APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1984, p. 88-93 Vol. 48, No. 10099-2240/84/070088-06$02.00/0Copyright ©D 1984, American Society for Microbiology

Isolation and Characterization of an Anaerobic, CellulolyticBacterium, Clostridium cellulovorans sp. nov.

ROBERT SLEAT,'t ROBERT A. MAH,l* AND RALPH ROBINSON2Division of Environmental and Occupational Health Sciences, School of Public Health, University of California, Los

Angeles, California 90024,1 and Department of Microbiology and Cell Science, University of Floridta, Gainesville, Florida326112

Received 13 February 1984/Accepted 21 March 1984

A new anaerobic, mesophilic, spore-forming cellulolytic bacterium is described. Cellulose is cleared within24 to 48 h around colonies formed in cellulose agar roll tubes. Cells stain gram negative and are nonmotile rodswhich form oblong spores either centrally or subterminally in a clostridial swelling. Colonies are irregular withan opaque edge and a center devoid of both vegetative cells and spores. Cellulose, xylan, pectin, cellobiose,glucose, maltose, galactose, sucrose, lactose, and mannose serve as substrates for growth. H2, C02, acetate,butyrate, formate, and lactate are produced during fermentation of cellulose or cellobiose. The temperatureand pH for optimum growth are 37°C and 7.0, respectively. The DNA composition is 26 to 27 mol% guanineplus cytosine. This bacterium resembles "Clostridium lochheadii" in morphological and some biochemicalcharacteristics but is not identical to it. The name Clostridium cellulovorans sp. nov. is proposed. The typestrain is 743B (ATCC 35296).

The potential for converting cellulosic wastes into indus-trial substrates has stimulated current interest in cellulosefermentation. The thermophilic degradation of cellulose isespecially interesting because axenic cultures of bacteriaferment cellulose directly to ethanol and organic acids (4, 15,19) and because the rate of cellulolysis is presumably morerapid at elevated temperatures (3). However, mesophilic,anaerobic, cellulolytic bacteria (14) were recently shown todegrade cellulose at rates comparable to those of thermophil-ic bacteria (24). Previously, Hungate (10) isolated a mesophi-lic, spore-forming bacterium, "Clostridium lochheadii",which used cellulose in agar roll tubes after 24 h of incuba-tion. This rapid rate of cellulolysis was not achieved withother anaerobic organisms subsequently isolated. Unfortu-nately, Hungate's culture was lost, although there have beenunconfirmed reports of its reisolation (22). During our cur-rent investigation, an anaerobic, mesophilic digestor whichfermented woody biomass yielded a bacterium which lysedcellulose in roll tubes within 24 to 48 h. This isolate wasmorphologically similar to "C. lochheadii" but differed incertain metabolic characteristics. We report here the isola-tion and characterization of this extremely cellulolytic bacte-rium.

MATERIALS AND METHODSIsolation of bacterial strains. Strain 743B was isolated from

a batch methanogenic fermentation of finely divided hybridpoplar wood (ca. 0.8 mm in size) (12). The inoculum wasdecimally diluted in agar roll tube medium with pebble-milled cellulose as substrate. The final culture volume was 5ml contained in roll tubes (16 by 150 mm; Bellco Glass, Inc.,Vineland, N.J.), sealed with no. 0 butyl rubber stoppers(Arthur H. Thomas Co., Philadelphia, Pa.) and outgassedwith N2-C02 (80:20). Cultures were incubated vertically inthe dark at 37°C. Zones of clearing appeared around thecellulose-fermenting colonies within 1 to 2 days. Colonieswere picked and diluted in cellulose agar medium. This

* Corresponding author.t Present address: BioTechnica Limited, 5 Chiltern Close, Cardiff

CF4 50L, United Kingdom.

procedure was repeated three times before cellulolytic colo-nies were transferred by serial decimal dilution into cellobi-ose agar medium. Well-isolated colonies were picked fromcellobiose agar back to pebble-milled cellulose agar medium.

Culture methods. The anaerobic techniques of Hungate(11) were used throughout these experiments. During thepreparation of culture medium and anaerobic stock solutionsof reagents, an anaerobic glovebox (Coy Lab Products, AnnArbor, Mich.) was also used. Nutritional and metabolicexperiments were performed in Balch tubes (18 by 150 mm;Bellco) containing 10 ml of the appropriate broth medium.Avicel (type PH-105, lot no. 5150-128; FMC Co., Philadel-phia, Pa.) utilization experiments were performed in 120-mlserum vials (Wheaton Scientific Co., Millville, N.J.) contain-ing 20 ml of broth medium. Growth on pectin (SigmaChemical Co., St. Louis, Mo.), xylan (Sigma), gum arabic(Sigma), protein, and starch (Difco Laboratories, Detroit,Mich.) was tested in roll tubes (16 by 150 mm; Bellco)containing 5 ml of agar medium. Transfer of inocula andmedium components was made with 1- or 10-ml Brunswickdisposable plastic syringes (Sherwood Medical Industries,Deland, Fla.) fitted with 21- or 23-gauge needles (Sherwood).Both syringes and attached needles were repeatedly flushedwith 02-free gas before use.

Culture media. N2-CO2 (80:20) was used as the gas atmo-sphere for all culture studies except the fermentation balanceand Avicel degradation experiments, in which 100% N2 wasused. A cellulose slurry was prepared by pebble-milling 15 gof Whatman no. 1 filter paper in 450 ml of distilled water for72 h in a 0.3-gallon (ca. 1.14-liter) high-alumina grinding jar(American Scientific Products, McGaw Park, Ill.) at ca. 100rpm. Pebble-milled cellulose medium contained:K2HPO4. 3H20, 1 g; NH4Cl, 1 g; KCl, 0.5 g;MgSO4 * 7H20, 0.5 g; L-cysteine hydrochloride monohy-drate, 0.15 g; Trypticase (BBL Microbiology Systems,Cockeysville, Md.), 0.5 g; yeast extract (Difco), 0.5 g;cellulose slurry, 330 ml; clarified rumen fluid (6), 20 ml; tracemetal solution (6), 20 ml; and resazurin 0.001 g. Distilledwater was added to bring the final volume to 1 liter. Themedium was solidified by the addition of 15 g of agar (Difco)per liter. The pH was adjusted to 7.0 with 4 M NaOH, and

88

CLOSTRIDIUM CELLULOVORANS SP. NOV. 89

the medium was boiled under O0-free N2. After reduction of

resazurin, the medium was cooled for 10 min at room

temperature under 100% N2. The flask was stoppered and

transferred to an anaerobic glovebox where the medium was

dispensed with a Repipet Junior Dispenser (Labindustries,Berkeley, Calif.) into roll tubes (4.9 ml per tube) and

stoppered. After removal from the glovebox, the tubes were

outgassed with N2-CO2 and sterilized by autoclaving. Just

before inoculation, 0.05 ml of sterile anaerobic 10% (wt/vol)Na2CO3 and 1.5% (wt/vol) Na2S 9H2O was added to each

tube. The final pH was ca. 7.2. Cellobiose agar medium was

similarly prepared except that the cellulose slurry was

replaced by an equal volume of distilled water. After auto-

claving, 0.1 ml of a sterile anaerobic 0.5 M cellobiosesolution was added to each tube.

Modified (M) medium was used for the maintenance andnutritional characterization of the isolates. M medium dif-fered from pebble-milled cellulose medium by omission ofTrypticase, rumen fluid, and cellulose. Stock cultures were

usually maintained in agar roll tubes containing 5 ml of Mmedium plus 1% (wt/vol) pebble-milled cellulose. Whenother growth substrates were added, the cellulose slurry wasreplaced by an equal volume of distilled water. Pectin,xylan, gum arabic, skim milk, and starch were added to Mmedium before autoclaving to give a final concentration of1% (wt/vol). Other substrates were added as sterile anaero-

bic stock solutions at a final concentration of 10 mM (finalvolume per tube, 10 ml).

Fermentation products from 10 mM cellobiose and 10 g ofAvicel per liter were determined in M broth medium bufferedat pH 7 with 50 mM PIPES [piperazine-N,N'-bis(2-ethane-sulfonic acid)] (Calbiochem-Behring Corp., La Jolla, Calif).The final gas phase was 100% N2, and Na2CO3 was omitted.

Morphological, biochemical, and physiological tests. Gramstain reaction, lysis by KOH, and hydrolysis of L-alanine-4-nitroanilide were determined by the methods of Carlone etal. (2). Morphological observations of cultures grown on Magar with pebble-milled cellulose and M broth containingcellobiose were made by both phase-contrast and electronmicroscopy. Substrate utilization was examined by inoculat-ing the test medium with 0.1 ml of an overnight M brothculture grown on cellobiose. Growth was measured byreading the optical density of a culture with a Bausch &Lomb spectronic 710 spectrophotometer (Bausch & Lomb,Inc., Rochester, N.Y.) at 600 nm, by detecting H2 formationby gas-liquid chromatography, and by observation ofcolo-nies in roll tubes. Cultures in both broth and agar media were

transferred at least twice on the same substrate. Starchhydrolysis, gelatin liquefaction, and catalase production were

determined by the methods of Smibert and Krieg (23). A 0.1-ml inoculum of a 24-h PIPES-cellobiose-grown culture was

used for fermentation balance determinations. For Aviceldegradation, 0.5 ml of a 72-h PIPES-Avicel-grown inoculumwas used. Unless otherwise stated, all cultures were incubat-ed at 37°C. Temperature and pH optima were examined in Mbroth containing cellobiose. The pH was adjusted withNa2CO3, and a 0.1-ml overnight M broth cellobiose-grownculture served as the inoculum.

Analytical techniques. Culture head space gases were

analyzed by gas chromatography as previously described(1). Volatile and nonvolatile fatty acids and alcohols were

measured with a Varian Aerograph series gas chromato-graph equipped with a flame ionization detector. Volatilefatty acids were analyzed by the following procedure. A 1-mlsample of culture fluid was transferred to a 1.5-ml cappedplastic centrifuge tube (Beckman Instruments Inc., Palo

Alto, Calif.) held in ice; 0.1 ml of 10 M H3PO4 was added toeach sample. After centrifugation at 11,600 x g for 15 min ina Microfuge 11 (Beckman), 2 [L of the supernatant liquid wasinjected into the gas chromatograph. A glass column (10 ft[ca. 3.05 m] by 2-mm inner diameter) packed with 15%SP1220-1% H3PO4 on 100/120 Chromosorb W AW (Supelco)served as the support material, and the carrier gas washelium (40 ml/min). The column was held at 140°C for 2 minand then raised to a final temperature of 180°C at a rate of10°C/min. The injector and detector temperatures were 170and 200°C, respectively. Nonvolatile fatty acids were deter-mined after the methyl derivatives were prepared by themethod of Holdeman et al. (8); 2 pl of the chloroform extractwas injected into the gas chromatograph as previouslydescribed for the volatile fatty acid determination. Samplesfor alcohol analysis were prepared by centrifugation at11,600 x g for 15 min in a Microfuge 11 centrifuge (Beck-man) to remove cells. Again, 2 pll of supernatant fluid wasinjected into the gas chromatograph. For alcohol determina-tion, a glass column (10 ft by 2 mm inner diameter) packedwith 5% Carbowax 20M on 60/80 Carbopack B (Supelco)was used. The carrier gas was helium (40 ml/min). Thecolumn was operated isothermally at 110°C, and the injectorand detector temperatures were 170 and 200°C, respectively.Lactate was also determined enzymatically with lactatedehydrogenase (lactic acid diagnostic kit; Sigma). Formatewas determined by the method of Lang and Lang (13);reducing sugars were determined with dinitrosalicylic acidreagent (18), and glucose was determined by the DirectGlucose Test Set (Stanbio Lab Inc., San Antonio, Tex.).Residual Avicel was measured gravimetrically by the meth-od of Weimer and Zeikus (24).

Microscopy. A Zeiss Universal research microscope (Carl

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0 --" -

FIG. 1. Cellulose roll tube culture showing empty center of thecolonies and surrounding zones of cellulose hydrolysis.

VOL. 48, 1984

90 SLEAT, MAH, AND ROBINSON

FIG. 2. Scanning electron photomicrograph of cell clump fromcellobiose-grown liquid culture. Bar, 5 p.m.

Zeiss, Oberkochen, West Germany) equipped with phaseoptics, epifluorescence, and an automatic photomicrograph-ic exposure system was used for phase-contrast photomi-crography. For thin-section electron microscopy, colonies ofM agar-grown cells on pebble-milled cellulose were fixed for20 min in 2% formaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2). After washing with buffer,they were fixed in buffered 1% OS04 for 1 h. Cells were thenenrobed in agar, dehydrated, and embedded in Spurr plastic.Thin sections were stained with uranyl and lead salts.Sections were examined with a Jeol 100CX electron micro-scope. For scanning electron microscopy, a clump of cellsgrown in M broth medium on cellobiose was fixed for 20 minin 2.5% glutaraldehyde in cacodylate buffer. It was thendehydrated in alcohol and critical point dried. After goldcoating, the colony was observed in a Hitachi S-450 scanningmicroscope.DNA isolation and analysis of base composition. Whole cell

DNA was extracted and purified by the method of Marmur(17). The buoyant density of the purified DNA was deter-mined by preparative ultracentrifugation in a cesium chlo-ride gradient (20). DNA from Escherichia coli B and Micro-coccus lysodeikticus were used as controls. The DNA baseratio (moles percent guanine plus cytosine [G+C]) wascalculated by the method of Schildkraut et al. (21).

Reagents, chemicals, and gases. All chemicals were reagentquality, except where otherwise noted. Gas mixtures werepurchased from Matheson (Searle Medical Products, Cuca-monga, Calif.). Other gases were purchased from LiquidCarbonics, Los Angeles, Calif. Avicel was a gift from FMCCorp.

RESULTSColonial and cellular features of strain 743B. Zones of

clearing in M medium containing pebble-milled celluloseappeared within 1 to 2 days. No distinct colony was visible atthis stage. After 3 to 4 days of incubation, colonies could bedetected by using a dissecting microscope. Deep colonieswere irregular with an opaque edge and empty center (Fig.1). Zones of cellulolysis may reach 35 mm in diameter uponprolonged incubation. The cellulose in agar roll tubes con-

taining high numbers of cellulolytic colonies was completelyhydrolyzed within a few days. Vegetative cells and sporeswere only found at the periphery of the colony and werecompletely absent from the center. As the colony enlarged,vegetative cells and spores previously located at the periph-ery of the colony disintegrated. Surface colonies spreadrapidly and were almost invisible except for a thin opaqueedge. Colonies in cellobiose-agar medium after 48 h ofincubation were 1 to 2 mm in diameter, creamy-white, andopaque with an entire margin. As the colony matured, itbecame rhizoid.

Cells of strain 743B always stained gram negative andwere L-alanine-4-nitroanilide and KOH negative. The cigar-shaped vegetative cells were 0.7 to 0.9 p.m by 2.5 to 3.5 p.m(Fig. 2). Vegetative cells elongated to form a presporangialcell, which then swelled to form the mature sporangium ofthe clostridial type (Fig. 3). The clostridial cells were 1.5 to2.0 p.m in width and 4 to 7 p.m in length and often spindleshaped. Oval to oblong spores were located either centrallyor subterminally (Fig. 3). The mature spores were 1.0 to 1.5p.m in width and 2 to 4 p.m in length (Fig. 4). Cultures ofstrain 743B held at 80dC for 1 min readily initiated growthafter inoculation into fresh medium. Cultures held at 80°C for5 or 10 min also grew, but after a long lag phase (1 to 4weeks).

Strain 743B was nonmotile, although peritrichous flagellawere observed under electron microscopy. Inclusion bodieswere also present (Fig. 3). Vegetative cells were resistant tosodium dodecyl sulfate treatment but lysed readily in thepresence of lysozyme-EDTA.Growth and nutrition. Strain 743B was an obligate anaer-

obe. Rumen fluid, Trypticase, and added vitamins were notrequired for growth. Nutritional requirements were met byadding yeast extract to the medium, although no growthactually occurred at the expense of yeast extract. In additionto cellulose, pectin, xylan, cellobiose, glucose, fructose,maltose, galactose, sucrose, lactose, and mannose were alsofermented. Gum arabic, rhamnose, melezitose, sorbitol,trehalose, xylose, arabitol, arabinose, glycerol, erythritol,lactate, and pyruvate were not fermented. Gelatin was notliquified, and starch and casein were not hydrolyzed. Strain743B was catalase negative and did not reduce sulfate.

FIG. 3. Thin section of cellulose agar-grown culture. Arrowsindicate spores within clostridial cell. Inclusion bodies are alsopresent. Bar, 5 p.m.

APPL. ENVIRON. MICROBIOL.

CLOSTRIDIUM CELLULOVORANS SP. NOV. 91

601

FIG. 4. Photomicrograph of mature spore and vegetative cells.Bar, 10 ,um.

Colonies growing in pectin roll tubes were yellow. Noyellow pigment was observed on cellulose in either solid orliquid medium. Growth in liquid medium containing a solu-ble carbohydrate was characterized by extensive floccula-tion of the culture. The viscosity of the culture fluid alsoincreased due to the production of extracellular material.Spore formation was inhibited during growth on solublecarbohydrates in liquid medium. Cellulose was readily fer-mented in medium buffered with PIPES, HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), MOPS(morpholinepropanesulfonic acid), or ACES [N-2-aceta-mido)-2-aminoethanesulfonic acid] organic buffers but wasinhibited in the presence of Tris or bis-Tris propane.The optimum temperature for growth was 37°C, and

growth occurred over the range 20 to 40°C (Fig. 5). Nogrowth was observed at 15°C on prolonged incubation. Theoptimum pH for growth was about 7, and the range was 6.4to 7.8 (Fig. 6).

Fermentation products. Strain 743B produced acetate,butyrate, lactate, formate, H2, and CO2 from cellobiose(Table 1) and cellulose (data not shown). Trace amounts ofethanol were also detected. Pyruvate and succinate were notdetected.

Cellulose utilization. Growth of strain 743B on pebble-milled cellulose in liquid medium was characterized by arapid evolution of gas bubbles. Gas formation was so vigor-ous that the cellulose occasionally floated to the surface ofthe culture medium. The maximutn rate of cellulose degrada-tion in carbonate-C02-buffered M medium (initial pH 7) was53 mg/liter per h. A maximum of 83 mg of Avicel wasdegraded in 20 ml of 50 mM PIPES-buffered M medium (Fig.7). H2 and butyrate production paralleled Avicel degrada-tion. No reducing sugars were detected. Upon further incu-bation, small quantities of reducing sugars (1 mM after 32days) were formed. The maximum rate of Avicel degradationwas 19 mg/liter per h.Mol% G+C. The mol% G+C of strain 743B was 26 to 27.

DISCUSSION

Cellulose as a lignocellulose component of wood is highlyresistant to microbial attack. This recalcitrance may beattributed in part to the crystalline structure of cellulose andto the presence of lignin, which acts as a physical barrier toenzymatic hydrolysis (5). However, Jerger et al. (12) demon-strated that size reduction greatly increased the digestibilityof hybrid poplar wood in batch methanogenic digestors. Anextremely cellulolytic, previously undescribed bacteriumwas isolated from such a woody biomass and is reported inthis paper.

0

00Eaa13NI

40

20

Temperature (°C)FIG. 5. Effect of temperature on H2 production of cellobiose-

grown culture. Maximal H2 production after 18 h of incubation.

Strain 743B was isolated directly from the digester samplewithout enrichment. Although present in low numbers, itwas extremely cellulolytic; zones of cellulolysis surroundingcolonies in cellulose agar roll tubes occurred rapidly (24 to 48h) and indicated that the cellulase complex was extracellular.Prior enrichment on a simple cellulose substrate (pebble-milled Whatman no. 1) may select for cellulolytic popula-tions with reduced ability to hydrolyze crystalline cellulose.Strain 743B readily utilized microcrystalline cellulose (Avi-cel), and this may reflect its ability to hydrolyze cellulosecomplexed as lignocellulose in native woody biomass (hy-brid poplar) methanogenic digestors.

Pebble-milled cellulose was hydrolyzed at a rate compara-ble to those reported for Clostridium thermocellum (24) andthe mesophilic spore-forming C strains (14). This supportsthe observation that rates of cellulolysis at mesophilic tem-peratures may be equivalent to those achieved at thermo-philic temperatures (14). Improved rates of cellulolysis at

0

.5EaN~I

pHFIG. 6. Effect of pH on H2 production of cellobiose-grown

culture. Maximal H2 production after 15 h of incubation.

VOL. 48, 1984

92 SLEAT, MAH, AND ROBINSON

TABLE 1. Products of cellobiose fermentation by strain 743B'Amt formed

Product (mol/100 mol ofcellobiose)b

Acetate ...................................... 52Butyrate ...................................... 176Lactate ...................................... 32Formate ...................................... 102Hydrogen ...................................... 148Carbon dioxide ................................. 156

a Determined after 105 h of incubation. The fermentation was complete asindicated by no further increase in H, on prolonged incubation. The followingwere calculated by the method of Gottschalk (7): carbon recovery, 92.5%;hydrogen recovery, 100%; O/R balance, 0.83.

b Values are means of four determinations.

mesophilic temperatures may be expected when the opti-mum conditions for cellulase production and cellulolysis aredetermined.

Strain 743B is a strictly anaerobic non-sulfate reducingsporeformer. These characteristics, in addition to its lowmol% G+C of 26 to 27, place it in the genus Clostridium. Itdiffers from other mesophilic cellulolytic clostridia (9, 14, 16)in fermentation products and in its unusual mode of sporula-tion. The colonial and cellular morphology and mode ofspore formation and spore morphology are similar to thosereported for "C. lochheadii" (10). Spores formed in cellu-lose roll tubes of strain 743B lyse upon aging. This results inan empty colony center and is considered typical of "C.lochheadii" (R. E. Hungate, personal communication). H2,C02, acetate, butyrate, formate, and ethanol are the mainfermentation products of "C. lochheadii" (10). Only traceamounts of lactate are detected. Strain 743B exhibits asimilar pattern except that lactate was a major product andethanol was produced in trace amounts. The production ofbutyrate as a major fermentation product differentiates strain743B and "C. Iochheadii" from all other cellulolytic clostrid-ia. "C. lochheadii" is nonmotile, does not require rumenfluid, and ferments cellobiose, glucose, maltose, and su-crose. Galactose, mannose, and lactose, substrates utilizedby strain 743B, are not fermented. Strain 743B furtherdiffered from "C. lochheadii" by its inability to utilize starchor exhibit proteolytic activity.We propose the name Clostridium cellulovorans sp. nov.

10

0)

01)

E

100

75

50

25

Time (days)FIG. 7. Fermentation of Avicel and production of acetate (Ac),

butyrate (But), and H2-

(cell.u'lo.vor.ans; L. noun. cellula, small cell; L. verb. voro,to devour; M.L. adj. cellulovorans, cell devouring) for strain743B.

Description of C. cellulovorans sp. nov. (i) Morphology.Cells stain gram negative and are non-motile rods 0.7 to 0.9by 2.5 to 3.5 ,um. Oblong spores 1 to 1.5 by 2 to 4 p.m occurcentrally or subterminally within a clostridial sporangiumthat is 1.5 to 2.0 to 4 to 7 ,um. Colonies in cellulose roll tubesare irregular with an opaque edge and an empty center.Colonies in cellobiose roll tubes are rhizoid.

(ii) Biochemistry and physiology. Obligate anaerobe. Fer-ments cellulose, xylan, pectin, cellobiose, glucose, fructose,galactose, sucrose, lactose, and mannose. H2, C02, acetate,butyrate, formate, and lactate are fermentation products.The temperature optimum for growth is 37°C. The range is 20to 40°C. The pH optimum for growth is 7.0. The range is 6.4to 7.8.

(iii) Growth requirements. Yeast extract required forgrowth. Rumen fluid and added vitamins not required.

(iv) Mol% G+C. The mol% G+C is 26 to 27.(v) Source. A batch methanogenic fermentation of finely

divided hybrid poplar wood.(vi) Type strain. The type strain is 743B (ATCC 35296).

The description of the type strain is the same as for thespecies given above.

ACKNOWLEDGMENTS

Samples of a batch methanogenic fermentation of finely dividedhybrid poplar wood were kindly provided by D. P. Chynoweth,Institute of Gas Technology, Chicago, Ill. We gratefully acknowl-edge the advice and encouragement of R. E. Hungate, University ofCalifornia, Davis, Calif., and we thank Tom Ferguson, HarveyNegoro, Bernard Ollivier, and Indra Mathrani for many helpfuldiscussions.

This work was supported by Gas Research Institute grants 5080-323-0423 and IFAS-GRI-FIA-MCS 2171 and by research grant DE-AT03-80ER10684 from the U.S. Department of Energy.

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