Caldicellulosiruptor O Wensensis Sp. Nov., an Anaerobic, Extremely Thermophilic, Xylanolytic Bacterium

lnternational Journal of Systematic Bacteriology (1998), 48, 91-97 Printed in Great Britain

Caldicellulosiruptor o wensensis sp. nov., an anaerobic, extremely thermophilic, xylanolytic bacterium

Chi-Yu Huang,' Bharat K. Patel,' Robert A. Mah' and Larry Baresi3

Author for correspondence : Chi-Yu Huang. Tel : + 1 3 10 825 2569. Fax : + 1 3 10 794 2 106. e-mail : [email protected]

1 Department of An anaerobic, extremely thermophilic, xylanolytic, non-spore-forming Environmental Health bacterium was isolated from a sediment sample taken from Owens Lake, Sciences, School of Public Health, University of California, and designated strain OLT (T = type strain). Strain OLT had a Gram- California, Los Angeles, CA negative reaction and occurred as short rods which sometimes formed long 90095-1772, USA chains containing a few coccoid cells. It grew at 50-80 "C, with an optimum at 2 Faculty of Science and 75 "C. The pH range for growth was 55-900 with an optimum at about pH 7.5. Technology, G rif f it h When grown on glucose at optimal conditions, its doubling time was 7-3 h. In University, Nathan 41 11, Brisba ne, Austra Ii a addition to glucose, the isolate utilized sucrose, xylose, fructose, ribose, xylan, starch, pectin and cellulose. Yeast extract stimulated growth on carbohydrates 3 Department of Biology, California State University, but was not obligately required. The end products from glucose fermentation Northridge, CA, USA were lactate, acetate, ethanol, H, and CO,. The G+C content of strain OLT was 36.6 mol%. The 165 rDNA sequence analysis indicated that strain OLTwas a member of the subdivision containing Gram-positive bacteria with DNA G+C content of less than 55 mol% and clustered with members of the genus Caldicellulosiru~tor. Because stra i n OLT is ph y logene t icaI I y and phe no typ icaI I y different from other members of this genus, it is proposed to designate this isolate Caldicellulosiruptor owensensis sp. nov. Strain OLT is the type strain (= ATCC 7001673.

Keywords: Caldicellulosiruptor owensensis, thermophile, xylanolytic bacterium

INTRODUCTION in the subdivision containing Gram-positive bacteria with a DNA G + C content of less than 55 mol% (26). Thermophilic and extremely thermophilic micro- The newly described genus, Caldicellulosiruptor, is organisms have gained a great deal of attention assigned to the phylogenetic cluster ' D ' and is the least recently (3). Enzymes from these micro-organisms are studied. The seven strains in cluster D were isolated of special interest since they are not usually denatured exclusively from hot springs and all degrade cellulose. by high temperatures and are even active at elevated Of these, only Caldicellulosiruptor saccharolyticus (for- temperatures (3, 35). Furthermore, members of the merly designated Caldocellum saccharolyticus) was thermoanaerobic saccharolytic group may be com- taxonomically validated (25). mercially useful for producing chemicals and fuels We describe in this paper, a new extremely thermo- such as ethanol and lactate from plant biomass (15). philic, xylanolytic anaerobic bacterium isolated from Xylan is a major component of plant hemicellulose; sediments of Owens Lake, California, USA. Pheno- after cellulose, it is the next most abundant renewable typic and phylogenetic characteristics indicate that it is polysaccharide in nature. Several species of xylano- a new species of the genus Caldicellulosiruptor. We lytic, thermophilic anaerobes have been isolated and designated it Caldicellulosiruptor o wensensis sp. nov. identified as members the genera Clostridium, Thermo- anaerobacter, Thermoanaerobacterium and Caldicellu- METHODS losiruptor (4, 16, 33). 16s rRNA sequence analysis of Sampling procedure. Sediment samples were collected from these four genera showed individual coherent clusters a small freshwater pond located within the dry Owens Lake bed in California. The samples were kept under anaerobic The GenBank accession number for the 165 rDNA sequence of strain OLT is conditions in serum bottles, transported to the laboratory at U80596. UCLA at ambient temperatures, and stored at 4 "C until

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Fig. 7. (a) Phase-contrast photomicrograph of cells from exponential growth phase of strain OLT. Cocci denoted by arrow. Bar, 10 pm. (b) Transmission electron micrograph of a thin section of whole cells of strain OLT. Bar, 1 pm. (c) Transmission electron micrograph of negatively stained strain OLT showing the presence of lophotrichous flagella. Bar, 1 pm. used. The temperature and pH of the sample site was 32 "C isolate was considered pure as judged by uniform colony and 9.0, respectively. morphology and microscopic appearance. Enrichment and isolation. An enrichment culture medium Physiological tests. The modified Hungate anaerobic tech- modified from Angelidaki et al. (2) designated CBM was nique (12, 18) was used throughout these studies. Unless used. CBM consisted of (per litre distilled water) yeast indicated, all tests were performed in triplicate. extract (Difco), 1 g; xylan, 5 g; NH,Cl, 1 g; NaC1, 0.1 g; For pH and temperature optima studies, CBM with xylose MgCl, . 6H,O, 0.1 g; CaC1,. 2H,O, 0.05 g; K,HPO,, 0.4 g; instead of xylan, was used. The effect of pH on growth was resazurin, 0.0005 g; 1 ml vitamin mixture, and 10 ml trace determined in xylose medium containing four different mineral solution (10). The vitamin solution contained (mg buffers at a final concentration of 50 mM : Walpole's Acetate 1-l): biotin, 2; folic acid, 2; pyridoxine hydrochloride, 10; Buffer (3 l), pH range 3.7-5.1 ; MES sodium salt, pH range thiamine hydrochloride, 5 ; riboflavin, 5 ; nicotinic acid, 5 ; 5.5-6.5; Tris (Trizma base), pH range 6-5-7.5; and Na,CO,, m-calcium pantothenate, 5; vitamin B1,, 0.1 ; p-amino- pH range 7.5-9.5. A stream of N, instead of N,/CO, (70: 30) benzoic acid, 5 ;and lipoic acid, 5. The trace mineral solution was used while dispensing medium. The pH values of the contained (per litre): H,SeO,, 0.01 g; MnC1,.4H2O, 0.1 g; medium after sterilization were 3.8-9.5. Cultures were FeSO,. 7H,O, 0.1 g; CoCl,. 6H,O, 0.15 g; ZnCl,, 0.1 g; incubated in the optimal temperature. H,BO,, 0.01 g; Na,MoO,. 2H,O, 0.01 g; CuC1,. 2H,O, 0.02 g ; NiSO, .6H,O, 0.02 g; AlC1,. 6H,O, 0.04 g; NaWO,, Gram reaction was determined using a Difco Gram Stain kit 0.03 g; and disodium EDTA dihydrate, 0.5 g. CBM was according to the manufacturer's recommended protocol boiled and 3.0 g NaHCO, was added after cooling under a (Difco). The effect of sodium chloride (0-3%) on growth stream of oxygen-free N,-CO, (70:30, YO v/v) gas. The was determined in CBM containing xylose. medium was dispensed in 10 ml aliquots into culture tubes To determine substrate utilization, other carbohydrates under a stream of N,-CO, (70%:30%), stoppered with replaced xylan in the CBM-based medium. All carbo- rubber septa, sealed with aluminium cap seals (Bellco Glass) hydrates were added from sterile anaerobic stock solutions and autoclaved at 140 "C for 20min. Prior to use, to a final concentration of 0-5'/o. Growth was positive if the Na,S. 9H,O was injected from a sterile stock solution of 25 g optical density was higher than the control carbohydrate- 1-1 into each tube to give a final concentration of 2-5mg 1-l. free CBM tubes. The ability of the isolate to utilize glucose For initiating enrichments, 0.5 ml of sample sediment slurry or xylose as the sole carbon source was tested in CBM was added to the CBM medium. Tubes were incubated at lacking xylan and yeast extract. The type strain C. saccharo- 75 "C for up to 1 week without shaking until xylan lyticus (ATCC 43494) used to compare the substrate spec- solubilization and an increase in microbial numbers was trum was obtained from the American Type Culture observed. Such enrichment cultures were subcultured four Collection. Generation time of the isolate was determined at more times and serially diluted in roll tubes containing CBM the optimum pH and temperature in CBM containing fortified with 2 YOagar (Difco). The roll tubes were incubated glucose (final concentration of 0.5 YO). at 60 "C for up to 1 week. Single colonies were picked using For antibiotic inhibition studies, CBM containing xylose sterile Pasteur pipettes and inoculated into fresh CBM was used. The ability of the isolate to grow aerobically was medium. This process was repeated four times before the determined in CBM which lacked xylan, sodium sulfide and

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N,: CO,, but contained glucose and air. Aerobic cultures programs were run on a Sun SPARC workstation and MEGA were incubated in Triple Baffled Nephelo culture flasks was run on a Compaq notebook (Contura model 410CX). (Bellco Glass) at 75 "C and 200 r.p.m. in an Environ-shaker (Lab-Line Instruments). The reduction of nitrate (10 mM), RESULTS sulfate (10 mM), sulfite (5 mM), and thiosulfate (20 mM) by the isolate was determined in xylan-free glucose CBM Colony and cell morphology medium under anaerobic conditions. Enrichment cultures were obtained after 1 week at Spore formation was induced using the methods of Schink & Zeikus (29) and Cook et al. (6). Endospore presence was 75 "C. A population of heterogeneous rods and fila- determined from 1-week- and 1-month-old cultures using ments were observed in the enrichment cultures. In roll the staining method described by Schaeffer & Fulton (30). tubes, 0.5-2 mm colonies developed after 1 week at Analytical methods. Growth was measured either by in- 60 "C. Zones of clearance were observed around the serting culture tubes directly into a Perkin-Elmer Junior colonies indicating that xylan solubilization had oc- model 35 spectrophotometer and measuring OD,,, or by curred. A single colony in the final serial dilution was counting cells using a Petroff Hausser counting chamber. picked and designated strain OLT. Cells of strain OLT Volatile fatty acids were analysed using a Hewlett-Packard were straight rods measuring 0.5-0.8 pm in diameter 5890A gas chromatograph equipped with a flame ionization and 2-5 pm in length, and occurred singly, in pairs, or detector and a Supelco 10% SP-1000, 1 YO H,PO, on in chains (Fig. la). Small coccoid cells were con- 100/120 Chromosorb W-AW packed 6 ft x 2 mm i.d. glass sistently observed during exponential growth, perhaps column maintained at 150 "C. Alcohols were also analysed because of unequal cell division. Colonies of strain by using a gas-liquid chromatograph equipped with a flame ionization detector and a Supelco 5% Carbowax 20M on 60/80 Carbopack B glass column at 120 "C. Lactate was measured enzymically (1 1). H, and CO, were measured 0.15 using a Carle AGC series 100 gas chromatograph equipped (a) with an SO/lOO silica gel-packed stainless steel column (Supelco) and a thermal conductivity detector. Sulfide was estimated using the method described by Cord-Ruwisch (5). Electron microscopy. Cells were fixed with cold 2.5% 0.10 ' glutaraldehyde in 0-1 M Na,HPO,-KH,PO, (pH 7.2) buffer overnight and post-fixed with 1 O/O osmium tetroxide (OsO,). They were then dehydrated yith ethanol and embedded in Spurr. Approximately 1000 A (100 nm) thick sections were stained with uranyl acetate and lead citrate and examined 0.05 . under a JEOL JEM-100 CX electron microscope at an accelerating voltage of 80 kV. Negative staining of cells for n electron microscopy was achieved with 1 % (w/v) uranyl 7 acetate. DNA base composition. DNA was extracted by the method 0.00 of Pitcher et al. (23). The buoyant density of purified DNA 40 50 60 70 80 90 was measured by ultracentrifugation in a CsCl density gradient (24). The G + C content of the DNA was calculated L by using the formula of Schildkraut et al. (28). .- 0.15' 16s rDNA sequence studies. Purification of genomic DNA, 'cU (b) amplification and purification of the 16s rRNA gene (16s rnQ rDNA) from strain OLT were performed by previously described techniques (1 7,27). The purified PCR product was sequenced directly on an ABI automated DNA sequencer by 0.10 - using a Prism Dyedeoxy Terminator Cycle Sequencing kit and protocols recommended by the manufacturer (Applied Biosystems). Ten sequencing primers were used to obtain the sequence which covered approximately 80 % of both strands (17, 27). Using the sequence editor, ae2, the 16s rDNA 0.05 - sequence of strain OLTwas aligned with the 16s rRNA gene sequences of various members of the bacterial phyla obtained from the Ribosomal Database Project (19) and from EMBL. Positions of sequence and alignment uncertainty were omitted from the analysis, and pairwise evolutionary distances for 1280 nucleotides were computed. Phylogenetic 0.00 --o$, ' I analysis was performed using programs which form part of the PHYLIP package and include DNADIST (Jukes & Cantor option), NEIGHBOR-JOINING and DNAPARS (9). Tree topology was re-examined by using 100 boot-strapped data sets for which a script file with the following PHYLIP programs was used : SEQBOOT,DNADIST, FITCH and CONSENSE.Programs in the phylogenetic package MEGA (14) were also used. PHYLIP

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F . - Cellulolytic thermophile str. Rt8B7 - Cellulolytic thermophile str. Rt8Bl5 - -Cellulolytic thermophile str. Ok9B1

Dictyoglomus thermophilum str. H-6-12 ...... , ...... , ...... , .. , ...... , ...... I ...... Fig. Phylogenetic dendrogram based on - Thermoanaerobacter thermohydrosulfuricus str. E 100-69 3. 165 rDNA sequence data indicating the Thermoanaerobacter thermocopriae str. JT-3 position of strain OLT within the radia- tion of members of the genus of Caldicellulosiruptor and related taxa. All the sequences used in the analysis, with the exception of the sequence for the cellulolytic strain SP83 (EMBL accession no. X93020) (22), were obtained from the Ribosomal Database Project, version 5.0 (19). 0.10 Sca le bar ind icates evol ut ionary distance.

OLT in roll tubes were circular with smooth edges, up OLT were acetate, lactate, ethanol, CO, and H,. to 2 mm in diameter, opaque, yellowish and were Nitrate, sulfate, sulfite and elemental sulfur were not convex. Strain OLT was non-motile and had a Gram- reduced. negative reaction, but the cell wall was typical of Strain OLTwas resistant to D-cycloserin (100 pg ml-l), Gram-positive bacteria as seen by electron microscopy erythromycin (200 pg ml-l), and tetracycline (100 pg (Fig. 1b). Electron microscopic examination of nega- ml-l). Growth was inhibited by penicillin G, strep- tively stained young cells showed the presence of tomycin, chloramphenicol, ampicillin at a concen- lophotrichous flagella (Fig. lc). Spores were not tration of 100 pg ml-'. The strain tolerated 0.5 YO but observed under any conditions tested. was inhibited by 1 YONaCl. DNA base composition. The genomic DNA base com- Growth characteristics and physiology position of strain OLT as determined by the buoyant Strain OLT was a thermophilic, strictly anaerobic, density of purified DNA in a CsCl density gradient was chemoorganotrophic bacterium. The optimal growth 36.6 mol% G + C. temperature of this strain was 75 "C and no growth 165 rDNA sequence analysis. Using ten primers, 1539 was observed below 50 "C or above 80 "C (Fig. 2a). nucleotides corresponding to E. coli positions 8-1 542 Strain OLT had a broad pH range for growth, 5.5-9.0, according to the nomenclature of Winker & Woese with an optimum of 7.5 (Fig. 2b). When grown under (34), were sequenced. Phylogenetic analysis indicated optimal conditions in xylan-free CBM-glucose me- that strain OLT was a member of the low-G+C- dium, strain OLT had a doubling time of 7.3 h and content sub-branch of the Gram-positive group. lowered the pH by 0.5 units. Presence of either yeast Further analysis indicated that strain OLT was extract or vitamins was not required but stimulated specifically related to the 11 members of the Caldi- growth. cellulosiruptor cluster with a mean similarity of 94 YO. Strain OLT could grow on a wide variety of carbon Currently the only taxonomically validated member of sources including arabinose, cellulose, cellobiose, dex- the cluster is C. saccharolyticus, with which strain OLT trin, fructose, galactose, glucose, glycogen, inositol, was related at a similarity level of 94%. Fig. 3 is a lactose, mannitol, mannose, maltose, pectin, raffinose, dendrogram that was generated by the neighbour- rhamnose, ribose, starch, sucrose, tagatose, xylan and joining method from the evolutionary distance matrix xylose but not acetate, arbutin, trypticase peptone, and shows this relationship. erythritol, glycerol, lactate, melibiose, methanol, pyruvate, sorbitol and trehalose. Autotrophic growth on DISCUSSION H,/CO, (80:20, %) was not observed. Yeast extract Most thermophilic microbes were isolated from geo- could also serve as a sole carbon and energy source. thermal environments associated with volcanic activity The end products of glucose fermentation by strain (8, 13, 35, 36). However, other thermally heated

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Table 1. Substrate spectrum of strain OLT and other group, including G + C content and fermentation end- Ca ldicellulosiruptor species products. The taxonomic nature of this cluster is still

...... 1 .... ill-defined and with the exception of C. saccharolyticus, Strains OLT and C. saccharolyticus were grown on CBM none of the other members has been taxonomically medium supplied with each substrate tested. Data for C. validated (25). lactoaceticus is from (20). + , Positive; -, negative; ND,not de tennined. Another phylogenetically closely related species to strain OLT is Anaerocellum thermophilurn (32). Strain Substrate Strain C. saccharolyticus C. lactoaceticus OLT differed in carbon utilization from A. thermo- OLT philurn and C. saccharolyticus. Sequencing of the 16s rRNA gene and phylogenetic analysis confirmed the Acetate - placement of strain OLT as a member of the Caldi- Cellobiose + cellulosiruptor cluster. Comparison of the phenotypic Cellulose + traits of strain OLTwith the widely studied members of CM-cellulose + Caldicellulosiruptor, namely C. saccharolyticus and ' C. Sigmacell 100 + lactoaceticus' (25, 20), indicated that strain OLT was Fructose + nutritionally more versatile and that C. lactoaceticus Galactose + was the least versatile (Table 1). Based on this study we Glucose + propose that strain OLT is the type strain of a new Inositol + species of the genus Caldicellulosiruptor, Caldicellulo- Lactose + sirup t or ow ensensis. Mannitol + Pyruva te - Raffinose + Ribose + Description of Caldicellulosir up tor o wensensis Sucrose + - Trehalose Caldicellulosiruptor owensensis (0.wen.sen'sis. N. L. Xylose + adj. owensensis from Owens Lake, CA, USA). Cells are non-motile straight rods that are 2-5 pm by 0.5-0-8 pm, and occur singly, in pairs, or in chains. environments including the deep subsurface aquifers, Lophotrichous flagella. Gram staining reaction was man-made thermal environments and naturally solar negative. Endospores were not found in any tested heated environments are also reported to harbour conditions. Colonies (diameter, < 2 mm) are circular thermophilic bacteria (1, 21). Strain OLT was isolated with smooth edges, convex, opaque and yellowish. from a shallow freshwater pond located in the Owens Growth occurred over the temperature range 50-80 "C Lake bed area. Owens Lake bed is a largely dry, with an optimum of 75 "C. No growth was detected at evaporite deposit at the eastern base of Sierra Nevada and below 45 "C or above 80 "C. Alkalitolerant. pH in California. Prior to 1910, the lake was fed by range, 5.5-9-0 with an optimum at about pH 7.5. excessive run-off and melted snow from the nearby Growth factors found in either yeast extract or vitamin mountains. After 1910, water was diverted by the Los solutions were not required for growth. Growth was Angeles aqueduct, and by 1921 the lake entered its inhibited by penicillin G, streptomycin, chlorampheni- present state of periodic flooding and drying. Like the col and ampicillin at 100 pg ml-l. Cells are resistant to rest of the lake, the sampling site is subjected to periods D-cycloserine (100 pg ml-l), erythromycin (200 pg of drying. It may therefore reach high temperatures ml-l) and tetracycline (100 pg ml-l). Growth strictly during summer, creating a favourable environment for anaerobic. Chemoorganotrophic. Growth with arabi- growth of thermophilic microbes. nose, cellobiose, cellulose, dextrin, fructose, galactose, Strain OLT is an extreme thermophile and is a member glucose, glycogen, inositol, lactose, mannitol, man- of the domain Bacteria based on 16s rRNA sequence nose, maltose, pectin, raffinose, rhamnose, ribose, analysis (8). Furthermore, strain OLT degraded xylan starch, sucrose, tagatose, xylan, xylose and yeast and hence could be a member of the genera Clos- extract. Does not grow on acetate, amygdalin, arbutin, tridiurn, Therrnoanaerobacter, Thermoanaerobacteriurn erythritol, glycerol, lactate, melezitose, melibiose, or Caldicellulosiruptor. Strain OLT was not a spore- methanol, pyruvate, sorbitol, trehalose, trypticase former and hence could not be included as a member peptone or HJCO,. The fermentation products from of the genera Clostridium or Therrnoanaerobacter (6,7, glucose are acetate, lactate, ethanol, H, and CO,. The 16). Strain OLT did not produce sulfur from thio- bacterium does not reduce nitrate, sulfate, sulfite and sulfate and hence could be excluded from the genus thiosulfate. The G+C content is 36.6 mol% (as Therrnoanaerobacteriurn (1 6). Strain OLT degraded determined by buoyant density of purified DNA by cellulose and hence possess a similar trait in common CsCl gradient centrifugation). Type strain OLT with members of the Caldicellulosiruptor group (25). In ( = ATCC 7001 67) was isolated from the Owens Lake addition, strain OLT had other similarities to this in California, USA.

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ACKNOWLEDGEMENTS 16. Lee, Y. E., lain, M. K., Lee, C. Y, Lowe, S. E. & Zeikus, 1. G. (1993). Taxonomic distinction of saccharolytic thermophilic We thank Jean-Louis Garcia for his advice in naming the anaerobes : description of Thermoanaerobacterium xylano- organism, and Felipe Alatriste-Mondragon for technical lyticum gen. nov., sp. nov., and Thermoanaerobacterium assistance. We also thank Birgitta Sjostrand for performing saccharolyticum gen. nov., sp. nov. ; reclassification of the electron microscopy. Funding came in part from the Thermoanaerobium brockii, Clostridium thermosulfuro- Australian Research Council (to B. K. C. P.) and is gratefully genes, and Clostridium thermohydrosulfuricum E 100-69 as acknowledged. Thermoanaerobacter brockii comb. nov., Thermoanaero- bacterium thermosulfirigenes comb. nov., and Thermo- REFERENCES anaerobacter thermohydrosulfuricus comb. nov., respectively; and transfer of Clostridium thermohydrosulfuricum 1. Andrews, K. T. & Patel, B. K. C. (1996). 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