APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1976, p. 562-568 Copyright © 1976 American Society for Microbiology

Vol. 31, No. 4 Printed in U.S.A.

Ultrastructure of Rumen Bacterial Attachment to Forage Cell Walls DANNY E. AKIN

Field Crop Utilization and Marketing Research Laboratory, Richard B. Russell Agricultural Research Center, Agricultural Research Service, Athens, Georgia 30604

Received for publication 31 December 1975

The degradation of forage cell walls by rumen bacteria was investigated with critical-point drying/scanning electron microscopy and ruthenium red staining/ transmission electron microscopy. Differences were observed in the manner of attachment of different morphological types of rumen bacteria to plant cell walls during degradation. Cocci, constituting about 22% of the attached bacteria, appeared to be attached to degraded plant walls via capsule-like substances averaging 58 nm in width (range, 21 to 84 nm). Many bacilli appeared to adhere to forage substrates without distinct capsule-like material, although unattached bacteria with capsules were observed occasionally. Certain bacilli appeared to be attached to degraded tissue via small amounts of extracellular material, but others apparently had no extracellular material. Bacilli with a distinct morphology due to an irregularly folded, electron-dense outer layer or layers (about 15 nm thick) and without fibrous extracellular material constituted about 37% of the attached bacteria and were observed to adhere so closely to degraded plant walls that the bacterial shape conformed to the shape of the degraded zone. In the rumen ecosystem, bacteria appeared to adhere to plant substrates during degradation by capsule-like material and by small amounts of extracellular material, as well as by other means not observable by electron microscopy.

Capsular and slime materials (21) have been shown to be involved in the association of bacteria with inert substances (13) and to cellulosic substrates (4, 19). The technique of ruthenium red staining (15) has been useful to show the fibrillar nature of capsule-like substances (13, 18, 20). Recently, the technique of critical-point drying (3) of bacteria, used to study extracellular material, showed a fibrous nature to the capsule-like polymer surrounding bacteria (5). Investigations of the rumen microfloral degradation of forage tissue have revealed the presence of capsule-like material adjoining large cocci to plant walls (1, 2). Additionally, bacteria from pure cultures of Ruminococcus albus were shown to attach to cellulose by fibrillar structures external to the bacterial cell wall (19). Research from this laboratory indicated that differences exist in the mode of attachment of rumen bacteria to forage tissue (2). The objectives of this work were to (i) investigate further the ultrastructural nature of rumen bacterial attachment to forage tissue, and (ii) delineate the manner of attachment of different morphological types of rumen bacteria to forage tissue, by means of critical-point drying/ scanning electron microscopy and ruthenium red staining/transmission electron microscopy.

MATERIALS ANI) METHOI)S Inoculum. For in vitro studies, rumen digesta obtained from a cannulated steer was strained through cheesecloth into a previously warmed vacuum bottle for transportation to the laboratory. The rumen fluid was strained again through cheesecloth and mixed 1:2 with McDougall carbonate buffer (17) previously bubbled with CO2 at 39 C. For in vivo studies, rumen bacteria that were associated with partially degraded leaf blades were removed from the fistulated steer and studied in situ. Substrate. For in vivo studies, substrates consisted of leaf blades of Coastal bermudagrass (Cynodon dactylon I L.l Pers.) frozen immediately after harvest at 4 weeks of age and maintained at -30 C. Leaf blades were cut into sections 2 to 5 mm long with a new, cleaned razor blade to reduce drag across the tissue cross section. These leaf sections were then inoculated with the rumen bacteriabuffer inoculum at 39 C with continuous CO2 bubbling. For in vivo studies, leaf blades from the partially digested hay that was predominately Coastal bermudagrass were examined. Scanning electron microscopy. Leaf blade sections digested in vitro or in vivo were fixed in 4% glutaraldehyde (in 0.1 M cacodylate buffer, pH 7.0 to 7.2) for 16 to 24 h and postfixed in 1.5% buffered osmium tetroxide for about 4 h at 4 C. Samples were dehydrated in a series of ethanol-water washes (25%

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through 100%; vol/vol) and critical-point dried in liquid CO2 without transitional solvents. Samples were then coated for conductive purposes with goldpalladium (60-40) alloy wire and observed with the scanning electron microscope (SEM) at about 15 kV. Transmission electron microscopy. A pretreatment of 0.1 M cacodylate buffer with 0.15% (wt/vol) ruthenium red for 30 min at room temperature was used for one group of samples of degraded leaves; all samples were then prepared for the transmission electron microscope (TEM) as described previously (2) with the inclusion of 0.15% (wt/vol) ruthenium red in both fixatives according to the formula used by Cagle et al. (6). Other samples were prepared without ruthenium red staining for comparative purposes. Sizes of capsules and external layers of the bacteria were determined from enlargements of thin sections with a particle-size analyzer. Capsular widths for cocci were determined by averaging the three unattached sides of 23 randomly chosen bacteria. The percentages of morphological types of attached bacteria were determined from bacteria associated with degraded zones in forage walls observed in 15 micrographs.

RESULTS Most interpretations were made from in vitro-digested leaf blade sections. However, the same morphological types of bacteria were found in both in vitro- and in vivo-digested specimens, and the differences in the mode of attachment by these morphological types were similar in both types of digestion. Ultrastructure shown by the SEM. Many bacteria, particularly the cocci, appeared to be attached by extracellular, fibrous structures to plant walls undergoing degradation (Fig. 1 [F]). That many of these bacteria were actively involved with plant tissue degradation was indicated by degraded zones surrounding the bacteria (Fig. 1, inset). However, rumen bacteria, particularly bacilli, without the extracellular, fibrous structures were often observed in degraded zones of plant walls (Fig. 2 IB]). These observations suggested that some bacilli involved with the attachment and degradation of plant tissues have no extracellular material or insufficient amounts to be observed with the SEM using critical-point-dried samples. Ultrastructure shown by the TEM. In thin sections of ruthenium red-stained, degraded leaf sections, numerous bacteria were attached to the plant wall by a capsule-like material (Fig. 3, 4, 5). Often, the width of the side of the capsule attached to the plant material was longer than the unattached side, indicating a stretching of the extracellular material (Fig. 4, insets). Many round-appearing bacteria were attached to cell walls. Cocci were distinguished from bacilli that had been sectioned perpendic-

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ularly to the long axis of the cell by similarity in morphology and electron denseness to capsules and cell walls of known cocci (as revealed by round bacteria dividing by binary fission, Fig. 3 [C]). No bacilli that resembled these dividing cocci in capsular and cell wall morphology were seen in thin sections. Although most cocci-degrading plant walls appeared to be attached via capsules, the capsules varied in size (Fig. 5 [C]), averaging 58 nm wide and ranging from 21 to 84 nm. These encapsulated cocci constituted about 22% of the attached bacteria. Distinct capsules were not observed with bacilli that were degrading plant material, and extracellular material was present only in small amounts (Fig. 4) or absent (Fig. 6 [B]) between bacteria and plant walls. On occasion, large capsules were observed surrounding bacilli, but these bacteria were not attached to plant walls (Fig. 6, arrow). Bacilli with a distinctive morphology appeared to be attached without external fibrous material to plant walls (Fig. 5, 7, 8 [B]). These bacteria had an electron-dense, irregularly folded outer layer (or layers) about 15 nm thick, which was separated from the plasma membrane by an electron-transparent layer (Fig. 7, 8). The electron-dense layer was present in specimens not treated with ruthenium red, but fixed with OsO and stained with uranyl acetate and lead citrate; however, the layer usually appeared to be more intensely stained in ruthenium red-stained specimens. These bacilli (with dimensions of 1 to 2 by 0.3 to 0.5 Am for unattached cells) were closely associated with degraded regions, and the bacterial shape was modified to conform with the shape of the degraded zones (Fig. 5 IB], 8). This type of microbe is numerous in degraded plant wall regions and constituted about 37% of the bacteria attached to plant cell walls. Diffuse, fibrillar material external to the irregularly folded outer layer of an unattached bacterium was found in one sample of in vivo-degraded tissue (Fig. 7, inset), but usually the extracellular coat material was negligible or perhaps contained in the thin electron-dense layer. Examination at high magnification revealed a gramnegative-type cell wall morphology (Fig. 8, inset) with, in one case, a small layer, not densely stained, external to the outer membrane (Fig. 8, arrow). Capsule-like material was not apparent between bacterial and plant cell walls. I)ISCUSSION Hungate (12) reported that the cellulolytic ruminococci possessed "abundant" capsules. Akin et al. (2) reported that large cocci from

FIG. 1. Scanning electron micrograph of critical-point-dried leaf digested in vitro with rumen bacteria. Bacteria appear to be attached to plant cell walls via fibrous structures (F) typical of critical-point-dried extracellular material. A coccus (inset) with fibrous structures (arrow) adjoining bacterial and plant wall lies in a degraded zone. x8,000; inset, x13,500. FIG. 2. Scanning electron micrograph of critical-point-dried leaf digested in vitro with rumen bacteria. Some bacilli (B) that lack extracellular material are within depressions of the plant wall. Two cocci appear to be joined by extracellular material (arrow). x15,000. 564

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mixed bacterial populations of the rumen were attached to plant structures by a capsule-like material. Additionally, Patterson et al. (19), using critical-point drying and the TEM, showed that extracellular fibrils from cells of a pure culture of R. albus were involved with attachment of the bacteria to cellulose. Leatherwood (14), using the SEM for cultures grown on cellulose, reported the presence of "tubelike" appendages on R. albus. Specimens observed in this study by criticalpoint drying/SEM and ruthenium red staining/ TEM revealed a fibrous nature to the capsulelike material that was apparently involved in the adhesion of cocci to plant walls during degradation. The average capsule width of 58 nm may be an underestimation due to shrinkage during TEM preparation of the highly hydrated (21) capsule. However, many bacilli lacking capsule-like, fibrous material in the critical-point-dried preparations appeared to be within depressions in the plant wall. That many of the bacilli lacked or possessed small amounts of extracellular material was confirmed by the TEM. Cheng and Costerton (7), using ruthenium red staining, described 10 different morphological types of external layers in bacteria (not attached to a substrate) from a mixed rumen microflora. Although some layers were small, these authors (7) reported that all bacteria examined had at least some external coat material. Conversely, Akin et al. (2) reported that in thin sections not stained with ruthenium red, some bacilli were attached to plant walls with-

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out apparent extracellular material; this was confirmed by the observations reported herein. Apparently, an adhesive layer not detected with the methodology used herein or other attractive forces (16) enabled certain rumen bacilli to adhere to the plant substrate during degradation. Bacilli with a distinct morphology due to an electron-dense, irregularly folded outer layer (or layers) were seen closely attached to plant walls undergoing degradation (Fig. 8). These bacteria possessed a gram-negative-type cell wall structure (10) without external, fibrous coat material in most cases. Costerton et al. (9) showed that Bacteroides succinogenes, a prevalent cellulolytic species in the rumen (12), in pure culture possessed an irregular outer membrane with small and irregular amounts of coat material similar to the bacilli mentioned above. These authors (9) concluded that the irregular folding, observed with two other bacteria, with foldings of different extents among species, was an artifact of fixation or embedding based on freeze-etch data. The bacilli reported herein to have the irregular layer(s) appeared to be of a certain dimension (1 to 2 by 0.3 to 0.5 Am), whereas bacteria of other dimensions and shapes lacked this morphology. The shapes of these bacteria appeared at times to be modified by close contact with substrate or other bacteria. The irregular shape of the bacilli in contact with plant cell walls is an unexplained phenomenon; it is not known if the thinness of the peptidoglycan layer, which has been reported for B. succinogenes (10), could be responsible. It

FIG. 3. Thin section of ruthenium red-stained leaf degraded in vitro by rumen bacteria. Cocci (as shown by cells dividing by binary fission IC]) are attached to the plant wall by capsule-like material (arrows). x20,OOO. FIG. 4. Enlargment of Fig. 3. A bacillus (B) appears to adhere to the plant wall by a small amount of extracellular material, whereas the coccus (C) is attached via a structure resembling capsule-like material. Stretching of the extracellular material is indicated in a coccus shown by the TEM (lower inset) and in a bacillus shown by the SEM (upper inset) (arrows). x40,OOO; lower inset, x25,500; upper inset, x12,000. FIG. 5. Thin section of ruthenium red-stained leaf degraded in vitro by rumen bacteria. Cocci (C) and bacilli (B) with an irregular shape appear to be attached to the plant wall. The extracellular material around the cocci varies in width. The bacilli possess an electron-dense outer layer but lack capsule-like material; their shape is altered when in contact with plant material (arrows). x18,000. FIG. 6. Thin section of ruthenium red-stained leaf degraded in vitro by rumen bacteria. A bacillus (B) in close proximity to an epidermal cell lacks any evidence of extracellular material but is degrading the cell wall material. The inset shows a bacillus with a diffuse fibrous capsule (arrow) from an in vivo-degraded section; this microbe is not attached to plant material. x46,000; inset, x28,900. FIG. 7. Thin section of ruthenium red-stained leaf degraded in vitro by rumen bacteria. Bacteria (B) with electron-dense, irregularly folded outer layers are attached closely to plant material; the bacteria appear to possess a pliable cell wall as shown by the modification in cell wall close to other bacteria (arrow). No apparent fibrous extracellular material is visible. The inset shows a morphologically similar, but unattached bacterium from the in vivo degradation of leaf tissue showing a diffuse fibrous capsule-like material (inset, arrow). x32,000; inset, x28,900. FIG. 8. Thin section of ruthenium red-stained leaf degraded in vitro by rumen bacteria. The bacterium with irregular folding of the outer layer is attached without apparent fibrous extracellular material, although a thin layer is seen on one side (arrow). The attached side of the bacterium is very irregular, with portions of the bacillus (B) extending into the plant wall (P). The inset reveals the double membrane (arrows) of the cell wall structure characteristic of gram-negative bacteria. x80,OOO; inset, x 150,000.

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is doubtful that these irregular shapes are artifacts of TEM preparation since, at times, extensions of the bacterial cells seem to be within the degraded zones of rigid plant walls. Factors other than capsules or slime have been reported to be responsible for bacterial attachment to substances (8, 16). Certain of the bacilli described herein appeared to have a thin, irregular, electron-dense layer that might be responsible for attachment like the "primary acid polysaccharide" reported for initial adhesion of a marine bacterium to solid surfaces (11). Other bacilli appeared to be in close proximity to plant walls undergoing degradation without noticeable extracellular material detectable by the TEM. However, that the fixation, dehydration, and embedding steps could have obscured particular extracellular substances of these bacteria cannot be completely ruled out. The observations reported herein reveal differences in the manner of attachment of various morphological types of rumen bacteria to forage material during degradation. ACKNOWLEDGMENTS I thank Billy D. Nelson, Southeast Louisiana Experiment Station, Franklinton, La., for the forage samples and Henry E. Amos, Field Crops Laboratory, Athens, Ga., for assistance in collecting and preparing the rumen fluid inoculum.

LITERATURE CITED 1. Akin, D. E., and H. E. Amos. 1975. Rumen bacterial degradation of forage cell walls investigated by electron microscopy. Appl. Microbiol. 29:692-701. 2. Akin, D. E., D. Burdick, and G. E. Michaels. 1974. Rumen bacterial interrelationships with plant tissue during degradation revealed by transmission electron

microscopy. Appl. Microbiol. 27:1149-1156. 3. Anderson, T. F. 1950-51. Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci. 13:130-134. 4. Berg, B., B. V. Hofstein, and G. Pettersson. 1972. Electron microscopic observations on the degradation of cellulose fibres by Celluibrio fulvus and Sporocytophaga myxococcoides. J. Appl. Bacteriol. 35:215-219. 5. Cagle, G. D. 1974. Critical point drying: rapid method

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for the determination of bacterial extracellular polymer and surface structures. Appl. Microbiol. 28:312316. Cagle, G. D., R. M. Pfister, and G. R. Vela. 1972. Improved staining of extracellular polymer for electron microscopy: examination of Azotobacter, Zoogloea, Leuconostoc, and Bacillus. Appl. Microbiol. 24:477-487. Cheng, K. J., and J. W. Costerton. 1975. Ultrastructure of cell envelopes of bacteria of the bovine rumen. Appl. Microbiol. 29:841-849. Corpe, W. A. 1970. Attachment of marine bacteria to solid surfaces, p. 73-87. In R. S. Manly (ed.), Adhesion in biological systems. Academic Press Inc., New York. Costerton, J. W., H. N. Damgaard, and K. J. Cheng. 1974. Cell envelope morphology of rumen bacteria. J. Bacteriol. 118:1132-1143. Costerton, J. W., J. M. Ingram, and K. J. Cheng. 1974. Structure and function of the cell envelope of gramnegative bacteria. Bacteriol. Rev. 38:87-110. Fletcher, M., and G. D. Floodgate. 1973. An electronmicroscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces. J. Gen. Microbiol. 74:325-334. Hungate, R. E. 1966. The rumen bacteria, p. 8-90. In R. E. Hungate (ed.), The rumen and its microbes. Academic Press Inc., New York. Jones, H. C., I. L. Roth, and W. M. Sanders III. 1969. Electron microscopic study of a slime layer. J. Bacteriol. 99:316-325. Leatherwood, J. M. 1973. Cellulose degradation by Ruminococcus. Fed. Proc. 32:1814-1818. Luft, J. H. 1971. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat. Rec. 171:347368. Marshall, K. C., R. Stout, and R. Mitchell. 1971. Mechanism of the initial events in the sorption of marine bacteria to surfaces. J. Gen. Microbiol. 68:337-348. McDougall, E. I. 1948. Studies on ruminant saliva. I. The composition and output of sheep's saliva. Biochem J. 43:99-109. Pate, J. L., and E. J. Ordal. 1967. The fine structure of Chondrococcus columnaris. III. The surface layers of Chondrococcus columnaris. J. Cell Biol. 35:37-51. Patterson, H., R. Irvin, J. W. Costerton, and K. J. Cheng. 1975. Ultrastructure and adhesion properties of Ruminococcus albus. J. Bacteriol. 122:278-287. Springer, E. L., and I. L. Roth. 1973. The ultrastructure of the capsules of Diplococcus pneumoniae and Klebsiella pneumoniae stained with ruthenium red. J. Gen. Microbiol. 74:21-31. Wilkinson, J. F. 1958. The extracellular polysaccharides of bacteria. Bacterial. Rev. 22:46-73.

Ultrastructure of rumen bacterial attachment to forage cell walls.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1976, p. 562-568 Copyright © 1976 American Society for Microbiology Vol. 31, No. 4 Printed in U.S.A. Ul...
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