L-lactic Acid Production Research Articles

PH shifting adaptive evolution stimulates the low pH tolerance of Pediococcus acidilactici and high L-lactic acid fermentation efficiency

L-lactic acid fermentation at low pH reduces the use of neutralizers during fermentation and the generation of solid wastes in purification processes. Most lactic acid bacteria exhibit weak tolerance and poor cell viability at low pH. This study proposes a pH shifting adaptive evolution method to improve the low-pH tolerance of an engineered Pediococcus acidilactici strain. In the first stage, cells were cultured at a moderate pH to maintain the cell viability, then shifted to a low pH to enhance low-pH tolerance. Long-term pH shifting evolution culture of the engineered P. acidilactici between the moderate and low pH resulted in a 43 % increase in L-lactic acid production at pH 4.6 (110.4 g/L) and a 2.1-fold increase at pH 4.4 (80.7 g/L) compared to the parental strain when using wheat straw as a feedstock. This pH-shifting adaptive evolution strategy provides an effective tool for improving the low-pH tolerance of lactic acid bacteria.

Production of L-lactic acid from methanol by engineered yeast Pichia pastoris

Lactic acid (LA) serves as a widely used platform compound and has received significant attention as a raw material for synthesis of biodegradable polylactic acid. Currently, LA is mainly produced through microbial fermentation, but its high costs undermine its competitive advantage against other materials, necessitating the development of novel production routes. Methanol bioconversion represents an emerging low-carbon circular economy, where LA could become an outstanding representative product. This study successfully established an efficient methanol-based LA synthesis route in Pichia pastoris. Through systematic metabolic engineering strategies, including screening lactate dehydrogenase, modification of cofactor preference, blocking LA consumption pathway, and mitochondrial LA synthesis compartmentalization, 4.2 g/L L-LA was produced in fed-batch fermentation by using methanol as the sole carbon source. Through multi-dimensional and spatial engineering of enzyme, a cell factory was developed for efficient synthesis of L-LA, highlights the significant potential of the low-carbon synthesis route for L-LA via methanol bioconversion.

Integrating use of sugarcane bagasse with sugarcane juice for efficient l-lactic acid production through fed-batch simultaneous saccharification and fermentation: Process configurations and kinetic analysis

The present study investigated the utilization of sugarcane juice (SJ) and sugarcane bagasse (SB) for L-lactic acid (L-LA) production by a newly isolated Enterococcus gallinarum TSKKU D-6. SJ was first investigated as a single substrate, yielding 43.9 ± 2.9 g-LA/L, with a fermentation efficiency of 72.0 ± 0.1 %, based on sucrose content in SJ. On the other hand, simultaneous saccharification and fermentation (SSF) of SB as a single substrate yielded 68.2 ± 2.7 g-LA/L, with a fermentation efficiency of 74.1 ± 0.0 %, based on cellulose content in SB. SSF of SJ and SB as co-substrates was subsequently carried out to increase the acid production, where SJ was first fermented, followed by the addition of SB to enhance LA titer. Effects of mixing and SB addition strategy on the kinetics and economic feasibility of L-LA production were investigated using a bioreactor equipped with a single and dual impellers, as well as the use of dual impellers under fed-batch SSF. The use of single and dual impellers gave similar L-LA titers of 95.9 ± 1.4 and 102.6 ± 0.6 g/L, respectively. Nevertheless, the use of dual impellers shortened the processing time, enhancing LA productivity from 0.37 to 1.12 g/(L·h). As a result, the economic yield and productivity were improved by 7.1 % and 187.5 %, respectively. Better still, performing fed-batch SSF with the use of dual impellers further improved the economic productivity by 8.7 %. This study demonstrated that integrating use of SB as a low-cost substrate considerably enhanced the acid production and lessened the use of SJ. It was also demonstrated that mixing, and process configuration are critical factors determining the process performance.

CRISPR/Cas9-based genome engineering in the filamentous fungus Rhizopus oryzae and its application to L-lactic acid production.

The filamentous fungus Rhizopus oryzae is one of the main industrial strains for the production of a series of important chemicals such as ethanol, lactic acid, and fumaric acid. However, the lack of efficient gene editing tools suitable for R. oryzae makes it difficult to apply technical methods such as metabolic engineering regulation and synthetic biology modification. A CRISPR-Cas9 system suitable for efficient genome editing in R. oryzae was developed. Firstly, four endogenous U6 promoters of R. oryzae were identified and screened with the highest transcriptional activity for application to sgRNA transcription. It was then determined that the U6 promoter mediated CRISPR/Cas9 system has the ability to efficiently edit the genome of R. oryzae through NHEJ and HDR-mediated events. Furthermore, the newly constructed CRISPR-Cas9 dual sgRNAs system can simultaneously disrupt or insert different fragments of the R. oryzae genome. Finally, this CRISPR-Cas9 system was applied to the genome editing of R. oryzae by knocking out pyruvate carboxylase gene (PYC) and pyruvate decarboxylase gene (pdcA) and knocking in phosphofructokinase (pfkB) from Escherichia coli and L-lactate dehydrogenase (L-LDH) from Heyndrickxia coagulans, which resulted in a substantial increase in L-LA production. In summary, this study showed that the CRISPR/Cas9-based genome editing tool is efficient for manipulating genes in R. oryzae.

Adaptive evolution of Paecilomyces variotii enhanced the biodetoxification of high-titer inhibitors in pretreated lignocellulosic feedstock

High inhibitor concentrations in lignocellulose feedstock negatively affect the degradation rate of biodetoxification strains. This study designed two adaptive laboratory evolutions in solid substrate and liquid medium to boost the biodetoxification capacity of P. variotii to high titers of lignocellulose-derived inhibitors, resulting in two evolved strains AC70 and ZW70. The results showed that the evolutionary adaptation in liquid medium could better boost the acetic acid assimilation compared to that on solid substrate. Transcriptional analysis revealed that the evolved strains exhibited a significant upregulation of adh, acs, ach1, and ackA directly related to the initial steps of acetate and furan aldehydes metabolisms. ZW70 strain can effectively remove the high concentration inhibitors cocktail from the hydrolysates derived from pretreated wheat straw and furfural residues. The biodetoxified hydrolysates by ZW70 were successfully used for cellulose chiral L-lactic acid production with the titers of ∼110 g/L, which were over 20 % higher than that detoxified by parental strain.

Overexpression of the transcriptional activators Mxr1 and Mit1 enhances lactic acid production on methanol in Komagataellaphaffii

A bio-based production of chemical building blocks from renewable, sustainable and non-food substrates is one key element to fight climate crisis. Lactic acid, one such chemical building block is currently produced from first generation feedstocks such as glucose and sucrose, both requiring land and water resources. In this study we aimed for lactic acid production from methanol by utilizing Komagataella phaffii as a production platform. Methanol, a single carbon source has potential as a sustainable substrate as technology allows (electro)chemical hydrogenation of CO2 for methanol production. Here we show that expression of the Lactiplantibacillus plantarum derived lactate dehydrogenase leads to L-lactic acid production in Komagataella phaffii, however, production resulted in low titers and cells subsequently consumed lactic acid again. Gene expression analysis of the methanol-utilizing genes AOX1, FDH1 and DAS2 showed that the presence of lactic acid downregulates transcription of the aforementioned genes, thereby repressing the methanol-utilizing pathway. For activation of the methanol-utilizing pathway in the presence of lactic acid, we constructed strains deficient in transcriptional repressors Nrg1, Mig1-1, and Mig1-2 as well as strains with overrepresentation of transcriptional activators Mxr1 and Mit1. While loss of transcriptional repressors had no significant impact on lactic acid production, overexpression of both transcriptional activators, MXR1 and MIT1, increased lactic acid titers from 4 g L−1 to 17 g L−1 in bioreactor cultivations.

Dilute acid-assisted microbubbles-mediated ozonolysis of Eucheuma denticulatum phycocolloid for biobased L-lactic acid production

Biobased L-lactic acid (L-LA) appeals to industries; however, existing technologies are plagued by limited productivity and high energy consumption. This study established an integrated process for producing macroalgae-based L-LA from Eucheuma denticulatum phycocolloid (EDP). Dilute acid-assisted microbubbles-mediated ozonolysis (DAMMO) was selected for the ozonolysis of EDP to optimize D-galactose recovery. Through single-factor optimization of DAMMO treatment, a maximum D-galactose recovery efficiency (59.10 %) was achieved using 0.15 M H2SO4 at 80 °C for 75 min. Fermentation with 3 % (w/v) mixed microbial cells (Bacillus coagulans ATCC 7050 and Lactobacillus acidophilus-14) and fermented residues achieved a 97.67 % L-LA yield. Additionally, this culture approach was further evaluated in repeated-batch fermentation and showed an average L-LA yield of 93.30 %, providing a feasible concept for macroalgae-based L-LA production.

Competition between biodetoxification fungus and lactic acid bacterium in the biorefinery processing chain for production of cellulosic L-lactic acid

Biodetoxification fungus selectively degrades toxic inhibitors generated from pretreatment of lignocellulose without consuming fermentable sugars. However, one barrier for practical application is the sustained cell viability in the consequent fermentation step to compete the fermentable sugars with fermenting strains, resulting in sugar loss and reduced target product yield. This study investigated the competitive growth property between the biodetoxification fungus Paecilomyces variotii FN89 and the L-lactic acid bacterium Pediococcus acidilactici ZY271 under varying temperature and lactic acid osmatic stress. The results show that the L-lactic acid bacterium Ped. acidilactici ZY271 showed less thermotolerance to Pae. variotii FN89 at high temperature of 45 °C to 50 °C in both synthetic medium and wheat straw hydrolysate. In the higher temperature environment, the growth of the biodetoxification strian failed to compete with the lactic acid fermentation strain and was quickly eliminated from the fermentation system. The high temperature fermentation facilitated a fast transition from the detoxification stage to the fermentation stage for higher production of L-lactic acid.

Identification of the Microbiota in Coconut Water, Kefir, Coconut Water Kefir and Coconut Water Kefir-Fermented Sourdough Using Culture-Dependent Techniques and Illumina-MiSeq Sequencing.

The principal objective of this study was to isolate and identify the microorganisms present in commercial kefir grains, a novel kefir-fermented coconut water (CWK) and a novel coconut water kefir-fermented sourdough using phenotypic identification and Sanger sequencing and examine the microbial diversity of CWK and CWK-fermented sourdough throughout the fermentation process using the MiSeq Illumina sequencing method. The phenotypic characterisation based on morphology identified ten isolates of LAB, five AAB and seven yeasts from kefir (K), CWK and CWK-fermented sourdough (CWKS). The results confirm the presence of the LAB species Limosilactobacillus fermentum, Lactobacillus. plantarum, L. fusant, L. reuteri and L. kunkeei; the AAB species Acetobacter aceti, A. lovaniensis and A. pasteurianus; and the yeast species Candida kefyr, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, C. guilliermondii and C. colliculosa. To the best of our knowledge, the identification of Rhodotorula from kefir is being reported for the first time. This study provides important insights into the relative abundances of the microorganisms in CWKS. A decrease in pH and an increase in the titratable acidity for CWK- and CWK-fermented sourdough corresponded to the increase in D- and L-lactic acid production after 96 h of fermentation. Significant reductions in the pHs of CWK and CWKS were observed between 48 and 96 h of fermentation, indicating that the kefir microorganisms were able to sustain highly acidic environments. There was also increased production of L-lactic acid with fermentation, which was almost twice that of D-lactic acid in CWK.

Strain engineering of Bacillus coagulans with high osmotic pressure tolerance for effective L-lactic acid production from sweet sorghum juice under unsterile conditions

Open unsterile fermentation of the low-cost non-food crop, sweet sorghum, is an economically feasible lactic acid biosynthesis process. However, hyperosmotic stress inhibits microbial metabolism and lactic acid biosynthesis, and engineering strains with high osmotic tolerance is challenging. Herein, heavy ion mutagenesis combined with osmotic pressure enrichment was used to engineer a hyperosmotic-tolerant Bacillus coagulans for L-lactic acid production. The engineered strain had higher osmotic pressure tolerance, when compared with the parental strain, primarily owing to its improved properties such as cell viability, cellular antioxidant capacity, and NADH supply. In a pilot-scale open unsterile fermentation using sweet sorghum juice as a feedstock, the engineered strain produced 94 g/L L-lactic acid with a yield of 91 % and productivity of 6.7 g/L/h, and optical purity of L-lactic acid at the end of fermentation was 99.8 %. In short, this study provided effective and low-cost approach to produce polymer-grade L-lactic acid.

An innovative temperature control strategy to improve optically pure L-lactic acid production from food waste

Fermentation of food waste to lactic acid (LA) is a strategy for resource recovery. However, the poor yield and optical activity (OA) of L-lactic acid (L-LA) are major bottlenecks for such efforts. This study focuses on enhancing L-LA yield and optical purity from the food waste without sterilization and inoculation at pilot-scale. First, the effects of temperature on LA production were explored. A higher LA yield but lower purity was obtained under mesophilic conditions, whereas the opposite results were represented at high temperature. On the basis of these findings, a novel two-stage temperature regulation strategy was developed that combined the benefits of both temperature conditions. This strategy not only ensured a L-LA yield of 0.371 g/g volatile solid, but also achieved a high optical purity of 98.0%. The underlying mechanism is that high temperature could enrich L-lactic acid bacteria (LAB) and inhibit LAB producing both L- and D-LA. Overall, this innovative strategy may be well-suited for generating optically pure L-LA from food waste fermentation.

Brewer’s spent grain as a self-sufficient feedstock for homofermentative production of optically pure L-lactic acid using Lactobacillus rhamnosus

Brewer’s spent grain (BSG), accounting for 85% of the total by-product from breweries, was used as a feedstock for L-lactic acid production in the present study. BSG was enzymatically hydrolysed and promoted to be a self-sufficient feedstock for L-lactic acid production by using it as a sole source of carbon, protein and minerals. Process parameters like glucose concentration (10–20 g/L), glucose-to-protein source ratio (1:1 – 5:1), source of protein and inoculum concentration (3 – 10% v/v) were selected to divert the carbon source and enhance L-lactic acid concentration, which would otherwise result in bacterial biomass production. Yeast extract and whey permeate resulted in high bacterial growth, whereas self-sustained protein (SSP) in BSG resulted in higher L-lactic acid production. Further, the glucose-to-protein source ratio was maintained at its lowest level for better glucose conversion to L-lactic acid. Glucose concentration strongly influenced L-lactic acid production. Therefore glucose concentration in the batch fermentation process was further increased from 60 to 120 g/L. A maximum L-lactic acid concentration of 114.4 g/L and productivity of 5.14 g/L·h was achieved with an initial glucose concentration of 120 g/L, and the rest of the process parameters such as glucose to protein source ratio of 1:1, inoculum concentration of 10% v/v and SSP in BSG were maintained at its optimum level. Finally, L-lactic acid in the fermentation broth was purified and analysed for its similarity with commercially available L-lactic acid using proton-NMR and FTIR spectroscopy. Thus, the present study valorised BSG by producing L-lactic acid under a biorefinery approach.

Thermophilic biocatalysts for one-step conversion of citrus waste into lactic acid

Agri-food residues offer significant potential as a raw material for the production of L-lactic acid through microbial fermentation. Weizmannia coagulans, previously known as Bacillus coagulans, is a spore-forming, lactic acid-producing, gram-positive, with known probiotic and prebiotic properties. This study aimed to evaluate the feasibility of utilizing untreated citrus waste as a sustainable feedstock for the production of L-lactic acid in a one-step process, by using the strain W. coagulans MA-13. By employing a thermophilic enzymatic cocktail (Cellic CTec2) in conjunction with the hydrolytic capabilities of MA-13, biomass degradation was enhanced by up to 62%. Moreover, batch and fed-batch fermentation experiments demonstrated the complete fermentation of glucose into L-lactic acid, achieving a concentration of up to 44.8 g/L. These results point to MA-13 as a microbial cell factory for one-step production of L-lactic acid, by combining cost-effective saccharification with MA-13 fermentative performance, on agri-food wastes. Moreover, the potential of this approach for sustainable valorization of agricultural waste streams is successfully proven.Key points• Valorization of citrus waste, an abundant residue in Mediterranean countries.• Sustainable production of the L-( +)-lactic acid in one-step process.• Enzymatic pretreatment is a valuable alternative to the use of chemical.Graphical

Biorefinery-based sustainability assessment of macroalgae waste valorization to polylactic acid: Exergy and exergoeconomic perspectives

This study evaluated the exergetic and exergoeconomic performance of three L-lactic acid (L-LA) production scenarios. All three scenarios produced L-LA from red macroalgae waste, Eucheuma cottonii residue (ECR) with the same species of enzymes, and lactic acid bacteria (LAB). Scenario 1 is the biorefinery process for the L-LA. Scenario 2 is the biorefinery process for the L-LA with fertilizer production and wastewater treatment. Scenario 3 is the polylactic acid (PLA) biorefinery process with fertilizer production and wastewater treatment. From the study, the total production rate for L-LA was 5194 kg/hr, with a total consumption rate of 12079 kg/hr for ECR. In addition, the production rates of organic fertilizer and reused water were 1609 and 1233 kg/hr, respectively, for scenario 2. Then, the production rate of the PLA was 4142 kg/hr for scenario 3. From the comparison, the energy consumption was increased due to the increased demand for process and equipment (from 28272.53 kW to 30296.15 kW). From the exergetic analysis aspect, the highest contributor to the exergy loss was the simultaneous saccharification and fermentation (SSF) unit, totaling 18875.62 kW. For the exergoeconomic aspect, the SSF unit also contributed the highest exergy destruction cost rate (average 70%). The study results provide theoretical guidance for exergetic and economic performance improvement in future L-LA and PLA production.

Continuous fermentation using high cell density cell recycle system for L-lactic acid production.

For complete utilization of high glucose at ∼100 g/L, a high cell density (HCD) continuous fermentation system was established using Lb. delbrueckii NCIM 2025 for the bioproduction of lactic acid (LA). An integrated membrane cell recycling system coupled with the continuous bioreactor, aided to achieve the highest 34.77 g/L h LA productivity and 0.94-0.98 g/g yield. ∼34 times higher productivity was observed (in comparison to batch fermentation conducted in this study), when the continuous operations were carried out at the maximum dilution rate and wet cell weight i.e. 0.36 h-1 and 230 g/L, respectively. These results show the potential of this method for large-scale lactic acid production because it not only produces high titers but also ensures that glucose is used effectively. The method's superior performance in comparison to earlier studies suggests it as an affordable and sustainable alternative for the production of LA.

Integrated Soybean Hull Biorefinery with Citric Acid-Catalyzed Hydrothermal Pretreatment for L-Lactic Acid and Xylooligosaccharide Production

Integrated Soybean Hull Biorefinery with Citric Acid-Catalyzed Hydrothermal Pretreatment for L-Lactic Acid and Xylooligosaccharide Production

Bioconversion of spray corn husks into L-lactic acid with liquid hot water pretreatment

Agricultural by-products like rice husk, bran, and spray corn husks, often utilized as feed, are considered less desirable. This study aims to enhance the utilization rate of these materials by subjecting then to liquid hot water (LHW) pretreatment, followed by enzymatic hydrolysis to produce fermentable sugars. We investigated the production of L-lactic acid using two methods: simultaneous saccharification fermentation (SSF) and separate hydrolysis fermentation (SHF), following varying intensities of LHW pretreatment. The results showed that the optimal enzymatic hydrolysis efficiency was achieved from spray corn husks under the pretreatment conditions of 155 °C and 15 min. SHF was generally more effective than SSF. The glucose L-lactic acid conversion rate in SHF using spray corn husks can reach more than 90 %. Overall, this work proposed a novel, environmental-friendly strategy for efficient and for L- lactic acid production from spray corn husks.

Development of a sugar platform and fermentation media from residues from alfalfa biorefining

Biorefining of fresh alfalfa for protein production leads to a solid fibrous residue (pulp), and a liquid fraction (brown juice) after recovery of proteins by precipitation. In this study, potential uses of these side streams were investigated. Treatment of the pulp with 6% sodium chlorite resulted in 79.8% removal of lignin, while 89.1% of the glucan and 75.4% of the xylan was retained. Subsequent enzyme hydrolysis using cellulase at a loading of 10 FPU/g dry substrate for 48 h, converted 81.0% of the glucan to glucose and 79.3% of the xylan to xylose. Concentrations of 35.2 g/L glucose and 13.0 g/L xylose were obtained in the hydrolysate after using a biomass loading of 10% w/v, and deionized water for suspension during saccharification. The brown juice was investigated as a medium for fermentative production of L-lactic acid using the bacterium Lactococcus lactis KF 147. Fermentation of the brown juice without further additions yielded 12.8 g/L lactic acid. When the brown juice was supplemented with increasing amounts of pulp hydrolysate, from 15% to 70% v/v, the lactic acid yield increased to 17.4 and 28.8 g/L, respectively. Overall, these results show that converting the alfalfa pulp to a sugar platform for fermentation is feasible, and the brown juice may serve as a nutrient additive in the fermentation process.

Time-resolved transcriptomic and proteomic profiling of Heyndrickxia coagulans during NaOH-buffered L-lactic acid production.

The L-lactic acid (L-LA) fermentation process, based on sodium hydroxide neutralization, demonstrates environmental friendliness during product extraction. However, lactate fermentation is hindered by the pronounced stress effect of sodium lactate on the strain compared with calcium lactate. In this study, we performed time-resolved transcriptomic and proteomic analyses of Heyndrickxia coagulans DSM1 during NaOH-buffered L-LA production. The expression levels of the glycolytic genes demonstrated an initial increase followed by a subsequent decrease, whereas the tricarboxylic acid cycle genes exhibited an initial decrease followed by a subsequent increase throughout the fermentation process. Moreover, we identified clusters of genes consisting of transcription factors and ATP-binding cassette (ABC) transporters that demonstrate a progressive elevation of expression levels throughout the fermentation process, with significant upregulation observed at later stages. This investigation yields valuable insights into the response mechanisms of H. coagulans during NaOH-buffered L-LA fermentation and presents potential targets for metabolic engineering.

Detection and elimination of trace d-lactic acid in lignocellulose biorefining chain: Generation, flow, and impact on chiral lactide synthesis.

High chiral purity of lactic acid is a crucial indicator for the synthesis of chiral lactide as the primary intermediate chemical for ring-open polymerization of high molecular weight polylactic acid (PLA). Lignocellulose biomass is the most promising carbohydrate feedstock for commercial production of PLA, but the presence of trace d-lactic acid in the biorefinery chain adversely affects the synthesis and quality of chiral lactide. This study analyzed the fingerprint of trace d-lactic acid in the biorefinery chain and found that the major source of d-lactic acid comes from lignocellulose feedstock. The naturally occurring lactic acid bacteria and water-soluble carbohydrates in lignocellulose feedstock provide the necessary conditions for d-lactic acid generation. Three strategies were proposed to eliminate the generation pathway of d-lactic acid, including reduction of moisture content,conversion of water-soluble carbohydrates to furan aldehydes in pretreatment, and conversion to l-lactic acid by inoculating engineered l-lactic acid bacteria. The natural reduction of lactic acid content in lignocellulose feedstock during storage was observed due to the lactate oxidase-catalyzedoxidation of l- and d-lactic acids. This study provided an important support forthe production of cellulosic l-lactic acid with high chiral purity.