SYMPOSIUM ON MICROBIAL DIGESTION IN RUMINANTS: IDENTIFICATION OF GROUPS OF ANAEROBIC BACTERIA ACTIVE IN THE RUMEN 1

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1 I SYMPOSIUM ON MICROBIAL DIGESTION IN RUMINANTS: IDENTIFICATION OF GROUPS OF ANAEROBIC BACTERIA ACTIVE IN THE RUMEN 1 T is now evident that a complete ecological analysis of the rumen microbial population (Hungate, 1960) is of basic importance to a more complete understanding of the metabolism of ruminants. Part of this analysis involves the collection of information on the identity, nutrition and metabolic functions of the individual microbial species involved. At the present time the best means of obtaining information on the individual species of bacteria is via studies on pure cultures. Pure culture studies already have led to important qualitative findings on the rumen fermentation concerning nutritional requirements, protein and lipid metabolism, and intermediary metabolism, and have yielded some interesting implications regarding the rumen environment. Some of the information would be difficult to obtain or to interpret by the use of the mixed microbial population. When one is studying various metabolic functions of pure cultures of tureen bacteria, it is of prime importance to know the identity of the individual strain studied. Organisms must be identified and classified in order for our knowledge to progress in an orderly fashion. In the present paper, I will discuss some of the features which at the present time seem to be of most utility in the identification of rumen non-sporeforming anaerobic bacteria at the genus or species level. Among the more recent publications concerning various characteristics of species of rumen bacteria the following may be of interest. Hungate (1960) has discussed the ecological analysis of the tureen population. Species of rumen bacteria were discussed by Bryant (1959). Their cu ture and enumeration (Hobson, 1961), nitrogen metabolism (Hobson, 1959: Bryant, 1961), nutrition (Bryant and Robinson, 1962), and the utility of pure culture studies (Bryant, 1960) have been discussed. Sneath (1957) and Ainsworth and Sneath (1962) discussed 1 Presented at 54th meeting of the American Society of Animal Science, November 24, ~ Dairy Cattle Research Branch, AHRD, ARS, ARC, Beltsville, Maryland. MARVIN P. BRYANT" United States Department o] Agriculture the philosophy of bacterial classification. These articles give reference to many excellent earlier discussions. General Considerations The adequate identification of a bacterial strain as to species depends on the determination of a large number of features and the indication that these features correlate reasonably well with those found by the workers who previously described and classified the organism. Identification is made difficult if the classification is based on few or poor features and usually this is also true if it is based on the study of only a few strains. The features used in classifying a given organism are subject to the viewpoint of the classifier. However, the main reason for classification is its high content of information, its predictive value, and the worker should try not to bias the predictive value of his classification toward any one viewpoint, e.g., pathogenicity or, as might be done in the case of rumen organisms, energy sources that are important in the rumen fermentation. A part of one paragraph from Sneath (1957) concerning genera and species of bacteria is quoted. "At present, we have almost nothing to guide us on the rank which should be given to a taxon of bacteria--it is a matter of personal preference. The theoretical method is to lay down that strains of any species should have a certain percentage of common features, but until more work is done upon this it is desirable to observe two working rules. The first is that a mutant should not be given specific rank. Bacteriologists do not do this, but they are sometimes tempted to give specific rank to strains which might be naturally occurring mutants differing from the common type in only a single feature. The second is that one should be chary of separating into different genera bacteria which undergo genetic exchange." The ideal in classifying bacteria would be to give equal weight to each of many features determined (Sneath, 1957); however, in the 801

2 802 BRYANT case of most bacteria so far classified, this has not been possible. Among rumen bacteria more weight has usually been given to gross morphology, major fermentation products, relations to oxygen, and ability to utilize certain energy sources than to many other features. In the first two cases we hope this is justified by the probability that they are usually the resultant of many more basic features of the cell. In the case of certain energy sources, e.g., cellulose digestion in the genus Ruminococcus, it was probably not justified (Hungate, 1957; Bryant, 1959). The rumen contains a great variety of genera and species of bacteria, many of which may constitute a significant proportion of the total population. Most of these are non-sporeforming anaerobes that are difficult to grow and study. The present state of their classification is quite poor. The more strains and the more features of these bacteria that can be studied, the better position one will be in to properly classify them, and the easier it will be for future workers to identify them and to have an idea of the probability of the occurrence of featues not determined. Methodology The methods used in determining characteristics of bacteria are extremely important (Society of American Bacteriologists, 1957). One should attempt to use methods similar to those used by the workers who previously established the characteristics. It is well to include previously studied strains of similar bacteria as controls; and this is particularly important if a previously used method seems inappropriate and different conditions are used. Many examples could be cited from the literature where confusion has been caused by failure to take these precautions. A point which is particularly troublesome today is the tendency to draw an imaginary line in identification between rumen anaerobes and anaerobes from other environments even though it is known that very similar organisms function in other habitats, e.g., Butyrivibrio (Brown and Moore, 1960), Ruminococcus or similar cellulolytic cocci (Baker and Harriss, 1947; Hall, 1952), anaerobic spirochetes (Veldkamp, 1960), Selenomonas (Macdonald et al., 1959), Methanobacterium ruminantium (Smith, 1961) have been found in other habitats and it is probable when further studies are made using selective methods other similar organisms will be found in habitats such as the oral cavity and intestinal tract of non- ruminant animals, sewage sludge and sediments in natural waters. Strains of many different species of anaerobic bacteria are available on request from Beltsville and many other laboratories and culture collections. The method of isolation has a great bearing on the ease with which an organism can be identified. The greatest difficulty is likely to be encountered if relatively non-selective, nondifferential, culture media such as that of Bryant and Robinson (1961a) are used. Two or more carbohydrates such as glucose, cellobiose, maltose or starch are usually added to these media as energy source and a great number of ruminal species will grow using one or more of these substances. When relatively selective or differential media for isolation involving energy sources such as cellulose (Hungate, 1950), lactate (Hobson et al., 1958), glycerol or glycerides (Hobson and Mann, 1961), or saponin (Gutierrez et al., 1959a) are used, relatively few species are encountered in large numbers and identification should be easier. The methanogenic bacteria have not been encountered as well isolated colonies after direct culture from rumen fluid except with highly specialized and difficult techniques and under these conditions only one species, Methanobacterium ruminantium, appears to have been encountered (Smith and Hungate, 1958; unpublished data of Robinson and Bryant, 1962). There is considerable nutritional and physiological information now available on tureen species which should be exploited in a search for good selective or differential culture media for primary isolation and accurate enumeration of physiological groups or species of rumen bacteria. Development of such media would greatly facilitate rapid identification. Many culture media used in the past to determine characteristics of rumen bacteria have contained rumen fluid because many strains would not grow well in media commonly used to grow other anaerobic bacteria or because the workers hoped to use conditions at least approximating the ruminal environment. However, different batches of rumen fluid vary considerably in concentration of various solutes and it is sometimes not readily available. Also, for a more adequate classification and identification, it will be beneficial if some future workers place more emphasis on features of use in classification in addition to features we believe to be of significance regarding rumen function. Recent advances in our knowledge of the

3 ANAEROBIC BACTERIA IN RUMEN 803 nutrition of rumen bacteria (see Bryant and Robinson, 1962, for many references) indicate that media without rumen fluid and containing well standardized ingredients may be used for most strains. Table 1 gives ingredients of a general medium which might be modified in appropriate ways depending on the feature to be determined and the species being studied. The medium includes an appropriate energy source at a level appropriate to the feature being determined. Trypticase and yeast extract can often be replaced by a B-vitamin mix because many species do not require organic nitrogen sources and heroin and vitamin K are required by few species. Minerals are included but many of them, e.g., the latter three, can be excluded if the crude nitrogen sources are included. The volatile acids are not required by some species and are not required by others if trypticase is included. Some species do not require CO2 so that phosphate buffer and N2, H2, or other gas that does not affect ph could be used in place of the H2CO~ - NaHCO~ buffer. Resorufin, TABLE 1. A GENERAL PURPOSE MEDIUM, CONTAINING STANDARDIZED INGREDIENTS, WHICH MAY BE USED FOR THE GROWTH OF A WIDE VARIETY OF RUMEN BACTERIAL SPECIES AND WHICH MIGHT BE MODIFIED IN APPROPRIATE MANNERS AND BE USED FOR DETERMINATION OF MANY FEATURES ~ Ingredients Final concentration 1. Energy source--glucose, cellobiose, maltose, glycerol or lactic acid, etc % 2. Organic nitrogen and vitamin sources Trypticase or equivalent % Yeast extract % Hemin 0.1 mg. % Menadione 0.1 rag. % 3. Minerals b 4. Resazurin or phenosafranine % 5. Acetic acid, glacial 0.17% (v/v) 6. Isobutyric, isovaleric, DL-a-methylbutyric and n-valeric acids, "pure," each 0.01% (v/v) 7. Add water, adjust to ph with NaOH, make to final volume minus volumes of carbonate and reducing agent solutions. 8. Autoclav 15 psi for min. and cool under Oe-free conditions. 9. Add sterile, O~-free solution Na~CO3. e 10. Add sterile COd-free solution of one or more reducing agent(s). Cysteine'HC1.H~O 0.05% Na2S-9H % Na2S2Oa % a See Hungate (1950), King and Smith (1955), Snfith and Hungate (1958), Kistner (1960), Bryant and Robinson (1961a), Blackburn and Hobson (1962), Brfiggemann et al. (1962), and Fulghum and Moore (1963), or retcrence therein fur various modifications for preparing media using the principles of the Hungate anaerobic technique. b An appropriate mineral concentration (final concentration) would be 0.09% (each) (NH~)2SO4, KI-LPO~ and NaCI, 0.002% (each) CaC12 and MgC12"6H20, 0.001% MnC12"4H~O, % CoCI~-6H~O, and 0.01% FeSO~-7H~O. e The level of NaI-ICOa or Na2CO~ added depends on the gaseous phase of the medium and the ph desired. For ph of about add about 0.4, 0.3, 0.06 and 0.03% of Na2COa for gaseous ohases containing 100, 50, 10, and 5% of CO2. the reduction product of resazurin, is the preferred oxidation-reduction indicator for most species; however, phenosafranine is much better for studies on the methanogenic bacteria and, perhaps, others which require an extremely low oxidation-reduction potential to initiate growth. Among the reducing agents, Na2S and Na2S203 are quite inhibitory for some species and this is particularly so in media containing little or no organic nitrogen; however, Na2S is stimulatory to ruminococci and possibly other species and both Na2S and Na2S2Os give a lower oxidation-reduction potential than cysteine. 3 Some of the difficulties in identification of rumen bacteria which have been encountered are discussed below. The shape and size of cells within a single culture or within a group of strains may vary considerably even when observed under one set of conditions. For example, in a relatively young culture of Bacteroides ruminicola, cells may vary in shape from coccoid to very long rods. Some strains are mainly coccoid, others mainly rodshaped and others in between. Strains of Bacteroides succinogenes show similar variations and in addition some strains may be smaller, more slender, somewhat curved rods and show sharply pointed, rather than rounded, ends. The apparent difference between the direct microscopic studies such as those of Warner (1962) indicating that the most numerous tureen bacteria are gram negative and coccoid, and cultural results, such as those at Beltsville, is probably at least partially due to the coccoid shape of many cells which actually belong to species of rod-shaped bacteria. Some strains of species composed of more or less curved rods may show very little or no curve, e.g., strains of Succinivibrio and Butyrivibrio. Cells of many species tend to swell and lyse shortly after maximum growth is obtained and to determine their shape it is necessary to examine cultures after short incubation periods. There is considerable variation in size of cells of strains within some species. Probably the most striking example of this is among strains of Selenomonas ruminantium. Some are small and their size and a It is possible that excellent colony counts from rumen contents could be obtained with a similar medium containing agar, particularly if organic nitrogen and energy sources were maintained at a low level (Bryant and Robinson, 1961a). Using a medium similar to that in table 2 containing agar, glucose, tryptiease, heroin, minerals, volatile fatty acids, cysteine and a CO2 gaseous phase and a chemically defined medium with B-vitamins and methionine replacing the trypticase, similar colony counts of a strain of B. ruminicola were obtained when the inoculum was grown in either a rumen fluid liquid medium or the defined medium minus agar (Bryant, Pittman and White, unpublished data, 1962).

4 804 BRYANT shape can be confused with other species of curved rods. Most strains are larger but very few are as large as serologically similar selenomonads in rumen contents as shown by Hobson et al. (1962) using fluorescent antibody techniques. Whether or not a strain is motile is of great importance in its identification, yet many strains which are actually motile have been recorded as nonmotile. Two of the reasons for this are that in some groups the motility rapidly diminishes on contact with air (Bryant, 1952) and in some cases motility is not detected when cells are grown in media containing more than a small amount of carbohydrate. Bryant and Small (1956) used a very simple method in demonstrating motility which seems to be satisfactory for all species studied. It involves the observation of wet mounts of young cultures immediately after preparation from the water of syneresis of stab inoculated rumen fluid agar slants containing a low level of sugar. A pasteur pipette is used for the transfer. A phase contrast or darkfield microscope is of great value both in motility studies and determining shape. Demonstration of the type of flagellation is also extremely important among the motile species but is in some cases extremely difficult to detect or if detected, to be sure of the arrangement or point of attachment of flagella. Leifson (1960) gives an excellent discussion of flagella and the staining technique. The gram reaction is another important characteristic but must be interpreted with great caution. Some species are strongly gram positive and some are definitely negative but others are only weakly positive even in young cultures and the genus Ruminococcus might include both positive and negative strains. If a strain appears to be gram negative it is well not to rule out the possibility that it has been previously described as gram positive and that a detailed study of young cultures with well controlled methods would show it to be gram positive. Also, some of the apparently gram negative species with large cells, e.g., Selenomonas and Peptostreptococcus elsdenii, have occasionally been described as showing some gram positive cells or gram positive intracellular granules. In the future, more detailed cytological studies involving various staining procedures and electron microscopy will probably be of great value. For example, cell wall studies might indicate that Bacteroides succinogenes is actually not a true bacterium but a member of the order Myxobacteriales as suggested by Hungate (1950). It might be more closely related to the genus Cytophaga than to Bacteroides. After the morphology has been adequately studied, probably the next most important group of features in identification are the fermentation products. The products of fermentation of cellulose, glucose, cellobiose, or starch have usually been determined, although in some cases, lactate, glycerol or H2 have been used, often because the strain does not ferment sugars. Few of the more numerous rumen anaerobes actively ferment amino acids, at least in the absence of carbohydrate. Some of the points which should be considered in determining fermentation products follow. Many species produce succinic acid as a major fermentation product and require a relatively high level of CO2 in order to ferment an energy source such as glucose (Anderson and Ordal, 1961; White et al., 1962). In some strains of rumen bacteria the amount of CO2 - HCO~ available may greatly affect the proportions of various fermentation products produced. Paynter and Elsden (personal communication, 1960) showed that a strain of Selenomonas ruminantium produced more lactic and less propionic and acetic acids from glucose when the gaseous phase was N2. More propionic and acetic acids were produced when the gaseous phase was CO2. Gill and King (1958) and Lee and Moore (1959) showed that less butyrate and more lactate were produced from glucose by a strain of Butyrivibrio when grown in a purified medium as compared to a rumen fluid medium. Many other factors such as growth rate (Rosenberger and Elsden, 1960), ph, the amount of iron, hydrogen acceptors and certain B-vitamins may affect the nature of the products produced by a single strain. Wood (1961) gives an excellent discussion on fermentation of carbohydrates by bacteria. Satisfactory fermentation balances, i.e., good recovery of carbon and oxidation-reduction balances corresponding to the substrates fermented, are important but have not been obtained with many rumen organisms. This might be very difficult with some species if methods involving growth are used. Some species probably synthesize much of their protoplasm from the carbohydrate energy source, CO2 and volatile fatty acids, rather than using preformed, exogenous cell monomers such as amino acids (see Bryant and Rob-

5 inson, 1963, for references), and cells produced often rapidly lyse. Therefore, a significant amount of the carbon of the energy source(s) might have to be recovered as the many possible products of cell lysis. The use of resting cell suspensions and low levels of C14-energy source might help one to obtain more accurate analyses. The study of enzyme systems by the use of C 14 labeled compounds (Baldwin et al., 1962) and other methods of enzymology will probably become increasingly useful in identification of rumen bacteria. For example, propionate is produced via a symmetrical molecule, probably succinate, in Veillonella (Johns, 1951a) and Selenomonas (Paynter and Elsden, 1960, personal communication) but is produced via another mechanism in Peptostreptococcus elsdenii ( Ladd and Walker, 1959). Energy sources utilized for growth are important in identification but often are quite variable within a species. Precautions should be taken to ensure that labile energy sources such as certain carbohydrates are not modified during preparation of test media and that acid production; growth, or other criteria of utilization are not confused by utilization of other materials such as amino acids or organic acids often present in the basal medium. If acid production is used as a criterion, it is well to use a lowly buffered medium. In the case of the many organisms requiring a high level of CO2 for growth, we have found a medium containing a 10% CO2--90% N2 gaseous phase and low phosphate to be satisfactory (Bryant et al., 1958) although considerably poorer growth is often obtained as compared to the same medium with more CO2. In the case of materials such as pectates and organic acids, criteria other than acid production may have to be used. These criteria might include growth, gas production, disappearance of substrate or production of specific end products. To obtain rapid fermentation of succinate in propionibacteria, Johns (1951b) found it necessary to include sugar and a low buffer capacity in the medium and to quantitatively analyze for succinate. The ph limits and optimum for growth initiation are good features for identification of many bacteria but are difficult to determine with many rumen anaerobes because of their large CO2 requirement. However, the final ph after growth in a lowly buffered medium, such as that indicated above for carbohydrate fermentation studies but with a non!imiting level ANAEROBIC BACTERIA IN RUMEN 805 of a readily fermented sugar added, gives quite valuable information. For example, selenomonas strains almost invariably give a lower final ph than motile curved rods with which they might be confused. Many other physiological tests are of some use in identifying rumen bacteria (Society of American Bacteriologists, 1957). A few of these are indole, sulfide, and NH3 production and NO3 reduction. It is hoped that in the future more workers will determine the source(s) of products such as sulfide and ammonia. Also, in the case of the reduction of ions such as NO~ and SO: it would be of great value in identification as well as in understanding rumen reactions to know whether assimilatory or dissimilatory, i.e., respiratory, reactions are involved (Nason, 1962 ; Peck, 1962). Serological tests have not been used to any great extent in identifying rumen bacteria; and where they have been used, there was often indication of many antigenic types within species (personal communication on ruminococci, E. Hall, 1953; unpublished data on B. ruminicola, Bryant, 1957; see Hobson et al., 1962 for further references, particularly on the use of fluorescent antibody techniques). Several laboratories are now engaged in serological studies, and it is possible that group or species specific antigens will be found. The term "anaerobic", as usually used in connection with rumen organisms, refers to their inability to initiate growth from relatively small inocula unless the oxidation-reduction potential of the medium is low. It has seldom been used to indicate the organisms inability to utilize O~ as a terminal hydrogen acceptor (see McBee et al., 1958, for a discussion of oxygen relations). Indeed, it seems probable that some rumen anaerobes have at least a limited ability to use 02. This feature requires more study. Colony types may be of use under some conditions (Hungate, 1950); however, when 2% of agar is used in isolation in order to stop movement of motile organisms through or over the surface of the agar and to prevent collapse of the agar in roll tubes, visible differences tend to disappear. Filamentous, woolly colonies are typical of Lachnospira. However, other species may show this type of colony (Blackburn and Hobson, 1962) and strains of Butyrivibrio fibrisolvens may undergo smooth to rough dissociation after isolation and show similar colonies. Pigment production is of very limited use.

6 806 BRYANT TABLE 2. SOME NUTRITIONAL FEATURES WHICH MAY BE OF USE IN IDENTIFICATION OF GROUPS OF SOME CULTURABLE ANAEROBIC RUMEN BACTERIA" Casein hydrolysate-nh3 medium e NH3 medium d Group b Growth NH3 A VFA C 1~ A.A.e Growth VFA A 1 good good -- 2 good E -- E poor good E 3 good E + -- poor good -- 5 good good good -- 6 ~ good poor good -- to to to to to none E 7 ~ good E poor good -- 8 good E -- E fair (?) good E 11 little 12 good E -[--}- E fair good E to to to to to to to none poor none good -~ good none 16 good E good good E none E none to? 18 good -[-+ -- good E 19 good good none 20 & good E 4- E poor good E 21 to to to to to to none? none? to to a Often based on few strains, see Bryant and Robinson (1962) or references therein. b See table 3. e Medium included minerals, vitamin-free casein hydrolysate (enzymatic)-nh8 was removed when NH~ + salts were deleted; B-vitamins; hemin; CO~-I~ICO8-; appropriate sugar energy source; cysteine and S-; acetate (A); n-valerate, isovalerate, 2-methylbutyrate, and isobutyrate (VFA). E=essential, -b to-}-+-{-~degree of stimulation. a Same as c but minus casein hydrolysate. e Efficiency of uptake of protein hydrolysate-c 1~ in comparison to heterotrophic organisms such as /~. coli which can synthesize all cell monomers from NHa, minerals and carbohydrate energy source. e Among those so far tested, methionine allows good growth when casein hydrolysate is deleted (Pittman and Bryant, unpublished data, 1962) % of strains require heme. g Strains studied only presumptively identified. Visible yellow pigments were first used in the description of R. flavefaciens Sijpesteijn; however, some strains of many other species produce a similar pigment and many strains of R. flavefaciens do not visibly show it. As more laboratories become equipped with good split-beam recording spectrophotometers, absorption and difference spectra of pigments in whole cells or cell fractions will be easily and rapidly obtained; and the characteristics of these spectra may be useful in identification. Cytochromes of different types have aiready been partially characterized in two species of rumen anaerobes (Wolin et al., 1960; White et al., 1962). Nutritional characters such as those shown in table 2 are useful in identification and B-vitamin requirements might be of some use. However, the latter appear to be of little use in the genus Ruminococcus (Bryant and Robinson, 1961b). B. succinogenes requires Na~- (Bryant et al., 1959). Other rumen species have not been studied for this feature but it might be useful because it is easily deter- to E ++ to + E ++ to mined and few other bacteria are known to exhibit it. Some Features and Differentiation of Species Table 3 shows some of the currently more useful features for identification of some bacterial groups known to function in the rumen and table 2 indicates some nutritional features that may be of utility. 4 Some more or less well described rumen bacteria are not included in the summary shown in table 3. The review of Bryant (1959) indicates others. Some more recent references to other species and additionai features of the organisms shown in table 3 include the following: Allison and Bryant (1963), Allison et al. (1962a, b), Blackburn and Hobson (1962), Bladen et al. (1961a, b), Brown and Moore (1960), Briiggemann et al. (1962), Bryant et al. (1959, 1960, 1961), Bryant and Robinson (1961a, b,c; 1962, 1963), Bryant and Small (1960), Butterworth et al. (1960), Caldwell et al. (1962), Clarke (1959, 1961), Coleman (1960), Dehority (1963), Dryden et al. (1962), Eadie et al. (1959), Fulghum and Moore (1963), Giesecke (1960, 1962), Gilchrist and Kistner (1962), Gordon and Moore (1961), Gutierrez et al. (1959a, b), I-Iobson and Mann (1961), Hobson et al. (1962), Hobson and Purdnm (1959, 196I), Howard et al. (1960), Kistner and Gouws (1962), Lee and Moore (1959), Lev (1958), Macdonald et al. (1959h, Phillipson et al. (1962), Provost and Doetsch (1960), Stellmach-Helwig (1961), Walker (1961), Wegner and Foster (1960, 1961), White et al. (1962), Williams and Doetsch (1960), Wolin et al. (1960).

7 ANAEROBIC BACTERIA IN RUMEN 807.,~ = o 6' ~ c~ c? ~a ~1~ ~ ~ ~ ~ ~ I ~0 9 o~ I 8 8 ~ s I I I I~ 1 I I I I I I I I I I ~r.. r..q ~ z~ I I I I I ~J I i j i 1 I I I I + ~ I + x~l I I I +o~ I I I ~.~ v v U3 0 r~ ~3.?.4

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9 ANAEROBIC BACTERIA IN RUMEN 809 r..) -to ~ +"-. II -HD o~ c~ ur I +l ~ +o +0 ~< # ~+1 +[ 8~ I I+ [ 4-1 I I I I I+ I + I I + +l +t I + I I I I +1 I I I I 4 < I I +i +1 I,.@ + +@~ I+ +~ +,,~, o +~ t ~ l o f [ ['~- I I "-" 4-1 +l [ ~.,..a u 9 ~11 > '~ II +1 ~ ~ec~ ~.~ _~ ~ ~.~ ~=~. ~ ~ ~ ='-~ ~ ~ =~ W~

10 810 BRYANT A few examples of differentiation of groups will be given. Certain strains of the nonmotile gram negative bacteroides species (table 3, no. 6-9) and Eubacterium ruminantium (table 3, no. 2), which might be recorded as gram negative, could be difficult to differentiate on the basis of gross morphology and certain other characteristics. No. 7, however, utilizes only starch or maltose as energy source while all of the others utilize glucose. Many strains of groups 2 and 6 utilize xylan but the other groups do not. Only no. 8 utilizes cellulose. Groups 6, 7, and 8 produce a large amount of succinate, little or no butyrate, show a net take up of CO2 and do not produce gas in the fermentation of carbohydrate while 2 and 9 produce little or no succinate, considerable butyrate, have a net production of CO2, and some strains produce H2. Turning to the nutritional features shown in table 1, organisms 2, 6, 7 and 8 can be further differentiated. No. 2 and 8 require NH3 and volatile fatty acids other than acetate even when casein hydrolysate is present and do not require factors in casein hydrolysate or heine. No. 7 is the same except it does not require volatile fatty acids. Strains of no. 6 do not require NH3 or volatile fatty acids when enzymatic hydrolysate of casein is present, usually require or are greatly stimulated by the casein hydrolysate, and usually require heme. Thus, with the exception of organisms no. 2 and 9, the few features given in tables 1 and 3, exclusive of morphological features, show from 3 to 8 features of use in differentiating these 5 groups. Close attention to morphology, gram reaction and physiological features not included in the tables would be necessary to differentiate group no. 2 from certain strains of no. 9. Another example of a possibly difficult differentiation is strains of groups no. 12 and 15 (table 3). They are very similar in most features so far studied and some strains are similar in morphology. However, they are easily differentiated on the basis of type of flagellation, and this difference is enough to indicate that they should be placed in different genera (see the genera of the family Spirillaceae Migula in Breed et al., 1957). With reference to only the few features shown in tables 2 and 3, it is evident, as it is with other bacteria, that most species of rumen bacteria must be defined to include many variable features (Bryant, 1959). This is true from the standpoint of practicality--we are concerned wih a great number of species without splitting them into more--and probably is also true from the standpoint of a "natural classification". One might consider it convenient for the ruminologist if all strains of a species such as Butyrivibrio fibrisolvens could be defined to indicate that all strains ferment xylan and all produce H2 and large amounts of butyric acid in the glucose fermentation as was done in the original description of the species (Bryant and Small, 1956). These characteristics do apply to most strains of the genus and are highly significant in rumen metabolism. However, if this precedent were followed, it is now evident that one would have to establish an unwieldy number of other species in the genus and further work, especially from the geneticists viewpoint, would probably show that most of them were only biotypes of the originally described species. 5 Summary and Conclusions A complete ecological analysis of the rumen microbial population is necessary for a more complete understanding of ruminant metabolism. The identification of the individual species is an important part of this analysis. The identification of most functional, nonsporeforming, anaerobic, rumen bacteria is difficult because they are difficult to grow, because of wide variations in some morphological, physiological and other features within species, because we do not have enough data on enough features or on enough strains to establish the degree of intra-species variation, and because workers have often used poor methods and inadequate controls in establishing their features. The same points apply to the classification and identification of many similar non-rumen organisms. Some of the more useful groups of features in their identification on the bases of present information (in approximate order of utility) are morphology, fermentation products, energy sources, certain nutritional features, cultural, serological, and other physiological features. Some pitfalls to be avoided in determining features and some suggestions for future work have been indicated. Individuals attempting to isolate and identify these bacteria should expect the ex- ~Ravin (1960) discussed mutation, genetic recombination and the viewpoints of the geneticist and the taxonomist concerning bacterial species. The author knows of no information that is available on genetic recombination or on the composition of DNA in rumen bacteria. These types of information are becoming increasingly useful in bacterial taxonomy.

11 ANAEROBIC BACTERIA IN RUMEN 811 penditure of considerable time and effort and should use similar previously identified strains as controls. Consultation with workers experienced in the field often can greatly expedite the work. The author knows of no one group of workers who are now placing major long term emphasis on the classification and identification of rumen non-sporeforming anaerobic bacteria. This is a situation which needs correction from the standpoints of agriculture, sanitation, medical science and comparative biology and biochemistry. Literature Cited Ainsworth, G. C. and P. H. A. Sheath Microbial Classification. Cambridge Univ. Press, Cambridge. Allison, M. J. and M. P. Bryant The biosynthesis of branched-chain amino acids from branched-chain fatty acids by rumen bacteria. Arch. Biochem., 101:269. Allison, M. J., M. P. Bryant and R. N. Doetsch. 1962a. Studies on the metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. 1. Incorporation of isovalerate into leucine. J. Bact. 83:523. Allison, M. J., M. P. Bryant, I. Katz and M. Keeney. 1962b. Studies on the metabolic function of branched-chain volatile fatty acids. II. Biosynthesis of higher branched-chain fatty acids and aldehydes. J. Bact. 83:1084. Anderson, R. L. and E. J. Ordal COs-dependent fermentation of glucose by Cytophaga suecinicans. J. Bact. 81:139. Baker, F. and S. T. Harriss Microbial digestion in the rumen (and caecum) with special reference to the decomposition of structural cellulose. Nutr. Abstr. and Rev. 17:3. Baldwin, R. L., W. A. Wood and R. S. Emery Conversion of lactate-c 1~ to propionate by the rumen microflora. J. Bact. 83:907. Blackburn, T. H. and P. N. Hobson Further studies on the isolation of proteolytic bacteria from the sheep tureen. J. Gen. Microbiol. 29:69. Bladen, H. A., M. P. Bryant and R. N. Doetsch. 1961a. The production of isovaleric acid from leucine by Bacteroides ruminicola. J. Dairy Sci. 44:173. Bladen, H. A., M. P. Bryant and R. N. Doetsch. 1961b. A study of bacterial species from the tureen which produce ammonia from a protein hydrolysate. Appl. Microbi01. 9 : 175. Bragg, P. D. and R. E. Reeves Studies oh the carbohydrate metabolism of a gram-negative anaerobe (Bacteroides symbiosis) used in the cultures of Entamoeba histolytica. J. Bact. 83:76. Breed, R. S., E. G. D. Murray and N. R. Smith Bergey's manual of determinative bacteriology. (Tth ed.). The Williams and Wilkins Co., Baltimore, Maryland. Brown, D. W. and W. E. C. Moore Distribution of Butyrivibrio fibrisolvens in nature. J. Dairy Sci. 43:1570. Briiggemann, J., D. Gieseck and K. Drepper Die Beeinflussung von Zusammensetzung und Leistung der Pansenflora durch Verabreichung unterschiedlicher Stickstoffquellen. Ztschr. Tierphysiologie Tiererni~hrung Futtermittelk 17:162. Bryant, M. P The isolation and characteristics of a spirochete from the bovine rumen. J. Bact. 64:325. Bryant, M. P Bacterial species of the rumen. Bact. Rev. 23:125. 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13 ANAEROBIC BACTERIA IN RUMEN 813 Stellmach-Helwig, Ruth Morphologische und physiologische Eigenschaften einiger aus Schafpansen isolierten Bakteriensfiimme. Arch. Mikrobiol. 38:40. Veldkamp, H Isolation and characteristics of Treponema zuelzerae nov. spec., an anaerobic, free-living spirochete. Antonie van Leewenhoek 26:103. Walker, D. J Isolation and characterization of a hemicellulose-fermenting bacterium from the sheep rumen. Australian J. Agr. Res. 12:171. Warner, A. C. I Enumeration of tureen microorganisms. J. Gen. Microbiol. 28:119. Wegner, G. H. and E. M. Foster Fatty acid requirements of certain rumen bacteria. J. Dairy Sci. 43 : 566. Wegner, G. H. and E. M. Foster Incorporation of insobutyrate-l-c 1~ and valerate-l-c 14 into phospholipid by Bacterioides succinogenes, a cellulolyfic bacterium from the rumen. Bact. Proc. p. 170 (Abstr.). White, D. C., M. P. Bryant and D. R. Caldwell Cytochrome-linked fermentation in the Bacteroides ruminicola. J. Bact. 84:822. Williams, P. P. and R. N. Doetsch Microbial dissimilation of galactomannan. J. Gen. Microbiol. 22 : 635. Wolin, M. J., E. A. Wolin, N. Jacobs and G. Wein- berg A new cytochrome-producing anaerobic vibrio. Bact. Proc. p. 77 (Abstr.). Wood, W. A Fermentation of carbohydrates and related compounds. In I. C. Gunsalus and R. Y. Stanier (ed). The Bacteria, Vol. II: Metabolism. Academic Press, Inc., New York. p. 59.