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.

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-catalyzed oxidation of  l- and  d-lactic acids. This study provided an important support for the production of cellulosic  l-lactic acid with high chiral purity.

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.

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.

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.