BackgroundPhenolic 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.ResultsWe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0333-9) contains supplementary material, which is available to authorized users.

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BackgroundPhenolic 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.ResultsThe 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.ConclusionThe 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.

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.

Compared with conventional production of biodiesel from oilseeds, in situ reactive extraction of oilseeds with acyl acceptors is an attractive approach, which consists of sustainable, economically attractive biotechnological processes for biodiesel synthesis. In this study, the presence of lipase activity in germinating oilseeds was detected and the lipase activity increased to a maximum after 4 days of germination. At that time, the germinated seeds featured an only slightly lower oil content relative to the ungerminated seeds. As such, an environmentally friendly and low-cost in situ self-catalytic process for biodiesel production was developed. The biodiesel was prepared by reactive extraction of germinated Jatropha curcas L. seeds with methanol without any other catalyst added. In this process, nhexane was used as reactive extraction solvent. The highest fatty acid methyl esters (FAMEs) yield of 87.6% could be achieved under the optimum conditions: a n-hexane/germinated seed ratio of 2.5 ml g À1 , a methanol/germinated seed oil (contained approximately 90% FFA) molar ratio of 1.5 : 1, a reaction temperature of 35 C, a germinated seed water content of 2.9% and a reaction time of 8 h. This simple reactive extraction process without additional catalyst can be a potential route for biodiesel production, which may greatly reduce the processing steps and costs.

BACKGROUND: Ultrasound-assisted fractionation of DDGS under mild conditions was proposed for extracting xylan from dried distillers' grains with solubles (DDGS) and recovering protein-rich DDGS residue as animal feed. The composition and properties of xylan fractions and the DDGS residue obtained by ultrasound-assisted extraction and those by a conventional method of alkaline extraction without ultrasound-assistance were compared. RESULTS: Compared with the result for the conventional method, a similar xylan yield of 73.8% in the extracted liquid, but higher protein content of 26.2% in the DDGS residue were obtained by the ultrasound-assisted process with NaOH at ultrasound power of 900W and 30 ∘ C for 20 min. The ultrasound-assisted process has a significant impact on the sugar composition and polysaccharide side chain and polymerization degree of the xylan fractions. The DDGS residue produced by the ultrasound-assisted process had a fine porous structure as well as greater protein and essential amino acid content. The enzymatic hydrolysis of cellulose in DDGS residue reached 70% in the initial 3 h. CONCLUSION: Ultrasound-assisted fractionation was an efficient process for comprehensive utilization of xylan and protein in the DDGS. The xylan fractions with lower polymerization degree were perfect feedstock for xylooligosaccharide production by enzymatic hydrolysis. The DDGS residue with adequate nutrition composition and efficient digestibility had potential advantages as an alternative animal feed to DDGS.

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