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.

Efficient polymalic acid production from corn straw hydrolysate by detoxification of phenolic inhibitors.

Inhibitory compounds generated from lignocellulose pretreatment would inhibit Poly (malic acid) (PMA) production by Aureobasidium pullulans, but the tolerance mechanism of A. pullulans to lignocellulosic inhibitor is poorly understood. In this study, the cellular response of A. pullulans to lignocellulosic inhibitor stress was studied. Among the three groups of inhibitors (furans, weak acids and phenolic aldehydes), phenolic aldehyde was the dominant inhibitor for PMA production. Phenolic aldehyde was mainly converted into phenolic alcohol by A. pullulans, and phenolic alcohol also exhibited severe inhibition on PMA production. Furthermore, the effect of detoxification methods on inhibitor-removal and PMA fermentation was investigated, both CaCO3 and overliming presented poor detoxification effect, whereas resin H103 could remove both furan derivatives and phenolic compounds efficiently, thereby producing 26.27 g/L of PMA with a yield of 0.30 g/g in batch fermentation. This study will be beneficial for the development of PMA production from lignocellulosic biomass.

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.

Co-expression of Xylose Transporter and Fructose-Bisphosphate Aldolase Enhances the Utilization of Xylose by Lactococcus lactis IO-1.

The raw material cost of lactic acid fermentation accounts for the main part of the production cost, and this necessitates the exploration of the efficient use of cheap raw materials in lactic acid production. We compared the outcomes of the homologous expressions of xylose transporters (xylFGH, xylE, araE, and xylT), 6-phosphofructokinase (pfkA), fructose-bisphosphate aldolase (fbaA), and their co-expression in Lactococcus lactis IO-1 on lactic acid production using xylose as the raw material. We found that the production rate of lactic acid on xylose fermentation by L. lactis IO-1 overexpressing fbaA was the highest (14.42%). Among the xylose transporters investigated, XylT had the strongest xylose transport capacity in L. lactis IO-1, with an increase in the lactic acid production rate by 10.38%. The genes near the overexpression of fbaA or xylT in the metabolic pathway were more upregulated than the distant genes. The co-expression of fbaA and xylT increased the production rate of lactic acid by 27.84% on xylose fermentation by L. lactis IO-1. This work presents a novel strategy for the simultaneous enhancement of the expression of important genes at the beginning and midway of the xylose metabolic pathway of L. lactis IO-1, which could greatly improve the target 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.

One-pot d-lactic acid production using undetoxified acid-pretreated corncob slurry by an adapted Pediococcus acidilactici.

Inhibitor tolerance is still a bottleneck for lactic acid bacteria in lignocellulose biorefinery, while it is hard to obtain one engineered strain with strong tolerance to all inhibitors. Herein, a robust adapted d-lactic acid producing strain Pediococcus acidilactici XH11 was obtained by 111 days' long-term adaptive evolution in undetoxified corncob prehydrolysates. The adapted strain had higher inhibitors tolerance compared to the parental strain, primarily due to its increased conversion capacities of four typical aldehyde inhibitors (furfural, HMF, vanillin, and 4-hydroxybenzaldehyde). One-pot simultaneous saccharification and co-fermentation was successfully achieved using the whole slurry of acid-pretreated corncob without solid-liquid separation and detoxification, by applying the adapted P. acidilactici XH11. Finally, 61.9 g/L of d-lactic acid was generated after 96 h' fermentation (xylose conversion of 89.9 %) with the overall yield of 0.48 g/g dry corncob. This study gave an important option for screening of industrial strains in cellulosic lactic acid production processes.

Evaluation of enhancing effect of soybean oil on polymalic acid production by Aureobasidium pullulans HA-4D.

Polymalic acid (PMA) is a water-soluble polyester produced by Aureobasidium pullulans. In this study, the physiological response of A. pullulans after the addition of vegetable oils was investigated. Soybean oil (SBO) is pivotal for shortening fermentation time and achieving high PMA titer. With the addition of 1% (w/v) SBO, the titer and productivity of PMA was, respectively, increased by 34.2% and 80%. SBO acted as a chemical stimulatory agent rather than a carbon source, the enhancement on PMA production was attributed to the component of fatty acid. SBO induced the dimorphism (yeast-like cells and mycelia) of A. pullulans, in vitro enzyme activities indicated that the TCA oxidative branch for malic acid synthesis might be strengthened, which could generate more ATP for PMA synthesis, and the assay of intracellular energy supply validated this deduction. This study provided a new sight for recognizing the regulatory behavior of SBO in A. pullulans.

One-pot fermentation for erythritol production from distillers grains by the co-cultivation of Yarrowia lipolytica and Trichoderma reesei.

A co-fermentation process involving Yarrowia lipolytica and Trichoderma reesei was studied, using distillers grains (DGS) as feedstocks for erythritol production. DGS can be effectively hydrolyzed by cellulase in the single-strain culture of T. reesei. One-pot solid state fermentation for erythritol production was then established by co-cultivating Y. lipolytica M53-S with the 12 h delay inoculated T. reesei Rut C-30, in which efficient saccharification of DGS and improved production of erythritol were simultaneously achieved. The 10:1 inoculation proportion of Y. lipolytica and T. reesei contributed to the maximum erythritol production of 267.1 mg/gds under the optimal conditions including initial moisture of 55%, pH of 5.0, NaCl addition of 0.02 g/gds and DGS mass of 200 g in 144 h co-cultivation. Being compared with the attempts to produce erythritol from other raw materials, the one-pot SSF with DGS is proposed to be a potential strategy for efficient and economical erythritol production.

Cyclic l-lactide synthesis from lignocellulose biomass by biorefining with complete inhibitor removal and highly simultaneous sugars assimilation.

Cyclic chiral lactide is the monomer chemical for polymerization of high molecular weight polylactic acid (PLA). The synthesis of cyclic l-lactide starts from poly-condensation of l-lactic acid to a low molecular weight prepolymer and then depolymerized to cyclic l-lactide. Lignocellulose biomass is the most promising carbohydrate feedstock for lactic acid production, but the synthesis of cyclic l-lactide from l-lactic acid produced from lignocellulose has so far not been successful. The major barriers are the impurities of residual sugars and inhibitors in the crude cellulosic l-lactic acid product. Here we show a successful cyclic l-lactide synthesis from cellulosic l-lactic acid by lignocellulose biorefining with complete inhibitor removal and coordinated sugars assimilation. The removal of inhibitors from lignocellulose pretreatment was accomplished by biodetoxification using a unique fungus Amorphotheca resinae ZN1. The nonglucose sugars were completely and simultaneously assimilated at the same rate with glucose by the engineered l-lactic acid bacterium Pediococcus acidilactici. The l-lactic acid production from wheat straw was comparable to that from corn starch with high optical pure (99.6%), high l-lactic acid titer (129.4 g/L), minor residual total sugars (~2.2 g/L), and inhibitors free. The cyclic l-lactide was successfully synthesized from the regularly purified l-lactic acid and verified by detailed characterizations. This study paves the technical foundation of carbon-neutral production of biodegradable PLA from lignocellulose biomass.

Direct conversion of cheese whey to polymalic acid by mixed culture of Aureobasidium pullulans and permeabilized Kluyveromyces marxianus.

Cheese whey is an abundant and low-cost feedstock with lactose as its main component, but the inability to metabolize lactose prevents Aureobasidium pullulans from using cheese whey directly. In this study, Kluyveromyces marxianus was permeabilized to obtain nonviable but biocatalytic cells for lactose hydrolysis, and the mixed culture of A. pullulans and permeabilized K. marxianus was conducted for polymalic acid (PMA) production from cheese whey. In the mixed culture, PMA titer varied directly to ß-galactosidase activity of K. marxianus, but inversely to cell viability of K. marxianus, and ethanol permeabilized K. marxianus was the most compatible with A. pullulans for PMA production. 37.8 g/L PMA was produced in batch fermentation, and PMA titer was increased to 97.3 g/L in fed-batch fermentation, with a productivity of 0.51 g/(L·h) and a yield of 0.56 g/g. This study paved an economical and environmentally friendly way for PMA production from cheese whey.

Increasing sodium lactate production by enhancement of Na+ transmembrane transportation in Pediococcus acidilactici.

Fermentative production of sodium lactate generally is a low efficient process because of the high Na+ osmatic stress on lactic acid bacterium cells. In this study, the homogeneous genes encoding Na+/H+ antiporters were screened and overexpressed in Pediococcus acidilactici for the enhancement of Na+ transmembrane transportation. The function of the gene RS02775 was identified and its overexpressing in P. acidilactici resulted in the significantly improved sodium lactate production. The recombinant not only accelerated the sugar consumption, but also achieved the record high titer of sodium lactate by 121.1 g/L using pure sugars and 132.4 g/L using wheat straw. The transcription analysis shows that the overexpression of Na+/H+ antiporter significantly upregulated the transcription of the sugar phosphorylation genes of P. acidilactici under high Na+ stress. This study provides an effective method for high titer production of sodium lactate using both pure sugars and lignocellulose feedstocks.

An oxidoreductase gene ZMO1116 enhances the p-benzoquinone biodegradation and chiral lactic acid fermentability of Pediococcus acidilactici.

p-Benzoquinone (BQ) is a lignin-derived inhibitor to microbial strains. Unlike the furan inhibitors, p-benzoquinone is recalcitrant to traditional detoxification methods. This study shows a biological degradation of p-benzoquinone and a simultaneous D-lactic acid fermentation by an engineered Pediococcus acidilactici strain. The overexpression of an oxidoreductase gene ZMO1116 from Zymomonas mobilis encoding oxidoreductase was identified to improve the D-lactic acid fermentability of P. acidilactici against p-benzoquinone. The gene ZMO1116 was integrated into the genome of P. acidilactici and enabled the engineered P. acidilactici to convert p-benzoquinone into less toxic hydroquinone (HQ), resulting in the improved p-benzoquinone tolerance. Simultaneous saccharification and co-fermentation (SSCF) was conducted using the pretreated and biodetoxified corn stover containing p-benzoquinone, the D-lactic acid production of the engineered strain (123.8 g/L) was 21.4 % higher than the parental strain (102.0 g/L). This study provides a practical method on robust p-benzoquinone tolerance and efficient cellulosic chiral lactic acid fermentation from lignocellulose feedstock.

One-step utilization of non-detoxified pretreated lignocellulose for enhanced cellulolytic enzyme production using recombinant Trichoderma reesei RUT C30 carrying alcohol dehydrogenase and nicotinate phosphoribosyltransferase.

Cell growth of Trichoderma reesei is greatly inhibited by furan derivatives (furfural and HMF) generated during pretreatment of lignocellulose, and the cellulase production is hence suppressed. In this study, a novel recombinant strain of T. reesei with high tolerance to furans was constructed by homologously co-expressing nicotinate phosphoribosyltransferase and alcohol dehydrogenase. We observed that furfural had a stronger inhibitory effect than HMF and cellulase production was decreased by 35% in T. reesei with the stress of 2.5 mM furfural. The activities of nicotinate phosphoribosyltransferase and alcohol dehydrogenase increased 8.6-fold and 2.9-fold in the recombinant strain, respectively. Furfural was effectively converted into furfuryl alcohol which was then depleted, thus the production of cellulase could be recovered when the recombinant strain was grown in 5% (w/v) two-step stem explosion pretreated rice straw without detoxification. This work presents an important strategy for efficient enzyme production in T. reesei from non-detoxified pretreated lignocellulose feedstocks.

A short-chain dehydrogenase plays a key role in cellulosic D-lactic acid fermentability of Pediococcus acidilactici.

Phenolic aldehydes from lignocellulose pretreatment are strong inhibitors of cell growth and metabolism of cellulosic lactic acid bacteria. Their low solubility and recalcitrance highly reduce the removal efficiency of various detoxification methods. This study shows a simultaneous conversion of phenolic aldehydes and fermentation of D-lactic acid by Pediococcus acidilactici using corn stover feedstock. Vanillin was found to be the strongest phenolic aldehyde inhibitor to P. acidilactici. The overexpression of a short-chain dehydrogenase encoded by the gene CGS9114_RS09725 from Corynebacterium glutamicum was identified to play a key role in D-lactic acid fermentability of P. acidilactici. The engineered P. acidilactici with the genome integration of CGS9114_RS09725 showed the accelerated vanillin reduction and improved cellulosic D-lactic acid production. This study reveals that vanillin conversion is crucial for D-lactic acid fermentation, and the direct expression of a specific vanillin reduction gene in lactic acid bacterium efficiently improves cellulosic D-lactic acid production.

Engineering Pediococcus acidilactici with xylose assimilation pathway for high titer cellulosic l-lactic acid fermentation.

Xylose-assimilating pathways were constructed in the parental Pediococcus acidilactici strain and evolutionarily adapted to yield a highly stable co-fermentation strain for l-lactic acid production. The phosphoketolase pathway (PK) was blocked for reduction of acetic acid generation by disrupting phosphoketolase (pkt) gene. The pentose phosphate pathway (PPP) was reconstructed for xylose assimilation by integrating four heterologous genes encoding transketolase (tkt), transaldolase (tal), xylose isomerase (xylA) and xylulokinase (xylB) into the P. acidilactici chromosome. The xylose-assimilating ability of the constructed strain was significantly improved by long term adaptive evolution. The engineered strain was applied to the simultaneous saccharification and co-fermentation (SSCF) under high solids loading of wheat straw. The l-lactic acid titer, productivity and xylose conversion reached the record high at 130.8±1.6g/L, 1.82±0.0g/L/h, and 94.9±0.0%, respectively. This study provided an important strain and process prototype for production of high titer cellulosic l-lactic acid.

Constructing xylose-assimilating pathways in Pediococcus acidilactici for high titer d-lactic acid fermentation from corn stover feedstock.

Xylose-assimilating pathway was constructed in a d-lactic acid producing Pediococcus acidilactici strain and evolutionary adapted to yield a co-fermentation strain P. acidilactici ZY15 with 97.3g/L of d-lactic acid and xylose conversion of 92.6% obtained in the high solids content simultaneous saccharification and co-fermentation (SSCF) of dry dilute acid pretreated and biodetoxified corn stover feedstock. The heterologous genes encoding xylose isomerase (xylA) and xylulokinase (xylB) were screened and integrated into the P. acidilactici chromosome. The metabolic flux to acetic acid in phosphoketolase pathway was re-directed to pentose phosphate pathway by substituting the endogenous phosphoketolase gene (pkt) with the heterologous transketolase (tkt) and transaldolase (tal) genes. The xylose-assimilating ability of the newly constructed P. acidilactici strain was significantly improved by adaptive evolution. This study provided an important strain and process prototype for high titer d-lactic acid production from lignocellulose feedstock with efficient xylose assimilation.