Vol. 40, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1980, p. 809-8200099-2240/80/10-0809/12$02.00/0

Attack on Lignified Grass Cell Walls by a FacultativelyAnaerobic Bacterium

DANNY E. AKIN

Field Crops Laboratory, Richard B. Russell Agricultural Research Center, Agricultural Research-Scienceand Education Administration, U.S. Department ofAgriculture, Athens, Georgia 30613

A filamentous, facultatively anaerobic microorganism that attacked lignifiedtissue in forage grasses was isolated from rumen fluid with a Bermuda grass-containing anaerobic medium in roll tubes. The microbe, designated 7-1, dem-onstrated various colony and cellular morphologies under different growth con-ditions. Scanning electron microscopy revealed that 7-1 attacked lignified cellwalls in aerobic and anaerobic culture. 7-1 predominately degraded tissuesreacting positively for lignin with the chlorine-sulfite stain (i.e., sclerenchyma inleaf blades and parenchyma in stems) rather than the more resistant acidphloroglucinol-positive tissues (i.e., lignified vascular tissue and sclerenchymaring in stems), although the latter tissues were occasionally attacked. Turbidi-metric tests showed that 7-1 in anaerobic culture grew optimally at 39°C at a pHof 7.4 to 8.0. Tests for growth on plant cell wall carbohydrates showed that 7-1grew on xylan and pectin slowly in aerobic cultures but not with pectin and onlyslightly with xylan in anaerobic culture. 7-1 was noncellulolytic as shown by filterpaper tests. The microbe used the phenolic acids sinapic, ferulic, and p-coumaricacids as substrates for growth; the more highly methoxylated acids were usedmore effectively.

Microorganisms with the ability to attack lig-nocellulose have been reported. For example,Thermomonospora fusca can utilize pulpingsubstrates with up to 18% lignin (10). However,the slow utilization of lignocellulose by thermo-philic actinomycetes, mesophilic bacteria, andwhite-rot fungi has been reported to be a hin-drance to the utilization of cellulosic waste,which requires chemical or physical pretreat-ment to expose the structural polysaccharides(4). Indeed, the biodegradation of lignin andlignocellulose has been reported to be a majorproblem in the commercial use of cellulosicwastes (5, 8). In general, anaerobic bacteria arenot considered to have the ability to utilize lignin(4).Lignin reduces the availability of structural

carbohydrates in forage cell walls to rumen mi-croorganisms (19). Chemical bonding betweenphenolic acids (p-coumaric and ferulic acidspresent presumably as lignin precursors) and ,B-glucans and 8l-xylans in ryegrass has been re-ported (18). Histochemical and biochemicalstudies (21) showed that lignified tissues variedin the type of lignin present in the cell walls, andthese cell walls varied in their reaction to delig-nification with KMnO4 (3). In electron micros-copy studies (1), a filamentous microbe thatattacked sclerenchyma (a lignified tissue) in leafblades of the digesta removed from a cannulated

steer was found, implying that attack on ligno-cellulose occurred under anaerobic conditions.The objective of the present work is to report

the isolation from runen fluid of a filamentous,facultatively anaerobic bacterium capable of at-tacking lignified tissues in forage grasses.

MATERIALS AND METHODSIsolation of 7-1. Rumen digesta from a perma-

nently cannulated steer maintained predominately onBermuda grass hay was strained through four layersof cheesecloth into a vacuum bottle and then trans-ported to the laboratory. Tenfold dilutions of thestrained rumen fluid were made with the anaerobicdilution solution of Bryant and Burkey (6). For isola-tion, 0.3 ml of the 103 to 105 dilutions was placed intotest tubes (15 by 150 mm) with 5 ml of anaerobicmedium. The isolation medium consisted of the basalmedium of the rumen fluid-glucose-cellobiose agar ofBryant and Burkey (6) with reducing agents as modi-fied by Bryant and Robinson (7). The medium wasmodified to include 1 to 3% (wt/vol) 8-week- old,freeze-dried and ground coastal Bermuda grass (CBG)as the sole substrate. This grass was chosen becauseof its high fiber content (68% neutral detergent fiber,37% acid detergent fiber), high lignin content (6%),and low in vitro dry matter digestibility (52%) (F. E.Barton, personal communication). Therefore, this sub-strate would be conducive for selecting microbes thatdegrade lignified tissue.

Roll tubes were prepared by the Hungate method(15), and the inoculated, rolled tubes were incubated

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at 35°C. Suspected filamentous, lignified tissue-de-grading microbes were subcultured by excising entirecolonies, suspending the cells in anaerobic dilutionsolution, and then inoculating roll tubes (containing1% CBG and 0.2% glucose as substrates) with 1 ml ofthe suspension. After growth for 5 weeks, colonieswere again selected for subculture on the basis of a

filamentous nature and overgrowth of lignified fiberfragments in the substrate. Colonies were subculturedtwo more times, and Gram-stained smears were ex-

amined for purity. The isolate was designated 7-1 andfurther studied for degradation of lignified tissues. Aculture has been deposited in the ARS Culture Collec-tion, Northern Regional Research Center and assignedthe accession number NRRL B-4370.

Microscopic studies of colony and cellularmorphologies. For scanning electron microscopy(SEM) of colony morphology, 7-1 was grown in theanaerobic roll tube medium used for subculturing andalso on aerobic medium. The aerobic medium was

made as slants with 15% each of solutions 1 and 2described by Bryant and Burkey (6), 65% distilledwater, 1% 8-week-old CBG, 0.2% glucose, 0.5% yeastextract, and 2% agar. Tubes of each of the mediahaving isolated colonies grown for 2 to 3 weeks werefilled with 4% glutaraldehyde in 0.1 M cacodylatebuffer (pH 7.2) and fixed overnight at 5°C. Fixedcolonies in agar blocks were excised and mounted on

SEM stubs with conductive silver paint. Specimenswere then postfixed over osmium tetroxide vapor,sputter-coated with gold-palladium alloy (60:40), andexamined by SEM at 15 kV. Plant and microbialsamples were prepared for transmission electron mi-croscopy as described previously (2). For light micro-scope study of cellular morphology, Gram-strainedsmears of 7-1 growing in broth or on agar were ex-

amined.Tests for optimal pH and temperature. The

semisynthetic anaerobic basal medium of Caldwell andBryant (9) was used with 0.2% cellobiose as sole carbonand energy source. (Earlier turbidity tests had shownthat cellobiose supported good growth.) Working sam-ples of the anaerobic medium were adjusted to pH 6,7, 8, or 9, and 7 ml of broth was dispensed under CO2into tubes (6 by 125 mm) for each pH. These tubeswere fitted with Hungate septa and caps (no. 2047;Bellco Biological Glassware and Equipment, Inc.).Media were autoclaved under fast exhaust. The tubeswere matched for identical absorbance on a Bauschand Lomb Spectronic-20 spectrophotometer beforeuse in growth studies. The tubes were then inoculated,using 25-gauge needles, with 0.1 ml of a 72-h CBGbroth culture of 7-1. Duplicate tubes at each pH werethen incubated at 25, 30, 35, 39, or 45°C for 72 h.Growth was evaluated by absorbance at 520 nm. In-oculated, matched tubes were read against blanks ofuninoculated media. Confirmation tests were run foroptimal pH using 0.2 ml of a 48-h broth culture andmethods similar to that described above.

Tests for utilization of celi wall-type carbohy-drates and phenolic acids. Anaerobic turbidimetricgrowth studies were carried out using the Caldwelland Bryant (9) basal broth medium adjusted beforeautoclaving to pH 6.7 (rumen pH) or 7.6 (optimalgrowth pH) in matched tubes fitted with Hungatesepta. Aerobic growth studies were carried out on the

APPL. ENVIRON. MICROBIOL.

basal broth for tryptone-yeast extract-glucose (TYG)medium (12), but without glucose, and adjusted to pH7.6 in matched tubes with screw caps loosened one-quarter turn. Carbohydrates, at a level of 0.2%, in-cluded purified xylan (Koch-Light), pectin (purifiedby dialysis to remove contaminating sugars and freezedried), cellulose (Solka Floc or ground Whatman no.1 filter paper), cellobiose, and glucose. Basal mediumwithout carbohydrates was also tested to ensure thatgrowth was due to the carbohydrate tested.

Sinapic acid (3,5-dimethoxy-4-hydroxycinnamicacid), ferulic acid (4-hydroxy-3-methoxycinnamicacid), and p-coumaric acid (p-hydroxycinnamic acid)at a level of 0.2% were tested for the ability of 7-1 touse these phenolic acids as the sole carbon and energysources both aerobically and anaerobically. Since thisamount of acid (0.2%) did not go into solution for allthe acids, all media were filtered through a 0.45-,umfilter before 7 ml was dispensed into each of thematched tubes. Basal medium with 0.2% glucose andbasal medium without any added substrate were in-cluded as growth controls.

Duplicate matched tubes were then inoculated with0.2 ml of an active culture of 7-1 in CBG brothmedium, incubated at 39°C, and read for absorbancedaily as described. Absorbance at 0 h was subtractedfrom the final absorbance to correct for initial differ-ences in the inocula.

Isolate 7-1 was also tested for its ability to degradefilter paper both aerobically and anaerobically. Fivemilliliters of basal anaerobic or aerobic medium-con-taining strips (12.5 by 144 mm) of Whatman no. 541filter paper was inoculated with 0.2 ml of an activeculture of 7-1 and incubated at 39°C for 28 days.Spectrophotometric (ultraviolet) analysis for

loss of sinapic acid from broth cultures. Stand-ards, uninoculated aerobic medium with and withoutsinapic acid, and inoculated aerobic medium with sin-apic acid were analyzed with a Varian spectrophotom-eter (model 635). The standards were prepared in thesame manner as the media, but with distilled wateronly. Samples were diluted 1:200 or 1:300 with distilledwater, placed in 10-mm cuvettes, and scanned from350 to 180 nm. Three trials were run, and spectra werecompared for differences in peak heights at the ab-sorbance maxima for sinapic acid.

Microscopic evaluation of fiber digestion bymicroorganisms. All microscopic investigations ofthe degradation of lignified tissue were carried outusing blades and stem sections prepared from a singleharvest of 5-week-old CBG. The central portion of thefourth internode and associated leaf blades were cutinto 2- to 3-mm sections. Representative blade andstem samples were sectioned freehand for light mi-croscopy, and the sections were stained for lignin withacid phloroglucinol and chlorine-sulfite (16).

Evaluation for microbial attack on lignified tissueswas carried out by incubating the microbes (0.2 to 0.5ml of an active culture) with 10 sections each of theblades and stems in 7 ml of basal anaerobic mediumcontained in tubes (16 by 125 mm) as described above.All tests were conducted with control blades and stemsincubated in uninoculated medium. After incubation,blades and stems were fixed without washing for SEMas described (1).

Several tests were conducted to evaluate the attack

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of 7-1 on the various types of lignified tissues in bladesand stems in basal anaerobic medium or in basalmedium supplemented with cellobiose. One test eval-uated sections incubated for 28 days. Another test wasincluded to compare the degradation of tissues by 7-1after 7 days of incubation in aerobic and anaerobicmedia.

In another test, 20 sections each of blades and stemswere incubated in uninoculated basal anaerobic me-dium or in medium inoculated with 0.5 ml of a brothculture of 7-1 to pretreat tissues for 72 h. The pre-treated and control sections were then autoclaved toinhibit further action of 7-1, and the sections werewashed with distilled water at 5°C for 3 days. Threeof each of the pretreated and control blades and stemswere prepared for SEM. Rumen fluid inoculum wasprepared by straining digesta from a cannulated steerthrough 12 layers of cheesecloth, mixing 1 part of thestrained fluid to 2 parts of McDougall carbonate bufferas described previously (1). Pretreated and controlblades and stems were then placed into each of two50-ml glass centrifuge tubes and inoculated with 30 mlof the rumen fluid suspension. The tubes were gassedwith C02, capped with a one-way valve, and incubatedat 39°C. Representative blades and stems from eachof the tubes were removed after 6, 24, and 48 h ofdigestion and prepared for SEM, and freehand sec-tions of stems were prepared for light microscopy.

Another test was conducted to compare the relativedegradation of lignified tissues by the cellulolytic fun-gus Trichodenna viride QM6a (17); the lignocellulose-degrading, thermophilic actinomycete T. fusca 190Th(ATCC 27730) (10); strained rumen fluid; and 7-1. Forthis test, blade and stem sections were placed intomatched tubes (16 by 125 mm) containing the optimalmedium for cellulose or lignocellulose degradation or,for rumen fluid, the environmental conditions approx-imating those of the rumen.

For T. viride, the medium described by Mandelsand Weber (17), but without cellulose, was used, andtubes were inoculated with a mat of mycelium andspores from a viable culture maintained on potato-dextrose-agar. Inoculated tubes were incubated at28°C on a rotary shaker at 75 rpm. For T. fusca, themedium of Crawford et al. (12) was used, but withoutpulping fines, and tubes were inoculated with hyphaeand spores from a 5-day-old culture maintained onTYG slants. Inoculated tubes were then incubated at53°C on a reciprocal shaker at 75 rpm. For rumenmicrobial degradation of tissues, 0.2 ml of rumen di-gesta strained twice through cheesecloth was inocu-lated into Caldwell and Bryant's basal broth medium(9) in anaerobic tubes, and the tubes were incubatedat 39°C. For 7-1, 0.2 ml of a 96-h broth culture wasinoculated into the anaerobic broth medium (pH 7.5)with and without cellobiose, and tubes were incubatedat 39°C. All tubes were read against medium blanks at520 nm for absorbance after 24 and 48 h. The tubeswere incubated for 8 days, and then blades and stemswere prepared for SEM.

RESULTS

Colony morphology. Anaerobic coloniesfrom roll tubes were flat, lacked aerial hyphae,

and showed an irregular periphery; they oftenovergrew lignified fragments of CBG in the me-dium (Fig. la). The colony periphery at highermagnification showed extremely long (severalmicrometers), unbranched filaments often frag-menting into shorter forms; often coccoid formsand rods of a few micrometers were seen (Fig.lb and inset). Conversely, colonies from aerobicculture demonstrated a different morphology.These colonies were raised and had entire edges(Fig. lc). Cells of diverse lengths were presentthroughout the colony (Fig. ld); however, ingeneral, the aerobically cultured cells weremarkedly shorter than the filaments present inthe anaerobic cultures.Cellular morphology. Because of the ex-

treme variations in filament lengths under var-ious growth conditions, further studies were un-dertaken to assess colony purity and to evaluatethe filamentation. Gram-stained smears of col-onies subcultured for purity several times con-sistently showed this difference in filamentlength when grown in the anaerobic and aerobicmedia prepared in this manner. Tests for growthwith CBG anaerobic broth at various pH levelsshowed that cells grown at pH near 7.5 hadfilaments that were markedly shorter than thoseof cells grown near pH 6.7. (Although the pHlevels were adjusted to 7.5 or 6.7, the final levelsafter autoclaving were 7.3 and 6.4, respectively.)Further tests confirmed these observations (Fig.2a and b). Further, cross-inoculation from CBGmedium at pH 6.7 and 7.5 to the medium at pH7.5 and 6.7, respectively, showed a reversal ofthe original morphology consistent with struc-tures shown in Fig. 2a and b. After 48 h ofincubation, 10 of the longest filaments grown atpH 7.5 averaged 12 ± 5 ,im, whereas at pH 6.7filaments averaged 67 ± 45 ,um in length. Thesefilaments were not necessarily the longest be-cause of difficulty in finding the ends of the cells.Cells of 7-1 grown in aerobic broth (TYG, pH7.5) or on TYG slants for up to 14 days revealedfilaments of 5 to 10 ,Im (Fig. 3), but extremelylong filaments were infrequently found. It wasnot ruled out that changes in redox potential perse affected filament length. However, the differ-ences in the colony and cellular morphologiesshown in Fig. 1 could also have been due to pHdifferences in the media. Gram-stained smearsof both anaerobically and aerobically grown cul-tures revealed an array of cellular forms includ-ing gram-positive and -negative areas in fila-ments of various lengths (Fig. 2 and 3). Fre-quently, long, gram-negative filaments werepresent in the anaerobic broth. The relativelyslow growth (e.g., 24 h for visible growth onTYG), the consistent change in filament lengthwith pH, and the finding of similar structures

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FIG. 1. (a) SEM of 7-1 grown for 2 weeks in anaerobic roll tubes. The colony is flat, the center is cracked,and the periphery (arrows) is irregular. A fiber fragment (F) in the CBG substrate was overgrown by 7-1. Bar,0.5 mm. (b) SEM of the periphery of an anaerobic colony of 7-1 showing long, unbranched filaments (arrow).The filaments fragmented into shorter cells of various lengths (inset, arrows). Bar, 2 ,um. (c) SEM of 7-1 grownfor 3 weeks on aerobic slants. The colony is raised from the agar and shows a definite edge. Bar, 0.5 mm. (d)SEM of an aerobic colony of 7-1 with cells of various lengths, but lacking long filaments. Bar, 2 ,um.

FIG. 2. Gram-stained smears of 16-h cultures of 7-1 grown in anaerobic CBG broth at differentpH levels.Bar, 10 ,um. (a) pH 7.5; microbial cells are about 5 to 10 ,um long, with some cells (arrow) having light anddark areas ofstaining. (b)pH 6.7; long filaments with gram-positive and -negative areas (arrows) are present.

FIG. 3. Gram-stained smear of 7-1 grown aerobically on TYG slants for 5 days. Cells of various lengthsare present, but long filaments are lacking. Short, gram-negative filaments with gram-positive areas (arrows)are present. Bar, 10 fim.

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(i.e., gram-positive and -negative cells of variouslengths) in both aerobic and anaerobic mediaindicate that isolate 7-1 apparently is a pureculture that demonstrates variable and unusualmorphologies.Tests for optimal anaerobic growth con-

ditions. Turbidimetric tests for growth on 0.2%cellobiose broth revealed that optimal growthoccurred at 390C with a pH range of 7.4 to 8.0.Growth was not apparent below pH 6, with theexception of slight growth at 390C after 74 h.Growth did not occur at 450C.Electron microscopy observations of lig-

nified tissue degradation. Preliminary studiesto differentiate types of lignin in cell walls ofblades and stems were undertaken by light mi-croscopy. Cell walls stained for lignin by usinglight microscopy are indicated by SEM in Fig. 4.In blades the inner bundle sheath and xylemtissues stained positively for lignin with acidphloroglucinol, whereas the sclerenchyma andto a lesser extent portions of the inner sheathgave a chlorine-sulfite-positive reaction (Fig. 4a).The parenchyma bundle sheath gave a positivebut more transient reaction with chlorine-sulfite.In stems, the thick-walled sclerenchyma ring,the bundle sheath and xylem cells, and the epi-dermis were acid phloroglucinol positive,whereas chlorine-sulfite-lignin was present inthe parenchyma cells, with the intensity of stain-ing increasing from the center to outer part ofthe tissue (Fig. 4b). Blades and stems incubatedin uninoculated medium (i.e., controls) for 8 dayswere intact and undegraded (Fig. 4a and b).Transmission electron microscopy examina-

tion of a colony from a roll tube overgrowing alignified fiber fragment in the medium such asshown in Fig. la revealed that 7-1 cleared thecell walls, especially the intercellular layer, ofsclerenchyma cells, leaving only a small residueof electron-dense cell wall material (Fig. 5).These cells were easily identified as blade scler-enchyma by their cell wall thickness and ar-rangement within the tissue (see Fig. 6 of Akin[1]). Incubation of 7-1 with blades and stems atoptimal growth conditions (pH 7.5, 3900) inanaerobic broth (both with and without 0.2%cellobiose as an additional energy source)showed degradation of lignified tissue; occasion-ally even the most resistant lignified vasculartissue was degraded (Fig. 6). Similar tissues weredegraded with or without cellobiose added, butevaluation for growth after 48 h did show in-creased turbidity with added cellobiose (absorb-ance at 520 nm of 0.56 versus 0.14).No differences in tissue degradation were

found in a study of anaerobically and aerobicallygrown cultures of 7-1 for 7 days. Short filaments

attacked sclerenchyma in blades (Fig. 7). Infre-quently, long filaments were associated with de-graded parenchyma cells, indicating that thesediverse filamentous forms could degrade ligni-fied cell walls aerobically.Because 7-1 demonstrated an ability to attack

lignified tissues, a comparison of lignified tissuedegradation was made by using various micro-organisms reported to have the ability to de-grade plant cell walls or cell wall components.T. viride did not degrade any of the blade tis-sues, not even the most easily digested phloemand mesophyll; however, hyphae were presenton the plant sections (Fig. 8). T. fusca separatedthe inner bundle sheath of blades into individualcells, caused a layering of the sheath wall, andpartially degraded the sclerenchyma cells (Fig.9a); T. fusca showed only slight attack on pa-renchyma cells in the stems, and filaments didoccasionally appear to associate with cells of theacid phloroglucinol-positive ring (Fig. 9b). Ru-men microorganisms extensively removed theunlignified cell walls in blades, leaving a residuein which the inner bundle sheath cells, the xy-lem, most of the abaxial sclerenchyma, and thecuticle were present and maintained the struc-tural organization (Fig. 10a); a large part of theparenchyma bundle sheath remained (notshown). In stems, the acid phloroglucinol-posi-tive tissues and the most intensely staining pa-renchyma cells resisted degradation (Fig. 10b).

Isolate 7-1 did not extensively degrade theunlignified tissue in blades and left a residuewith some mesophyll and parenchyma bundlesheath, but deformation of the inner sheath andseparation of sclerenchyma into cells occurred(Fig. lla). In stems, 7-1 degraded virtually allbut the acid phloroglucinol-positive tissues (Fig.llb); long filaments extended into cells of thering (Fig. llb, inset).A comparison of pretreated and control stems

incubated for 72 h and then digested with rumenbacteria for up to 48 h showed that pretreatmentwith 7-1 resulted in more digestion of the par-enchyma cells, even after 6 h (Fig. 12a and b).Measurements of parenchyma tissue remainingin control and pretreated stem cross-sections,which had been sectioned for light microscopy,showed that about three times more tissue re-mained in the control stems after rumen micro-bial digestion for both 6 and 48 h.One study was conducted to test the degra-

dation of lignified tissues by 7-1 after 28 days ofincubation in anaerobic broth. Both scleren-chyma and the inner bundle sheath in the bladesshowed degradation by cells of various lengths,and inner sheath cells were disrupted into layers(Fig. 13). Mesophyll and parenchyma bundle

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FIG. 4. SEM of sections of CBG blade and stem incubated for 8 days in basal anaerobic medium withoutinoculation, showing the intact structure ofcontrol cell walls. Cell walls stained for lignin by light microscopy-histochemistry are indicated by SEM. Bar, 50 pm. (a) Blade. Chloride-sulfite-positive lignin was presentprimarily in the sclerenchyma (S), adaxial part of the inner bundle sheath (arrow), and to a lesser extent in

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MICROBIAL ATTACK ON LIGNIFIED CELL WALLS 815

sheath cells were distorted. In stems, digestionwas primarily limited to parenchyma cells withdigestion up to the first or second layers of cellsadjacent to the ring. Most lignified vascular andring tissues were not degraded.

Utilization of cell wall-type carbohy-drates and phenolic acids. Anaerobic andaerobic media containing xylan, pectin, cello-biose, and Solka Floc or ground filter paper weretested for their ability to support growth (asmeasured by turbidity) of 7-1. The maximumabsorbance and the day this maximum wasreached are shown for aerobic growth after 28days (Table 1). Growth on Solka Floc, which isinsoluble and settles to the bottom, was evalu-ated by reading at 520 nm, swirling the tube tosuspend the cellulose, and allowing the celluloseto settle totally before reading again. Thismethod indicated no growth of 7-1 on cellulose.However, definite growth did occur on xylan andpectin, although growth on pectin was slow.Anaerobic growth studies at pH 7.5 using thesesame carbohydrates (except ground filter paperwas substituted for Solka Floc) showed that onlycellobiose supported growth. However, whencultured anaerobically at pH 6.7 on xylan, thebacterium showed slight but definite growth (ab-sorbance at 520 nm = 0.10) in two tests; thebacterium did not grow on pectin. Tests forcellulose degradation using filter paper strips inaerobic and anaerobic media incubated for 28days indicated that 7-1 was noncellulolytic.Growth studies (measured by turbidity) using

phenolic acids as sole carbon and energy sourcesfor 7-1 are shown for aerobic and anaerobicmedia (Table 2). Basal medium and basal me-dium plus 0.2% glucose were included in thesetests as negative and positive growth indicators,respectively. In aerobic tubes the sinapic acidbroth changed from yellow to dark brown after

a few days in both uninoculated controls andinoculated tubes. Growth as shown by the in-creased absorbance was confirmed by the pres-ence of short gram-negative filaments withgram-positive areas. In anaerobic medium, al-though discoloration was not noted, the sinapicacid, and ferulic acid to some extent, precipitatedand thus interfered with the absorbance due tobacterial growth. However, smears again showedthe gram-negative filaments with gram-positivezones, indicating that 7-1 grew on ferulic acid.Spectrophotometric (ultraviolet) spectra

of sinapic acid. Tests for loss of sinapic andferulic acids in anaerobic broth were not possiblebecause of the precipitate. However, the aerobicmedium with sinapic acid was analyzed. Sinapicacid standards showed absorbance maxima at308, 244, and 200 nm. The 308-nm peak wasmarkedly lower in tubes in which the browndiscoloration was apparent and therefore wasnot included in comparison for loss of sinapicacid due to microbial growth. Absorbance max-ima at 244 and 200 nm were present in uninoc-ulated and inoculated media tubes. Peak heightsof inoculated broth at 244 and 200 nm werereduced 7.5 and 7.0%, respectively, from thosefor uninoculated broth. This analysis, in con-junction with turbidimetric growth studies, in-dicated that growth of 7-1 was due to utilization(in some form) of sinapic acid.

DISCUSSIONAttempts have been made to classify 7-1, but

the unusual morphology and the responses tocertain biochemical and histological tests do notpermit placing 7-1 into an established classifi-cation at present. Although 7-1 has not beenidentified, the various cellular morphologies (i.e.,gram-positive and -negative filaments, frag-

the rest of the inner sheath (I). The parenchyma bundle sheath (B) also gave a positive but more transientreaction with chlorine-sulfite. Acidphloroglucinol-positive lignin was present in the inner bundle sheath andxylem (X) tissues. (b) Stem. Chlorine-sulfite-positive lignin was present in the parenchyma cell walls (P), withthe stain intensity increasing centrifugally from the center, and in the walls ofcells underneath the epidermis.Acid phloroglucinol-positive tissues included the epidermis (E), sclerenchyma ring (R), and xylem (X); sheathcells (arrows) of the vascular bundles.

FIG. 5. TEM of sclerenchyma cells in CBG blades from a fiber fragment overgrown with 7-1 in anaerobicroll tube medium similar to (F) in Fig. 1. Extensive clearing of cell walls indicates loss of wall material,especially in the intercellular areas (arrows). Electron-dense areas indicate remnants of remaining cell wall(19. Bar, 2 pm.

FIG. 6. SEM of lignified xylem (X) cells and metaxylem vessels (17 ofstem vascular bundles degraded afteranaerobic incubation for 4 days. The inset shows an enlargement ofmicroorganisms at arrow associated withdegraded xylem cells. Bar, 5 p±m; inset bar, 2 ,pm.

FIG. 7. SEM of blade sclerenchyma degraded by 7-1 after 7 days of aerobic incubation. Rods and shortfilaments (arrows) with narrow threads associated with degraded zones in cell walls. The inset shows lowmagnification ofa stem with a long filament (arrow) within degradedparenchyma cells. Bar, 2 ,um; inset bar,10 p,m.

FIG. 8. SEM of blade incubated for 8 days with T. viride QM6a. Although hyphae (arrows) overgrew theblade, no plant tissues were degraded. Bar, 10 p.m.

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MICROBIAL ATTACK ON LIGNIFIED CELL WALLS 817

TABLE 1. Utilization of carbohydrates for aerobicgrowth (turbidity) by isolate 7-1a

Maximum Day ofCarbohydrate absorbance Dayor

arbneabsorbanceNone (basal medium) 0.07 21Cellobiose 0.76 4Pectin 0.72 28Xylan 0.32 6Cellulose (Solka Floc) 0.06 7

a Medium was TYG (but without glucose) with car-bohydrates at the 0.2% level, pH 7.5, 39°C. Turbiditywas measured at 520 nm against a blank of uninocu-lated medium. Absorbance is that remaining aftersubtracting the absorbance at 0 h to correct for differ-ences due to inoculum.

TABLE 2. Utilization ofphenolic acids for growth(turbidity) by isolate 7-1a

Maximum Day ofCarbon source absorbance axobnueabsorbance

AerobicbNone (basal medium) 0.07 21Glucose 0.92 3p-Coumaric acid 0.19 28Ferulic acid 0.26 28Sinapic acid 0.77 24

Anaerobic'None (basal medium) 0Glucose 0.48 3p-Coumaric acid 0.05 6Ferulic acid 0.45 24Sinapic acid 0.23 17da Turbidity was measured at 520 nm against a blank

ofuninoculated medium. Absorbance is that remainingafter subtracting the absorbance at 0 h to correct fordifferences due to inoculum.

b TYG basal medium, but without glucose, pH 7.5,390C.

'Basal medium of (9), pH 7.5, 390C.d May not reflect true absorbance because of precip-

itated acid.

ments of shorter filaments and cocco-rods) arerepresentative of this culture. The filaments,although variable in length with changes in pH,were useful in tracing the microbial attack ofcell walls by SEM. Isolate 7-1 grown underoptimal conditions of pH (7.4 to 8.0) and tem-perature (39°C) degraded structurally intact, lig-nified cell walls that microbes in rumen fluidalone did not degrade. 7-1 was isolated from therumen population, but the suboptimal pH (6.7)of the rumen for growth of 7-1 may have pre-vented the extensive degradation of lignified cellwalls by rumen fluid detectable by SEM in thisstudy. The data indicated that growth on hem-icellulose-type constituents (i.e., xylan and pec-tin) was slow in aerobic culture and slight tolacking in anaerobic culture. This fact plus thefact that 7-1 was noncellulolytic by all methodstested herein indicated that much of the availa-ble (i.e., unlignified) constituents in forage cellwalls would not be used by 7-1. Indeed, compar-isons of digestion by SEM confirmed that rumenmicroorganisms were markedly more efficient indegrading unlignified tissues than was 7-1. Al-though degradation of tissues by 7-1 was notextensive, pretreatment with 7-1 before diges-tion with rumen microorganisms disrupted un-lignified and some lignified tissues and made thecell walls more readily available to rumen micro-organisms.Although other bacteria (14) and notably a

thermophilic actinomycete (10) have been re-ported to degrade lignocellulose, 7-1 degradedlignified cell walls of CBG in anaerobic andaerobic culture. The anaerobic degradation ofintact lignified tissues indicates a unique role forthis microbe. Comparison by SEM of the attackof CBG cell walls by the lignocellulolytic acti-nomycete T. fusca and 7-1 showed that bothmicroorganisms degraded lignified tissues, al-though the degree of digestion was not extensiveafter 8 days. Notably, lignified tissues such asthe inner bundle sheath and sclerenchyma were

FIG. 9. SEM of blade and stem sections incubated for 8 days with T. fusca 190Th. (a) Blade. The innerbundle sheath is separated into cells which show a layered wall (arrows). Sclerenchyma cells (C) weredegraded, and the actinomycete overgrew the vascular bundle. Bar, 10 iLm. (b) Stem. T. fusca degraded aportion of the parenchyma cells (P), whereas the sclerenchyma ring (R) and vascular bundles (1' resisteddegradation. The microbe formed a mycelial mat (M). The inset shows a hypha (arrow) in a hole between cellsof the resistant ring cells. Bar, 50 ,um; inset bar, 2 pin.

FIG. 10. SEMofblade and stem sections incubated for 8 days with rumen fluid. (a) Blade. The sclerenchyma(S), inner bundle sheath (I), and xylem (X) resisted digestion and appear intact for the most part. Bar, 10 ,um.(b) Stem. The vascular bundles (V), sclerenchyma ring (R), epidermis (E), and the most intensely stainingparenchyma cells (arrows) near the ring resisted digestion. Bar, 50 nm.

FIG. 11. SEM of blade and stem sections incubated for 8 days with 7-1. (a) Blade. The inner bundle sheath(I) is deformed whereas the sclerenchyma (arrows) is separated into individual cells. A large portion ofunlignified tissue remains. Bar, 10 tum. (b) Stem. The epidermis (E), the sclerenchyma ring (R), and vascularbundles (V) resisted degradation. The inset shows a filament (arrow) in a hole in the ring cells. Bar, 50 rmn;inset bar, 2 ,um.

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~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ '

t.S*ss,<"*'u' ;._,# |;zAM2

FIG. 12. SEM of control andpretreated (with 7-1) stem incubated for 6 h with rumen microorganisms. Bar,50 p.m. (a) Control stem, incubated for 72 h in uninoculated anaerobic medium before rumen microbialdigestion. A coating from the rumen fluid occurred on undegraded cell walls. (b) Stem pretreated for 72 h with7-1 in anaerobic medium before rumen microbial digestion. The parenchyma tissue (P) was partiallydegraded, and many protozoa (arrows) were within the stem section.

FIG. 13. SEM of blade incubated anaerobically for 28 days with 7-1. The sclerenchyma tissue (S) wasextensively degraded, and many rods and filaments (arrows) were present within disrupted cells. The innerbundle sheath (I) was partially degraded with the cells showing a layered wall (double arrows). Bar, 2 ,um.

often separated into individual cells, thus indi-cating that the intercellular substances weremore available to enzymatic degradation bythese microorganisms. T. fusca has been re-

ported to use the cellulose but not the ligninmoiety of lignocellulose pulps included as sub-strates (11). Anaerobic and aerobic culturing of7-1 on cell wall-type carbohydrates and phenolic

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MICROBIAL ATTACK ON LIGNIFIED CELL WALLS 819

acids representing lignin moieties suggested an-other mode of attack in which the acids suppliedcarbon and energy for growth. Ultraviolet spec-trophotometry showed that the amount of acidsdecreased in the spent broth, indicating thatsinapic acid was removed during growth. How-ever, the mechanism of degradation of the phe-nolic acids was not studied. Gas-liquid chroma-tography of each of the phenolic acids used assubstrates showed a purity of greater than 99%(M. E. Snook, personal communication), furtherindicating that growth was due to the phenyl-propanoid unit per se and not a contaminatingenergy source.SEM showed that 7-1 predominately attacked

the chlorine-sulfite-positive tissues (i.e., scler-enchyma in blades, parenchyma in stems),whereas attack on the acid phloroglucinol-posi-tive tissue (i.e., lignified vascular tissue and xy-lem) was markedly less. Chlorine-sulfite gives apositive reaction for syringyl-type lignin, i.e., 3,5-dimethoxypropane units (20). The primary at-tack on this type of lignified tissue agreed withaerobic culturing in which sinapic acid sup-ported greater growth than phenolic acids withone or no methoxyl groups substituted on thearomatic ring. A similar trend was present inanaerobic growth studies of 7-1 in which the lessmethoxylated acids supported less microbialgrowth. Furthermore, chlorine-sulfite-positivetissues are more easily degraded into individualcells by oxidation with the delignifying agentKMnO4 (3). The greater ease of delignificationand the attack on chlorine-sulfite-positive tis-sues by 7-1 suggested that these tissues are lessrigidly complexed than the acid phloroglucinol-positive tissues.

Filamentous bacteria in rumen fluid degradingsclerenchyma cells such as those reported (1)have been found by using transmission electronmicroscopy in six other grass species; further,sclerenchyma cells were essentially undegradedin the absence of these bacteria (unpublisheddata). Isolate 7-1 probably is able to exist in therumen despite its low number and relativelyslow growth rate because of its ability to usesubstrates other rumen bacteria cannot use. Asoluble form of substrate may be available to 7-1 from lignin-carbohydrate-complexes report-edly solubilized from plant cell walls in rumenfluid (13).Research is needed to test the potential of 7-

1 to use lignocelluloses at higher rates. Further-more, research with 7-1 and other microorga-nisms of known specificities for lignin-carbohy-drate degradation could be useful in clarifyingthe role of the various types of lignin in limitingthe availability of structural polysaccharides tomicrobial degradation.

ACKNOWLEDGMENTSI thank John F. Munnell, College of Veterinary Medicine

University of Georgia, for use of the scanning electron micro-scope; F. E. Barton II, Russell Research Center, for back-ground data on forages; John J. Evans, Russell ResearchCenter, for assistance with ultraviolet spectrophotometry; M.E. Snook, Russell Research Center, for data on phenolic acidpurity; and W. W. Hanna, Georgia Coastal Plains ExperimentStation, Tifton, for forage samples.

LITERATURE CITED

1. Akin, D. E. 1976. Ultrastructure of rigid and lignifiedforage tissue degradation by a filamentous rumen mi-croorganism. J. Bacteriol. 125:1156-1162.

2. Akin, D. E., D. Burdick, and G. E. Michaels. 1974.Rumen bacterial interrelationships with plant tissueduring degradation revealed by transmission electronmicroscopy. Appl. Microbiol. 27:1149-1156.

3. Barton F. E., II, and D. E. Akin. 1977. Digestibility ofdelignified forage cell walls. J. Agric. Food Chem. 25:1299-1303.

4. Bellamy, W. D. 1974. Single cell proteins from cellulosicwastes. Biotechnol. Bioeng. 16:869-880.

5. Bellamy, W. D. 1977. Cellulose and lignocellulose diges-tion by thermophilic Actinomyces for single-cell proteinproduction. Dev. Ind. Microbiol. 18:249-254.

6. Bryant, M. P., and L. A. Burkey. 1953. Cultural meth-ods and some characteristics of some of the more nu-merous groups of bacteria in the bovine rumen. J. DairySci. 36:205-217.

7. Bryant, M. P., and I. M. Robinson. 1961. An improvednonselective culture medium for ruminal bacteria andits use in determining diurnal variation in numbers ofbacteria in the rumen. J. Dairy Sci. 44:1446-1456.

8. Bryant, M. P., V. H. Varel, R. A Frobish, and H. R.Isaacson. 1977. Biological potential of thermophilicmethanogenesis from cattle wastes, p. 347-359. In H. G.Schlegel and J. Barnea (ed.), Microbial energy conver-sion. Pergamon Press, Inc., Elmsford, N.Y.

9. Caldwell, D. R., and M. P. Bryant. 1966. Mediumwithout rumen fluid for nonselective enumeration andisolation of rumen bacteria. Appl. Microbiol. 14:794-801.

10. Crawford, D. L. 1974. Growth of Thermomonosporafusca on lignocellulosic pulps of varying lignin content.Can. J. Microbiol. 20:1069-1072.

11. Crawford, D. L., and R. L. Crawford. 1976. Microbialdegradation of lignocellulose: the lignin component.Appl. Environ. Microbiol. 31:714-717.

12. Crawford, D. L., E. McCoy, J. M. Harkin, and P.Jones. 1973. Production of microbial protein fromwaste cellulose by Thermomonospora fusca, a thermo-philic actinomycete. Biotechnol. Bioeng. 15:833-843.

13. Gaillard, B. D. E., and G. N. Richards. 1975. Presenceof soluble lignin-carbohydrate complexes in the bovinerumen. Carbohydr. Res. 42:135-145.

14. Hajny, G. J., C. H. Gardner, and G. J. Ritter. 1951.Thermophilic fermentation of cellulosic and lignocellu-losic materials. Ind. Eng. Chem. 43:1384-1389.

15. Hungate, R. E. 1969. A roll tube method for cultivationof strict anaerobes, p. 117-132. In J. R. Norris and D.W. Ribbons (ed.), Methods in microbiology, vol. 3B.Academic Press, Inc., New York.

16. Jensen, W. A. 1962. Carbohydrates and cell wall constit-uents, p. 205. In W. A. Jensen (ed.), Botanical histo-chemistry. W. H. Freeman & Co., San Francisco.

17. Mandels, M., and J. Weber. 1969. The production ofcellulases, p. 391-414. In R. F. Gould (ed.), Cellulasesand their applications. American Chemical SocietyPublications, Washington, D.C.

18. Morrison, I. M. 1974. Structural investigations on thelignin-carbohydrate complexes of Lolium perenne. Bio-

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chem. J. 139:197-204.19. Prins, R. A. 1977. Biochemical activities of gut micro-

organisms, p. 73-183. In R. T. J. Clarke and T. Bauchop(ed.), Microbial ecology of the gut. Academic Press,Inc., New York.

20. Sarkanen, K. V., and C. H. Ludwig. 1971. Definition

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and nomenclature, p. 1-18. In K. V. Sarkanen and C.H. Ludwig (ed.), Lignins: occurrence, formation, struc-ture and reactions. Wiley-Interscience, New York.

21. Stafford, H. A. 1962. Histochemical and biochemicaldifferences between lignin-like materials in Phleumpra-tense L. Plant Physiol. 37:643-649.

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