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[Display omitted] • Liquid fraction containing glycerol was used for recycled corn stover pretreatment. • Dry cell weight of P. pastoris reached ∼ 40 g/L with glycerol as sole carbon source. • The titer of human lysozyme reached 1.3 g/L by P. pastoris with methanol induction. • Mixing pretreated corn stover with cassava as co-substrate to produce biobutanol. • Final butanol titer of 13.9 g/L was produced from mixed substrates under SSF. Glycerol organosolv pretreatment (GOP) is considered an efficient method to deconstruct lignocellulose for producing fermentable sugars. Herein, the liquid fraction containing glycerol after GOP was utilized for recycled pretreatment of corn stover (CS) for four cycles. Enzymatic yield of glucose after recycled pretreatment was enhanced by 2.4–3.5 folds compared with untreated CS. Meanwhile, residual glycerol was used as carbon source for cultivation of Pichia pastoris to obtain high cell-density, and a final titer of 1.3 g/L human lysozyme was produced by P. pastoris under low temperature methanol induction strategy. Additionally, the pretreated CS was mixed with cassava as fermentable substrates for butanol production by wild-type Clostridium acetobutylicum ATCC 824. Final butanol production of 13.9 g/L was obtained from mixed substrates (25%:75% of CS/cassava) at 10% solids loading by simultaneous saccharification and fermentation. Overall, integration of residual glycerol utilization and butanol production by microbial fermentation provided an efficient strategy for biorefinery. [ABSTRACT FROM AUTHOR]

The techniques for enhancing butanol production in ABE fermentation by Clostridium acetobutylicum generally focus on adding electron carrier to strengthen NADH synthesis, repressing hydrogenase by aerating CO, supplementing butyrate, etc. However, those methods suffer from the problems of total solvent decrease, high purification cost, using expensive supplemental substances, etc. In this study, we added small amount of electron receptors (Na 2 SO 4 /CaSO 4 , 2 g/L) into ABE fermentation broth: to alter electron/proton distributions in the intracellular electron transport shuttle system, directing more electron/proton pairs into NADH synthesis route; to stimulate intracellular accumulation of those amino acids favorable for cells survival/butanol synthesis. In ABE fermentation in a 7 L fermentor, adding 2 g/L Na 2 SO 4 could raise butanol concentration to a higher level of 12.96 g/L, which was 34.8% higher than that of the control. Addition of tiny amount cheap electron receptor would provide a new way to enhance bio-butanol production. [ABSTRACT FROM AUTHOR]

A novel glucose/acetate co-substrate system incorporating with C. acetobutylicum / S. cerevisiae co-culturing and glucose limitation was proposed and applied in ABE fermentations by C. acetobutylicum . With this strategy, acetone production was enhanced while NADH dependent butanol synthesis rate was adequately restricted by glucose limitation via decreasing initial glucose concentration in the medium and adaptively adding acetate/ S. cerevisiae during solventogenic phase. Under these conditions, acetone production ability of C. acetobutylicum could be fully utilized by delaying butanol inhibition occurrence time; favorable environment for acetone production was created while incomplete ABE fermentation due to glucose limitation avoided. The proposed strategy could arbitrarily control or maximize acetone concentration and acetone/butanol ratio (A/B) in the ranges of 6–12 g/L and 0.5–1.0, the maximum values could reach the levels of 11.74 g/L and 1.02 which would potentially promote ABE fermentative products flexibility and bio-acetone productions using renewable biomass. The theoretical analysis of A/B ratio based on carbon mass and energy balances showed that, the maximum A/B ratio of 1.04 could be achieved when 60 g/L glucose and 12 g/L acetate were consumed by C. acetobutylicum , which coincided with the experimental data (1.02) well. [ABSTRACT FROM AUTHOR]

In this study, an efficient acetone-butanol-ethanol (ABE) fermentation strategy integrating Clostridium acetobutylicum/Saccharomyces cerevisiae co-culturing system with exogenous butyrate addition, was proposed and experimentally conducted. In solventogenic phase, by adding 0.2 g-DCW/L-broth viable S. cerevisiae cells and 4.0 g/L-broth concentrated butyrate solution into C. acetobutylicum culture broth, final butanol concentration and butanol/acetone ratio in a 7 L anaerobic fermentor reached the highest levels of 15.74 g/L and 2.83 respectively, with the increments of 35% and 43% as compared with those of control. Theoretical and experimental analysis revealed that, the proposed strategy could, 1) extensively induce secretion of amino acids particularly lysine, which are favorable for both C. acetobutylicum survival and butanol synthesis under high butanol concentration environment; 2) enhance the utilization ability of C. acetobutylicum on glucose and over-produce intracellular NADH for butanol synthesis in C. acetobutylicum metabolism simultaneously; 3) direct most of extra consumed glucose into butanol synthesis route. The synergetic actions of effective amino acids assimilation, high rates of substrate consumption and NADH regeneration yielded highest butanol concentration and butanol ratio in C. acetobutylicum under this stress environment. The proposed method supplies an alternative way to improve ABE fermentation performance by traditional fermentation technology. [ABSTRACT FROM AUTHOR]

[Display omitted] • Com-Phe stress changed the intracellular levels of reducing power and ROS. • Response mechanism of C. acetobutylicum to Com-Phe was elucidated by RNA-seq. • First report on evaluation of the toxicity of Com-Phe on solventogenic clostridia. • Strategy for sustainable and cleaner biobutanol production was provided. Lignocellulosic biomass is the most abundant bioresource to consider as an alternative feedstock to produce environmentally friendlier biobutanol as an advanced biofuel. However, the pretreatment for lignocellulose depolymerization suffers from toxic inhibitors formation and wastewater generation. The phenolic compounds, including phenolic aldehydes (Phe-Ad) and phenolic acids (Phe-Ac), derived from lignin degradation during the acid pretreatment of lignocellulose such as corn stover, are the main inhibitors for butanol fermentation. However, the overall impacts of the co-existence of Phe-Ad and Phe-Ac (Com-Phe) on solventogenic clostridia metabolism were not investigated thus far. Here, toxic effects of Com-Phe on butanol fermentation were firstly evaluated by analyzing apparent kinetics, reducing power, and reactive oxygen species (ROS). The lower intracellular reducing power level and higher ROS result in a 56.3 % decrease of butanol titer after 12 h stress. Subsequently, the response mechanism of Clostridium acetobutylicum to Com-Phe stress was elucidated by RNA-seq focusing on physiological processes with carbon metabolism and product biosynthesis. Some key genes related to heat shock proteins (HSP) and antioxidative activity were up-regulated under Com-Phe stress, which might be an adaptive mechanism of cells to alleviate its toxicity. Phenolics also altered two-component systems, sporulation, and cell division processes. This is the first report on systematic evaluation of the toxicity of Com-Phe on solventogenic clostridia. The proposed response mechanism provides valuable guidance (such as regulation of sugar transport, HSP-related genes, glutathione metabolism, etc.) for developing robust strains and promotes cleaner biobutanol production from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]