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Int J Syst Evol Microbiol 57 (2007), 2267-2271; DOI  10.1099/ijs.0.65124-0
© 2007 International Union of Microbiological Societies

Paenibacillus humicus sp. nov., isolated from poultry litter compost

Ivone Vaz-Moreira1, Cátia Faria1, M. Fernanda Nobre2, Peter Schumann3, Olga C. Nunes4 and Célia M. Manaia1

1 Escola Superior de Biotecnologia, Universidade Católica Portuguesa, 4200-072 Porto, Portugal
2 Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7b, D-38124 Braunschweig, Germany
4 LEPAE – Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto, 4200-465 Porto, Portugal

Correspondence
Célia M. Manaia
cmmanaia{at}esb.ucp.pt


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Two bacterial strains, PC-142 and PC-147T, isolated from poultry litter compost, were characterized with respect to their phenetic and phylogenetic characteristics. The isolates were endospore-forming rods that were reddish in colour after Gram staining. They were catalase- and oxidase-positive, were able to degrade starch and gelatin and grew at 15–40 °C and pH 5.5–10.0. The predominant fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0, the major respiratory quinone was menaquinone MK-7, the cell-wall peptidoglycan was of the A1{gamma} type and the G+C content of the DNA was 58 mol%. The 16S rRNA gene sequence analysis and phenetic characterization indicated that these organisms belong to the genus Paenibacillus, with Paenibacillus pasadenensis SAFN-007T as the closest phylogenetic neighbour (97.5 %). Strains PC-142, PC-147T and P. pasadenensis SAFN-007T represent a novel lineage within the genus Paenibacillus, characterized by a high DNA G+C content (58–63 mol%). The low levels of 16S rRNA gene sequence similarity with respect to other taxa with validly published names and the identification of distinctive phenetic features in the two isolates indicate that strains PC-142 and PC-147T represent a novel species of the genus Paenibacillus, for which the name Paenibacillus humicus sp. nov. is proposed. The type strain is PC-147T (=DSM 18784T =NBRC 102415T =LMG 23886T).

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains PC-142 and PC-147T are AM411529 and AM411528, respectively.

RAPD patterns and an extended phylogenetic tree for strains PC-142 and PC-147T and related taxa are available as supplementary figures with the online version of this paper.


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In a study designed to characterize culturable heterotrophic bacteria in composts, endospore-forming rods affiliated to the genus Paenibacillus were isolated from poultry litter compost. This compost was produced from chicken wastes from aviaries, meat meal, bird bones, vegetable ash and grape husks. The material was stored for 5–6 months until it had composted. The composting process was conducted in piles, under a covered area, for a period of 45 days. Two isolates from the compost had a randomly amplified polymorphic DNA (RAPD) profile that was distinct from those of other bacterial isolates (identified as members of the genera Staphylococcus, Bacillus and Pseudomonas; data not shown) recovered from the same compost. On the basis of 16S rRNA gene sequence analysis, the two isolates, designated strains PC-142 and PC-147T, were presumed to be members of the genus Paenibacillus and were further characterized by using a polyphasic approach.

The bacterial isolates were recovered on plate count agar (PCA) at 30 °C, purified by subculturing on the same medium and then cryopreserved at –80 °C in a nutritive broth supplemented with 15 % (v/v) glycerol. PCA was used for culture maintenance, and biochemical and physiological tests were performed in modified Luria–Bertani medium (Tiago et al., 2004Go) incubated at 30 °C. Colony and cell morphology, Gram-staining, cytochrome c oxidase and catalase activities, motility and flagellation were assessed according to the methodologies of Murray et al. (1994)Go and Smibert & Krieg (1994)Go. The pH range for growth was examined in culture medium containing 12 mM MES (Sigma), to adjust the pH to 5.5, and 12 mM CAPS (Sigma), to adjust the pH to 9.0 and 10.0. The NaCl tolerance and temperature ranges for growth were assayed, respectively, in culture medium supplemented with 1, 3 and 5 % NaCl (w/v) or at 15, 36, 40 and 45 °C. The ability of the strains to grow in the presence of 0.001 % (w/v) lysozyme was tested in tryptic casein soy broth medium. Anaerobic growth was tested under a N2-saturated atmosphere in the presence of 0.1 % KNO3 (w/v). The same medium, under aerobic conditions, was used to assay nitrate reduction. The production of extracellular amylases, gelatinases and tweenases (Tween 80) was tested as described previously (Tiago et al., 2004Go). Other biochemical tests were performed using API 20E and API 20NE galleries, and the nutritional pattern was determined using API 50 CH and API 20NE kits, according to the instructions of the manufacturer (bioMérieux) at 30 °C. The API 50 CH carbon-source utilization kit was used both with the medium recommended for testing acid production (50 CHB/E; bioMérieux) and with mineral medium B (Barreiros et al., 2003Go) supplemented with 4 mM (NH4)2SO4, vitamins (p-aminobenzoic acid, folic acid, nicotinic acid, pantothenic acid, biotin, cyanocobalamin, inositol, riboflavin, pyridoxine, thiamine) (40 µg l–1) and nitrogenated bases (adenine, cytosine, inosine, thymine and uracil) and amino acids (methionine, phenylalanine, tryptophan, arginine, histidine, alanine, glycine, proline and tyrosine) (5 mg l–1).

The DNA G+C content, respiratory quinones and fatty acid methyl ester composition were determined as described previously (Vaz-Moreira et al., 2007Go) using the methods of Mesbah et al. (1989)Go, Tindall (1989)Go and Kuykendall et al. (1988)Go, respectively. The diaminopimelic acid isomer was determined by one-dimensional TLC on cellulose plates (Merck) by using the solvent system of Rhuland et al. (1955)Go.

The sequence of the 16S rRNA gene was determined after PCR amplification of total DNA extracts as described previously (Rainey et al., 1996Go). The 16S rRNA gene sequence was compared with others available in the GenBank/EMBL/DDBJ database by using BLASTN (NCBI) and FASTA (EMBL-EBI) and was aligned with reference sequences. Phylogenetic analysis was conducted using MEGA, version 3.1 (Kumar et al., 2004Go). Levels of sequence relatedness were estimated using the model of Jukes & Cantor (1969)Go, and the phylogenetic tree was created using the neighbour-joining method. Other methods (minimum evolution and maximum parsimony) were used to assess the tree stability. A total of 1322 nucleotide positions in each 16S rRNA gene were included in the analysis. Non-homologous and ambiguous nucleotide positions were excluded from the calculations.

After 24 h incubation at 30 °C on PCA, isolates PC-142 and PC-147T formed flat, very smooth, translucent colonies that tended to produce a swarming effect. These strains were unable to produce growth on Bacillus cereus medium (Pronadisa) agar after 7 days incubation. They were able to produce acid from, and assimilate, several sugars (see Table 1Go and the species description). The major fatty acid methyl esters were anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0, which accounted for more than 60 % of the total. Other minor components (representing more than 4 %) were iso-C17 : 0, anteiso-C17 : 0 and C16 : 0. The DNA G+C content for PC-147T and PC-142 was, respectively, 58.3±0.3 and 58.1±0.2 mol%. Menaquinone MK-7 was the major respiratory quinone in both strains. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid, suggesting the presence of peptidoglycan type A1{gamma} (Schleifer & Kandler, 1972Go). Strains PC-142 and PC-147T differed only in the ability to produce acid from three carbon sources (D-lactose, D-melibiose and D-turanose); they had similar fatty acid methyl ester compositions and DNA G+C contents and their 16S rRNA gene sequences were identical. These findings were confirmed by the similarity of the RAPD patterns obtained for isolates PC-142 and PC-147T (see Supplementary Fig. S1, available in IJSEM Online).


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  		Table 1. Distinctive characteristics of strains PC-142 and PC-147T and P. pasadenensis NBRC 101214T

Strains PC-142, PC-147T and P. pasadenensis NBRC 101214T tested positive for beta-galactosidase, for the hydrolysis of gelatin and aesculin and for the assimilation of the following carbon sources: D-glucose, D-mannitol, N-acetylglucosamine, maltose, potassium gluconate, D-xylose, methyl beta-D-xylopyranoside, D-galactose, D-fructose, methyl {alpha}-D-glucopyranoside, amygdalin, arbutin, salicin, D-cellobiose, D-lactose, D-melibiose, sucrose, trehalose, D-melezitose, D-raffinose, starch, glycogen, gentiobiose and D-turanose. All of the strains tested negative for the following: Gram staining, nitrate reduction, arginine dihydrolase, urease, indole production and glucose fermentation. All were unable to assimilate the following carbon sources: caprate, adipate, malate, citrate, phenylacetate, erythritol, D-arabinose, D-ribose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, methyl {alpha}-D-mannopyranoside, inulin, xylitol, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconate and 5-ketogluconate. Strains PC-142 and PC-147T were negative for anaerobic growth, lysine and ornithine decarboxylase and tryptophan deaminase activities, citrate utilization and H2S production and for growth at 45 °C, with 5 % NaCl and on Bacillus cereus medium (P. pasadenensis NBRC 101214T not tested). PC-142 and PC-147T were unable to use the following carbon sources: (fermentation/oxidation) D-mannitol, inositol, D-sorbitol, L-rhamnose, sucrose, D-melibiose, amygdalin and L-arabinose; (acid production) D-glucose, D-mannitol, inositol, D-sorbitol, L-rhamnose, L-arabinose, D-arabinose, glycerol, erythritol, D-ribose, D-xylose, L-xylose, D-adonitol, methyl beta-D-xylopyranoside, D-galactose, D-mannose, L-sorbose, dulcitol, methyl {alpha}-D-mannopyranoside, methyl {alpha}-D-glucopyranoside, N-acetylglucosamine, arbutin, inulin, D-melezitose, D-raffinose, starch, glycogen, xylitol, gentiobiose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, 2-ketogluconate and 5-ketogluconate (data not shown for P. pasadenensis NBRC 101214T). Strain PC-142 showed weak acid production for D-lactose, D-melibiose and D-turanose; strain PC-147T was unable to produce acids from these carbon sources (data not shown for P. pasadenensis NBRC 101214T).

 
The diagnostic diamino acid of the peptidoglycan, the menaquinone and the fatty acid methyl ester composition of isolates PC-142 and PC-147T confirm the results of the 16S rRNA gene sequence comparative analysis, identifying these organisms as members of the genus Paenibacillus. On the basis of the 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of isolates PC-142 and PC-147T are Paenibacillus pasadenensis SAFN-007T (97.5 %), Paenibacillus kobensis DSM 10249T (94.3 %) and Paenibacillus mendelli C/2T (93.8 %) (Fig. 1Go; an extended version of this tree is available as Supplementary Fig. S2). In the light of these results, the nutritional and chemotaxonomic properties of strains PC-142 and PC-147T were compared with those of P. pasadenensis NBRC 101214T (Osman et al., 2006Go) (Tables 1Go and 2Go). The fatty acid methyl ester patterns obtained for PC-142, PC-147T and P. pasadenensis NBRC 101214T confirm the taxonomic relatedness of these organisms and their affiliation to the same genus (Table 2Go). However, the DNA G+C contents of these three organisms (58.3±0.3 mol% for PC-147T, 58.1±0.2 mol% for PC-142 and 63.4±0.6 mol% for P. pasadenensis NBRC 101214T) demonstrate the taxonomic distinctiveness of the two isolates. Amongst the highest DNA G+C contents reported for this genus were those obtained for Paenibacillus validus (54–55 mol%; buoyant density method) (Heyndrickx et al., 1995Go) and Paenibacillus panacisoli (Ten et al., 2006Go). Strains PC-142 and PC-147T and P. pasadenensis NBRC 101214T form a high-G+C lineage within the genus Paenibacillus.


Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the nearest neighbours of strain PC-142 and PC-147T. Bootstrap percentages were generated from 1000 resamplings; only values greater than 50 % are shown. Bar, 1 substitution per 200 nucleotide positions. An extended version of this tree is available as Supplementary Fig. S2 in IJSEM Online.

 

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  		Table 2. Fatty acid methyl ester compositions of strains PC-142 and PC-147T and P. pasadenensis NBRC 101214T

 
As strains PC-142 and PC-147T show less than 98.5 % 16S rRNA gene sequence similarity with respect to their closest phylogenetic neighbour, P. pasadenensis, DNA–DNA hybridizations to demonstrate their separate species status were considered unnecessary in view of the results of recent investigations (Stackebrandt & Ebers, 2006Go). Characteristics that serve to distinguish PC-142 and PC-147T from their closest phylogenetic neighbour, P. pasadenensis NBRC 101214T, are presented in Table 1Go. On the basis of the data presented, strains PC-142 and PC-147T represent a novel species of the genus Paenibacillus, for which the name Paenibacillus humicus sp. nov. is proposed.

Description of Paenibacillus humicus sp. nov.
Paenibacillus humicus (hu'mi.cus, L. n. humus earth, soil and, in earth sciences or agriculture, humus; L. suff. -icus -a -um suffix used with the sense of belonging to; N.L. masc. adj. humicus pertaining to humus).

Cells are motile rods (2.0 µm long and 0.5 µm wide) with peritrichous flagella. Gram staining is negative. Terminal, ellipsoidal endospores are produced in swollen sporangia. On nutritive medium, such as PCA or Luria–Bertani agar, colonies are white/translucent, very smooth and flat. Swarming is observed. Catalase- and oxidase-positive. Growth occurs in the presence of 3 % NaCl and at 15–40 °C and pH 5.5–10. No growth occurs under anaerobic conditions, at 45 °C or in the presence of 5 % NaCl. Nitrate is not reduced. Tests positively for beta-galactosidase, Voges–Proskauer (API 20NE) and growth in the presence of 0.001 % lysozyme. Starch, gelatin, Tween 80 and aesculin are hydrolysed. The following carbon sources are assimilated: starch, amygdalin, arbutin, D-cellobiose, D-fructose, D-galactose, gentiobiose, D-glucose, glycogen, D-lactose, maltose, D-mannitol, D-melezitose, D-melibiose, methyl {alpha}-D-glucopyranoside, methyl beta-D-xylopyranoside, N-acetylglucosamine, potassium gluconate, D-raffinose, salicin, sucrose, trehalose, D-turanose and D-xylose. Acid is produced from amygdalin, D-cellobiose, maltose, sucrose and trehalose. The peptidoglycan cell wall is of the A1{gamma} type. The major cellular fatty acid components are anteiso-C15 : 0 (approx. 40 %), iso-C15 : 0 (17–21 %) and iso-C16 : 0 (11–13 %). The major respiratory quinone is MK-7 and the DNA G+C content is 58 mol%.

The type strain, PC-147T (=DSM 18784T =NBRC 102415T =LMG 23886T), was isolated from final compost produced from poultry litter.


   ACKNOWLEDGEMENTS
 
The authors acknowledge Luisa Barreiros (Faculdade de Engenharia da Universidade do Porto) for her helpful collaboration, Elisabete Silva (Escola Superior de Tecnologia de Viseu, Instituto Politécnico de Viseu) for providing the poultry litter compost and Milton S. da Costa (Departamento de Bioquímica da Universidade de Coimbra) for help with the fatty acid methyl ester analyses.


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