Characterization of Two Novel Saccharolytic, Anaerobic Thermophiles, Thermoanaerobacterium Polysaccharolyticum Sp
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International Journal of Systematic and Evolutionary Microbiology (2001), 51, 293–302 Printed in Great Britain Characterization of two novel saccharolytic, anaerobic thermophiles, Thermoanaerobacterium polysaccharolyticum sp. nov. and Thermoanaerobacterium zeae sp. nov., and emendation of the genus Thermoanaerobacterium Isaac K. O. Cann,† Peter G. Stroot,‡ Kevin R. Mackie, Bryan A. White and Roderick I. Mackie Author for correspondence: Roderick I. Mackie. Tel: j1 217 244 2526. Fax: j1 217 333 8809. e-mail: r-mackie!uiuc.edu Department of Animal Two anaerobic, thermophilic, Gram-positive, non-spore forming bacteria with Sciences, 132 Animal an array of polysaccharide-degrading enzymes were isolated from the leachate Sciences Laboratory, 1207 W. Gregory Drive, of a waste pile from a canning factory in Hoopeston, East Central Illinois, USA. University of Illinois at The results of 16S rDNA sequence homology indicated that their closest Urbana–Champaign, relatives belong to the saccharolytic, thermophilic and anaerobic genera of Urbana, IL 61801, USA Thermoanaerobacterium and Thermoanaerobacter. Although, the evolutionary distances between these bacteria and their closest relatives are greater than 11%, there is no defining phenotypic characteristic for the creation of a new genus. It is proposed that these bacteria should be placed in the genus Thermoanaerobacterium, which requires emendment of the genus description with regard to the reduction of thiosulfate to sulfur, because neither isolate is capable of this reduction. Thermoanaerobacterium polysaccharolyticum reduces thiosulfate to sulfide, whereas Thermoanaerobacterium zeae is unable to reduce thiosulfate. The cells of both isolates are rod-shaped and exist as single cells or sometimes in pairs. Cells are motile by means of flagella. Growth occurs between 45 and 72 SC, with optimum temperature of 65–68 SCatpH68. The pH range for growth is from 4 to 8 at a temperature of 65 SC. Both organisms ferment glucose, arabinose, maltose, mannose, rhamnose, sucrose, trehalose, xylose, cellobiose, raffinose, melibiose and melezitose. The major end products of fermentation with glucose are ethanol and CO2, with lesser amounts of acetate, formate, lactate and hydrogen. The DNA GMC contents of Thermoanaerobacterium polysaccharolyticum sp. nov. and Thermoanaerobacterium zeae sp. nov. are 46 and 42 mol%, respectively. The type strains are KMTHCJT (l ATCC BAA-17T l DSM 13641T) and mel2T (l ATCC BAA-16T l DSM 13642T), respectively. Keywords: thermophilic, bacteria, Thermoanaerobacterium, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae INTRODUCTION into organisms capable of growth at high tempera- tures. During the last two decades, many reports have The importance of thermostable biomolecules in the described the isolation of novel thermophilic orga- growing field of biotechnology has spurred research nisms from both the domains Archaea and Bacteria ................................................................................................................................................................................................................................................................................................................. † Present address: New England Biolabs Inc., 32 Tozer Road, Beverly, MA 01915–5510, USA. ‡ Present address: Department of Agricultural Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801, USA. The GenBank accession numbers for the 16S rDNA sequence of strains KMTHCJT and mel2T are U40229 and U75993, respectively. 01442 # 2001 IUMS 293 I. K. O. Cann and others (Cayol et al., 1995; Cook et al., 1996; Engle et al., oven and cooled under a stream of oxygen-free carbon 1995, 1996; Fiala & Stetter, 1986; Freier et al., 1988; dioxide prepared by passage through a column of heated Jones et al., 1983; Liu et al., 1996b; Schink & Zeikus, copper filings. This was then dispensed in 9n0 ml amounts 1983; Wiegel & Ljungdahl, 1981). Most of the into 15i180 mm Balch tubes in an anaerobic chamber saccharolytic, anaerobic thermophiles belonging to (Coy Laboratory Products). Dispensed medium was auto- the bacterial domain have received attention for their claved for 15 min at 110 mC. After cooling, 0n2ml1n25% cysteine ; HCl\Na#S (sterile), 0n05 ml B vitamin solution potential in the bioconversion of substrates of plant (0n2 g thiamin ; HCl, 0n2 g calcium -pantothenate, 0n2g origin to end products such as lactate and ethanol, riboflavin, 0n2 g pyridoxine ; HCl, 0n01 g p-amino benzoic compounds with potential for the production of bulk acid, 0n25 g biotin, 0n25 g folic acid and 0n01 g vitamin B"#) chemicals and fuels (Lee et al., 1993b). and 0n7ml7%(w\v) NaHCO$ (filter-sterilized) were in- jected into each tube. The initial samples were incubated at Members of the genera Thermoanaerobacterium and 60 mC until visible growth was observed and passed through Thermoanaerobacter have been isolated from unique a series of serial dilutions until a uniformly pure culture was areas such as deep surface oil wells (Cayol et al., 1995), obtained, as determined by microscopic examination of wet geothermally heated water outlets (Cook et al., 1996) mounts and Gram-stained preparations. and hot springs (Wiegel & Ljungdahl, 1981). Several T thermostable enzymes have either been purified or Strain mel2 was isolated using the enrichment procedure described for strain KMTHCJT, except that melibiose was cloned from the members of this group of organisms. the carbon and energy source. Aliquots from tubes showing For example, polysaccharide-hydrolysing enzymes growth were streaked on plates of the same defined medium. from this group are thermostable endoxylanases (Lee A colony was picked and grown in the defined medium based et al., 1993a; Liu et al., 1996a) which can be used in on melibiose and passed through several serial dilutions biomass conversion and the pulp and paper industry. until a uniformly pure culture was obtained. Several thermophilic bacteria have recently been iso- Physiological properties. The isolates were grown on rep- lated from the leachate of a waste pile from a canning resentative carbon sources (glucose, arabinose, lactose, factory in Illinois, USA. In this paper, two unique, maltose, rhamnose, sucrose, trehalose, xylose, cellobiose, non-spore-forming, Gram-positive Thermoanaero- mannose, raffinose, sorbose, melibiose, melezitose, pyruvate, bacterium members capable of using a wide range of mannitol, fumarate, malate or citrate) to determine whether carbohydrate sources for the heterofermentative pro- different substrates could be used as sole carbon source. Bacterial growth was monitored by determining the OD'!! duction of lactate or ethanol are described. A recent (Spectronic 70; Bausch and Lomb). In addition, ability to publication describes the cloning and sequencing of a grow on cracked corn, ground corn cob and ground corn thermostable multidomain mannanase from one of the stalk was evaluated. The carbon sources were suspended novel isolates (Cann et al., 1999). separately under anaerobic conditions in distilled water and sterilized by autoclaving. They were then added to sterile defined medium to a final concentration of 0n5% (w\v). METHODS Nitrate reduction, catalase and indole production tests were Sample site. A canning factory situated in Hoopeston, carried out as described by Smibert & Krieg (1994). The Illinois, USA, which seasonally processes sweet corn and defined medium described above with glucose as energy other vegetables, was selected for study. Organic waste from source was used to grow both bacteria above 37 mCto the canning process is dumped 1 mile west of town. The determine their respective optimum temperatures for waste pile and surrounding run-off were characterized by growth. In the optimum pH studies, the isolates were grown active gas evolution, low pH (5n5), high concentrations of at 68 mC in the defined medium with glucose as the carbon source and the initial pH adjusted over the ranges 5n0–8n0 for short-chain fatty acids (31n6 mM acetate, 8n2 mM propi- T T onate, 26n0 mM butyrate and 6n4 mM valerate) as well as strain KMTHCJ and 1n0–9n0 for strain mel2 . The reduction heat generation. Temperature was not recorded, but of thiosulfate, sulfate and sulfur at a 20 mM concentration exhibited a steep gradient away from the centre of the waste was tested. Excess iron (0n5 mM) in the form of FeCl# ; 4H#O pile. A subsurface sample of the leachate from this waste pile was added to the Balch tubes in order to detect sulfide was collected in screw-cap flasks and transported under precipitation. At our incubation temperature, it was necess- anaerobic conditions and ambient temperature to the ary to use a low cysteine ; HCl concentration of 0n05% laboratory. The sample was kept at 4 mC until used to (w\v), in order to prevent precipitation of the excess iron inoculate enrichment medium. with the breakdown of the cysteine ; HCl. Microscopic T examination was used to check for sulfur formation. Enrichments. To isolate strain KMTHCJ , aliquots (0n2 ml) Reductive acetogenesis was examined by including 80% H# of samples were inoculated into a volume of a minimal and 20% CO# in the headspace above the basal medium medium containing raffinose as sole added carbon and " without glucose and measuring the change in OD'!! energy source [0 25 g Trypticase-peptone l− (BBL Micro- n " compared to the basal medium. biology Systems), 5 g raffinose l− , 50 ml Pfennig’s mineral −" solution (g l :KH#PO%, 10; MgCl# ; 6H#O, 6n6; NaCl, 8n0;