APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1982, p. 913-9220099-2240/82/100913-10$02.00/0Copyright C 1982, American Society for Microbiology

Vol. 44, No. 4

Changes in Viability, Cell Composition, and Enzyme LevelsDuring Starvation of Continuously Cultured (Ammonia-

Limited) Selenomonas ruminantiumRONALD W. MINK,t JOHN A. PATTERSON, AND ROBERT B. HESPELL*

Department ofDairy Science, Microbiology Division, University of Illinois, Urbana, Illinois 61801

Received 5 February 1982/Accepted 9 June 1982

Under nitrogen (ammonia)-limited continuous culture conditions, the ruminalanaerobe Selenomonas ruminantium was grown at various dilution rates (D). Theproportion of the population that was viable increased with D, being 91% at D =0.5 h-1. Washed cell suspensions were subjected to long-term nutrient starvationat 39°C. All populations exhibited logarithmic linear declines in viability that wererelated to the growth rate. Cells grown at D = 0.05, 0.20, and 0.50 lost about 50%viability after 8.1, 4.6, and 3.6 h, respectively. The linear rates of decline in totalcell numbers were dramatically less and constant regardless of dilution rate. Allmajor cell constituents declined during starvation, with the rates of decline beinggreatest with RNA, followed by DNA, carbohydrate, cell dry weight, and protein.The rates ofRNA loss increased with cells grown at higher D values, whereas theopposite was observed for rates of carbohydrate losses. The majority of thedegraded RNA was not catabolized but was excreted into the suspending buffer.At all D values, S. ruminantium produced mainly lactate and lesser amounts ofacetate, propionate, and succinate during growth. With starvation, only smallamounts of acetate were produced. Addition of glucose, vitamins, or both to thesuspending buffer or starvation in the spent culture medium resulted in greaterlosses of viability than in buffer alone. Examination of extracts made fromstarving cells indicated that fructose diphosphate aldolase and lactate dehydro-genase activities remained relatively constant. Both urease and glutamate dehy-drogenase activities declined gradually during starvation, whereas glutaminesynthetase activity increased slightly. The data indicate that nitrogen (ammonia)-limited S. ruminantium cells have limited survival capacity, but this capacity isgreater than that found previously with energy (glucose)-limited cells. Apparentlyno one cellular constituent serves as a catabolic substrate for endogenousmetabolism. Relative to losses in viability, cellular enzymes are stable, indicatingthat nonviable cells maintain potential metabolic activity and that generalized,nonspecific enzyme degradation is not a major factor contributing to viability loss.

A number of factors, both environmental andintrinsic to the microorganism in question, canaffect microbial survival under conditions oftotal nutrient starvation (11, 12, 36, 46). Onefactor of particular importance is the nature ofthe nutrient which limits growth before starva-tion. Postgate and Hunter have shown that nitro-gen-limited cells of Aerobacter (Enterobacter)aerogenes exhibited better survival capabilitiesthan did carbon-limited populations (36). Unfor-tunately, there have been few similar investiga-tions with other microorganisms because of thetendency of most researchers to study starvationof microorganisms using batch cultures, whichare not growth limited until the late exponential

t Present address: W. Alton Jones Cell Science Center,Lake Placid, NY 12946.

phase, and often the limiting nutrient(s) is un-known. In addition, there have been numerousstudies on the survival of aerobes, but relativelyfew on strictly anaerobic bacteria, particularlythose which are found in the rumen, cecum, orlarge intestine of mammals. Within the rumen,the levels of many nutrients are constantlychanging due to the discontinuous ingestion offeedstuffs and microbial degradation of thesematerials. Analyses for organic or inorganicnitrogen (27, 32, 34, 53) and for carbohydrates(23, 45; J. A. Z. Leedle, Ph.D. thesis, Universi-ty of Illinois, Urbana, 1981) have shown thatlarge variations can occur in rumen contents,depending upon the time after feeding, the typeof diet fed, and the composition of the microbialpopulation present. These variations not onlyaffect the growth of various rumen bacterial

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914 MINK, PATTERSON, AND HESPELL

species, but ultimately affect the abilities of themicroorganisms to survive, as reflected in thelarge changes seen in the viable to direct cellcounts in rumen contents (J. A. Z. Leedle,Ph.D. thesis, University of Illinois, Urbana,1981).

In previous studies, we examined the survivalabilities of the ruminal anaerobes Megasphaeraelsdenii (29) and Selenomonas ruminantium (30)that had been grown under energy (glucose)limitation using steady state continuous cultureconditions. In the current study, we examinedwith continuous culture conditions the effects ofnitrogen (ammonia) limitation and growth rateon the viability and cell composition of S. rumin-antium during nutrient starvation. In addition,the stability of selected glycolytic and ammonia-assimilating enzymes in these cells was exam-ined.

MATERIALS AND METHODSOrganism and culture conditions. Selenomonas ru-

minantium strain D (22) was obtained from the culturecollection of the Department of Dairy Science, Uni-versity of Illinois, Urbana. Stock cultures were main-tained on carbohydrate maintenance agar slants (6)and transferred twice a month. All medium prepara-tion and experimental manipulations were carried outanaerobically as previously described (30). Continu-ous cultures were grown in an anaerobic chemostatmaintained at 39 ± 0.5°C (20). Steady state conditionswere verified by constant absorbance measurements(660 nm) and constant cell RNA levels measured asdescribed below. Verification of ammonia-limitedgrowth was made by measurement of both glucose andammonia levels in spent culture media using the glu-cose oxidase (Sigma Chemical Co., bulletin 510) andindophenol methods (9, 19), respectively.The growth medium contained (amount per 100 ml):

NH4CI, 10 mg; NaCl, 60 mg; KCI, 60 mg; MgSO4, 26mg; CaCl2 * 2H20, 20 mg; hemin, 20 jig; salts andtrace elements, 0.1 ml; resazurin, 100 ,ug; sodiumacetate (anhydrous), 246 mg; glucose (1 M), 5.0 ml;potassium phosphate (0.5 M), 1.4 ml; S. ruminantiumvitamins, 0.5 ml; Na2S * 9H20, 50 mg; and Na2CO3,400 mg. Salts and trace elements, glucose, potassiumphosphate, vitamins, and sodium carbonate were pre-pared and added as previously described (30). Heminwas added as an aqueous solution of 0.01% (wt/vol)hemin-25% (vol/vol) ethanol-0.28% (wt/vol) KOH.Sodium sulfide was added as an anaerobic aqueous2.5% (wt/vol) solution.Starvation buffer and cell collection and washing. The

starvation buffer (S buffer) was a mineral salts buffercontaining 1.5 mM dithiothreitol as the reducing agentand was prepared anaerobically as previously de-scribed (25). Cell culture samples were collected bycentrifugation (8,800 x g, 10 min, 15°C). The cellswere washed twice at room temperature by centrifuga-tion using S buffer followed by a final resuspension inS buffer to the same cell concentration as the originalgrowth medium. Suspensions (1.5 liters) of S. rumin-antium were starved in 2-liter serum flasks and incu-bated with shaking (39°C, N2 atmosphere).

Total and viable cell counts. Total cell counts weredetermined by a direct microscopic count using Pet-roff-Hausser chambers with samples of cultures dilut-ed 1:10 or 1:20 in anaerobic dilution solution. Viablecell counts were determined by serial 10-fold dilutionin anaerobic dilution solution followed by platingvarious dilutions on recovery medium. The plateswere incubated (under C02, 39°C, 5 days) in anaerobicincubation vessels (25), and the colonies on each platewere counted. Anaerobic dilution solution had thesame composition as the growth medium used, butlacked the glucose, vitamins, and sodium carbonate.Recovery medium composition was identical togrowth medium but also contained 1.5 g of agar per 100ml.

Cell analyses. Absorbance of cultures was measuredat 660 nm using cuvettes with a 1-cm light path. Dryweights were determined by drying (110°C, 15 h) thepellet of an anaerobically centrifuged (8,800 x g, 10min, 4°C) cell culture or suspension of known volume.Cell components were extracted as previously de-scribed (18) and assayed by the diphenylamine methodof Burton (8) for DNA, the orcinol method ofSchneider (41) for RNA, the method of Lowry et al.(26) for protein, and the phenol-sulfuric acid methodfor carbohydrate (1).

Analyses of growth media and suspending buffers.Fermentation acids were measured by gas-liquid chro-matography using either of two methods: (i) prepara-tion and analysis of butyl esters (39) or (ii) analysis offree acids (30). The starvation buffer was also analyzedfor material absorbing at 260 and 280 nm to measurelevels of nucleotide bases or nucleic acids or both (52).Ribose or RNA or both in the buffer were measured bythe orcinol method described above.Enzyme activities. Cell suspensions were centrifuged

(10,000 x g, 10 min, 4°C) and washed twice in 50 mMTris-hydrochloride-1% (wt/vol) KCl-1 mM dithio-threitol (TKD) buffer (pH 7.4) and resuspended inTKD buffer under argon to 1% (vol/vol) of theiroriginal volume. Cell-free extracts were prepared bypassing cell suspensions twice through a French pres-sure cell (20,000 lb/in2) at 4°C under argon. Altercentrifugation (15,000 x g, 20 min, 4°C), the cell-freeextract supernatant was stored under argon on iceuntil used (1 to 2 h). Protein in the cell-free extractswas measured as described above. Fructose diphos-phate aldolase (FDA) activity was measured by thecoupled NAD reduction reaction of glyceraldehyde-3-phosphate dehydrogenase as described by Hespell andCanale-Parola (16). One unit of FDA activity wasdefined as the amount of enzyme required to reduceone nanomole of NAD per minute. Lactate dehydrog-enase (LDH) activity was determined as the rate ofNADH oxidation. The reaction mixture was preparedin 3-ml cuvettes by mixing 1.8 ml of 28 mM Tris-hydrochloride (pH 7.0), 0.1 ml of 4 mM NADH, and0.025 to 0.05 ml of a 1:10 dilution (in TKD buffer) ofcell-free extract (5 to 50 mg of protein per ml). Thereaction was initiated by the addition of 0.1 ml of 0.1 Msodium pyruvpte.One unit of LDH was defined as the amount of

enzyme required to oxidize one nanomole of NADHper minute. The urease assay was carried out usingWarburg-type incubation vessels with a center wellcontaining 0.05 ml of 2.0 M KOH (CO2-free). Thereaction chamber contained 0.5 ml of 25 mM KH2PO4

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CHANGES IN S. RUMINANTIUM DURING STARVATION 915

(pH 7.2), 0.1 ml of 250 mM tetrasodium EDTA (pH7.2), 0.2 ml of water, and 0.1 ml of [14C]urea (10 mM,150,000 dpm/,umol). The side arm contained 0.05 to 0.1ml of cell suspensions (50 to 500 mg of protein). Theassay was begun Oy mixing the cells in the side armwith the contents of the reaction chamber. After 20min at 30°C, 0.1 ml of 3.0 N H2SO4 was added to theside arm and mixed with the contents of the reactionchamber to stop the assay. The KOH in the center wellwas assayed for 14CO2 using a Beckman scintillationcounter (model LS230) with Aquasol-2 (New EnglandNuclear Corp.) as the scintillation cocktail. One unit ofurease was defined as the amount of enzyme requiredto hydrolyze one nanomole of urea per minute. Gluta-mate dehydrogenase (GDH) activity was measured asthe rate of NADPH oxidation by the procedure ofMeers et al. (28) as modified by Smith et al. (42). Oneunit of GDH activity was defined as the amount ofenzyme required to oxidize one nanomole of NADPHper minute. Glutamine synthetase (GS) activity wasmeasured in whole-cell suspensions by the forwardreaction assay described by Bender et al. (2) asmodified by Smith et al. (42). One unit of GS activitywas defined as the amount of enzyme required to formone nanomole of -y-glutamyl hydroxamate per minute.

Statistical analyses. Cell composition data were sub-jected to linear or nonlinear regression (least squares)analysis or both as described previously (30).

RESULTSCell composition and viability before starva-

tion. For experiments to determine the effects ofstarvation on ammonia-limited continuously cul-tured S. ruminantium strain D, the organism wasgrown at three different dilution rates (D = 0.05,0.20, and 0.50 h-1). There were no large differ-ences in total cell numbers per milliliter betweendilution rates. However, the proportion of thepopulation that was viable was directly relatedto the dilution rate and ranged from 30.1% forcells grown at D = 0.05 h-1 to 91.1% for cellsgrown at D = 0.50 h-1 (Table 1). Analyses ofcellular components indicated that there wereonly small differences between dilution rates forcellular dry weight and levels of protein andDNA. In contrast, faster-grown cells tended tocontain greater levels of RNA and lesser levelsof carbohydrate than slower-grown cells. Analy-ses of cell suspensions before and after washingin the S buffer (but before starvation) indicatedno significant loss of total or viable cell num-bers, cell integrity, or any particular cellularcomponent.Changes in cell numbers during starvation. The

survival of cell suspensions subjected to starva-tion was measured in terms of both total andviable cell numbers (Fig. 1). In general, total cellnumber declined modestly, about 30% after 48 hof starvation, while viability declined muchmore rapidly, to less than 1% of the initial levelduring the same period. Populations grown ateach of the three dilution rates exhibited a

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916 MINK, PATTERSON, AND HESPELL

Time (Hours)FIG. 1. Changes in the viable (0) and total (O) cell

numbers of ammonia-limited S. ruminantium duringstarvation. The data are the grand means of twoexperiments each of populations grown at D = 0.05,0.20, and 0.50 h-1. The data are expressed as thepercentage of the values at 0 h (Table 1).

steady decline in viability. The populationsgrown at D = 0.05 h-1 consistently exhibited thebest survival, and those grown at D = 0.50 h-1exhibited the poorest. Regression analysis of thecell number data indicated that the loss in viabil-ity was log-linear for all three populations andthat the 50% survival times (ST50) were 8.07,4.61, and 3.56 h for populations grown at D =

0.05, 0.20, and 0.50 h-1, respectively (Table 2).However, no major differences in the rates ofloss of total cell numbers were observed be-

tween populations grown at different dilutionrates.Changes in cell composition. The decreases in

cell dry weight during starvation were morerapid than decreases in total cell numbers, indi-cating an overall decline in individual cell mass

(Table 2). Total cell numbers remained consis-tently high, and relatively little cell debris was

seen in the suspending buffer until quite late intothe starvation time. Of the cellular constituents,DNA was generally lost relatively quickly, al-though those losses were still at far slower ratesthan the declines in viability (Table 2 and Fig. 2).Protein levels showed no indication of selectivedegradation, as their losses closely paralleledthose of dry weight. Losses in carbohydratewere relatively low, but appeared to be relatedto the growth rate at which the organism was

grown, with the most rapid losses observed inthe cells grown at the lowest dilution rate (D =

0.05 h-1). Of all of the cellular constituents,RNA losses were the greatest for each starvingpopulation and were directly related to thegrowth rate before starvation. An analysis of thestarvation buffer indicated that most, if not all,of the lost RNA was apparently expelled outsideof the cell, based on the measurement of orcinol-reactive material and material absorbing at 260nm found in the buffer (data not shown). Similarresults were found with starvation of glucose-limited cells (30). However, the levels of orcinol-reactive material in the buffer of cell suspensionsgrown at D = 0.20 h-1 did show a subsequentdecline after 24 h of starvation (data not shown),which could not be accounted for in terms ofincrease in mass or in any component of the cellsuspension.Fermentation acids produced. S. ruminantium

during ammonia-limited growth produced pri-marily lactate, regardless of dilution rate (Table3). The tendency for higher acid levels at lowerdilution rates was primarily a reflection of in-creased glucose utilization rather than differ-ences in molar ratios of acids produced per moleof glucose. The exception to this observation

TABLE 2. Rates of change in population parameters of S. ruminantium strain D (ammonia-limited) duringstarvation

Rate of change in no. ofD (h-1) cellsa ST b (h) Rate of change of cellular componentSa

Total Viable Dry wt DNA RNA Protein Carbohydrate

0.05 -0.0051 -0.0858 8.07 -0.0099 -0.0198 -0.0492 -0.0097 -0.02050.20 -0.0069 -0.1502 4.61 -0.0133 -0.0504 -0.0637 -0.0106 -0.00940.50 -0.0060 -0.1944 3.56 -0.0117 -0.0501 -0.0729 -0.0069 -0.0048a The data were subjected to linear regression analysis (see the text), and the rates are the fractional change

per hour as determined from the slopes of the regression lines. All regression lines were significant at P < 0.005or better.

b ST50 is the time for 50% of the initial viable population to become nonviable, calculated from the fractionalturnover rate (m) of the regression line of viable cell numbers where ST50 = In (2/m).

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CHANGES IN S. RUMINANTIUM DURING STARVATION 917

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6 12 18 24 30 36 42 48

Time (Hours)

FIG. 2. Changes in cellular dry weight, DNA,RNA, protein, and carbohydrate of S. ruminantiumduring starvation. The data are the grand means ± thestandard deviation of two experiments each of popula-tions grown at D = 0.05, 0.20, and 0.50 h-V. The dataare expressed as a percentage of the levels at 0 h(Table 1).

occurred with succinate, which was produced atrates of 0.30, 0.18, and 0.13 mol per mol ofglucose degraded for D = 0.05, 0.20, and 0.50h-1, respectively. During starvation, fermenta-tion acids were monitored, and only acetate wasfound in the suspending buffer. The levels ofacetate present increased during starvation, withacetate levels being 90 to 140 nmol/ml after 36 h

of starvation. Although differences in acetate

levels between starving populations grown at

different dilution rates were observed, they werenot significant. The acetate present in the buffercould not be attributed to contamination fromthe high levels of acetate present in the spentgrowth medium, since 0-h suspensions had no

significant levels of lactate present despite thefact that spent growth medium contained as

much or more lactate than acetate.Enzyme activities during starvation. In an ef-

fort to determine if premature loss of catabolicor biosynthetic enzyme activities may have con-

tributed to losses in viability, selected enzymes

were assayed in starving suspensions of S. ru-

minantium. The glycolytic enzymes, FDA andLDH, exhibited essentially no significantchanges in activity during the entire 48 h of theexperiment, although whole-cell protein levelsexhibited a slight decline (Fig. 3). The nitrogenmetabolism enzymes, urease, GDH, and GS,were also analyzed, and although changes inactivity were observed (Fig. 4), those changeswere not great in comparison to viabilitychanges (Fig. 1). Total activities of both ureaseand GDH decreased, but specific activities ofthese enzymes did not. GS, on the other hand,exhibited a slight increase in total activity,which would reflect a significant increase inspecific activity. Similar responses were ob-served with all populations regardless of thedilution rate at which the cells had been previ-ously grown.

Effect of nutrients on survival. To determinethe sparing effects, if any, of selected nutrientson survival during starvation, S. ruminantiumwas grown at D = 0.20 h-1 and suspended in Sbuffer alone or supplemented with vitamins orglucose or both or resuspended in the spentgrowth medium after washing. The results (Ta-ble 4) indicated that none of the treatments hadany positive survival effects over starvation in Sbuffer alone, and in fact, they may have has-tened death in some cases. The initial death ratefor cells resuspended in buffer supplementedwith both glucose and vitamins appeared to bemore than three times greater (56.5% loss in

TABLE 3. Levels of fermentation acids in the medium during ammonia-limited growth of S. ruminantiumstrain Da

Fermentation Level of acid (,imol/ml) for the following dilution rates:

acid 0.05 h-1 0.20 h-1 0.50 h-

Acetate 7.65 + 1.08 3.81 + 2.69 4.23 0.97Propionate 5.28 2.08 3.17 1.39 1.47 0.66

Lactate 43.30 1.03 36.20 5.25 25.60 0.41

Succinate 12.20 1.14 5.32 2.87 3.00 1.33

a Cells were grown in continuous culture at the indicated dilution rate as described in the text. The data are the

means t standard deviation of two experiments at each D value. Glucose utilization was 41.0, 29.8, and 23.8

FmoUml for cells grown at D = 0.05, 0.20, and 0.50 h-1.

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918 MINK, PATTERSON, AND HESPELL

100

50

0

150

100 .

50 -

0 6 12 18 24 30 36 42 48GrowingCulture Time (Hours)

FIG. 3. Changes in whole-cell protein and activi-ties of FDA (fructose-1,6-diphosphate aldolase) andLDH (lactate dehydrogenase) of an S. ruminantiumstrain (ammonia limited) during starvation. The dataare expressed as the percentage of the values at 0 h.The 0-h values are as follows (per ml of cell suspen-sion): whole-cell protein, 299 ,ug; FDA, 12.1 U; andLDH, 680 U. All data are the means ± the standarddeviation of seven experiments (two replicates of D =

0.05, 0.20, 0.50, and one at D = 0.35).

viability) than for cells starved in S buffer alone(15.8% loss in viability).

DISCUSSIONWhen compared with the other bacteria (Ta-

ble 5), the survival of ammonia-limited, continu-ously cultured S. ruminantium was relativelypoor, having ST50 values of 3.5 to 8 h. Yet, thesurvival of S. ruminantium was significantlybetter (ST50 = 0.5 to 2.5 h) than when it wasgrown under glucose limitation before starvation(30). With the ammonia-limited populations, thefastest-grown cells (D = 0.50 h-') had thehighest ratio of viable to total cells in the culture,but also had the poorest survival. This may

simply have reflected decreased availability ofdead cell materials available for cryptic growth.This explanation alone would seem unlikely,however, as cells starved in spent growth medi-um, which was likely to contain an abundance ofproducts from lysed or dying cells, exhibited no

greater survival ability than those starved in Sbuffer (Table 4). The likelihood of a role ofcryptic growth is further diminished by the ob-

servation that both total cell numbers and dryweight declined slowly (Table 2). In addition,these losses in cell mass and numbers wereslower than those which were observed withglucose-limited cells, which survive even morepoorly and have greater cell lysis (30). An alter-nate explanation of short survival times of S.ruminantium could be that the slower-growncells survived better because they were betteradapted to starvation by their slow growth rate.Yet, just the opposite results were obtained withstarvation studies of Aerobacter aerogenes (36)and Megasphaera elsdenii where faster-growncells were found to survive better (29).A relatively rapid loss of cellular DNA com-

pared with dry weight occurred during the initial12 h of starvation (Table 2 and Fig. 2). Sinceonly a minor portion of the DNA loss could beattributed to cell lysis, it is probable that theDNA loss was indicative of the degradation ofexcess genome copies. However, such degrada-tion probably played an insignificant role inproviding the cell with endogenous substrates towithstand starvation, as the DNA represented arelatively small amount of organic materialavailable to the cell (Table 1). Nevertheless, thepossibility exists that such degradation, if un-controlled, could have caused cell death vialethal lesions in the DNA.The cellular component which exhibited the

most rapid decline among all populations wasRNA (Table 2 and Fig. 2). Although this sug-gests that RNA is a preferred substrate forendogenous metabolism, the analysis of the star-vation buffer indicated that most of the RNAappears to be partially broken down and re-leased into the starvation buffer without beingcompletely catabolized. Similar results were ob-served previously with the starvation of glucose-limited S. ruminantium cells (30). However,since the latter had lower initial levels of RNA,the RNA was depleted in a shorter time despitethe fact that both types of cells degraded RNA atsimilar rates on an absolute scale. Thus, itappears likely that glucose-limited cells wouldbegin degradation of essential RNA sooner thanthe cells in this study and that this may account,in part, for their poorer survival. RNA degrada-tion is a common phenomenon among bacteriaduring starvation and has been reported forPeptococcus prevotii (37), Aerobacter aero-genes (44), Pseudomonas aeruginosa (15),Streptococcus lactis (47), Escherichia coli (21),Megasphaera elsdenii (29), and Bdellovibriobacteriovorus (17), to name a few examples. Theprimary site of RNA degradation is generallyattributed to ribosomes due to the minimal needfor protein synthesis under starvation conditions(24). In contrast to RNA, protein degradation instarving S. ruminantium was at a rate less than

Whole Cell Protein

I- I

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CHANGES IN S. RUMIIVANTIUM DURING STARVATION 919

150K

Glutomate Dehydrogenose

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Glutomine Synthetase0

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6 12 18Time (Hours)

24 30 36 42 4e

FIG. 4. Changes in urease, GDH, and GS of S.ruminantium (ammonia limited) during starvation. Thedata are expressed as the percentage of the values at 0h. The 0-h values were: whole-cell protein, 299 ,ug;urease, 24.0 U; GDH, 15.2 U; and GS, 81.6 U. All dataare the means ± the standard deviation of sevenexperiments (two replicates of D = 0.05, 0.20, 0.50,and one at D = 0.35).

or equal to that of dry weight loss, indicatinglittle preferential degradation of this component(Table 2).Carbohydrate loss in starving S. ruminantium

varied inversely with the dilution rate at whichthe population was grown. At the higher dilutionrates, the loss was less rapid than that of dryweight, suggesting that the degradation was notpreferential. On the other hand, cells grown at D= 0.05 h-' displayed a carbohydrate degrada-tion rate double that of dry weight loss (Table 2).Often, bacterial species under ammonia limita-tion accumulate carbohydrate reserves such asglycogen, and this is also the case with S.ruminantium (51). Starvation studies of glyco-gen-rich versus glycogen-poor populations ofother organisms have revealed that the formerexhibit better survival overall (13, 43). The car-

bohydrate levels present in this particular Selen-omonas strain are lower regardless of growthrate than in some other strains of Selenomonas(R. Mink, unpublished data; 51) and are lowerthan that ofM. elsdenii (29) or other large rumenbacteria or protozoa (10). Nevertheless, thiscarbohydrate pool present in Selenomonas maywell play a critical role in its survival, as the

slower-grown cells survived best and containedthe highest levels of carbohydrate.During growth, considerable energy uncou-

pling occurred due to the presence of excessglucose and having ammonia as the limitingfactor for growth. In going from lower to higherdilution rates, the cell yields were slightly high-er, while both glucose utilization and acid pro-duction decreased (Table 3). This suggests thatat higher dilution rates, growth was more effi-cient and there was less energetic uncoupling. Incontrast, the environment drastically changedduring starvation, and the cells most likely be-came energy deficient. Acid production shiftedto the more energy-efficient acetogenesis andaway from inefficient lactate production. Similarresults were seen with the starvation of glucose-limited S. ruminantium (30) and, to a lesserextent, in M. elsdenii (29). However, with thestarvation of glucose-limited S. ruminantium,acetate production was higher than was ob-served in this study. This may reflect a morecontrolled, energetically coupled endogenousmetabolism in the ammonia-limited cells duringstarvation.Peptococcus prevotii, another anaerobe, pro-

duces a combination of acids plus C02, H2, andammonia during starvation as well as withgrowth during which the organism ferments nu-cleosides, purines, and ribose (37). Thus, thisorganism is already substantially prepared to,and does, ferment RNA as an endogenous sub-strate during starvation. P. prevotii, therefore,does not have to make a major metabolic changeat the onset of starvation. This may account forits better survival (Table 5) over S. ruminan-tium. The failure of S. ruminantium to formsuccinate or propionate during starvation mayhave been due to the inability of the cells toincorporate CO2. Propionate formation is de-rived by decarboxylation of succinate which isformed by incorporation of CO2 into phospho-enolpyruvate to form oxaloacetate (33, 40). Suc-cinate is then ultimately formed by reductionreactions. However, we utilized starvation con-ditions with a C02-free atmosphere, and theamount of CO2 contributed by the cell in theproduction of acetate may have been insufficientto drive the formation of oxaloacetate (andtherefore succinate and propionate) significant-ly. Indeed, the addition of CO2 to the starvationenvironment might have improved survival byvirtue of the possibility that propionate produc-tion has been suggested to produce up to threeATPs per propionate in addition to the ATPsformed during glycolysis (19). If so, then surviv-al of S. ruminantium would be enhanced in therumen environment which contains substantiallevels of bicarbonate derived from microbialfermentation and saliva input.

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920 MINK, PATTERSON, AND HESPELL

TABLE 4. Effect of nutrients on survival of S.ruminantiuma

% ViabilityStarvation condition

3h 12h

Buffer 84.2 6.4Buffer plus glucose 75.9 6.1Buffer plus vitamins 59.1 4.2Buffer plus glucose plus vitamins 43.5 6.8Spent growth medium 62.8 8.8

a The organism was grown in an ammonia-limitedcontinuous culture at D = 0.20 h-1 and then washed,resuspended, and starved in S buffer, with or withoutadditions, or in spent growth medium. Glucose andvitamins, when added, were present at 11.1 mM and0.5% (vol/vol), respectively. The data are expressed asthe percentage of viable cell numbers at 0 h (1.66 x109/ml) and are the means of two experiments.

A possible explanation for the rapid death ofS. ruminantium during starvation could be theloss of essential enzyme activities. The apparentlevel of LDH remained high during starvation,but its activity is controlled by pyruvate whichacts as a homotropic activator (50). One wouldexpect pyruvate to be low in starving cells inview of its need in biosynthetic reactions offattyacid and amino acid synthesis, as well as thecatabolism to acetate for energy. The high activ-ity observed may have been an artifact of theassay, which contained added pyruvate. Thus,in the cells, LDH may have been inactive, andthis is consistent with the lack of lactate produc-tion by starving cells. The apparent increase ofGS during starvation could be expected, sinceGS tends to be more important under low-nitrogen conditions than is GDH (42). Yet, ure-ase, which usually parallels GS activity, inexpli-cably showed no increase in activity. Theseparticular enzymes were by no means all of theenzymes needed by the cells to maintain viabili-ty, but they could be considered representativeof the cells' enzymes. The fact that none werepreferentially degraded implicates a cause otherthan nonspecific enzyme breakdown in the lossof viability during starvation.The addition of glucose or vitamins or both to

starving cell suspensions did not aid survival(Table 4). When added, substantial amounts ofglucose were fermented by the starving cells(data not shown). This suggests that neither a

shortage of energy nor the inability to get atendogenous substrates due to a lack of cofactorswas the cause of lost viability. Similar resultswere seen with glucose-limited S. ruminantium(29); however, unlike their ammonia-limitedcounterparts, the glucose-limited cells survivedbetter in spent growth medium.Why do microorganisms exhibit such marked

differences (Table 5) in survival? We and others

TABLE 5. Survival times of various bacterialspeciesa

Organism ST50 (h)b Reference

Selenomonas 3.6-8.1 Current studyruminantium (Nlimited)

S. ruminantium (C 0.5-2.5 30limited)

Megasphaera elsdenii 3-5 29(C limited)

Bdellovibrio 10 18bacteriovorus

Peptococcus prevotii 10-12 31Methanospirillum ca. 25 4

hungatiiEscherichia coli 36 14Sarcina lutea 65 7Chromatium vinosum 120 (dark) 49Rhodospirillum 344 (light) 5rubrum

12 (dark)Nocardia corallina 480 38Arthrobacter 2,400 3

crystallopoietesa Starvations were at physiological temperatures (30

to 370C) in nonnutrient buffers.b Time required for half of the initial population to

die.

have alluded to the roles of such environmentalvariables as growth rate, limiting nutrient, andcomposition or complexity of the growth medi-um, but apparent intrinsic differences betweenmicroorganisms may be more important. Someorganisms may be intrinsically better equippedto efficiently turn over cellular constituents andto scavenge for needed materials both intracellu-larly and extracellularly. Various bacteria mayhave different ATP requirements to maintainessential cell functions (35, 48). The specifictype of energy metabolism an organism pos-sesses may affect its survival ability. For exam-ple, microorganisms that rely on oxidative phos-phorylation may obtain energy more efficientlyfrom catabolism of cell materials than thosewhose sole source is substrate-level phosphor-ylation. Thus, aerobes appear to survive muchbetter than anaerobes (Table 5). Photosyntheticscan survive in an anaerobic nonorganic environ-ment much longer if a source of light for energygeneration is available (Table 5). S. ruminantiumhas high rates of loss in dry weight (Table 2) thatreflect a high loss of carbon, and the organism isrelegated to substrate phosphorylation for ener-gy. Both factors probably contribute greatly toits short survival, whereas extremely low carbonlosses and aerobic metabolism probably formthe basis of the very long survival of Arthro-bacter crystallopoietes (Table 5) (3).

S. ruminantium tends to be more numerous in

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CHANGES IN S. RUMINANTIUM DURING STARVATION 921

the rumens of animals that are fed high-energydiets containing much grain. Because these dietscontain substantial amounts of soluble carbohy-drates and are more frequently fed to the ani-mals (usually lactating cows or beef lot steers) athigh intake levels, more continuous levels ofsoluble sugars are available for the growth of S.ruminantium. Thus, increased S. ruminantiumnumbers may result, in part because the orga-nism is not significantly subjected to nutrientstarvation which would lead to rapid death asour studies have shown. On the other hand,high-forage diets are often fed to animals lessfrequently and contain less soluble carbohy-drates. With these latter diets, S. ruminantium isless numerous, probably because of several fac-tors, including increased viability losses due tonutrient starvation resulting from lack of solublesugars in the rumen fluid. Although potentialnutrients would be associated with the particu-late plant matter with high-forage diets, S. ru-minantium apparently does not attach to thesematerials and does not degrade either celluloseor xylans. However, regardless of the type ofdiet fed, one would expect that if dietary nitro-gen limited microbial growth, greater viable cellnumbers of S. ruminantium (and other ruminalbacteria?) would be sustained as ammonia-limit-ed cells survive better. Finally, it may be notedthat during starvation, S. ruminantium rapidlyloses viability, but apparently maintains enzymestability and catabolic potential. If a similarsituation exists for most other ruminal bacteria,then in the rumen, fermentative capacity may bemore related to total cell numbers than to actualviable cell numbers.

ACKNOWLEDGMENTS

The discussions with Marvin Bryant and Carl Davis weregreatly appreciated.

This research was supported in part by grants from theNational Science Foundation (PCM 79-10776) and from theUniversity of Illinois Agricultural Experiment Station (ILL36-349).

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