JOURNAL OF BACTERIOLOGY, Apr. 1979, p. 109-1170021-9193/79/04-0109/09$02.00/0

Vol. 138, No. 1

Change from Homo- to Heterolactic Fermentation byStreptococcus lactis Resulting from Glucose Limitation in

Anaerobic Chemostat CulturesTERENCE D. THOMAS,t* DEREK C. ELLWOOD, AND V. MICHAEL C. LONGYEAR

Microbiolokical Research Establishment, Porton Down, Salisbury, England

Received for publication 20 November 1978

Lactic streptococci, classically regarded as homolactic fermenters of glucoseand lactose, became heterolactic when grown with limiting carbohydrate concen-trations in a chemostat. At high dilution rates (D) with excess glucose present,about 95% of the fermented sugar was converted to L-lactate. However, as D waslowered and glucose became limiting, five of the six strains tested changed to aheterolactic fermentation such that at D = 0.1 h-1 as little as 1% of the glucosewas converted to L-lactate. The products formed after this phenotypic change infermentation pattern were formate, acetate, and ethanol. The level of lactatedehydrogenase, which is dependent upon ketohexose diphosphate for activity,decreased as fermentation became heterolactic with Streptococcus lactis ML3.Transfer of heterolactic cells from the chemostat to buffer containing glucoseresulted in the nongrowing cells converting nearly 80% of the glucose to L-lactate,indicating that fine control of enzyme activity is an important factor in thefermentation change. These nongrowing cells metabolizing glucose had elevated(ca. twofold) intracellular fructose 1,6-diphosphate concentrations ([FDP]m.) com-pared with those in the glucose-limited heterolactic cells in the chemostat.[FDP]ui was monitored during the change in fermentation pattern observed inthe chemostat when glucose became limiting. Cells converting 95 and 1% of theglucose to L-lactate contained 25 and 10 mM [FDP]in, respectively. It is suggestedthat factors involved in the change to heterolactic fermentation include both[FDP]in and the level of lactate dehydrogenase.

Group N streptococci (Streptococcus cre-moris, S. lactis, and S. diacetylactis) play a vitalrole in many commercial milk fermentations, inwhich their primary function is to convert lac-tose to lactic acid (18). Lactic streptococci areuseful because they possess limited metabolicdiversity and usually convert about 95% of thefermented sugar to L-lactate (23, 26). This homo-lactic fermentation of either lactose or glucoseoccurs in batch culture when organisms aregrown anaerobically near pH 7 at 30°C. In con-trast, heterolactic fermentation was observedduring growth on galactose (26) and duringgrowth on lactose of variants defective in eitherlactate dehydrogenase (LDH; 21) or the lactosephosphotransferase system and/or phospho-fB-D-galactosidase (7, 26), suggesting that theseorganisms have pathways which are not nor-mally expressed. The alternative products re-ported include acetate, acetoin, C02, ethanol,formate, and glycerol. Unsuccessful attempts

t Present address: New Zealand Dairy Research Institute,Palmerston North, New Zealand.

have been made with lactic streptococci to divertglucose fermentation products away from lactateby using procedures which cause other "homo-fermentative" lactic acid bacteria to become het-erolactic. These procedures included growthwithout pH control (23) and aerobic growth onlow concentrations of glucose (3).Uptake of lactose and glucose by lactic strep-

tococci involves phosphoenolpyruvate-depend-ent phosphotransferase systems, and glucose 6-phosphate is then metabolized to pyruvate viathe glycolytic pathway. There is no evidence forother pathways of glucose metabolism havingquantitative significance in lactic streptococci(for review, see 18). The galactose 6-phosphatemoiety from the lactose molecule is metabolizedvia the D-tagatose 6-phosphate pathway (2) totriose phosphate intermediates. Pyruvate reduc-tion to lactate in these streptococci involves anLDH whose activity in vitro is markedly de-pendent upon either fructose 1,6-diphosphate(FDP; 6, 17, 25) or tagatose 1,6-diphosphate (25).It has been suggested (7, 8, 26, 29) that theintracellular FDP concentration ([FDP]in) may

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110 THOMAS, ELLWOOD, AND LONGYEAR

regulate LDH activity in vivo and, hence, thebalance of fermentation products, but there isno experimental evidence for this with the lacticstreptococci.The purpose of this investigation was to ex-

amine regulation of end-product formation byusing intact cells since, as Sanwal (24) haspointed out, proof is rare that mechanisms stud-ied in vitro are actually the means of physiolog-ical regulation in vivo. Attempts were made tovary the intracellular concentration of LDH ac-tivator by using glucose-limited chemostat cul-tures and to examine the effect on end-productformation. Glucose was chosen as the growthsugar to simplify measurement of the intracel-lular concentration ofLDH activator, and it wasassumed that FDP was the only activator pres-ent.

MATERIALS AND METHODSOrganisms. S. lactis strains ML3, MIb, and H1 and

S. cremoris strains 158, 266, and AM2 were from thecollection held at the New Zealand Dairy ResearchInstitute.Growth media. S. lactis strains were grown in a

chemically defined medium containing, per liter: (i)glucose, 5 g, unless otherwise specified; (ii) aminoacids-cystine, glycine, histidine, isoleucine, leucine,methionine, phenylalanine, proline, threonine, tryp-tophan, tyrosine, and valine, 0.1 g each (L isomer);lysine and serine, 0.2 g each (L isomer); DL-alanine, L-asparagine, DL-aspartic acid, DL-glutamic acid, and L-glutamine, 0.3 g each; and L-arginine, lg; (iii) bases-adenine, 5 mg; guanine, 1 mg; uracil, 5 mg; and xan-thine, 5 mg; (iv) vitamins-p-aminobenzoic acid, 0.2mg; biotin, 0.1 mg; folic acid, 0.1 mg; nicotinic acid, 1mg; pantothenic acid, 1 mg; pyridoxal hydrochloride,0.2 mg; pyridoxine hydrochloride, 1 mg; riboflavin, 0.1mg; and thiamine hydrochloride, 0.1 mg; (v) othercompounds-Tween 80, 0.2 g; disodium EDTA, 4 mg;FeSO4. 7H20, 1 mg; MnCI2*4H20, 1 mg; MgSO4.7H20, 2.5 g; Na2HPO4, 2.5 g; and KH2PO4, 1 g. Thismedium was sterilized by passage through membranefilters (0.22-pm pore size; Millipore Corp.).

S. cremoris strains were grown in a complex me-dium containing, per liter; tryptone, 4 g; proteosepeptone (no. 3), 2 g; beef extract, 2 g; yeast extract, 2g (all products of Difco Laboratories); glucose, 5 g;MgCl2.6H20, 0.1 g; and K2HPO4, 3.5 g. This mediumwas autoclaved in two parts, and the glucose part wasthen added aseptically to the bulk medium.Growth conditions. Cultures were grown in Por-

ton-type chemostats (16) of 500-ml working capacity.The temperature was maintained at 30°C, and the pHwas controlled at 6.5 ± 0.1 by automatic addition ofNaOH. Anaerobic conditions were maintained bypassing a 95% N2-5% C02 mixture through the stirredculture (9). Medium was pumped into the fermentorat the appropriate dilution rate (D, proportion ofculture volume replaced per hour), and the culturewas allowed to reach equilibrium (i.e., grown for atleast 10 mean generation times at each D) before beingsampled.

J. BACTERIOL.

Culture purity was checked daily by microscopicexamination and by streaking on nutrient agar plateswhich were incubated at 30 and 37°C both aerobicallyand anaerobically in an N2-CO2 (95:5) atmosphere. Inaddition, cultures were tested periodically for lysis bytheir homologous phages and for agglutination withgroup N antisera (Wellcome Research Laboratories,Beckenham, England).Sampling procedures. (i) Residual glucose

and metabolic products. Because of the high gly-colytic rate of growing lactic streptococci (ca. 0.4 ,umolof glucose utilized per mg [dry weight] of bacteria permin), it is important that cells are rapidly removedfrom chemostat culture samples required for residualglucose and end-product analyses. Therefore, culturesamples (ca. 10 ml) were cooled rapidly to about 10°Cand passed through Millipore membranes (0.8-pum poresize) such that the total time between the sampleleaving the chemostat and the end of ifitration was<15s.

(ii) Preparation of cell extracts. From the gly-colytic rate it can be calculated that, at an [FDP]i. of25 to 30 mM, the cellular content of FDP could bemetabolized by celLs, held in the absence of exogenousglucose, within a few seconds. Therefore, an extremelyrapid sampling and extraction procedure was required,which avoided the delays involved in methods usingcell separation by filtration or centrifugation (22).Perchloric acid (0.3 ml, 9.2 N) was placed in a plasticsyringe (5-ml capacity), the barrel was inserted, andair was displaced from the syringe. A sterile needle (11cm long, 1.65-mm ID; with a tap) was fitted, the tapwas closed, and the syringe barrel was drawn back andfixed at the 5-ml mark, leaving a vacuum in the sy-ringe. The needle was inserted through a septum intothe chemostat culture. When the tap was opened,culture was sucked into the syringe where turbulentmixing with the HCl04 occurred. The average timebetween cells leaving the fermentor and mixing withHCl04 was ca. 30 ms, and the final HCl04 concentra-tion was 0.6 N. Samples were held for 2 min at roomtemperature and then in an ice bath for 10 min beforeaddition of 0.23 g of K2CO3. Neutralized (pH 7.0 to 7.5)samples were centrifuged and supematants werestored at -90°C until assayed. With these procedures,glycolytic intermediates were stable and extractionwas maximized.To examine the effect of sampling time on [FDP],,

(see Table 4), 10-ml culture samples were membranefiltered (0.8-pm pore size, 47-mm diameter; MilliporeCorp.), and the filters plus retained cells were placedin 2 ml of 0.6 N HCl04 at 0°C. The time taken forthese manipulations was progressively increased sothat the interval between cells leaving the fermentorand suspension in HCl04 varied from 20 to 60 s.Assay of residual glucose and metabolic prod-

ucts. (i) Glucose. Glucose ws determined with Glu-costat reagents (Worthington Biochemicals).

(ii) Lactate. L-Lactate was measured enzymati-cally (Lactate UV Test, Boehringer Mannheim Corp.),and total lactate was determined colorimetrically (1).

(iii) Volatile products. Culture filtrates were an-alyzed for volatile products with a gas chromatograph(Pye Unicam series 104) and glass columns (1.5 m by4 mm ID) packed with (a) 10% polyethylene glycol

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CHANGE IN END PRODUCTS OF S. LACTIS 111

adipate on Phasesep CL Aw 80-100 mesh and (b)Poropak Q. After sample injection, column (a) washeld at 100°C for 4 min and then programmed at 3°C/min to 200°C and held for 15 min. Column (b) washeld at 150°C for 6 min and then programmed at12°C/min to 195°C. The carrier gas was 02-free N2flowing at 40 ml/min. Detection was by flame ioniza-tion for column (a) and by Katharometer for column(b). Quantitation was carried out by a reporting inte-grator (Hewlett-Packard HP 3380A) programmed be-forehand with authentic standards. Samples (0.25 ml)for acetate and ethanol analysis were acidified (6 NH2SO4, 20 pl) just before injection into column (a). Forformate analysis, filtrate samples (1 ml) were madealkaline (ca. pH 8.0) with NaOH, dried, and dissolvedin 0.5 N H2SO4 (0.1 ml), and samples (1 to 5 uIl) wereinjected into column (b).

(iv) Nonvolatile acids. Culture filtrates (0.5 ml)were dried in vacuo before heating in 2 N methanolichydrochloric acid (1 ml) in a sealed tube at 65°C for 2h. The methyl esters were removed by ether extraction(three 3-ml extractions) and dissolved in dichloro-methane after removal of the ether in a stream of N2.Portions (1 to 5 !d) were then applied to column (a).

Uninoculated media were included as blanks for allend-product analyses, and the volume increase due tothe addition of alkali for pH control in chemostatcultures was corrected for. The detection limits forglucose, formate, acetate, and ethanol were 5, 75, 50,and 50,g/ml, respectively. In a search for other endproducts, column temperatures were varied from thosegiven above.Assay of glycolytic intermediates in cell ex-

tracts. The need for extremely rapid sampling andextraction of cells, without separation from the growthmedium, has three important consequences for theassay of glycolytic intermediates: (i) the concentrationof these compounds in extracts is low, such that assayrequires enzymatic analysis by fluorimetry; (ii) culturefiltrates must be assayed to check for release of inter-mediates from cells, and the appropriate subtractionsfrom culture extract samples must be made; (iii) anygrowth medium constituents which fluoresce underthe assay conditions will give high backgrounds. Al-though the defined medium used in this study wassatisfactory in this regard, the relatively high fluores-cence of the complex broth precluded assay ofFDP inextracts from cultures growing in this medium.

Intermediates were assayed with a Perkin-Elmerfluorescence spectrophotometer (model MPF-2A) andNADH-coupled indicator systems as previously de-scribed (5, 28). Corrections were made for the differ-ence in fluorescence between NADH in solution andenzymatically bound NADH. Values for [FDP]i,, werecalculated on the basis that 1 g (dry weight) of cellswas equivalent to 1.67 ml of intracellular fluid (cyto-plasm) (28).LDH activity. Cells were disrupted by shaking for

2 min at 4°C with glass beads in a Mickle disintegrator.After centrifugation (35,000 x g for 10 min), NAD-dependent LDH activity was immediately assayed insupernatant samples, using saturating pyruvate (10mM), FDP (0.2 mM), and NADH (0.3 mM) as de-scribed previously (25). Activity was proportional to

enzyme concentration, and assays on duplicate cellextracts were in good agreement. Protein was deter-mined by the biuret method (15).

Experiments with nongrowing cells. Culturesamples (50 ml) were removed from the chemostatand centrifuged, and the cells were washed and resus-pended in 10 ml of phosphate buffer (20 mM, pH 6.5)at 3 to 6 mg (dry-weight equivalent) of bacteria perml. Portions (2 ml) of this suspension were placed ina Radiometer pH-stat apparatus (TTA 31 titrationassembly linked to an ABU 12 Autoburette, pH meter26, Titrator 11, and Titrigraph SBR 2c). The stirredsuspension was adjusted to pH 6.50 and maintained at30°C under an atmosphere of 02-free N2. Glucose (50pl, 0.2 M) was added, and the rate of alkali (0.1 NNaOH) addition required for pH control at 6.50 wasrecorded until alkali addition ceased (6 to 12 min).The total volume of alkali added was measured, thesuspension was then centrifuged, and the supernatantwas assayed for L-lactate and residual glucose. Formeasurement of [FDP]in in cells metabolizing glucose,suspensions (5 ml) were similarly maintained and glu-cose (100 il, 0.2 M) was added. When half of theglucose had been utilized, FDP was rapidly extractedfrom cells by the evacuated-syringe/HCl04 procedureand assayed by methods described above.

Other procedures. Bacterial density was deter-mined directly in all chemostat cultures by filtrationof three 10-ml samples, using preweighed Milliporemembranes (0.8-,um pore size, 47-mm diameter). Afterwater washing, the membranes plus retained cells weredried to constant weight at 100°C; variation betweentriplicates was <±3%. Amino acids were assayed witha Technicon TSM amino acid analyzer. Cells wereassayed for glucoamylase-specific glycogen by themethod of Hamilton (11).

RESULTS

Growth characteristics of lactic strepto-cocci in the chemostat. Batch growth of S.lactis ML3 in the fermentor pot (30°C, pH 6.5)gave mean generation times of 52 and 35 min inchemically defined and complex broth media,respectively. In continuous culture, steady-statepopulations were maintained at dilution rates(D = 0.8 h-1 for defined medium; D at least 1.0h-' for complex medium) similar to the maxi-mum values predicted from batch cultures. Thelimiting growth factor in chemostat cultures wasusually glucose (or lactose). In some S. lactiscultures, arginine may also have become limiting(Fig. 1), although cell density was proportionalto glucose concentration in glucose-limited cul-tures. Analyses indicated that the other aminoacids were in excess at all D values when themedium contained 0.5% glucose. When excessglucose was present at D = 0.6 to 1.0 h-', thespecific rate of glucose utilization (14) was 0.38to 0.44 ,umol/mg (dry weight) of bacteria per minwith the six strains of lactic streptococci. Nodetectable glycogen was found in cells growing

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112 THOMAS, ELLWOOD, AND LONGYEAR

at D = 0.2 to 0.8 h-' in the presence of excessglucose.

Cell growth on the wall ofthe fermentor vesselwas not apparent with any ofthe six strains usedin this study. With one strain (S. lactis ML3),however, a clumping phenomenon was observedin all growth media (i.e., complex or defined;sugar excess or limiting) which was a function ofthe growth rate. This strain grew as diplococciat D > 0.75 hIf and D < 0.35 h-', but at inter-mediate values the cells aggregated to form largeclumps containing several thousand celLs.Fermentation products in chemostat cul-

tures with glucose or lactose limitation.Growth of S. lactis ML3 in the presence of excessglucose gave homolactic fermentation, and at D= 0.76 and 0.64 h-' about 190 mmol of lactatewas formed per 100 mmol of glucose fermented(Table 1). At D = 0.56 h-1, the residual glucoseconcentration in the culture was very low, andproducts other than lactate became detectable.

02 04 0-6 08Dilution rate ( h-')

FIG. 1. Cell density and fermentation pattern ofS.lactis ML3 growing in a chemostat in a chemicallydefined medium containing 25 mM glucose and 5.5MM L-arginine.

TABLE 1. Residual glucose and fermentationproducts with S. lactis ML3 growing in a chemostata

mmol of product/100 mmol ofD Residual glucose fermented Carbon

(h-') glucose recovery(mM) L-Lac- For- Acetate Etha- (%)tate mate nol

0.76 5.11 187 NDb ND ND 940.64 1.55 193 ND ND ND 970.56 0.03 185 16 13 9 1030.48 ND 134 29 26 53 980.36 ND 38 134 73 88 950.25 ND 15 141 99 94 950.18 ND 7 125 111 103 960.11 ND 2 156 103 87 90a Culture conditions: 30°C, pH 6.5, N2-CO2 (95:5)

atmosphere, chemically defined medium containing 28mM glucose.

'ND, Not detectable (see text).

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As the dilution rate was progressively reducedin the absence of detectable glucose, the propor-tion of these products (formate, acetate, andethanol) progressively increased until at D =0.11 h-' only 1% of the glucose was fermented tolactate. Data similar to those shown in Table 1were obtained when the dilution rate was pro-gressively increased from an initial low value.The carbon recovery was close to 100%, allow-

ing for the incorporation of glucose carbon intocellular material (normally about 3% with theseorganisms [13]). Gas-liquid chromatography re-vealed no trace of other products such as acet-aldehyde, acetoin, acetolactate, butylene glycol,diacetyl, fumarate, pyruvate, and succinate. En-zymatic assays for L-lactate were in good agree-ment with the chemical assay for total lactate,indicating that no D-lactate was formed.When the glucose in the growth medium was

replaced by lactose (14 mM), a diversion of endproducts similar to that found with glucose (Ta-ble 1) was observed when lactose became limit-ing (data not shown).Fermentation products in chemostat cul-

tures with excess glucose. To determinewhether the change in fermentation productswas a function of growth rate or sugar limitation,the glucose concentration in the defined mediumwas increased to 0.22 M so that an excess (>25mM) was present in the culture at all dilutionrates. L-Lactate production was only slightlysuppressed with S. lactis ML3 as the dilutionrate was decreased. At D = 0.2 and 0.1 h-1, forexample, 89 and 83%, respectively, of the glucosefermented were converted to L-lactate (Table 2).The limiting nutrient in these cultures was ar-ginine.Molar growth yields. The only known en-

ergy sources for S. lactis are carbohydrates andarginine (which is converted to ornithine withthe production of 1 mol of ATP per mol ofarginine utilized). The cell density of S. lactisML3 growing in chemostat cultures reflects notonly the residual glucose and arginine concen-trations, but also the change from homo- toheterolactic fermentation (Fig. 1). At D = 0.78and 0.67 h-', when glucose was still in excess andfermentation was homolactic, the molar growthyields (Ygl,.e) calculated from the data plottedin Fig. 1 were, respectively, 35.9 and 36.1 g (dryweight) of bacteria per mol ofglucose fermented.When glucose became limiting, arginine was ca-tabolized (Fig. 1), and a corresponding amountof ornithine appeared in the culture (data notshown). At D = 0.35 to 0.16 h-f, when fernen-tation was heterolactic, the apparent Yglu518value was 46.8 g/mol. Subtraction of the theo-retical contribution from arginine catabolismgave a corrected Yglucose of 43.8 g/mol. Therefore,

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CHANGE IN END PRODUCTS OF S. LACTIS 113

TABLE 2. Regulation of end-product formation in S.lactis ML3a

Chemostat Nongrowing cellsb

Conver- Conver-Expt D sion of [FP]siDHon0f of

(h1) g sp act glucosecFDP]into L-laC- to L-lac-tate (%) tate (%)

i 0.76 94 25.2 12.4 99 26.80.64 97 25.5 14.1 103 25.20.56 93 21.2 18.1 99 27.40.48 67 17.6 19.6 100 ND0.36 19 15.0 8.9 81 ND0.25 8 14.1 4.9 77 24.20.18 4 11.0 4.1 78 28.70.11 1 10.3 4.6 83 25.9

ii 0.20 89 27.7 10.4 99 ND0.10 83 19.8 9.2 ND

a Experiment i was described in Table 1; the me-dium contained 28 mM glucose, and chemostat cul-tures became glucose limited at D values below 0.56h-' (percent conversion of glucose to lactate derivedfrom data in Table 1). In experiment ii, the mediumcontained 0.22 M glucose so that glucose was alwaysin excess (>25 mM).

b Cells were removed from the chemostat and placedin buffer (see text). ND, Not determined.

'Intracellular FDP concentration (30-ms samplingtime).

d LDH activity (micromoles of NADH oxidized permilligram of protein per minute).

GLUCOSE

NADH NAD

PTRUVATE L-(40-LACTATE

CoA. TW*, Co2

.t-_ - TPP

IC Pyruvate* CoA ~~~ac~

lMATE .cetyl- CeA - 1

NADH 1|

MAD

CA LCoA CoA TPP C0

ACETALDENYDE

NADH

, MNAD

ETHANOL

WACETYTL- ACETOIN

NADH NAD

_ P

(.ADP

ATP

ACETATE

FIG. 2. Alternative pathways for pyruvate metab-olism in homofermentative lactic streptococci. CoA,Coenzyme A.

associated with the change to heterolactic fer-mentation was a YO,.. increase from ca. 36 to44 g/mol, which is consistent with the amountof acetate produced (Table 1) by the pathwayshown in Fig. 2. These Ygiuco8e values were notcorrected for maintenance energy requirementswhich appear to be low with these organisms.For S. lactis ML, which remained homolacticwith limiting glucose (Table 3), Ygluco did notincrease at low D values.Distribution of heterolactic potential

among the lactic streptococci. Two addi-tional strains of S. lactis (H1 and ML8) and threestrains of S. cremoris (AM2, 158, and 266) wereexamined in experiments similar to that de-scribed for S. lactis ML3 in Table 1. Fermenta-tion with all S. cremoris strains and with S.lactis Hi became heterolactic with the produc-tion of formate, acetate, and ethanol (data notshown). The changes with S. lactis HI weresimilar to those observed with S. lactis ML3,and, in the first steady state in which glucosewas limiting (D = 0.51 h-') fonnate, acetate, andethanol were produced. Diversion to these prod-ucts increased as D was lowered until at D = 0.1h-' about 7% of the glucose was fermented toL-lactate. All S. cremoris strains remained hom-olactic in the first two steady states in whichglucose was limiting (D = 0.6 and 0.4 h-'), but atlower D values lactate formation was suppresseduntil at D = 0.1 h-1 less than 20% of the glucosewas fermented to L-lactate.

In striking contrast to the above-mentionedfive strains, S. lactis ML8 remained essentiallyhomolactic when cultures were glucose limitedat low D values (Table 3). Small quantities ofacetate were produced, but no formate orethanol was detected. With this strain the recov-ery of glucose carbon was consistently near 85%in separate experiments. Gas-liquid chromatog-raphy revealed no trace of the missing carbon.

TABLE 3. Fermentation characteristics of S. lactisML8 growing in a chemostata

mmol of product/D Residual 100 mmol of glu- [FDP], c LDH s

(h-') glucose cose fennentedb (mM) actd(MM)L-Lactate Acetate

0.61 3.42 171 ND 28.7 15.10.45 1.59 167 1 24.3 16.90.29 ND 166 6 28.9 11.60.15 ND 178 6 25.7 10.90.06 ND 164 10 24.2 8.8a Culture conditions as for Table 1.b Formate and ethanol were not detectable (ND).Intracellular FDP concentration (30-ms sampling

time).d Micromoles of NADH oxidized per milligram of

protein per minute.

fOR

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114 THOMAS, ELLWOOD, AND LONGYEAR

Does the switch to heterolactic fermen-tation involve phenotypic or genotypicchange? A chemostat can provide a stronglycompetitive environment for the selectivegrowth of different genotypes (see 10). There-fore, to distinguish between these two possibili-ties, the kinetics of the transition process fromhetero- to homolactic fermentation were exam-ined with S. lactis ML3 (Fig. 3). Conditions in aheterolactic chemostat culture were changed byincreasing D from 0.1 to 0.66 h-1, at which pointit was anticipated that fermentation would even-tually become homolactic. At intervals after thetransition, culture samples were assayed for glu-cose, lactate, formate, acetate, and ethanol. Ex-cess glucose was present in the culture within0.5 h of the transition. The rate of washout ofproducts other than lactate approximated thetheoretical washout rate (Fig. 3). This indicatesthat a phenotypic change occurred upon adjust-ment of the growth conditions such that theheterolactic cells immediately changed to hom-olactic fermentation. This transition would havebeen much slower had cells with a heterolactic

loof

9

cm

:E

'a

aL)

c 0

.2

>1(L

5

11a,

I

homniolaufic

2 4 6Time after transition ( h )

FIG. 3. Kinetics of transition from hetero- tohomolactic fermentation with S. lactis ML3. A steady-state chemostat culture atD = 0.1 h-5 (glucose limited,converting only 2% of the available glucose to L-lactate) was changed at time zero to D = 0.66 h-1:-----, theoretical washout ofproducts other than lac-tate at D = 0.66 h-', *, actual conversion ofglucoseto products other than lactate at D = 0.66 h-1.

genotype been selected in the glucose-limitedcultures (10).Regulation of end-product formation.

(i) Glucose metabolism of cells after re-moval from chemostat. Growing S. lactis ML3cells were removed from chemostat cultures ateach dilution rate and incubated anaerobicallywith glucose in a pH-stat apparatus (see Mate-rials and Methods). To these nongrowing cellsglucose was added, and the rate of alkali additionrequired for pH control at 6.50 was recorded.This rate, which corresponds to the rate of glu-cose utilization, was linear in all suspensions andvaried between 0.21 and 0.15 ,umol/mg (dryweight) of bacteria per min for cells from chem-ostat cultures growing at D = 0.67 and 0.10 h-1,respectively (with nongrowing cells, the glucoseutilization rate is about half that determined forcells growing in chemostat cultures with excessglucose). After the alkali demand had stopped,suspensions were assayed for L-lactate and resid-ual glucose. Glucose was undetectable in all sus-pensions, and the conversion ofglucose to lactateis given in Table 2. With cells derived fromhomolactic chemostat cultures, L-lactate ac-counted for ca. 100% of the glucose fermented inthe nongrowing cell suspensions. Cells from het-erolactic chemostat cultures (D = 0.36 to 0.11h-1) converted about 80% of the glucose to L-lactate suggesting that fine control of enzymeactivity has an important influence on the bal-ance of end products.

(ii) Intracellular concentration of FDP.With the rapid sampling and extraction proce-dure, the [FDP]in in S. lactis ML3 cells growingwith excess glucose was ca. 25 mM (Table 2).Although the [FDP]in declined as fermentationbecame heterolactic, the cells fermenting only1% of the glucose to lactate still contained 10mM FDP. Similar data were obtained in a sep-arate experiment (Table 4, Fig. 1). Upon addi-tion of excess glucose to a culture at D = 0.2 h-'(Table 2) the [FDP], increased to 28 mM, andfermentation became homolactic. With S. lactisML8, which did not deviate from L-lactate pro-duction, the [FDP]u, remained high in glucose-limited cultures at low D values (Table 3).Sampling times of up to 60 s had no effect on[FDP],. values measured with glucose-excess S.lactis ML3 cultures, but with glucose-limitedcells the sampling time was obviously critical(Table 4). Nongrowing cells, derived from chem-ostat cultures at all D values, contained similar[FDP]in (ca. 26 mM) while fermenting glucose toL-lactate (Table 2).Attempts were made to measure the intracel-

lular concentrations of other glycolytic inter-mediates (glucose 6-phosphate, phosphoenol-

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CHANGE IN END PRODUCTS OF S. LACTIS 115

TABL~E 4. Effect of sampling timea on [FDP]in in S.lactis ML3b

D Residual [FDP]I. (mM)(W'l) glucose

(mM) 0.03 se 20s 40 s 60 s

0.78 6.85 26.5 27.9 26.4 25.10.67 2.23 27.1 24.50.54 NDd 22.6 16.40.45 ND 21.3 11.70.35 ND 18.2 8.50.26 ND 16.8 6.60.16 ND 12.1 4.50.10 ND 10.1 3.6 0.9 0.3

a Time between cells leaving chemostat culture andsuspension in 0.6 N HC104.

b Other data from this experiment are shown in Fig.1.

Sampling time.d ND, Not detectable.

pyruvate, and pyruvate) by using the rapid pro-cedure which involved sampling without sepa-ration of cells from the medium followed byfluorometric enzymatic analysis. However,growing cells released small quantities of glucose6-phosphate and pyruvate into the medium sothat the concentration of these compounds inculture extracts could not be measured abovethe background level present in the cell-freeculture samples. Phosphoenolpyruvate was ob-scured since the enzymatic assay procedure in-volved the intermediate formation of pyruvate.Although pyruvate was released from cells, themaximum concentration was too low for detec-tion by gas-liquid chromatography.

(iii) LDH activity. The specific activity ofLDH in S. lactis ML3 increased when D wasreduced from 0.76 to 0.48 h-', but further reduc-tion in D resulted in a sharp decline in LDHactivity (Table 2). A less-marked decline in ac-tivity was found with S. lactis ML8 (Table 3).LDH specific activities were not affected byincreasing the FDP concentration in the assaysystem from 0.2 to 2 mM. It seemed possiblethat LDH activity was inhibited by adeninenucleotides (4) and that the balance of thesecompounds changed in glucose-limited cells.However, AMP, ADP, and ATP had little effecton the in vitro activity of LDH from S. lactisML3 when tested at concentrations up to 10mMin standard assay conditions with 0.05, 0.2, and1 mM FDP.

DISCUSSIONL-Lactate was the only significant end product

when lactic streptococci were grown with excessglucose in a chemostat at rates ranging from D= 0.8 to 0.1 h-' (corresponding to mean genera-

tion times of 0.9 to 6.9 h). However, with areduced glucose concentration in the growthmedium, glucose became growth limiting, andend products were diverted from lactate to for-mate, acetate, and ethanol. Similar results wereobtained with Lactobacillus casei (8) and S.mutans (9, 29). The likely pathways leading tothe formation of these compounds from pyru-vate in the lactic streptococci are shown in Fig.2. The products suggest that, under conditionsof limiting energy supply (glucose), the cellsobtained ATP from pyruvate by a mechanisminvolving pyruvate formate-lyase. This is con-sistent with the Ygl ,0. increase from 36 to 44 g/mol which accompanied the change to hetero-lactic fermentation. Glucose-limited S. lactiscells not only derived additional ATP from thischange but also induced the enzymes for argi-nine catabolism (T. D. Thomas, unpublisheddata), which generates ATP. The product ratiosexpected from "phosphoroclastic" cleavage ofpyruvate by pyruvate formate-lyase are formate-acetate plus ethanol (1:1). Since the observedratios always showed a higher-than-expectedproportion of acetate plus ethanol, it is possiblethat acetate was also produced from pyruvatevia the intermediate fornation of an acetalde-hyde-TPP complex. The absence of detectableacetoin in heterolactic cultures is perhaps sur-prising in view of the pathways present in lacticstreptococci (Fig. 2). Pyruvate formate-lyase hasbeen found in S. faecalis (19) and S. mutans(30), but this highly 02-sensitive enzyme has yetto be demonstrated in lactic streptococci. Of thesix strains used in the present study, only one(S. lactis ML8) remained homolactic under glu-cose limitation, although small amounts of ace-tate were produced, suggesting the absence ofpyruvate formate-lyase in this organism.

Regulation of pyruvate metabolism may in-volve fine control of enzyme activity by effectorsand/or control of enzyme synthesis, althoughanother possible explanation for the change toheterolactic fermentation upon glucose limita-tion is the selection of cells with a differentgenotype. Continuous cultures are known to pro-vide a highly selective environment (10), andaccumulation of lactose-negative mutants inchemostat cultures of lactic streptococci hasbeen demonstrated (20). However, kinetic ex-periments (Fig. 3) indicated that, when hetero-lactic cells were supplied with excess glucose,homolactic fermentation was immediately re-sumed, indicating that the change was entirelyphenotypic.The specific activity of the FDP-activated

LDH of S. lactis ML3 increased asD was initiallylowered, but the level then decreased, as lactate

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116 THOMAS, ELLWOOD, AND LONGYEAR

production was suppressed, to about 25% of themaximum (Table 2). With S. lactis ML8 theLDH level also fell (to a lesser extent), but thisstrain remained homolactic (Table 3). Transferof heterolactic S. lactis ML3 cells, having re-duced LDH levels, from the chemostat to abuffer containing glucose resulted in 80% con-version of glucose to L-lactate. Although thisindicated that fine control of enzyme activitywas important, nongrowing cells containingmaximal LDH specific activity converted nearly100% of the glucose to L-lactate, suggesting thatthe level of LDH did have some effect. Thespecific activity of the other enzymes likely tobe involved in pyruvate metabolism (Fig. 2) mayalso change in the different growth environ-ments, but these were not determined.

Factors which may regulate the activity ofenzymes involved in pyruvate metabolism in-clude substrate concentrations, activator and in-hibitor concentrations, and the internal pH. Theonly LDH activator likely to be present in cellsgrowing on glucose is FDP. The change fromheterolactic fermentation in the chemostat to80% conversion of glucose to lactate by nongrow-ing S. lactis ML3 cells was accompanied by atwofold increase in [FDP]in (Table 2). A decreaseof similar magnitude accompanied the transitionfrom homo- to heterolactic metabolism withcells growing in a chemostat, and [FDP]i, fell toca. 10 mM (Table 2). Crow and Pritchard (6)found that the FDP concentration required forhalf-maximal velocity of LDH from S. lactis C10in vitro was 4.4 mM in the presence of Pi. Thissuggests that the [FDP]in in heterolactic cellswould be sufficient to give LDH catalytic activ-ity in vivo. The present results suggest thatchanges in [FDP]in alone are not sufficient toexplain fully the diversion of end products. Inaddition, the [FDP]i, for lactic streptococcigrowing in batch cultures on glucose and galac-tose were similar, and yet fermentation of thesesugars was homo- and heterolactic, respectively(T. D. Thomas, unpublished data). It is con-cluded that the combined effect of changes in[FDP]in and LDH level may largely explain thechanges in fermentation pattern observed inchemostat cultures. However, additional factorsmay also influence the in vivo activity of LDHand/or the other enzymes involved in pyruvatemetabolism. The pool size of NADH and pyru-vate may vary with growth conditions. Pi is apotent inhibitor ofLDH from lactic streptococci(6, 17, 25) and increased the FDP concentrationrequired for half-maximal velocity in vitro by2,000-fold (6) so that changes in the intracellularlevel of Pi may affect LDH activity in vivo.Glucose limitation may induce pyruvate for-

mate-lyase synthesis or could conceivably resultin a shortage of ATP and lead to a fall inintracellular pH. It is interesting that S. lactisML8 maintained a high [FDP]in with limitingglucose (Table 3), and it is possible that thisability, together with the relatively smaller de-crease in LDH specific activity, was responsiblefor the homolactic behavior of this strain underglucose limitation.Yamada and Carlsson (29) grew S. mutans JC

2 in a chemostat at D = 0.12 h-1 under glucoseand nitrogen limitation and observed homo- andheterolactic fermentation, respectively. Cells inboth cultures had similar LDH levels, butwhereas [FDP]in was high in homolactic cells,this LDH activator was apparently absent fromheterolactic cells which contained high levels ofphosphoenolpyruvate. It was concluded thatchanges in [FDP]in regulate LDH activity invivo, and suppression of lactate production wasattributed to an effect on this single enzyme (29).Later work, however, indicated that pyruvateformate-lyase was induced in glucose-limitedcells and that this enzyme was inhibited byglyceraldehyde 3-phosphate (30). In these stud-ies (29), cells were removed from the chemostatand centrifuged before FDP extraction. Fromthe data for S. lactis (Table 4) it would beanticipated that these relatively slow procedureswould allow extensive FDP metabolism in glu-cose-limited S. mutans during sampling. Theneed for rapid sampling and extraction of me-tabolites, which may otherwise be quickly de-pleted, has often been stressed (see 12 and 22).When nongrowing cells of S. lactis depletedexogenous glucose, intracellular FDP disap-peared and there was a corresponding increasein the levels of phosphoglycerates and phospho-enolpyruvate (28).

Previous reports on lactic streptococci havesuggested that fermentation of glucose and lac-tose under anaerobic conditions is invariablyhomolactic. Brown and Collins (3) attempted todivert end products by growing lactic strepto-cocci in batch cultures with "low concentra-tions" (2 to 5 mM) of glucose, reasoning that thecells would have low [FDP],i. However, fermen-tation remained homolactic, presumably be-cause glucose uptake by these organisms in-volves a high-affinity phosphoenolpyruvate-de-pendent phosphotransferase system (Km 15 ,uM;27) so that glucose only became limiting nearthe point of exhaustion from the batch cultures.The present investigation demonstrates theunique potential of the chemostat, since it isdoubtful whether this heterolactic metabolismcould be expressed by lactic streptococci growingin batch cultures on glucose or lactose.

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CHANGE IN END PRODUCTS OF S. LACTIS 117

ACKNOWLEDGMENTSWe thank Ivor Whitlock for excellent technical assistance.The hospitality of the Microbiological Research Establish-

ment and the award of a Travelling Fellowship by the NuffieldFoundation are gratefully acknowledged (T.D.T.).

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3. Brown, W. V., and E. B. Collins. 1977. End productsand fermentation balances for lactic streptococci grownaerobically on low concentrations of glucose. Appl. En-viron. Microbiol. 33:38-42.

4. Brown, A. T., and C. L Wittenberger. 1972. Fructose-1,6-diphosphate-dependent lactate dehydrogenase froma cariogenic streptococcus: purification and regulatoryproperties. J. Bacteriol. 110:604-615.

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6. Crow, V. L., and G. G. Pritchard. 1977. Fructose 1,6-diphosphate-activated L-lactate dehydrogenase fromStreptococcus lactis: kinetic properties and factors af-fecting activation. J. Bacteriol. 131:82-91.

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15. Herbert, D., P. J. Phipps, and R. E. Strange. 1971.Chemical analysis of microbial cells, p. 209-344. In J.R. Norris, and D. W. Ribbons (ed.), Methods in micro-biology, vol. 5B. Academic Press Inc., New York.

16. Herbert D., P. J. Phipps, and D. W. Tempest. 1965.The chemostat: design and instrumentation. Lab. Pract.14:1150-1161.

17. Jonas, H. A., R. F. Anders, and G. R. Jago. 1972.Factors affecting the activity of lactate dehydrogenaseof Streptococcus cremoris. J. Bacteriol. 111:397-403.

18. Lawrence, R. C., T. D. Thomas, and B. E. Terzaghi.1976. Reviews in the progress of dairy science: cheesestarters. J. Dairy Res. 43:141-193.

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20. McDonald, I. J. 1975. Occurrence of lactose-negativemutants in chemostat cultures of lactic streptococci.Can. J. Microbiol. 21:245-251.

21. McKay, L. L., and K. A. Baldwin. 1974. Altered metab-olism in a Streptococcus lactis C2 mutant deficient inlactic dehydrogenase. J. Dairy Sci. 57:181-186.

22. Niven, D. F., P. A. Collins, and C. J. Knowles. 1977.Adenylate energy charge during batch culture of Be-neckea natriegens. J. Gen. Microbiol. 98:95-108.

23. Platt, T. B., and E. M. Foster. 1958. Products of glucosemetabolism by homofermentative streptococci underanaerobic conditions. J. Bacteriol. 75:453459.

24. Sanwal, B. D. 1970. Allosteric controls of amphibolicpathways in bacteria. Bacteriol. Rev. 34:20-39.

25. Thomas, T. D. 1975. Tagatose-1,6-diphosphate activationof lactate dehydrogenase from Streptococcus cremoris.Biochem. Biophys. Res. Commun. 63:1035-1042.

26. Thomas, T. D. 1976. Regulation of lactose fermentationin group N streptococci. Appl. Environ. Microbiol. 32:474-478.

27. Thompson, J. 1978. In vivo regulation of glycolysis andcharacterization ofsugar:phosphotransferase systems inStreptococcus lactis. J. Bacteriol. 136:465-476.

28. Thompson, J., and T. D. Thomas. 1977. Phosphoenol-pyruvate and 2-phosphoglycerate: endogenous energysource(s) for sugar accumulation by starved cells ofStreptococcus lactis. J. Bacteriol. 130:583-595.

29. Yamada, T., and J. Carlsson. 1975. Regulation of lac-tate dehydrogenase and change of fermentation prod-ucts in streptococci. J. B3acteriol. 124:55-61.

30. Yamada, T., and J. Carlsson. 1976. The role of pyruvateformate-lyase in glucose metabolism of Streptococcusmutans, p. 809-819. In H. M. Stiles, W. J. Loesche, andT. C. O'Brien (ed.), Proceedings, microbial aspects ofdental caries. Special supplement of microbiology ab-stracts, vol. 3. Information Retrieval Inc., Washington,D.C.

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