JOURNAL OF BACTERIOLOGY, May 1969, p. 668-676Copyright © 1969 American Society for Microbiology

Vol. 98, No. 2Printed in U.S.A.

Effects of Carbon Dioxide on Growth and MaltoseFermentation by Bacteroides amylophilusDANIEL R. CALDWELL, MARK KEENEY, AND PETER J. VAN SOEST1

Animal Husbandry Research Division, U.S. Department of Agriculture, Beltsville, Maryland 20705, and Depart-ment of Dairy Science, University of Maryland, College Park, Maryland 20741

Received for publication 6 December 1968

The requirement of carbon dioxide for growth of Bacteroides amylophilus isquantitatively similar to that of certain other rumen bacteria. Carbon dioxidecould be replaced by bicarbonate, but not by formate or certain amino acids. Labelfrom '4CO2 was incorporated into the succinate produced during maltose fermenta-tion by B. amylophilus, and during glucose fermentation by B. ruminicola, andduring cellobiose fermentation by B. succinogenes. All of the incorporated label couldbe associated with the carboxyl function of the molecule. The depression in radio-activity per micromole of carbon in the succinate formed from the fermentation otuniformly labeled 14C-maltose by B. amylophilus was greater than would be ex-pected if all of the succinate formed was produced via a direct CO2 fixation path-way(s) involving phosphoenolpyruvate or pyruvate; the radioactivity per micro-mole of carbon suggests that as much as 60% of the total succinate results from a

pathway(s) involving direct CO2 fixation. Maltose fermentation by B. amylophiluswas dependent upon CO2 concentration, but CO2 concentration could not be shownto influence either the fermentation end-product ratios or the proportion of totalsuccinate formed attributable to CO2 fixation.

Carbon dioxide is recognized as an importantruminal metabolite: it is required for growth ofmany species or rumen bacteria (5, 23); it is asubstrate for quantitatively important ruminalprocesses as diverse as methane formation (14,30, 32) and amino acid biosynthesis (1, 2, 21,26, 33); and it is an end product from rumenfermentation of carbohydrate (6). Although car-bon dioxide is recognized as an important ruminalmetabolite, the details of its uptake and utilizationby particular bacterial species of the rumen areless thoroughly understood.

Evidence of a carbon dioxide requirement forcarbohydrate fermentation by predominant rumi-nal bacteria was first provided by White et al.(31). These authors showed that carbon dioxideoxidized the b-type cytochrome of Bacteroidesruminicola, and suggested that carbon dioxidewas involved in the formation of succinate duringglucose fermentation by that organism. Directevidence for the involvement of carbon dioxide information of succinate by ruminal bacteria wassubsequently obtained by Scardovi (27), whoshowed nearly stoichiometric incorporation oflabel from carbon dioxide into the succinate

1 Present address: Department of Animal Science, CornellUniversity, Ithaca, N.Y. 14850.

produced during glucose fermentation by Suc-cinivibrio dextrinosolvens. Recent work of Hop-good and Walker (19, 20) has shown the incor-poration of label from carbon dioxide into thecarboxyl carbon of the succinate produced by anorganism similar to Ruminococcus flavefaciens.Since many rumen bacteria produce succinate asa major fermentation end product, study of themechanisms of succinate formation by these or-ganisms should contribute to knowledge of rumi-nal carbohydrate fermentation, of energy metabo-lism of anaerobic bacteria, and carbon dioxidemetabolism. It was with these objectives that thepresent study of carbon dioxide incorporationduring carbohydrate fermentation by B. am-ylophilus, B. ruminicola, and B. succinogenes,three succinogenic rumen Bacteroides species, andof some effects of carbon dioxide on maltosefermentation by B. amylophilus, was initiated.

MATERIALS AND METHODSStrain sources and maintenance. Most of the strains

studied were strains of B. amylophilus which were iso-lated from bovine rumen contents by Leonard Slyter,Agricultural Research Service, Beltsville, Md. Toconfirm that these strains were, in fact, B. amylophilus,the strains were characterized by use of previouslydescribed presumptive identification procedures (6,

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7, 11, 13), and by measurement of the end productsof their fermentation of starch and maltose. Allstrains were gram-negative, nonmotile, nonspore-forming, rounded-end rods which fermented starchand maltose to produce succinate, acetate, and for-mate as major acidic fermentation end products. Allstrains failed to ferment either glucose or cellulose,did not produce gas from glucose, and failed toproduce hydrogen sulfide. These characteristics are inaccord with those of B. amylophilus, Hamlin andHungate, 1956 (16). In some experiments, strainsrepresentative of related rumen Bacteroides specieswere included for comparative purposes. Thesestrains included B. ruminicola subsp. brevis strainGA33 (12) and B. succinogenes strain S85 (9). Allstrains were maintained by periodic loop transfer inRGCA (&, 10) agar slants.

Culture techniques and growth conditions. TheHungate anaerobic technique (22) was employedthroughout the study. Organisms were cultured in13-mm rubber-stoppered test tubes. For studies ofthe effect of carbon dioxide concentration on growth,the tubes employed were calibrated by insertingstoppers into air-filled dry tubes, carefully markingthe point on the tubes corresponding to the end ofthe stoppers, filling the tubes to the marks with water,and quantitatively transferring the tube contents intodry 10-ml graduated cylinders calibrated in 0.1-mldivisions. The average volume of 10 tubes was 8.07ml (,, 0.11). The average volume of medium used was4 0.1 ml. The difference between the average totalvolume of the tubes and the volume of completedinoculated medium used was taken as the gaseousspace. Calculations of the quantity of inorganiccarbon available to the organisms during growth inthe presence of various carbon dioxide-nitrogen mix-tures were made from a consideration of the availablegaseous space and the percentage of carbon dioxidein the mixture.The composition of the medium used for routine

growth experiments is shown in Table 1. The brothmedium used for culture of B. ruminicola and B.succinogenes was identical to that described in Table1 except that either 1.0% (w/v) glucose or 0.5%(w/v) cellobiose replaced maltose as an energy sourcefor these organisms. The nature of the gaseous phasesand the buffers employed varied from experiment toexperiment. However, all media in which carbondioxide concentration was critical were prepared andinoculated under oxygen-free nitrogen. Carbon diox-ide was introduced into these media by rapidlychanging the gaseous phase, after inoculation andprior to incubation, from nitrogen to an appropriatenitrogen-carbon dioxide mixture. When bicarbonatewas utilized in place of carbon dioxide gas, thebicarbonate was incorporated into media preparedand dispensed under nitrogen by additions of smallquantities (0.05 to 0.20 ml) of sterile Na2CO3 solu-

tion previously prepared and stored under nitrogen.All media in which the effects of carbon dioxide con-

centration were studied contained 0.05 M phosphatebuffer (pH 7.0). The addition of carbon dioxide or

an equivalent amount of Na2CO3 solution to these

TABLE 1. Composition of the defined medium usedfor routine growth experiments with

Bacieroides amylophilus

Component Concn (w/v)

Maltose ....... .............. 0.5Minerals............................ -a

Vitamins............................ b

Volatile fatty acids........ c

Resazurin ............................ 0.0001Hemin ...0.0001Methionine .......................... 0.045(NH4)2SO4......0.......O .09L-Cysteine*HClXH20 ................. 0.05Distilled-demineralized water.Buffer............................ d

Gaseous phase...

a The minerals used and their final medium con-centrations were as follows: NaCl, 1.2 X 10-2 M;KH2PO4, 6.5 X 10-3 M; CaCl2, 1.8 X 10-4 M;MgCl2.7H20, 9.0 X 1076 M; MnC2-4H20, 5 X10-5 M; CoCl2-6H20, 4.2 X 10-6 M; and FeSO47H20, 3.6 X 10- M.bThe vitamins used and their final medium con-

centrations were as follows: thiamine hydro-chloride, 5.9 X 10-6 M; nicotinamide, 1.6 X 10-5 M;riboflavine, 5.6 X 10-6 M; calciUm-D-pantothenate,4.2 X 10-6 M; pyridoxal, 9.9 X 10-6 M; p-amino-benzoic acid, 7.3 X 10-7 M; biotin, 2.0 X 10-7 M;folic acid, 1.1 X 10-v M; lipoic acid, 2.4 X 10-v M;and cyanocobalamin, 3.7 X 10-9 M.

c The volatile fatty acids used were as follows:acetic, 2.8 X 10-2 M; propionic, 9 X 10-3 M; n-butyric, 4.5 X 10-3 M; isobutyric, in-valeric, iso-valeric, and DL-2-methylbutyric, 9 X 10-4 M each.

d The buffer and gaseous phase were varied fromexperiment to experiment as described in the text.

media did not significantly alter the initial mediumpH.

The medium ingredients, with Na2CO3, cysteine,and carbohydrate deleted, were sterilized by auto-claving under an appropriate oxygen-free gaseousphase. Cysteine, Na2CO3, and carbohydrate, all pre-pared and sterilized as separate solutions, were addedto the other medium ingredients, and the completedmedium was anaerobically dispensed into test tubes.Experimental media were inoculated with 0.1 to 0.5ml of broth medium which had been previouslyinoculated from an RGCA slant. Cultures wereincubated at 37 to 39 C, and growth was measuredas turbidity (optical density) of cultures at 600 nmversus uninoculated tubes.

Chemical procedures. Acidic fermentation endproducts were separated with the silicic acid columnand methods of Bruno and Moore (4); 25% n-butylalcohol was used as the second solvent. The quantityof acid in elution fractions from the column was de-termined by titration of the fractions with sodiumethoxide standardized against potassium hydrogenphthalate. Quantitative recovery of acetate, formate,succinate, and lactate was obtained when known

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quantities of these compounds were applied to thecolumn. Liquid scintillation counts of appropriatefractions of eluate were used to determine the recov-ery of label in fermentation end products producedduring fermentation of uniformly labeled "IC-maltoseor maltose-1-14C and also to demonstrate incorpora-tion of label from 14CO2 into succinate. Succinatewas separated from lactate by thin-layer chromatog-raphy on Brinkmann silica gel H by use of a develop-ment solvent composed of 95% ethyl alcohol, ammo-nium hydroxide, and distilled water (90:4:10).Quantitative recovery of label from succinate-2,3-14Cand lactate-1-14C was obtained when known stand-ards of these compounds were subjected to the sepa-ration procedure.

Succinate was degraded by use of the Phares andLong (25) modification of the Schmidt reaction at atemperature between 76 and 80 C. The carbon dioxideresulting from the degradation was collected in anethanolamine-ethylene glycol trap (24) and was sub-sequently counted by liquid scintillation procedures.Radocheical procedures. Uniformly labeled 14C-

maltose, succinate-2,3-"4C, succinate-1,4-14C, lactate-1-14C, and Na214CO3 were obtained from New EnglandNuclear Corp., Boston, Mass. Maltose-1_14C wasobtained from the Calbiochem, Los Angeles, Calif.All radiochemicals were maintained in either 50 or95% (v/v) aqueous ethyl alcohol solutions and wereadded in 0.1-ml amounts to individual tubes of media.Radiochemical measurements were made by liquidscintillation procedures with either a Nuclear-Chicagomodel 725 or a Beckman model 2000 system. Disin-tegration rates obtained were corrected for differencesin time, quenching, and background, and are ex-pressed as disintegrations per minute per milliliter ofmedium, unless otherwise indicated.

RESULTSCarbon dioxide requirement of B. amylophflus.

Table 2 shows the growth response of 10 strainsof B. amylophilus as a function of carbon dioxideconcentration. No growth of any strain was ob-tained in the absence of carbon dioxide after anincubation period of 1 week. Growth was initi-ated at a total available carbon dioxide concen-tration between 4.5 X 10-1 and 9.0 x 10-' M, butwas significantly more extensive, at the 5% levelof significance, at concentrations greater than or

equal to 1.2 X 10-2 M. Maximal optical densitywas obtained within a 12-hr period after inocula-tion. The data in Table 3 show that 2 X 10-2 Mbicarbonate replaced the carbon dioxide require-ment of B. amylophilus strain 1441, but neither2 X 10-2 M formate nor a mixture of alanine,glutamic acid, asparagine, arginine, and lysine,supplied at concentrations of 2 X 10-2 M each,would replace carbon dioxide. Neither formatenor the amino acid mixture was inhibitory togrowth of B. amylophilus strain 1441 when carbondioxide was substituted for nitrogen as thegaseous phase.

Metabolic fate of carbon dioxide. A substantialloss of carbon dioxide from the gaseous phaseoccurred during growth of B. amylophilus strain1441 in the defined medium in the presence of1.8 x 102 M carbon dioxide (Table 4). As muchas 70% of the original carbon dioxide was lostfrom the gaseous phase. Figure 1 shows theincorporation of label from carbon dioxide into

TABLE 2. Growth of Bacteroides amylophilus strains in the defined medium- containing variousconcentrations of carbon dioxide

Percentage of carbon dioxide in gaseous phaseStrain

5 10 20 30 40

78 0.04 (5)b 0.03 (6) 0.70 (8) 0.69 (10) 0.60 (8)1022 0 (168) 0.04 (7) 0.42 (8) 0.60 (8) 0.76 (8)1024 0 (168) 0.03 (6) 0.55 (10) 0.55 (8) 0.66 (9)1055 0 (168) 0.04 (7) 0.60 (9) 0.64 (9) 0.63 (10)1058 0.03 (5) 0.59 (7) 0.74 (7)1059 0 (168) 0 (168) 0.58 (8) 0.64 (8) 0.0.749)1413 0.05 (5) 0.07 (10) 0.68 (12) 0.88 (9) 0.92 (12)1417 0.04 (5) 0.07 (5) 0.69 (9) 0.87 (9) 0.94 (9)1441 0.02 (5) 0.02 (5) 0.05 (5) 0.81 (9) 0.88 (10)1444 0 (168) 0.03 (5) 0.06 (6) 0.72 (9) 0.88 (10)

a All media contained 0.05 M phosphate buffer, pH 7.0. Media were prepared and dispensed into tubesunder oxygen-free nitrogen. Carbon dioxide was added by rapidly changing the gaseous phase afterinoculation from nitrogen to an appropriate nitrogen-carbon dioxide mixture. The medium volume andgaseous space were 4 ml each. The 10%/ carbon dioxide gaseous phase thus corresponds to a total avail-able carbon dioxide concentration of 4.5 X 10- M, if all of the available carbon dioxide dissolved in theliquid phase.

b Cultures were incubated at 37 to 39 C. Results are given as the maximal optical density at 600 nm.Each value is the average of duplicate tubes. Figures in parentheses are hours of incubation requiredfor maximal optical density. Cultures incubated for 168 hr without carbon dioxide showed no growth.

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succinate produced during maltose fermentationby B. amylophilus strain 1441. Similar resultswere obtained with nine other strains of B.amylophilus. Figures 2 and 3 show the incorpora-tion of label from carbon dioxide into succinateproduced during glucose fermentation by B.

TABLE 3. Growth of Bacteroides amylophilus strain1441 in the defined medium containingcompounds suspected of replacing its

carbon dioxide requirement

Gaseous phase'Medium

Carbon dioxide Nitrogen

Amino acidb..... 0.51 (8)c 0 (168)Formate......... 0.51 (8) 0 (168)Bicarbonate ...... 0.69 (8) 0.88 (10)

a The gaseous phase of the medium was eitheroxygen-free nitrogen or 100% carbon dioxide. Allmedia contained 0.05 M phosphate buffer (pH 7.0)and were prepared as described in Table 2. Bi-carbonate was incorporated into individual tubesof medium as sterile Na2CO3 solution prepared,maintained, and added to the medium undernitrogen. The gaseous phase in carbon dioxide-containing media was changed after inoculationjust prior to incubation.

b Values for amino acid-containing media areaverages of duplicate tubes. All other values arethe average of six tubes.

c Results are given as the maximal opticaldensity at 600 nm. Figures in parentheses are hoursof incubation at 37 to 39 C required for maximaloptical density.

TABLE 4. Loss of carbon dioxide from the gaseousphase during growth of Bacteroides amylophilus

strain 1441 in the defined mediumacontaining 40% carbon dioxide

Carbon dioxide peak heightbExpt Percentage

Incubated Incubated losscontrol tubes inoculated tubes

1 67 29 572 69 18 723 79 25 68

a All media contained 0.05 M phosphate buffer,pH 7.0. The media were prepared and the gaseousphase was changed as described in Table 2. Bothcontrol and inoculated tubes were incubated for20 hr at 37 to 39 C.

b Gas samples were collected through the stop-pers with a gas-sampling syringe. The values arearbitrary peak height units per milliliter of gas-eous phase as measured with a Fisher Hamiltongas partitioner equipped with a thermal conduc-tivity detector. Each value is the average ofduplicate determinations.

ruminicola strain GA33 and during cellobiosefermentation by B. succinogenes strain S85. Thedata in Table 5 show the results of Schmidtdegradation of chromatographically isolated suc-cinate produced during growth of strains of B.amylophilus, B. ruminicola, and B. succinogenesin the presence Of '4CO2 ; the results from Schmidtdegradation of position-labeled succinate are alsoshown. Under the reaction conditions employed,an average of 30% (23 to 37%) of the originallabel in succinate-J , 4-"'C was recovered in thetrap, and contamination from the internal carbonsof succinate was less than 2.0%. The percentagesof original label recovered from degradation ofsuccinate isolated from the medium after growthof B. amylophilus, B. succinogenes, and B. rumini-cola are similar to those obtained from degrada-tion of carboxyl-labeled succinate. The labelassociated with the carboxyl carbon is sufficientto account for all of the label incorporated intosuccinate.

Quantitative importance of carbon dioxide fixa-tion in succinate formation by B. amylophilus.Attempts were made to determine the extent towhich carbon dioxide fixation could account forthe succinate formed during maltose fermentationby B. amylophilus. Table 6 shows the radioactivityper micromole of carbon in uniformly labeled"4C-maltose; the radioactivity per micromole ofcarbon in the succinate formed from maltoseduring growth of strain 1441 in the definedmedium containing 5 X 10-2 M phosphate bufferand a gas phase of 100% carbon dioxide is also

0z

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5

3

0

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4 x

1 0 30 50FRACTION NUMBER

FIG. 1. Incorporation of radioactivity into succinateduring growth of Bacteroides amylophilus 1441 in thedefined medium containing 14CO2. X, Separation ofvaleric (V), propionic (P), acetic (A), formic (F), andsuccinic (S) acids as measured by titration; 0, radio-activity perfraction as determined by liquid scintillationcounts.

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16

10

4

10 30 50FRACTION NUMBER

FiG. 2. Incorporation of radioactivity into succinateduring growth of Bacteroides ruminicola subsp. brevisGA33 in the defined medium containing 14CO2. X,Separation of valeric (V), propionic (P), acetic (A),formic (F), and succinic (S) acids as determined bylitration; 0, radioactivity per fraction as determinedby liquid scintillation counts.

(AIn

VI0D-

c0

5

3

10 30 50FRACTION NUMBER

FIG. 3. Incorporation of radioacthity into succinateduring growth of Bacteroides succinogenes S-85 in thedefined medium containing 14CO2. X, Separationi ofvaleric (V), propionic (P), acetic (A), formic (F), andsuccinic (S) acids as measured by titration; 0, radio-activity per fraction as measured by liquid scintillationcounts.

shown. Although there was some variation fromexperiment to experiment, the radioactivity permicromole of carbon in succinate was consider-ably less than the radioactivity per micromole ofcarbon in maltose. The radioactivity per micro-mole of carbon in succinate was less than wouldbe expected if all of the succinate was formedby a direct fixation of carbon dioxide, e.g., by apathway involving carboxylation of phosphoenol-pyruvate or pyruvate. Table 7 shows the radio-

activity per micromole of carbon in the succinateformed during growth of B. amylophilus strain1441 in the defined medium containing a constantamount of maltose-1-14C and varying concentra-tions of carbon dioxide. Although there was somevariation between experiments, carbon dioxideconcentration could not be shown to influencethe radioactivity per micromole of carbon in thesuccinate formed during growth.Some effects of carbon dioxide on maltose fer-

mentation by B. amylophilus. Table 8 shows therecovery of label from uniformly labeled 4C-maltose in fermentation end products and in cellsproduced during growth of B. amylophilus strainsin the defined medium containing 7.4 X 102 Mbicarbonate buffer and a gas phase of 100%carbon dioxide. More than 90% of the maltoseradioactivity was recovered in nongaseous acidicfermentation end products. The remainder wasrecovered in cells. The percentages of maltoselabel in particular fermentation end productswere similar for most strains studied. No evidencefor substantial net carbon dioxide formationform maltose was found. Only traces of labelwere found in the gaseous phase after growth ofB. amylophilus strain 1441 under a gas phase of100% carbon dioxide in the presence of uni-formly labeled 14C-maltose. Table 9 shows theeffects of carbon dioxide concentration on maltosefermentation by B. amylophilus strain 1441. Dur-ing growth in the defined medium under a gasphase of 20% carbon dioxide, the quantity ofnmaltose fermented was reduced relative to thatfermented in the same medium with a gas phaseof 100% carbon dioxide, but the proportion offermented maltose carbon in particular fermenta-tion end products could not be shown to beinfluenced by carbon dioxide concentration. Atleast 90% of the radioactivity in the succinatefraction isolated from the medium after growthwas associated with succinate. No evidence forlactate or ethyl alcohol formation by B.amylophilus strain 1441 could be found undereither condition of growth.

DISCUSSION

The present study confirms the previously rec-ognized requirement of carbon dioxide for growthof B. amylophilus (16). The requirement is quan-titatively similar to that of other rumen bacteriawhose carbon dioxide requirement has beencritically studied (9, 12, 15). The ability ofbicarbonate to replace carbon dioxide as a growthfactor is presumably due to the conversion ofbicarbonate to carbon dioxide.The inability of formate to replace carbon

dioxide as a growth factor for B. amylophilus is of

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TABLE 5. Schmidt reaction degradation ofposition-labeled succinate and succinate isolated after growth ofrumen Bacteroides species in the presence of 14CO2

Disintegrations per mla

PrepnSuccinate before Carbon dioxide in Percentage recovereddegradation trap

Succinate-1,4-14C....................... 21,900 6,575 30.Ob (24-37)Succinate-2,3-14C....................... 148,600 721 0.48c (0.2-0.9)Succinate isolated from

B. amylophilus 1024................... 11,700 3,198d 28B. amylophilus 1055................... 9,093 3,767 41B. amylophilus 1058................... 11,307 4,783 42B. amylophilus 1059................... 3,285 1,273 39B. amylophilus 1413................... 6,457 4,249 65B. amylophilus 1417................... 3,987 1,359 29B. amylophilus 1444................... 6,229 2,673 43B. amylophilus 78..................... 9,378 3,789 40B. ruminicola GA33................... 9,577 4,262 44B. succinogenes S85................... 5,564 2,710 48

a Standards and degraded samples of position-labeled succinate were counted for 2 min. All samplesof material isolated from the organisms were counted for 20 min.

b Average value of 20 degradations. The range is indicated by the figures in parentheses.c Average value of 10 degradations. The range is indicated by the figures in parentheses.d All values for degradation of succinate isolated from organisms are average of two or three degrada-

tions. The succinate was isolated by column chromatography from the medium after growth.

TABLE 6. Radioactivity in the succinate producedduring growth of Bacteroides amylophilus strain

1441 in the defined medium containinguniformly labeled 14C-maltose and a100% carbon dioxide gaseous phase

Excpt Carbon)a DPM/mlb Radioactivity

Ext fjmoles/ml perymole S/MC

1 15,1 10,516 698 0.502 17.8 10,416 585 0.423 13.5 10,568 782 0.564 25 8 18,372 713 0.515 16 6 10,983 662 0.476 16.7 10,501 630 0.457 15.6 11,203 718 0.518 18.2 9,516 522 0.379 15.9 9,555 602 0.4310 16.7 8,807 529 0.38

Maltose 175 245,200 1,401

a Micromoles of carbon in succinate calculatedfrom duplicate titrations of the succinic acid iso-lated by column chromatography from the me-dium after growth.

b Disintegrations per minute per milliliter. Eachvalue is the average of duplicate liquid scintillationcounts of the succinate isolated by column chro-atography from the medium after growth.

c The ratio of the radioactivity per micromole ofcarbon in succinate relative to the radioactivityper micromole of carbon in maltose.

TABLE 7. Effect of carbon dioxide concentration oncount rate per micromole of carbon in the succinateformed during growth of Bacteroides amylophilus

strain 1441 in the defined phosphate-bufferedmedium containing maltose-i-14C

Count rate per micromole of succinatecarbona

Carbon dioxide

Expt 1 Expt 2

10 24720 352 37840 359 68180 373 450100 525 365

a Disintegrations per minute per micromole ofcarbon in succinate isolated from the medium asdescribed in Table 6. Each value was determinedfrom duplicate titrations and liquid scintillationcounts of the succinate isolated after growth bycolumn chromatography.

interest, since Pittman (unpublished data, 1966)showed that label from uniformly labeled 14C-glucose fermented by B. ruminicola can appear incarbon dioxide under conditions which do notallow detection of formate. The inability of for-mate to replace carbon dioxide as a growth factorfor B. amylophilus, and subsequent observations

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TABLE 8. Recovery of label from uniformly labeled14C-maltose in cells and fermentation end products

produced during growth of Bacteriodesamylophilus in the defined mediuma

Percentage of original radioactivity recoveredb

St ma inHV- Acetate Formate Suc- Cells FEP Total

FAC cinate

1024 3.3 28.9 25.1 46.9 5.3 104.2 109.51055 2.5 24.8 18.1 48.7 5.3 94.1 99.41058 2.6 30.1 17.7 44.5 5.9 94.9 100.81059 3.3 33.6 15.8 43.9 6.0 93.8 99.81417 3.9 33.6 12.0 43.2 7.6 92.7 100.31441 3.3 32.1 16.4 42.1 7.3 93.9 101.21444 2.5 15.4 25.5 54.7 7.8 92.4 99.778 1.7 27.1 14.6 48.7 6.5 92.1 98.9

aThe medium contained a 7.4 X 102 MNaHCO3 , 100% carbon dioxide-equilibrated buf-fer system.bEach value is calculated from quadruplicate

liquid scintillation counts of column elution frac-tions corrected for time, quenching, and back-ground. The original radioactivity supplied as uni-formly labeled 14C-maltose, obtained from theaverage quadruplicate counts, was 186,800 disinte-grations per min per ml.

The letters refer to the column fraction con-taining volatile fatty acids with chain lengthslonger than or equal to propionate. The lettersFEP refer to fermentation end products.

that label from '4C-formate exogenously suppliedto growing cells of B. amylophilus strain 1441 failsto appear in succinate, suggest that B. amylophiluslacks an extracellular formic dehydrogenase-likesystem. It is possible, however, that lack of utiliza-tion of exogenously supplied formate by B.amylophilus may result from the inability offormate to enter the cell, and that formate can beconverted to carbon dioxide intracellularly.The failure of certain amino acids which appear

to spare or replace the CO2 requirement of certainother rumen bacteria (2, 21, 26, 33) is of interest.The present study confirms previous results show-ing that the addition of these amino acids to mediacontaining carbon dioxide, ammonia, and maltosedoes not enhance growth (3). The failure of thesecompounds to affect growth in carbon dioxide-containing media presumably results from theintracellular synthesis of these substances fromother metabolites. Previous studies have shownthat at least 90% of the cellular nitrogen of B.amylophilus may be derived from ammonia (17).The failure of these amino acids to replace thecarbon dioxide requirement of B. amylophilusindicates that the carbon dioxide requirement ofthis organism results from the involvement of

TABLE 9. Some effects of carbon dioxideconcentration on uniformly labeled

14C-maltose fermentation byBacteroides amylophilus

strain 1441a

Total Percentage radioactivityradio- recovered"

Carbon Maltose activity ___-__ -__-___dioxide fetmentedb inaii

tfernenta- Suc- Ace- For- Resid-

products cmate tate mate uald

% snmoles

20 (I)e 6.9 126,900 49.9 32.5 9.8 7.620 (II) 7.7 135,700 48.7 29.9 16.0 2.9100 (I) 14.6 258,900 47.8 33.8 16.2 2.5100 (II) 11.3 200,500 48.6 33.5 15.3 2.4

a The media contained 0.05 M phosphate buffer(pH 7.0) and were prepared as described in Table2.

6 Maltose was measured colorimetrically by useof the anthrone reagents and methods of Scottand Melvin (29). The maltose concentration of themedium was 1.46 X 1r2 M.

e Each percentage value was calculated fromthe average of four replicate liquid scintillationcounts. The percentage refers to the proportionof acidic fermentation end product radioactivityattributable to a particular end product.

d Residual radioactivity refers to fermentationend product radioactivity not associated withsuccinate, acetate, or formate.

e The Roman numerals refer to different experi-ments.

carbon dioxide in critically important metabolicprocesses other than, or in addition to, aminoacid biosynthesis, e.g., carbohydrate fermenta-tion, but the present experiments fail to showwhether or not carbon dioxide is involved inamino acid biosynthesis by B. amylophilus.The present results confirm previous reports

that a substantial loss of carbon dioxide from themedium occurs during growth of B. amylophilus(16). The results further show that label from"CO2 is incorporated, apparently specifically, intothe carboxyl function of the succinate formedduring maltose fermentation. These results, com-bined with the demonstration of a similar in-corporation of label from "CO2 into the carboxylcarbon of the succinate formed during glucosefermentation by B. ruminicola subsp. brevis strainGA33, and during cellobiose fermentation by B.succinogenes strain S85, provide direct evidencefor reactions involving carbon dioxide fixation insuccinate formation by rumen Bacteroides species.The present results, combined with previousstudies showing the incorporation of label from4CO2 into the succinate formed during glucose

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EFFECT OF CARBON DIOXIDE ON B. AMYLOPHILUS

fermentation by R. flavefaciens (19, 20) and byS. dextrinosolvens (27, 28), indicate that reactionsinvolving the fixation of label from "CO2 intothe succinate are commonly found among suc-cinogenic rumen bacteria.The extent of dilution of label, on a per micro-

mole of carbon basis, in the succinate formedduring fermentation of uniformly labeled 1"C-maltose by B. amylophilus in the presence ofnonlabeled carbon dioxide is greater than wouldbe expected if all of the succinate formed wereproduced by the condensation of carbon dioxidewith phosphoenolpyruvate or pyruvate. However,the recovery of radioactivity shows that as muchas 60% of the total succinate formed may resultfrom a pathway involving the fixation of one moleof carbon dioxide per mole of succinate formed.These results indicate that a pathway involvingcarbon dioxide fixation can account for a largefraction of the total succinate formed by B.amylophilus, but further study is required todetermine whether such a pathway can accountfor all of the succinate formed, or whether analternative pathway(s) for succinate formation ispresent in this organism. Such a pathway, ifpresent, must contribute more than one non-labeled carbon per molecule of succinate formedto account for the low radioactivity per micromoleof succinate carbon, relative to that per micro-mole of carbon in maltose. Preliminary studiesfailed to show that label from acetate-J-'4C or-2-14C or from propionate-2-'4C, all exogenouslysupplied to growing cells, was incorporated intosuccinate, but the possibility that other nonlabeledmedium components are converted to succinatecannot be excluded. Further, more critically con-trolled studies are required to assess the quantita-tive importance of carbon dioxide fixation tosuccinate formation by succinogenic rumen bac-teria, and to understand more clearly the mecha-nisms of succinate formation in these organismsThe present results show that carbohydrate

fermentation in B. amylophilus is dependent uponcarbon dioxide. During growth in a nonlimitingconcentration of carbon dioxide, virtually all ofthe original radioactivity in maltose was re-covered in cells and in nonvolatile acidic fermen-tation end products. These results, combined withdirect measurements of the gaseous phase aftergrowth of B. amylophilus strain 1441 in the pres-ence of uniformly labeled 14C-maltose, suggestthat little, if any, net formation of carbon dioxidefrom maltose occurs during growth. The ap-parently complete utilization of maltose in thepresent experiments contrasts with previous re-sults showing that a substantial portion of mal-tose, even when it is supplied at growth-limiting

concentrations, remains unfermented by B.amylophilus (3, 18). The apparent discrepancybetween these results and those of the presentstudy might be explained by differences in growthconditions or by formation of anthrone-reactingsubstances other than maltose during growth ofB. amylophilus.

During growth in a limiting concentration ofcarbon dioxide, the quantity of maltose fermentedby B. amylophilus was reduced, but neither thepercentage of the carbon of fermented maltoseassociated with a particular nonvolatile acidicfermentation end product nor the radioactivityper micromole of carbon in the succinate formedduring fermentation of maltose-1-_4C was influ-enced by carbon dioxide concentration. The latterresult suggests that variation in carbon dioxideconcentration would not alter the extent of thecontribution of a carbon dioxide-fixing mecha-nism for succinate formation to the total suc-cinate formed.A dependent relationship between carbohydrate

fermentation and carbon dioxide appears com-mon in succinogenic rumen bacteria, but furtherstudy of the carbohydrate fermentation schemesof these organisms, and of the relationship(s)between the carbohydrate fermentation and theelectron transport systems of these bacteria, isrequired to understand the mechanisms of energyrelease and utilization by these organisms and theeffect of the latter processes on the rumen fer-mentation.

LITERATURE CITED

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CALDWELL, KEENEY, AND VAN SOEST

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