Improved Fermentation Performance Research Articles

Citrate-buffered Yamanaka medium allows to produce high-yield bacterial nanocellulose in static culture using Komagataeibacter strains isolated from apple cider vinegar.

Bacterial nanocellulose (BNC) is a sustainable, renewable, and eco-friendly nanomaterial, which has gained great attentions in both academic and industrial fields. Two bacterial nanocellulose-producing strains (CVV and CVN) were isolated from apple vinegar sources, presenting high 16S rRNA gene sequence similarities (96%-98%) with Komagataeibacter species. The biofilm was characterized by scanning electron microscopy (SEM), revealing the presence of rod-shaped bacteria intricately embedded in the polymeric matrix composed of nanofibers of bacterial nanocellulose. FTIR spectrum and XRD pattern additionally confirmed the characteristic chemical structure associated with this material. The yields and productivities achieved during 10 days of fermentation were compared with Komagataeibacter xylinus ATCC 53524, resulting in low levels of BNC production. However, a remarkable increase in the BNC yield was achieved for CVV (690% increase) and CVN (750% increase) strains at day 6 of the fermentation upon adding 22mM citrate buffer into the medium. This effect is mainly attributed to the buffering capacity of the modified Yakamana medium, which allowed to maintain pH close to 4.0 until day 6, though in combination with additional factors including stimulation of the gluconeogenesis pathway and citrate assimilation as a carbon source. In addition, the productivities determined for both isolated strains (0.850 and 0.917gL-1 d-1) compare favorably to previous works, supporting current efforts to improve fermentation performance in static cultures and the feasibility of scaling-up BNC production in these systems.

Saccharomyces cerevisiae strains performing similarly during fermentation of lignocellulosic hydrolysates show pronounced differences in transcriptional stress responses.

The need for sustainable alternatives to oil-based production of biochemicals and biofuels is undisputable. Saccharomyces cerevisiae is the most commonly used industrial fermentation workhorse. The fermentation of lignocellulosic hydrolysates, second-generation biomass unsuited for food and feed, is still hampered by lowered productivities as the raw material is inhibitory for the cells. In order to map the genetic responses of different S. cerevisiae strains, we performed RNA sequencing of a CEN.PK laboratory strain, two industrial strains (KE6-12 and Ethanol Red), and two wild-type isolates of the LBCM collection when cultivated anaerobically in wheat straw hydrolysate. While the response to inhibitors of S. cerevisiae has been studied earlier, this has in previous studies been done in aerobic conditions. The transcriptomic analysis highlights different evolutionary adaptations among the different S. cerevisiae strains and suggests novel gene targets for improving fermentation performance and robustness.

Analyzing the occurrence of foaming in batch fermentation processes using multiway partial least square approaches

AbstractThis article presents an application of multiway partial least squares (MPLS) methods to develop interpretative correlation models to monitor the foaming occurrence and improve batch fermentation. We choose the exhaust differential pressure as a quality variable to quantify the foaming occurrence and consider three‐dimensional datasets of different batches, process variables, and measurements. We integrate batch‐wise unfolding (BWU) and observation‐wise unfolding (OWU) of plant datasets with standard, dynamic, and kernel PLS methods. We find that dynamic PLS (DPLS) with OWU and time‐lagged quality variables to be the most efficient, accurate, and easy to implement. The BWU approach is useful for analyzing the differences between batches and identifying abnormalities and outliers, while the OWU quantifies the variation within a given batch. With OWU, the DPLS method with one unit of time lag in the quality variable is the most effective, accurate, and easy to implement. With both BWU and OWU, we identify the quantitative effects of process variables on the quality variable and providence guidance to improve fermentation performance.

Peptide extract from spent yeast improves resistance of Saccharomyces cerevisiae to oxidative stress

Yeast cells face various stress factors during industrial fermentations, since they are exposed to harsh environmental conditions, which may impair biomolecules productivity and yield. In this work, the use of an antioxidant peptide extract obtained from industrial spent yeast was explored as supplement for Saccharomyces cerevisiae fermentation to prevent a common bottleneck: oxidative stress. For that, a recombinant yeast strain, producer of β-farnesene, was firstly incubated with 0.5 and 0.7 g/L peptide extract, in the presence and absence of hydrogen peroxide (an oxidative stress inducer), for 1–5 h, and then assayed for intracellular reactive oxygen species, and growth ability in agar spot assays. Results showed that under 2 mM H2O2, the peptide extract could improve cells growth and reduce reactive oxygen species production. Therefore, this antioxidant effect was further evaluated in shake-flasks and 2-L bioreactor batch fermentations. Peptide extract (0.7 g/L) was able to increase yeast resistance to the oxidative stress promoted by 2 mM H2O2, by reducing reactive oxygen species levels between 1.2- and 1.7-fold in bioreactor and between 1.2- and 3-fold in shake-flask fermentations. Moreover, improvements on yeast cell density of up to 1.5-fold and 2-fold, and on biomolecule concentration of up to 1.6-fold and 2.8-fold, in bioreactor and shake-flasks, respectively, were obtained. Thus, culture medium supplementation with antioxidant peptide extracted from industrial spent yeast is a promising strategy to improve fermentation performance while valuing biomass waste. This valorization can promote a sustainable and eco-friendly solution for the biotechnology industry by the implementation of a circular economy model.Key points• Peptide extract from spent yeast applied for the first time on yeast fermentation.• Antioxidant peptide extract enhanced S. cerevisiae oxidative stress resistance.• Fermentation performance under stress improved by peptide extract supplementation.

Immobilization of lager yeast by hydrocolloids as supporting matrix for improving fermentation performance of high gravity brewing

The objective of this study was to investigate the effects of six kinds of hydrocolloids supplementations, namely κ-carrageenan, gellan gum, pectin, gum ghatti, karaya, and guar gum as the supporting matrix, on the fermentation kinetics of lager yeast during high gravity beer fermentation. Results showed that hydrocolloids supplementations significantly accelerated fermentation and resulted in higher consumption of reducing sugar and free amino nitrogen, and higher production of ethanol and flavor volatiles. Compared to the control, wort fermentability and ethanol production were increased by 19.3% and 25.0%, respectively, by κ-carrageenan supplementation. Furthermore, volatile compounds formation and aroma complexity (25 new volatile compounds) of green beers were improved by hydrocolloids supplementation, and the reduction in acetic acid and acetaldehyde contents was observed, which suggested favorable flavor characteristics. Results of SEM images revealed that hydrocolloids supplementation induced the immobilization of yeast cells through flocculation, providing a protective effect on the physiological characterization of lager yeast during high gravity brewing.

New Saccharomyces cerevisiae-Kluyveromyces marxianus fusant shows enhanced alcoholic fermentation performance.

Kluyveromyces marxianus has a promising enological potential because of strong extracellular pectinase activity and the copious production of specific higher alcohols and esters. However, K. marxianus is unable to complete alcoholic fermentation on its own. For this reason it is only used in co- or sequential fermentation in conjunction with Saccharomyces spp. Here we applied the protoplast fusion technique to two commercial Saccharomyces cerevisiae strains (Lalvin EC1118® and Lalvin QA23®) and K. marxianus isolate IWBT Y885, to obtain fusants that possess pre-selected, enologically relevant features of K. marxianus, but with improved fermentation performance. After an extensive selection process, performed using non-auxotrophic markers, we identified one fusant, BF2020, that shows enhanced fermentation performance compared to the parental strain K. marxianus Y885, but that still exhibits strong pectinase activity and may be suitable for industrial production. The genome of the selected hybrid was further investigated and characterized by RAPD-PCR analysis and whole genome sequencing.

Hydrothermally processed polyethylene as starting point for fermentative production of triglycerides

Plastic waste accumulation in the environment is an exponentially growing problem that needs to be urgently addressed to avoid the catastrophic damage of terrestrial and oceanic ecosystems. In this work, currently undervalued plastic carbon content was aimed to be exploited by converting it into triglycerides (TGs), following a two-stage approach. Polyethylene (PE) was first treated by hydrothermal processing (HTP) to obtain PE oils that were later used as carbon source for fermentation with Yarrowia lipolytica and transformed into TGs. HTP PE oil obtained at 425ºC for 120 min was found as an optimal carbon source for the biotechnological stage. Under these optimized conditions, 75% of PE mass was transformed into a PE oil that could be used by Yarrowia as carbon source to grow and produce TGs. An oil concentration of 15 g/l in fermentation media in the biotechnological stage improved fermentation performance as well in comparison to 10 g/l concentration: biomass growth and TGs production yield were increased by 130% regarding the control. Thereby, an alternative approach towards turning one of the most widespread plastics as PE into TGs is proposed in this work.

Increasing Ethanol Tolerance and Ethanol Production in an Industrial Fuel Ethanol Saccharomyces cerevisiae Strain

The stress imposed by ethanol to Saccharomyces cerevisiae cells are one of the most challenging limiting factors in industrial fuel ethanol production. Consequently, the toxicity and tolerance to high ethanol concentrations has been the subject of extensive research, allowing the identification of several genes important for increasing the tolerance to this stress factor. However, most studies were performed with well-characterized laboratory strains, and how the results obtained with these strains work in industrial strains remains unknown. In the present work, we have tested three different strategies known to increase ethanol tolerance by laboratory strains in an industrial fuel–ethanol producing strain: the overexpression of the TRP1 or MSN2 genes, or the overexpression of a truncated version of the MSN2 gene. Our results show that the industrial CAT-1 strain tolerates up to 14% ethanol, and indeed the three strategies increased its tolerance to ethanol. When these strains were subjected to fermentations with high sugar content and cell recycle, simulating the industrial conditions used in Brazilian distilleries, only the strain with overexpression of the truncated MSN2 gene showed improved fermentation performance, allowing the production of 16% ethanol from 33% of total reducing sugars present in sugarcane molasses. Our results highlight the importance of testing genetic modifications in industrial yeast strains under industrial conditions in order to improve the production of industrial fuel ethanol by S. cerevisiae.

Pre-gelatinization and cellulase addition improve fermentation performance and antioxidant activity of black rice wine.

Black rice contains a variety of bioactive substances that contribute to the high nutritional value of black rice wine (BRW). However, the dense bran layer of black rice retards the fermentation rate and reduces the dissolution of active components. Hence, this study aims to investigate the effects of pre-gelatinization (PG) before cooking and cellulase (CE) addition during fermentation on the fermentation performance of BRW and its antioxidant activity. PG combined with CE treatments (PGCE) increases the alcohol content, free amino acid content, volatile flavor content and total antioxidant activity of BRW by 90.81%, 15.36%, 38.05% and 19.56%, respectively, compared with the control group. Scanning electron microscopy, low-field nuclear magnetic resonance and texture properties analysis indicate that PG treatment increases gelatinization degree of starch during cooking, decreases bound water content in cooked black rice and promotes unbound water release. CE destroys the aleurone layer structure, facilitates the release of unbound water and the exposure of rice starch, thus increasing the reaction area and extravasation content significantly, which is beneficial to microbial growth and fermentation. Incomplete aleurone layer also promotes the dissolution of anthocyanins, phenols and other active substances, increasing the antioxidant activities of BRW. PG and CE treatments reduce the fermentation time and improve the quality of BRW by destroying the black rice structure. © 2022 Society of Chemical Industry.

Synergistic effects of inhibitors and osmotic stress during high gravity bioethanol production from steam-exploded lignocellulosic feedstocks

High gravity second-generation bioethanol production (solid loading ≥30% w w−1) suffers from prolonged fermentation times and low ethanol productivities due to high concentrations of toxic by-products (inhibitors) and high monosaccharide concentrations. To better understand inhibition during high gravity bioethanol production, this study focused on ethanol production from a variety of different steam-exploded feedstocks with different inhibitor concentrations. The effects of inhibitors on enzymatic hydrolysis and fermentation were investigated by selective removal of inhibitors by means of biological detoxification. The degree of lignocellulose depolymerization and inhibitor formation during steam explosion varied significantly between different feedstock types. Subsequent enzymatic hydrolysis and fermentation indicated that lignocellulosic inhibitors had no significant effect on cellulolytic enzymes, but showed strong inhibition on fermentation performance. Biological detoxification of furan aldehydes and organic acids had no impact on monosaccharide yield during enzymatic hydrolysis, but reduced the fermentative lag phase by up to 48 h. While the detoxification significantly improved fermentation performance, the maximal ethanol yield of 52.6 g L−1 with wheat straw or miscanthus could not be increased. A key finding of this study was that the effect of lignocellulosic inhibitors is greatly increased in environments with high osmotic stress. This synergistic effect is hypothesized to be caused by an inhibitor-induced redox imbalance, impeding the cells’ ability to counteract osmotic stress.

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation.

D-Allulose is an ultra-low calorie sweetener with broad market prospects. As an alternative to Izumoring, phosphorylation-dephosphorylation is a promising method for D-allulose synthesis due to its high conversion of substrate, which has been preliminarily attempted in enzymatic systems. However, in vitro phosphorylation-dephosphorylation requires polyphosphate as a phosphate donor and cannot completely deplete the substrate, which may limit its application in industry. Here, we designed and constructed a metabolic pathway in Escherichia coli for producing D-allulose from D-fructose via in vivo phosphorylation-dephosphorylation. PtsG-F and Mak were used to replace the fructose phosphotransferase systems (PTS) for uptake and phosphorylation of D-fructose to fructose-6-phosphate, which was then converted to D-allulose by AlsE and A6PP. The D-allulose titer reached 0.35 g/L and the yield was 0.16 g/g. Further block of the carbon flux into the Embden-Meyerhof-Parnas (EMP) pathway and introduction of an ATP regeneration system obviously improved fermentation performance, increasing the titer and yield of D-allulose to 1.23 g/L and 0.68 g/g, respectively. The E. coli cell factory cultured in M9 medium with glycerol as a carbon source achieved a D-allulose titer of ≈1.59 g/L and a yield of ≈0.72 g/g on D-fructose.

Reduced sensitivity of lager brewing yeast to premature yeast flocculation via adaptive evolution

Malt-induced premature yeast flocculation (PYF) is a sporadic problem within the brewing industry. The use of PYF malts is concomitant with a number of negative impacts on beer quality, including incomplete fermentation and/or flavor defects. Although malt-induced PYF is widely acknowledged, actions taken so far have proved insufficient to solve the PYF-related issues. To limit the detrimental effects of PYF malts on beer production, an adaptive laboratory evolution (ALE) process was applied in this study to an industrial lager brewing yeast strain (TT02), in an attempt to generate variant strains with improved fermentation performance in PYF wort. Through a batch fermentation-based adaptation process, evolved variants were isolated and screened for their phenotypic and metabolic traits. The investigation focused mainly on the tendency to remain in suspension, fermentation capacity and final acetaldehyde concentration. We successfully obtained a variant (TT02-30 T) with improved fermentation properties. The improvement was seen in worts prepared from different types of PYF malt as well as normal malt. Furthermore, ALE of lager brewing yeast in PYF wort yielded a wide array of mutations. Several changes in the genomes (copy number variation in flocculin encoding gene FLO1 and a missense SNP in a putative mitochondrial membrane protein coding gene FMP10) of the variant strains relative to the original strain were observed. These could potentially contribute to the improved yeast suspension during fermentation. Importantly, mutational enrichment in genes related to ion binding in PYF-evolved strains suggests the involvement of the yeast ion transportation process in dealing with the PYF stress. Our study demonstrates the possibility of attenuating yeast sensitivity to PYF malts over time through adaptive laboratory evolution via spontaneous mutation.

Light-Controlled Fermentations for Microbial Chemical and Protein Production.

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Effects of Hydroxycinnamates and Exogenous Yeast Assimilable Nitrogen on Cider Aroma and Fermentation Performance

Cider apple cultivars typically contain high concentrations of tannins (phenolic compounds) and/or acids. The phenolic content of some cider apples far exceeds that of white wine grapes, yet modern global cider styles are often fermented using yeast strains originally selected and commercialized for white wine production. The potential for phenolic compounds in cider apple juice to influence fermentation rate or aromas generated during cider fermentation is not typically considered in yeast strain selection. The objective of this study was to determine the impact of ferulic acid, p-coumaric acid, and chlorogenic acid (at concentrations reported in apples) on fermentation kinetics and cider aroma, and the interactive effects of yeast assimilable nitrogen (YAN) on these outcomes. Our hypothesis was that phenolic compounds present in high-tannin cider apples would slow down fermentation, but not alter aroma, and that added YAN would moderate these effects. Ferulic acid negatively affected fermentation performance , but p-coumaric and chlorogenic acids did not. A sensory sorting task showed that p-coumaric acid led to distinct differences in grouping by aroma, while ferulic acid resulted in distinct aroma descriptors. Finally, addition of YAN improved fermentation performance for low concentrations of ferulic acid, and it affected the aroma of cider amended with p-coumaric acid. Supplemental data for this article is available online at at [publisher’s weblink].

Effects of inhibitory compounds derived from lignocellulosic biomass on the growth of the wild-type and evolved oleaginous yeast Rhodosporidium toruloides

The presence of toxic compounds in lignocellulosic hydrolysates is one of the main problems affecting the efficiency of hydrolysate-based fermentation processes. Understanding the effects of biomass-derived inhibitors on the performance of the microbial strain is essential to develop strategies able to result in an improved fermentation performance. In the present study, efforts were done to elucidate the effects of the main biomass-derived inhibitors on the growth of the oleaginous yeast Rhodosporidium toruloides. Furfural, 5-hydroxymethylfurfural (5-HMF), acetic acid, levulinic acid, benzoic acid, p-coumaric acid, ferulic acid, vanillic acid, vanillin and syringaldehyde were the inhibitory compounds investigated. Assays were performed using a wild-type and an evolved strain of R. toruloides. For the individual inhibitors, more than 3 mM benzoic acid completely inhibited the growth of both strains, while acetic acid and levulinic acid were less toxic, presenting IC50 values of 16 and 15 mM, respectively. In a subsequent step, assays were performed in media containing different combinations of inhibitory compounds and a Placket-Burman experimental design was used to identify the most severe inhibitors in mixture, as well as combination of inhibitors, affecting the yeast growth. Results revealed that in mixture, furfural was the most potent inhibitor affecting the growth of R. toruloides, followed by vanillin and 5-HMF. The combination of furfural, 5-HMF, vanillin, vanillic acid and ferulic acid resulted in the most severe inhibition to this yeast. The evolved strain showed an improved ability to tolerate toxic compounds, suggesting that the adaptive laboratory evolution is a potential strategy to obtain oleaginous yeasts with improved ability to grow in biomass hydrolysates.

Ethanol production from gas fermentation: Rapid enrichment and domestication of bacterial community with continuous CO/CO2 gas

Industrial production processes will produce a large amount of inorganic carbon (CO/CO2), which can be used as a carbon substrate for gaseous fermentation to reduce environmental pollution and promote the development of renewable energy. To solve the bottleneck that the strains used for the gaseous fermentation cannot adapt well to the mixed gas (CO, CO2 and H2) and the low ethanol production, the mixed gas was aerated continuously into the culture with rabbit faeces to enrich ethanol-producing strains. After CO/CO2 domestication, the structure of the microbial community changed significantly. Blautia, which can use CO/CO2 to produce ethanol, became the dominant bacteria, and the relative abundance significantly increased by 5.4 times to 41.1%. What's more, 14.07% of CO/CO2 was converted to organic carbon through fermentation by the domesticated bacteria, and the ethanol concentration reached 1.41 g L−1. The results indicated that with the continuous CO/CO2 domestication, the bacteria could adapt the syngas better and showed an improved fermentation performance.

Wheat gluten hydrolysates promotes fermentation performance of brewer\u2019s yeast in very high gravity worts

The effects of wheat gluten hydrolysates (WGH) and their ethanol elution fractions obtained on XAD-16 resin on physiological activity and fermentation performance of brewer’s yeast during very-high-gravity (VHG) worts fermentation were investigated. The results showed that the addition of WGH and their elution fractions in VHG worts significantly enhanced yeast biomass and viability, and further increased the fermentability, ethanol yield and productivity of yeast. Supplementation with 40% ethanol fraction exhibited the highest biomass (6.9 g/L dry cell), cell viability, fermentability (82.05%), ethanol titer (12.19%, v/v) and ethanol productivity during VHG worts fermentation. In addition, 40% ethanol fraction supplementation also caused the most consumption of amino acid and the highest accumulation of intracellular glycerol and trehalose, 15.39% of increase in cell-membrane integrity, 39.61% of enhancement in mitochondrial membrane potential (MMP), and 18.94% of reduction in intracellular reactive oxygen species (ROS) level in yeast under VHG conditions. Therefore, WGH supplementation was an efficient method to improve fermentation performance of brewer’s yeast during VHG worts.

Sequential fed batch extractive fermentation for enhanced bioethanol production using recycled Spathaspora passalidarum and mixed sugar composition

The simultaneous ethanol production and removal during sequential cell recycle fed batch fermentation provides a complementary route to produce this biofuel from sugar mixtures, which may greatly improve yields and productivity from lignocellulosic hydrolysates. Spathaspora passalidarum is a wild-type strain able to naturally convert glucose, fructose, xylose and arabinose into ethanol. Therefore, the present work has focused on 2G bioethanol production by S. passalidarum aiming at the consumption of all sugars released after pre-treatment and enzymatic hydrolysis of sugarcane bagasse in a single fermentation step. The fermentation strategy with sequential cell recycle, fed-batch mode and ethanol removal in situ was performed on a hemicellulosic hydrolysate medium supplemented with molasses. This strategy gave improved fermentation performance and enabled the co-fermention of all sugars under microaerobic conditions. The maximum ethanol yield and productivity was 0.482 g.g−1 and 9.5 g·L-1·h−1, respectively, showing a process efficiency of 94.3%. The selective ethanol removal enables the operation of the bioreactor at low levels of ethanol (20–30 g·L-1), even with high sugar concentration inputs, accelerating the fermentation performance and avoiding inhibitory effects on yeast metabolism. Applying the cell recycle strategy, S. passalidarum was able to increase its robustness, as shown by a 10-fold increase in ethanol productivity, and it was also able to tolerate a high acetic acid concentration (4.5 g·L-1) during long-term fermentations. These results demonstrate that the bioprocess strategy has a strong potential to improve bioethanol production of rich mixed sugar from lignocellulosic hydrolysates in a single fermentation step.

Target-site directed rational high-throughput screening system for high sophorolipids production by Candida bombicola

In this study we have established a rational and high performance high-throughput screening system to select for a sophorolipids (SLs) high-producing strain of Candida bombicola. Introduction of mutagen combination, relaxation culture and multi-stress significantly improved both mutation and positive mutation rates. A high-performing strain, Ncbio 5, was selected out of 6212 mutants. Final SLs titer, productivity and yield of this strain in 5 L bioreactors were 26.9%, 27.0% and 35.0% higher than in the original strain, respectively. The improved fermentation performance in Ncbio 5 is contributed by the longer production period, higher SLs productivity and more efficient oil utilization. The strategy adopted herein to optimize the high-throughput screening system should be readily extendable to other similar systems.

Improvement and Metabolomics-Based Analysis of d-Lactic Acid Production from Agro-Industrial Wastes by Lactobacillus delbrueckii Submitted to Adaptive Laboratory Evolution.

To decrease d-lactic acid production cost, sugarcane molasses and soybean meal, low-cost agro-industrial wastes, were selected as feedstock. First, sugarcane molasses was used directly by Lactobacillus delbrueckii S-NL31, and the nutrients were released from soybean meal by protease hydrolysis. Subsequently, to ensure intensive substrate utilization and enhanced d-lactic acid production from sugarcane molasses and soybean meal, adaptation of L. delbrueckii S-NL31 to substrates was performed through adaptive laboratory evolution. After two-phase adaptive laboratory evolution, the evolved strain L. delbrueckii S-NL31-CM3-SBM with improved cell growth and d-lactic acid production on sugarcane molasses and soybean meal was obtained. To decipher the potential reasons for improved fermentation performance, a metabolomics-based approach was developed to profile the differences of intracellular metabolism between initial and evolved strain. The in-depth analysis elucidated how the key factors exerted influence on d-lactic acid biosynthesis. The results revealed that the enhancement of glycolysis pathway and cofactor supply was directly associated with increased lactic acid production, and the reinforcement of pentose phosphate pathway, amino acid metabolism, and oleic acid uptake improved cell survival and growth. These might be the main reasons for significantly improved d-lactic acid production by adaptive laboratory evolution. Finally, fed-batch simultaneous enzymatic hydrolysis of soybean meal and fermentation process by evolved strain resulted in d-lactic acid levels of 112.3 g/L, with an average production efficiency of 2.4 g/(L × h), a yield of 0.98 g/g sugar, and optical purity of 99.6%. The results show the applicability of d-lactic acid production in L. delbrueckii fed on agro-industrial wastes through adaptive laboratory evolution.