Comparison of Maintenance Energy Expenditures and Growth

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1979, p /79/ /07$0.00/0 Vol. 37, No. 3 Comparison of Maintenance Energy Expenditures and Growth Yields Among Several Rumen Bacteria Grown on Continuous Culture JAMES B. RUSSELLt* AND R. L. BALDWIN Department ofanimal Science, University of California, Davis, Davis, California Received for publication 4 January 1979 Maintenance energy expenditures were mesured for five rumen bacteria, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Bacteroides ruminicola, Megasphaera elsdenii, and Streptococcus bovis, by using a complex medium with glucose as the carbon source. Large differences (as high as 8.5-fold) in maintenance energy expenditures were seen among these bacteria. The suggestion is made that maintenance requirements could be a significant determinant of bacterial competition in the rumen. Theoretical maximum growth yields, calculated from double reciprocal plots of yield versus dilution rate, were compared to theoretical YATPM' values in order to estimate minimum molar adenosine 5'-triphosphate yields from glucose for each bacterium. Results showed that relative yield among the bacteria was growth rate dependent. At high dilution rates, both S. ruminantium and S. bovis produced lactate as their principal fermentation product. At lower dilution rates very little lactate was formed and growth yields increased. Acetate and ethanol were the predominant fermentation products of S. bovis at low dilution rates. Other workers have shown that S. ruminantium produces acetate and propionate at low growth rates. With the advent of continuous culture techniques in the late 1940's (16, 19), it became apparent that cell yield could vary with growth rate, and the idea was introduced that energy could be used for growth-related functions and for functions not necessarily related to growth (7). Since this time, the term "maintenance energy" has commonly been used to describe the use of energy for non-growth related functions. Theoretical equations describing maintenance energy were introduced by Marr et al. (15). However, these equations were not easily adapted to experimental estimation of maintenance energy. The theoretical equations introduced somewhat later by Pirt (0), however, have proven to be easily adaptable to the growth of bacteria in continuous culture. The Pirt double reciprocal plot of yield versus dilution rate is commonly used to quantitate maintenance energy expenditures. Bauchop and Elsden (1), in 1960, postulated that production of microbial mass was related to the amount of adenosine 5'-triphosphate (ATP) formed during catabolism. Because grams dry weight of cells produced per mole of ATP appeared to be relatively constant for several species, the term YATP was introduced. The comt Present address: Department of Animal Science, University of Illinois, Urbana, IL monly used value for YATP was 10.5 g, dry weight, of cells/mol of ATP. Subsequent work has shown that YATP is not a constant (6, 5-30) and varies with growth rate, the composition of the medium, and the presence of inhibitory compounds. Because natural environments usually contain very low concentrations of one or more substrates necessary for microbial cell synthesis, microbial growth rates rarely reach maximum potential in nature (17). At low growth rates, maintenance energy expenditures can make up a significant portion of total energy utilization (0). Based on this, it seems reasonable to suppose that variations in maintenance energy can affect the relative success of different microbial species inhabiting the same environment. Because soluble substrate concentrations are usually low and because microbial density and diversity are high in the rumen (11), bacteria must compete for substrates. It was postulated that if large differences in maintenance energy existed among rumen bacteria, maintenance energy could be a significant determinant of relative success of microbes in the rumen. One of the goals of the following research was to ascertain whether such significant differences in maintenance energy and growth yields exist among rumen bacteria.

2 538 RUSSELL AND BALDWIN Additionally, because ruminants are dependent on the supply of microbial protein as an amino acid source, yield of bacteria produced from the diet is of great importance to ruminant animal production. In nonrumen bacteria, bacterial growth yields are known to vary widely among different species (6), and it was felt that comparison of rumen bacteria growth yields would provide information useful to the selection of optimal ruminant diets. MATERIALS AND METHODS Media. The media used were essentially the same as the media described in the previous paper () except only glucose was used as the energy source. The medium used in Fig. 7 was modified slightly as described in the text. Organisms. Megasphaera elsdenii B159, Butyrivibrio fibrisolvens A38, Selenomonas ruminantium HD4, Bacteroides ruminicola GA33, and a newly identified Streptococcus bovis were used (J. B. Russell, W. N. Sharp, and R. L. Baldwin, J. Anim. Sci., in press). Cell growth. The conditions of cell growth were essentially the same as described in the previous paper Ṡampling and substrate analysis. Samples used for glucose analysis were removed from the culture vessel through a 16-gauge needle and were immediately passed through a 0.45-um membrane filter (Millipore Corp., New Bedford, Mass.). Separation of cells from medium was always accomplished in less than 30 s. Substrate samples were stored at -15 C until analyzed. Another 50 ml (M. elsdenii, B. fibrisolvens, and S. ruminantium) or 5 ml (B. ruminicola and S. bovis) of sample was removed from the culture vessel and was analyzed for bacterial dry weight by using the membrane filter procedure of Isaacson et al. (1). The formaldehyde treatment used in this procedure was found to quickly inhibit bacterial growth. Another portion was removed and checked for optical density in a Gilford model 40 spectrophotometer at 600 nm. Bacterial dry weight was found to agree well with optical density recordings. ph was monitored and always remained between 6.60 and Glucose was assayed by a method previously described (9). L- and D-lactic acid were measured by the method of Hohorst (10). Rabbit muscle L-lactate dehydrogenase (EC ) and Lactobacillus leichmannii D-lactate dehydrogenase (EC ) were used (Sigma Chemical Co., St. Louis, Mo.). Acetate and ethanol were determined by gas chromatography by using an Aerograph Hy Fi 600D chromatograph. A 15% FFAP, mesh, 6-foot column was used for acetate, while a 5% FFAP, mesh, 5-foot (1.54-m), Porapak column (Varion Aerograph, Walnut Creek, Calif) was used for ethanol. Ammonia was determined by using the method of Markham (14), except that magnesium oxide was used instead of NaOH. Slopes and intercepts of plots presented were estimated by using linear regression (4). RESULTS Approach. The same medium was used for the growth of all five rumen bacteria, so that APPL. ENVIRON. MICROBIOL. comparisons independent of medium differences could be made. Because several of the bacteria are quite fastitious in their nutritional requirements (4), a rather rich medium was chosen. Minimal media would have made substrateproduct analysis less difficult, but it was felt that the use of minimal media could have biased comparative interpretation by causing specific nutritional deficiencies not normally seen in the rumen. The medium employed gave high maximum growth rates (>0.48 h-1) for all five bacteria. B. ruminicola. A double reciprocal plot of yield versus dilution rate (growth rate) is shown in Fig. 1 for the growth of B. ruminicola on glucose. The slope of this plot represents the maintenance coefficient of the culture and was found by linear regression to be g of glucose per g of bacteria per h. The theoretical growth yield, yield corrected for maintenance energy, was calculated from the intercept of the ordinate and was found to be 50% (90 g of cells per mol of glucose). Since linearity of this plot was very good (correlation coefficient = 0.99), it seems likely that the ATP yield from glucose remained constant over these dilution rates. A shift in fermentation products affecting ATP yield from glucose would be expected to produce a nonlinear plot. B. fibrisolvens. The maintenance coefficient for B. fibrisolvens was found to be g of glucose per g of bacteria per h (Fig. ), while the theoretical maximum growth yield was calculated from the intercept to be 40% (7 g of cells per mol of glucose). The correlation coefficient of this plot was 0.93 and indicated a constant ATP yield from glucose over this range of dilution rates. M. elsdenii. In Fig. 3, growth yields for M. elsdenii based on glucose utilization were found 1; 3-.c F-. co o I. -_CD o YDILUTION RATE (hours) FIG. 1. Growth of B. ruminicola on glucose. The slope of the regression line, the maintenance coefficient, is g ofglucose per g of bacteria per h. The reciprocal of the intercept, the theoretical growth yield, is 50% (90 g of cells per mol of glucose). The correlation coefficient is 0.99.

3 VOL. 37, 1979 MAINTENANCE ENERGY EXPENDITURES FOR BACTERIA 539 to be very high (as high as 58% at relatively low is not known for M. elsdenii, the assumption growth rates). Analyses of membrane-filtered was made that carbon skeletons of amino acids medium showed increases in ammonia over ini- would be used with the same energetic efficiency tial concentration. This increase in ammonia as carbohydrate. The protein equivalent column suggested that protein as well as glucose was shown in Table 1 was calculated by multiplying being used as a carbon or energy source by M. the increase in ammonia nitrogen by 6.5. The elsdenii. Such ammonia increases were not seen carbohydrate equivalent was calculated by mulwith the other four bacteria. tiplying the protein equivalent by 86%. This 86% Ammonia corrected yields are presented in factor was used because approximately 14% of Table 1. Since ATP yield per ammonia formed the weight of an average amino acid is derived from the amino group and cannot be used for ATP generation. An ammonia-corrected plot for M. elsdenii is shown in Fig. 4. The maintenance coefficient of 0o this plot was calculated to be g of glucose..----a- -vw ^ per g of bacteria per h, and the theoretical a xa growth yield was estimated at 4% (83 g of cells per mol of glucose). A high degree of linearity in this corrected plot is evidenced by the high o 1 t correlation coefficient, S. ruminantium A non-linear douhale reciprocal plot for the growth of S. ruminantium is in Fig. 5. It is not surprising that a presented 4 1 ;0 ; 1' non-linear plot was obtained because several DILUTION RATE (hor) workers (1,, 3) have shown that fermenta- FIG.. Growth of B. fibrisolvens on glucose. The slope of the regression line, the maintenance coefficient, is g of glucose per g of bacteria. The 4. reciprocal of the intercept, the A~~~ theoretical growth yield is 40% (7 g of cells per mol of glucose). The correlation coefficient is a= A- 1- I.. ^^A *A w-. o 1- _0 A.~ /DILUTION RATE (hours) FIG. 4. Growth of M. elsdenii on glucose with a 0 correction for ammonia production. The slope of the regression line, the maintenance coefficient, is V/DILUTION RATE (hours) g ofglucose per g of bacteria per h. The reciprocal of the intercept is 4% (8 g of cells per mol ofglucose). FIG. 3. Growth ofm. elsdenii on glucose. The correlation coefficient is TABLE 1. M. elsdenii growth yields corrected for ammonia production Increase in Protein Carbohy- Total carbo-. Yield (g of drate /a Glu/ml hydrate used Bwat(emiga/li) Ml) (mg/ml) bactel(a/g Mlent (m/ (gm) (g(mg/m mgm) carbohydrate) of (h-') NH3-N (mg/ equivalent b a Computed by multiplying increase in NH3 nitrogen by 6.5. b Computed by multiplying protein equivalent by 86%. l

4 540 RUSSELL AND BALDWIN 0 0) ' 0 I A, A* AN ~~ /DILUTION RATE ( hours ) FIG. 5. Growth of S. ruminantium on glucose. The slope of the dotted regression line, the maintenance coefficient, is 0.01 g of glucose per g of bacteria per h. The reciprocal of the intercept, the theoretical growth yield, is 58% (105 g of cellsper mol ofglucose). The correlation coefficient is tion products vary with growth rate in this bacterium. Lactate is produced at high growth rates with a gradual shift to acetate and propionate as growth rate decreases (1). Lactate levels were measured and lactate to glucose ratios are presented in Table. At the three lowest dilution rates, production of lactate from glucose was very low. If one assumes that ATP yield was relatively constant over these dilution rates, a maintenance coefficient of 0.01 g of glucose per g of bacteria per h can be estimated. Similarly, theoretical maximum growth yield was calculated to be 58% (105 g of cells per mol of glucose). These three low dilution rates in Fig. 5 showed linearity (correlation coefficient = 0.96). S. bovis. A non-linear double reciprocal plot also occurred when the S. bovis data were plotted (Fig. 6). Lactate levels were measured, and their ratios to glucose are presented in Table 3. At high dilution rates nearly all of the glucose was converted to lactate, but at low dilution rates only small amounts of lactate were produced. To ascertain the residual fermentation products, a second incubation was performed by using a medium without volatile fatty acids or Trypticase and much reduced levels of yeast extract (0.1 g/liter). This restricted medium made the measurement of volatile fatty acids and the calculation of a carbon balance feasible. The carbon balance for this incubation is presented in Table 4. As dilution rate and lactate concentration decreased, ethanol and acetate became significant fermentation products. In spite of the possibility that some ethanol may have been bubbled from the medium, overall carbon recovery based on glucose utilization and yeast extract added to the medium was always greater than 84%. Higher chain volatile fatty acids were APPL. ENVIRON. MICROBIOL. not detected. The double reciprocal plot of this secondary incubation is presented in Fig. 7. Calculation of a maintenance coefficient for S. bovis is complicated by the fermentation shift described above. In Table 3, lactate levels were quite low at the four lowest dilution rates, and the double reciprocal plot through these points (Fig. 6) appeared to be linear. The slope of this TABLE. Lactate production in S. ruminantium Ratio of lactate to Yield (g of bac- glucose (g of lac- Dilution rate (h-1) teria/g of glu- tate/liter to g of cose) glucose metabolized/liter % ) - It A ka "'~~A ~A---~A ` /DILUTION RATE ( hours ) FIG. 6. Growth of S. bovis on glucose. The slope of the dotted regression line, the maintenance coefficient, is g ofglucose per g of bacteria per h. The reciprocal of the intercept, the theoretical growth yield, is 40%o (7 g of cells per mol of glucose). The correlation coefficient is TABLE 3. Lactate production in S. bovis Yield (g of glu- Ratio of lactate to glucose metaboe(h) cose/gofrbactea na) lized (g/liter to g/ Diluionate(h-'Yioeldg ofbacue ~~~~liter)

5 VOL. 37, 1979 TABLE 4. MAINTENANCE ENERGY EXPENDITURES FOR BACTERIA 541 Carbon balance for the growth of S. bovis in restricted medium Dilution rate Cells (g/li- Lactate (g/ Acetate (g/ COa (g/li- Ethanol COb (g/li- Carbon re- Glucose (h-') ter) liter) liter) ter) (g/liter) ter) covered liter) (g/ used' (g/li- ter) a Calculated from acetate production. b Calculated from ethanol production. 'Yeast extract was also added to the medium at 0.1 g/liter. 7. low molar ATP yields of an anaerobic environment. The theoretical maximum growth yield of S. ruminantium was determined to be 105 g of cells A.5 A' per mol of glucose. If one assumes that 4 mol of C 4 5\. 1 ATP per mol of glucose is formed in acetate and 5- ~ ~ ~ propionate production by this organism, a YATP value of 6.3 can be calculated. This value is 4 somewhat higher than a previous estimate of 0 for this bacterium (6). However, this value is It less than the theoretical value of 3 proposed by 0Wo ^,o lt Stouthamer for the growth of bacteria in complex media (5). A YATPm" of 3 also predicts 1/DILUTION RATE (ha FIG. 7. Growth of S. bovis in a restricte ducsn) that at least 3. mol of ATP per mol of glucose ~d glucose must be formed in S. ruminantium since values medium. lower than 3. would give estimates of YATP greater than 3. Similar minimum estimates of linear portion was g of glucose per g of ATP formation from glucose would be.8,., bacteria per h, and the intercept was 440% (7 g.5, and. for B. ruminicola, B. fibrosolvens, of cells per mol of glucose). The cc )rrelation M. elsdenii, and S. bovis, respectively. It is probable that the actual yields of ATP per mol of coefficient was Significant growth yield differences between glucose are significantly higher than these minthe complex medium and the restricted[medium imnum estimates. can be observed by comparing Fig. 6 and 7. Comparison of the data presented in Table 5 These differences are much too great to be ac- indicates that S. ruminantium and B. fibrosolcounted for by a theoretical conversio)n of glu- vens had relatively small maintenance require- a ments compared to M. elsdenii, B. ruminicola, cose to cell material (14) and may iindicate relative inefficiency in biosynthesis by S bovis. and S. bovis. The high maintenance coefficients Quantitation of these differences is connplicated in the latter three organisms may help explain by the fermentation shifts described at)ove. the relative lack of success of these microbes on diets containing low levels of soluble substrates. DISCUSSION Both S. bovis and M. elsdenii are prominent The maintenance coefficients presentted in Ta- only when ruminants are fed high grain diets ble 5 are within range with those calcu. ldated for (11). B. fibrisolvens has been known to make up aerobic bacteria (0, 5, 7, 8). How,ever, the a significant number of total colony counts in maintenance coefficients derived for B '. fibrisol- animals fed very poor forage diets (13). A low vens and S. ruminantium were sub. stantially maintenance requirement would be consistent lower than the maintenance coefficie!nt deter- with success under these conditions. The very mined for the anaerobic growth of A erobacter low maintenance energy coefficient seen with S. aerogenes in complex medium (8). T'hese low ruminantium and the relatively high one oban ener- served for B. ruminicola cannot alone be used maintenance coefficients may indicate getic adaptation of these strict anaerotdes to the to explain some whole animal observations. S.

6 +ne^a na;+asto 54 RUSSELL AND BALDWIN ruminantium was unsuccessful on diets containing low concentrations of soluble substrates (), while straw diets produce high numbers of bacteria identified B. ruminicola (3). Other factors such resistance to low ph (Russell et al., in press), substrate preference (1), and substrate affinity () could be involved. The plots shown in Fig. 8 indicated that relative yield among rumen bacteria can be growthrate dependent and that a simple comparison of theoretical growth yields is not always adequate. Comparison of the theoretical growth yields for B. ruminicola and B. fibrisolvens (Table 5) indicated that at high growth rates the yield of cells would be greater for B. ruminicola. However, at low growth (less than h-1) this situation is reversed (Fig. 8). B. fibrisolvens exhibits higher growth yields due to low main-.a..1- mainte B. fibr noted yields,, atgot ae maxim proxin yields founde not nlybymintnancenrgy,but * u 019 _8 n FIG. APPL. ENVIRON. MICROBIOL. also by the observation that there is a switch to fermentation products yielding less ATP at high growth rates. In these two organisms growth rates above 0.5 h-' resulted in decreased growth yields. The data presented illustrate the complexity of predicting bacterial competitions and overall bacterial yield in a natural environment such as the rumen. It should be noted that the data presented in this experiment were collected using a very rich medium with glucose as the carbon source. Because composition of the medium has shown to affect maintenance coefficients (6, 5, 6, 8), further work is obviously needed. The possibility of strain differences within a species should also be considered. LITERATURE CITED ;e expenauilulres. simiiuar reversat uue to 1. Bauchop, T., and S. R. Elsden The growth of anance was seen between M. elsdenii and microorganisms in relation to their energy supply. J. risolvens (Fig. 8). However, it should be Gen. Microbiol. 3: that M. elsdenii only exhibited higher. Bryant, M. P The characteristics of strains of raehn04-1 isolated from bovine TSelenomonas rumen contents. J. at growth rates greater than 0.4 h'. The Bacteriol. 7: uum growth rate of each bacterium is ap- 3. Bryant, M. P., N. Small, C. Bouma, and H. Chu iately 0.50 h'1. Comparison of growth Bacteroides ruminicola n. sp. and Succinomonas amyfor S. ruminantium and S. bovis is con- lolytica, the new genus and species. Species of acid-producing anaerobic bacteria succinic of the bovine rumen. Zd not only by maintenance energy, but J. Bacteriol. 76: * 4. Bryant, M. P., and L. M. Robinson Some nutritional characteristics of predominant culturable rums.minal bacteria. J. Bacteriol. 84: Caldwell, D. R., and M. P. Bryant Medium 3- /A_*K-- - ~ without rumen fluid for nonselective enumeration and A w v- isolation of rumen bacteria. Appl. Microbiol. 14: Hempfling, W. P., and S. E. Mainzer Effects of M. elsdenii varying the carbon source limiting growth on yield and ** B. ruminicola maintenance characteristics of Escherichia coli in con- 1 A1B. fibrisolvens tinuous culture. J. Bacteriol. 13: Herbert, D., R. Elsworth, and R. 0. Telling The continuous culture of bacteria: a theoretical and experimental study. J. Gen. Microbiol. 14: O 4 S Hishinuma, F., S. Kanegasaki, and T. Takahashi. /DILUTION RATE (hours Ruminal fermentation and sugar concentrations: a model experiment with Selenomonas ruminantium. 8. Comparison of growth yields among M. Agric. Biol. Chem. 3: elsdenici, B. ruminicola, and B. fibrisolvens. 9. Hobson, P. N., and R. Summers ATP pool and growth yield in Selenomonas ruminantium. J. Gen. Microbiol. 47: TABLE 5. Comparison of maintenance coefficients 10. Hohorst, H.-J Lactate, p In H. U. Berg- theoretical maximum growth yields meyer (ed.), Methods of enzymatic analysis. Academic aznd Press Inc., New York. Maintenance Theoretical 11. Hungate, R. E The rumen and its microbes, p. 8- Org,anisin coefficient (g of maxunum 81 and 8. Academic Press Inc., New York. glucose/g of bac- growth yield (g 1. Isaacson, H. R., F. C. Hinds, M. P. Bryant, and F. N. teria/h) of cells/mol of Owens Efficiency of energy utilization by mixed glucose) rumen bacteria in continuous culture. J. Dairy Sci. 58: B. rumiinicola B. fibrisolvens Margherita, S. S., R. E. Hungate, and H. Storz Variation in rumen M. elsd!enii 0.187a 8 Butyrivibrio strains. J. Bacteriol ~~~85 87: S. ruminantium O.15105b 14. Markham, R A steam distillation apparatus suita- S. bovis 7 ble for micro-kjeldahl analysis. Biochem. J. 36:790- a 791. Estiiimated for glucose plus protein hydrolysate 15. A. G., E. H. Manr, Nelson, and D. J. Clark The utilizati ion. b Esti. imated..maintenance requirement of Escherichia coli. Ann. from low dilution rate data points. N.Y. Acad. Sci. 10:

7 VOL. 37, Monod, J La technique de culture continu, theorie et applications. Ann. Inst. Pasteur (Paris) 79: Neijssel, 0. M., and D. W. Tempest The regulation of carbohydrate metabolism in Klebsiella aerogenes NCTC 418 organisms, growing in chemostat culture. Arch. Microbiol. 106: Neijssel, 0. M., and D. W. Tempest Bioenergetic aspects of aerobic growth of Klebsiella aerogenes NCTC 418 in carbon limited and carbon sufficient chemostat culture. Arch. Microbiol. 107: Novick, A., and L Szilard Description of the chemostat. Science 11: Pirt, S. J The maintenance energy of bacteria in growing cultures. Proc. R. Soc. Lond. Ser. B 163: Russell, J. B., and R. L Baldwin Substrate preferences in rumen bacteria: evidence of catabolite regulatory mechanisms. Appl. Environ. Microbiol. 36: Russell, J. B., and R. L Baldwin Comparison of substrate affinities among several rumen bacteria: a possible determinant of rumen bacterial competition. Appl. Environ. Microbiol. 37: Scheifinger, C. C., M. J. Latham, and J. J. Wolin Relationship of lactate dehydrogenase specificity and growth rate to lactate metabolism by Selenomonas MAINTENANCE ENERGY EXPENDITURES FOR BACTERIA 543 ruminantium. Appl. Microbiol. 30: Snedecor, G. W., and W. G. Cochran Statistical methods (5th ed.), p Iowa State University Press, Ames. 5. Stouthamer, A. H A theoretical study on the amount of ATP required for synthesis of cell material. Antonie van Leeuwenhoek J. Microbiol. Serol. 9: Stouthamer, A. H., and C. Bettenhaussen Utilization of energy for growth and maintenance in continuous and batch cultures of microorganisms. Biochim. Biophys. Acta 301: Stouthamer, A. H., and C. W. Bettenhaussen Determination of the efficiency of oxidative phosphorylation in continuous cultures of Aerobacter aerogenes. Arch. Microbiol. 10: Stouthamer, A. H., and C. W. Bettenhaussen Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium. Arch. Microbiol. 111: Stouthamer, A. H., and C. W. Bettenhaussen A continuous culture study ofan ATPase-negative mutant of Escherichia coli. Arch. Microbiol. 113: Stouthamer, A. H Energetic aspects of the growth of microorganisms. Symp. Soc. Gen. Microbiol. 8: Downloaded from on May 8, 018 by guest