JOURNAL OF BACTERIOLOGY, May 1987, p. 2150-21570021-9193/87/052150.08$02.00/0Copyright © 1987, American Society for Microbiology

Vol. 169, No. 5

Physiological Adaptations of Anaerobic Bacteria to Low pH:Metabolic Control of Proton Motive Force in Sarcina ventriculi

STEVE GOODWINt AND J. GREGORY ZEIKUSt*Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received 3 July 1986/Accepted 5 February 1987

Detailed physiological studies were done to compare the influence of environmental pH and fermentation endproduct formation on metabolism, growth, and proton motive force in Sarcina venticuli. The kinetics of endproduct formation during glucose fermentation in unbuffered batch cultures shifted from hydrogen-acetateproduction to ethanol production as the medium pH dropped from 7.0 to 3.3. At a constant pH of 3.0, theproduction of acetate ceased when the accumulation of acetate in the medium reached 40 mmol/liter. At aconstant pH of 7.0, acetate production continued throughout the entire growth time course. The in vivohydrogenase activity was much higher in cells grown at pH 7.0 than at pH 3.0. The magnitude of the protonmotive force increased in relation to a decrease of the meditim pH from 7.5 to 3.0. When the organism wasgrown at pH 3.0, the cytoplasmic pH was 4.25 and the organism was unable to exclude acetic acid or butyricacid from the cytoplasm. Addition of acetic acid, but not hydrogen or ethanol, inhibited growth and resultedin proton motive force dissipation and the accumulation of acetic acid in the cytoplasm. The results indicatethat S. ventriculi is an acidophile that can continue to produce ethanol at low cytoplasmic pH values. Both theability to shift to ethanol production and the ability to continue to ferment glucose while cytoplasmic pH valuesare low adapt S. ventriculi for growth at low pH.

Sarcina ventriculi is an obligate anaerobic bacterium thatis capable of growth on sugars over a wide pH range, from2.0 to 10.0 (6). Smit (24) and Kluyver (16) demonstrated thatethanol and carbon dioxide are major end products ofglucose metabolism. Initial biochemical studies (3; J. P.Arbuthnott, T. Bauchop, and E. A. Dawes, Biochem J.76:12, 1960) demonstrated that S. ventriculi is capable ofboth ethanol production and hydrogen production. Fermen-tation studies (5) later showed that ethanol, acetate, andhydrogen are important end products. Stephenson andDawes (25) demonstrated that S. ventriculi possesses both aferredoxin-dependent "clostridiumlike" phosphoroclasticmechanism and a thiamine-dependent "yeastlike" decarbox-ylation mechanism for pyruvate cleavage.

Analysis of glucose fermentation end product balancessuggested that at a low pH value (i.e., 4.7), the two pathwaysof pyruvate metabolism are of equal importance (5, 25). Atneutral pH values, the phosphoroclastic pathway dominates,resulting in an increased proportion of acetate and hydrogenproduction. The glucose fermentation end product balancesat pH values below 4.7 have not been detailed. The distri-bution of end products for arabinose-grown cells is similar tothat for glucose grown cells, and end products other thanethanol have been suggested to limit growth (4, 7).

Little to nothing is known about how S. ventriculi adaptsto low pH or about the influence of the pH on the protonmotive force (PMF) within the organism. Stephenson andDawes (25) suggested that the accumulation of acetate at lowmedium pH values may lead to the concentration of aceticacid internally and cause inhibition of the phosphoroclastic

* Corresponding author.t Present address: Department of Microbiology, University of

Massachusetts, Amherst, MA 01003.t Present address: Michigan Biotechnology Institute and Depart-

ment of Biochemistry and Molecular Biology, Michigen State Uni-versity, East Lansing, MI 48824.

pathway. However, the distribution of acetate between themedium and the cytoplasm has not been reported. Otherauthors have suggested that the accumulation of acetate atlow pH may limit growth in other organisms by causingacetate to accumulate in the cytoplasm (23, 28). Baronofskyet al. (1) have shown that the accumulation of acetate at lowpH by Clostridium thermoaceticum leads directly to a dissi-pation of the PMF and a lowering of the cytoplasmic pH.

S. ventriculi represents one of the few described anaerobicbacterial species that is able to grow at extremely low pHvalues. We recently documented that the niche for thisspecies includes the pH 4.9 sediments of Crystal Bog, Wis.(8). The anaerobic digestion processes in this bog, whileslow, appear optimized to the in situ pH; artificial loweringof the pH below 4.9 caused inhibition of hydrogen metabo-lism and the accumulation of ethanol.The purpose of the present paper is twofold. First, we

wanted to determine the effects of low pH on the metabolismand PMF of S. ventriculi. Second, we wanted to test thehypothesis that the mechanism by which S. ventriculi adaptsto low pH differs dramatically from that described foracidophilic aerobes because this acidophilic anaerobe lackssufficient chemical energy for pumping protons out of thecytoplasm in the presence of weak organic acids and underconditions of low pH.

MATERIALS AND METHODS

Chemicals, gases, and isotopes. All chemicals used werereagent grade or better and were obtained from SigmaChemical Co., St. Louis, Mo., or Mallinckrodt, Inc., Paris,Ky. All gasses used were at least 99.9% pure and werepassed over copper-filled Vycor furnaces (Sargent WelchScientific Co., Skokie, Ill.) to remove oxygen. Valinomycin,gramicidin S, nigericin, and N,N'-dicyclohexylcarbodiimidewere purchased from Sigma. [3H]tetraphenylphosphoniumbromide (diluted to 250 ,uCi/2.5 ml of distilled H20; 24

2150

on January 7, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ADAPTATIONS OF SARCINA VENTRICULI TO LOW pH 2

Ci/mmol) and ["4C]sorbitol (50 ,uCi/2.5 ml of distilled H20;304 mCi/mmol) were purchased from Amersham Corp.,Arlington Heights, Ill. ['4C]salicylate (250 ,uCi/2.5 ml; 56.5mCi/mmol) was purchased from ICN Pharmaceuticals Inc.,Irvine, Calif. Radiolabeled weak organic acids ['4C]acetate(54 mCi/mmol), [14C]propionate (6.2 mCi/mmol), and[14C]butyrate (1.8 mCi/mmol) were purchased from NewEngland Nuclear Corp., Boston, Mass.Organism and culture conditions. S. ventriculi JK was

isolated and characterized previously (8). It was routinelycultured in 26-ml anaerobic pressure tubes that contained 10ml of glucose complex medium and a nitrogen headspace.The glucose complex medium contained 30 g of glucose perliter, 5 g of peptone per liter, 5 g of yeast extract per liter,and 0.1 ml of a titanium-nitrotriacetic acid solution (20). The2-liter controlled pH culture vessel contained 1 liter ofglucose complex medium, and the pH was controlled by theaddition of 4 N NaOH. The fermentation system consisted ofa multigen fermentor (BioFlo model C30), an automatic pHcontroller (model pH-40; New Brunswick Scientific Co.,Inc., Edison, N.J.), and a glass electrode (Ingold Electron-ics, Inc., Andover, Mass.). Cultures were routinely grown at370C.Measurement of growth and end products. Cell dry weight

was determined by filtration of a known volume of samplethrough a porosity membrane (pore size, 0.22 ,um; MilliporeCorp., Bedford, Mass.) and dried at 600C to constant weight.For the determination of protein, 1 ml of culture wascentrifuged at 3,000 x g for 5 min, the supernatant wasremoved, and the cell pellet was suspended in 0.5 ml of 0.1N NaOH. Cells were digested for 20 min at 100°C. Thesolution was neutralized, and protein was determined by themethod of Bradford (3). Glucose was determined by thehexokinase method (Sigma diagnostic kit). The compositionof cells varied with growth pH and cell-protein-to-dry-weightratio was not constant at different pH values. Cells grown atpH 7.0 were less compact and floated compared with cellsgrown at pH 3.0. Therefore, protein was used to estimategrowth kinetics, whereas cell yields were determined by dryweights and required larger samples.Carbon dioxide was determined by using a model 417 gas

chromatograph (Packard Instrument Co., Inc., DownersGrove, Ill.) equipped with a Carbosieve-B column (120/140mesh) (Supelco Inc., Bellefonte, Pa.) and a thermal conduc-tivity detector. The column was operated at 95°C withhelium as the carrier gas at a flow rate of 60 cm3/min. Totalcarbon dioxide was calculated by using Henry's Law and thein situ pH. The hydrogen concentration was determined byinjecting a 0.4-ml gas sample into a model 750 GowMac gaschromatograph (GowMac Instrument Co., Bridgewater,N.J.) equipped with a Spherocarb column (GowMac) andflame ionization detection. Nitrogen was used as a carriergas at 25 cm3/min, and the chromatograph was operated at1500C.Ethanol and acetate were determined by flame ionization

gas chromatography. Samples (1 ml) of culture were centri-fuged at 3,000 x g for 5 min. Supernatant (250 RI) was placedinto 0.5-ml chilled centrifuge tubes, acidified with 25 ,lI of 2N H3PO4, and centrifuged at 3,000 x g for 5 min. Acetatewas quantified by injecting 2 ,ul of the supernatant into amodel 419 Packard gas chromatograph equipped with aChromosorb 101 (80/100 mesh) column (Supelco). The col-umn was operated at 200°C, and the detector and injectortemperatures were 240 and 220°C, respectively. Ethanol wasmeasured by injecting 2 pI of the supernatant into a model419 Packard gas chromatograph equipped with a Super Q

(80/100 mesh) column (Alltech Associates, Inc., Deerfield,Ill.). The column was operated at 180°C, while detector andinjector temperatures were both 200°C. The carrier gas washelium at a flow rate of 100 cm3/min.

Determination of PMF. Determination of the proton gradi-ent (ApH) and the membrane potential (A*4) was based on thedistribution of both the weak acid ['4C]salicylic acid (14) andthe lipophilic cation [3H]tetraphenylphosphonium bromide(15) into culture suspensions of cells growing on glucosecomplex medium. These procedures are reviewed byMaloney et al. (18). Corrections for label binding were basedon the extent of label accumulation in control cells treatedwith 5% butanol for 60 min (15, 17).

Cell and supernatant fractions from 1-ml culture samplestreated with radiotracers were rapidly separated by centrif-ugation through 0.3 ml of silicone oil (55:45 mixture of Hysol550 and Hysol 556; Hysol Co., Olean, N.Y.) in 1.5-mlcentrifuge tubes at 3,000 x g for 5 min. Samples werecentrifuged aerobically immediately after being removedfrom the anaerobic pressure tubes. Radioactivity in cellpellets and supernatant samples was determined after incu-bation in Instagel scintillation cocktail (Packard) at 5°C for24 h. Radioactivity was determined on a TriCarb 4530(Packard) liquid scintillation counter programmed with a3H-14C dual-label quench curve. All analyses were per-formed in triplicate, and all experiments were repeated aminimum of three times.The internal cell pellet volumes were measured as outlined

by Kashket et al. (15) from the difference of included(3H20-permeable) and excluded (14C-sorbitol-impermeable)volumes of pellets treated as described above.PMF inhibitor studies. Inhibitor studies were performed on

mid-exponential-phase cells grown on glucose complex me-dium. Cells (10-ml samples) were treated with 10 pI of a 20mM solution of inhibitor in anaerobic ethanol. This resultedin final inhibitor concentrations of approximately 20 pM.Cells were then incubated at 37°C for an additional 30 minbefore the PMF was determined. Three samples (1 ml each)were removed and centrifuged through silicone oil. Thesepellets were stored for the determination of protein.[3H]tetraphenylphosphonium bromide and [14C]salicylate(1.5 ,uCi each) were added to the remaining medium, and thecultures were incubated for an additional 10 min. Three 1-mlsamples were centrifuged through silicone oil to separate thecells from the medium. The radioactivity of both the mediumand the cell pellet was determined as described above. Theremaining culture medium (4 ml) was aerobically mixed with5% butanol for determination of pellet-bound label. The pHof the remaining culture medium was recorded.

Influence of end products on growth and PMF. To deter-mine the influence of end products and other organic acidson growth, mid-exponential-phase cultures were treatedwith ethanol, acetate, hydrogen, or butyrate. Anaerobicsolutions were prepared at the same pH as the cell cultures,except for hydrogen, which was added as a gas at partialpressures up to 1 atm (101.29 kPa). To determine theinfluence of organic acids on PMF, anaerobic solutions ofsodium acetate and sodium butyrate were added to mid-exponential-phase cultures at the pH of the medium to finalconcentrations of 25, 50, and 100 mmol/liter. After incuba-tion for 30 min, the PMF was determined as describedabove.

In vivo hydrogenase assay. The activity of hydrogenasewas determined by using an in vivo assay (22). The methodmeasures the hydrogenase-dependent incorporation of 3H2gas into 3H20. This method has been shown to estimate the

2151VOL. 169, 1987

on January 7, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

2152 GOODWIN AND ZEIKUS

S

UUE

_zia

a.UU-III

4 8 12 16 20TIME (hours)

FIG. 1. Relationship of growth and metabolite transformationduring glucose fermentation by S. ventriculi in batch culture withoutpH control. The organism was cultured in 56-ml pressure vialscontaining 20 ml of glucose complex medium.

catalytic potential of hydrogenase in both hydrogen-producing and hydrogen-consuming microorganisms. Theincorporation of 3H2 gas was shown to be linear for 60 minand proportional to the quantity of culture added utder theconditions used in this study.

RESULTS

knd product formation and pH relationships. Experimentswere initiated to determine whether the kinetics of reducedend product formation were altered in relation to changes inmedium pH during the growth of S. ventriculi. Figure 1illustrates a representative time course of growth, substrateconsumption, and end product formation in neutral unbuf-fered rnedium. The production of acetic acid and hydrogenwas stoichiometrically coupled and caused a rapid decreasein mediunm pH. After 10 h, the medium pH reached aconstant value of 3.3, but growth (measured as proteinproduction) and glucose consumption continued for another10 h. Once the pH reached 3.3, acetate and hydrogenproduction decreased notably, but ethanol and carbon diox-ide production continued until 20 h after the start of thereaction.

Further experiments were conducted to develop a buff-ered medium that would support the growth of S. ventriculiover a wide range of pH values (Table 1). The organism wasgrown in 20 ml of glucose complex medium in 56-ml pressurevials. Additions of 100 mM potassium phosphate, sodiumcitrate, or sodium succinate were made at pH values from2.0 to 7.0. Growth was positive if more than 200 jig ofprotein per liter was produced during a 36-h incubation at376C. Phosphate, citrate, and succinate are multiprotic ionsand allowed for growth at pH values below but not above

TABLE 1. Relationship of environmental pH to growth ofS. ventriculi in buffered glucose complex medium

pH supporting growthBuffer PKa Speciesa (mM)

7 6 5 4 3 2

Phos'phate pK3 12.32 - 3P04 (0.16%7)pK2 7.21pK, 2.23

Citrate pK3 6.40pK2 4.76pK, 3.13

H2PO4- (94.27%)HP042- (5.81%)

- - - + + + H3C6H507 (10,30%)H2C6H507'- (76.37%)HC6H5072- (13.27%)

Succinate pK2 5.48 - - - - + + H2C4H404 (93.92%)pKj 4.19 HC4H404- (6.06%)

a Species are given at the highest pH supporting growth.

their second pKa. These buffers may not support growthabove their second pKa because in the -2 valence state theymay have caused the chelation of an essential component ofthe medium.The growth physiology of S. ventriculi was further inves-

tigated in pH-controlled batch cultures because there was nocombination of buffers that would allow growth at both pH3.0 and 7.0. Figures 2 and 3 compare the glucose fermenta-tion time courses at pH 3.0 and 7.0. A change in the fer-mentation pH altered the kinetics of end product formationas well as the final end product concentrations. At pH 7.0,acetate production continued until growth ceased (24 h),whereas at pH 3.0, acetate production ceased at 18 h. Thefinal ethanol concentration reached 90 mmol/liter at pH 7.0but was 150 mmollliter at pH 3.0.

'a

I2afIaa.

an

ina%CD

pIH 7

12TIME (hours)

U

aU

gU

2

Ua

FIG. 2. Glucose fermentation time course of S. ventriculi in pH7.0 controlled batch culture. The organism was cultured in 2-litervessels containing 1 liter of glucose complex medium, and the pHwas maintained by constant addition of NaOH.

J. BACTERIOL.

on January 7, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ADAPTATIONS OF SARCINA VENTRICULI TO LOW pH

g

o0

MA

a.

0

a

coaIL

pH 3

a.L" o

o eNo E_Ca -

W0a xm CD

a 44la.Vmf

E

e

aa

0

a10aes

3

TIME (hours)

FIG. 3. Glucose fermentation time course of S. ventriculi in pH3.0 controlled batch culture. The organism was cultured in 2-litervessels containing 1 liter of glucose complex medium, and the pHwas maintained by constant addition of NaOH.

Experiments were designed to determine the influence ofpH on the production of reduced end products (ethanol andhydrogen). The ratio of reduced end products to carbondioxide was used because carbon dioxide production doesnot vary for the two pathways of pyruvate metabolism.Table 2 shows the response of varying the fermentation pHfrom 7.0 to 3.0 on the ratio of reduced end products tocarbon dioxide. Notably, the amount of hydrogen produceddropped in response to a lowering of the pH, whereas theamount of ethanol produced increased. The organism wasgrown in a 2-liter fermentation vessel containing 1 liter ofglucose complex medium. The pH was maintained by addingNaOH. The cells were incubated for 36 h at 37°C.

Studies were initiated to determine the influence of me-dium pH on in vivo hydrogenase activity because hydrogenand acetate production appeared to be stoichiometrically

TABLE 2. Influence of environmental pH on fermentationproduct ratios and cell yield of S. ventriculi grown

on glucose complex mediuma

pH Cell yield (g [dry End product ratios

Pwt]/mol of Glc) H2/CO2 Ethanol/CO2

7.0 32.3 0.71 0.656.0 30.2 0.62 0.685.0 27.7 0.55 0.734.0 23.9 0.46 0.773.0 24.2 0.42 0.79a Carbon and electron recoveries ranged from 93 to 102%. Cell yields were

determined during mid-exponential growth, and end product ratios weredetermined after 36 h.

Time (minutes)FIG. 4. Comparison of in vivo hydrogenase activity in cells of S.

ventriculi grown at constant external pH 7.0 versus pH 3.0. Cellswere grown in glucose complex medium to mid-exponential phase(16 h). Samples (10 ml) were transferred by syringe into 56-mlpressure vials containing a N2 atmosphere and 3H2 gas and hydrog-enase measured by 3H20 formation (6.3 x 106 dpm represent 0.1,umol of hydrogen).

coupled and this activity was inhibited at low pH. The invivo hydrogenase activity of S. ventriculi was twofold higherat pH 7.0 than at pH 3.0 (Fig. 4). The activity was linear for60 min and was directly proportional to the amount ofenzyme present (i.e., cell protein added). Control experi-ments were performed with a hydrogenase inhibitor, carbonmonoxide, to establish the validity of the assay procedure. A100% carbon monoxide headspace completely inhibited invivo hydrogenase and hydrogen production by exponentiallygrowing cultures of S. ventriculi at pH 5.0.PMF and pH relationships. Experiments were initiated to

determine whether alteration of reduced end product forma-tion during changes in medium pH was related to alterationof cytoplasmic pH and cellular PMF. No information con-cerning the cytoplasmic pH or PMF was previously availablefor S. ventriculi, and it was not known how these parametersresponded to decreasing medium pH. Initial studies involvedthe use of Clostridium pasteurianum as a control organismbecause measurements of cytoplasmic pH as a function ofmedium pH had been previously determined for this orga-nism (21).To ensure that the PMF of S. ventriculi was properly

quantified, cells were also treated with inhibitors known toaffect one or both components of the PMF (Table 3). Bothorganisms were grown to mid-exponential phase, and 10 mlof culture was removed to assay the electrochemical param-eters by the [14C]salicylate and [3H]tetraphenylphosphonium

TABLE 3. Comparison of PMF inhibitors on the cellularelectrochemistry of C. pasteurianum and S. ventriculi

S. ventriculi C. pasteurianumInhibitora

,ApH A&q (mV) PMF (mV) ApH Aqi (mV) PMF (mV)

Noneb 1.3 46 122 0.8 136 183Nigericin 0.3 7 28 0.1 20 27Gramicidin 0.0 13 13 0.1 0 4DCCDC 1.1 25 92 0.2 0 11

a The inhibitor was added dissolved in 10 Fl of ethanol to final concentra-tions of 20 FM.

b Ethanol (10 pl) without inhibitor was used as a control.c Abbreviation: DCCD, N,N'-dicyclohexylcarbodiimide.

VOL. 169, 1987 2153

on January 7, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

2154 GOODWIN AND ZEIKUS

TABLE 4. Influence of environmental pH on growth andelectrochemical parameters of S. ventriculi in

pH-controlled batch cultures

Medium pH Growth" Internal pH ApHb Aq; (mV)- PMF (mV)d

10.0 -

9.0 +7.5 + 7.6 0.1 46 50 ± 47.0 + 7.1 0.1 63 67 ± 24.2 + 5.1 0.9 55 112 ± 0.43.0 + 4.3 1.3 38 114 ± 42.0 +1.0 -

a Positive growth indicates that the organism produced >200 ±Lg of proteinper liter.

I The ApH was determined from the distribution of [14C]salicylic acid.c The A* was determined from the distribution of [3H]tetraphenyl-

phosphonium bromide.d The values for PMF are averages of triplicate analyses + 1 standard

deviation.

methods. Nigericin, a protonophore, decreased the protongradient from 1.35 to 0.33. Gramicidin completely abolishedthe proton gradient. N,N'-dicyclohexylcarbodiimide dra-matically decreased the PMF of C. pasteurianum but hadlittle effect on the PMF of S. ventriculi. Control experimentsalso established that compounds which decreased the PMFof S. ventriculi also inhibited the growth of the organism(data not shown). In addition, the distribution of salicylicacid, acetic acid, and benzoic acid were compared and theproton gradients measured were comparable for all threeacids (data not shown). Because these three weak acids havedifferent molecular weights and structures, the proton gra-dients reported here measured with salicylic acid are furthersubstantiated.The results of PMF determinations for S. ventriculi as a

function of medium pH during growth in pH-controlledbatch cultures are presented in Table 4. The cells weregrown in 2-liter pH-controlled vessels that contained 1 literof glucose complex medium. The pH was maintained by theaddition ofNaOH. S. ventriculi grew at pH 2.0 but not at pH

go

.5

40

2 4 8 10 12 14 16 18 20 22

TIME (hours)FIG. 5. Influence of exogenous weak organic acids on growth of

S. ventriculi in glucose complex medium at pH 4.2. Cells were

grown in 28-ml pressure tubes containing 10 ml ofmedium at pH 3.1.Sodium acetate, sodium propionate, or sodium butyrate (40 mM) atthe pH of the culture was added at 6.5 h. The pH remained constantduring the course of the experiments.

TABLE 5. Distribution of weak organic acids across thecytoplasmic membrane of S. ventribuli at pH 4.2a

Concn ratios (internal/external)Acid PKa

Measuredb Predictedc

Acetic acid 4.76 4.7 4.2Butyric acid 4.82 3.9 3.9

a Cells were grown in batch culture at pH 4.2. The distribution of['4Clsalicylic acid gave a cytoplasmic pH value of 5.4.bMeasured ratios were determined from the distribution of '4C-labeled

weak organic acids.c Predicted ratios were calculated from the measured cytoplasmic pH and

the appropriate pKa.

10.0 when the medium pH was held constant. Notably, thePMF increased from 50 to 114 mV as the medium pH wasdecreased from 7.5 to 3.0. As the medium pH was decreasedfrom 7.5 to 3.0, the measured cytoplasmic pH decreased to4.25. The proton gradient (ApH) increased greatly at lowmedium pH values. This increase in the proton gradient wasonly partially compensated for by a small decrease in themembrane potential (Ad). The cytoplasmic pH of culturesgrown at pH 2.0 could not be determined because the pKa ofthe probe was too high.End product and PMF relationships. Studies were initiated

to determine whether physiological concentrations of endproducts inhibited growth and altered cellular electrochem-istry of S. ventriculi cultured at a constant pH of 4.2. Growthand PMF of S. ventriculi were not significantly altered by theaddition of 1 atm of hydrogen or carbon dioxide or by theaddition of 100 mmol of ethanol per liter to cultures (data notshown). Exogenous additions of ethanol were not seen toaffect growth until concentrations of 180 mmol/liter werereached.On the other hand, addition of exogenous organic acids

dramatically altered growth and cellular electrochemistry.The influence of acetic acid, butyric acid, and propionic acidto cells growing at pH 4.2 is shown in Fig. 5. These acids, at40 mmol/liter, did not change the medium pH but totallyinhibited growth. The addition of acetate, but not butyrate orpropionate, in concentrations as low as 10 mmol/liter prior toinoculation of cultures at pH 3.0 also completely inhibitedgrowth (data not shown).

Experiments were performed to determine the distributionof these acids across the cytoplasmic membrane. The protongradient was determined from the distribution of ['4C]salicyl-ate and used to predict the distribution of acetic acid andbutyric acid. These predictions were compared with themeasured distribution of the appropriate radiolabeled acid.The experiments (cells grown at pH 4.2) demonstrated thatthese three weak acids equilibrated with the proton gradientrepresented by a cytoplasmic pH of 5.4 (Table 5). S.ventriculi was not capable of excluding these acids from the

TABLE 6. Influence of exogenous acetate addition on cellularelectrochemistry of S. ventriculi grown at pH 4.2

in glucose complex medium

Addition (mM) ApH A1i (mV) PMF (mV)

NaCl controls (25 to 1.1 18 83100)

Acetate25 0.9 26 8050 0.5 16 45100 0.2 30 43

J. BACTERIOL.

on January 7, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ADAPTATIONS OF SARCINA VENTRICULI TO LOW pH

pH 3.0glucose

(0.51 EV 29.49 mE)

A pH = 1.3AI = 38 (NV)

Ys = 24.2 (g dry wV ml gic)

(29.83 mM/ 0.17 mE)CH3COOHA p = L.1AO =07(NV)

Ts = 32.3 (g dry wt/ mel Sic)FIG. 6. General model of metabolic control of PMF in S. ventriculi at pH 3.0 versus pH 7.0.

cytoplasm at low pH. The total concentration of acetic acidwithin the cytoplasm was fivefold higher than in the mediumwhen the cells were grown to mid-exponential phase at pH4.2.To determine whether the protonated form of organic

acids resulted in the dissipation of the proton gradient,experiments were designed to measure the influence oforganic acid addition on the PMF (Table 6). Cells weregrown to mid-exponential phase at pH 4.2 in glucose com-plex medium at 37°C. Culture samples were removed, andelectrochemical parameters were determined by radiotracertechniques. Cells were incubated in the presence of sodiumacetate for 30 min at 37°C and pH 4.2. Addition of acetic acidresulted in the dissipation of the proton gradient within 30min. The decrease in the proton gradient was directlyproportional to the acetic acid concentration.

DISCUSSIONIn general, these data indicate that the broad pH range of

growth of S. ventriculi (pH 9.0 to 2.0) is a direct consequenceof physiological adaptations to large changes in cytoplasmicpH values (>7.6 to <4.3). The mechanism for physiologicaladaptation of S. ventriculi to low pH does not involve themaintenance of the extremely large proton gradients ob-served in aerobic species. S. ventriculi metabolically con-trols its PMF in relation to internal pH values by alteringpyruvate metabolism from hydrogen-acetate production atnear-neutral pH to ethanol production at low pH. Thisprevents acetic acid from dissipating the PMF.

Figure 6 provides a generalized model that summarizesand predicts the major metabolic controls that adapt S.ventriculi for growth under constant conditions of 60 mmolof glucose per liter, 30 mmol of acetate per liter, and a pH of7.0 versus 3.0. The sensitivity of the measured PMF toknown inhibitors confirms the validity of the electrochemis-try measurements. The validity of the measured cytoplasmicpH is further confirmed by the comparable distribution of

several weak-acid probes of differing molecular weights andstructures. The decrease of cytoplasmic pH as a function ofdecreasing medium pH observed for S. ventriculi is in linewith the results observed for several other anaerobic bacte-ria, such as C. pasteurianum (21), Streptococcus cremoris(26), Streptococcus lactis (12, 15), Clostridium acetobutyl-icum (11), and C. thermoaceticum (1).The cellular electrochemistry of S. ventriculi is strongly

influenced by the medium pH at which growth occurs. Theexperiments demonstrate that the PMF increases as a func-tion of decreasing medium pH, and this supports theacidophilicity of the organism. This phenomenon is mostnotable in the proton gradient. Interestingly, very littlechange in the membrane potential was observed over a widerange of medium pH values. The membrane potential wasalways negative internally, and there were no conditionsunder which the membrane potential inverted. S. ventriculiis clearly an acidophile, as a consequence of prolific growthat pH 2.0 to 3.0; however, it is not an obligate acidophile.

This response to the variation in pH is in sharp contrast tothe response of aerobic acidophiles. When grown at pH 2.0,Thermoplasma acidophila maintained an internal pH be-tween 6.4 and 6.9 (9). This leads to a large proton gradient,which is compensated for by an inverted membrane potentialof 120 mV (internal positive) (10). The internal pH ofThiobacillus ferro-oxidans remained between 6.8 and 6.2 asthe external pH was varied between 1.0 and 8.0 (6). Similarfindings were obtained for Thiobacillus acidophilus (19, 29).Because anaerobes derive substantially smaller amounts

of energy from a given substrate than aerobes do (13, 27),anaerobes may have adapted to low pH by mechanisms thatdo not involve energy-dependent proton pumps. This hypo-thesis is supported by the present data but remains to befully tested. The PMF of Staphylococcus aureus has beenshown to be approximately 110 mV lower when the organismis growing anaerobically than when it is growing aerobically(12).During growth at low pH, S. ventriculi is not capable of

VOL. 169, 1987 2155

on January 7, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

2156 GOODWIN AND ZEIKUS

excluding weak organic acids produced by fermentation.The physiological mechanisms for adapting to growth at lowpH are therefore related to the ability of the organism tometabolically control its PMF as a function of internal pH byaltering the activity of carbon and electron flow pathwaysfrom acetate-hydrogen to ethanol production. This alterationof carbon and electron flow involves both the external pHand the external accumulation of acetic acid. The ratio ofethanol to acetic acid produced increases with decreasingmedium pH. It should be noted that this ratio changes duringgrowth even at constant pH, so that at pH 3.0, no acetate isproduced during the later stages of growth, indicating com-plete dominance of the thiamine decarboxylase pathway.The results indicate that the shift in fermentation pathwaymust be related to the lowering of cytoplasmic pH and theconcentration of acetic acid internally.That both the accumulation of acetic acid in the cytoplasm

and the lowering of cytoplasmic pH are involved in themetabolic control of fermentation is supported by the exper-imental results. The fact that nonmetabolite weak organicacids can inhibit growth suggests that part of the response ofthe PMF to the accumulation of organic acid is the transloca-tion of protons across the cytoplasmic membrane by theprotonated form of the acid and the dissipation of the protongradient. The fact that acetic acid is an effective inhibitor ofthe PMF suggests that the accumulation of this metabolitewithin the cytoplasm also contributes to the observed re-sponse of the PMF to the production of weak organic acidsduring fermentation.The ability of S. ventriculi to metabolize glucose at cyto-

plasmic pH values at least as low as 4.3 raises severalinteresting questions concerning the enzymes of this organ-ism. Stephenson and Dawes (25) determined the influence ofpH on pyruvate decarboxylation and hydrogen production incrude cell extracts. In these experiments, the optimum pHfor the evolution of hydrogen from pyruvate was 6.8, and nohydrogen was evolved below pH 5.0. The optimum activityof the decarboxylation was between pH 5.0 and 6.0, and noactivity was observed below pH 4.5. Although the in vivohydrogenase activity measured was indeed lower at pH 3.0than at pH 7.0, activity was still quantifiable at pH 3.0 (witha measured cytoplasmic pH of 4.3). These findings questionthe assays used for in vitro determinations of pH optima andactivity of specific enzymes. The possibility exists that thepH optimum of enzymes in colloidal suspensions character-istic of the cytoplasm varies from the pH optimum reportedfor in vitro assays of crude enzyme preparations withnonphysiological buffers whose dynamic range may causechelation of metal cofactors (e.g., nickel or iron) needed formeasurement of hydrogenase, pyruvate dehydrogenase, orother enzyme activities. Localized pH variation within thecytoplasm may also explain the discrepancy between in vitroand in vivo enzyme activities.

ACKNOWLEDGMENTS

We thank Eva Kashket for helpful advice concerning the mea-surement of PMF and Timothy Paustian for excellent technicalsupport.

This research was supported by Department of Energy grantDE-FG-02-85ER13376. S.G. was supported by a Public HealthService Cellular and Molecular Biology training grant from theNational Institutes of Health.

LITERATURE CITED1. Baronofsky, J. J., W. J. A. Scheurs, and E. R. Kashket. 1984.

Uncoupling by acetic acid limits growth of and acetogenesis by

Clostridium thermoaceticum. Appl. Environ. Microbiol.48:1134-1139.

2. Bauchop, T., and E. A. Dawes. 1959. Metabolism of pyruvic acidand formic acid by Zymosarcina ventriculi. Biochim. Biophys.Acta 36:294-296.

3. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

4. Bringer, S., G. Durst, H. Sahm, and R. K. Finn. 1984. Studies ongrowth and product formation of an anarabinose-utilizing strainof Sarcina ventriculi. Biotechnol. Bioeng. Symp. 14:269-278.

5. Canale-Parola, E. 1970. Biology of sugar-fermenting sarcinae.Bacteriol. Rev. 34:82-97.

6. Cox, J. C., D. G. Nicholls, and W. J. Ingledew. 1979.Transmembrane electrical potential and transmembrane pHgradient in the acidophile Thiobacillus ferro-oxidans. J. Bacte-riol. 153:371-374.

7. Finn, R. K., S. Bringer, and H. Sahm. 1984. Fermentation ofarabinose to ethanol by Sarcina ventriculi. Appl. Environ.Microbiol. 19:156-166.

8. Goodwin, S., and J. G. Zeikus. 1987. Ecophysiological adapta-tions of anaerobic bacteria to low pH: analysis of anaerobicdigestion in acidic bog sediments. Appl. Environ. Microbiol.53:57-64.

9. Hsung, J. C., and A. Haug. 1975. Intracellular pH ofThermoplasma acidophila. Biochim. Biophys. Acta 389:477-482.

10. Hsung, J. C., and A. Haug. 1977. Membrane potential ofThermoplasma acidophila. FEBS Lett. 73:47-50.

11. Huang, L., L. N. Gibbins, and C. W. Forsberg. 1985.Transmembrane pH gradient and membrane potential in Clos-tridium acetobutylicum during growth under acetogenic andsolventogenic conditions. Appl. Environ. Microbiol. 50:1043-1047.

12. Kashket, E. R. 1981. Proton motive force in growing Strepto-coccus lactis and Staphylococcus aureus cells under aerobicand anaerobic conditions. J. Bacteriol. 146:369-376.

13. Kashket, E. R. 1981. Effects of aerobiosis and nitrogen sourceon the proton motive force of growing Escherichia coli andKlebsiella pneumoniae. J. Bacteriol. 146:377-384.

14. Kashket, E. R., and S. L. Barker. 1977. Effects of potassiumions on the electrical gradients across the membrane of Strep-tococcus lactis. J. Bacteriol. 130:1017-1023.

15. Kashket, E. R., A. G. Blanchard, and W. C. Metzger. 1980.Proton motive force during the growth of Streptococcus lactiscells. J. Bacteriol. 143:128-134.

16. Kluyver, A. J. 1931. The chemical activities of microorganisms,p. 38. University of London Press, London.

17. Lolkema, J. S., K. J. Hellingwerf, and W. N. Konings. 1982. Theeffect of 'probe binding' on the quantitative determination ofproton motive force in bacteria. Biochim. Biophys. Acta681:85-96.

18. Maloney, P. C., E. R. Kashket, and T. H. Wilson. 1975. Methodsfor studying transport in bacteria. Methods Membr. Biol.5:1-49.

19. Matin, A., B. Wilson, E. Zychlinsky, and M. Matin. 1982. Theproton motive force and the physiological basis of pH mainte-nance in Thiobacillus acidophilus. J. Bacteriol. 150:582-591.

20. Moench, T. T., and J. G. Zeikus. 1983. An improved preparationmethod for a titanium (III) media reductant. J. Microbiol.Methods 1:199-202.

21. Riebling, R., R. Thauer, and K. Jungermann. 1975. The internal-alkaline pH gradient, sensitive to uncoupler and ATPase inhib-itor, in growing Clostridium pasteurianum. J. Biochem.55:445-453.

22. Schink B., F. S. Lupton, and J. G. Zeikus. 1983. Radioassay forhydrogenase activity in viable cells and documentation of aer-obic hydrogen-consuming bacteria living in extreme environ-ments. Appl. Environ. Microbiol. 45:1491-1500.

23. Schwartz, R. D., and F. A. Keller. 1982. Acetic acid productionby Clostridium thermoaceticum in pH-controlled batch fermen-tations at acidic pH. Appl. Environ. Microbiol. 43:1385-1392.

24. Smit, J. 1930. Die Garungssarcine. Monographie Pflanzen-

J. BACTERIOL.

on January 7, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ADAPTATIONS OF SARCINA VENTJICULI TO LOW pH

forschung Heft, vol. 14. Gustav Fischer, Jena, German Demo-cratic Republic.

25. Stephenson' M. P., and E. A. Dawes. 1971. Pyruvic acid andformic acid metabolism in Sarcina ventriculi and the role offerredoxin. J. Gen. Microbiol. 69:331-343.

26. ten Brink, B., and W. N. Konings. 1982. Electrochemical protongradient and lactate concentration gradient in Streptococcuscremoris cells gr6wn in batch culture. J. Bacteriol. 152:682-686.

27. Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy

cohservation in chemotrophic anaerobic bacteria. Bacteriol.Rev. 41:100-180.

28. Wang, G., and D. I. C. Wang. 1984. lu6cidation of growthinhibition and acetic acid ptoduction by Closvridium thermoace-ticum. Appl. Environ. Microbiol. 47:294-298.

29. Zychlinsky, E., and A. Matin. 1983. Effect of starvation oncytoplasmic pH, proton motive force, and viability of anacidophilic bacterium Thiobacillus acidophilus. J. Bacteriol.153:371-374.

VOL. 169, 1987 2157

on January 7, 2020 by guesthttp://jb.asm

.org/D

ownloaded from