Efficient production and secretion of pyruvate from Halomonas sp. KM-1 under aerobic conditions
© Kawata et al. 2016
Received: 8 March 2016
Accepted: 11 March 2016
Published: 18 March 2016
The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 can utilize both hexose and pentose sugars for the intracellular storage of bioplastic poly-(R)-3-hydroxybutyric acid (PHB) under aerobic conditions. In this study, we investigated the effects of the sodium nitrate concentration on PHB accumulation in the KM-1 strain. Unexpectedly, we observed the secretion of pyruvate, a central intermediate in carbon- and energy-metabolism processes in all organisms; therefore, pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals and is employed for the production of crop-protection agents, polymers, cosmetics, and food additives. We then further analyzed pyruvate productivity following changes in culture temperature and the buffer concentration. In 48-h batch-cultivation experiments, we found that wild-type Halomonas sp. KM-1 secreted 63.3 g/L pyruvate at a rate of 1.32 g/(L·h), comparable to the results of former studies using mutant and recombinant microorganisms. Thus, these data provided important insights into the production of pyruvate using this novel strain.
Pyruvate is a central intermediate in carbon and energy metabolism in all organisms; therefore, pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals and is employed for production of crop-protection agents, polymers, cosmetics, and food additives (Li et al. 2001). Chemical production of pyruvate has been achieved by dehydration and decarboxylation of tartrate (Howard and Fraser 1932). However, this process is not cost-effective (Li et al. 2001); hence, biotechnological pyruvate production has attracted attention as a potential alternative method of pyruvate synthesis. To date, the successful biotechnological production of pyruvate from glucose has primarily been achieved using bacteria, such as Escherichia coli (Causey et al. 2004; Yokota et al. 1994; Zhu et al. 2008) and Corynebacterium glutamicum (Wieschalka et al. 2012), and yeasts, such as Saccharomyces cerevisiae (van Maris et al. 2004) and Torulopsis glabrata (Liu et al. 2007; Miyata and Yonehara 1999).
Halophilic bacteria are utilized in industrial chemical-production processes owing to their unique properties, such as contamination-free culture conditions and a tolerance for high substrate concentrations (Quillaguamán et al. 2010; Yin et al. 2015). In addition, alkaliphilic bacteria are utilized to produce pure organic acids (Calabia et al. 2011; Yokaryo and Tokiwa 2014). Therefore, alkaliphilic and halophilic bacteria are appropriate candidates for industrial production of pure organic acids.
The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 was recently isolated and found to store bioplastic poly-(R)-3-hydroxybutyric acid (PHB) intracellularly under aerobic conditions (Kawata and Aiba 2010). In addition, the KM-1 strain secretes pure (R)-3-hydroxybutyric acid ([R]-3-HB) under microaerobic conditions (Kawata et al. 2012a). The KM-1 strain has potential for use in industrial fermentation applications, with some specific advantages over other microorganisms, particularly for the industrial-scale production of organic acids. First, sterilization of culture medium is not required to eliminate contamination by other bacteria because the KM-1 strain can be cultured in modified SOT medium that is alkaline (pH 9.4), with moderate salinity. Moreover, modified SOT medium is an inexpensive, simple, and chemically defined medium. Second, biotechnological production using the KM-1 strain is not subject to strict safety regulations because it is neither pathogenic nor recombinant. Third, the KM-1 strain can utilize a variety of carbon sources; surprisingly the KM-1 strain can utilize C6 and C5 sugars in parallel, without glucose catabolite repression (Kawata et al. 2012a, 2013). In addition, this strain has already been employed to produce PHB from biodiesel waste glycerol and saccharified wood (Kawata and Aiba 2010; Kawata et al. 2013). Finally, because the KM-1 strain is tolerant of moderately high osmolality, a high concentration of the substrate, e.g., 20 % glucose, may be used to facilitate high productivity of pure organic acids. Thus, the KM-1 strain has been evaluated for potential applications in the industrial-scale production of PHB and (R)-3-HB (Kawata et al. 2012a, 2013, 2014).
In the metabolic pathway of PHB synthesis, the carbon source (glucose) is converted to phosphoenolpyruvate (PEP), and PEP is then converted to pyruvate by pyruvate kinase (PK). Pyruvate is decarboxylated by the pyruvate dehydrogenase complex (PDC) to produce acetyl-CoA, which is then used for subsequent reactions. When the sodium nitrate concentration is increased, the entire metabolic pathway of PHB synthesis would likely be activated; thus, higher PHB productivity would be expected. Interestingly, however, in our preliminary work, we found that increased sodium nitrate concentrations resulted in reduced PHB productivity and enhanced pyruvate secretion. Therefore, in this study, we further explored this observation and investigated changes in pyruvate production with different nitrate concentrations, culture temperatures, and buffer salinities in simple batch cultivations of the KM-1 strain under aerobic conditions.
Materials and methods
In this study, we used Halomonas sp. KM-1, which was deposited at the National Institution of Technology Evaluation as FERM BP-10995 (Kawata and Aiba 2010). KM-1 cells were cultivated using 20 mL modified unsterilized SOT medium supplemented with 20 % (w/v) glucose, with initial sodium hydrogen carbonate and sodium carbonate concentrations of 0.2 M (pH 9.4), in 200-mL Erlenmeyer flasks with rotational shaking at 200 rpm at 33 °C (Kawata et al. 2012a).
Optimal pyruvate production conditions were investigated by varying the sodium nitrate concentration (15.0, 20.0, 25.0, and 30.0 g/L), culture temperature (33, 37, 40, and 43 °C), and sodium hydrogen carbonate and sodium carbonate concentration (0.2, 0.3, 0.4, 0.5, and 0.6 M; maintaining pH 9.4). All experiments were performed in triplicate.
Analysis of PHB, glucose pyruvate, and pH levels
To measure PHB content, samples were collected every 12 h, unless otherwise stated. PHB contents were analyzed by gas chromatography (Kawata et al. 2012a; Monteil-Rivera et al. 2007) using a commercial PHB sample (Fluka, Buchs, Switzerland) used as a standard. Glucose and pyruvate contents were analyzed by high-performance liquid chromatography (HPLC), as described by the manufacturer (Aminex HPX-87H; Bio-Rad, Tokyo, Japan) with commercially available, pure samples of d-glucose and pyruvate (Wako, Osaka, Japan) used as standards. The pH levels of the different media were analyzed using a portable D-71 pH meter with a Micro ToupH electrode 9618S-10D (Horiba, Kyoto, Japan).
Effects of changing the sodium nitrate concentration on the production of pyruvate and PHB by KM-1 cells
In the same experiment, analysis of glucose contents in the medium by HPLC revealed that glucose was consumed at a constant rate until 60 h of cultivation, as expected based on the PHB-production levels observed (Fig. 1b, d). In particular, for samples supplemented with 20.0, 25.0, and 30.0 g/L sodium nitrate, the speed of glucose consumption was generally similar, whereas the maximum PHB production decreased as the sodium nitrate concentration increased (Fig. 1b, d). Surprisingly, we also observed an increase in pyruvate concentrations in the medium as the amount of sodium nitrate increased (Fig. 1c). Thus, we expected that some of the consumed glucose was converted to pyruvate. We selected 30.0 g/L sodium nitrate for use in further experiments.
Effects of changing the culture temperature on pyruvate production by KM-1 cells
Effects of changing the sodium bicarbonate buffer concentration on pyruvate production by KM-1 cells
The amounts of CDM and PHB measured were similar up through 24 h. Subsequent increases in CDM and PHB levels were blocked in samples with 0.2, 0.3, or 0.4 M buffer at 36 h. However, for samples cultured in 0.5 or 0.6 M buffer, the CDM and PHB levels continued to increase until 60 h (Fig. 3a, b). Similarly, glucose consumption was suppressed in samples cultured in 0.2 or 0.3 M buffer after 36 h and in samples cultured in 0.4 M buffer after 48 h; glucose consumption was not halted in samples cultured in 0.5 or 0.6 M buffer until 72 h (Fig. 3d). Because the pyruvate concentration exceeded more than the individual buffer concentration, the pH in the medium decreased to below 8.0, after which the pyruvate concentration did not increase (Fig. 3c, e). For samples cultured with 0.5 or 0.6 M buffer, after the consumption of glucose, the increase in the pyruvate concentration was blocked after 60 h (Fig. 3c, d), suggesting that the secreted pyruvate may have been taken back up by KM-1 cells. The highest pyruvate levels produced by the KM-1 strain were 63.3 g/L with a productivity of 1.32 g/(L·h) at 48 h and 67.0 g/L with a productivity of 0.93 g/(L·h) at 72 h in simple aerobic batch cultivations, using 0.4 M sodium bicarbonate buffer (Fig. 3c).
Schügerl (2000) suggested that organic acid accumulation may suppress growth and byproduct formation during organic acid fermentation. Moreover, this inhibitory effect may be related to the decrease in pH observed as acids accumulate and subsequent acid-dependent toxicity, particularly at low pH values, at which acids will be preferentially in the undissociated state. The same conditions were employed for pyruvate production in the current study (Fig. 3c). The maximum pyruvate production levels for samples incubated with 0.2, 0.3, or 0.4 M buffer were 47.1, 57.9, and 67.0 g/L, respectively. If the maximum pyruvate production was controlled by the buffer concentration, then pyruvate production in samples containing 0.3 or 0.4 M buffer should have exceeded the observed pyruvate levels (Fig. 3c), as should the pyruvate concentrations observed in samples containing 0.5 or 0.6 M buffer (Fig. 3c). Thus, higher salinity may interfere with the growth of KM-1 cells and pyruvate production.
In this study, pyruvate secretion changes by strain Halomonas sp. KM-1 were investigated in the presence of different nitrate concentrations, culture temperatures, and buffer salinities in simple batch cultivations under aerobic conditions. To increase PHB productivity, nitrate concentrations were increased, but PHB productivity was not increased, and pyruvate secretion by KM-1 cells was unexpectedly observed (Fig. 1b, c). These results showed that increased sodium nitrate concentrations promoted pyruvate secretion by the KM-1 strain. Some soil bacteria can use nitrate respiration under aerobic conditions (Carter et al. 1995), and Halomonas type bacterium Halomonas elongata can reduce nitrate and grow anaerobically (Vreeland et al. 1980). Based on this phenomenon, the nitrate concentration is associated with pyruvate production; however, the association between pyruvate production and nitrate concentration observed in this study has not been previously described.
The KM-1 strain could be cultured at pH 6.5–10.5, with an optimum pH of 9.4 (Kawata and Aiba 2010; Kawata et al. 2014). When the sodium carbonate buffer concentration in the medium was 0.2 M, pyruvate production resulting in pyruvate concentrations of >0.2 M (17.6 g/L) caused the pH to decrease, blocking additional pyruvate production (Fig. 2e). The maximal pyruvate production was 47.1 g/L (0.53 M) after culturing KM-1 cells at 40 °C for 72 h, when the optimal culture temperature was investigated.
Pyruvate production by various microorganisms using different procedures
Halomonas sp. KM-1
This work (72 h)
Halomonas sp. KM-1
This work (48 h)
E. coli ALS1059
Zhu et al. (2008)
E. coli TC44
Causey et al. (2004)
E. coli TBLA-1
Yokota et al. (1994)
S. cerevisiae TAM
van Maris et al. (2004)
C. glabrata (T. glabrata) ACII-33
Miyata and Yonehara (1999)
C. glabrata (T. glabrata) RS23
Mutant, continuous culture
Liu et al. (2007)
C. glutamicum ELB-P
Wieschalka et al. (2012)
Liu et al. (2007) reported an enhancement of pyruvate production by the osmotic-tolerant mutant C. glabrata RS23 strain. The KM-1 strain has a relatively high tolerance for salinity until the NaCl concentration reaches 10 % (w/v; 1.7 M) (Kawata et al. 2012b). Thus, the wild-type KM-1 strain exhibits a natural tolerance of moderately high osmolality; accordingly, a substrate concentration of up to 20 % glucose can be used to facilitate high pyruvate production. Thus, future studies are needed to achieve pyruvate production at an industrial scale using the osmotic-tolerant mutant of the KM-1 strain, which may result in higher pyruvate productivity under high osmotic culture conditions, using a fed-batch culture system.
In this study, the alkaliphilic, halophilic bacterium Halomonas sp. KM-1 secreted 63.3 g/L pyruvate with a productivity of 1.32 g/(L·h) by 48 h of aerobic batch cultivation in sodium nitrate- and glucose-rich medium, without sterilization. Pyruvate production was related to the nitrate concentration; this mechanism has not been published previously. Pyruvate production in this study occurred at a relatively high level, and the KM-1 strain offered some advantages, such as contamination-free culture and the capacity for high substrate concentrations. Thus, these results suggest that the KM-1 strain may be a suitable candidate for industrial pyruvate production.
Conceived and designed the experiments: YK, TN, IM, JT. Performed the experiments: YK. Analyzed the data: YK, TN, IM, JT. Contributed reagents/materials/financing support/analysis tools: YK, TN, IM, JT. Wrote main manuscript: YK, TN. Preapared figures and tables: YK, IM. All authors read and approved the final manuscript.
This study was partly supported by JSPS KAKENHI (Grant No. 15K07821). The authors thank Noriko Shimozono and Rewa Yanagisawa for providing technical assistance. The authors also thank Cactus Communications, Inc. for English language editing.
The authors declare that they have no competing interests. This article does not contain any studies with human participants or animals performed by any of the authors.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Calabia BP, Tokiwa Y, Aiba S. Fermentative production of l-(+)-lactic acid by an alkaliphilic marine microorganism. Biotechnol Lett. 2011;33:1429–33. doi:10.1007/s10529-011-0573-0.View ArticlePubMedGoogle Scholar
- Carter JP, Hsaio YH, Spiro S, Richardson DJ. Soil and sediment bacteria capable of aerobic nitrate respiration. Appl Environ Microbiol. 1995;61:2852–8.PubMedPubMed CentralGoogle Scholar
- Causey TB, Shanmugam KT, Yomano LP, Ingram LO. Engineering Escherichia coli for efficient conversion of glucose to pyruvate. Proc Natl Acad Sci USA. 2004;101:2235–40. doi:10.1073/pnas.0308171100.View ArticlePubMedPubMed CentralGoogle Scholar
- Howard JW, Fraser WA. Preparation of pyruvic acid. Org Synth Coll. 1932;1:475–80.Google Scholar
- Kawata Y, Aiba S. Poly(3-hydroxybutyrate) production by isolated Halomonas sp. KM-1 using waste glycerol. Biosci Biotechnol Biochem. 2010;74:175–7. doi:10.1271/bbb.90459.View ArticlePubMedGoogle Scholar
- Kawata Y, Kawasaki K, Shigeri Y. Efficient secreted production of (R)-3-hydroxybutyric acid from living Halomonas sp. KM-1 under successive aerobic and microaerobic conditions. Appl Microbiol Biotechnol. 2012a;96:913–20. doi:10.1007/s00253-012-4218-6.View ArticlePubMedGoogle Scholar
- Kawata Y, Shi LH, Kawasaki K, Shigeri Y. Taxonomic characterization and metabolic analysis of the Halomonas sp. KM-1, a highly bioplastic poly(3-hydroxybutyrate)-producing bacterium. J Biosci Bioeng. 2012b;113:456–60. doi:10.1016/j.jbiosc.2011.11.018.View ArticlePubMedGoogle Scholar
- Kawata Y, Jin YX, Nojiri M. Efficient secretion of (R)-3-hydroxybutyric acid from Halomonas sp. KM-1 cultured with saccharified Japanese cedar under microaerobic conditions. Biores Technol. 2013;140:443–5. doi:10.1016/j.biortech.2013.05.015.View ArticleGoogle Scholar
- Kawata Y, Ando H, Matsushita I, Tsubota J. Efficient secretion of (R)-3-hydroxybutyric acid from Halomonas sp. KM-1 by nitrate fed-batch cultivation with glucose under microaerobic conditions. Biores Technol. 2014;156:400–3. doi:10.1016/j.biortech.2014.01.073.View ArticleGoogle Scholar
- Li Y, Chen J, Lun SY. Biotechnological production of pyruvic acid. Appl Microbiol Biotechnol. 2001;57:451–9.View ArticlePubMedGoogle Scholar
- Liu L, Xu Q, Li Y, Shi Z, Zhu Y, Du G, Chen J. Enhancement of pyruvate production by osmotic-tolerant mutant of Torulopsis glabrata. Biotechnol Bioeng. 2007;97:825–32. doi:10.1002/bit.21290.View ArticlePubMedGoogle Scholar
- Miyata R, Yonehara T. Breeding of high-pyruvate producing Torulopsis glabrata with acquired reduced pyruvate decarboxylase. J Biosci Bioeng. 1999;88:173–8.View ArticlePubMedGoogle Scholar
- Monteil-Rivera F, Betancourt A, Van Tra H, Yezza A, Hawari J. Use of headspace solid-phase microextraction for the quantification of poly(3-hydroxybutyrate) in microbial cells. J Chromatogr A. 2007;1154:34–41. doi:10.1016/j.chroma.2007.03.121.View ArticlePubMedGoogle Scholar
- Quillaguamán J, Guzmán H, Van-Thuoc D, Hatti-Kaul R. Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects. Appl Microbiol Biotechnol. 2010;85:1687–96. doi:10.1007/s00253-009-2397-6.View ArticlePubMedGoogle Scholar
- Schügerl K. Integrated processing of biotechnology products. Biotechnol Adv. 2000;18:581–99. doi:10.1016/S0734-9750(00)00051-3.View ArticlePubMedGoogle Scholar
- Van Maris AJ, Geertman JM, Vermeulen A, Groothuizen MK, Winkler AA, Piper MD, van Dijken JP, Pronk JT. Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast. Appl Environ Microbiol. 2004;70:159–66. doi:10.1128/AEM.70.1.159-166.2004.View ArticlePubMedPubMed CentralGoogle Scholar
- Vreeland RH, Litchfield CD, Martin EL, Elliot E. Halomonas elongata, a new genus and species of extremely salt-tolerant bacteria. Int J Syst Evol Microbiol. 1980;30:485–95. doi:10.1099/00207713-30-2-485.Google Scholar
- Wieschalka S, Blombach B, Eikmanns BJ. Engineering Corynebacterium glutamicum for the production of pyruvate. Appl Microbiol Biotechnol. 2012;94:449–59. doi:10.1007/s00253-011-3843-9.View ArticlePubMedGoogle Scholar
- Yin J, Chen JC, Wu Q, Chen GQ. Halophiles, coming stars for industrial biotechnology. Biotechnol Adv. 2015;33:1433–42. doi:10.1016/j.biotechadv.2014.10.008.View ArticlePubMedGoogle Scholar
- Yokaryo H, Tokiwa Y. Isolation of alkaliphilic bacteria for production of high optically pure L-(+)-lactic acid. J Gen Appl Microbiol. 2014;60:270–5. doi:10.2323/jgam.60.270.View ArticlePubMedGoogle Scholar
- Yokota A, Terasawa Y, Takaoka N, Shimizu H, Tomita F. Pyruvic acid production by an F1-ATPase-defective mutant of Escherichia coli W1485lip2. Biosci Biotechnol Biochem. 1994;58:2164–7. doi:10.1271/bbb.58.2164.View ArticlePubMedGoogle Scholar
- Zhu Y, Eiteman MA, Altman R, Altman E. High glycolytic flux improves pyruvate production by a metabolically engineered Escherichia coli strain. Appl Microbiol Biotechnol. 2008;74:6649–55. doi:10.1128/AEM.01610-08.Google Scholar