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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
2 Graduate School, Chinese Academy of Sciences, Beijing 100049, PR China
Correspondence
Xiuzhu Dong
dongxz{at}sun.im.ac.cn
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ABSTRACT |
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An electron micrograph of an ultrathin section of strain GWT is available with the online version of this paper.
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). Currently most investigations concerning anaerobic degradation of organic materials have focused on the metabolism of carbohydrates, especially in the fermentative biohydrogenesis process. However, many industrial and agricultural wastes also contain a considerable amount of proteinaceous materials and fat. Although part of this can be digested by some saccharide users, the protein-specific degraders are needed to remove proteinaceous materials efficiently from the wastewater.
In recent years, a few protein-specific anaerobic degraders, such as Clostridium thiosulfatireducens (Hernández-Eugenio et al., 2002), ‘Clostridium tunisiense’ (Thabet et al., 2004) and Proteiniphilum acetatigenes (Chen & Dong, 2005), have been isolated from upflow anaerobic sludge blanket (UASB) reactor sludge. They were all proteolytic, chemo-organotrophic anaerobic bacteria characterized by using only proteinaceous compounds, but not any tested carbohydrates, as a carbon and energy source. During a study on the microbial composition of a fermentative hydrogen-producing reactor, several hydrogen-producing anaerobic bacterial strains were isolated with a rich medium (glucose-peptone-yeast extract), among which was a proteinaceous compound-specific strain. Phylogenetically the strain was affiliated to the phylum of low G+C Gram-positive bacteria but was distantly related to all the existing species. Hence a novel mesophilic, protein-utilizing, hydrogen-producing anaerobe is described in this paper.
A pre-reduced PY medium (Holdeman et al., 1977) was used for isolation and routine cultivation. Granular sludge from a laboratory-scale UASB reactor for treating food industry wastewater and producing hydrogen was used as inoculum. Granules were crushed with a mortar in an anaerobic glove box (Forma scientific 1209) and then inoculated into the pre-reduced PY broth under 100 % N2. After the enrichments were incubated at 37 °C for 24 h, the Hungate roll-tube technique (Hungate, 1969) was performed. Single colonies in the roll-tube were picked and transferred to the same broth and incubated at 37 °C for 2 days. The roll-tube procedure was repeated several times until a pure culture of strain GWT was obtained. The purity of the culture was examined under a light microscope.
Substrate utilization studies were performed in a basal medium containing different substrates as follows: Casamino acids, peptone, yeast extract and tryptone (0.2 %, final concentration); sugars, fatty acids (20 mM, final concentration) and amino acids (0.2 %, final concentration). The basal medium contained (l–1): 1 g NH4Cl, 0.3 g KH2PO4, 0.3 g K2HPO4, 0.6 g NaCl, 0.1 g CaCl2.2H2O, 0.1 g MgCl2.6H2O and 1 mg resazurin. The pH was adjusted to 7.5 with 1 M NaOH.
Cell morphology was examined under a light (Olympus BH-2) and electron (Hitachi H-600A) microscope. For electron microscopy studies, bacterial cells grown in PY at 37 °C for 24 h were negatively stained with uranyl acetate. For ultrathin section examination of the cell wall, bacterial cells were fixed with osmic acid and embedded in araldite; the samples were then sliced and stained with lead citrate (Reynolds, 1963).
The generation time of strain GWT was determined by monitoring the OD600 of the PY culture at 37 °C at 1 h intervals up to 48 h. The temperature profile was determined in PY using a water bath with temperature controller between temperatures of 15 to 55 °C at 1 °C intervals. The pH range for growth was determined for the culture in PY broth at various pH values adjusted with HCl or NaOH (1 mol l–1). Growth was determined by measuring the OD600 of cultures at 1, 3 and 7 days. Biochemical traits were determined using conventional methods. All tests were performed in duplicate. The fermentation products, short-chain fatty acids, alcohols and gases were measured using a gas chromatograph (GC-14B Shimadzu) as described previously (Chen & Dong, 2004). The diagnostic isomers of diaminopimelic acid and amino acids in the cell wall were determined with established TLC procedures (Lechevalier & Lechevalier, 1980). Cellular fatty acids were extracted, methylated and analysed using the standard MIDI (Microbial Identification) system (Miller, 1982; Sasser, 1990).
Genomic DNA was extracted and purified using the method of Marmur (1961). The G+C content of the DNA was determined by the thermal denaturation method (Marmur & Doty, 1962) using a DU800 spectrophotometer (Beckman) with Escherichia coli K-12 as the reference. The 16S rRNA gene was amplified and sequenced according to Chen & Dong (2004). The sequencing was performed by Sangon Biological Engineering Technology Service, Shanghai, China, using ABI PRISM Big Dye Terminator cycle sequencing ready reaction kits (Perkin Elmer) and an ABI PRISM 377XL DNA sequencer. The 16S rRNA gene sequence of strain GWT was submitted to GenBank and EMBL to search for similar sequences using the BLAST algorithm (Altschul et al., 1990). Sequences with higher similarities were retrieved from the database and aligned and similarity analysis was performed using the CLUSTAL_X program (Thompson et al., 1997). Phylogenetic trees were constructed using the neighbour-joining, maximum-likelihood and maximum-parsimony methods implemented in the program MEGA3 (Kumar et al., 2004). The resultant tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings.
Cells of strain GWT were non-motile short rods (supplementary Fig. S1 in IJSEM Online), 0.5–0.6 µmx1.4–3.8 µm, and arranged singly or in clumps. The cells stained Gram-negative in all the growth phases; however, a Gram-positive bacterial cell wall ultrastructure was revealed by electron microscopy (Fig. 1). Spores were never observed. Colonies on PY agar were white, smooth, circular and entire and slightly convex and reached 0.5 mm after cultivation at 37 °C for 72 h.
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The cell wall hydrolysate of strain GWT was rich in L-lysine, but no diaminopimelic acids were detected. The cellular fatty acids of strain GWT were characterized mainly by saturated fatty acids, predominantly C14 : 0 (15.58 %), C16 : 0 (25.40 %) and C18 : 0 (12.03 %); C18 : 1
9c (11.20 %), C16 : 17c (6.18 %), isoC17 : 1 I (9.49 %) and isoC15 : 0 (4.30 %) were also relatively abundant.
The genomic DNA G+C content of strain GWT was determined as 38.0 mol%.
To ascertain the phylogenetic position of the novel strain, the complete 16S rRNA gene sequence (1523 bp) of strain GWT was compared with the most similar sequences and those of the representatives of the ‘Clostridia’ retrieved from GenBank. On the basis of a consensus 1367 bp of the 16S rRNA gene sequence, a phylogenetic tree rooted with Peptostreptococcus hydrogenalis GIFU 7662T was constructed. Phylogenetic analysis showed that strain GWT was affiliated to the low G+C Gram-positive bacteria phylum, and belonged to cluster XII of the ‘Clostridia’ (Collins et al., 1994). The closest relatives were Clostridium purinilyticum, Clostridium acidurici and Eubacterium angustum, with sequence similarity levels of 89.7 , 88.9 and 88.8 %, respectively. The similarity levels of the 16S rRNA gene sequence with that of other members in phylogenetic cluster XII ranged between 86 and 88 %. Trees constructed by neighbour-joining, UPGMA and minimum evolution showed the same topology. The 16S rRNA sequence of strain GWT is not closely related to that of previously described taxa and represents a novel genus.
Strain GWT also showed distinct phenotypic features distinguishing it from representative members in the same cluster (Table 1). Clostridium purinilyticum, Clostridium acidurici and Eubacterium angustum grew exclusively on purines, such as uric acid and adenine (Dürre et al., 1981; Cato et al., 1986; Beuscher & Andreesen, 1984), however strain GWT could not use these substances at all. Also, the three species were not able to use proteinaceous materials as sole carbon and energy sources. Moreover, strain GWT was also different from the species Caloranaerobacter azorensis and Thermohalobacter berrensis in the same phylogenetic branch in the optimal growth temperature and substrate range: while strain GWT is mesophilic and a protein-consumer, the other two species grow optimally at 65 °C and are sugar-consumers (Wery et al., 2001; Cayol et al., 2000).
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Description of Proteiniborus gen. nov.
Proteiniborus (Pro.tei.ni'bo.rus. N.L. n. proteinum protein; Gr. adj. boros gluttonous; N.L. masc. n. Proteiniborus protein-consumer).
Gram-positive, non-motile, non-spore-forming rod. Anaerobic and mesophilic. Cell wall peptidoglycan contains abundant L-lysine but not diaminopimelic acids. Cellular fatty acids consist mainly of saturated fatty acids, predominantly C14 : 0, C16 : 0 and C18 : 0. Chemo-organotrophic. Yeast extract and peptone can be used as energy sources. The fermentation products from PY include ethanol, acetic acid, hydrogen and carbon dioxide. Nitrate but not thiosulfate is reduced. The G+C content of the genomic DNA of the known strain is 38.0 mol%. The type species of the genus is Proteiniborus ethanoligenes, a member of cluster XII of the ‘Clostridia’.
Description of Proteiniborus ethanoligenes sp. nov.
Proteiniborus ethanoligenes (e.tha.no.li'ge.nes. N.L. n. ethanol-is ethanol; Gr. v. gennao produce; N.L. part. adj. ethanoligenes ethanol-producing).
Morphology and general characteristics are as described for the genus. Cells are 0.5–0.6 µm wide and 1.4–3.8 µm long. Colonies on PY agar are white, smooth, circular and entire and slightly convex and reach 0.5 mm after cultivation at 37 °C for 72 h. Growth occurs between 20 and 48 °C (optimum 37 °C) and at pH 6.4–10.0 (optimum 8.5–8.8). Besides yeast extract and peptone, weak growth is also observed on tryptone and Casamino acids. Ethanol, acetic acid and hydrogen are the main products from yeast extract and peptone, and a trace amount of propionic acid is also produced. The following substrates are not used: L-serine, L-threonine, L-alanine, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-valine, L-glutamine, L-arginine, L-tyrosine, tryptophan, L-isoleucine, L-proline, aspartate, L-cysteine, L-arabinose, cellobiose, aesculin, D-fructose, D-galactose, D-glucose, glycogen, inulin, D-lactose, maltose, mannose, melibiose, raffinose, rhamnose, ribose, sucrose, salicin, sorbose, starch, trehalose, D-xylose, adonitol, amygdalin, dulcitol, erythritol, inositol, mannitol, sorbitol, ribitol, methanol, ethanol, l-propanol, citrate, fumarate, malate, succinate, malonate, hippurate, sodium gluconate, butane diacid, β-hydroxybutyric acid, phenylacetic acid, cellulose and xylan. Milk is not curdled. Indole is produced. No NH3 is produced from yeast extract or peptone. Methyl red and Voges–Proskauer tests are negative. Casein is degraded and nitrate is reduced. Gelatin and DNA are not hydrolysed.
The type strain is GWT (=CGMCC 1.5055T=JCM 14574T), isolated from granular sludge from a laboratory-scale UASB hydrogen-producing reactor used to treat food industry wastewater.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Beuscher, H. U. & Andreesen, J. R. (1984). Eubacterium angustum sp. nov., a Gram-positive anaerobic, non-sporeforming, obligate purine fermenting organism. Arch Microbiol 140, 2–8.[CrossRef]
Cato, E. P., George, W. L. & Finegold, S. M. (1986). Genus Clostridium Prazmowski 1880, 23AL. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1141–1200. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Cayol, J.-L., Ducerf, S., Patel, B. K. C., Garcia, J.-L., Thomas, P. & Ollivier, B. (2000). Thermohalobacter berrensis gen. nov., sp. nov., a thermophilic, strictly halophilic bacterium from a solar saltern. Int J Syst Evol Microbiol 50, 559–564.[Abstract]
Chen, S. & Dong, X. (2004). Acetanaerobacterium elongatum gen. nov., sp. nov., from paper mill wastewater. Int J Syst Evol Microbiol 54, 2257–2262.
Chen, S. & Dong, X. (2005). Proteiniphilum acetatigenes gen. nov., sp. nov., from a UASB reactor treating brewery wastewater. Int J Syst Evol Microbiol 55, 2257–2261.
Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. E. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44, 812–826.
Dürre, P., Andersch, W. & Andreesen, J. R. (1981). Isolation and characterization of an adenine-utilizing, anaerobic sporeformer, Clostridium purinolyticum sp. nov. Int J Syst Bacteriol 31, 184–194.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Hernández-Eugenio, G., Fardeau, M.-L., Cayol, J.-L., Patel, B. K. C., Thomas, P., Macarie, H., Garcia, J.-L. & Ollivier, B. (2002). Clostridium thiosulfatireducens sp. nov., a proteolytic, thiosulfate- and sulfur-reducing bacterium isolated from an upflow anaerobic sludge blanket (UASB) reactor. Int J Syst Evol Microbiol 52, 1461–1468.[Abstract]
Holdeman, L. V., Cato, E. P. & Moore, W. E. C. (1977). Anaerobe Laboratory Manual, 4th edn. Blacksburg, VA: Virginia Polytechnic Institute and State University.
Hungate, R. E. (1969). A roll tube method for cultivation of strict anaerobes. Methods Microbiol 3B, 117–132.[CrossRef]
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA 3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Lechevalier, M. P. & Lechevalier, H. A. (1980). The chemotaxonomy of actinomyces. In Actinomycete Taxonomy, Special Publication 6, pp. 227–291. Arlington, VA:Society for Industrial Microbiology.
Lettinga, G. (1995). Anaerobic digestion and wastewater treatment system. Antonie Van Leeuwenhoek 67, 3–28.[CrossRef][Medline]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
Miller, L. T. (1982). Single derivation method for routine analysis of bacterial whole-cell fatty acid-methyl esters, including hydroxy acids. J Clin Microbiol 16, 584–586.
Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17, 208–212.
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. Newark, DE: MIDI.
Thabet, O. B. D., Fardeau, M.-L., Joulian, C., Thomas, P., Hamdi, M., Garcia, J.-L. & Ollivier, B. (2004). Clostridium tunisiense sp. nov., a new proteolytic, sulfur-reducing bacterium isolated from an olive mill wastewater contaminated by phosphogypse. Anaerobe 10, 185–190.[CrossRef][Medline]
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Wery, N., Moricet, J.-M., Cueff, V., Jean, J., Pignet, P., Lesongeur, F., Cambon-Bonavita, M.-A. & Barbier, G. (2001). Caloranaerobacter azorensis gen. nov., sp. nov., an anaerobic thermophilic bacterium isolated from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 51, 1789–1796.[Abstract]
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