D-lactic Acid Production Research Articles

Evaluation of a Burkholderia ambifaria strain from plants as a novel promising probiotic in dental caries management

ABSTRACT Background Probiotics serve as a novel preventive or therapeutic approach for dental caries owing to their ability to reverse dysbiosis and restore a healthy microbiota. Here, we identified Burkholderia ambifaria AFS098024 as a probiotic candidate isolated from plants. Methods The safety of B. ambifaria was evaluated by hemolytic activity, D-lactic acid production and antibiotic susceptibility. In vitro biofilm model derived from the saliva of caries-free and caries-active donors and in vivo rat caries model were used to assess the efficacy of B. ambifaria in caries prevention and treatment. Results B. ambifaria was safe as a probiotic candidate and it could integrate with in vitro biofilm model. It significantly reduced the biomass and lactate production of biofilms from caries-active donors and disrupted biofilm structures. B. ambifaria effectively reduced the severity of carious lesions in rat molars, regardless of the inoculation sequence. Molars pretreated or treated with B. ambifaria demonstrated notably higher enamel volumes. Additionally, colonization of rat molars by B. ambifaria persisted for 6 weeks. Conclusion The B. ambifaria strain used in this study holds promise as a probiotic for inhibiting dental caries, both in vitro and in vivo.

Simultaneous saccharification and fermentation for d-lactic acid production using a metabolically engineered Escherichia coli adapted to high temperature

BackgroundEscherichia coli JU15 is a metabolically engineered strain capable to metabolize C5 and C6 sugars with a high yield of d-lactic acid production at its optimal growth temperature (37 °C). The simultaneous saccharification and fermentation process allow to use lignocellulosic biomass as a cost-effective and high-yield strategy. However, this process requires microorganisms capable of growth at a temperature close to 50 °C, at which the activity of cellulolytic enzymes works efficiently.ResultsThe thermotolerant strain GT48 was generated by adaptive laboratory evolution in batch and chemostat cultures under temperature increments until 48 °C. The strain GT48 was able to grow and ferment glucose to d-lactate at 47 °C. It was found that a pH of 6.3 conciliated with GT48 growth and cellulase activity of a commercial cocktail. Hence, this pH was used for the SSF of a diluted acid-pretreated corn stover (DAPCS) at a solid load of 15% (w/w), 15 FPU/g-DAPCS, and 47 °C. Under such conditions, the strain GT48 exhibited remarkable performance, producing d-lactate at a level of 1.41, 1.42, and 1.48-fold higher in titer, productivity, and yield, respectively, compared to parental strain at 45 °C.ConclusionsIn general, our results show for the first time that a thermal-adapted strain of E. coli is capable of being used in the simultaneous saccharification and fermentation process without pre-saccharification stage at high temperatures.

Comprehensive Safety Assessment of Lacticaseibacillus paracasei subsp. paracasei NTU 101 Through Integrated Genotypic and Phenotypic Analysis

Probiotics, as defined by the World Health Organization, are live microorganisms that, when consumed in sufficient quantities, provide health benefits to the host. Although some countries have approved specific probiotic species for use in food, safety concerns may still arise with individual strains. Lacticaseibacillus paracasei subsp. paracasei NTU 101 (NTU 101), isolated from the gut of healthy infants, has demonstrated various probiotic effects and shown safety in a prior 28-day animal feeding study. To further verify its safety and mitigate potential risks, we performed a comprehensive genotypic and phenotypic safety evaluation in accordance with the European Food Safety Authority guidelines for safety assessment through whole genome sequencing and related literature. In this research, minimum inhibitory concentration testing identified NTU 101’s resistance to chloramphenicol; however, subsequent gene analysis confirmed no associated risk of resistance. Assessments of safety, including biogenic amine content, hemolytic activity, mucin degradation, and D-lactic acid production, indicated a low level of risk. Additionally, a repeated-dose 90-day oral toxicity study in Sprague-Dawley rats revealed no toxicity at a dose of 2000 mg/kg body weight, further supporting the strain’s safety for consumption. Based on these comprehensive analyses, NTU 101 is considered safe for regular consumption as a health supplement.

From agricultural biomass to D form lactic acid in ton scale via strain engineering of Lactiplantibacillus pentosus

Worsening environmental conditions make lactic acid a sustainable alternative to petroleum-based plastics. This study created a genetically-engineered strain Lactiplantibacillus pentosus PeL containing a disrupted L-lactate dehydrogenase gene to produce high yield and optically pure D-lactic acid. Cellobiose was identified as the optimal sugar in the single carbon source test, yielding the highest lactic acid. In 5-L fermentation tests, pretreated wood chips hydrolysate was the best lignocellulosic substrate for PeL, resulting in a D-lactic acid yield of 900.7 ± 141.4 mg/g of consumed sugars with an optical purity of 99.8 ± 0.0 %. Gradually scaled-up fermentations using this substrate were achieved in 100-, and 9,000-L fermenters; PeL produced remarkably high D-lactic acid yields of 836.3 ± 11.9 and 915.9 ± 4.4 mg/g of consumed sugars, with optical purities of 95.0 ± 0.0 % and 93.8 ± 0.2 %, respectively. This study is the pioneer in demonstrating economical and sustainable ton-scale production of D-lactic acid.

Experimental evolution reveals an effective avenue for d-lactic acid production from glucose-xylose mixtures via enhanced Glk activity and a cAMP-independent CRP mutation.

d-Lactic acid holds significant industrial importance due to its versatility and serves as a crucial component in the synthesis of environmentally friendly and biodegradable thermal-resistant poly-lactic acid. This polymer exhibits promising potential as a substitute for nonbiodegradable, petroleum-based plastics. The production of d-lactic acid from lignocellulosic biomass, a type of biorenewable and nonfood resources, can lower costs and improve product competitiveness. Glucose and xylose are the most abundant sugar monomers in lignocellulosic biomass materials. Despite Escherichia coli possessing native xylose catabolic pathways and transport, their ability to effectively utilize xylose is often hindered in the presence of glucose. Here, the E. coli strain Rec1.0, previously engineered to overcome carbon catabolite repression, was selected as the initial strain for reengineering to produce d-lactic acid. An adaptive evolution approach was employed to achieve highly efficient fermentation of glucose-xylose mixtures. The resulting strain, QJL010, could produce d-lactic acid of 87.5 g/L with a carbon yield of 0.99 mol/mol. Notably, the consumption rates of glucose and xylose reached 0.75 and 0.82 g/gDCW/h, respectively. Further analysis revealed that increased Glk activity, resulting from glk mutations (A142V and R188H), along with their upregulated expression, contributed to an elevated glucose consumption rate. Additionally, a CRP G141D mutation, cAMP-independent, stimulated the expression of the xylR, xylE, and galABC* genes, resulting in an accelerated xylose consumption rate. These findings provide valuable support for the utilization of E. coli platform strains in the production of value-added chemicals from lignocellulosic biomass.

Third-generation D-lactic acid production using red macroalgae Gelidium amansii by co-fermentation of galactose, glucose and xylose

Macroalgae biomass has been considered as a promising renewable feedstock for lactic acid production owing to its lignin-free, high carbohydrate content and high productivity. Herein, the D-lactic acid production from red macroalgae Gelidium amansii by Pediococcus acidilactici was investigated. The fermentable sugars in G. amansii acid-prehydrolysate were mainly galactose and glucose with a small amounts of xylose. P. acidilactici could simultaneously ferment the mixed sugars of galactose, glucose and xylose into D-lactic acid at high yield (0.90 g/g), without carbon catabolite repression (CCR). The assimilating pathways of these sugars in P. acidilactici were proposed based on the whole genome sequences. Simultaneous saccharification and co-fermentation (SSCF) of the pretreated and biodetoxified G. amansii was also conducted, a record high of D-lactic acid (41.4 g/L) from macroalgae biomass with the yield of 0.34 g/g dry feedstock was achieved. This study provided an important biorefinery strain for D-lactic acid production from macroalgae biomass.

Production of fermentable sugar, ethanol, D-lactic acid, and biochar from starch-rich traditional Chinese medicine decoction residues

Production of fermentable sugar, ethanol, D-lactic acid, and biochar from starch-rich traditional Chinese medicine decoction residues

Harnessing valorization potential of whey permeate for D-lactic acid production using lactic acid bacteria

Harnessing valorization potential of whey permeate for D-lactic acid production using lactic acid bacteria

Dietary D-xylose promotes intestinal health by inducing phage production in Escherichia coli

Elimination of specific enteropathogenic microorganisms is critical to gut health. However, the complexity of the gut community makes it challenging to target specific bacterial organisms. Accumulating evidence suggests that various foods can change the abundance of intestinal bacteria by modulating prophage induction. By using pathogenic Escherichia coli (E. coli) ATCC 25922 as a model in this research, we explored the potential of dietary modulation of prophage induction and subsequent bacterial survival. Among a panel of sugars tested in vitro, D-xylose was shown to efficiently induce prophages in E. coli ATCC 25922, which depends, in part, upon the production of D-lactic acid. In an enteric mouse model, prophage induction was found to be further enhanced in response to propionic acid. Dietary D-xylose increased the proportion of Clostridia which converted D-lactic acid to propionic acid. Intestinal propionic acid levels were diminished, following either oral gavage with the dehydrogenase gene (ldhA)-deficient E. coli ATCC 25922 or depletion of intestinal Clostridia by administration of streptomycin. D-Xylose metabolism and exposure to propionic acid triggered E. coli ATCC 25922 SOS response that promoted prophage induction. E. coli ATCC 25922 mutant of RecA, a key component of SOS system, exhibited decreased phage production. These findings suggest the potential of using dietary components that can induce prophages as antimicrobial alternatives for disease control and prevention by targeted elimination of harmful gut bacteria.

Characterization of the Pro101Gln mutation that enhances the catalytic performance of T. indicus NADH-dependent d-lactate dehydrogenase

NADH-dependent d-lactate dehydrogenases (d-LDH) are important for the industrial production of d-lactic acid. Here, we identify and characterize an improved d-lactate dehydrogenase mutant (d-LDH1) that contains the Pro101Gln mutation. The specific enzyme activities of d-LDH1 toward pyruvate and NADH are 21.8- and 11.0-fold greater compared to the wild-type enzyme. We determined the crystal structure of Apo-d-LDH1 at 2.65Å resolution. Based on our structural analysis and docking studies, we explain the differences in activity with an altered binding conformation of NADH in d-LDH1. The role of the conserved residue Pro101 in d-LDH was further probed in site-directed mutagenesis experiments. We introduced d-LDH1 into Bacillus licheniformis yielding a d-lactic acid production of 145.9g L-1 within 60h at 50°C, which was three times higher than that of the wild-type enzyme. The discovery of d-LDH1 will pave the way for the efficient production of d-lactic acid by thermophilic bacteria.

Development of a Novel D-Lactic Acid Production Platform Based on Lactobacillus saerimneri TBRC 5746.

D-Lactic acid is a chiral, three-carbon organic acid, that bolsters the thermostability of polylactic acid. In this study, we developed a microbial production platform for the high-titer production of D-lactic acid. We screened 600 isolates of lactic acid bacteria (LAB) and identified twelve strains that exclusively produced D-lactic acid in high titers. Of these strains, Lactobacillus saerimneri TBRC 5746 was selected for further development because of its homofermentative metabolism. We investigated the effects of high temperature and the use of cheap, renewable carbon sources on lactic acid production and observed a titer of 99.4g/L and a yield of 0.90g/g glucose (90% of the theoretical yield). However, we also observed L-lactic acid production, which reduced the product's optical purity. We then used CRISPR/dCas9-assisted transcriptional repression to repress the two Lldh genes in the genome of L. saerimneri TBRC 5746, resulting in a 38% increase in D-lactic acid production and an improvement in optical purity. This is the first demonstration of CRISPR/dCas9-assisted transcriptional repression in this microbial host and represents progress toward efficient microbial production of D-lactic acid.

EFFECTS OF MEDIA COMPOSITIONS ON AMYLASE AND D-LACTIC ACID PRODUCED BY A POTENTIAL STARCH-UTILIZING BACTERIUM

The high demand for amylase and lactic acid in various industries made enzyme and acid production from cheap substrates very attractive. This study investigated the effect of major medium constituents on amylase and D-lactic acid production by the potential starch-utilizing and optical pure D-lactic acid-producing Lactobacillus sp. SUTWR 73, to provide data for the efficient application of the lactic acid bacterium. Its amylase and D-lactic acid production was considerably affected by the addition of organic nitrogen tryptone to certain cassava starch concentrations as a carbon-source. While the other medium compositions: yeast extract and cheap nitrogen sources (soy protein, defatted rice bran, and spent brewery yeast) compared to tryptone as equal total nitrogen contents, did not have any effect on either metabolite production. The medium containing cassava starch (4.0%, w/v) and tryptone (4.5%, w/v) can be satisfactorily used for both products. The initial pH of the production medium was another factor affecting metabolite production. A suitable initial pH was 8.0 and 7.0-10.0 for amylase and D-lactic acid production, respectively. Amylase activity was detected at the early log growth phase while the D-lactic acid was produced at the late log phase. Understanding the relationship between amylase and D-lactic acid production from this study is important for designing an economical operating system for the direct production of D-lactic acid from cheap cassava starch without adding any commercial amylase as a substrate in pretreatment resulting in reduction costs of the production process.

Transcriptional analysis of the molecular mechanism underlying the response of Lactiplantibacillus plantarum to lactic acid stress conditions

Lactic acid bacteria (LAB) present various benefits to humans; they play key roles in the fermentation of food and as probiotics. Acidic conditions are common to both LAB in the intestinal tract as well as fermented foods. Lactiplantibacillus plantarum is a facultative homofermentative bacterium, and lactic acid is the end metabolite of glycolysis. To characterize how L. plantarum responds to lactic acid, we investigated its transcriptome following treatment with hydrochloride (HCl) or dl-lactic acid at an early stage of growth. Bacterial growth was more attenuated in the presence of lactic acid than in the presence of HCl at the same pH range. Bacterial transcriptome analysis showed that the expression of 67 genes was significantly altered (log2FC > 2 or < 2). A total of 31 genes were up- or downregulated under both conditions: 19 genes in the presence of HCl and 17 genes in the presence of dl-lactic acid. The fatty acid synthesis-related genes were upregulated in both acidic conditions, whereas the lactate racemization-related gene (lar) was only upregulated following treatment with dl-lactic acid. In particular, lar expression increased following l-lactic acid treatment but did not increase following HCl or d-lactic acid treatment. Expression of lar and production of d-lactic acid were investigated with malic and acetic acid; the results revealed a higher expression of lar and production of d-lactic acid in the presence of malic acid than that in the presence of acetic acid.

Engineering Flocculation for Improved Tolerance and Production of d-Lactic Acid in Pichia pastoris.

d-lactic acid, a chiral organic acid, can enhance the thermal stability of polylactic acid plastics. Microorganisms such as the yeast Pichia pastoris, which lack the natural ability to produce or accumulate high amounts of d-lactic acid, have been metabolically engineered to produce it in high titers. However, tolerance to d-lactic acid remains a challenge. In this study, we demonstrate that cell flocculation improves tolerance to d-lactic acid and increases d-lactic acid production in Pichia pastoris. By incorporating a flocculation gene from Saccharomyces cerevisiae (ScFLO1) into P. pastoris KM71, we created a strain (KM71-ScFlo1) that demonstrated up to a 1.6-fold improvement in specific growth rate at high d-lactic acid concentrations. Furthermore, integrating a d-lactate dehydrogenase gene from Leuconostoc pseudomesenteroides (LpDLDH) into KM71-ScFlo1 resulted in an engineered strain (KM71-ScFlo1-LpDLDH) that could produce d-lactic acid at a titer of 5.12 ± 0.35 g/L in 48 h, a 2.6-fold improvement over the control strain lacking ScFLO1 expression. Transcriptomics analysis of this strain provided insights into the mechanism of increased tolerance to d-lactic acid, including the upregulations of genes involved in lactate transport and iron metabolism. Overall, our work represents an advancement in the efficient microbial production of d-lactic acid by manipulating yeast flocculation.

Simultaneous and rate-coordinated conversion of lignocellulose derived glucose, xylose, arabinose, mannose, and galactose into D-lactic acid production facilitates D-lactide synthesis

D-lactide is the precursor of poly(D-lactide) (PDLA) or stereo-complex with poly(L-lactide) (PLLA). Lignocellulosic biomass provides the essential feedstock option to synthesize D-lactic acid and D-lactide. The residual sugars in D-lactic acid fermentation broth significantly blocks the D-lactide synthesis. This study showed a simultaneous and rate-coordinated conversion of lignocellulose derived glucose, xylose, arabinose, mannose, and galactose into D-lactic acid by adaptively evolved Pediococcus acidilactici ZY271 by simultaneous saccharification and co-fermentation (SSCF) of wheat straw. The produced D-lactic acid achieved minimum residual sugars (∼1.7 g/L), high chirality (∼99.1%) and high titer (∼128 g/L). A dry acid pretreatment eliminated the wastewater stream generation and the biodetoxification by fungus Amorphotheca resinae ZN1 removed the inhibitors from the pretreatment. The removal of the sugar residues and inhibitor impurities in D-lactic acid production from lignocellulose strongly facilitated the D-lactide synthesis. This study filled the gap in cellulosic D-lactide production from lignocellulose-derived D-lactic acid.

Low acyl gellan gum immobilized Lactobacillus bulgaricus T15 produce d-lactic acid from non-detoxified corn stover hydrolysate

Straw biorefinery offers economical and sustainable production of chemicals. The merits of cell immobilization technology have become the key technology to meet d-lactic acid production from non- detoxified corn stover. In this paper, Low acyl gellan gum (LA-GAGR) was employed first time for Lactobacillus bulgaricus T15 immobilization and applied in d-lactic acid (D-LA) production from non-detoxified corn stover hydrolysate. Compared with the conventional calcium alginate (E404), LA-GAGR has a hencky stress of 82.09 kPa and excellent tolerance to 5-hydroxymethylfurfural (5-HMF), ferulic acid (FA), and vanillin. These features make LA-GAGR immobilized T15 work for 50 days via cell-recycle fermentation with D-LA yield of 2.77 ± 0.27 g/L h, while E404 immobilized T15 can only work for 30 days. The production of D-LA from non-detoxified corn stover hydrolysate with LA-GAGR immobilized T15 was also higher than that of free T15 fermentation and E404 immobilized T15 fermentation. In conclusion, LA-GAGR is an excellent cell immobilization material with great potential for industrial application in straw biorefinery industry.Graphical

Construction of a machine-learning model to predict the optimal gene expression level for efficient production of D-lactic acid in yeast.

The modification of gene expression is being researched in the production of useful chemicals by metabolic engineering of the yeast Saccharomyces cerevisiae. When the expression levels of many metabolic enzyme genes are modified simultaneously, the expression ratio of these genes becomes diverse; the relationship between the gene expression ratio and chemical productivity remains unclear. In other words, it is challenging to predict phenotypes from genotypes. However, the productivity of useful chemicals can be improved if this relationship is clarified. In this study, we aimed to construct a machine-learning model that can be used to clarify the relationship between gene expression levels and D-lactic acid productivity and predict the optimal gene expression level for efficient D-lactic acid production in yeast. A machine-learning model was constructed using data on D-lactate dehydrogenase and glycolytic genes expression (13 dimensions) and D-lactic acid productivity. The coefficient of determination of the completed machine-learning model was 0.6932 when using the training data and 0.6628 when using the test data. Using the constructed machine-learning model, we predicted the optimal gene expression level for high D-lactic acid production. We successfully constructed a machine-learning model to predict both D-lactic acid productivity and the suitable gene expression ratio for the production of D-lactic acid. The technique established in this study could be key for predicting phenotypes from genotypes, a problem faced by recent metabolic engineering strategies.

Galactitol Transport Factor GatA Relieves ATP Supply Restriction to Enhance Acid Tolerance of Escherichia coli in the Two-Stage Fermentation Production of D-Lactate

Escherichia coli is a major contributor to the industrial production of organic acids, but its production capacity and cost are limited by its acid sensitivity. Enhancing acid resistance in E. coli is essential for improving cell performance and production value. Here, we propose a feasible strategy for improving cellular acid tolerance by reducing ATP supply restriction. Transcriptome assays of acid-tolerant evolved strains revealed that the galactitol phosphotransferase system transporter protein GatA is an acid-tolerance factor that assists E. coli in improving its resistance to a variety of organic acids. Enhanced GatA expression increased cell survival under conditions of lethal stress due to D-lactic acid, itaconic acid and succinic acid by 101.8-fold, 29.4-fold and 41.6-fold, respectively. In addition, fermentation patterns for aerobic growth and oxygen-limited production of D-lactic acid were identified, and suitable transition and induction stages were evaluated. GatA effectively compensated for the lack of cellular energy during oxygen limitation and enabled the D-lactic acid producing strain to exhibit more sustainable productivity in acidic fermentation environments with a 55.7% increase in D-lactic acid titer from 9.5 g·L−1 to 14.8 g·L−1 and reduced generation of by-product. Thus, this study developed a method to improve the acid resistance of E. coli cells by compensating for the energy gap without affecting normal cell metabolism while reducing the cost of organic acid production.

Enhanced cellulosic d-lactic acid production from sugarcane bagasse by pre-fermentation of water-soluble carbohydrates before acid pretreatment

After several rounds of milling process for sugars extraction from sugarcane, certain amounts of water-soluble carbohydrates (WSC) still remain in sugarcane bagasse. It is a bottleneck to utilize WSC in sugarcane bagasse biorefinery, since these sugars are easily degraded into inhibitors during pretreatment. Herein, a simple pre-fermentation step before pretreatment was conducted, and 98 % of WSC in bagasse was fermented into d-lactic acid. The obtained d-lactic acid was stably preserved in bagasse and 5-hydroxymethylfurfural (HMF) generation was sharply reduced from 46.0 mg/g to 6.2 mg/g of dry bagasse, after dilute acid pretreatment. Consequently, a higher d-lactic acid titer (57.0 g/L vs 33.2 g/L) was achieved from the whole slurry of the undetoxified and pretreated sugarcane bagasse by one-pot simultaneous saccharification and co-fermentation (SSCF), with the overall yield of 0.58 g/g dry bagasse. This study gave an efficient strategy for enhancing lactic acid production using the lignocellulosic waste from sugar industry.

Microbial D-lactic acid production, In Situ separation and recovery from mature and young coconut husk hydrolysate fermentation broth

The bioplastic industry's demand for D-lactic acid is increasing due to the widespread use of polylactic acid (PLA). Presently, D-lactic acid production exploits lignocellulosic biomass as carbon sources for fermentation gaining major attention. Mature coconut husk (MCH) and young coconut husk (YCH) hydrolysates were used as fermentation broth for D-lactic acid production by Lactobacillus coryniformis subsp. torquens. Yeast extract was added and broth pH was controlled by NaOH in both hydrolysates to facilitate the D-lactic acid production. A significant increase in D-lactic acid production with 14.93 and 19.88 g/L, in MCH and YCH was observed, respectively. However, the D-lactic acid produced existed in the form of lactate salt, which requires multiple separation and purification steps. In situD-lactic acid separation and recovery using ion exchange resin (IRA-67) of 0.4–1.0 g was investigated, and the result showed that 0.6 g of resin effectively separate D-lactic acid while maintaining broth pH in the optimum range. The total recovery of D-lactic acid from IRA-67 resin was 0.67 g for MCH and 0.90 g for YCH hydrolysate broth, with 90% recovering efficiency. The recovered D-lactic acid was considered to have the least fermentation impurities and potentially be used in PLA synthesis.