High-Efficiency Conversion of Pyruvate to Acetoin by Lactobacillus plantarum during ph-controlled and Fed-Batch Fermentationst

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1 APPLID AND NVIRONMNTAL MICROBIOLOGY, Aug. 1987, p Vol. 53, No /87/ $2./ Copyright 1987, Americn Society for Microbiology High-fficiency Conversion of Pyruvte to Acetoin by Lctobcillus plntrum during -Controlled nd Fed-Btch Fermenttionst THOMAS J. MONTVILL,* AMY HAN-MING HSU, AND MARY. MYR Deprtment of Food Science, New Jersey Agriculturl xperiment Sttion, Cook College, Rutgers-The Stte University of New Jersey, New Brunswick, New Jersey 893 Received 12 Mrch 1987/Accepted 4 My 1987 The influence of on the type nd concentrtion of metbolites produced from pyruvte by Lctobcillus plntrum ATCC 814 ws exmined in -controlled fermentors t vlues of 4.5 to 6.5. Specific growth rtes, cell dry weights, nd dicetyl concentrtions were highest t 5.5, with vlues of.78 h-1, 19 mg/liter, nd 1.2 mm, respectively. While the conversion efficiency (millimoles of cetoin formed per millimoles of pyruvte utilized) ws highest (94.6%) t 4.5, cetoin levels were similr (2 mm) between 4.5 nd 5.5. Feeding sttionry-phse cells exogenous pyruvte incresed cetoin levels to 78 mm. Lctobcillus plntrum is homofermenttive bcterium tht ferments lctose nd glucose lmost entirely to lctic cid, but lso produces trce (prts per million) quntities of cetoin, dicetyl, nd 2,3-butnediol (13, 14). Reltive insensitivity to bcteriophge ttck; tolernce to low, het, nd slt; generlly recognized s sfe sttus in foods (3); nd the bility to ferment more thn 2 different sugrs (4) recommend this species for use in biotechnologicl processes. Homofermenttive species cn be shifted to heterofermenttive metbolism (16, 3). This suggests tht the distribution of pyruvte to vrious end products, the key to the diversity of microbil end products (22), cn be mnipulted experimentlly. Dicetyl nd cetoin synthesis cn be incresed to improve the flvor of diry products (5-8, 13-15), but true hyperproduction (i.e., millimolr rther thn prts per million levels) would constitute flvor defect nd hs not been exmined. However, high-level production of cetoin would hve pplicbility in other food systems. Microbilly produced cetoin could be used directly, s precursor to tetrmethylpyrzine (1), or oxidized to dicetyl (21) for use s nturl flvor. It might lso be reduced to 2,3-butnediol (21, 31) nd used s fuel. Thus, if pyruvte dissimiltion could be shifted from lctte to the cetoin, dicetyl, 2,3-butnediol pthwy, number of industrilly importnt fermenttions might ensue. The regultion of pthwys from pyruvte to cetoin is complex nd not entirely cler. The ddition of citrte enhnces dicetyl synthesis by inducing citrse nd cette synthetse (24) nd increses cetoin synthesis in L. plntrum, but it is inhibitory to growth nd cell mss synthesis (27). Pyruvte lso stimultes dicetyl nd cetoin synthesis (13, 15, 27). Lctobcillic strter cultures form cetoin nd dicetyl in proportion to the concentrtion of pyruvte dded (1). In L. plntrum ATCC 814 test tube cultures, pyruvte utiliztion sturtes t bout 2 mm nd results in cetoin production t 3% of the theoreticl yield; the use of higher pyruvte concentrtions simply increses residul pyruvte levels (27). The objectives of the studies reported herein were to determine the influence of on the distribution of pyruvte to lctte, dicetyl, nd cetoin in L. plntrum nd to demonstrte the temporl correltion of * Corresponding uthor. t Mnuscript D of the New Jersey Stte Agriculturl xperiment Sttion cetoin synthesis with pyruvte utiliztion. We lso report the use of regenerble fed-btch fermenttion to increse finl cetoin concentrtions. (Some of this informtion ws presented t the 86th Annul Meeting of the Americn Society for Microbiology, Wshington, D.C., 23 to 28 Mrch 1986). MATRIALS AND MTHODS Culture conditions. L. plntrum ATCC 814, obtined from the Americn Type Culture Collection (Rockville, Md.), ws mintined in Lctobcillus MRS gr slnts (Difco Lbortories, Detroit, Mich.) t 4 C nd trnsferred monthly. We used modifiction of the medium of Crig nd Snell (9), designted CS-T nd contining (per liter of distilled wter): yest extrct, 1 g; KH2PO4, 6 mg; K2HPO4, 6 mg; MnSO4 4H2, 2 mg; MgSO4 7H2, 2 mg; FeSO4. 7H2, 1 mg; NCl, 1 mg; Tween 8, 5 ml; nd supplemented with 8 mm sodium pyruvte (Sigm Chemicl Co., St. Louis, Mo.). The medium ws djusted to 4.5, 5., 5.5, 6., or 6.5 with HCl. The medi were dispensed into screw-cp test tubes nd flsks for inocultion nd into the fermentor vessel. These were utoclved for 2 min t 121C. The inocul for the fermentors were prepred by three successive trnsfers in CS-T medium with incubtion t 37C for 24 h. A 3 to 5% (vol/vol) portion of the third culture ws used to inoculte Mouse fermentor (Queue Systems, Prkersburg, W.V.). The fermentor ws operted t 2 rpm, 25-ml/min nitrogen overly, 37C, with mintined within.5 unit of the setpoint by cid (.5 N HCl) nd bse (.25 N NOH) delivery pumps clibrted to monitor the totl volumes used. Smples (2 ml) were drwn septiclly, nd cell density ws mesured s A66 t 1-h intervls until the sttionry phse, nd t lest every 6 h therefter. Cells were then removed by centrifugtion. During the controlled fermenttions, these superntnts were stored in tightly cpped test tubes t -8C until needed for gs chromtogrphic nlysis. During the fed-btch fermenttion, mintined t 5.3, the superntnts were filtered (.45-iim pore size) nd nlyzed immeditely by high-pressure liquid chromtogrphy s described below. When residul pyruvte levels plteued, concentrted filter-sterilized sodium pyruvte ws septiclly dded to the fermentor vessel to bring the totl

2 VOL. 53, 1987 pyruvte concentrtion to circ 9 mm. Concentrted sterile CS-T medium ws fed t the point when no more cetoin ws being produced nd no more pyruvte ws being utilized. Microscopic checks nd periodic plting on MRS medium were used to verify culture purity. Specific growth rtes were clculted from the liner portion of the growth curves. Cell dry weights were clculted from the A6w by the method of Koch (23). Pyruvte utiliztion rtes (millimoles per hour) nd cetoin production rtes (millimoles per hour) were clculted for periods of liner chnge. The cid ddition rte (millimoles per hour) nd pyruvte conversion efficiency (percent theoreticl) were lso clculted for ech fermenttion. Metbolite nlysis. Pyruvte nd lctte were quntified with Vrin high-pressure liquid chromtogrph (model 221; Vrin Corp., Wlnut Creek, Clif.) equipped with model 21 solvent delivery system, model 25 UV detector set t 22 nm, Polypore H ction-exchnge column (22 cm by 4.6-mm inner dimeter) equipped with reverse-phse C18 gurd column (Brownlee Lbortories, Snt Clr, Clif.), nd.9 N H2SO4 s the mobile phse (27). The detector ws reset to 192 nm for cetoin determintions (M.. Meyer, M.S. thesis, Rutgers-The Stte University of New Jersey, New Brunswick, 1987). Dicetyl nd cetoin levels in the -controlled fermenttions were quntified by gs chromtogrphic nlysis of culture superntnt extrcts. The extrcts were prepred by dding 1,ul of hexnl (s n internl stndrd) to 5 ml of culture superntnt, extrcting with 2 ml of ethyl ether, blending on vortex mixer for 1 min, nd dding 1 g of nhydrous sodium sulfte to brek the emulsion. The solvent lyer ws trnsferred to n ir-tight tube nd sved t 4 C until nlysis. xtrcts were injected onto gs chromtogrph (model 583A; Hewlett-Pckrd Co., Avondle, P.) equipped with mdoel 1885A printing integrtor nd flme ioniztion detector nd modified to ccept wide-bore cpillry column (3 m by.75-mm inner dimeter) pcked with Supelcowx 1 (Supelco, Inc., Bellefonte, P.). Stisfctory seprtion of dicetyl nd cetoin ws obtined when the oven temperture ws held t 5 C for 7 min, incresed to 12 C t 1 C/min, nd then held t 12 C for 1 min. Dicetyl nd cetoin concentrtions were clculted from seprte correspondence fctor versus concentrtion stndrd curves for ech vlue exmined s described elsewhere (A. H.-M. Hsu, M.S. thesis, Rutgers-The Stte University of New Jersey, New Brunswick, 1987). RSULTS -controlled fermenttions. When L. plntrum ws cultured in CS-T medium mintined t 4.5, 5., 5.5, 6., or 6.5 in seprte fermentor runs, mny differences in fermenttion prmeters were observed. Cell growth ws rpid (,u =.78 h-1) t 5.5 nd slowed mrkedly with decresing (Fig. 1). The growth rte on the lkline side of the optimum lso declined, but not s severely. Cell dry weights of 24-h-old cultures followed similr pttern. Dicetyl levels were lso highest t 5.5, but only 1.2 mm ws formed fter 48 h of fermenttion. No dicetyl ws detected in cultures grown t 4.5, 6., or 6.5. The optimum for cetoin synthesis ws much broder. While the 5.5 fermenttion hd the most cetoin t 24 h, by 48 h cetoin levels in fermentors t 4.5 nd 5.5 incresed to lmost equl levels. Less cetoin ws formed during fermenttions mintined t 6. nd 6.5. ven though there ws considerble utiliztion of pruvte not ccounted for by ACTOIN PRODUCTION BY L. PLANTARUM 1799 increses in cetoin production t these vlues between 24 nd 48 h, no cette or ethnol ws detected. Lctic cid production ws similr (12.9 to 13.9 mm) in cultures mintined t 5. to 6.5, but no lctte ws formed in the 4.5 culture. The reltionship between residul pyruvte concentrtions t 24 h nd ws inversely relted to cetoin production. Pyruvte utiliztion nd cetoin levels t 24 h were used to clculte conversion efficiencies (percent theoreticl, bsed on 2 mol of pyruvte forming 1 mol of cetoin). The conversion efficiencies were 94.6, 46., 4., nd % t 4.5, 5., 5.5, nd 6., respectively. The conversion efficiency for the 6.5 fermenttion could not be clculted becuse the residul pyruvte concentrtion ws higher thn the initil level. Fed-btch experiments. Cells grew rpidly (,u =.48 h-1) nd entered the sttionry phse fter 8 h during the first stge of fed-btch fermenttion mintined t 5.3. Pyruvte ws rpidly utilized nd cetoin ws produced by sttionry-phse cells, but pyruvte ctbolism continued for lmost 24 h fter cetoin synthesis plteued (Fig. 2). Lctte levels incresed lte in the exponentil phse to 8 mm nd remined constnt until dy 5. Low levels (.8 mm) of dicetyl were formed during stge I; no dditionl dicetyl synthesis occurred during the rest of the fermenttion. When pyruvte utiliztion plteued, the culture ws fed dditionl pyruvte (stge II). Acetoin synthesis resumed during this second period of pyruvte utiliztion, but leveled off t n cetoin concentrtion of 38 mm. Pyruvte ctbolism continued without n increse in cetoin. No cette or ethnol ws detected t ny point in this experiment. Lctte levels dropped t the end of stge II. Pyruvte dded t 5.2 dys ws utilized firly rpidly, but no mrked increse in the rte of cetoin synthesis occurred during stge III. The cell density continued to decrese slowly owing to dilution by the cid used for mintennce. While there ws no chnge in cetoin or pyruvte concentrtions between dy 8 nd 1, the requirement for continued cid ddition for control indicted ongoing metbolic ctivity. Becuse the residul pyruvte concentrtion ws high (57 mm), dditionl pyruvte ws not fed. Insted, CS-T medium ws prepred in concentrted form so tht, upon ddition to the fermentor, it would be diluted to single-stretch medium. Upon ddition of the fresh CS-T medium, the culture grew exponentilly (pu =.24 h-1) fter short lg period. Although cells grew more slowly thn during stge I, they reched higher finl cell density. Lctte ws produced lte in the exponentil phse. In the erly sttionry phse, pyruvte ws ctbolized nd cetoin concentrtions incresed to finl concentrtion of 78 mm. DISCUSSION The ctbolism of pyruvte yielded primrily cetoin, low levels of lctte, nd, under some conditions, dicetyl, but no cette or ethnol under the conditions used in these studies. This contrsts with the report of Collins nd Bruhn (7) tht resting "Streptococcus dicetilctis" (now clssified s Streptococcus lctis) cells convert 73% of pyruvte to cette nd only 1% to dicetyl nd cetoin. Hickey et l. (21) reported tht L. plntrum ctbolizes pyruvte to cette, cetoin, nd lctte t 6:2:1 rtio. Similr incrementl cetoin-to-lctte rtios occurred during stges I nd IV of the fed-btch experiment. The bsence of cette in our cultures is probbly due to mintennce of dissolved oxygen t %, since the presence of oxygen is required for the production of cette by L. plntrum (28).

3 18 MONTVILL T AL. APPL. NVIRON. MICROBIOL. - C, 2cn -. 3: 2 c._ U C Acetoln-24hr A Acetoin-48hr Ā, 2 - q D O Pyruvte-24hr A Pyruvts-48hr o A un v -I I I I, I _1, FIG. 1. Influence of culture on fermenttion chrcteristics of L. plntrum ATCC 814. Cultures were grown in CS-T medium contining 8 mm sodium pyruvte in fermentors t 37 C, 2 rpm, dissolved oxygen = %, nd the vlues given. Specific growth rtes (,u) nd cell density (s cell dry weight [CDW]) t 24 h (A), dicetyl levels t 48 h (B), nd cetoin (C) nd residul pyruvte (D) concentrtions fter 24 nd 48 h were determined s described in the text. The conversion efficiency (moles of cetoin produced per moles of pyruvte utilized) for cetoin synthesis ws highest t 4.5. L. plntrum ATCC 814 hs both L(+) nd D(-) NAD-dependent lctte dehydrogenses (nldhs), C nd C , respectively (11, 17, 18). These nldhs re nonllosteric (2) nd hve optim between 7. nd 8.5 (11, 12, 25). Thus, t 4.5, intrcellulr cidifiction would decrese nldh ctivity, no lctte would be produced (s ws the cse in the 4.5 fermenttion), nd pyruvte could be converted to cetoin with incresed efficiency. The production of lctte in the fermenttions t 5. to 6.5 reduced their conversion efficiencies. During the fed-btch fermenttion t 5.3, lctte ccumulted only during periods of cell growth (stges I nd IV) nd ws ctbolized lte in stge 2. Lctte ctbolism suggests scvenging metbolism in which lctte recycle to pyruvte provides n lternte energy source, s proposed by Snoswell (29). This conversion of lctte to pyruvte undoubtedly occurs vi NAD-independent LDHs (ildh; C ) since nldhs re not reversible under physiologicl conditions (17). Both L(+) nd D(-) ildhs hve been reported for L. plntrum nd hve optim of 5.8 to 6.6 (12). Gsser's (18) inbility to detect ildh ctivity in L. plntrum ATCC 814 might hve been cused by the use of n inpproprite electron cceptor in the in vitro ssy. High cetoin concentrtions occurred t vlues of 4.5 to 5.5 fter 48 h. The optim observed here were slightly broder thn the optiml for cetoin nd dicetyl production for Lctobcillus csei, which is between 4.5 nd 5. (2). Indirect ssy of cetoin production by dried cells of Lctobcillus rbinosus lso demonstrted n cidic optimum (26). Acetoin production ws much greter thn dicetyl production, in greement with previous reports (14, 21). Dicetyl reductse, which hs optiml ctivity t 4.5 to 5. (2), my be responsible for the low dicetyl levels found t low vlues. The pprent conversion efficiencies during periods of liner pyruvte utiliztion nd cetoin production in the fed-btch fermenttion were high (Tble 1), but intervening periods when no cetoin ws produced lowered the overll conversion efficiency for the 13-dy fed-btch fermenttion to bout 28%. The rtes of pyruvte utiliztion declined with

4 VOL. 53, 1987 ACTOIN PRODUCTION BY L. PLANTARUM ( -._ ( -i J.2 C Is C, it Incubtion Time (dy) FIG. 2. Time course dt for fed-btch fermenttion of L. plntrum ATCC 814. L. plntrum ws inoculted (stge I) into CS-T medium contining 8 mm pyruvte nd mintined t 5.3 in fermentor t 37 C, 2 rpm, nd dissolved oxygen = %. When pyruvte utiliztion plteued, dditionl pyruvte ws septiclly dded (i.e., t the beginning of stges II nd III). When pyruvte nd cetoin levels becme reltively constnt (dys 8 to 1), the culture ws fed concentrted CS-T medium to regenerte the cells nd strt stge IV. The cid dded ws.5 N HCl. successive feedings of sttionry-phse cells. Pyruvte utiliztion by L. plntrum is enhnced by n dditionl energy source (2, 14, 21) which my be depleted during the course of the fed-btch fermenttion nd regenerted during stge IV. Higher thn theoreticl yields cn be ttributed to endogenous pyruvte production. Lctte production in medi lcking fermentble 'crbohydrtes hs been ttributed to TABL 1. Rtes of pyruvte uitiliztion nd cetoin production by L. plntrum ATCC 814 during the 5.3 fed-btch fermenttion Pyruvte Acetoin Conversion Stge utiliztion production efficiency (% (mm/h) (mm/h) theoreticl) I (dys -2) II (dys 2-5) III (dys 5-1) IV (dys 1-14) Rtes were clculted from the liner periods in Fig. 2 to derive the conversion efficiency. ctbolism of yest extrct components (27). Since this must occur through pyruvte s n intermedite, the high residul pyruvte in the 6.5 fermenttion nd the higher thn theoreticl conversion efficiency in stge II of the fed-btch fermenttion might be cused by similr route. Acetoin concentrtions were incresed by using fed-btch fermenttion; to our knowledge, the 78 mm cetoin concentrtion observed here constitutes the highest level reported for L. plntrum. The conversion of cidic intrcellulr pyruvte to neutrl cetoin my provide the cell with detoxifiction mechnism s hypothesized by Hrvey nd Collins (19). The fed-btch fermenttion suggests tht this conversion is regulted. The mechnism of this regultion is the subject of ongoing investigtions in this lbortory. ACKNOWLDGMNTS This reserch ws supported by stte nd U.S. Htch Act funds nd by grnt from the Rutgers University Reserch Council. We thnk Greg Koch for expert nocturnl ssistnce during the fed-btch experiments.

5 182 MONTVILL T AL. LITRATUR CITD 1. Abo-lng, I. G., nd C. F. Hegzi Fctors influencing the production of cetoin nd dicetyl by lctic cid bcteri in skim milk. Nhrung 25: Brnen, A. L., nd T. W. Keenn Dicetyl reductse of Lctobcillus csei. Cn. J. Microbiol. 16: Chssy, B. M Prospects for improving economiclly significnt Lctobcillus strins by genetic engineering. Trends Biotechnol. 3: Chen, K. H., R. F. McFeeters, nd H. P. Fleming Complete heterolctic cid fermenttion of green bens by Lctobcillus cellobiosis. J. Food Sci. 48: Christensen, M. D., nd C. S. Pedersen Fctors ffecting dicetyl production by lctic cid bcteri. Appl. Microbiol. 6: Collins,. B Biosynthesis of flvor compounds by microorgnisms. J. Diry Sci. 55: Collins,. B., nd J. C. Bruhn Roles of cette nd pyruvte in the metbolism of Streptococcus dicetilctis. J. Bcteriol. 13: Collins,. B., nd R. A. Speckmn Influence of cetldehyde on growth nd cetoin production by Leuconostoc citrovorum. J. Diry Sci. 57: Crig, J. A., nd.. Snell The comprtive ctivities of pntethine, pntothenic cid, nd coenzyme A for vrious microorgnisms. J. Bcteriol. 61: Demin, A. L., M. Jckson, nd N. R. Trenner Thiminedependent ccumultion of tetrmethylpyrzine ccompnying muttion in the isoleucine-vline pthwy. J. Bcteriol. 94: Dennis, D., nd N.. Kpln D- nd L-lctic cid dehydrogenses in Lctobcillus plntrum. J. Biol. Chem. 235: Doelle, H. W Nicotinmide denine dinucleotidedependent nd nicotinmide denine dinucleotide-independent lctte dehydrogenses in homofermenttive nd heterofermenttive lctic cid bcteri. J. Bcteriol. 18: l-gendy, S. M., H. Abdel-Glil, Y. Shhin, nd F. Z. Hegzi Acetoin nd dicetyl production by Lctobcillus plntrum ble to use citrte. J. Food Prot. 46: l-gendy, S. M., H. Abdel-Glil, Y. Shhin, nd F. Z. Hegzi Acetoin nd dicetyl production by homofermenttive nd heterofermenttive lctic cid bcteri. J. Food Prot. 46: l-gendy, S. M., H. Abdel-Glil, Y. Shhin, nd F. Z. Hegzi Acetoin nd dicetyl production by Lctobcillus csei. J. Food Prot. 46: Fordyces, A. M., V. L. Crow, nd T. D. Thoms Regultion of product formtion during glucose or lctose limittion in nongrowing cells of Streptococcus lctis. Appl. nviron. Microbiol. 48: APPL. NVIRON. MICROBIOL. 17. Grvie,. I Bcteril lctte dehydrogenses. Microbiol. Rev. 44: Gsser, F lectrophoretic chrcteriztion of lctte dehydrogenses in the genus Lctobcillus. J. Gen. Microbiol. 62: Hrvey, R. J., nd. B. Collins Roles of citrte nd cetoin in the metbolism of Streptococcus dicetilctis. J. Bcteriol. 12: Hensel, R., U. Myr, H. Fujiki, nd. Kndler Comprtive studies of lctte dehydrogenses in lctic cid bcteri. ur. J. Biochem. 8: Hickey, M. W., A. J. Hillier, nd G. R. Jgo Metbolism of pyruvte nd citrte in lctobcilli. Aust. J. Biol. Sci. 36: Ingrhm, J. L.,. Mrole, nd F. C. Neidrdt Polymeriztion, biosynthesis, fueling, nd trnsport, p In Growth of the bcteril cells. Sinur Assocites, Inc., Sunderlnd, Mss. 23. Koch, A Growth mesurement, p In P. Gerdrdt, R. G.. Murry, R. N. Costilow,. W. Nester, W. A. Wood, N. R. Krieg, nd G. B. Phillips (ed.), Mnul of methods for generl bcteriology. Americn Society for Microbiology, Wshington, D.C. 24. Mellerick, D., nd T. M. Cogn Induction of some enzymes of citrte metbolism in Leuconostoc lctis nd other heterofermenttive lctic cid bcteri. J. Diry Res. 48: Mizushim, S., T. Hiym, nd K. Kithr Quntittive studies on the glycolytic enzymes of Lctobcillus plntrum. III. Intrcellulr ctivities of reverse rections of D- nd L- lctte dehydrogenses during glucose fermenttion. J. Gen. Appl. Microbiol. 1: Mot, A. G., nd H. C. Lichstein Fctors ffecting the formtion of cetylmethylcrbinol by Lctobcillus rbinosus. J. Bcteriol. 66: Montville, T. J., M.. Meyer, nd A. H. M. Hsu Influence of crbon substrte on lctic cid, cell mss, nd dicetylcetoin production in Lctobcillus plntrum. J. Food Prot. 5: Murphy, M. G., nd S. Condon Comprison of erobic nd nerobic growth in glucose medium. Arch. Microbiol. 138: Snoswell, A. M Flvins of Lctobcillus rbinosus. Aust. J. xp. Biol. 37: Thoms, T. D., D. C. llwood, nd V. M. C. Longyer Chnge from homo- to heterolctic fermenttion by Streptococcus lctis resulting from glucose limittion in nerobic chemostt cultures. J. Bcteriol. 138: Ui, S., N. Mtsuym, H. Msud, nd H. Murki Mechnisms for the formtion of 2,3-butnediol stereoisomers in Klebsiell pneumonie. J. Ferment. Technol. 62: