Effects of and Medium Modifications on Acetone-Butanol-Ethanol Production From Switchgrass.

The presence of lignocellulose-derived microbial inhibitory compounds (LDMICs) in lignocellulosic biomass (LB) hydrolysates is a barrier to efficient conversion of LB hydrolysates to fuels and chemicals by fermenting microorganisms. Results from this study provide convincing evidence regarding the effectiveness of metabolically engineered NCIMB 8052 for the fermentation of LB-derived hydrolysates to acetone-butanol-ethanol (ABE). The engineered microbial strain (_SDR) was produced by the integration of an additional copy of a short-chain dehydrogenase/reductase (SDR) gene (3904) into the chromosome of NCIMB 8052 wildtype, where it is controlled by the constitutive thiolase promoter.

Ribozyme-Mediated Downregulation Uncovers DNA Integrity Scanning Protein A (DisA) as a Solventogenesis Determinant in .

Carbon catabolite repression (CCR) limits microbial utilization of lignocellulose-derived pentoses. To relieve CCR in NCIMB 8052, we sought to downregulate catabolite control protein A (CcpA) using the M1GS ribozyme technology. A CcpA-specific ribozyme was constructed by tethering the catalytic subunit of RNase P (M1 RNA) to a guide sequence (GS) targeting CcpA mRNA (M1GS).

Three-stage repeated-batch immobilized cell fermentation to produce butanol from non-detoxified sugarcane bagasse hemicellulose hydrolysates.

To enable the production of butanol with undiluted, non-detoxified sugarcane bagasse hemicellulose hydrolysates, this study developed a three-staged repeated-batch immobilized cell fermentation in which the efficiency of a 3D-printed nylon carrier to passively immobilize Clostridium saccharoperbutylacetonicum DSM 14923 was compared with sugarcane bagasse. The first stage consisted of sugarcane molasses fermentation, and in the second stage, non-detoxified sugarcane bagasse hemicellulose hydrolysates (SBHH) was pulse-fed to sugarcane molasses fermentation. In the next four batches, immobilized cells were fed with undiluted SBHH supplemented with molasses, and SBHH-derived xylose accounted for approximately 50% of the sugars.

Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation.

The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in , was successfully disrupted. The levansucrase null mutant produced 2.

Chromosomal integration of aldo-keto-reductase and short-chain dehydrogenase/reductase genes in Clostridium beijerinckii NCIMB 8052 enhanced tolerance to lignocellulose-derived microbial inhibitory compounds.

In situ detoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression of Cbei_3974 and Cbei_3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, in Clostridium beijerinckii NCIMB 8052 (Cb), resulting in two strains: Cb_3974 and Cb_3904. Expression of Cbei_3974 led to 2-fold increase in furfural detoxification relative to Cb_3904 and Cb_wild type.

Metabolic engineering of to improve glycerol metabolism and furfural tolerance.

Background: Inefficient utilization of glycerol by () is a major impediment to adopting glycerol metabolism as a strategy for increasing NAD(P)H regeneration, which would in turn, alleviate the toxicity of lignocellulose-derived microbial inhibitory compounds (LDMICs, e.g., furfural), and improve the fermentation of lignocellulosic biomass hydrolysates (LBH) to butanol.

Resolving mismatches in the flexible production of ethanol and butanol from eucalyptus wood with vacuum fermentation.

In flexible ethanol-butanol plants, low tolerance to butanol by solventogenic clostridia (and resulting dilute fermentation) results in considerable number of empty fermentors whenever production focuses on ethanol. This research identified scenarios in which vacuum fermentation (in-situ vacuum recovery) may be applied to solve this problem. We conducted ethanol (Saccharomyces cerevisiae) and ABE (Clostridium beijerinckii NCIMB 8052) batch vacuum fermentations of eucalyptus hydrolysates according to the distribution of sugars in a flexible plant.

Development of a high-throughput assay for rapid screening of butanologenic strains.

We report a Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)-dependent spectrophotometric assay for quantifying the amount of butanol in growth media, an advance that will facilitate rapid high-throughput screening of hypo- and hyper-butanol-producing strains of solventogenic Clostridium species. While a colorimetric nitroblue tetrazolium chloride-based assay for quantitating butanol in acetone-butanol-ethanol (ABE) fermentation broth has been described previously, we determined that Saccharomyces cerevisiae (Sc) ADH used in this earlier study exhibits approximately 13-fold lower catalytic efficiency towards butanol than ethanol. Any Sc ADH-dependent assay for primary quantitation of butanol in an ethanol-butanol mixture is therefore subject to "ethanol interference".

Investigation of relationship between 2,3-butanediol toxicity and production during growth of Paenibacillus polymyxa.

Understanding the capacity of Paenibacillus polymyxa DSM 365 to tolerate increasing concentrations of 2,3-butanediol (2,3-BD) is critical to engineering a 2,3-BD-overproducing strain. Hence, we investigated the response of P. polymyxa to high 2,3-BD concentrations.

Use of Cupriavidus basilensis-aided bioabatement to enhance fermentation of acid-pretreated biomass hydrolysates by Clostridium beijerinckii.

Lignocellulose-derived microbial inhibitors (LDMICs) prevent efficient fermentation of Miscanthus giganteus (MG) hydrolysates to fuels and chemicals. To address this problem, we explored detoxification of pretreated MG biomass by Cupriavidus basilensis ATCC(®)BAA-699 prior to enzymatic saccharification. We document three key findings from our test of this strategy to alleviate LDMIC-mediated toxicity on Clostridium beijerinckii NCIMB 8052 during fermentation of MG hydrolysates.

Unorthodox methods for enhancing solvent production in solventogenic Clostridium species.

While production of biofuels from renewable resources is currently receiving increased attention globally, concerns on availability and sustainability of cheap substrates for their production are growing as well. Lignocellulose-derived sugars (LDS) remain underutilized and merit consideration as a key feedstock. Among other obstacles such as low yield and low solvent titer, mitigation of stresses stemming from lignocellulose-derived microbial inhibitory compounds (LDMICs) that severely impair cell growth and solvent production is a major area of research interest.

Identification, purification and characterization of furfural transforming enzymes from Clostridium beijerinckii NCIMB 8052.

Generation of microbial inhibitory compounds such as furfural and 5-hydroxymethylfurfural (HMF) is a formidable roadblock to fermentation of lignocellulose-derived sugars to butanol. Bioabatement offers a cost effective strategy to circumvent this challenge. Although Clostridium beijerinckii NCIMB 8052 can transform 2-3 g/L of furfural and HMF to their less toxic alcohols, higher concentrations present in biomass hydrolysates are intractable to microbial transformation.

Allopurinol-mediated lignocellulose-derived microbial inhibitor tolerance by Clostridium beijerinckii during acetone-butanol-ethanol (ABE) fermentation.

In addition to glucans, xylans, and arabinans, lignocellulosic biomass hydrolysates contain significant levels of nonsugar components that are toxic to the microbes that are typically used to convert biomass to biofuels and chemicals. To enhance the tolerance of acetone-butanol-ethanol (ABE)-generating Clostridium beijerinckii NCIMB 8052 to these lignocellulose-derived microbial inhibitory compounds (LDMICs; e.g.

Evaluation of industrial dairy waste (milk dust powder) for acetone-butanol-ethanol production by solventogenic Clostridium species.

Readily available inexpensive substrate with high product yield is the key to restoring acetone-butanol-ethanol (ABE) fermentation to economic competitiveness. Lactose-replete cheese whey tends to favor the production of butanol over acetone. In the current study, we investigated the fermentability of milk dust powder with high lactose content, for ABE production by Clostridium acetobutylicum and Clostridium beijerinckii.

Elucidating and alleviating impacts of lignocellulose-derived microbial inhibitors on Clostridium beijerinckii during fermentation of Miscanthus giganteus to butanol.

Fermentation of liquid hot water (LHW) pretreated Miscanthus giganteus (MG) by Clostridium beijerinckii NCIMB 8052 was investigated towards understanding the toxicity of lignocellulose-derived inhibitors to solventogenic Clostridium species vis-à-vis butanol production. While C. beijerinckii NCIMB 8052 did not grow in undiluted MG hydrolysate-based fermentation medium, supplementation of this medium with Calcium carbonate enabled the growth of C.

Glycerol supplementation of the growth medium enhances in situ detoxification of furfural by Clostridium beijerinckii during butanol fermentation.

Lignocellulose-derived microbial inhibitors such as furfural and 5-hydroxymethyl furfural adversely affect fermentation of lignocellulosic biomass hydrolysates to fuels and chemicals due to their toxicity on fermenting microbes. To harness the potential of lignocellulose as a cheap source of fermentable sugars, in situ detoxification of furfural and other lignocellulose-derived microbial inhibitors is essential. To enhance in situ detoxification and tolerance of furfural by Clostridium beijerinckii NCIMB 8052 during acetone-butanol-ethanol (ABE) fermentation, the effect of glycerol on NADH/NADPH generation and ABE production by furfural (4, 5, and 6 g/L)-challenged cultures was investigated in this study.

Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 to elucidate role of furfural stress during acetone butanol ethanol fermentation.

Background: Furfural is the prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulose biomass to monomeric sugars, but the response of acetone butanol ethanol (ABE) producing Clostridium beijerinckii NCIMB 8052 to this compound at the molecular level is unknown. To discern the effect of furfural on C. beijerinckii and to gain insight into molecular mechanisms of action and detoxification, physiological changes of furfural-stressed cultures during acetone butanol ethanol (ABE) fermentation were studied, and differentially expressed genes were profiled by genome-wide transcriptional analysis.

Use of proteomic analysis to elucidate the role of calcium in acetone-butanol-ethanol fermentation by Clostridium beijerinckii NCIMB 8052.

Calcium carbonate increases growth, substrate utilization, and acetone-butanol-ethanol (ABE) fermentation by Clostridium beijerinckii NCIMB 8052. Toward an understanding of the basis for these pleiotropic effects, we profiled changes in the C. beijerinckii NCIMB 8052 proteome that occur in response to the addition of CaCO(3).

Microbial production of a biofuel (acetone-butanol-ethanol) in a continuous bioreactor: impact of bleed and simultaneous product removal.

Acetone butanol ethanol (ABE) was produced in an integrated continuous one-stage fermentation and gas stripping product recovery system using Clostridium beijerinckii BA101 and fermentation gases (CO(2) and H(2)). In this system, the bioreactor was fed with a concentrated sugar solution (250-500 g L(-1) glucose). The bioreactor was bled semi-continuously to avoid accumulation of inhibitory chemicals and products.

Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation.

The ability of fermenting microorganisms to tolerate furan aldehyde inhibitors (furfural and 5-hydroxymethyl furfural (HMF)) will enhance efficient bioconversion of lignocellulosic biomass hydrolysates to fuels and chemicals. The effect of furfural and HMF on butanol production by Clostridium acetobutylicum 824 was investigated. Whereas specific growth rates, μ, of C.

Acetone production in solventogenic Clostridium species: new insights from non-enzymatic decarboxylation of acetoacetate.

Development of a butanologenic strain with high selectivity for butanol production is often proposed as a possible route for improving the economics of biobutanol production by solventogenic Clostridium species. The acetoacetate decarboxylase (aadc) gene encoding acetoacetate decarboxylase (AADC), which catalyzes the decarboxylation of acetoacetate into acetone and CO(2), was successfully disrupted by homologous recombination in solventogenic Clostridium beijerinckii NCIMB 8052 to generate an aadc ( - ) mutant. Our fermentation studies revealed that this mutant produces a maximum acetone concentration of 3 g/L (in P2 medium), a value comparable to that produced by wild-type C.

Bioproduction of butanol in bioreactors: new insights from simultaneous in situ butanol recovery to eliminate product toxicity.

Simultaneous acetone butanol ethanol (ABE) fermentation by Clostridium beijerinckii P260 and in situ product recovery was investigated using a vacuum process operated in two modes: continuous and intermittent. Integrated batch fermentations and ABE recovery were conducted at 37 °C using a 14-L bioreactor (7.0 L fermentation volume) containing initial substrate (glucose) concentration of 60 g/L.

Bioproduction of butanol from biomass: from genes to bioreactors.

Butanol is produced chemically using either the oxo process starting from propylene (with H2 and CO over a rhodium catalyst) or the aldol process starting from acetaldehyde. The key problems associated with the bioproduction of butanol are the cost of substrate and butanol toxicity/inhibition of the fermenting microorganisms, resulting in a low butanol titer in the fermentation broth. Recent interest in the production of biobutanol from biomass has led to the re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture and for manipulating the culture to achieve better product specificity and yield.