon 930 (Kontron Instruments) double-beam spectrophotometer (Matthies et al., 2001).
DNA G+C content.
Cells were disrupted by French press. The DNA was purified on hydroxyapatite (Cashion et al., 1977). The hydrolysis of DNA with P1 nuclease, the HPLC analysis of hydrolysate, and the calculation of DNA G+C content were done according to established protocols (Tamaoka & Komagata, 1984; Mesbah et al., 1989). Analysis was obtained commercially at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany).
Phylogenetic analysis.
Almost full-length 16S rRNA gene fragments were pre-amplified and sequenced as described previously (Ihssen et al., 2003). Public databases (GenBank, EMBL) were searched for closely related sequences using the program BLAST (Altschul et al., 1997). Alignments of sequences, distance matrix calculations and construction of phylogenetic trees were performed using the program package ARB (Department of Microbiology, Technical University Munich, Germany; http://www.arb-home.de). For calculation of phylogenetic trees, neighbour-joining, parsimony and maximum-likelihood algorithms were applied to 16S rRNA gene sequences; all sequences were more than 1400 nt long. A consensus tree was constructed when consistent branching was obtained with all three methods (Ludwig et al., 1998).
N2O production by isolates in sterile soil microcosms under gut-simulated conditions.
Field fresh, autoclaved soil was utilized in soil microcosms. Soil samples (31 g fresh weight) were weighed into 125 ml infusion flasks (Merck ABS); the water content of the soil was adjusted to 55 % with double-distilled water. The flasks were sealed with a screw-cap and butyl-rubber stopper, flushed with 100 % argon, and autoclaved. The physico-chemical environment of the earthworm gut was simulated in soil microcosms as described previously (Horn et al., 2003). Supplements in these microcosms were: NaCl, 130 mM; sodium phosphate buffer, pH 6·8, 10 mM; NH4Cl, 10 mM; NaNO3, 3 mM; NaNO2, 1 mM; glucose, 10 mM; tryptone, 0·2 g l–1; and soytone, 0·2 g l–1. ED1T-containing microcosms also contained 2 mM acetate. Microcosms of the denitrifiers ED1T and ED5T were inoculated with 3x106 cells (g dry weight soil)–1; microcosms of the fermenter MH21T and the fermenter/nitrate-dissimilator MH72 were inoculated with 3x107 cells (g dry weight soil)–1. These cell numbers were based on most-probable-number counts of denitrifiers and fermenters/nitrate-dissimilators [6x106 and 1x107 (g dry weight gut section)–1, respectively] obtained from the gut of earthworms (Ihssen et al., 2003). Rates reported for the production of N2O by the isolates were corrected by values obtained with uninoculated control microcosms [which approximated 17 pmol N2O h–1 (g dry weight soil)–1]. Unless otherwise indicated, microcosms were performed in triplicates.
Analytical methods.
N2 and high concentrations of N2O were analysed with a Hewlett Packard model 5890 series II gas chromatograph equipped with a thermal conductivity detector and either a Molecular Sieve column (Alltech) for N2 or a Chromosorb 102 column (Alltech) for N2O (Karsten & Drake, 1995, 1997). Low concentrations of N2O (<300 p.p.m.) were analysed with a Hewlett Packard gas chromatograph equipped with an electron capture detector and a Porapak Q-80/100 column (Supelco) (Karsten & Drake, 1997). Soluble organic compounds were determined by HPLC (Karsten & Drake, 1995); the detection limit for sugars and organic acids was approximately 0·1 mM. Nitrate, nitrite and ammonium were determined colorimetrically (Harrigan & Mc Cance, 1966; Cataldo et al., 1975; Gadkari, 1984) with a UVIKON 930 spectrophotometer (Kontron Instruments). Established methods were utilized to determine the classical properties (e.g. enzymological and Gram reactions) of the isolates (Cowan, 1974; Bergey et al., 1990; Smibert & Krieg, 1994). Growth was measured as optical density at 660 nm (OD660). Uninoculated medium served as reference. Values are means of duplicate or triplicate analyses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). ED1T and ED5T were derived from single colonies obtained by plating 0·1 ml of 10–6 and 10–4 dilutions of a gut homogenate, respectively, onto solidified anoxic Medium B with 2 mM glucose. For the isolation of MH21T and MH72, enrichment cultures that were obtained by inoculating liquid anoxic Medium G and D, respectively, with 10–6 and 10–5 dilutions of gut homogenates, respectively, were streaked onto solidified anoxic Medium D. Isolated colonies were screened for the production of N2O in liquid media and restreaked three times on solidified media to guarantee purity.
Morphological and cytological characteristics
See description of species (below) for information on ED1T, ED5T and MH21T. Cells of ED1T had polar flagella (Fig. 1a), formed multicellular aggregates and sometimes displayed connecting filaments (Fig. 1b) (Kuhner et al., 2000; Matthies et al., 2001; Küsel et al., 2001); outer and cytoplasmic membranes were observed (Fig. 1c). ED5T formed chains consisting of 3–14 cells that were sometimes tethered to one another by connecting filaments (Supplementary Figure, in IJSEM Online). Cells of MH21T were also sometimes tethered by connecting filaments (Fig. 1d), had a three-layered cell wall (Fig. 1e) and formed terminal spores (Fig. 1f).
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Phylogenetic analysis
The 16S rRNA gene sequence (1487 nt) of strain ED1T was phylogenetically closely related to the 16S rRNA gene sequences of Ferribacterium limneticum, ‘Dechloromonas aromatica’ and Dechloromonas agitata (97 % sequence similarities to that of ED1T) (Fig. 2a). ED1T had a 99 % 16S rRNA gene sequence similarity to that of its closest phylogenetic relative, Dechloromonas sp. SIUL, which is shown in Fig. 2(a) as a representative for several other unclassified strains of Dechloromonas (Coates et al., 1999).
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). However, different treeing methods yielded inconsistent branchings, indicating that the phylogeny of the genus Flavobacterium is uncertain.
The 16S rRNA gene sequence of MH21T (1512 bp) was phylogenetically most closely related to the 16S rRNA gene sequences of Paenibacillus borealis and Paenibacillus chibensis (95–96 % sequence similarity to that of MH21T) and clustered with the 16S rRNA gene sequences of other type strains of the genus Paenibacillus (Fig. 2c).
The 16S rRNA gene sequence (1461 nt) of MH72 was phylogenetically most closely related to the 16S rRNA gene sequences found in the Paenibacillus cluster (Fig. 2c). MH72 had a 99 % 16S rRNA gene sequence similarity to that of its closest phylogenetic relative, Paenibacillus terrae (Yoon et al., 2003).
DNA G+C content
Strains ED1T, ED5T, MH21T and MH72 had a DNA G+C content of 61·2, 34·6, 42·6 and 46·0 mol%, respectively.
Effects of temperature and pH on growth
See description of species (below) for information on ED1T, ED5T and MH21T. MH72 grew optimally at 35 °C; growth occurred at 5–35 °C. Growth occurred at pH 5·2–8·5 and was optimal at pH 7·3–7·7. MH72 had a doubling time of 2·7 h (under optimal conditions).
Substrate range and fermentation product profiles
Of the compounds tested, only organic acids were growth-supportive for ED1T (Supplementary Table, in IJSEM Online). Vanillate, ferulate, syringate and trimethoxybenzol were not growth-supportive for ED1T. Many sugars and other compounds, including polymers, were growth-supportive for ED5T, MH21T and MH72 (Supplementary Table). Maximum optical densities (OD660) observed for ED1T, ED5T, MH21T and MH72 were approximately 0·4, 0·6, 1·0 and 1·3, respectively.
MH21T and MH72 grew by fermentation when an exogenous terminal electron acceptor was not available. Formate (4·5 mM), acetate (2·0 mM) and ethanol (6·0 mM) were produced when glucose (6·5 mM) was fermented by MH21T (the recovery of glucose-derived carbon and reductant in these products was approximately 50 and 62 %, respectively; the gas phase was not evaluated). Formate (4·7 mM), acetate (2·2 mM), 2,3-butanediol (2·2 mM), ethanol (6·1 mM), H2 (4·5 mM) and CO2 (25 mM) were produced when glucose (9·0 mM) was fermented by MH72 (the recovery of glucose-derived carbon and reductant was approximately 100 and 75 %, respectively).
Alternative electron acceptors and the production of N2O
ED1T, ED5T and MH72 grew facultatively. ED1T and ED5T reduced nitrate to N2, during which N2O was produced as a transient intermediate (Fig. 3a, b). Stationary denitrifying cultures of ED1T initially produced N2O upon transfer into fresh medium, while exponential cultures initially produced mainly N2. A fragment of the nitrous oxide reductase gene nosZ has been sequenced from both ED1T and ED5T (M. A. Horn, A. Schramm & H. L. Drake, unpublished data), corroborating the ability of these organisms to reduce N2O to N2. ED1T also utilized fumarate, sulfate, chlorate or perchlorate as electron acceptor. ED5T did not utilize sulfate or Fe3+ as electron acceptor. MH72 reduced nitrate to nitrite and produced N2O as a side-product under anoxic conditions (Fig. 3c). Sulfate or Fe3+ was not dissimilated by MH72. MH21T reduced Fe3+ to Fe2+, and the recovery of electrons from glucose in Fe2+ was 0·5–1 %. Under these conditions, MH21T produced formate and acetate, but almost no ethanol. MH21T reduced nitrite (1 mM) to N2O; nitrite concentrations above 2 mM inhibited growth. Sulfate or nitrate was not dissimilated by MH21T.
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Cytochromes
Redox spectra of membranes from denitrifying and aerobic cultures of ED1T and ED5T were similar. Membranous fractions and cell-free extracts of ED1T and ED5T contained c-type cytochromes with absorption maxima at approximately 425, 523, 552 nm and 425, 525, 554 nm, respectively. Spreading (i.e. the width) of the 
-peak at 425 nm and the shoulders of the 
- and 
-peaks suggest that membranous b-type cytochromes were also present in ED1T and ED5T.
Membranes from aerobic cultures of MH72 contained a- and b-type cytochromes (Fig. 4a); in contrast, cell-free extracts of MH72 had absorption maxima characteristic of b-type cytochromes (data not shown). Membranes and cell-free extracts of nitrate-dissimilating cells of MH72 only contained b-type cytochromes (Fig. 4b and data not shown). Cell-free extracts and membranes from aerobic cultures of MH21T contained b-type cytochromes with absorption maxima at 426, 534 and 560 nm.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-Proteobacteria. The closest phylogenetic relatives of ED1T with validly published names were Ferribacterium limneticum and D. agitata, both of which have a 97 % 16S rRNA gene sequence similarity to that of ED1T. In contrast to ED1T, Ferribacterium limneticum is an obligate anaerobe that reduces Fe3+ (Cummings et al., 1999). D. agitata is a facultative aerobe that contains c-type cytochrome(s), reduces chlorate and perchlorate, but is not able to grow with Casamino acids or by denitrification (Achenbach et al., 2001). The collective properties of ED1T indicate that it represents a novel species of the genus Dechloromonas, for which the name Dechloromonas denitrificans sp. nov. is proposed.
ED5T is a facultative aerobe that grew by denitrification in mineral medium. The closest phylogenetic relatives of ED5T, Flavobacterium flevense and Flavobacterium johnsoniae (Bernardet et al., 1996), have 16S rRNA sequence similarities of less than 96 % to that of ED5T. In contrast to ED5T, the type strain of Flavobacterium johnsoniae is a strict aerobe that has oxidase activity (Reichenbach, 1989; Bernardet et al., 1996). Flavobacterium flevense is negative for the flexirubin reaction and cannot grow on gelatin (Van der Meulen et al., 1974; Bernardet et al., 1996). There are conflicting reports on the abilities of Flavobacterium flevense and Flavobacterium johnsoniae to reduce nitrate (Van der Meulen et al., 1974; Reichenbach, 1989; Bernardet et al., 1996); the reduction of nitrate to N2 or N2O has not been reported for these organisms. ED5T yielded a positive flexirubin reaction, which is typical for non-marine species of Flavobacterium (Reichenbach, 1989; Bernardet et al., 1996). The collective properties of ED5T indicate that it represents a novel species of the genus Flavobacterium, for which the name Flavobacterium denitrificans sp. nov. is proposed.
MH21T was phylogenetically placed in the genus Paenibacillus in the Firmicutes. The closest phylogenetic relatives of MH21T, P. borealis and P. chibensis, have a less than 97 % 16S rRNA gene sequence similarity to that of MH21T, and have a DNA G+C content that is at least 10 % higher than that of MH21T (Shida et al., 1997; Elo et al., 2001) (Table 1). P. borealis has a temperature optimum that is 7 °C lower than that of MH21T (Elo et al., 2001). In contrast to the anaerobic ability of MH21T, P. chibensis is a strict aerobe (Shida et al., 1997). The production of N2O and the reduction of Fe3+ under anoxic conditions have not been reported for P. borealis and P. chibensis (Shida et al., 1997; Elo et al., 2001). The collective properties of MH21T indicate it represents a novel species of the genus Paenibacillus, for which the name Paenibacillus anaericanus sp. nov. is proposed.
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), is 99 % similar to that of MH72. MH72 produced N2O when nitrate was used as a terminal electron acceptor and contained a- and b-type cytochromes. Information on the production of N2O, fermentation products, cytochromes and cellular ultrastructure are not provided in the species description of P. terrae (Yoon et al., 2003). Nonetheless, given the similarity of the 16S rRNA gene sequences and other properties of MH72 and P. terrae (Table 1), it is proposed that MH72 represents a new strain of P. terrae.
Production of N2O in the gut of the earthworm
Earthworms and earthworm gut content produce N2O (Karsten & Drake, 1997; Matthies et al., 1999). Depending on the isolate, the initial per cell rates at which the earthworm gut isolates produced N2O under gut-simulated conditions were 50–300 % (mean 75 %) of that obtained in TSB-based liquid media (Ihssen et al., 2003), suggesting that (i) the isolates produce N2O under in situ conditions and (ii) the nutritional contents of either the gut or TSB foster similar N2O-producing activities by the isolates.
The denitrifiers ED1T and ED5T produce N2O at cellular rates that are one to two orders of magnitude greater than those of the fermentative MH21T and MH72 (Ihssen et al., 2003), suggesting a predominance of denitrifiers with respect to the production of N2O in the earthworm gut. However, the numbers of cultured fermenters in the earthworm gut per gram dry weight of gut content are approximately ten-fold greater than the number of cultured denitrifiers (Ihssen et al., 2003), suggesting that fermentative micro-organisms could be more important to the in situ production of N2O in the earthworm gut than is suggested by the estimated per cell production of N2O from isolates. Indeed, when microcosms that simulated the gut environment were inoculated with a ten-fold excess of fermentative microbes (i.e. MH21T or MH72) than denitrifiers (i.e. ED1T or ED5T), the microcosms inoculated with the fermentative isolates produced N2O at rates that were only six-fold lower than those of denitrifier-inoculated microcosms. Thus, the direct contribution of fermenters to the production of N2O by earthworms may not be negligible.
A major fermentation product of isolates MH21T and MH72 was acetate. Acetate is a common product of fermenters (e.g. Bulthuis et al., 1991; Schlegel & Jannasch, 1992; Kuhner et al., 2000), and is a substrate for denitrifiers such as ED1T, suggesting that a mutualistic interaction of certain denitrifiers and fermenters might occur in the gut of earthworms. The production of nitrite via the dissimilatory reduction of nitrate is a characteristic trait of many fermenters (Stouthamer, 1988). Nitrite is a precursor for N2O during denitrification (Zumft, 1992) and greatly stimulates the production of N2O by earthworms (Matthies et al., 1999). Thus, the nitrite-dependent production of N2O by denitrifiers in the earthworm gut might also be enhanced by the nitrite-forming activities of certain fermenters.
Description of Dechloromonas denitrificans sp. nov.
Dechloromonas denitrificans (de.ni.tri'fi.cans. L. prep. de away from; L. n. nitrum soda; N.L. n. nitras nitrate; N.L. v. denitrifico to denitrify; N.L. part. adj. denitrificans denitrifying).
Colonies are yellowish and 0·5–1 mm in diameter. Cells are Gram-negative, facultative, short rods, 1·7x0·5 µm, motile with a polar flagellum and sometimes form connecting filaments. Membranes contain c-type cytochromes; b-type cytochromes might also occur. Grows from 5 to 36 °C and pH 6·1 to 8·3, with optimal growth at 30 °C and pH 7. Doubling time under optimal conditions is 6·5 h. O2, 
, 
, 
, 
, 
and fumarate are used as electron acceptors. Fe3+ is not used as an electron acceptor. N2O is produced as an intermediate during the reduction of 
to N2. Utilizes acetate, propionate, butyrate, iso-butyrate, iso-valerate, lactate, pyruvate, succinate, malate, glutamate and Casamino acids as electron donors. Formate, H2, methanol, ethanol, propanol, cellobiose, glucose, fructose, xylose, lactose, galactose, mannose, arabinose, pectin, vanillate, ferulate, syringate and trimethoxybenzol are not growth-supportive. The DNA G+C content is 61·2 mol%. Phylogenetically most closely related to Dechloromonas agitata.
The type strain (MH21T=DSM 15890T=ATCC BAA-844T) was isolated from the gut of the earthworm Aporrectodea caliginosa (collected from garden soil in Bayreuth, Germany).
Description of Flavobacterium denitrificans sp. nov.
Flavobacterium denitrificans (de.ni.tri'fi.cans. L. prep. de away from; L. n. nitrum soda; N.L. n. nitras nitrate; N.L. v. denitrifico to denitrify; N.L. part. adj. denitrificans denitrifying).
Colonies are flat, circular, entire and yellow. Cells are facultative, rods, 0·8–3·0x0·3–0·9 µm, can form chains (3–14 cells), sometimes form connecting filaments, stain Gram-negative, motile and have an outer membrane. Membranes contain c-type cytochromes; b-type cytochromes might also occur. Grows from 10 to 30 °C and pH 5·5 to 8·2, with optimal growth at 25 °C and pH 7. Doubling time under optimal conditions is 7·3 h. Utilizes arabinose, cellobiose, fructose, fumarate, gelatin, glucose, glutamate, inulin, lactose, maltose, mannitol, mannose, N-acetylglucosamine, pectin, starch, succinate and xylose as electron donors. 1-Butanol, 1-propanol, acetate, butyrate, chitin, citrate, ethanol, ethanolamine, glycerol, glycolate, i-butyrate, inositol, i-valerate, lactate, oxalate, propionate, raffinose, saccharose, sorbitol and tartrate are not growth-supportive. Uses ammonium as nitrogen source. Flexirubin reaction-, arginine dihydrolase- and catalase-positive. Oxidase- and lysine decarboxylase-negative. Does not deaminate phenylalanine, hydrolyse urea or form indole. Grows at 2 % but not 5 % NaCl. O2, 
and 
are used as electron acceptors. N2O is produced as an intermediate during the reduction of 
to N2. 
and Fe3+ are not used as electron acceptors. Does not grow by fermentation. The DNA G+C content is 34·6 mol%. Phylogenetically most closely related to Flavobacterium johnsoniae.
The type strain (ED5T=DSM 15936T=ATCC BAA-842T) was isolated from the gut of the earthworm Aporrectodea caliginosa (collected from garden soil in Bayreuth, Germany).
Description of Paenibacillus anaericanus sp. nov.
Paenibacillus anaericanus (an.ae.ri.ca'nus. Gr. pref. an no/not; Gr. n. aer air; Gr. adj. ikanos capable; N.L. masc. adj. anaericanus capable of anaerobic growth).
Colonies are flat, smooth, circular and entire. Cells are facultative, motile rods with flagella, 2·0–5·0x0·5–1·0 µm, grow in chains, are linked by connecting filaments, form terminal to subterminal, ellipsoidal spores, stain Gram-negative and have a three-layered cell wall with no outer membrane. Cells contain b-type cytochromes. Grows at 5–40 °C, with an optimum at 30–35 °C. Grows at pH 5·8–8·6, with an optimum at pH 7·7. Doubling time under optimal conditions is 5 h. Grows aerobically on arabinose, cellobiose, chitin, fructose, glucose, glycerol, lactose, maltose, mannitol, mannose, N-acetylglucosamine, raffinose, saccharose, starch and xylose. 1-Butanol, 1-propanol, acetate, butyrate, citrate, ethanol, ethanolamine, fumarate, gelatin, glutamate, glycolate, i-butyrate, inositol, inulin, i-valerate, lactate, oxalate, pectin, propionate, sorbitol, succinate and tartrate are not growth-supportive. Catalase- and oxidase-positive. Grows at 2 % but not 5 % NaCl. Formate, acetate and ethanol are formed when glucose is fermented. 
and 
are not disssimilated. Low amounts (1 mM) of 
are reduced to N2O. The DNA G+C content is 42·6 %. Phylogenetically most closely related to Paenibacillus borealis.
The type strain (MH21T=DSM 15890T=ATCC BAA-844T) was isolated from the gut of the earthworm Aporrectodea caliginosa (collected from garden soil in Bayreuth, Germany).
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, nitrate; 
, nitrite; 
, N2O; 
, N2; 
, optical density (OD660).