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Clostridium Aldrichii S P . Nov., a Cellulolytic Mesophile Inhabiting A INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1990, p. 268-272 Vol. 40, No. 3 0020-7713/90/030268-05$02.OO/O Copyright 0 1990, International Union of Microbiological Societies Clostridium aldrichii sp. nov., a Cellulolytic Mesophile Inhabiting a Wood-Fermenting Anaerobic Digester? JUNCHANG C. YANG,l* DAVID P. CHYNOWETH,l DONNA S. WILLIAMS,, AND ANMING L12$ Bioprocess Engineering Research Laboratory, Agricultural Engineering Department,' and Microbiology and Cell Science Department,2 University of Florida, Gainesville, Florida 3261 1 An anaerobic, mesophilic, spore-forming, cellulolytic bacterium was repeatedly isolated from a wood- fermenting anaerobic digester. Cells of this organism were gram-positive rods, motile with a bundle of polar flagella, and formed subterminal oblong spores. The colonies in agar had an irregular shape with many platelike structures and were greyish white. Cellulose, xylan, and cellobiose served as substrates for growth. Acetate, propionate, butyrate, isobutyrate, isovalerate, lactate, succinate, H,, and CO, were products of cellobiose fermentation. The optimal temperature and pH for growth were 35°C and 7, respectively. The DNA composition was 40 mol% G+C. The name Clostridium aldn'chii sp. nov. is proposed. The type strain is P-1 (= OGI 112, = ATCC 49358). Woody biomass generally was not considered to be bio- 10 ml; resazurin (0.5 mg/ml), 1 ml; L-cysteine hydrochloride, degradable under anaerobic conditions until Zeikus and 0.5 g; Na,S . 9H20, 0.2 g. The agar concentration was 1.5% Ward (22) showed that a methane fermentation occurs in the (wthol). The pH was adjusted to 7.0 with NaHCO,. The heartwood of live trees and Chynoweth and co-workers (4, medium was prepared and dispensed with continuous flush- 9) showed that the carbohydrates of poplar and other hard- ing of the gas mixture mentioned above. Medium was woods are degraded to methane by a domestic sludge dispensed into serum tubes (1) (18 by 150 mm; Bellco Glass, digester inoculum without pretreatment other than reduction Inc., catalog no. 2048-00150) or serum bottles, according to of particle size to the millimeter range. These observations need, stoppered with butyl rubber stoppers (Bellco, catalog have stimulated research to determine the organisms and no. 2048-11800), sealed with aluminum seals (Wheaton In- interactions involved with the possible goal of improving the dustries; 20-mm tear-off, catalog no. 224193), and sterilized extent and rate of these depolymerization reactions. Sleat at 121°C for 20 min. and co-workers (18, 19) have isolated two new cellulolytic, The cellulose slurry was prepared as follows: 30 g of Clostridium species from wood methane fermentation. Our Whatman no. 1 filter paper was ground with 1,000 ml of laboratory has begun to isolate predominant cellulolytic distilled water for 72 h in a 5-liter alumina grinding jar with pectinolytic, and xylanolytic bacteria from poplar-fed wood pebbles (20 to 30 mm in diameter). The cellulose slurry was digesters. The first of these, a new cellulolytic Clostridium added (50 ml/liter) to the basic medium to make the cellulose species, has been repeatedly isolated from high dilutions of medium or cellulose agar medium (CAM). The amount of the the samples over a period of 2 years and is described below. cellulose slurry added was varied according to the purpose The name Clostridium aldrichii is proposed for this organ- of the culture. The cellulose concentration in the medium ism. was typically in the range of 0.2 to 0.4% (wthol). A Whatman no. 1 filter paper strip (50 by 5 mm) was sub- MATERIALS AND METHODS merged in the 4.5-ml basal medium tube as filter paper strip Samples and treatments. Strict anaerobic methods (7) were medium (CSM) to determine the highest dilution of the used for sample collection, medium preparation, inocula- mixed population exhibiting cellulolytic activity. tion, and identification. Samples were collected from the Other substrates, including cellobiose, pectin, xylan, and effluent of a continuously stirred tank, bench-scale, meso- glucose, were added to the basal medium at a final concen- philic (35°C)) poplar-fed digester. The effluent was blended tration of 1% (wthol). The media and methods of Holdeman for less than 1 min in a homogenizer under oxygen-free et al. (6) were used for biochemical tests and fermentation N,-CO, (80:20). The treated sample was diluted in basal product analysis. medium up to lo-'' in order to exceed the highest dilution Isolation, purification, and enumeration. Serial dilutions of with cellulolytic activity. The first sample was collected 65 sample (0.5 ml) were inoculated in triplicate into CAM (4.5 days after the digester was started. ml per tube) for roll tubes and CSM for cellulolytic activity Culture media. The basal medium contained (per liter): detection. The tubes were incubated at 35°C until colonies NH,C1,2 g; KCl, 0.5 g; K,HP04,1.65 g; MgSO4 * 7H,O, 0.5 surrounded by clear zones appeared in CAM or until the g; Trypticase (BBL Microbiology Systems), 0.5 g; yeast submerged filter paper strips were digested in the CSM. extract (BBL), 0.5 g; clarified rumen fluid, 200 ml; clarified Cellulolytic colonies in the highest-dilution roll tube were woody-biomass digester sludge, 100 ml; mineral solution picked and suspended in cellulose medium, which was used "aCl(6 g), (NH4),S04 (6 g), CaCl, (0.6 g), H,O (1,000 mOl, to inoculate CAM roll tubes. The tubes were incubated at 150 ml; trace mineral solution (l),10 ml; vitamin solution (l), 35°C until cellulolytic colonies appeared again. This transfer procedure was repeated several times until a pure culture * Corresponding author. was obtained. To confirm the purity, the isolate was trans- t Florida Agricultural Experiment Station Journal Series no. ferred to cellobiose or glucose agar medium in roll tubes and R-00436. incubated at 35°C. After ascertaining that only one morpho- $ Present address: Chengdu Institute of Biology, Academia Sin- logical type of colony appeared, a single colony was picked ica, Chengdu, Sichuan, People's Republic of China. and transferred back to CAM roll tubes to check its cellu- 268 VOL.40,1990 C. ALDRZCHZZ SP. NOV., A CELLULOLYTIC MESOPHILE 269 FIG. 1. (A) Ultrathin section of P-1 cells, showing gram-positive-type cell wall structures (an enlarged cross section is shown at bottom right). (B) Ultrathin section showing the round particle between two cells. lolytic activity. The colony and cell morphologies from the a Balzers model MED 010 vacuum evaporator. Grids were secondary growth of the clear-zoned colonies in CAM were examined by transmission electron microscopy, compared and were identical to those of the previous isolate Physiological and biochemical identifications. Basal me- (8). dium with cellobiose was used for the measurements of Numbers of cellulolytic bacteria were estimated from the optimal growth temperature and pH. Cell density was deter- reciprocal of the highest dilution of the inoculated samples in mined by measurement of optical densities of cultures with a which the filter paper strip was digested in CSM and in which spectrophotometer (Perkin-Elmer model 35) at 610 nm. clear-zoned colonies appeared in CAM. Optimal temperature and pH measurements were deter- Morphological studies and microscopy. Morphological ob- mined twice, and each measurement was made in triplicate. servations of the cells from 24- to 48-h growth broth cultures Nutrient requirements were tested in the medium with and single colonies were made by both light microscopy cellobiose as the only carbon and energy source and with (Nikon Labophot equipped with phase optics, epifluores- different combinations of yeast extract, Trypticase, clarified cence, and an automatic photomicrographic Nikon FX-35A rumen fluid, digester sludge, and vitamin solution. Two- camera with UFX-I1 control) and electron microscopy (Phil- day-old cultures grown on cellobiose basal agar slants were ips EM-301 transmission electron microscope). Gram stains, washed with the salt medium to serve as inocula for these the KOH test (S), and the ~danine-4-nitroanilide(LANA) tests. The fermentation products were analyzed after incu- test (2) were used to complement the Gram reaction. Me- bation for 2 days in PY basal medium (6) with cellobiose as dium without Trypticase, yeast extract, clarified rumen fluid, the substrate. Biochemical tests were performed in PY basal and digester sludge but with the vitamin solution and cello- medium with carbohydrates, enzymes, and other substrates. biose was used to induce spore formation. A fresh culture (1 Fermentation product analyses and biochemical tests were to 2 days) in cellobiose medium was used for electron done in the laboratory of W.E. C. and L. V. H. Moore, microscopic observation of flagella. Virginia Polytechnic Institute and State University, Blacks- To prepare specimens for electron microscopy, glutaral- burg. dehyde was injected into the fresh broth culture to a final Analysis of DNA base composition. Resuspended cells from concentration of approximately 1.3%. Cells were immedi- a centrifuged pellet were treated with 5% (voVvol) Triton ately centrifuged, and the supernatant was replaced with 1% X-100, and DNA was extracted and purified by the method glutaraldehyde in 0.025 M sodium cacodylate buffer (pH of Marmur (13). DNA base compositions were determined 7.2). Cells were postfixed in buffered 1% osmium tetroxide from the buoyant density of DNA in CsCl gradients by the followed by 1% aqueous uranyl acetate, dehydrated in a method of Preston and Boone (16). DNAs from Micrococcus graded ethanol to acetone series, and embedded in Spurr lysodeikticus and Escherichia coli were used as standards. low-viscosity resin. Sections were cut with a diamond knife The mol% G+C of the DNA was calculated by the method of on an LKB Ultrotome I11 and poststained with uranyl Schildkraut et al.
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