Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source

Journal of Bioscience and Bioengineering VOL. xxx No. xxx, xxx, xxxx www.elsevier.com/locate/jbiosc

Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source Mio Kawai,1 Asami Tsuchiya,2 Junya Ishida,2 Nobuo Yoda,2 Shino Yashiki-Yamasaki,3 and Yoshio Katakura3, * Graduate School of Science and Engineering, Kansai University, 3-3-35 Yamate, Suita, Osaka 564-8680, Japan,1 Food Science & Technology Research Laboratories, R&D Division, Meiji Co., Ltd., 1-29-1 Nanakuni, Hachiouji, Tokyo 192-0919, Japan,2 and Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate, Suita, Osaka 564-8680, Japan3 Received 13 September 2019; accepted 14 November 2019 Available online xxx

We report a method for suppression of lactate production by lactic acid bacteria (LAB) in culture. LAB produce lactate to regenerate NADD that is consumed during glycolysis. Glucose suppresses NADD regeneration pathways other than lactate dehydrogenase and non-glycolytic ATP production pathways. Therefore, the carbon source was changed to sucrose, and fed-batch culture was performed to limit the glycolytic flux and thus suppress lactate production. As a result, lactate productivity (i.e., the amount of lactate produced per amount of grown cell) in the sucrose/fed-batch culture was decreased compared to that in glucose/batch culture, in all five LAB strains examined. The productivity level decreased to 24% and 46% in Lactobacillus reuteri JCM 1112 and Lactococcus lactis JCM 7638, respectively. Metabolic flux analysis of Lactobacillus reuteri JCM 1112 revealed increased contributions of the mannitol production pathway to NADD regeneration and the arginine deiminase pathway to ATP production in the sucrose/fed-batch culture. Ó 2019, The Society for Biotechnology, Japan. All rights reserved. [Key words: Lactic acid bacteria; Fed-batch culture; Sucrose; Redox balance; Glucose repression; Flux analysis]

Lactic acid bacteria (LAB) acquire ATP by metabolizing sugars to pyruvate through glycolytic pathway. In this process, NADþ is consumed by the action of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and regenerated through conversion of pyruvate to lactate by lactate dehydrogenase (LDH) (1). Therefore, LAB produce lactate as they grow. However, excessive lactate accumulation leads to a large decrease in culture pH. Furthermore, even when the pH level is controlled via alkali addition, LAB growth is still inhibited due to ATP consumption to discharge the protons that are dissociated from lactate at high lactate concentrations (2e4). High LAB densities are thus difficult to achieve in culture, and strategies to suppress lactate production by LAB are needed. Using a hot-water extract of chlorella powder as growth medium (5), we previously reduced lactate productivity of LAB to a quarter or less of that with the MRS medium, a routinely used LAB cultivation medium. An analysis of intracellular metabolites of Lactococcus lactis subsp. lactis NBRC 12007 indicated significantly suppressed lactate production in the chlorella medium. Moreover, two ATP generation mechanisms, arginine deiminase (ADI) pathway and decarboxylation of glutamate and GABA, were activated, which are usually repressed by glucose. Unlike in glycolysis, LAB produce ATP without consuming NADþ through these

* Corresponding author. Tel./fax: þ81 6 6368 0809. E-mail addresses: [email protected] (M. Kawai), asami.tsuchiya@ meiji.com (A. Tsuchiya), [email protected] (J. Ishida), nobuo.yoda@ meiji.com (N. Yoda), [email protected] (S. Yashiki-Yamasaki), katakura@ kansai-u.ac.jp (Y. Katakura).

pathways. If ATP is generated via these alternative pathways, lactate production is suppressed, since NADþ regeneration via LDH is unnecessary. However, most of the alternative ATP production pathways are repressed by glucose (6e9). The chlorella medium contained almost no glucose, and the main carbon source was sucrose. Hence, these pathways were derepressed, and lactate production was subsequently suppressed. Moreover, further suppression of lactate production is also possible in case LAB regenerate NADþ via alternative mechanisms instead of using LDH, and produce ATP via pathways other than glycolysis. However, we noticed that unless the NADþ regeneration capacities of these alternative pathways are comparable to that of LDH, NADþ may not be regenerated in sufficient amounts for glycolysis. Therefore, we cultured LAB in fed-batch mode to limit the flux through glycolysis. The aim of this study was thus to suppress lactate production by LAB via fed-batch mode of operation with sucrose as carbon source, and reveal underlying suppression mechanisms via comparison of metabolic fluxes from batch and fed-batch cultivations.

MATERIALS AND METHODS Bacterial strains and growth conditions Lactobacillus reuteri JCM 1112, Lactobacillus gasseri NCIMB 11718 and Lactobacillus paracasei ATCC 334 was cultivated at 37  C. Lactobacillus plantarum NCIMB 8826 and Lactococcus lactis JCM 7638 were cultivated at 30  C. LAB were grown in MRS medium including 20 g,L1 glucose or sucrose (20 g sucrose or glucose, 10 g Bacto peptone (Becton, Dickinson and Company (BD), Tokyo, Japan), 10 g Bacto beef extract (BD), 5 g Bacto yeast extract (BD), 1 g

1389-1723/$ e see front matter Ó 2019, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2019.11.009

Please cite this article as: Kawai, M et al., Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.11.009

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KAWAI ET AL.

J. BIOSCI. BIOENG., TABLE 1. Lactate production in batch and fed-batch culture using glucose or sucrose as carbon source.

Strain Lactobacillus reuteri JCM 1112

Culture method Batch Fed-batch

Lactococcus lactis JCM 7638

Batch Fed-batch

Lactobacillus gasseri NCIMB 11718

Batch Fed-batch

Lactobacillus plantarum NCIMB 8826

Batch Fed-batch

Lactobacillus paracasei ATCC 334

Batch Fed-batch

Carbon source Glucose Sucrose Glucose Sucrose Glucose Sucrose Glucose Sucrose Glucose Sucrose Glucose Sucrose Glucose Sucrose Glucose Sucrose Glucose Sucrose Glucose Sucrose

m (h1) 1.01 1.14 0.18 0.19 0.40 0.39 0.11 0.12 0.49 0.46 0.13 0.18 0.33 0.31 0.18 0.18 0.39 0.30 0.15 0.18

                   

YX/S (g-cell,g1)

0.02 0.08 0.02 0.02 0.07 0.06 0.01 0.03 0.17 0.09 0.02 0.02 0.06 0.04 0.01 0.01 0.17 0.02 0.04 0.06

0.08 0.08 0.19 0.18 0.06 0.09 0.12 0.12 0.08 0.09 0.10 0.11 0.10 0.11 0.14 0.16 0.08 0.09 0.13 0.16

                   

0.01 0.00 0.03 0.01 0.01 0.01 0.03 0.02 0.03 0.02 0.02 0.01 0.02 0.02 0.02 0.01 0.01 0.01 0.03 0.04

rL (g,g-cell1,h1) 4.33 2.79 0.70 0.19 7.26 7.27 1.20 1.01 5.41 5.66 1.54 1.44 2.32 2.11 1.01 0.93 3.58 2.61 1.44 1.40

                   

YL/X (g,g-cell1)

Relative YL/X (%)

                   

100 57 91 24 100 103 60 46 100 111 105 72 100 97 80 74 100 94 104 84

0.25 0.03 0.02 0.14 1.07 1.74 0.30 0.24 1.24 2.36 0.14 0.37 0.11 0.27 0.16 0.16 0.81 0.39 0.54 0.45

4.29 2.45 3.89 1.02 18.2 18.7 10.9 8.42 11.1 12.3 11.7 8.01 7.03 6.80 5.61 5.17 9.25 8.70 9.60 7.77

0.16 0.01 0.35 0.16 1.17 0.12 1.73 0.10 1.30 2.72 0.72 1.39 0.61 0.01 0.58 0.60 1.69 0.72 1.04 0.09

m, specific growth rate; YX/S, dry-cell yield on the basis of consumed glucose or sucrose; rL, specific lactate production rate; YL/X, lactate yield on the basis of dry-cell. Each value indicates a slope (Fig. S1), and the 95% confidence limit in the regression analysis. Relative YL/X is a relative value with respect to YL/X in glucose batch culture (which was taken as 100% for each strain).

polyoxyethylene(20)sorbitan monooleate, 2 g triammonium citrate, 8.3 g sodium acetate trihydrate, 0.1 g magnesium sulfate heptahydrate, 0.05 g manganese (II) sulfate pentahydrate, 2 g dipotassium hydrogenphosphate per liter). For metabolic flux analysis of Lactobacillus reuteri JCM 1112, a synthetic medium including 17 amino acid, 5 nucleic acid, 14 vitamin, 8 inorganic salt types as well as trace elements (10e13) was used. No asparagine and glutamine were added to this medium, whereas aspartic acid, glutamic acid and arginine were added at final concentrations of 358, 391 and 2500 mg L1, respectively. Nucleic acids (adenine, guanine, thymine, hypoxanthine and cytosine) were present at 5.0 mg L1 concentration each. Those reagents for which no vendor is listed above were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Culture Static preculture was performed for 15 h at the optimum growth temperature of each LAB species (as given above) in the MRS medium containing 20 g L1 glucose or sucrose. The bacterial cells obtained from preculture were washed twice with physiological saline. An appropriate amount of cells was then inoculated into a medium bottle (250 mL, equipped with a pH sensor) containing 100 mL of MRS medium without carbon source to obtain a turbidity reading of OD660 ¼ 1. The cultivation was performed at each optimum temperature at 500 rpm on a magnetic stirrer (8 mmf  25 mm). Culture pH was maintained at 6.5 using 3 M NaOH. For fed-batch culture, MRS medium containing an appropriate concentration of glucose or sucrose was used as feed medium. The feeding began immediately after inoculation, and was achieved using a peristaltic pump MP-2000 (Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The feed rate was calculated according to the following formula. FðtÞ ¼

m $V0 $X0 SF $YX=S

expðm $ tÞ

(1)

where F is the feed rate (L,h1), m* is the target specific growth rate (h1), V0 is the initial volume (L), X0 is the cell concentration (g-cell,L1), SF is the glucose or sucrose concentration of the fed-medium (g-sugar,L1), YX/S is the cell yield factor on the basis of sugar (g-cell,g-sugar1), and t is the culture time (h). The flow rate of the peristaltic pump was updated every hour, and the flow rate from t [h] to tþ1 [h] was set to (F(t)þF(tþ1))/2 [L,h1]. Calculation of specific rates and yields The specific growth rate was calculated from the slope of lnVX time profile. The specific production rate was calculated from the slope of the plot of product amount (VP) versus accumulated cell mass (!VXdt). The cell yield factor based on sugar was calculated from the slope of the plot of increased cell mass versus consumed substrate at each time point (SDX vs SDS plot). The yield of lactate on the basis of dry-cell YL/X [g$g-cell1] was calculated by dividing the specific lactate production rate by the specific growth rate. The confidence limit of YL/X at 95% was calculated as (rDmmDr)/Dm2, where 95% confidence limits of m and r are Dm and Dr, respectively. In other words, (rDr)/ (mDm) y r/m(rDmmDr)/Dm2. Details of calculations are presented in Fig. S1. Analysis The cell concentration was determined by measuring optical density at 660 nm (OD660), and OD660 value of 1 was assumed to correspond to 0.25 g of dry cells in one L. Glucose, sucrose and lactate were analyzed enzymatically using the biosensor BF-5 (Oji Scientific Instruments, Hyogo, Japan). Mannitol and fructose concentrations were quantified by using the sugar analyzer SU-300 (DKK Toa, Tokyo, Japan; column, PCI-520 (anion-exchange type); developing solvent, 0.2 M NaOH; flow rate, 0.6 mL,min1; detection, pulse amperometry method). Acetate was

analyzed via gas chromatography (GC-4000 Plus, GL Sciences, Tokyo, Japan) using InertCap FFAP (0.25 mm I.D.  30 m L; GL Sciences). The oven temperature was initially set to 60  C, kept at this temperature for 5 min, then was gradually increased to 240  C at a rate of 10  C,min1, and was kept at 240  C for 30 min. Carrier gas (He) flow rate was 55 mL,min1. Elemental analysis of dry cell was performed by using a 2400 Series II CHNS/O Elemental Analyzer (PerkinElmer, Waltham, MA, USA). Polysaccharides were isolated according the following procedure: supernatant was mixed with an equal volume of ethanol, and incubated at 20  C for 1 h. The pellet was collected using centrifugation (at 4  C, 15,000  g, 5 min), washed with a mixture of water and ethanol at 1:1 ratio, and dried under reduced pressure for 5 min. The precipitate including the exopolysaccharide was completely dissolved in water (at 4  C, overnight), and quantified using the phenol sulfuric acid method with glucose as a standard (14). Amino acids concentrations were determined using an L-8900 amino acid analyzer (Hitachi High-Tech Science Corporation, Tokyo, Japan).

RESULTS Suppression of lactate production in fed-batch cultures of LAB using sucrose as carbon source Lactate production levels in batch and fed-batch cultures with glucose or sucrose MRS medium were compared to verify suppression of lactate production by sucrose feeding. Five LAB strains with the ability to catabolize sucrose as a carbon source and sequenced genomes, Lactobacillus reuteri JCM 1112, Lactobacillus plantarum NCIMB 8826, Lactobacillus gasseri NCIMB 11718, Lactobacillus paracasei ATCC 334 and Lactococcus lactis JCM 7638, were used. In batch cultures, specific growth rate, m [h1], and specific lactate production rate, rL [g$g-cell1$h1], were determined using data from the logarithmic growth phase (between 0.5 and 4 h of cultivation). The maximum specific growth rate of Lactobacillus reuteri in batch culture exceeded 1.0 h1, whereas those of other strains remained between 0.3 and 0.5 h1. The target specific growth rate (m*) in fed-batch was set to 0.2 h1, which is sufficiently lower than the maximum specific growth rate of each strain in batch culture to suppress NADþ consumption in glycolysis. The cell yield factor based on sugar (YX/S), and the feed sugar concentration (SF) were set to 0.2 gcell$g1 and 20 g L1, respectively. For the four strains other than Lactobacillus reuteri, the observed specific growth rates in fedbatch mode were lower than the target rate (0.2 h-1) (Table 1). This may be due to the fact that the observed YX/S values of the other four strains were lower than the set value (0.2 g-cell$g1).

Please cite this article as: Kawai, M et al., Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.11.009

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Lowest lactate yield factors based on cell (YL/X) were obtained from sucrose fed-batch cultures for all five strains. Lactate yield factors were also converted to a relative scale to facilitate comparison between strains and fermentation conditions. For this purpose, the relative lactate yield factor from glucose batch culture of each strain was set to 100%, and yield factors for each strain were calculated with respect to these values. The lowest value, 24%, was obtained from the sucrose fed-batch culture of Lactobacillus reuteri. Even in batch culture of Lactobacillus reuteri, relative YL/X was suppressed down to 57% when sucrose was the carbon source. In fed-batch culture of Lactococcus lactis, relative YL/X levels were suppressed to 60% and 46% for glucose and sucrose, respectively. In general, changing the operational mode from batch to fed-batch significantly reduced YL/X level, whereas changing the carbon source from glucose to sucrose was not as effective. In Lactobacillus gasseri, Lactobacillus plantarum, and Lactobacillus paracasei, lactate yield factors in the sucrose fedbatch culture decreased to 72, 74, and 84% of that in glucose batch culture, respectively, yet the reduction levels for Lactobacillus reuteri and Lactococcus lactis were relatively higher. Cell yield factors (YX/S) in fed-batch mode were higher than those in the batch culture of all strains (Table 1). Metabolic analysis of Lactobacillus reuteri in the synthetic medium The use of sucrose rather than glucose as a carbon source suppressed lactate production in fed-batch culture of Lactobacillus reuteri. The choice of carbon source only slightly affected lactate production in batch culture, except for Lactobacillus reuteri. Lactate production of all strains in MRS medium was suppressed in the sucrose fed-batch culture when compared to the glucose batch culture. Highest level of suppression was observed for Lactobacillus reuteri. In order to investigate suppression mechanisms, Lactobacillus reuteri was cultivated in a synthetic medium, and the metabolic fluxes under four tested culturing conditions were analyzed. Dry cell concentration corresponding to OD660 ¼ 1, and the carbon content per dry cell were determined for each culture condition to calculate carbon balance. Lactate production was suppressed when sucrose was used as the carbon source in fed-batch culture with the synthetic medium as well (Table 2). Lactate production in sucrose fed-batch culture was less than that in glucose fedbatch culture. Furthermore, lactate production level in the sucrose fed-batch culture was suppressed to 6% of the glucose batch culture, whereas that in MRS medium was 24%. Lactate, fructose, mannitol, ethanol, acetoin, diacetyl, acetate, and exopolysaccharide levels in culture supernatants were analyzed based on the reported metabolic pathways of Lactobacillus reuteri (15,16). The amount of produced carbon dioxide was calculated based on produced amounts of acetate and ethanol. Under all culture conditions, carbon recovery level reached almost 100% (Table 3). The metabolic fluxes from different culture conditions were compared by setting the relative carbon content of the consumed sugar to 100%. In sucrose batch culture, 19% of the consumed carbon was metabolized to lactate. This value decreased to 10% in sucrose fed-batch culture, and to 58% or 60% in glucose

3

TABLE 3. Carbon balance of Lactobacillus reuteri (mmol carbon). Batch Sucrose

Glucose

Sucrose

10.1 5.81 0.27 0.40 0.00 0.20 1.63 0.01 0.77 0.81 98

34.8 6.58 7.00 10.5 2.11 1.10 3.91 0.00 0.97 3.01 101

2.10 1.25 0.11 0.10 0.00 0.12 0.00 0.00 0.34 0.00 92

2.49 0.25 0.69 1.07 0.09 0.00 0.00 0.01 0.47 0.05 106

Sugar Lactate Fructose Mannitol Ethanol Acetoin Acetate Exopolysaccharide Biomass CO2a Carbon recovery (%) a

Fed-batch

Glucose

Calculated using (ethanolþacetate)/2.

batch or fed-batch cultures, respectively (Fig. 1). Ethanol was produced only when sucrose was used as the carbon source. Although fructose and mannitol were produced under all conditions, the flux leading to these compounds was larger with sucrose as the carbon source. Acetate was produced both in glucose and sucrose batch, but not fed-batch cultures. The flux leading to biomass production was higher in fed-batch cultures. Amino acid consumption and production of Lactobacillus reuteri The concentrations of amino acids in culture supernatants were analyzed to investigate which NADþ regeneration and ATP production mechanisms other than LDH and glycolysis, respectively, were used (Table S2). Only the concentrations of Arg, Orn, and ammonia changed significantly during the first 2.5 h of culture (Table S2). Table 4 summarizes the values obtained by dividing the change in amounts of these substances by the amount of cells produced during this period. Consumption of arginine per amount of cells produced in fedbatch culture was four and two times higher than that in batch culture when sucrose and glucose were used as carbon sources, respectively. In the fed-batch culture, Lactobacillus reuteri likely produced ATP via the ADI pathway, since almost equimolar ornithine and approximately two molar equivalents of ammonia were produced with consumption of arginine. DISCUSSION The NADþ/NADH redox balance is essential for maintenance of cellular metabolism. NADþ is reduced to NADH by GAPDH in glycolysis. In order to drive the glycolytic system toward energy production, NADþ must be regenerated. Respiring cells regenerate NADþ from NADH using oxygen as an electron acceptor (17,18). However, NADþ must be regenerated via other pathways under anaerobic conditions. For this purpose, yeast cells utilize alcohol dehydrogenase (ADH) and produce ethanol, whereas LAB utilize LDH and produce lactate. Other pathways for NADþ regeneration may be used as well, yet the regeneration capacities of these pathways are insufficient to compensate for consumption of NADþ by GAPDH. In energy metabolism of yeast, aerobic fermentation

TABLE 2. Lactate production of Lactobacillus reuteri in the synthetic medium. Operational mode Batch Fed-batch

Carbon source Glucose Sucrose Glucose Sucrose

Cell concn. at OD660¼1 (g-cell,L1)

Carbon content (g,g-cell1)

0.26 0.27 0.28 0.28

0.41 0.42 0.39 0.40

m (h1) 0.27 0.33 0.15 0.19

   

0.03 0.04 0.05 0.03

YX/S (g-cell,g1) 0.06 0.03 0.16 0.20

   

0.01 0.01 0.05 0.02

rL

(g,g-cell1,h1) 2.64 2.06 0.70 0.12

   

0.71 0.07 0.19 0.03

YL/X (g,g-cell1)

Relative YL/X (%)

   

100 64 48 6

9.77 6.24 4.66 0.63

1.54 0.55 0.78 0.06

m, specific growth rate; YX/S, dry-cell yield on the basis of consumed glucose or sucrose; rL, specific production rate of lactate; YL/X, yield of lactate on the basis of dry-cell. Each value shows the slope and the 95% confidence limit in the regression analysis. Relative YL/X is a relative value with respect to YL/X in glucose batch culture (which was taken as 100% for each strain).

Please cite this article as: Kawai, M et al., Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.11.009

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KAWAI ET AL.

J. BIOSCI. BIOENG.,

FIG. 1. Carbon flux in Lactobacillus reuteri. Values shown within the frame are relative carbon fluxes ordered from top to bottom as follows: glucose-batch, sucrose-batch, glucosefed-batch and sucrose-fed-batch. G6P, glucose-6-phosphate; 6 PG, 6-phosphogluconate; Xu5P, xylulose-5-phosphate; AcP, acetyl phosphate; AcCoA, acetyl-CoA; F6P, fructose-6phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone-phosphate; GA3P, glyceraldehyde-3-phosphate.

occurs even with sufficient oxygen amounts and sugar concentrations above 0.1 g L1 (corresponding to a specific growth rate of about 0.25 h1) (19e22). This is due to the higher rate of NADþ consumption than NADþ regeneration rate via respiration. Hence, NADþ is regenerated by ADH when the flux through glycolysis increases. Fed-batch mode of operation is thus used to suppress ethanol production and maximize the cell yield in industrial production of baker’s yeast (19e22). In LAB, LDH action is the main NADþ regeneration mechanism. However, LAB can also utilize other NADþ regeneration mechanisms. Some LAB can regenerate NADþ by using oxygen as an electron acceptor (17,18). NADþ can be also regenerated via pathways producing 2,3-butanediol, acetoin and diacetyl. These pathways are activated upon disruption of LDH gene (23e25). Lactobacillus reuteri has the ability to regenerate NADþ by converting glycerol to 3-hydroxypropionaldehyde, then producing 1,3propanediol using 1,3-propanediol oxidoreductase (26). When glycerol was co-fed with glucose at a molar ratio of three exponentially, lactate production by Lactobacillus reuteri JCM 1112 was reduced to one-third of that in the batch culture using glucose as a carbon source (27). These alternative NADþ regeneration pathways are also repressed by glucose (6e9,27). In batch cultures with high glucose concentrations, the flux through glycolysis increases, and alternative pathways are therefore repressed. As a result, most of the required NADþ amount is regenerated via LDH, and thus a large amount of lactate is produced. Indeed, Lactococcus lactis was previously reported to shift from homo fermentation to mixed acid TABLE 4. Consumption or production of amino acids per amount of cells produced (mmol,g-cell1). Culture method

Carbon source

Arginine consumed

Ornithine produced

Ammonia produced

Batch

Glucose Sucrose Glucose Sucrose

6.9 4.9 12.3 17.4

8.2 1.2 12.0 18.2

12.1 3.0 21.3 28.5

Fed-batch

fermentation producing ethanol, acetate and formic acid when specific growth rate was kept low, and lactate production was suppressed (28,29). These findings also suggest that limiting the glycolytic flux leads to suppression of lactate production. Here, we conducted cultivations in fed-batch mode to reduce glycolytic flux, and thereby increased the relative contributions of alternative pathways. Moreover, repression of alternative pathways by glucose was avoided by using sucrose as the carbon source instead. Lactate productivity (i.e. the amount of produced lactate per grown cell) of all five LAB strains (with ability to catabolize sucrose) decreased in sucrose fed-batch culture. However, the level of decrease was dependent on the strain. Highest level of suppression of lactate production was observed for Lactobacillus reuteri. We hypothesize that the following two factors are responsible for this suppression effect. First, mannitol dehydrogenase (MDH) likely contributed to NADþ regeneration instead of LDH. Lactobacillus reuteri can oxidize NADH to NADþ via conversion of fructose (derived from sucrose by sucrose phosphorylase) to mannitol by MDH (30). By using sucrose as carbon source, Lactobacillus reuteri was able to regenerate NADþ via LDH as well as MDH. In this case, 43% and 30% of the sugar carbon consumed in fed-batch and batch cultures were used to produce mannitol, respectively (Fig. 1). Carbon flux from consumed sugar to lactate thus decreased to 10% and 19% in batch and fedbatch cultures, respectively. Suppression of lactate production was suggested to occur due to MDH-mediated regeneration of a significant part of NADþ that is consumed by GAPDH. Moreover, sucrose phosphorylase can phosphorylate the glucose moiety of sucrose with inorganic phosphate instead of ATP (31). Therefore, degradation of one sucrose molecule by sucrose phosphorylase instead of using glucose as carbon substrate can save one ATP molecule that is required for glycolysis, and thereby contribute to the suppression of lactate production. Second, the contribution of the ADI pathway to ATP production increased in fed-batch culture. In the ADI pathway, arginine is degraded to citrulline and ammonia by arginine deiminase, and

Please cite this article as: Kawai, M et al., Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.11.009

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TABLE 5. Amount of ATP produced in batch and fed-batch cultures (mmol). Operational mode

Carbon source

Glycolysis

Acetate production

Batch

Glucose Sucrose Glucose Sucrose

3.14 5.77 0.63 0.24

0.81 1.95 0.00 0.00

Fed-batch

ADI Total pathway 0.32 0.06 0.37 0.63

4.27 7.78 1.00 0.87

ADI/ total 0.08 0.01 0.37 0.72

Amount of ATP obtained from glycolysis was calculated by doubling the molar amount of hexose which entered glycolysis. It was assumed that the molar amount of hexose entering glycolysis pathway is given by subtracting the amount flowing to mannitol, fructose and exopolysaccharide from that of consumed hexose sugar. Molar amount of ATP obtained from acetate production was assumed to be equal to the molar amount of produced acetate. Total ATP is the sum of ATP obtained via glycolysis, acetate production and ADI pathway.

citrulline is degraded to carbamoyl phosphate and ornithine by ornithine carbamoyltransferase. The carbamoyl phosphate is decomposed into ammonia and carbon dioxide by carbamoyl phosphate kinase, and one molecule of ATP is produced in this process (32,33). According to KEGG (http://www.genome.jp/kegg/), Lactobacillus reuteri genome includes the genes of all enzymes involved in the ADI pathway. Arginine was significantly consumed in fed-batch compared to batch cultures, and approximately equimolar ornithine and nearly two molar equivalents of ammonia were produced (Table 4). Hence, the ADI pathway was activated in fed-batch mode. We made several assumptions to elucidate the contribution of the ADI pathway to total ATP production. First, we assumed that the molar amount of ATP obtained via the ADI pathway was equal to the molar amount of ornithine produced in the medium. We also assumed that the molar amount of hexose entering the glycolysis pathway is given by subtracting the amount flowing to mannitol, fructose, and exopolysaccharide from that of consumed hexose sugar. Molar amount of ATP obtained from glycolysis was then calculated by doubling this value. Moreover, we also assumed that one mol of ATP could be obtained by producing one mol of acetate. The ratio of the ATP obtained from the ADI pathway to the total amount of ATP produced in sucrose and glucose fed-batch cultures were 0.72 and 0.37, respectively. The ratios obtained from batch cultures were 0.01 and 0.08 (Table 5). The ADI pathway was previously reported as inactive in the absence of fermentable sugars, and repressed with high sugar concentrations (6). Here, we considered the ADI pathway to be active, since fermentable sugar was continuously supplied at low concentration in fed-batch cultures. Furthermore, arginine consumption per cell increased when sucrose rather glucose was used as carbon source in the fed-batch culture. This finding indicates that the repression of the ADI pathway by glucose was reduced with sucrose as a carbon source. In conclusion, the production of lactate per cell was suppressed in sucrose fed-batch culture since (i) a significant part of NADþ was regenerated by MDH with sucrose as a carbon source, and (ii) ATP was generated via the ADI as well as glycolysis pathways in fedbatch culture. Lactococcus lactis, whose lactate production in sucrose fed-batch culture was suppressed to 46% of that in glucose batch culture, has only genes related to arginine deiminase pathway, and not the MDH gene. The other three strains have none of these genes. This suggests that the suppression of lactate production depends on the use of the strain-specific alternative pathways. In addition to MDH described above, LAB can utilize various NADþ regeneration mechanisms involving enzymes such as alcohol dehydrogenase, 2,3-butanediol dehydrogenase, succinate dehydrogenase, and membrane-bound NADH oxidase. LAB can also utilize alternative ATP production pathways other than the ADI pathway, e.g., the decarboxylation of glutamate and aspartate, in

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which one molecule of ATP is obtained via F1F0-ATPase (34e36). The strains for which no marked suppression of lactate production was observed in sucrose fed-batch culture here possibly lack these alternative pathways, or the required environmental conditions for their activation were not achieved. Lactate production of strains other than Lactobacillus reuteri could be suppressed further if these strain-specific conditions are provided, e.g., by supplying oxygen to strains with membrane-bound NADH oxidase. We have found that the lactate production levels of some LAB strains were suppressed more strongly when galactose or maltose was used as carbon source rather than sucrose (data not shown). This suggests that the glucose repression of alternative pathways may be avoided by feeding non-glucose sugar. Lactate production thus may be suppressed not only in Lactobacillus reuteri, but other LAB strains as well, by selecting a suitable carbon source for each strain, and providing the conditions for activation of these alternative pathways. The suppression of lactate production by feeding non-glucose sugar will further contribute to high cell density culture of LAB as well as efficient production of useful substances from LAB. Supplementary data to this article can be found online at https://doi.org/10.1016/j.jbiosc.2019.11.009. ACKNOWLEDGMENTS We thank Prof J. Hayashi from Department of Chemical, Energy and Environmental Engineering, Kansai University for their help with elemental analysis of dry cell. This work was partially supported by Grant-in-Aid for Challenging Exploratory Research (no. 26660073) from Japan Society for the Promotion of Science (JSPS). References 1. Makarova, K., Slesarev, A., Wolf, Y., Sorokin, A., Mirkin, B., Koonin, E., Pavlov, A., Pavlova, N., Karamychev, V., Polouchine, N., Shakhova, V., and Grigoriev, I.: And other 38 authors.: comparative genomics of the lactic acid bacteria, Proc. Natl. Acad. Sci. USA, 103, 15611e15616 (2006). 2. Kashket, E. R.: Bioenergetics of lactic acid bacteria: cytoplasmic pH and osmotolerance, FEMS Microbiol. Rev., 46, 233e244 (1987). 3. Pieterse, B., Leer, R. J., Schuren, F. H., and van der Werf, M. J.: Unravelling the multiple effects of lactic acid stress on Lactobacillus plantarum by transcription profiling, Microbiology, 151, 3881e3894 (2005). 4. O’Sullivan, E. and Condon, S.: Relationship between acid tolerance, cytoplasmic pH, and ATP and Hþ-ATPase levels in chemostat cultures of Lactococcus lactis, Appl. Environ. Microbiol., 65, 2287e2293 (1999). 5. Kawai, M., Harada, R., Nobuo, Y., Fukusaki, E., Yamasaki-Yashiki, S., and Katakura, Y.: Suppression of lactate production of lactic acid bacteria by using sucrose, J. Biosci. Bioeng. https://doi.org/10.1016/j.jbiosc.2019.06.017 (29 July 2019) [Epub ahead of print]. 6. Montel, M. C. and Champomier, M. C.: Arginine catabolism in Lactobacillus sake isolated from meat, Appl. Environ. Microbiol., 53, 2683e2685 (1987). 7. Rimaux, T., Vrancken, G., Vuylsteke, B., De Vuyst, L., and Leroy, F.: The pentose moiety of adenosine and inosine is an important energy source for the fermented-meat starter culture Lactobacillus sakei CTC 494, Appl. Environ. Microbiol., 77, 6539e6550 (2011). 8. Stuart, M. R., Chou, L. S., and Weimer, B. C.: Influence of carbohydrate starvation and arginine on culturability and amino acid utilization of Lactococcus lactis subsp. lactis, Appl. Environ. Microbiol., 65, 665e673 (1999). 9. Zotta, T., Parente, E., and Ricciardi, A.: Aerobic metabolism in the genus Lactobacillus: impact on stress response and potential applications in the food industry, J. Appl. Microbiol., 122, 1365e2672 (2017). 10. Exterkate, F. A.: Accumulation of proteinase in the cell wall of Streptococcus cremoris strain AM1 and regulation of its production, Arch. Microbiol., 120, 247e254 (1979). 11. Otto, R., Klont, B., and Konings, W. N.: The relation between growth rate and electrochemical proton gradient of Streptococcus cremoris, FEMS Microbiol. Lett., 16, 69e74 (1983). 12. Poolman, B. and Konings, W. N.: Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport, J. Bacteriol., 170, 700e707 (1988). 13. Mitsunaga, H., Meissner, L., Büchs, J., and Fukusaki, E.: Branched chain amino acids maintain the molecular weight of poly(g-glutamic acid) of Bacillus

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Please cite this article as: Kawai, M et al., Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.11.009