Annals of Microbiology (2019) 69:1537–1546 https://doi.org/10.1007/s13213-019-01538-8 ORIGINAL ARTICLE D-Lactic acid fermentation performance and the enzyme activity of a novel bacterium Terrilactibacillus laevilacticus SK5–6 Budsabathip Prasirtsak1 & Sitanan Thitiprasert2 & Vasana Tolieng2 & Suttichai Assabumrungrat3 & Somboon Tanasupawat4 & Nuttha Thongchul2 Received: 15 July 2019 /Accepted: 20 November 2019 /Published online: 7 December 2019 # The Author(s) 2019 Abstract Purpose The aim of this study was to prove that Terrilactibacillus laevilacticus SK5-6, a novel D-lactate producer, exhibited a good fermentation performance comparing to the reference D-lactate producer Sporolactobacillus sp. Methods Glucose bioconversion for D-lactate production and the activity of five key enzymes including phosphofructokinase (PFK), pyruvate kinase (PYK), D-lactate dehydrogenase (D-LDH), L-lactate dehydrogenase (L-LDH), and lactate isomerase (LI) were investigated in the cultivation of T. laevilacticus SK5–6andS. laevolacticus 0361T. Results T. laevilacticus SK5–6 produced D-lactate at higher yield, productivity, and optical purity compared with S. laevolacticus 0361T. T. laevilacticus SK5–6, the catalase-positive isolate, simultaneously grew and produced D-lactate without lag phase while delayed growth and D-lactate production were observed in the culture of S. laevolacticus 0361T. The higher production of D- lactate in T. laevilacticus SK5–6 was due to the higher growth rate and the higher specific activities of the key enzymes observed at the early stage of the fermentation. The low isomerization activity was responsible for the high optical purity of D-lactate in the cultivation of T. laevilacticus SK5–6. Conclusion The lowest specific activity of PFK following by PYK and D/L-LDHs, respectively, indicated that the conversion of fructose-6-phosphate was the rate limiting step. Under the well-optimized conditions, the activation of D/L-LDHs by fructose- 1,6-phosphate and ATP regeneration by PYK drove glucose bioconversion toward D-lactate. The optical purity of D-lactate was controlled by D/L-LDHs and the activation of isomerases. High D-LDH with limited isomerase activity was preferable during the fermentation as it assured the high optical purity. Keywords D-Lactic acid . Terrilactibacillus laevilacticus . Fermentation . Glycolytic enzymes . Lactate isomerization . Lactate dehydrogenase The GenBank/EMBL/DBBJ accession number for the 16S rRNA of Introduction T. laevilacticus strain SK5-6 is LC222555 Lactic acid as a chiral compound provides essential roles in * Nuttha Thongchul industrial applications in food industries and life sciences. [email protected] Approximately 70% of lactic acid produced is utilized in the 1 Program in Biotechnology, Faculty of Science, Chulalongkorn food industry (Es et al. 2018). During the last decade, attention University, Phayathai Road, Wangmai, Pathumwan, Bangkok 10330, was given to the commercially valuable products by microbial Thailand biotechnology due to the raised issues in global energy and 2 Research Unit in Bioconversion/Bioseparation for Value-Added environmental concerns. This provides the great potential for Chemical Production, Biorefinery Cluster, Institute of Biotechnology producing green, biodegradable, and biocompatible polylactic and Genetic Engineering, Chulalongkorn University, Phayathai acid (PLA) for replacement of the plastic commodities from Raod, Wangmai, Pathumwan, Bangkok 10330, Thailand 3 petrochemical based feedstocks. The optical purity of lactic Department of Chemical Engineering, Faculty of Engineering, acid (both D- and L-lactic acid) plays a crucial role in control- Chulalongkorn University, Phayathai Road, Wangmai, Pathumwan, Bangkok 10330, Thailand ling the physical properties of PDLLA (Cingadi et al. 2015). Although microbial fermentation of L-lactic acid has long 4 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Phayathai been commercially established, there are relatively few studies Road, Wangmai, Pathumwan, Bangkok 10330, Thailand on D-lactic acid fermentation (Tsuge et al. 2014). Microbial 1538 Ann Microbiol (2019) 69:1537–1546 fermentation can produce three forms of lactic acid, e.g., L- incubated at 37 °C for 24–48 h prior to preparing the bacterial lactate, D-lactate, and DL-lactate, depending on the microbes suspension for inoculation. employed (John et al. 2009). A few wild-type strains such as Lactobacillus delbrueckii subsp. delbrueckii, Fermentation cultivation of the catalase-positive Sporolactobacillus inulinus, Sporolactobacillus laevolacticus, T. laevilacticus SK5–6 for D-lactic acid Sporolactobacillus terrae, Lactobacillus coryniformis subsp. torquens, and L. delbrueckii subsp. lactis QU41 and some D-lactic acid production by the catalase-positive metabolically engineered strains such as Lactobacillus T. laevilacticus SK5–6 was previously optimized for the flask plantarum, Escherichia coli, Corynebacterium glutamicum, cultivation and reported in Prasirtsak et al. (2017). The fer- Enterobacter asburiae,andSaccharomyces cerevisiae includ- mentation consisted of two phases, (i) the preculture phase for ing a novel genus Terrilactibacillus used in this study have cell growth and (ii) the production phase for producing D- been reported for the production of optical pure D-lactic acid lactic acid by cells obtained in the preculture phase. (Tashiro et al. 2011; Mimitsuka et al. 2012;Lietal.2013; T. laevilacticus SK5–6 was subcultured onto the GYP agar Zhao et al. 2014;Cingadietal.2015; Bai et al. 2016; slant and incubated at 37 °C for 24 h and suspended in the Prasirtsak et al. 2016). sterile NaCl solution (0.85% (w/v)). The optical density (OD) Terrilactibacillus genus is comprised of the catalase- at 600 nm of the bacterial suspension was adjusted to approx- positive bacteria that can be grown in the presence of oxygen imately 30–40 using sterile NaCl solution. The diluted sus- (Prasirtsak et al. 2016). It produces the higher cell biomass and pension was then used to inoculate into the sterile preculture lactic acid than other previously reported D-lactate producers medium at 1% (v/v) inoculum size. The preculture medium that usually lack catalase, and thus, required growth and lactic for the catalase-positive bacteria (pH 6.8) contained (in 1 L) acid production under anaerobic conditions (Prasirtsak et al. 10 g glucose, 15 g yeast extract, 4 g NH4Cl, 0.5 g KH2PO4, 2017). Owing to the above-mentioned evidences, 0.5 g K2HPO4, 5 g CaCO3, and 20 mL salt solution. The Terrilactibacillus laevilacticus SK5–6 was selected for further culture was incubated at 37 °C, 200 rpm for 4 h in a flask investigating the fermentation performance and understanding plugged with a C-type silicone stopper. Then, 25 mL of this its bioconversion of glucose to lactic acid from the specific preculture broth was transferred into 25 mL of sterile fermen- activities of the key enzymes in glycolytic pathway as well as tation medium for the catalase-positive isolate which lactate production and isomerization. It is believed that by contained (in liter) 240 g glucose and 160 g CaCO3 and con- understanding the regulation of the key enzymes involving tinued to incubate in a flask plugged with a T-type silicone in glucose bioconversion for D-lactate production, one can stopper in a W-zip pouch containing AnaeroPack-Anaero gas apply this knowledge in process optimization to achieve high pack (Mitsubishi Gas Chemical, Tokyo, Japan) at 37 °C, fermentation performance. 150 rpm for 48 h. Samples were taken every 12 h for analyses of OD600 reading, the remaining glucose, D-lactate and byproduct formation, and the optical purity of D-lactate prod- Materials and methods uct. Cell biomass was also collected for enzymatic activity assays. Microorganism and inoculum preparation Fermentation cultivation of the catalase-negative Terrilactibacillus laevilacticus SK5–6, a catalase-positive D- S. laevolacticus 0361T for D-lactic acid lactic acid producing bacterium, was used for D-lactic acid production and enzyme activity assay (Prasirtsak et al. S. laevolacticus 0361T, the catalase-negative D-lactate pro- 2017). The GenBank/EMBL/DBBJ accession number for ducer, was also used for lactic acid production in this study the 16S rRNA of strain SK5–6 is LC222555. A catalase- for a comparison purpose to T. laevilacticus SK5–6. The fer- negative D-lactic acid producing bacterium, mentation of S. laevolacticus 0361T consisted of two phases Sporolactobacillus laevolacticus NRIC 0361T, was obtained but slight differences in the optimized operating conditions from NODAI Culture Collection Center (NRIC) for use as the previously reported elsewhere (Prasirtsak et al. 2017; reference D-lactic acid producer. Both strains were maintained Thitiprasert et al. 2017). An active 48 h GYP agar slant grown on the GYP agar containing (per liter) 10 g glucose, 5 g yeast under anaerobic condition was used to prepare the bacterial extract, 5 g peptone, 0.25 g KH2PO4,0.25gK2HPO4,20g suspension using the same procedure mentioned above. The agar, 5 g CaCO3, and 10 mL salt solution. Salt solution bacterial suspension was inoculated into the sterile preculture consisted of (per 10 mL) 400 mg MgSO4·7H2O, 20 mg medium at 1% (v/v). The preculture medium for the catalase- MnSO4·5H2O, 20 mg FeSO4·7H2O, and 20 mg NaCl. The negative bacteria (pH 6.8) contained (in 1 L) 10 g glucose, 5 g pH of the GYP agar medium was adjusted to 6.80. Both iso- yeast extract, 5 g peptone,