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INTERNATIONALOURNALF SYSTEMATICACTERIOLOGY,ul y 1986, p. 435-443Copyright 0 986, International Union of Microbiological Societies0020-7713186103043 5-09$02 OO/O

Vol. 36, No. 3

Isolation and Characterization of Acidothermus cellulolyticus gemnov. , sp . nov. , a New Genus of Therm ophilic, A cidophilic,Cellulolytic B acteria

A. MOHAGHEGHI,' K . GROHMANN,' M . HIMMEL,l L. LEIGHTON,' AND D. M. UPDEGRAFF2*Fermentation Section, Biotechnology Branch, Solar Fuels Division, Solar Energy Research Institute,' and Department ofChemistry and G eochemistry, Colorado School of Mines,2 Golden, Colorado 80401

Twelve isolates of thermophilic, acidophilic, cellulolytic bacteria were obtained from three different primaryenrichment cultures from acidic hot springs at Yellowstone National Park, Wyo. The three isolates which hadthe highest cellulolytic activity, as shown by the diameter of clearing zones surrounding colonies on celluloseagar plates, were selected for intensive study. All were gram-variable, nonsporulating aerobic rods whichformed no pigment. They grew at 37 to 65"C, with optimum growth at 55C. The pH range for growth was 3.5to 7, with an optimum pH of 5. The guanine-plus-cytosine content of the deoxyribonucleic acid was 60.7 k 0.6mol%. The organisms are resistant to penicillin G at 100 pglml. They share several important features withThermus strains, namely heterotrophic, aerobic, and thermophilic mode of growth; morphological features;sensitivity to lysozyme; and presence of catalase. They differ in other important aspects, such as the pattern ofcarbon sources utilized for growth, the pH and temperature profiles of growth, the pattern of sensitivity toantibiotics, the guanine-plus-cytosine content of DNA, the composition of amino acids in the cell walls, and thestructure of the cell walls. Thermus species are very sensitive to penicillin G, whereas our strains are resistant.Our strains also are different, in important respects, from the genus Thermomicrobium. Therefore, wedesignate the organism Acidothermus ceZZuZoZyticus gen. nov., sp. nov., of which the type strain is our strain11B, ATCC 43068.

This research effort was undertaken as part of a researchprogram on the production of fuel alcohol from cellulosicbiomass materials. The driving hypothesis is that athermophilic, acidophilic, cellulolytic bacterium can begrown in coculture with other thermotolerant microorga-nisms to produce ethanol at a high rate from cellulosicwastes. Alternatively, the cellulolytic organism could beused to produce cellulase, which could be added to acellulose-containing culture of an organism which fermentsglucose to ethanol.Most of the thermophilic, cellulolytic microorganismsisolated to date are sporeforming anaerobes belonging to thegenus Clostridium. None of these are acidophilic, and allhave an optimum pH near 7. Thus, they would not grow wellunder the acidic conditions (pH 3 to 5 ) prevalent in anethanolic fermentation.

Aerobic, thermophilic, cellulolytic microorganisms in-clude several species of fungi (10) and a few species offilamentous bacteria belonging to the family A ct ino-m y c e t a c e a e (33) . Among the fungi, Mycel ioph thorathermophila is of considerable interest, since it grows wellon cellulose in submerged culture at 50C and pH 4.5 andsynthesizes cellulolytic enzymes (10).Thus, the organism isacidophilic, as well as cellulolytic and thermophilic.Rosenberg (24) screened 21 species of thermophilic andthermotolerant fungi for the ability to degrade cellulose andfound that 13 species had this ability. It was not determinedwhether these organisms could grow well in submergedculture under thermophilic and acidophilic conditions andproduce significant amounts of cellulase. Aspergi l lus

* Corresponding author.

fumigatus has been shown to grow well on cellulose insubmerged culture at 45C and pH 4.6 and to produceconsiderable amounts of cellulase (27).Among the thermophilic, aerobic bacteria, only a fewactinomycetes are actively cellulolytic, notably Thermo-monospora curvata (28) and Thermoactinomyces cellulosae(1, 29). However, neither of these organisms is acidophilic.The acidic hot springs at Yellowstone National Park,Wyo., provide an excellent source for the isolation ofacidophilic thermophilic bacteria. The distribution of pHamong the springs at Yellowstone is bimodal, with a largegroup of springs in the highly acidic range, pH 2 to 5 , andanother group in the near-neutral to alkaline range, pH 6 to10. Brock (2 ) isolated T h e r m u s a q u a t i c u s , B a c i l l u sacidocaldarius, and Sulfolobus species from Yellowstonesprings, and Jackson et al. (9) isolated Thermomicrobiumspecies. All of these organisms are obligate thermophiles,and B . acidocaldarius and Sulfolobus are acidaphilic; how-ever, none of these are known to be cellulolytic.Since the major objective of the screening program was toisolate microorganisms for potential application in the fer-mentation of cellulosic substrates, the following major con-straints were placed on enrichment conditions and proper-ties of these isolates: pH range, 3 to 6 (optimum pH of 5);temperature range, 50 to 60C; and secretion of the cellulaseenzyme complex. Selection for specific pH range was doneto match the pH range at which many industrial fermenta-tions (e.g., ethanol production) are carried out. Further-more, we assumed that pH profiles of cellulase enzymesproduced by such microorganisms would be similar to pHprofiles for growth of these microorganisms. The tempera-ture selected for enrichment is slightly higher than thehighest temperature (50C) at which yeasts seem to be able

435


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436 MOHAGHEGHI ET AL. INT. J. S YS T.BACTERIOL.to grow (17, 25); a similar assumption, with respect totemperature profiles of cellulolytic enzymes and growthtemperature, was made.

MATERIALS AND METHODSBacterial strains. Th e 12 isolates d escribed were obtainedfrom the upper Norris Geyser basin area in YellowstoneNational Park. Th e acidic springs selected had tem perature sof 45 to 65C and a pH range of 4 to 5.5. The strains wereisolated by procedures described below.All of the isolates form ed similar colony ty pes o n celluloseagar and D-cellobiose agar, and all showed identical mor-phology. T he type strain, 11B, has been de posited with theAmerican T ype C ulture Collection, Rockville, Md., with thenumber 43068. All strains have been successfully maintainedboth in a freeze-dried state and frozen at -70C after theaddition of 77 pl of dimethyl sulfoxide per ml of culturesuspension.Culture media. Enrichment cultures were prepared with a

low-p hosph ate basal salts medium (LPBM ) which containedthe following, in grams per liter: NH4C1, 1.0; KH2P04, 1.0;N a 2 H P 0 4 . 7 H 2 0 , 0.1; M g S 0 4 . 7 H 2 0 , 0 . 2 ; a n dCaC12 . 2H 20 , 0.2. This medium w as also supplemented withthe following, in grams per liter: ye ast extrac t (Difco Labo -ratories, Detroit, Mich.), 0.5; D-cellobiose, 0.5; and cellulo-sic substrate , 5 . Th e cellulosic sub strates used included filterpaper strips (Whatman number 1;Whatman Ltd. , England),Sigmacell 50 powdered cellulose (Sigma Chemical Co. , S t.Louis, Mo.), carboxymethyl cellulose (46 MF; HerculesInc., Wilmington, Del.), and amorphous phosphoric acid-treated swollen cellulose (28). Solid media containe d 25 g ofBacto-Agar (Difco) per liter. All media were adjusted to pH5.2 and sterilized by auto claving at 121C for 20 min.Isolation conditions. Portion s (9-ml each) of sterile LPBMcontaining 0.05% yeast extract, 1%Sigmacell 50, and filterpaper strips were put up in capp ed culture tubes. Each tubewas inoculated with 1 ml of a slurry of mud and decayingvegetation from acidic (pH 4.5 to 5.5) hot (45 to 65C)springs. Tu bes were placed in a field incuba tor within a fewminutes of collection and incubated for 1 to 2 weeks.Samples showing bacterial growth or signs of cellulosedecomposition were subcultured to fresh tubes of the samemedium.Pure culture isolation. The filter paper strips served as anindicator of cellulolytic activity and in some insta nces pro-vided a solid surfac e for the grow th of colonies of cellulolyticbacteria. Sam ples of the degrading paper strips were teasedoff with an inoculating loop and streake d on plates of LPBMsupplemented with 0.05% yeast extract and 0.5% amorphous(phosphoric acid-swollen) cellulose (3 1). Cellulolytic colo-nies were identified by the presence of a clear zone aroundthe colonies. Th ese colonies were generally found to containmore than a single species; therefore, they were streaked oncarboxymethyl cellulose and D-cellobiose agar containingLPBM supple men ted with yeast extract. Colonies werepicked from these plates and streaked out again on theswollen cellulose agar for the isolation of pure cultures.Microscopy. All of the cultu res were repeatedly exam ined(magnification, X 1,000) under phase-contrast illuminationwith a Nikon B iophot microscope. All isolated pure cultureswere examined when growth first appear ed, and at intervalsof 3 or 4 days thereafter for up to 2 weeks, to search formorphological developmental changes such as endosporeforma tion, motility, or rod-to-coccu s changes suc h as thosereported for Arthrobacter species.

Cells of s trains 10B , 11A, and ll B T , grown t o logarithmicphase in LPBM -yeast extract-D-cellobiose medium , we reexamined by electron microscopy. The cells were pelletedby centrifugation and washed twice with 0.1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfoniccid (HEPES)buffer (pH 6.8). The pellets were suspend ed in 4% glutaral-dehyde (electron microscopy grade; Sigma) fixative, buff-ered with HE PES buffer, and incuba ted at 20C for 1.5 h.Samples were then washed with three changes of HEPESbuffer, set in Noble M aga r, cut into 1-mm cube s, postfixedin 1%buffered osmium tetro xide for 2 h a t 20"C, and washedagain in HEP ES buffer. The sam ples were then stained for 2h in 2% uranyl acetate and washed three times in HEPESbuffer. Samples were dehy drated with an eth yl alcohol series(25 to 100% ethyl alcohol), followed by dehydra tion with anethanol-propylene oxide series (100% ethyl alcohol to 100%propylene oxide), after which the samples were em bedded inEpon-812 resin. A fter curing at 60C for 48 h, th e sampleswere s ec t ioned on a Re ichard t U l t r acu t m ode l E -4ultramicrotome. These thin sections (60 nm each) werestained with a 2% solution of uranyl acetate and stainedagain with Reynolds lead citrate solution. Sections wereexamined with a Phillips EM-400T transmission electronmicroscope operated under standard cond itons.Samples to be examined by scanning electron microsc opywere fixed with glutaraldehyde and osmium tetroxide,mounted on polylysine-coated cover slips, dehydrated withan acetone elution series, subjected to critical point drying,and coated with gold-palladium. The Au-Pd coating wasapproximately 19 nm thick. Sam ples were exam ined with aHitachi HHS-2R electron microscope at a 5-mm workingdistance with 15" of tilt. Magnifications were calibrated withcommercial replicas made from ruled diffraction gratingswith 15,240 lines per in. (1 in . = 2.54 cm) (number 64005;Ladd Research Industries, Burlington, Vt.).

Cell growth. Cell growth in fiquid cultures containing onlysoluble substrates was measured by determination of absor-banc e at 640 nm with a Spec tronic 20 (Bausc h & Lomb, Inc. ,Rochester, N.Y .) spectrophotometer and, in some cases,with a Petroff-Hauser bacteria-counting chamber and aphase-contrast microscope. Cultures containing cellulosewere extremely turbid ; in this case , growth was estimate d bya modified Lowry protein assay (15). The cell pellets weresuspended in Low ry reagent A, an d cellulose a nd cell debriswere removed b y centrifugation after lysis induced by heat-ing to 60C for 20 min. Th e lysates were coole d and c entri-fuged, the supernatants were mixed with Lowry reagent B,and the chromophore was developed as usual. The goodagreement between these two widely different methods isshown in Fig. 1.DNA composition analysis. Deoxyribonucleic acid (DNA )was isolated by the procedure of Marmur (16), modified.Cells were lysed by a lysozyme-ethylene diaminetetraacet-ate (EDTA)-sodium dodecyl sulfate (SDS) treatment. Ly-sates were then extracted twice with phe nol, and the DN Awas precipitated from aqueous layers by the addition of 2volumes of ethanol. After centrifugation, the pellets weredissolved in 50 mM Tris chloride-5 mM E DT A buffer (pH8 O) and treated successively with ribonuclease A (Sigma)and protease K (Sigma). The density of solutions wasadjusted to 1.68 g/ml by the addition of cesium ch loride, andDNA was purified by two cycles of equilibrium densitycentrifugation with a Beckman VTi 50 rotor a t 41,000 rpm for24 h in a Beckman L8-70 ultracentrifuge. Purified DNAsolutions w ere dialyzed against 0.1 x stan dard saline-citratebuffer (SSC)(IxSC is 0.15 M NaCl plus 0.015 M sodium


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VOL. 36, 1986 ACIDOTHERMCJS CELLULOLYT ICUS G E N . N O V . , SP . NOV. 437

1.0

0.8

0E02 0.60Ln

IIs 0.4c3

0.2

citrate), and the DNA melting temperature was determinedwith a Gilford spec trophoto meter eq uipped with the thermalprogramming accessory. T , was determined with Esche-richia coli B DNA (Sigma) as a standard as suggested byOwen and Hill (21). The guanine-plus-cytosine (G +C) con-tent of the DNA was also determined by analyt icalultracentrifugation with a Beckman model E ultracentrifugeby the methods of Vinograd and Hearst (32) and Szybalski(30). By these m ethod s, the appare nt buoyant density of theDN A sample is determ ined from its equilibrium position andfrom that of an internal standard DNA ( E . coli B) in theultracentrifuge cell. This condition was found, from serialphotographs, to take approximately 42 to 48 h at 44,000 rpmin an AN-D ro tor with RTIC temperature control at 25C.DNA samples were prepared in 1 mM Tris-buffered solu-tions of 56.0% cesium chloride (5.65 M) to a n optical densityat 260 nm equal to about 0.02. DN A G + C composition wasestimated with the relationships d escribed by De L ey (6).Amino acid analysis of cell wall preparations. Cells weregrown to mid-logarithmic phase in a batch culture on 1%glucose in LPB M and harv ested by low-speed centrifugation(7,000 rpm , GSA rotor) in a S orvall RC5B centrifuge at 4Cfor 15 min. The cell pellets were washed twice by centrifu-gation in deionized water. A portion of the pellets waslyophilized for am ino acid an alysis. Th e remaining pelletedfractions were suspended in 50 ml of deionized water andhomogenized in a Braun homogenizer a t 3,000 rpm for 8 minat 4C. Glass beads (450 to 500-pm diameter; Sigma) wereused to homogenize the cells. No visible lysis occurredduring this step. Cell suspensions were filtered and sus-pended in 150ml of 2% SDS-25 mM EDTA . Na3 solution.Light microscopy confirmed tha t lysis of the cells occurre dinstantly at room temperature. Also, the viscosity of thesuspensions increased dramatically. The cell suspensionswere placed for 30 min into a water bath held at 60C tocomplete the lysis, and the lysate s were centrifuged at 8,000rpm in a GSA ro tor at 25C. Th e pellets were suspen ded in 20ml of SDS-EDTA solution and stirred at room temperatureovernight. Sus pension s were maintained a t 60C for 30 minand pelleted again by centrifugation. The pellets were the nwashed o nce with 0.2% SD S solution and twice with deion-ized water, and fractions were then lyophilized for aminoacid analysis. T he remaining pellet fractions were su spende din 45 ml of 50 mM sodium phosphate buffer (pH 7.8) with 20mg of crystalline ribonu clease A (Sigma) and incubated fo r 2h at 37C. Th e mixtu res were the n incubated at 37C for 6 hwith 50 mg of trypsin (1:250, vol/vol; Difco) and pelleted bycentrifugation. The pellets were suspended in 1% SDSsolution, heated to 60C for 30 min, and pelleted again.Finally, the pellets were wa shed tw ice with deionized wa terand lyophilized. After dialysis with water and lyophilization,62-pg samples of the cell wall preparatio n were dissolved in5OO-pl portions of 4.7 N HC1 containing 0.05% (vol/vol)phenol and 0.5% (vol/uol) P-mercaptoethanol. The tubeswere e vac uate d, sealed, and the n hydrolyzed at 110C for 20h. Each hydrolysis was performed in duplicate for eachhydrolysis time. The amino acids and amino sugars wereanalyzed with a Beckman model 121 CL amino acid ana-lyzer. The amino acid com position was not normalized forlosses or apparent increases during hydrolysis.Nutritional studies and biochemical tests. The nutritionalrequirements of the three selected strains were determinedby adding the various nutritional supplements to mineralbase LPBM supplemented with trace elements at the follow-ing final concentrations, in milligrams per liter: FeS04 . 7H20,0.5; MnS04 . H 2 0 , 0.1; N a 2 M o 0 4 . 2 H 2 0 , 0.1; CuC12,

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0 .05; CoCl2 . 5 H 2 0 , 0.05; Z n S 0 4 . 7 H 2 0 , 0.05; a n dNa2B 407 10H20,0 .05. Major nutr ients were added at 0.5 to1%conce ntration . Each 5-ml tube of medium w as inoculatedwith 0.1 ml of a wash ed cell suspe nsion in distilled water.Routine biochemical te sts were carried out as described inth e Manual of Methods for General Bacteriology (7) andelsewhere (13).Volatile fatty acids were determined with a Hewlett-Packard model 5840A gas chromatograph equipped with aflame ionization detector and a Carbopack C/0.3% CW20M/0.1% H3P04 packed colum n (6 ft [ l t = 30.48 cm] by 2mm; Supelco). The column, injection port, and detectortemperatures were maintained at 125, 145, and 250"C, re-spectively. Nitrogen served as the carrier gas. The columnwas calibrated with volatile fatty acid stand ards obtainedfrom Supelco. Nonvolatile fatty acids were determined byhigh-pressure liquid chromatography using the HPX-87Hcolumn from Bio-Rad Labo ratories. T he high-pressure liquidchromatography system consisted of a Beckman model llOBpump, a Beckman-Altex model 210 injection valve, and aWaters model 450 variable wavelength detector adjusted to220 nm. The mobile phase was 10% (vollvol) ac etonitrile in0.0007 N H2SO4. Nonvolatile fatty acid standards wereobtained from Sigma.Effectsof temperature and pH on growth. Th e relationshipsamong growth rate, temperature, and pH were determinedby chem ostat stud ies in Biogen an d Bioflo (New BrunswickScientific Co., Inc., Edison, N.J.) fermentors with 2-literreactors ch arged with 1.2 liter of LPBM supplemented with1g of yeast extract per liter and 10g of D-cellobiose per liter.Antibiotic resistance. Antibiotic resistance tests were car-ried out in LPBM with 0.1% yeast extract and 0.5% D-cellobiose. The antibiotics were dissolved in water or anappropriate solvent as stock solutions, filter sterilized, and


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438 MOHAGHEGHI ET A L. I NT . J. S YS T.BACTERIOL.

FIG. 2. Scanning electron micrograph of strain l l B T cultures showing flocculated cells. Bar, 1 km.

added to th e culture medium at the following concentratio ns,in micrograms per ml: 0.1, 1,10,25,50 , an d 100.Tubes wereinoculated and incubated at 55C fo r 72 h. Growth wasfollowed by measurement of the absorbance at 640 nm .Measurement of cellulase activities. Cellulase activity inculture supernatants was measured by a filter paper assay(14) in 25 mM sodium ac etat e buffer (pH 4.8). Samples wereincubated for 1 h at 55 an d 1 h at 65C. Reducing sugarsformed from cellulose were determined by a dinitrosalicylicacid method (18). Reducing sugars produced by cellulosehydrolysis were charac terized further by high-pressure sizeexclusion chromatography (8).Induction of cellulase enzymes. Initial stu dies on cellulaseinduction were con ducted in batch c ultures with shake flasksof LPBM supplemented with 0.5% Sigmacell 50 powderedcellulose o r 0.5% amorphous cellulose at 55C. Cell growthand cellulase activities were m onitored as described abov e.These tests were followed up by batch experiments in theBiogen fermentor s et at 55C and maintained at pH 5.0 by theaddition of sodium hydroxide.

RESULTSEnrichment and isolation. Pure cultures w ere isolated fromthe enrich men t cultures grown on powdered cellulose mediaas described in Materials and Methods. The presence ofpersistently contaminating bacteria necessitated repeatedstreaking on two different kinds of agar. A total of 12 strainswere isolated from th ree different enric hme nts from acidichot springs (pH range, 4 to 5.5; temperature range, 50 to

60C). All of the isolates formed identical colony types onLPBM-yeas t extract-cel lu lose , D-cel lobiose, and car-boxymethyl cellulose agars. All produced cellulase , asshown by clear zones on amorphous cellulose aga r, and allwere similar morphologically. Three strains, 10B, 11A, andll B T , which had the highest cellulolytic activity a s deter-mined by clearing zone assay, were selected for intensivestudy.Cultural characteristics.All 12 strains formed small (1 o 3mm), smooth, circular, entire, creamy white colonies onagar plates containing LPBM -yeast extract-D-cellobioseand amorphous cellulose. Transfers to LPBM plus yeastextract agar with added carboxymethyl cel lu lose, D-cellobiose, or D-glucose yielded the same colony types.Morphology. Upon initial isolation on cellulose agar, allstrains formed sle nder rods and long , slender filaments, ca.0.4 by 5 to 20 Fm. Upon repeated transfer, many of thestrains formed a higher proportion of shorter rods, 0.5 by 2to 5 pm , and fewer long filaments. In liquid med ia, both rods(0.5 x 2 to 5 pm) and filaments were present in a ratio whichvaried from strain to strain, as did the propensity forflocculation. At no time were spor es or othe r resting bodiesproduced by any of the pure cultures. Motility was notobserved, nor were flagella seen in electron micrographs.The large rotund bodies desc ribed for T . aquaticus by Brockand Freeze (4)were never observed, Gram stains wereusually gram negative but occasionally gram positive. Noclear relationship between phases of growth and stainingproperties of these strains has been observed.Scanning and transmission electron micrographs of strain


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VOL.36 , 1986 ACIDOTHERMUS CELLULOLYTICUS G E N . N O V . , S P. N O V . 439

FIG. 3. Transmission electron m icrograph of a thin section of strain l l B T cells. The specimen was fixed with glutaraldehyde-osmiumtetroxide and stained with uranyl acetate and lead citrate. Bar, 0.1 pm.l lBTare shown in Fig. 2 and 3, respectively. Other electronmicrographs (data not shown) demonstrated identical cellwall structures for strains 10B and 1lA. Mesosomelikestructures and internal cell membranes are clearly visible inFig. 3. Also, the outermost side of the cell wall appears tohave a thin, electron-dense layer. Although the plasmamembrane is well defined, no outer cell membranes wereseen in thin sect ions of all three strains at any magnification.Thus, the cell walls are distinctly different from the typicalgram-netative cell walls of T . a q u a t i c u s (4) andThermomicrobium species (9, 23).Influence of temperature and pH on growth rate. Therelationships between temperature and specific growth ratein LPBM plus 0.1% yeast extract and 0.5% D-cellobiosemaintained at pH 5.5 and aerated with 1 volume of air pervolume of fermenter per min are shown in Fig. 4. All threestrains grew between 37 and 70C. From multiple experi-ments, the average temperature optima for growth werefound to be 60, 55, and 50C for strains 10B, llB T, and 11A,respectively. The maximum specific growth rates (k ) wereabout 0.15 per h for all strains, which is equivalent to ageneration time of 6.7 h.The effect of pH on growth determined in the chemostatwith the same medium at 55C is illustrated in Fig. 5. Strains10B and ll BT grew between pH 3 and pH 7, whereas strain11A grew from pH 4 to pH 7. The average pH optimum from

multiple experiments was approximately 5.0 for strains 10Band l lB T; strain 11A had a broad pH optimum, from 5 to 6.Cultures growing in liquid media with D-glucose or D-cellobiose produce organic acids, and the pH may drop from5 to 6 to as low as 3.4, resulting in the death of the culture.Succinic, maleic, and citric acids were found in growthsupernatants at concentrations less than 0.1% (wt/wt); aceticacid was found only in trace quantities.Nutritional studies. The nutritional studies, summarized inTable 1, show that many common nutrients, including cel-lulose, xylan, starch, D-cellobiose, D-maltose, D-raffinose,sucrose, D-trehalose, D-glucose, D-galactose, D-mannose,sorbitol, and D-mannitol, are capable of serving as a solesource of energy and carbon, with ammonium ion as thenitrogen source for all three strains studied. A few nutrientssupported growth of one or more of the three strains, and afew others (acetate, DL-lactate, citrate, and pectin) failed tosupport growth of any strain. The vitamin supplement,containing a mixture of vitamins, failed to stimulate growth.Amino acids, including vitamin-free Casamino Acids(Difco), asparagine, and glutamic acid, were definitelystimulatory. In fact, 0.1% Casamino Acids alone supportedfair growth, as did 0.1% yeast extract plus 0.1% tryptone.There was no growth in nutrient broth o r on nutrient agar atpH 5.5, and not only did citrate and acetate fail to supportgrowth, but they also proved to be inhibitory at 0.01 M


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440 MOHAGHEGHI ET AL. I NT . J . SYST.BACTERIOL.

n 20 40 60 80TEMPERATURE ("C)

FIG. 4. Plot of specific growth rates (p) at different tempera-tures. pH was maintained at 5.5.

concentration. Therefore, the organisms are prototrophic,requiring no vitamins or accessory growth factors.Biochemical tests. The following routine biochemical testswere carried out at 55C in liquid or solid media adjusted tothe pH range 5 to 6: motility, catalase, oxidase, sulfuroxidation, hydrogen sulfide production, nitrate reduction,gelatin liquefaction, casein hydrolysis, growth on egg yolk,indole production, hemolysis of blood agar, methyl red test,Voges-Proskauer test, growth in litmus milk, and citrateutilization (Simmons citrate). No variability in response ofthe three strains to these tests was observed. The catalasetest was positive, indole production was weakly positive,and all other tests yielded negative results. Shake cultures inLPBM plus 0.1% yeast extract and 0.1% D-glucose orD-cellobiose plus 7 g of purified agar (Oxoid Ltd., London,England) per ml yielded growth only near the surface of theagars when incubated in air; no growth was observed innitrogen, indicating that the organisms are obligate aerobes.Salt tolerance tests carried out with strains 10B, 11A, andllBT in LPBM-yeast extract-glucose medium showed thatgrowth was equally good with 0, 0.5, 1.0, 1.5, and 2.0%added NaC1, but growth was poor at 4% added NaC1. Thus,the organisms are moderately osmotolerant.

20

1500X- 10'Lv

5

0

?\

2 3 4 5 6 7 aP H

FIG. 5. Plot of specific growth rates at different pH values. Thetemperature was maintained at 55C.

TABLE 1. Nutritional studiesUtilization by strainasc:

10B 11A llBTCSubstrateStarchD-TrehaloseD-FructoseD-XyloseL- ArabinoseGlycerol

" +, Positive growth; ?, slight growth; -, no growth.The following substrates permitted growth (+) of all three strains:cellulose, xylan, D-cellobiose, D-maltose, D-raffinose, sucrose, D-glucose, D-galactose, D-mannose, D-sorbitol, and D-mannitol. The following substratespermitted no growth (-) of all three strains: acetate, citrate, DL-lactate, andpectin.T, Type strain.

Antibiotic resistance tests. Resistance to 12 antibiotics wasdetermined in liquid cultures with the following concentra-tions of antibiotics, in micrograms per ml: 0.1, 1, 10, 2 5 , 50,and 100. The concentrations of individual antibiotics atwhich no growth of all three strains occurred were asfollows, in micrograms per ml: vancomycin, 1; ampicillinand chloramphenicol, 10; bacitracin, 25 ; polymyxin B andrifampin, 50; and erythromycin and kanamycin, 100. Allthree strains were also resistant to penicillin G at 100 pg/ml.Variable response was observed for actinomycin D, whichprevented growth of strains 10B and 11 A at 10 pg/ml and ofstrain llBT at 25 kg/ml, and for tetracycline, which pre-vented growth of strains 10B and 11A at 25 pg/ml and ofstrain l l B T at 10 pglml.Amino acid analysis of cell wall preparations. The aminoacid and amino sugar compositions of hydrolysates of puri-fied cell wall fractions are presented in Table 2. The aminoacids cysteine, cystine, and tryptophan are not listed in thetable, since they are, to a large extent, destroyed by the

TABLE 2. Amino acid com position of cell wall hydrolysatesAmino acid composition(nmoY100 pl of purifiedcell wall fraction hydrolystate) of strain:

10B-1" 10B-2" 11A llBTAmino acid inhydro1ysate

AspartateThreonineSerineMuramic acidbGlutamateProlineGlycineAlanineValineDiaminopimelic acidMethionineIsoleucineLeucineTyrosinePhen ylalanineGlucosamineHistidineLysineArginine

1.81.77.13.28.11.83.48.82.510.50.71.22.80.91.37.9ND'1.51.6

2.62.39.66.19.72.14.510.63 O20.10.41.23.51.21.714.21.12.92.5

2.72.38.35.78.72.04.210.42.510.40.91.33.91 o1.713.71.01.72.3

5.95.613.05.314.14.010.220.56.214.72.04.69.42.33.519.21.62.55.1~~ ~~" Preparations 10B-1 and 10B-2 correspond to two different pellets which

A severe destruction (up to 75%) of muramic acid occurred during diluteND, Not detected.

formed during the last step of purification.acid hydrolysis.


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VOL. 36, 1986 ACIDOTHERMUS CELLULOLYTICUS GE N. NOV. , SP. N O V . 441TABLE 3. Properties of different species and strains of the gene ra Thermus, Thermomicrobium, and Acidothermus

Species Pigmentation Nutrition Temperaturegrowth pc) optimum p~ (mol ratio o/o) Reference(s)range for pH range for growth/ DNA G +CT. aquaticus YT-1 YellowThermus spp strain X-1 WhiteT . lavus AT-62 YellowT. thermophilus HB-8 YelowT. aquaticus (many strains)T. ruber RedThermomicrobium sp. (ATCC 27502) PinkA. cellulolyticus l l BT Whi t e

Some white,som e yellow

Pro o rop hND "Requires vitaminsand amino acidsRequires vitaminsand amino acidsN DRequires yeast extractPrototrophPrototroph

40-79170 6-9.5J7.5-7.840-80170 ND (adjusted to 8)40-80170 6-917.0-7.547-85165-72 5.1-9.617 . O-7.547-85 ND35-70160 8.Qb70-75b 8.3b37-7OJ55-60 3-715.5

65.4-67.46466.669

N D666460.7 2 0 .6

4232520511, 129This work

a ND, Not determined.Optimum.hydrolysis steps. The enrichment in amino acids common inpeptidoglycans and other cell wall components can beclearly seen. Diaminopimelic acid, glucosamine, andmuramic acid were notably enriched. The amino acids whichcan be ascribed to the cell wall peptidoglycan on the basis oftheir ratio to glutamic acid (>0.5), which is a known com-ponent of peptidoglycans in most gram-negative and gram-positive bacteria (19, 26), are serine, alanine, anddiaminopimelic acid, and perhaps glycine and leucine instrain llBT. The amounts of alanine, diaminopimelic acid,and glucosamine seemed to be consistently higher thanglutamic acid. Other dibasic amino acids which are commonin cell walls of bacteria were either not detected (e.g.,ornithine) or partially removed by the treatment (e.g., ly -sine). Galactosamine was also not detected.

400

0- 300E-c(u.-+:2 200Q,

mI$0LKc3 100

20 30 40TIM E (hours)

0 0 10

0.5

0,0.4-:

L-!=I-E

0.3 p2

-0,

-aJm3Q,-

0.2 ~E>v!=20.1 0aI-

0

FIG. 6. Correlation of strain l l B T cell growth and the productionof cellulase activity found in portions taken from the culturesupernatant and subjected to incubation with cellulose substrate at55 and 65C.

Base composition of DNA. G+C composition of DNAisolated from strains 10B, 11A, and l lBTwas determined bythermal melting studies and isopycnic banding centrifuga-tion. N o strain specificity was apparent, as the G+C contentwas found to be 61.1 f 0.2 mol% G+ C and 60.3 5 0.3 mol%G+C from T , values of 81.8 2 0.1"C and buoyant densityvalues of 1.7196 5 0.0003 g/ml, respectively, for the threestrains of Acidothermus cellulolyticus.Induction of cellulase activity. All three strains producedactive cellulase enzymes when grown on powdered or amor-phous cellulose in liquid cultures. An example of an induc-tion of cellulolytic activity secreted by the batch culture ofstrain ll BTgrown aerobically in the liquid LPBM containing0.5% powdered cellulose (Sigmacell 50) as a carbon sourceat 55C and pH 5.0 is shown in Fig. 6 . The cellulolyticactivity was secreted mainly during the late logarithmic andearly stationary phases of growth, and the pattern of induc-tion was similar for all three strains. The cellulase enzymecomplexes can completely degrade microcrystalline cellu-lose (Sigmacell 50 and a-cellulose [Sigma]); this indicatesthat the full complement of cellulolytic enzymes is beingsecreted. The products of cellulose hydrolysis are D-glucoseand D-cellobiose. No higher oligosaccharides were detectedby high-performance size exclusion chromatography. A de-tailed study on the induction and characterization ofcellulase enzymes produced by these microorganisms willappear in a future publication.

DISCUSSIONAs a result of screening, several isolates of thermophilic,

cellulolytic bacteria growing in the acidic pH range wereobtained.The microorganisms we isolated are a result of selectiveconditions applied during enrichment and isolation and donot represent major microbial populations in geothermal hotsprings, The number of thermophilic microorganisms whichwere discarded during the isolation process far exceeds thenumber of isolates we obtained. It should be noted, how-ever, that no thermophilic fungi, yeasts, or actinomyceteswere observed during enrichment and isolation. The major-ity of bacteria we observed during the initial stages ofisolation were sporulating rods resembling bacteria of thegenus Bacillus. The second surprising result was a unifor-mity of cellulolytic strains we isolated from various springs.The isolated thermophiles are all nonpigmented, gram-variable rods. These morphological features and theheterotrophic, obligately aerobic mode of growth are similar


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442 MOHAGHEGHI ET A L.TABLE 4. Sensitivity of Therrnus species to antibiotics andinhibitors

Sensitivity of":Antibiotic (&ml) T . aquaticusb T' f l a ~ ~ ~ ~ ~ ~ ~ s o ' i d. thermophilusd

Actinomycin D1Penicillin

110Novobiocin10100Chloramphenicol10100Streptomycin

10100Tetracycline10100C closerine10100SD S10100

- N D ~ I -

N D N D /-- ND/-

- +I+N D -I+

- ND/+ND ND/-

+-

+/+-I*

2 +/+-/ +

ND

N D

N D

N D

- at 144 pg/mla For symbols, see Table 1, footnote a . ND , Not determined.Solid medium; reference 4.Reference 25.Liquid medium; reference 20.

to those reported for Thermus and Thermomicrobium strainswhich were isolated from similar enviroments (4, 9, 20, 22).The characteristic features of Thermus and Thermo-microbium species are summarized in Tables 3 and 4 and arecompared with our isolates. Our isolates share with Thermusand Thermomicrobium strains several important features,namely morphology, sensitivity to lysozyme, and presenceof catalase. They differ in other important aspects, such asthe pattern of carbon sources utilized for growth, pH andtemperature profiles of growth, the pattern of sensitivity toantibiotics, the G+C content of DNA, the composition ofamino acids in cell walls, and the structure of the cell walls.Also, Thermus spp. are very sensitive to penicillin G, towhich our strains are resistant at 100 pg/ml, and the dibasicamino acid in cell walls of Thermus spp. is ornithine (22),whereas in our strains it is diaminopimelic acid. Serine isanother amino acid in cell walls of our isolates which is notpresent in the cell walls of Thermus spp. Finally, we couldnot detect D-galactosamine, which is present in cell walls ofThermus spp. The cell wall structure as revealed by trans-mission electron microscopy resembles the cell wall struc-ture of gram-positive bacteria since it lacks an outer mem-brane, which is an important characteristic of gram-negativebacteria of the genus Thermus (3). The genusThermomicrobium is morphologically distinctly differentfrom our isolates since it forms small, short, pleomorphiccells rather than the long slender filaments of our organism.Therefore, me must conclude that the strains we isolated

INT. J . S Y S T .BACTERIOL.belong to a new species in a new genus of thermophilicbacteria.Based on the experimental results presented above, webelieve that a new genus should be created to accommodatethis organism, for which we propose the nameAcidothermus. The type species of the genus is A .cellulolyticus. The type strain is our 11B, ATCC 43068.Description of Acidothermus gen. nov. (L . adj, acidus acid;Gr. n. thermus heat; M. L. masc. n. Acidothermus acid andheat [loving]). Morphology: slender rods and long, slenderfilaments, 0.4 by 5 to 20 ym, which have rounded ends. Doesnot form endospores, flagella, or rotund bodies. Nonmotile.Gram variable, but usually stains gram negative. Thin sec-tions show no outer cell membranes and thus are distinctlydifferent from the typical gram-negative cell walls ofThermus and Thermomicrobium species. The principal con-stituents of purified cell walls are diaminopimelic acid,glucosamine, muramic acid, serine, and alanine.Colony characteristics: on LPBM mineral salts agar with0.5 g of yeast extract per liter and 5.0 g of D-glucose orD-cellobiose per liter, creamy white, smooth, circular, en-tire, 1 to 3 mm.Liquid cultures: on same medium lacking agar show amoderate uniform turbidity. Some strains tend to flocculateand sediment out after 3 to 10 days of incubation.Physiology: obligate aerobes, prototrophic; grow on sev-eral carbohydrates, including D-glucose and D-cellobiose.Temperature characteristics: thermophilic, growth range37 to 70C. pH requirements: acidophilic; pH range forgrowth, 3.5 to 7.DNA base ratio, 60.7 2 0.6 mol% G+C.Source: acidic hot springs at Yellowstone National Park,

The type species is A . cellulolyticus.Description of A . cellulolyticus sp. nov. (L. M . cellulosacellulose, lytic Gr. v . break up; cellulolyticus cellulosedissolving).

pH 4 to 5.5, 45 to 65C.

Morphology: as described for the genus.Colony characteristics: as described for the genus.Temperature characteristics: grow from 37 to 70"C, withoptima between 50 and 60C.pH requirements: pH range 3.5 to 7, with an optimum near5.0.Physiology: obligate aerobes; prototrophic, grow on D-glucose, D-cellobiose, cellulose, xylan, D-galactose,maltose, sucrose, raffinose, D-mannose, D-mannitol, or D-sorbitol as sole source of carbon and energy, with ammo-nium ion or amino acids as a source of nitrogen. Grows onCasamino Acids (0.1%) plus tryptone (0.1%); no growth onnutrient broth, acetate, lactate, citrate, or pectin. Citrate andacetate inhibitory at 0.01 M. Resistant t o penicillin G at 100k.g/ml; sensitive to vancomycin and lysozyme. Catalasepositive. Actively digests cellulose.The type strain is strain 11B (ATCC 43068).Description of the type strain. Strain 11B (ATCC 43068T)has all of the characteristics given for the species. Inaddition, it utilizes D-fructose, L-arabinose, and glycerol as asole source of carbon, and it appears to be slightly moreresistant to actinomycin D and slightly more sensitive totetracycline than strains 10B and 11A.

ACKNOWLEDGMENTSWe thank T. J . Beveridge for performing thin-section transmis-sion electron microscopy and M . P. Tucker for assistin g with DNAthermal-melting analys is.


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VOL . 36, 1986 A C IDOT HE R MUS C E L L UL OL Y T IC US GE N. NO V. , SP. NOV. 443This work was supported by the Office of Alcohol Fuels of theDepartment of Energy under WPA no. 349.

LITERATURE CITED1. Bellamy, W. D. 1977. Cellulose an d lignocellulose digestion bythermophilic actinomyces for single-cell protein production.

Dev. Ind. Microbiol. 18:249-254.2. Brock, T. D. 1978. Thermo philic microo rganisms a nd life at hightemperatures. Springer-Verlag, New York.3. Brock, T. D., and M. R. Edwards. 1970. Fine structure ofThermus aquaticus, an extreme thermophile. J . Bacteriol.4. Brock, T. D., and H. Freeze. 1969. Thermus aquaticus gen. n.and sp. n. , a nonsporulating extreme thermophile. J. Bacteriol.98:289-297.5. Brock, T. D., and I. Yoder. 1971. Thermal pollution of a smallriver by a large university: bacteriological studies. Proc. Indian aAcad. Sci. 80:183-187.6. De Ley, J. 1970. Reexam ination of the association betweenmelting point, buoyant density, an d chemical base compositionof deoxyribonucleic acid. J. Bacteriol. 101:738-754.7. Gerhardt, P., R. G. E. Murray, R. N. Costilow, E. W. Nester,

W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.). 1981. Manualof methods for general bacteriology. American Society forMicrobiology, Washington, D.C.8 . Himmel, M. E ., M. D . Tucker, and K. K. Oh. 1983. Ap plicationsof high performance size exclusion chromatography in biomassprocessing. Biotechnol. Bioeng. Symp. 13583-595.9. Jackson, T. J., R. F. Ramaley, and W. G. Meinschein. 1973.Therrnomicrobiurn, a new genus of extremely thermophilicbacteria. Int. J. Syst. Bacteriol. 23:28-36.10 . Loginova, L. G., L. A. Belyakova, E. P. Guzkova, I. K.Yusupova, L. G. Burdenko, and L. M. Seregina. 1983.T h e r m o p h i l i c , c e l l u l o s e - d e c o m p o s i n g M y c e l i o p h t h o r athermophila. Mikrobiologiya 52:605-608.11. Loginova, L. G., and L. A. Egorova. 1975. An obligatelythermophilic bacterium Therrnus ruber from hot springs inKamchatka. Mikrbiologiya 44:661-665.12 . Loginova, L. G., L. A. Egorova, R. S. Golovacheva, and L. M.Seregina. 1984. Thermus ruher sp. nov. , nom. rev. Int. J . Syst .Bacteriol. 34:49&499.13 . MacFaddin, J. F. 1976. Biochemical tests for identification ofmedical bacteria. The Williams & Wilkins Co., Baltimore.14 . Mandels, M., and R. E. Andreotti. 1976. Measurement ofsaccharify ng cellulase. Biotechnol. Bioeng . Sym p. 6: 17-34.15 . Markwell, M. A. R., S. Hass, N. Tolbert, an d L. Bieber. 1981.Protein determination in membrane and lipoprotein samples:m a n u a l a n d a u t o m a t e d p r o c e d u r e s . M e t h o d s E n z y m o l .72:296303.16 . Marmur, J. 1961. A procedure for the isolation of deoxyribo-nucleic acid from microorganisms. J. Mol. Biol. 3:208-218.

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17 . McCracken, L. D., and C. H. Gong. 1982. Fermentation ofcellulose and hemicellulose carbohydrates by thermotolerantyeasts. Biotechnol. Bioeng. Symp. 12:91-102.18 . Miller, G. L. 1959. Use of dinitrosalicylic acid reagent fordetermination of reducing sugar. Anal. Chem. 31:426-428.19 . Mirelman, D. 1979. Biosynthesis and assembly of cell wallpeptidoglycan, p. 115-168. In M. Inouye (ed.), Bacterial outermembranes. John Wiley & Sons , Inc ., New York.20 . Oshima, T., and K. Imahori. 1974. Description of Therrnust h er rn oph i l u s ( Y o s h i d a a n d O s h i m a ) c o m b . n o v . , anonsporulating thermophilic bacterium from a Jap anese thermalspa. Int. J. Syst. Bacteriol. 24:102-112.21 . Owen, R. J., and L. R. Hill. 1979. The estimation of basecompositions, base pairing, and genome sizes of bacterialDN As, p. 277-296. In F. A. Sk inner and D. W. Lovelock (ed.),Identification methods for microbiologists. The Society forApplied Bacteriology Technical Series number 14. AcademicPress , Inc . , New York.22 . Pask-Hughes, R. D., and R. A. D. Williams. 1977. Yellow-pigmented strains of Thermus spp. from Icelandic hotsprings. J.Gen. Microbiol. 102:375-383.23 . Ramaley, R. F., and J . Hixson. 1970. Isola t ion of anonpigmented, thermophilic bacterium similar to Therrnusaquaticus. J . Bacteriol. 103527-528.24 . Rosenberg, S. L. 1978. Cellulose an d lignocellulose degradationby thermophilic and thermotolerant fungi. Mycologia 70: 1-13.25 . Saiki, T., R. Kimura, and K. Arima. 1972. Isolation andcharacterization of extremely thermophilic bacteria from hptsprings. Agric. Biol. Chem. 36:2357-2366.26 . Schleifer, K. H., and 0. Kandler. 1972. Peptidoglycan types ofbacterial cell walls and their taxonomic implications. Bacteriol.Rev. 36:407477.27 . Shaker, H. M., M. A. Farid, and A. I. El-Diwang. 1984.Optimization of the composition of the nutrient medium forcellulase and protein biosynthesis by thermophilic Aspergillusfurnigatus, NRC 272. Enzyme Microb. Technol. 6:212-216.28 . Stutzenberger, F. J. 1979. Degradation of cellulosic substancesby Thermornonospora curvata . Biotechnol. Bioeng. 219 9-91 3.29 . Su, T. M., and J. Paulavicius. 1975. Enzymatic saccharificationof cellulose by thermophilic actinom yces. Appl. Polym. Symp .30 . Szybalski, W. 1968. Equilibrium sedimentation of viruses, nu -cleic acids and other macromolecules in density gradients.Fractions 1:l-15.31 . Tansey, M. R . 1971. Agar diffusion a ssay of cellulolytic ability ofthermophilic fungi. Arch. Microbiol. 77:l-11.32 . Vinograd, J., and J. E. Hearst. 1962. Equilibrium sedimentationof macromolecules and viruses in a density gradient. Fortschr.Chem. Org. Naturst. 20:327422.33 . Zeikus, J. G . 1979. Thermo philic bacteria: ecology, physiologyand technology. Enzyme Microb. Technel. 1:243-252.

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