Vol. 56, No. 9APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1990, p. 2644-26490099-2240/90/092644-06$02.00/0Copyright C) 1990, American Society for Microbiology

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp.lactis 3022 Grown Aerobically in the Presence of Hemin or Cu2+

TSUTOMU KANEKO,* MASAHIRO TAKAHASHI, AND HIDEKI SUZUKICentral Research Institute, Meiji Milk Products Co., 1-21-3 Sakae-cho, Higashimurayama, Tokyo 189, Japan

Received 3 April 1990/Accepted 6 June 1990

Citr+ Lactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucosesubstrate to diacetyl and acetoin when grown aerobically with hemin and Cu2". The activity of diacetylsynthase was greatly stimulated by the addition of hemin or Cu2+, and the activity of NAD-dependent diacetylreductase was very high. Hemin did not affect the activities ofNADH oxidase and lactate dehydrogenase. Theseresults indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that thediacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH whenthe cells were grown aerobically with hemin or Cu2+. On the other hand, the YGIU value for the hemin-containing culture was lower than for the culture without hemin, because acetate production was repressedwhen an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of thedihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu2+ enhancedacetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase waslowered by the addition of lipoic acid. These results indicate that hemin or Cu2+ stimulates acetyl coenzyme A(acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA withhydroxyethylthiamine PP;. In addition, it appears that acetyl-CoA not used for diacetyl synthesis is convertedto acetate.

It has been shown that diacetyl and acetoin in lactic acidbacteria are not produced from carbohydrate unless anadditional source or pyruvate, such as citrate, is present (6,10, 19). This is because in homofermentative organisms,most of the pyruvate is converted to lactate by NAD-dependent lactate dehydrogenase to regenerate NAD. Inaddition, lactococci are generally considered to be faculta-tive anaerobes, albeit with a preference for anaerobic con-ditions. However, we recently showed that citrate-positive(Citr+) Lactococcus lactis subsp. lactis 3022 (Streptococcuslactis subsp. diacetylactis 3022) cells grown aerobicallycould produce a considerable amount of diacetyl in MRSmedium without citrate and that diacetyl production wasfurther increased by the addition of Cu2+, which is aneffective stimulator of diacetyl synthase activity (13, 15).These results indicated that under aerobic conditions, di-acetyl synthase could be effectively induced and then thecells could produce diacetyl from pyruvate formed via theglycolytic pathway.

Several workers (3-5, 29) also found that acetoin produc-tion was enhanced under aerobic conditions. However, ithas not yet been demonstrated whether most of the glucoseis converted to diacetyl and acetoin. To enhance the produc-tion of these flavor compounds instead of lactate, furtherstimulation of the activity of diacetyl formation from pyru-vate (diacetyl synthase) is required. In addition, an alterna-tive system to reoxidize NADH formed via glycolysis isrequired, because lactate production from pyruvate is re-pressed. In this study we found that hemin, a chloride ofheme, could greatly stimulate the activity of diacetyl syn-thase in Citr+ L. lactis subsp. lactis 3022 in the same manneras Cu2+ and that the activity of NAD-dependent diacetylreductase in the cells was at a markedly high level. This

* Corresponding author.

paper reports the effects of hemin or Cu2" on acetoinfermentation by Citr' L. lactis subsp. lactis 3022 cells.

MATERIALS AND METHODSMicroorganisms and growth conditions. All experiments

were made with Citr+ L. lactis subsp. lactis 3022 isolatedfrom cream cheese. MRS medium (9) without triammoniumcitrate and containing 0.003% silicon (by weight) as a foambreaker was used as the basal medium. In some experi-ments, 10 ,uM hemin, 0.1 mM CUC12, 73 U of catalase per ml,0.05 mM antimycin A, and 1.0 mM lipoic acid were added tothe basal medium. The cells were grown in a Sakaguchi flaskon a reciprocal shaker at 120 strokes per min at 30°C. Molargrowth yields (YGIU values) were determined by dividing thedry weights of cells by the number of moles of glucoseutilized. Growth was measured by determining the opticaldensity at 580 nm.

Bakers' yeast (Saccharomyces cerevisiae 8004) cells werealso prepared for the assay of cytochromes by incubation ina basal medium at 30°C for 24 h and by three washes withdistilled water.

Preparation of cell extract and enzyme assay. Preparationof cell extract and assays of the activities of NADH oxidaseand diacetyl reductase (EC 1.1.1.5) were carried out asdescribed previously (15). The activity of diacetyl formationfrom pyruvate (diacetyl synthase) was assayed as follows.The reaction mixture contained 20 mM sodium pyruvate, 0.2mM thiamine PP1, 0.2 mM MgCl2, 0.1 M phosphate buffer(pH 6.0), and 0.1 ml of cell extract in a total volume of 1.0ml. After incubation at 30°C for 30 min, the amount ofdiacetyl produced in the reaction mixture was determined.One unit of diacetyl synthase activity was defined as theamount of enzyme which catalyzes the production of 1 ,umolof diacetyl from pyruvate per min at 30°C.

Lactate dehydrogenase (EC 1.1.1.27) activity was mea-sured as follows. The reaction mixture contained 50 mMsodium pyruvate, 10 mM NADH, 0.1 M phosphate buffer

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(pH 6.0), and 0.5 ml of cell extract in a total volume of 5.5ml. After incubation at 30°C for 30 min, the reaction wasstopped by heating in a boiling-water bath for 1 min, and theamount of lactate produced in the reaction mixture wasdetermined by high-pressure liquid chromatography (seebelow). One unit of lactate dehydrogenase activity wasdefined as the amount of enzyme which catalyzes the pro-duction of 1 ,umol of lactate per min at 30°C.The protein concentration for the assay of specific activi-

ties was determined by the method of Lowry et al. (17) withbovine serum albumin as the standard.Hydrogen peroxide. Hydrogen peroxide was determined

by the luminol chemiluminescence technique (14) on aBiocounter M-2010 (Lumac, The Netherlands). A 100-,ulvolume of 0.1 ptM luminol in 0.2 M glycine-NaOH buffer(pH 9.9) was added to 100 pl of the sample. After 100 ,ul of2 mM K3Fe(CN)6 solution had been added to the mixture asa catalyst, the generated light signal was integrated for 10 s.Relative light units were recorded digitally, and the H202content of the sample was calculated from a standard curve.

Assays of cytochromes. Citr+ L. lactis subsp. lactis 3022cells and Saccharomyces cerevisiae 8004 cells (3 g [wetweight] each), which were washed three times with distilledwater, were finally suspended in 7 ml of distilled water. Thecells were analyzed for the presence of cytochromes byobtaining the absorption spectra from the cell suspension,using a spectrophotometer (model 624; Hitachi, Tokyo,Japan) equipped with opalescent plates, as described byShibata et al. (24).

Analyses. Glucose and end products such as lactate,acetate, formate, and ethanol were analyzed by an enzy-matic analysis method with test kits from Boehringer GmbH,Mannheim, Federal Republic of Germany. Lactate in thereaction mixture for the assay of lactate dehydrogenaseactivity was determined by a high-pressure liquid chroma-tography system (model L-6200; Hitachi) equipped with arefractometer (model RI SE-61; Shodex, Tokyo, Japan). Thecolumn used was a Polyspher OAKC (Cica-Merck, Tokyo,Japan) packed with a cation-exchange resin in the hydrogenionic form and protected by a Polyspher OAKC guardcolumn (Cica-Merck). The column was run at 35°C, and theflow rate of the mobile phase (0.02 N H2SO4) was set at 0.4ml/min.

Diacetyl and acetoin were determined by headspace gaschromatography on a dual flame ionization detector chro-matograph (Sigma 300; The Perkin-Elmer Corp., Norwalk,Conn.) with a steel column (1.8 m by 3 mm [outer diameter])packed with Unicarbon A200, 60/80 mesh (12). A 1-g portionof the sample was transferred into a headspace vial (Perkin-Elmer), which was closed with a rubber septum and analuminum cap. After equilibration at 80°C for 30 min, di-acetyl and acetoin were analyzed under the following con-ditions. After the column temperature had been held at 70°Cfor 4 min, the temperature was programmed to increase at arate of 30°C/min from 70 to 130°C. The nitrogen flow rate was30 ml/min, and the temperature of the injection port anddetector was set at 1500C.

RESULTS

Effects of hemin on growth, glucose utilization, and endproduct formation. There are great differences in growth,pH, glucose utilization, and end product formation by Citr+L. lactis subsp. lactis 3022 aerobic cultures with and withouthemin (Fig. 1). In a culture with hemin, 91.8% (89.2 mmol) ofthe glucose was consumed after a 24-h incubation. In a

culture without hemin, however, the cells could utilize only38.8% (37.6 mmol) of the glucose after a 48-h incubation.The growth rate and the production of diacetyl and acetionwere also markedly affected by the addition of hemin. Thecells in hemin-containing culture grew faster and producedmore biomass than those in the culture without hemin (2.27and 0.87 mg [dry weight] per ml, respectively, after a 15-hanaerobic incubation at 30°C), although the YG1U value of theformer culture was lower than that of the latter (37.5 and 47.2g [dry weight] per mol, respectively). (Fig. 1). Furthermore,the production of diacetyl and acetoin in the culture withhemin after a 48-h incubation was about 10-fold higher thanin the culture without hemin (Table 1).On the other hand, lactate production per mole of glucose

was inhibited in the culture with hemin, and, interestingly,the consumption of accumulated lactate and the productionof acetate were very rapid after the disappearance of glucosefrom the medium (Fig. 1). In hemin-containing culture,acetate production was repressed when an excess of glucosewas present. In addition, the increase in pH from 4.73 at 24h of incubation to 5.31 at 48 h of incubation was accompa-nied by the consumption of lactate (Fig. 1; Table 1). In thisstudy, formate and ethanol were not detected in eitherculture.

Influence of additives on growth, glucose utilization, andend product formation. The influence of Cu2' and lipoic acidon glucose utilization and end product formation werestudied when Citr+ L. lactis subsp. lactis 3022 cells weregrown aerobically (Tables 1 and 2). Cu2+ stimulated growth,glucose utilization, and diacetyl and acetoin productionwhen the cells were grown aerobically in the medium withsilicon used as a foam breaker. Growth, glucose utilization,and diacetyl and acetoin production were further increasedwhen both Cu2+ and hemin were added to the basal medium.In this case, 99.8 mmol of glucose was converted to 1.19mmol of diacetyl and 105.4 mmol of acetoin after 48 h ofincubation. In contrast, lactate production in the culturewith both Cu2' and hemin was lower than that in the culturewith just one these additions. Furthermore, acetate produc-tion at 48 h of incubation in the culture with both Cu2+ andhemin was also lower than in the cutlure supplemented withonly hemin. However, when lipoic acid was added to themedium with hemin or Cu2+, acetate production was mark-edly enhanced and diacetyl and acetoin production wasrepressed (Table 2). Lipoic acid also slightly repressed theglucose utilization and lactate production.On the other hand, 0.05 mM antimycin A did not reduce

the stimulatory effect of hemin on growth, glucose utiliza-tion, and acetoin production. In addition, no increases ingrowth rate, glucose utilization, and end product formation(particularly diacetyl and acetoin) were observed after theaddition of 73 U of catalase per ml (T. Kaneko, unpublishedresults).

Production and reduction of H202 by cells. Citr+ L. lactissubsp. lactis 3022 cells that had been washed three times indistilled water produced H202 when incubated aerobically at35°C for 60 min (3,870 and 5,670 ,ug of H202 per liter beforeand after incubation, respectively). In this case, H202 accu-mulation was repressed by the addition of 10 ,uM hemin tothe cell suspension (3,870 and 4,260 pug/liter before and afterincubation, respectively). However, catalase activity wasnot observed in the cells grown with hemin.Cytochrome spectra. Cytochrome spectra of S. cerevisiae

8004 cells and of Citr+ L. lactis subsp. lactis 3022 cellsgrown aerobically with hemin were determined (Fig. 2).Peaks at 520, 548, 559, and 600 nm, presumably due to

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Time (h)FIG. 1. (A and B) Growth of and diacetyl and acetoin production by Citr+ L. lactis subsp. lactis 3022 grown aerobically at 30°C in a basal

medium without (A) and with (B) 10 ,uM hemin. (C and D) Glucose utilization, pH change, and production of lactate and acetate by Citr+ L.lactis subsp. lactis 3022 grown aerobically at 30°C in a basal medium without (C) and with (D) 10 ,uM hemin.

cytochromes, were detected for S. cerevisiae 8004 cells, butno trace of cytochrome peaks was recognized for Citr+ L.lactis subsp. lactis 3022 cells grown aerobically with orwithout hemin.

Effect of hemin on the activities of several enzymes. Toinvestigate the reasons for the enhancement of diacetyl andacetoin production in the culture with hemin, we comparedthe activities of NADH oxidase, diacetyl synthase, anddiacetyl reductase for the cells grown aerobically in themedium with and without hemin (Table 3). There was a greatdifference in diacetyl synthase activity under the two dif-ferent conditions: diacetyl synthase activity in the cellsgrown with hemin was 4.7-fold higher than that in the cellsgrown without hemin. The diacetyl reductase activity in theformer cells was slightly lower than that in the latter,although both the activities were high. No great difference inNADH oxidase activity was observed.

The influence of hemin on the activities of several en-zymes was also studied by using a cell extract prepared fromthe cells grown aerobically in a basal medium. The activitiesof NADH oxidase, diacetyl reductase, and lactate dehydro-genase were not influenced by hemin. However, diacetylsynthase activity was increased 3.5-fold by the addition of 5p.M hemin to the reaction mixture. In contrast, lipoic acidlowered the activity of diacetyl synthase, regardless of thepresence of hemin or Cu2" (Table 4).

DISCUSSION

The ability to utilize 02 as an electron acceptor and toproduce H202 is widespread among lactic acid bacteria (2, 7,8, 20). An inhibitory effect due to the formation of H202 hasbeen indicated by decreases in growth rate and acid produc-tion under aerobic conditions (21). Since catalase containing

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TABLE 1. Effects of hemin and Cu2" on growth, pH, glucose utilization, and end product formation by Citr+ L. lactissubsp. lactis 3022 cells grown aerobicallya

Addition Incubation Loglo pH Amt (mmol) of Amt (mmol) of end product formed:lontime (h) 0D580 P glucose utilized (%) Lactate Acetate Diacetyl Acetoin

None 24 0.21 4.65 30.5 (31.4) 41.4 5.0 0.14 9.748 0.17 4.52 37.6 (38.8) 50.3 4.4 0.12 5.5

10 ,uM hemin 24 0.54 4.73 89.2 (91.8) 34.1 1.3 0.88 55.948 0.50 5.31 96.8 (99.7) 0.9 16.7 1.14 78.5

0.1 mM CuCl2 24 0.39 4.73 85.5 (88.0) 35.8 2.2 0.43 32.748 0.35 4.61 97.1 (99.9) 44.8 2.9 0.53 35.0

10 ,uM hemin + 0.1 mM CUC12 24 0.62 5.00 96.9 (99.8) 4.2 3.3 0.83 85.348 0.63 5.58 96.9 (99.8) 0.0 9.9 1.19 105.4

a Citr+ 1. lactis subsp. lactis 3022 cells were propagated in a basal medium at 30°C for 18 h. Next, 1 ml of this was inoculated into 100 ml of fresh basal mediumand incubated aerobically at 30°C. Cultivations in basal medium containing 10 pLM hemin, 0.1 mM CuC12, or 10 p.M hemin plus 0.1 mM CuC12 were also carriedout.

b OD580, Optical density at 580 nm.

heme has been found in some lactic acid bacteria grown inmedium with heated blood or hematin (11, 30, 31), onepossible explanation for the enhancement of growth inhemin-containing culture is that there is a system to detoxifyH202. Formation of H202 in lactic acid bacteria occursthrough the action of a flavoprotein, NADH oxidase (7, 8,18, 27). Citr+ L. lactis subsp. lactis 3022 also possessedNADH oxidase, although there was no marked difference inthe activity between the cells grown with and without hemin(Table 3). The cells produced H202 when incubated underaerobic conditions. In this case, H202 production was re-pressed by the addition of hemin to the cell suspension, sincehemin can decompose H202 (16). However, catalase activitywas not detected in the cells grown aerobically with hemin.The increases in growth, glucose utilization, and acetoinproduction were not observed by the addition of catalaseinto the medium. These results indicate that the eliminationof H202 formed in the culture is not the main reason for theenhancement of growth, although the decomposition ofH202 by hemin may be available for the cells.Another possible explanation for the enhancement of

growth is that there is a system for ATP formation fromoxidative phosphorylation, because cytochrome inductionoccurs exclusively under aerobic conditions and is depen-dent upon the environment for exogenous heme (4, 22, 23,

TABLE 2. Effects of lipoic acid on glucose utilization and endproduct formation by Citr+ L. lactis subsp. lactis 3022 cells

grown aerobically in the presence of hemin or Cu2+a

Amt Amt (mmol) of end products formed:

Addition (mmol) ofAddition glucoseutilized Lactate Acetate Diacetyl Acetoin

None 29.2 53.7 7.2 0.11 <5.01.0 mM lipoic acid 28.0 44.5 8.9 0.02 <5.010 ,uM hemin 98.7 31.1 9.0 0.98 61.310 puM hemin + 1.0 70.7 19.5 31.2 0.75 37.6mM lipoic acid

0.1 mM CuCl2 34.5 43.3 5.8 0.31 25.50.1 mM CuCl2 + 1.0 31.2 40.9 1Q.2 0.03 <5.0mM lipoic acida Citr+ L. lactis subsp. lactis 3022 cells were grown at 30°C for 24 h (see

Table 1, footnote a), except that the medium contained 1.0 mM lipoic acid, 10FLM hemin, 0.1 mM CuC12, and 10 p.M hemin plus 1.0 mM lipoic acid or 0.1mM CuCl2 plus 1.0 mM lipoic acid.

25). However, no trace of cytochrome peaks was recognizedin Citr+ L. lactis subsp. lactis 3022 cells grown aerobicallywith hemin (Fig. 2). Furthermore, antimycin A, which is aninhibitor of oxidative phosphorylation, did not reduce thestimulative effects of hemin on growth, glucose utilization,and acetoin production. These results indicate that thecytochrome-mediated respiration system is absent in thecells grown aerobically with hemin. Therefore, it appearedthat the cells grown aerobically with hemin or Cu2+ wouldhave an alternative system to reoxidize NADH which is

460 520 580 640 700

Wave Length (nm)

FIG. 2. Light absorption spectra of Citr+ L. lactis subsp. lactis3022 and S. cerevisiae 8004 cells. S. cerevisiae 8004 cells (A) weregrown in a basal medium at 30°C for 24 h. Citr' L. lactis subsp.lactis 3022 cells were grown aerobically at 30°C for 24 h in a basalmedium with (B) and without (C) 10 ,uM hemin. The cells wereprepared as described in Materials and Methods.

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TABLE 3. Effects of hemin on the activities of several enzymesof Citr+ L. lactis subsp. lactis 3022a

Addition to: Sp act (mU/mg of protein) of:Assay NADH Diacetyl Diacetyl Lactateno. Medium Reaction oxi- syn- reduc- dehydro-

mixture dase thase tase genase

1 None None 40 8.7 2,810 1002 None 5 F.M hemin 40 30.2 2,590 1003 10 ,uM hemin None 30 41.1 1,570 NDba Cell extracts were prepared after Citr+ L. lactis subsp. lactis 3022 cells

were grown aerobically in a basal medium with and without 10 ,uM hemin at30°C for 15 h. Assays 1 and 2 were performed with the same cell extract whichwas prepared from the cells grown aerobically in a basal medium withouthemin.

b ND, Not determined.

formed via glycolysis, since the lactate production per moleof glucose was markedly repressed.

Acetoin formation from diacetyl by diacetyl reductase iscoupled to the oxidation of NADH to NAD. In this study,the diacetyl reductase activity of Citr+ L. lactis subsp. lactis3022 was markedly high. Furthermore, the activity of di-acetyl synthase was greatly stimulated by the addition ofhemin or Cu2+ to the medium or reaction mixture (Tables 3and 4). These results indicate that the pyruvate formed viaglycolysis would be rapidly converted to diacetyl in aeratedculture with hemin or Cu2+ and that the diacetyl would thenbe converted to acetoin by the NAD-dependent diacetylreductase. In fact, most of the glucose was converted todiacetyl and acetoin more rapidly in the culture with bothhemin and Cu2+ than in the cutlure without these additives(Fig. 1; Table 1).However, growth, glucose utilization, and acetoin produc-

tion were apparently lower in the culture with both KCN andhemin than in the culture supplemented with only hemin,because KCN reduced the stimulatory effect of hemin ondiacetyl synthase activity (T. Kaneko, unpublished results).KCN is known to combine strongly with heme compounds(26). Therefore, the amount of diacetyl needed for thesynthesis of acetoin is likely to be lowered by the addition ofKCN, and the pathway of acetoin production from diacetylwould be essential to reoxidize NADH formed via glycolysiswhen Citr+ L. lactis subsp. lactis 3022 cells were grownaerobically with hemin or Cu2+.

Diacetyl is normally formed enzymatically by condensa-tion of hydroxyethylthiamine PP1 with acetyl coenzyme A(28). We defined the activity of diacetyl formation frompyruvate (diacetyl synthase) as the amount of enzyme whichcatalyzes the production of 1 p.mol of diacetyl from pyruvate

TABLE 4. Effects of lipoic acid on diacetyl synthase activity inthe presence of hemin or Cu2+

Diacetyl synthaseAddition sp acta (mU/mg

of protein)

None ................................................ 5.90.5 mM lipoic acid ....................................... 4.60.1 mM CuC12 ....................................... 22.80.1 mM CuCl2 + 0.5 mM lipoic acid ................... 17.210 ,uM hemin ..................... .................. 21.210 ,uM hemin + 0.5 mnM lipoic acid..................... 18.6

a Diacetyl synthase activities were assayed as described in Materials andMethods, except that the reaction mixture contained lipoic acid, hemin, orcu2+.

in the presence of thiamine PP1 (see Materials and Methods).Therefore, diacetyl synthase catalyzes both the formation ofacetyl-CoA from pyruvate and the condensation of acetyl-CoA with hydroxyethylthiamine PP1. In this study, formatewas not detected when the cells were grown aerobically withand without hemin, probably because pyruvate-formatelyase is sensitive to oxygen (1). Therefore, acetyl-CoAformation by Citr+ L. lactis subsp. lactis 3022 in aeratedculture would be catalyzed by the pyruvate dehydrogenasecomplex. Hemin or Cu2+ stimulated diacetyl and acetoinproduction, although acetate production was repressedwhen excess glucose was present (Fig. 1). However, in thepresence of lipoic acid, an essential cofactor of the dihydro-lipoamide acetyltransferase part of the pyruvate dehydroge-nase complex, hemin or Cu2+ enhanced acetate productionand then repressed diacetyl and acetoin production (Table2). Cogan et al. (5) also showed that acetate production inaerated culture depended on lipoic acid. In this study, theactivity of diacetyl synthase was lowered by lipoic acid,regardless of the presence of hemin or Cu2+ (Table 4).However, acetate production from acetyl-CoA was notstimulated by the addition of lipoic acid to the reactionmixture (T. Kaneko, unpublished results). These resultsindicate that hemin or Cu2+ would stimulate acetyl-CoAformation from pyruvate and that lipoic acid would inhibitthe condensation of acetyl-CoA with hydroxyethylthiaminePP1. Therefore, it appeared that acetyl-CoA not used fordiacetyl synthesis would be converted to acetate.When the cells were grown aerobically with hemin, the

amount of acetyl-CoA available for acetate synthesis wouldnot be sufficient in the presence of glucose, because most ofacetyl-CoA is utilized for diacetyl and acetoin production toregenerate NAD. The reduction of acetate production wouldbe the reason why the YGIu value in hemin-containing culturewas lower than in the culture without hemin. However, afterthe disappearance of glucose from the medium, the cellsutilized the lactate accumulated in the culture and producedacetate concomitantly when grown in the presence of hemin(Fig. 1). Presumably, since NADH formation is low in theabsence of glucose, the regeneration of NAD coupled withthe conversion of diacetyl to acetoin is not so stronglyrequired.

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