Upgrading dry acid pretreatment by post-hydrolysis for carbon efficient conversion of lignocellulose.

Dry acid pretreatment (DAP) as a promising process for industrial biorefinery provide an efficient bioconversion of cellulose without free wastewater, although the partial xylan and lignin degrade to inhibitors or recondense. A biorefinery strategy for carbon efficient conversion of lignocellulose into bioethanol, xylose, and reactive lignin was developed by upgrading DAP with post-hydrolysis. The results showed that lignocellulose after mild DAP (175 °C, acid dosage of 15 mg/g dry material) obtained higher xylan recovery and lower inhibitors than that of general DAP. Subsequently, post-hydrolysis, simultaneous saccharification and ethanol fermentation were performed at solids loading of 20 wt% without detoxification and sterilization, resulting in xylose and ethanol yield of 71.8 % and 67.6 %. The fractionated lignin presented more reactive ß-aryl ether linkages and less condensation than that from DAP. 66 % of lignocellulose carbon was recovered as ethanol, xylose and reactive lignin. This upgrading biorefinery strategy provided an easy-to-operate process for integrated utilization of lignocellulose.

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.

Overexpressing CCW12 in Saccharomyces cerevisiae enables highly efficient ethanol production from lignocellulose hydrolysates.

A Saccharomyces cerevisiae strain CCW12OE was constructed by overexpressing CCW12 in a previously reported strain WXY70 harboring six xylose utilization genes. CCW12OE produced an optimal ethanol yield of 98.8% theoretical value within 48 h in a simulated corn stover hydrolysate. CCW12OEwas comprehensively evaluated for ethanol production in Miscanthus, maize and corncob hydrolysates, among which a 96.1% theoretical value was achieved within 12 h in corncob hydrolysates. Under normal growth conditions, CCW12OE did not display altered cell morphology; however, in the presence of acetate, CCW12OE maintained relatively intact cell structure and increased cell wall thickness by nearly 50%, while WXY70 had abnormal cell morphology and reduced cell wall thickness by nearly 50%. Besides, CCW12OE had higher fermentation capacity than that of WXY70 in undetoxified and detoxified hydrolysates with both aerobic and anaerobic conditions, demonstrating that CCW12 overexpression alone exhibits improved stress resistance and better fermentation performance.

Physiological mechanism of improved tolerance of Saccharomyces cerevisiae to lignin-derived phenolic acids in lignocellulosic ethanol fermentation by short-term adaptation.

BACKGROUND: Phenolic acids are lignin-derived fermentation inhibitors formed during many pretreatment processes of lignocellulosic biomass. In this study, vanillic, p-hydroxybenzoic, and syringic acids were selected as the model compounds of phenolic acids, and the effect of short-term adaptation strategies on the tolerance of S. cerevisiae to phenolic acids was investigated. The mechanism of phenolic acids tolerance in the adapted yeast strains was studied at the morphological and physiological levels. RESULTS: The multiple phenolic acids exerted the synergistic inhibitory effect on the yeast cell growth. In particular, a significant interaction between vanillic and hydroxybenzoic acids was found. The optimal short-term adaptation strategies could efficiently improve the growth and fermentation performance of the yeast strain not only in the synthetic media with phenolic acids, but also in the simultaneous saccharification and ethanol fermentation of corncob residue. Morphological analysis showed that phenolic acids caused the parental strain to generate many cytoplasmic membrane invaginations with crack at the top of these sites and some mitochondria gathered around. The adapted strain presented the thicker cell wall and membrane and smaller cell size than those of the parental strain. In particular, the cytoplasmic membrane generated many little protrusions with regular shape. The cytoplasmic membrane integrity was analyzed by testing the relative electrical conductivity, leakage of intracellular substance, and permeation of fluorescent probe. The results indicated that the short-term adaptation improved the membrane integrity of yeast cell. CONCLUSION: The inhibition mechanism of phenolic acid might be attributed to the combined effect of the cytoplasmic membrane damage and the intracellular acidification. The short-term adaptation strategy with varied stressors levels and adaptive processes accelerated the stress response of yeast cell structure to tolerate phenolic acids. This strategy will contribute to the development of robust microbials for biofuel production from lignocellulosic biomass.

Transcriptome analysis of Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic aldehyde inhibitors from lignocellulose pretreatment.

BACKGROUND: Phenolic aldehydes generated from lignocellulose pretreatment exhibited severe toxic inhibitions on microbial growth and fermentation. Numerous tolerance studies against furfural, 5-hydroxymethyl-2-furaldehyde (HMF), acetate, and ethanol were reported, but studies on inhibition of phenolic aldehyde inhibitors are rare. For ethanologenic strains, Zymomonas mobilis ZM4 is high in ethanol productivity and genetic manipulation feasibility, but sensitive to phenolic aldehyde inhibitors. Molecular mechanisms of tolerance for Z. mobilis toward phenolic aldehydes are not known. RESULTS: We took the first insight into genomic response of Z. mobilis ZM4 to the phenolic aldehyde inhibitors derived from lignocellulose pretreatment. The results suggest that the toxicity to cells is caused by the functional group of phenolic aldehyde, similar to furfural and HMF, rather than aromatic groups or phenolic hydroxyl groups. Transcriptome response against 4-hydroxybenzaldehyde, syringaldehyde, and vanillin, representing phenolic groups H, S, and G, respectively, was investigated. The atlas of the important genes responsible for significantly enhanced and repressed genes at the genomic level was illustrated. 272 genes with twofold greater expressions than non-treated controls and 36 gene clusters in response to challenges of these phenolic aldehydes were identified. Several reductases encoded by ZMO1116, ZMO1696, and ZMO1885 were found to play the key roles in reducing phenolic aldehydes into the corresponding phenolic alcohols. Reduction of phenolic aldehydes by overexpression of ZMO1116, ZMO1696, and ZMO1885 in Z. mobilis ZM4 resulted in the increased inhibitor conversion and ethanol productivity, especially for 4-hydroxybenzaldehyde and vanillin. Several transporter genes such as ZMO0282, ZMO0283, ZMO0798, ZMO0799, and ZMO0800 was also displayed significantly increased expressions against the phenolic aldehydes. CONCLUSIONS: The genes encoding reductases are with potentials on phenolic aldehydes-tolerant genes contributing to the reduction of phenolic aldehydes into the corresponding phenolic alcohols forms for Z. mobilis ZM4. Overexpression of the key genes improved the conversion ratio and ethanol productivity of 4-hydroxybenzaldehyde and vanillin with high toxicity. New knowledge obtained from this research aids understanding the mechanisms of bacterial tolerance and the development of the next-generation biocatalysts for advanced biofuels production.

High tolerance and physiological mechanism of Zymomonas mobilis to phenolic inhibitors in ethanol fermentation of corncob residue.

Corncob residue as the lignocellulosic biomass accumulated phenolic compounds generated from xylitol production industry. For utilization of this biomass, Zymomonas mobilis ZM4 was tested as the ethanol fermenting strain and presented a better performance of cell growth (2.8 × 10(8) CFU/mL) and ethanol fermentability (54.42 g/L) in the simultaneous saccharification and fermentation (SSF) than the typical robust strain Saccharomyces cerevisiae DQ1 (cell growth of 2.9 × 10(7) CFU/mL, ethanol titer of 48.6 g/L). The physiological response of Z. mobilis ZM4 to the twelve typical phenolic compounds derived from lignocellulose was assayed and compared with that of S. cerevisiae DQ1. Z. mobilis ZM4 showed nearly the same tolerance to the phenolic aldehydes with S. cerevisiae DQ1, but the stronger tolerance to the phenolic acids existing in corncob residue (2-furoic acid, p-hydroxybenzoic acid, p-coumaric acid, vanillic acid, ferulic acid, and syringic acid). The tolerance mechanism of Z. mobilis was investigated in terms of inhibitor degradation, cell morphology and membrane permeability under the stress of phenolics using GC-MS, scanning and transmission electron microscopies (SEM and TEM), as well as fluorescent probes. The results reveal that Z. mobilis ZM4 has the capability for in situ detoxification of phenolic aldehydes, and the lipopolysaccharide aggregation on the cell outer membrane of Z. mobilis ZM4 provided the permeable barrier to the attack of phenolic acids.

Analysis of particle size reduction on overall surface area and enzymatic hydrolysis yield of corn stover.

Particle size of lignocellulose materials is an important factor for enzymatic hydrolysis efficiency. In this study, corn stover was milled and sieved into different size fractions from 1.42, 0.69, 0.34, to 0.21 mm, and the corresponding enzymatic hydrolysis yields were 24.69, 23.96, 25.34, and 26.97 %, respectively. The results indicate that the hydrolysis yield is approximately constant with changing corn stover particle sizes in the experimental range. The overall surface area and the inner pore size measurement show that the overall specific surface area was less than 2 % with the half reduction of particle size due to the greater inner pore surface area. The scanning electron microscope photographs gave direct evidence of the much greater inner pore surface area of corn stover particles. This result provided a reference when a proper size reduction of lignocellulose materials is considered in biorefining operations.

Inhibitor analysis and adaptive evolution of Saccharomyces cerevisiae for simultaneous saccharification and ethanol fermentation from industrial waste corncob residues.

Industrial waste corncob residues (CCR) are rich in cellulose and can be hydrolyzed directly without pretreatment. However, a poor fermentation performance was frequently observed in the simultaneous saccharification and ethanol fermentation (SSF) of CCR, although the furans and organic acid inhibitors were very low. In this study, the high level of water-insoluble phenolic compounds such as 2-furoic acid, ferulic acid, p-coumaric acid, guaiacol, and p-hydroxybenzoic acid were detected in CCR and inhibited the growth and metabolism of Saccharomyces cerevisiae DQ1. An evolutionary adaptation strategy was developed by culturing the S. cerevisiae DQ1 strain in a series of media with the gradual increase of CCR hydrolysate. The high ethanol concentration (62.68g/L) and the yield (55.7%) were achieved in the SSF of CCR using the adapted S. cerevisiae DQ1. The results provided a practical method for improving performance of simultaneous saccharification and ethanol production from CCR.

Simultaneous saccharification and high titer lactic acid fermentation of corn stover using a newly isolated lactic acid bacterium Pediococcus acidilactici DQ2.

A lactic acid bacterium with high tolerance of temperature and lignocellulose derived inhibitor was isolated and characterized as Pediococcus acidilactici DQ2. The strain used in the simultaneous saccharification and fermentation (SSF) for high titer lactic acid production at the high solids loading of corn stover. Corn stover was pretreated using the dry sulphuric acid pretreatment, followed by a biological detoxification to remove the inhibitors produced in the pretreatment. The bioreactor with a novel helical impeller was used to the SSF operation of the pretreated and biodetoxified corn stover. The results show that a typical SSF operation at 48 °C, pH 5.5, and near 30% (w/w) solids loading in both 5 and 50 L bioreactors was demonstrated. The lactic acid titer, yield, and productivity reached 101.9 g/L, 77.2%, and 1.06 g/L/h, respectively. The result provided a practical process option for cellulosic lactic acid production using virgin agriculture lignocellulose residues.