Applied Microbiology and Biotechnology

, Volume 72, Issue 3, pp 421–429

Variations in the energy metabolism of biotechnologically relevant heterofermentative lactic acid bacteria during growth on sugars and organic acids

Authors

  • T. Zaunmüller
    • Institut für Mikrobiologie und WeinforschungJohannes Gutenberg-Universität Mainz
  • M. Eichert
    • Institut für Mikrobiologie und WeinforschungJohannes Gutenberg-Universität Mainz
  • H. Richter
    • Institut für Mikrobiologie und WeinforschungJohannes Gutenberg-Universität Mainz
    • Institut für Mikrobiologie und WeinforschungJohannes Gutenberg-Universität Mainz
Mini-Review

DOI: 10.1007/s00253-006-0514-3

Cite this article as:
Zaunmüller, T., Eichert, M., Richter, H. et al. Appl Microbiol Biotechnol (2006) 72: 421. doi:10.1007/s00253-006-0514-3

Abstract

Heterofermentative lactic acid bacteria (LAB) such as Leuconostoc, Oenococcus, and Lactobacillus strains ferment pentoses by the phosphoketolase pathway. The extra NAD(P)H, which is produced during growth on hexoses, is transferred to acetyl-CoA, yielding ethanol. Ethanol fermentation represents the limiting step in hexose fermentation, therefore, part of the extra NAD(P)H is used to produce erythritol and glycerol. Fructose, pyruvate, citrate, and O2 can be used in addition as external electron acceptors for NAD(P)H reoxidation. Use of the external acceptors increases the growth rate of the bacteria. The bacteria are also able to ferment organic acids like malate, pyruvate, and citrate. Malolactic fermentation generates a proton potential by substrate transport. Pyruvate fermentation sustains growth by pyruvate disproportionation involving pyruvate dehydrogenase. Citrate is fermented in the presence of an additional electron donor to acetate and lactate. Thus, heterofermentative LAB are able to use a variety of unusual fermentation reactions in addition to classical heterofermentation. Most of the reactions are significant for food biotechnology/microbiology.

Keywords

Sugar fermentationLactic acid bacteriaHeterofermentativeOrganic acids

Introduction

Sugars are the characteristic substrates for lactic acid bacteria (LAB). Homofermentative LAB ferment hexoses by the reactions of glycolysis and lactate dehydrogenase to lactic acid. At slow growth and low glycolytic flux rates the homofermentative bacteria shift to mixed acid fermentation with formate, acetate, ethanol, and lactate as the products. The change is caused by regulation of the lactate dehydrogenase and pyruvate formate lyase activities, which are subject to control by the catabolic and anabolic flux rates and changes in the NADH/NAD ratios (Melchiorsen et al. 2002; Garrigues et al. 1997, 2001). Overall, limiting catabolism leads to mixed acid fermentation and nonlimiting catabolism with high glycolytic flux to homofermentative metabolism.

Heterofermentative LAB play an important role in production, preservation, and improvement of food, mainly of plant origin (for reviews, see Steinkraus 1993; Ross et al. 2002; Caplice and Fitzgerald 1999; Mills et al. 2005; Moreno-Arribas and Polo 2005). The products of the fermentation of sugars and organic acids are essential in this process. Sugars are the preferred substrates of the heterofermentative LAB. Heterofermentative LAB grow in plant-associated environments like degrading plant material (Leuconostoc strains), grape must and wine (Oenococcus oeni, Lactobacillus hilgardii, Lactobacillus brevis) or sourdough (Lactobacillus sanfranciscensis and Lactobacillus fermentum) (Rodas et al. 2005; Randazzo et al. 2005). The same biotopes also house facultatively heterofermentative LAB like Lactobacillus plantarum and Lactobacillus pentosus. The plant material contains, in addition to hexoses (glucose, fructose, mannose, and lactose), considerable amounts of pentoses (xylose and ribose). Hexoses and pentoses are fermented by the heterofermentative LAB by the phosphoketolase (or oxidative pentose–phosphate) pathway. Excess hexoses are also used for the production of exopolysaccharides like dextran, levan, and fructan by various groups of LAB (Cerning 1990). A second class of substrates fermented by many heterofermentative LAB is organic acids, which is common in plant material, and arginine. Thus, malate, citrate, or pyruvate are fermented either on their own or as a cosubstrate with hexoses (for reviews, see Mills et al. 2005; Salou et al. 1994; Lolkema et al. 1995; Wagner et al. 2005).

The review deals with peculiarities of the carbon metabolism of obligately heterofermentative LAB. Heterofermentative fermentation of hexose includes limiting steps, which are overcome by modifications of the fermentation reaction or by the use of electron acceptors like fructose, O2, pyruvate, or citrate. In the same way, fermentation reactions of organic acids and of arginine will be discussed. The role of the fermentation reactions for biotechnology will be addressed. Only obligately heterofermentative bacteria will be considered with emphasis on Leuconostoc mesenteroides and O. oeni.

Special features of hexose fermentation by O. oeni and L. mesenteroides

Fermentation and phosphoketolase pathway

The phosphoketolase pathway is optimized for the fermentation of pentoses, which are oxidized to pyruvate and acetyl-P (Fig.  1). The NADH produced is transferred to pyruvate, yielding lactate, and acetyl-P is converted to acetate. When hexoses are fermented by the same pathway, an extra four [H] [or two NAD(P)H] are released during conversion of the hexose to a pentose. In typical glucose fermentation, the extra two NAD(P)H are loaded onto acetyl-P/-SCoA, yielding ethanol instead of acetate. Shifting from pentoses to hexoses causes a drop in the growth rate by a factor of approx. 3 (Table 1) and a decrease in the growth yield by a factor of about 2 in accordance with the decrease in ATP yield (two ATP per pentose vs one ATP per hexose).
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Fig. 1

Fermentation of pentoses and hexoses by the phosphoketolase (oxidative pentose-P) pathway by O. oeni and L. mesenteroides. The additional four [H] or two NAD(P)H formed during hexose oxidation, and their reoxidation by the ethanol, erythritol, or glycerol pathways are shown in blue; the central phosphoketolase pathway for the degradation of pentoses in black. Minor and alternative pathways are shown with broken lines. Phosphoketolase (Xfp type) cleaves pentose-5P (xylulose-5P) or fructose-6P (genes 55 and 520 for O. oeni putative isoenzymes). Key enzymes and intermediates of the pathways are shown. AdhE Bifunctional acetaldehyde/ethanol dehydrogenase, LDH lactate dehydrogenase, Glyc-1P DH glycerol-1P dehydrogenase (genes 583 and 1,721 of O. oeni and L. mesenteroides, respectively; putative), Glyc-1P glycerol-1 phosphatase (genes 582 and 1,720 of O. oeni and L. mesenteroides, putative), Ery-4P erythrose-4P, Glyc-1P glycerol-1P, Fru-6P, fructose-1P, and GAP glyceraldehyde-3P

Table 1

Fermentation balances and growth parameters of O. oeni on sugars (with and without electron acceptors) under various conditions

Idealized reaction

 

ATP/gluc or ribosea (calculated)

YGlucoseb (g/mol)

μb(h−1)

Ribose

→1 Lac+1 ace

2

14.9c

0.101

Gluc

→1 Lac+1 EtOH+1 CO2

1

8.3

0.034

Gluc+2 fruc

→1 Lac+1 ace+2 Mtl+1 CO2

2 (0.7)d

13.9

0.096

Gluc+2 pyr

→3 Lac+1 ace+1 CO2

2

17.9

0.088

Gluc+2 cit

→3 Lac+3 ace+1 3CO2

2

NDe

0.084

Gluc+2 O2

→ 1 Lac+1 ace+2 H2O2+1 CO2

2

NDe

NDe

Lac Lactate, ace acetate, Gluc, glucose, fruc fructose, EtOH ethanol, Mtl mannitol, pyr pyruvate, and cit citrate

aAccording to Fig. 2

bValues from Richter et al. 2003a and unpublished report

cYRibose

dATP per hexose (glucose + fructose)

eNo comparable values

Studies with O. oeni, L. mesenteroides, and other heterofermentative LAB showed that the slow growth on glucose is caused by the low activity of the ethanol pathway in the reoxidation of the extra two NAD(P)H (Maicas et al. 2002; Richter et al. 2001, 2003a). The key enzymes of the phosphoketolase pathway including glucose-6P dehydrogenase and 6P-gluconate dehydrogenase have activities, which are distinctly above the activities required for maintaining glucose fermentation (Richter et al. 2001). The acetaldehyde dehydrogenase activity of the bifunctional acetaldehyde/ethanol dehydrogenase of the ethanol pathway, on the other hand, has activities, which are only at the same level, or lower, as the rate of glucose fermentation. When the cellular contents of HSCoA, and thus of acetyl-CoA, were limiting, the activity of acetaldehyde dehydrogenase dropped further by a factor of approx. 2 and limited catabolism and growth. Limitation of HSCoA (and consequently acetyl-CoA) can be produced by shortage of d-pantothenate (Richter et al. 2001), an essential precursor of HSCoA in O. oeni and other LAB (Garvie 1967; http://www.jgi.doe.gov). The result is a decrease in production of ethanol and a partial shift to alternative, HSCoA-independent production of erythritol (Veigha-Da-Cunha et al. 1992, 1993; Richter et al. 2001) (Fig. 1). The ethanol/erythritol shift is reversed by the supply of sufficient d-pantothenate (Richter et al. 2001). During growth on C5-sugars like ribose, when the ethanol pathway is not required, d-pantothenate depletion has no significant effect on the fermentation pattern. Overall, the results show that the reoxidation of the extra two NAD(P)H from glucose oxidation in the ethanol pathway limits heterofermentative growth on glucose due to low acetaldehyde dehydrogenase activity.

d-Pantothenate as a specific growth factor for hexose fermentation

d-Pantothenate is required as a growth factor and precursor for HSCoA biosynthesis in many LAB (Garvie 1967). HSCoA is needed for the activation of fatty acids in biosynthetic and catabolic pathways. Ethanol formation from acetyl-CoA in the heterofermentative pathway is inhibited during d-pantothenate (and HSCoA) limitation, before general growth and cell synthesis is affected (Richter et al. 2001). It was suggested that 4′-O-(β-d-glucopyranosyl)-d-pantothenic acid, which is found in tomato and fruit juice (“tomato juice growth factor”), is the actual growth factor (Amachi et al. 1970). A minimal concentration of 50 μg/l was required for efficient growth on glucose. The glycosylated d-pantothenate could be produced from uridine 5′-diphosphate-glucose by plant glycosyltransferases. d-Pantothenate, however, has the same biological activity (50 to 80 μg/l) (Richter and Unden, unpublished report). O. oeni and L. mesenteroides lack the gene products for the synthesis of d-pantothenate from the precursors 2-acetolactate, l-valine, and β-alanine, which explains the need for d-pantothenate (http://www.jgi.doe.gov). The genes encoding the enzymes for the subsequent conversion of d-pantothenate to HSCoA are present with the exception of a structural gene for a PanF type pantothenate permease. d-Pantothenate therefore plays a specific role in the heterofermentative metabolism of O. oeni and other LAB (Richter et al. 2001).

Endogenous alternative pathways for [H] reoxidation

Heterofermentative LAB use alternative pathways for reoxidizing [H] to bypass the limiting ethanol pathway. Part of the extra NAD(P)H can be consumed by reduction of erythrose-4P to erythritol-4P and formation of erythritol as an end product (Fig. 1). Erythritol fermentation was shown in O. oeni, L. mesenteroides, and L. sanfranciscensis (Veigha-Da-Cunha et al. 1993; Stolz et al. 1995; Richter et al. 2001). Erythrose-4P is obtained (together with acetyl-P) from fructose-6P by d-xylulose 5-phosphate/d-fructose 6-phosphoketolase (Xfp). Various heterofermentative LAB and Bifidobacterium are able to use fructose-6P as a minor substrate for phosphoketolase, in addition to the principle substrate xylulose-5P (Veigha-Da-Cunha et al. 1993; Meile et al. 2001; Yin et al. 2005; Mills et al. 2005). The activity of the erythritol pathway is low and does not significantly increase the rate of glucose fermentation (Richter et al. 2001). Under pantothenate limitation or in resting cells up to 0.2 mol erythritol are formed per mole glucose. The residual enzymes of the erythritol pathway (erythritol-4P dehydrogenase) were not identified, and in the genomes of O. oeni and L. mesenteroides there are no genes with similarity to the eryB and eryA genes of Brucella abortus encoding erythritol-1P dehydrogenase and erythritol kinase (Sangari et al. 2000; Sperry and Robertson 1975). Glycerol which is also a minor alternative product of NAD(P)H reoxidation, is obtained by reduction of glyceraldehyde-3P to glycerol-1P followed by dephosphorylation (Fig. 1) (Veigha-Da-Cunha et al. 1993). Candidate genes encoding glycerol-1P dehydrogenase and phosphatase are found in the genome of O. oeni and L. mesenteroides (http://www.jgi.doe.gov) (Fig. 1). Biochemically, the pathway resembles that of erythritol formation, and erythritol and glycerol could be formed by the same or similar enzymes.

Cofermentation of hexoses with electron acceptors

Pyruvate, citrate, O2, or fructose can be used as external acceptors for reoxidation of the extra NAD(P)H from hexose oxidation (Fig. 2). The external acceptors reoxidize NAD(P)H more rapidly than the ethanol, erythritol, and glycerol pathways and can replace the pathways largely. When excess pyruvate is available, the four [H] are transferred nearly completely by the highly active lactate dehydrogenase to this acceptor (Nuraida et al. 1992; Richter et al. 2003a). In a similar way citrate is used after conversion to pyruvate via oxaloacetate by the use of citrate lyase and oxaloacetate decarboxylase Mae (see Fig. 4) (Salou et al. 1994; Hache et al. 1999; Stolz et al. 1995). Molecular O2 is converted by an oxidase to H2O2 (Maicas et al. 2002). Fructose is reduced to mannitol (Salou et al. 1994; Richter et al. 2003a,b). In glucose/fructose cofermentation, fructose is used mostly as an electron acceptor, whereas glucose is channeled to the phosphoketolase pathway as demonstrated by the use of 13C-labeled glucose or fructose (Richter et al. 2003b). When glucose is fermented in the presence of external acceptors, most of the acetyl-P/-CoA is no longer required as electron acceptor and is excreted as acetate instead of ethanol (Table 1). As a consequence, the ATP and molar growth yields increase by a factor of up to 2 per glucose. In addition, the growth rates increase by factors of 2 to 3 and approach those of ribose, in support of the view that the ethanol pathway is rate limiting. In agreement with this explanation, stimulation of growth by acceptors like citrate is observed only in heterofermentation, but not in homofermentation (Starrenburg and Hugenholtz 1991; Drinan et al. 1976).
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Fig. 2

Alternative pathways of O. oeni and L. mesenteroides for reoxidation of the “extra” four [H] (or two NAD(P)H) derived from hexose→pentose conversion by external electron acceptors (O2, pyruvate, citrate, and fructose). The overall reactions are given; for details, see text

When fructose is present as the only hexose, it serves both as a substrate for the phosphoketolase pathway and as an electron acceptor (Richter et al. 2003a,b). Under fructose limitation most of the fructose is fermented by the heterofermentative pathway similar to glucose (Table 1). However, under fructose excess conditions and in resting cells, up to two to three of the fructose are used as electron acceptor and large amounts of mannitol are produced. Therefore, fructose is fermented either by the phosphoketolase or by a combined phosphoketolase/mannitol pathway. The heterofermentative pathway yields one ATP per hexose, whereas in the phosphoketolase/mannitol pathway the ATP yield decreases to two to three ATP per hexose. Channeling of fructose in either pathway is regulated at the level of phosphoglucose isomerase by 6P-gluconate and erythrose-4P (Richter et al. 2003b). Mannitol dehydrogenase from Leuconostoc pseudomesenteroides and other bacteria can be used, together with an appropriate electron donor, to produce d-mannitol from fructose in metabolically engineered bacteria (Gaspar et al. 2004; Kaup et al. 2003; Hahn et al. 2003).

Overall, growth of O. oeni and other heterofermentative LAB on hexoses is stimulated by the presence of electron acceptors, which provide a by-pass for NAD(P)H reoxidation in the ethanol pathway. The erythritol and glycerol pathways are only of limited capacity, whereas the pathways using the external acceptors O2, pyruvate, citrate, and fructose have a much higher capacity and significantly increase the growth rate of the bacteria. The redox reactions are cytoplasmic without involvement of electron transport or generation of a proton potential.

Growth by fermentation of organic acids and arginine

Fermentation of organic acids plays an important role in the energy metabolism of heterofermentative LAB (Radler 1958, 1966; Radler and Brohl 1984; Stolz et al. 1995). Fermentation of citrate and malate is widespread under LAB, whereas fermentation of fumarate and pyruvate is found only in some bacteria.

Malate (“malolactic”) fermentation

Fermentation of malate by heterofermentative LAB is of physiological significance in wine and fruit juice, which contain high amounts of this C4-dicarboxylic acid. Malate is metabolized to lactate and CO2 (l-malate→l-lactate+CO2). The free energy of the reaction is conserved by a chemiosmotic mechanism (Salema et al. 1996), which depends on an electrogenic malate transport (Lolkema et al. 1995; Poolman et al. 1991; Konings 2002) (Fig. 3). In O. oeni (growing around pH 4) the transport is effected by a carrier-mediated uptake of monoanionic malate vs a carrier-independent efflux of lactic acid. In Lactococcus lactis growing at less acidic conditions, the transport is mediated by the malate2−/lactate antiporter. Both transport processes result in the net translocation of one charge per malate and energization of the membrane. In addition, one proton is consumed by the decarboxylation in the cytoplasm, generating a ΔpH. The proton motive force (1 H+/malate) derived from both processes is used by the bacteria for maintenance of pH homeostasis and for the uptake of nutrients. Malolactate fermentation stimulates growth of the bacteria but is not sufficient as the sole energy source for growth (Pilone and Kunkee 1976; Salema et al. 1996). The reaction results in deacidification of the medium by conversion of a divalent to a monovalent carboxylic acid. The process is used in winemaking by applying starter cultures or the natural population of O. oeni (Mills et al. 2005; Moreno-Arribas and Polo 2005; Liu 2002; Coucheney et al. 2005; Lonvaud-Funel 1999).
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Fig. 3

Pathways and carriers for the fermentation of malate and citrate by heterofermentative LAB (O. oeni, L. mesenteroides, and Lactobacillus lactis). Important enzymes and carriers (MleP malate carrier, MleA malolactic enzyme, CitP or MaeP citrate/lactate antiporter, Mae oxaloacetate decarboxylase, and CL citrate lyase) and intermediates (HMal1 malate anion, Mal2− malate dianion, HLac lactic acid, HAc acetic acid, and OAA oxaloacetate) are indicated

Pyruvate fermentation

In addition to the use of pyruvate as an electron acceptor (see above), pyruvate was identified as a substrate for fermentation by O. oeni and L. mesenteroides (Wagner et al. 2005). Pyruvate fermentation enables growth of the bacteria, and the growth rates are comparable to those obtained by glucose fermentation. Pyruvate is decarboxylated by pyruvate dehydrogenase (PDH) to acetyl-CoA and NADH. Acetyl-CoA is used for ATP formation (via acetyl-P) and the NADH is transferred by lactate dehydrogenase to a second molecule of pyruvate. Acetate kinase provides one ATP per reaction, which is in agreement with the ΔG0′ value and the growth yields (Wagner et al. 2005).
$$ 2\;Pyruvate + H_{2} O \to Acetate + CO_{2} + lactate\quad {\left( {\Delta G^{\prime }_{0} = - 95.1\,kJ/reaction} \right)} $$
PDH represents a new type of pyruvate-metabolizing enzyme in heterofermentative bacteria in addition to the previously known LDH, pyruvate oxidase, and 2-acetolactate synthase (Fig. 4). The use of PDH in the fermentation pathways of anaerobic bacteria is remarkable because PDH is regarded to be typical for aerobic metabolism. O. oeni and L. mesenteroides contain a gram-positive type PDH, but none of the alternative enzyme pyruvates: ferredoxin oxidoreductase, pyruvate decarboxylase, or pyruvate-formate lyase (Wagner et al. 2005). The use of PDH makes sense in pyruvate fermentation by generating NADH, which can be reoxidized by lactate dehydrogenase. The genomic sequence of Propionibacterium acnes (Brüggemann et al. 2004) also contains the structural genes of PDH (GenBank accession number AE017283), suggesting that the anaerobic propionibacteria also use the PDH enzyme under anoxic conditions.
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Fig. 4

Reactions for the formation and degradation of pyruvate in O. oeni and L. mesenteroides. The enzymes and reactions are indicated. LDH Lactate dehydrogenase, PDH pyruvate dehydrogenase (PdhABCD), Pox pyruvate oxidase (see Sedewitz et al. 1984; Wagner et al. 2005), ACS acetolactate synthase (product of genes 1,721/1,638 of O. oeni or 1,736/1,737 of L. mesenteroides), Mae oxaloacetate (OAA) decarboxylase, and PyrK pyruvate kinase

Citrate fermentation

Many LAB, including O. oeni and L. mesenteroides, use citrate as substrate (electron acceptor) for cometabolism with sugars like glucose, fructose, lactose, or xylose providing NADH (citrate+2 [H]→lactate+acetate+CO2) (Salou et al. 1994; Schmitt et al. 1997; Hache et al. 1999; Starrenburg and Hugenholtz 1991; Drinan et al. 1976) (see Fig. 2). Only few LAB are able to grow on citrate as the sole substrate (Medina de Figueroa et al. 2000). Transport of citrate and of the products lactate and acetate is central to the function of citrate fermentation (Fig. 3). Citrate uptake is catalyzed by the CitP or MaeP carrier in an electrogenic precursor/product (Hcitrate2−/lactate) antiport, which generates an electrochemical gradient over the membrane (Ramos et al. 1994; Marty-Teysset et al. 1995, 1996; Konings 2002). The electrochemical gradient is (similar to that from malolactic fermentation) not sufficient for growth of the bacteria.

The product of citrate cleavage, oxaloacetate, is decarboxylated by soluble oxaloacetate decarboxylase, which is related to malate decarboxylase, yielding pyruvate (Marty-Teysset et al. 1996; Mills et al. 2005; Sender et al. 2004). Pyruvate is used as electron acceptor for NADH from hexose oxidation. Thus, citrate fermentation depends on the presence of an electron donor, i.e., hexose, which supplies NADH for pyruvate reduction. The lactate produced in this way is important for efficient driving of the citrate/lactate antiport (Salou et al. 1994; Konings 2002).

Part of the pyruvate derived from citrate is converted to acetoin and 2,3-butanediol by condensation of two molecules of pyruvate to 2-acetolactate (Fig. 4), followed by decarboxylation to acetoin and reduction to 2,3-butanediol (Ramos et al. 1994; Nielsen and Richelieu 1999). 2-Acetolactate can be converted to diacetyl by decarboxylation and chemical (nonenzymatic) oxidation with O2. Channeling of pyruvate into the acetoin pathway is significant in LAB when the bacteria are incubated with citrate in the absence of other carbon sources. In the presence of excess NADH (e.g., from hexoses) the pyruvate is reduced essentially to lactate (Ramos et al. 1994). 2-Acetolactate is the source of diacetyl, a flavor compound in wine and other products treated by LAB (Mills et al. 2005; Nielsen and Richelieu 1999; Schmitt et al. 1997; Bartowsky and Henschke 2004).

The cit gene clusters of L. mesenteroides and O. oeni comprise genes for citrate lyase (citDEF), citrate lyase ligase (citC), oxaloacetate decarboxylase (mae) and the citrate carrier (maeP or citM) (Mills et al. 2005; Martin et al. 2000, and references therein). The clusters also contain the citX and citG genes, which are homologous to the corresponding genes of Klebsiella for the synthesis of the phosphoribosyl-dephospho-SCoA prosthetic group of citrate lyase (Schneider et al. 2002). Putative genes for acetolactate synthase and acetolactate decarboxylase are found in O. oeni and L. mesenteroides (Fig. 4).

Arginine fermentation

Arginine fermentation by the arginine deiminase pathway (1 arginine→1 ornithine+1 CO2+1 NH3) yields one ATP per arginine from carbamoyl-P by substrate level phosphorylation. The pathway includes an arginine/ornithine antiporter (ArcD), arginine deiminase (ArcA), ornithine carbamoyl transferase (ArcB), and carbamate kinase (ArcC). The fermentation is found in obligately heterofermentative LAB as L. sanfranciscensis, some strains of O. oeni, L. buchneri, L. hilgardii, but also in the facultatively heterofermentative L. plantarum (Arena et al. 2002; De Angelis et al. 2002; Spano et al. 2004; Mira De Orduna et al. 2001; Tonon and Lonvaud-Funel 2000; Mills et al. 2005). For some of the bacteria the respective genes, enzymes, and fermentation products were demonstrated. Grape must and wine contain significant amounts of arginine, and arginine fermentation supports viability and growth of the bacteria (Mira De Orduna et al. 2001; Tonon and Lonvaud-Funel 2000), but growth rate and yield is low. Formation of NH3 and of citrulline is undesirable in wine due to the increase in pH and the formation of potentially toxic derivatives.

Overall, the heterofermentative LAB show during growth on hexoses a considerable number of variations in the classical phosphoketolase pathway, which serve to by-pass the rate limiting ethanol pathway for NAD(P)H reoxidation. The alternatives include the formation of small amounts of acetate plus erythritol or glycerol. In the presence of external electron acceptors like fructose, O2, pyruvate, or citrate, the extra NAD(P)H is transferred to the acceptors, resulting in the excretion of large amounts of acetate, which is no longer reduced to ethanol. At the same time the growth rates of the bacteria increase due to omission of the limiting pathway. In addition, the bacteria are able to ferment various organic acids (citrate, malate, and pyruvate) and arginine, but only part of the organic acids support growth when present as the sole substrate.

Acknowledgement

The work in the authors’ laboratory was supported by Innovationsstiftung Rheinland-Pfalz (Grant no. 15202-38 62 61/675).

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© Springer-Verlag 2006