Although microbes directly accepting electrons from a cathode have been applied for CO2 reduction to produce multicarbon-compounds, a high electron demand and low product concentration are critical limitations. Alternatively, the utilization of electrons as a co-reducing power during fermentation has been attempted, but there must be exogenous mediators due to the lack of an electroactive heterotroph. Here, we show that Clostridium pasteurianum DSM 525 simultaneously utilizes both cathode and substrate as electron donors through direct electron transfer. In a cathode compartment poised at +0.045 V vs. SHE, a metabolic shift in C. pasteurianum occurs toward NADH-consuming metabolite production such as butanol from glucose (20% shift in terms of NADH consumption) and 1,3-propandiol from glycerol (21% shift in terms of NADH consumption). Notably, a small amount of electron uptake significantly induces NADH-consuming pathways over the stoichiometric contribution of the electrons as reducing equivalents. Our results demonstrate a previously unknown electroactivity and metabolic shift in the biochemical-producing heterotroph, opening up the possibility of efficient and enhanced production of electron-dense metabolites using electricity.

BackgroundMetabolic engineering of cyanobacteria has enabled photosynthetic conversion of CO2 to value-added chemicals as bio-solar cell factories. However, the production levels of isoprenoids in engineered cyanobacteria were quite low, compared to other microbial hosts. Therefore, modular optimization of multiple gene expressions for metabolic engineering of cyanobacteria is required for the production of farnesyl diphosphate-derived isoprenoids from CO2.ResultsHere, we engineered Synechococcus elongatus PCC 7942 with modular metabolic pathways consisting of the methylerythritol phosphate pathway enzymes and the amorphadiene synthase for production of amorpha-4,11-diene, resulting in significantly increased levels (23-fold) of amorpha-4,11-diene (19.8 mg/L) in the best strain relative to a parental strain. Replacing amorphadiene synthase with squalene synthase led to the synthesis of a high amount of squalene (4.98 mg/L/OD730). Overexpression of farnesyl diphosphate synthase is the most critical factor for the significant production, whereas overexpression of 1-deoxy-d-xylulose 5-phosphate reductase is detrimental to the cell growth and the production. Additionally, the cyanobacterial growth inhibition was alleviated by expressing a terpene synthase in S. elongatus PCC 7942 strain with the optimized MEP pathway only (SeHL33).ConclusionsThis is the first demonstration of photosynthetic production of amorpha-4,11-diene from CO2 in cyanobacteria and production of squalene in S. elongatus PCC 7942. Our optimized modular OverMEP strain (SeHL33) with either co-expression of ADS or SQS demonstrated the highest production levels of amorpha-4,11-diene and squalene, which could expand the list of farnesyl diphosphate-derived isoprenoids from CO2 as bio-solar cell factories.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0617-8) contains supplementary material, which is available to authorized users.

Caproiciproducens galactitolivorans gen. nov., sp. nov., a bacterium capable of producing caproic acid from galactitol, isolated from a wastewater treatment plant A strictly anaerobic, Gram-stain-positive, non-spore-forming, rod-shaped bacterial strain, designated BS-1 T , was isolated from an anaerobic digestion reactor during a study of bacteria utilizing galactitol as the carbon source. Its cells were 0.3-0.5 mm62-4 mm, and they grew at 35-45 8C and at pH 6.0-8.0. Strain BS-1 T produced H 2 , CO 2 , ethanol, acetic acid, butyric acid and caproic acid as metabolic end products of anaerobic fermentation. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain BS-1 T represented a novel bacterial genus within the family Ruminococcaceae, Clostridium Cluster IV. The type strains that were most closely related to strain BS-1 T were Clostridium sporosphaeroides KCTC 5598 T (94.5 %), Clostridium leptum KCTC 5155 T (94.3 %), Ruminococcus bromii ATCC 27255 T (92.1 %) and Ethanoligenens harbinense YUAN-3 T (91.9 %). Strain BS-1 T had 17.6 % and 20.9 % DNA-DNA relatedness values with C. sporosphaeroides DSM 1294 T and C. leptum DSM 753 T , respectively. The major components of the cellular fatty acids were C 16 : 0 dimethyl aldehyde (DMA) (22.1 %), C 16 : 0 aldehyde (14.1 %) and summed feature 11 (iso-C 17 : 0 3-OH and/or C 18 : 2 DMA; 10.0 %). The genomic DNA G+C content was 50.0 mol%. Phenotypic and phylogenetic characteristics allowed strain BS-1 T to be clearly distinguished from other taxa of the genus Clostridium Cluster IV. On the basis of these data, the isolate is considered to represent a novel genus and novel species within Clostridium Cluster IV, for which the name Caproiciproducens galactitolivorans gen. nov., sp. nov. is proposed. The type species is BS-1 T (5JCM 30532 T and KCCM 43048 T ).

Butanol production by Clostridium beijerinckii NCIMB 8052 was investigated using both batch and continuous cultures containing suspended or immobilized cells. In the batch reactor, the initial addition of acetate and butyrate into the culture media was found not only to enhance solvent production but also to affect the ratio of acetone/butanol, which might result from the metabolic changes in solvent production. Furthermore, the addition of butyrate to the medium prevented strain degeneration during an extended subculturing, significantly induced butanol production (11.2 g/L butanol versus 0.45 g/L butanol with and without 36 mM butyrate, respectively), and shifted the acetone/butanol ratio to 1:3, which resulted in a higher yield (0.45 g of butanol/g of glucose) when compared to other studies. The beneficial effects of butyrate were also observed in continuous reactor tests containing suspended cells as the solvent production was maintained over more than 300 h of continuous operation. During a continuous butanol production with immobilized cells, using porous hydrophilic media and a dilution rate of 0.04 h−1, the overall butanol productivity and yield were 0.40 g L−1 h−1 and 0.44 g of butanol/g of glucose, respectively, which are approximately twice the values seen in a continuous reactor with suspended cells. Moreover, the butanol production was maintained over 150 days without apparent degeneration, even in the presence of high butanol concentrations (10−13 g/L). These results validate the effectiveness of producing butanol with an immobilized cell system supplemented with butyrate.

Hydrothermal pretreatment using liquid hot water, steam explosion, or dilute acids enhances the enzymatic digestibility of cellulose by altering the chemical and/or physical structures of lignocellulosic biomass. However, compounds that inhibit both enzymes and microbial activity, including lignin-derived phenolics, soluble sugars, furan aldehydes, and weak acids, are also generated during pretreatment. Insoluble lignin, which predominantly remains within the pretreated solids, also acts as a significant inhibitor of cellulases during hydrolysis of cellulose. Exposed lignin, which is modified to be more recalcitrant to enzymes during pretreatment, adsorbs cellulase nonproductively and reduces the availability of active cellulase for hydrolysis of cellulose. Similarly, lignin-derived phenolics inhibit or deactivate cellulase and β-glucosidase via irreversible binding or precipitation. Meanwhile, the performance of fermenting microorganisms is negatively affected by phenolics, sugar degradation products, and weak acids. This review describes the current knowledge regarding the contributions of inhibitors present in whole pretreatment slurries to the enzymatic hydrolysis of cellulose and fermentation. Furthermore, we discuss various biological strategies to mitigate the effects of these inhibitors on enzymatic and microbial activity to improve the lignocellulose-to-biofuel process robustness. While the inhibitory effect of lignin on enzymes can be relieved through the use of lignin blockers and by genetically engineering the structure of lignin or of cellulase itself, soluble inhibitors, including phenolics, furan aldehydes, and weak acids, can be detoxified by microorganisms or laccase.