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Hans P Blaschek - One of the best experts on this subject based on the ideXlab platform.

  • a comparative phenotypic and genomic analysis of Clostridium beijerinckii mutant with enhanced solvent production
    Journal of Biotechnology, 2021
    Co-Authors: Seung Oh Seo, Yong Su Jin, Hans P Blaschek
    Abstract:

    The acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia has a long history of industrial butanol production. The Clostridium beijerinckii mutant BA101 has been widely studied for ABE fermentation owing to its enhanced butanol production capacity. Here, we characterized the BA101 mutant under controlled environmental conditions in parallel with the parental strain C. beijerinckii NCIMB 8052. To investigate the correlation between phenotype and genotype, we carried out the genome sequencing of BA101. Through comparative genomic analysis, several mutations in the genes encoding transcriptional regulator, sensor kinase, and phosphatase were identified in the BA101 genome as well as other sibling mutants. Among them, the SNP in the Cbei_3078 gene encoding PAS/PAC sensor hybrid histidine kinase was unique to the BA101 strain. The identified mutations relevant to the observed physiological behaviors of BA101 could be potential genetic targets for rational engineering of solventogenic clostridia toward desired phenotypes.

  • bacterial genome editing with crispr cas9 taking Clostridium beijerinckii as an example
    Methods of Molecular Biology, 2018
    Co-Authors: Zhong Tian Zhang, Seung Oh Seo, Yong Su Jin, Hans P Blaschek, Pablo Jimenezbonilla, Yi Wang
    Abstract:

    CRISPR-Cas9 has been explored as a transformative genome engineering tool for many eukaryotic organisms. However, its utilization in bacteria remains limited and ineffective. This chapter, taking Clostridium beijerinckii as an example, describes the use of Streptococcus pyogenes CRISPR-Cas9 system guided by the single chimeric guide RNA (gRNA) for diverse genome-editing purposes, including chromosomal gene deletion, integration, single nucleotide modification, as well as "clean" mutant selection. The general principle is to use CRISPR-Cas9 as an efficient selection tool for the edited mutant (whose CRISPR-Cas9 target site has been disrupted through a homologous recombination event and thus can survive selection) against? the wild type background cells. This protocol is broadly applicable to other microorganisms for genome-editing purposes.

  • genomic transcriptional and phenotypic analysis of the glucose derepressed Clostridium beijerinckii mutant exhibiting acid crash phenotype
    Biotechnology Journal, 2017
    Co-Authors: Seung Oh Seo, Yi Wang, Yong Su Jin, Hans P Blaschek, Holger Janssen, Andrew T Magis, Nathan D Price
    Abstract:

    Clostridium beijerinckii is a predominant solventogenic bacterium that is used for the ABE fermentation. Various C. beijerinckii mutants are constructed for desirable phenotypes. The C. beijerinckii mutant BA105 harboring a glucose derepression phenotype was previously isolated and demonstrated the enhanced amylolytic activity in the presence of glucose. Despite its potential use, BA105 is not further characterized and utilized. Therefore, the authors investigate fermentation phenotypes of BA105 in this study. Under the typical batch fermentation conditions, BA105 consistently exhibits acid crash phenotype resulting in limited glucose uptake and cell growth. However, when the culture pH is maintained above 5.5, BA105 exhibits the increased glucose uptake and butanol production than did the wild-type. To further analyze BA105, the authors perform genome sequencing and RNA sequencing. Genome analysis identifies two SNPs unique to BA105, in the upstream region of AbrB regulator (Cbei_4885) and the ROK family glucokinase (Cbei_4895) which are involved in catabolite repression and regulation of sugar metabolism. Transcriptional analysis of BA105 reveals significant differential expression of the genes associated with the PTS sugar transport system and acid production. This study improves understanding of the acid crash phenomenon and provides the genetic basis underlying the catabolite derepression phenotype of C. beijericnkii.

  • markerless chromosomal gene deletion in Clostridium beijerinckii using crispr cas9 system
    Journal of Biotechnology, 2015
    Co-Authors: Yi Wang, Zhong Tian Zhang, Seung Oh Seo, Ki Joong Choi, Yong Su Jin, Hans P Blaschek
    Abstract:

    The anaerobic spore-forming, gram-positive, solventogenic clostridia are notorious for being difficult to genetically engineer. Based on CRISPR/Cas9 assisted homologous recombination, we demonstrated that clean markerless gene deletion from the chromosome can be easily achieved with a high efficiency through a single-step transformation in Clostridium beijerinckii NCIMB 8052, one of the most prominent strains for acetone, butanol and ethanol (ABE) production. This highly efficient genome engineering system can be further explored for multiplex genome engineering purposes. The protocols and principles developed in this study provided valuable references for genome engineering in other microorganisms lacking developed genetic engineering tools.

  • optimization of butanol production from tropical maize stalk juice by fermentation with Clostridium beijerinckii ncimb 8052
    Bioresource Technology, 2011
    Co-Authors: Yi Wang, Hans P Blaschek
    Abstract:

    Abstract Mixed sugars from tropical maize stalk juice were used to carry out butanol fermentation with Clostridium beijerinckii NCIMB 8052. Batch experiments employing central composite design (CCD) and response surface methodology (RSM) optimization were performed to evaluate effects of three factors, i.e. pH, initial total sugar concentration, and agitation rate on butanol production. Optimum conditions of pH 6.7, sugar concentration 42.2 g/L and agitation rate 48 rpm were predicted, under which a maximum butanol yield of 0.27 g/g-sugar was estimated. Further experiments demonstrated that higher agitation facilitated acetone production, leading to lower butanol selectivity in total acetone–butanol–ethanol (ABE). While glucose and fructose are more preferable by C. beijerinckii, sucrose can also be easily degraded by the microorganism. This study indicated that RSM is a useful approach for optimizing operational conditions for butanol production, and demonstrated that tropical maize, with high yield of biomass and stalk sugars, is a promising biofuel crop.

Hanspeter M Blaschek - One of the best experts on this subject based on the ideXlab platform.

  • gene transcription repression in Clostridium beijerinckii using crispr dcas9
    Biotechnology and Bioengineering, 2016
    Co-Authors: Yi Wang, Zhong Tian Zhang, Seung Oh Seo, Yong Su Jin, Patrick Lynn, Hanspeter M Blaschek
    Abstract:

    CRISPR-Cas9 has been explored as a powerful tool for genome engineering for many organisms. Meanwhile, dCas9 which lacks endonuclease activity but can still bind to target loci has been engineered for efficient gene transcription repression. Clostridium beijerinckii, an industrially significant species capable of biosolvent production, is generally difficult to metabolically engineer. Recently, we reported our work in developing customized CRISPR-Cas9 system for genome engineering in C. beijerinckii. However, in many cases, gene expression repression (rather than actual DNA mutation) is more desirable for various biotechnological applications. Here, we further demonstrated gene transcription repression in C. beijerinckii using CRISPR-dCas9. A small RNA promoter was employed to drive the expression of the single chimeric guide RNA targeting on the promoter region of amylase gene, while a constitutive thiolase promoter was used to drive Streptococcus pyogenes dCas9 expression. The growth assay on starch agar plates showed qualitatively significant repression of amylase activity in C. beijerinckii transformant with CRISPR-dCas9 compared to the control strain. Further amylase activity quantification demonstrated consistent repression (65-97% through the fermentation process) on the activity in the transformant with CRISPR-dCas9 versus in the control. Our results provided essential references for engineering CRISPR-dCas9 as an effective tool for tunable gene transcription repression in diverse microorganisms. Biotechnol. Bioeng. 2016;113: 2739-2743. © 2016 Wiley Periodicals, Inc.

  • Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable “Clean” Mutant Selection in Clostridium beijerinckii as an Example
    ACS synthetic biology, 2016
    Co-Authors: Yi Wang, Zhong Tian Zhang, Seung Oh Seo, Yong Su Jin, Patrick Lynn, Hanspeter M Blaschek
    Abstract:

    CRISPR-Cas9 has been demonstrated as a transformative genome engineering tool for many eukaryotic organisms; however, its utilization in bacteria remains limited and ineffective. Here we explored Streptococcus pyogenes CRISPR-Cas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer) as a representative attempt to explore CRISPR-Cas9 for genome editing in microorganisms that previously lacked sufficient genetic tools. By combining inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved gene deletion and integration with high efficiency in single steps. We further achieved single nucleotide modification by applying innovative two-step approaches, which do not rely on availability of Protospacer Adjacent Motif sequences. Severe vector integration events were observed during the genome engineering process, which is likely difficult to avoid but has never been reported by other researchers for th...

  • evidence for the presence of an alternative glucose transport system in Clostridium beijerinckii ncimb 8052 and the solvent hyperproducing mutant ba101
    Applied and Environmental Microbiology, 2005
    Co-Authors: Ji Eun Lee, Wilfrid J Mitchell, Martin Tangney, Hanspeter M Blaschek
    Abstract:

    The effects of substrate analogs and energy inhibitors on glucose uptake and phosphorylation by Clostridium beijerinckii provide evidence for the operation of two uptake systems: a previously characterized phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a non-PTS system probably energized by the transmembrane proton gradient. In both wild-type C. beijerinckii NCIMB 8052 and the butanol-hyperproducing mutant BA101, PTS activity declined at the end of exponential growth, while glucokinase activity increased in the later stages of fermentation. The non-PTS uptake system, together with enhanced glucokinase activity, may provide an explanation for the ability of the mutant to utilize glucose more effectively during fermentation despite the fact that it is partially defective in PTS activity.

  • continuous butanol fermentation and feed starch retrogradation butanol fermentation sustainability using Clostridium beijerinckii ba101
    Journal of Biotechnology, 2005
    Co-Authors: Thaddeus Chukwuemeka Ezeji, Nasib Qureshi, Hanspeter M Blaschek
    Abstract:

    Use of starch solution as feed for butanol bioconversion processes employing Clostridium beijerinckii BA101 may have added economic advantage over the use of glucose. Acetone butanol ethanol (ABE) was produced from 30 gL(-1) starch solution using a continuous process. The bioreactor was fed at a dilution rate of 0.02 h(-1) and starch solution/feed volume (3 L) was replaced every 72 h. The continuous reactor fed with cornstarch solution (feed temperature 19 degrees C) produced approximately 6.0 gL(-1) total ABE. Increasing the feed storage temperature to 37 degrees C improved ABE production to 7.2 gL(-1) suggesting that retrogradation was occurring more rapidly at 19 degrees C. In both these cases the fermentation drifted toward acid production after approximately 260 h, consistent with the retrogradation of starch overtime. The use of soluble starch, which is less prone to retrogradation, resulted in the production of 9.9 gL(-1) ABE at 37 degrees C feed storage temperature, as compared to 7.2 gL(-1) ABE when cornstarch was used. It should be noted that gelatinized starch retrogradation takes place after sterilization and prior to use of the feed medium, and does not occur during long-term storage of the raw corn material in the months leading up to processing. The degree of hydrolysis of gelatinized starch decreased from 68.8 to 56.2% in 3 days when stored at 37 degrees C. Soluble starch which does not retrograde demonstrated no change in the degree of hydrolysis.

  • recent advances in abe fermentation hyper butanol producing Clostridium beijerinckii ba101
    Journal of Industrial Microbiology & Biotechnology, 2001
    Co-Authors: N Qureshi, Hanspeter M Blaschek
    Abstract:

    This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone–butanol–ethanol, ABE) at elevated levels. This organism expresses high levels of amylases when grown on starch. C. beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27–29 g l−1. C. beijerinckii BA101 has been characterized for both substrate and butanol inhibition. Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g l−1. The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors. Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C. beijerinckii BA101, and solvent production as high as 165 g l−1 has been achieved. Immobilization of C. beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g l−1 h−1. Recent economic studies employing C. beijerinckii BA101 suggested that butanol can be produced at US$0.20–0.25 lb−1 by employing batch fermentation and distillative recovery. Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices. Journal of Industrial Microbiology & Biotechnology (2001) 27, 287–291.

Min Jiang - One of the best experts on this subject based on the ideXlab platform.

  • the draft genome sequence of Clostridium beijerinckii njp7 a unique bacterium capable of producing isopropanol butanol from hemicellulose through consolidated bioprocessing
    Current Microbiology, 2018
    Co-Authors: Yujia Jiang, Wenming Zhang, Weiliang Dong, Min Jiang, Tianpeng Chen, Min Zhang, Fengxue Xin
    Abstract:

    A wild type solventogenic Clostridium beijerinckii NJP7 capable of converting polysaccharides, such as hemicellulose, into butanol and isopropanol via a unique acetone-isopropanol-butanol (AIB) pathway was isolated and characterized. This represents the first wild type isopropanol-butanol generating bacterium which could achieve butanol production directly from lignocellulose through consolidated bioprocessing (CBP). Strain NJP7 was isolated from decomposite soil from Laoshan Nature Park, China, and its genome shows 98.6% identical to 89.5% of the Clostridium diolis submitted genome sequence. The assembled draft genome contains 5.76 Mb and 5101 predicted encoding proteins with a GC content of 29.73%. Among these annotated proteins, hemicellulase and the secondary alcohol dehydrogenase play key roles in achievement of AIB production from hemicellulose through CBP.

  • effects of ph and ferrous iron on the coproduction of butanol and hydrogen by Clostridium beijerinckii ib4
    International Journal of Hydrogen Energy, 2017
    Co-Authors: Chao Wang, Min Jiang, Xiangping Kong, Pan Chen, Fengxue Xing
    Abstract:

    Abstract Butanol and hydrogen gas are main products in butanol production by solventogenic Clostridia. Effects of pH and ferrous iron on hydrogen and butanol production by Clostridium beijerinckii IB4 were investigated in this work. With the increasing of the pH value, the hydrogen yields increased during acidogenic phase and decreased during solventogenic phase. Compared with the process without pH control, butanol and hydrogen increased by 15.43% and 11.77%, respectively, when pH was controlled at 5.2. Under the control of pH at 5.2 and supplementation of 250 mg/L of FeSO 4 ·7H 2 O, the maximum hydrogen quantity of 8.24 L/L was obtained, and hydrogen productivity achieved 187 mL/L/h with yield of 145 mL/g glucose, which increased by 65%, 46% and 37%, respectively. However, butanol yield was reduced slightly by 4.8%. The reducing power was enhanced in solventogenic phase with supplementation of 250 mg/L FeSO 4 ·7H 2 O, and the energy content of fuel produced in this process also remained stable. These results indicated that hydrogen production was enhanced by pH control and ferrous iron regulation, and butanol production performance was also maintained, which was favorable for coproduction of butanol and hydrogen in ABE fermentation.

  • efficient acetone butanol ethanol abe production by a butanol tolerant mutant of Clostridium beijerinckii in a fermentation pervaporation coupled process
    Biochemical Engineering Journal, 2016
    Co-Authors: Xiangping Kong, Jie Zhao, Ce Wei, Wanqin Jin, Min Jiang
    Abstract:

    Abstract Butanol inhibition is one of the major obstacles limiting the economic viability of acetone–butanol–ethanol (ABE) fermentation. In this study, a butanol-tolerant mutant ( Clostridium beijerinckii BT14) was generated by atmospheric and room temperature plasmas (ARTP). This mutant showed significant advantage over its parent strain in terms of butanol tolerance. Compared to its parent strain, batch fermentation by this mutant produced 25% higher butanol and 33% higher ABE solvents due to its efficient generation of intracellular NADH and high NADH-dependent butanol dehydrogenase activity. Furthermore, C. beijerinckii BT14 was applied to fed-batch fermentation with pervaporation (PV). As a results, C. beijerinckii BT14 grew to a high cell density and this process generated highly concentrated ABE solution with a high solvent productivity of 0.98 g/(L h) and glucose consumption rate of 2.64 g/(L h). Thus, this work provides an appropriate strategy to develop an efficient process for ABE production in the PV coupled fermentation.

  • enhanced acetone butanol ethanol production by Clostridium beijerinckii ib4 using ph control strategy
    Process Biochemistry, 2014
    Co-Authors: Min Jiang, Jianan Chen, Xiangping Kong, Junli Liu, Chunyan Yin, Wufang Chen, Pan Chen
    Abstract:

    Abstract PH is an essential factor for acetone/butanol/ethanol (ABE) production using Clostridium spp. In this study, batch fermentations by Clostridium beijerinckii IB4 at various pH values ranging from 4.9 to 6.0 were examined. At pH 5.5, the ABE production was dominant and maximum ABE concentration of 24.6 g/L (15.7 g/L of butanol, 8.63 g/L of acetone and 0.32 g/L of ethanol) was obtained with the consumption of 60 g/L of glucose within 36 h. However, in the control (without pH control), an ABE concentration of 14.1 g/L (11.0 g/L of butanol, 3.01 g/L of acetone and 0.16 g/L of ethanol) was achieved with the consumption of 41 g/L of glucose within 40 h. A considerable improvement in the productivity of up to 93.8% was recorded at controlled pH in comparison to the process without pH control. To better understand the influence of pH on butanol production, the reducing power capability and NADH-dependent butanol dehydrogenase activity were investigated, both of which were significantly improved at pH 5.5. Thus, the pH control technique is a convenient and efficient method for high-intensity ABE production.

  • butanol production from hemicellulosic hydrolysate of corn fiber by a Clostridium beijerinckii mutant with high inhibitor tolerance
    Bioresource Technology, 2013
    Co-Authors: Ting Guo, Min Jiang, Dafeng Liang, Dawei Zhu, Ping Wei, Pingkai Ouyang
    Abstract:

    A Clostridium beijerinckii mutant RT66 with considerable inhibitor-tolerance obtained by continuous culture was used for butanol production from non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). In fed-batch fermentation, 1.8 L of diluted SAHHC containing 10 g/L of reducing sugar was provided during the acidogenic phase and 0.2 L of concentrated SAHHC containing 300 g/L of reducing sugar was provided during the solventogenic phase. The mutant produced a total amount of solvents of 12.9 g/L, which consisted of 3.1 g/L of acetone, 9.3 g/L of butanol and 0.5 g/L of ethanol. A solvent yield of 0.35 g/g sugar and a productivity of 0.18 g/L h in 72 h were achieved. The remarkable inhibitor-tolerance of C. beijerinckii RT66 demonstrates that this may be an excellent strain for butanol production from ligocellulosic materials.

Eberhard Morgenroth - One of the best experts on this subject based on the ideXlab platform.

  • Lignocellulosic hydrolysates and extracellular electron shuttles for H2 production using co-culture fermentation with Clostridium beijerinckii and Geobacter metallireducens.
    Bioresource Technology, 2013
    Co-Authors: Xinyu Zhang, Kevin T. Finneran, Xiaofeng Ye, Julie L. Zilles, Eberhard Morgenroth
    Abstract:

    A co-culture of Clostridium beijerinckii and Geobacter metallireducens with AH2QDS produced hydrogen from lignocellulosic hydrolysates (biomass of Miscanthus prepared by hydrothermal treatment with dilute acids). This co-culture system enhanced hydrogen production from lignocellulosic hydrolysates by improving substrate utilization and diminishing acetate accumulation, despite the presence of fermentation inhibitors in the hydrolysates. The improvements were greater for xylose-rich hydrolysates. The increase in maximum cumulative hydrogen production for hydrolysates with glucose:xylose mass ratios of 1:0.2, 1:1 and 1:10 g/g was 0%, 22% and 11%, respectively. Alternative extracellular electron shuttles (EES), including indigo dye, juglone, lawsone, fulvic acids and humic acids, were able to substitute for AH2QDS, improving hydrogen production in the co-culture system using xylose as model substrate. Increased utilization of xylose-rich hydrolysates and substitution of alternative EES make the co-culture with EES system a more attractive strategy for industrial biohydrogen production.

  • interactions between Clostridium beijerinckii and geobacter metallireducens in co culture fermentation with anthrahydroquinone 2 6 disulfonate ah2qds for enhanced biohydrogen production from xylose
    Biotechnology and Bioengineering, 2013
    Co-Authors: Xinyu Zhang, Eberhard Morgenroth, Kevin T. Finneran, Julie L. Zilles
    Abstract:

    To enhance biohydrogen production, Clostridium beijerinckii was co-cultured with Geobacter metallireducens in the presence of the reduced extracellular electron shuttle anthrahydroquinone-2, 6-disulfonate (AH(2)QDS). In the co-culture system, increases of up to 52.3% for maximum cumulative hydrogen production, 38.4% for specific hydrogen production rate, 15.4% for substrate utilization rate, 39.0% for substrate utilization extent, and 34.8% for hydrogen molar yield in co-culture fermentation were observed compared to a pure culture of C. beijerinckii without AH(2)QDS. G. metallireducens grew in the co-culture system, resulting in a decrease in acetate concentration under co-culture conditions and a presumed regeneration of AH(2)QDS from AQDS. These co-culture results demonstrate metabolic crosstalk between the fermentative bacterium C. beijerinckii and the respiratory bacterium G. metallireducens and suggest a strategy for industrial biohydrogen production.

  • Anthrahydroquinone-2,6,-disulfonate (AH2QDS) increases hydrogen molar yield and xylose utilization in growing cultures of Clostridium beijerinckii
    Applied microbiology and biotechnology, 2011
    Co-Authors: Eberhard Morgenroth, Xinyu Zhang, Kevin T. Finneran
    Abstract:

    H2 production and xylose utilization were investigated using the fermentative culture Clostridium beijerinckii NCIMB 8052. Adding anthrahydroquinone-2,6-disulfonate (AH2QDS) increased the extent of xylose utilization by 56% and hydrogen molar yield by 24–37%. Enhanced hydrogen molar yield correlated with increased xylose utilization and increases in the acetate/butyrate product ratio. An electron balance indicated that AH2QDS shifted the electrons from the butyric acid pathway (NADH-dependent pathway) to the acetic acid pathway (non-NADH-dependent pathway), putatively creating a surplus of reducing equivalents that were then available for hydrogen production. These data demonstrate that hydrogen yield and xylose utilization can be manipulated by amending redox active molecules into growing cultures. This will impact biohydrogen/biofuel production by allowing physiological manipulations of growing cells for increased (or decreased) output of selected metabolites using amendments that are not consumed during the reactions. Although the current yield increases are small, they suggest a target for cellular alterations. In addition, increased xylose utilization will be critical to the fermentation of pretreated lignocellulosic feedstocks, which may have higher xylose content.

Thaddeus Chukwuemeka Ezeji - One of the best experts on this subject based on the ideXlab platform.

  • Metabolic engineering of Clostridium beijerinckii to improve glycerol metabolism and furfural tolerance
    Biotechnology for Biofuels, 2019
    Co-Authors: Chidozie Victor Agu, Victor Ujor, Thaddeus Chukwuemeka Ezeji
    Abstract:

    Background Inefficient utilization of glycerol by Clostridium beijerinckii ( Cb ) 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. To address this problem, we employed a metabolic engineering strategy to enhance glycerol utilization by Cb . Results By overexpressing two glycerol dehydrogenase (Gldh) genes ( dhaD1 and gldA1 ) from the glycerol hyper-utilizing Clostridium pasteurianum ( Cp ) as a fused protein in Cb , we achieved approximately 43% increase in glycerol consumption, when compared to the plasmid control. Further, Cb _ dhaD1  +  gldA1 achieved a 59% increase in growth, while butanol and acetone–butanol–ethanol (ABE) concentrations and productivities increased 14.0%, 17.3%, and 55.6%, respectively, relative to the control. Co-expression of dhaD1  +  gldA1 and gldA1  + dihydroxyacetone kinase ( dhaK ) resulted in significant payoffs in cell growth and ABE production compared to expression of one Gldh. In the presence of 4–6 g/L furfural, increased glycerol consumption by the dhaD1  +  gldA1 strain increased cell growth (> 50%), the rate of furfural detoxification (up to 68%), and ABE production (up to 40%), relative to the plasmid control. Likewise, over-expression of [( dhaD1  +  gldA1 ) dhaK ] improved butanol and ABE production by 70% and 50%, respectively, in the presence of 5 and 6 g/L furfural relative to the plasmid control. Conclusions Overexpression of Cp gldhs and dhaK in Cb significantly enhanced glycerol utilization, ABE production, and furfural tolerance by Cb . Future research will address the inability of recombinant Cb to metabolize glycerol as a sole substrate.

  • metabolic engineering of Clostridium beijerinckii to improve glycerol metabolism and furfural tolerance
    Biotechnology for Biofuels, 2019
    Co-Authors: Chidozie Victor Agu, Victor Ujor, Thaddeus Chukwuemeka Ezeji
    Abstract:

    Inefficient utilization of glycerol by Clostridium beijerinckii (Cb) 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. To address this problem, we employed a metabolic engineering strategy to enhance glycerol utilization by Cb. By overexpressing two glycerol dehydrogenase (Gldh) genes (dhaD1 and gldA1) from the glycerol hyper-utilizing Clostridium pasteurianum (Cp) as a fused protein in Cb, we achieved approximately 43% increase in glycerol consumption, when compared to the plasmid control. Further, Cb_dhaD1 + gldA1 achieved a 59% increase in growth, while butanol and acetone–butanol–ethanol (ABE) concentrations and productivities increased 14.0%, 17.3%, and 55.6%, respectively, relative to the control. Co-expression of dhaD1 + gldA1 and gldA1 + dihydroxyacetone kinase (dhaK) resulted in significant payoffs in cell growth and ABE production compared to expression of one Gldh. In the presence of 4–6 g/L furfural, increased glycerol consumption by the dhaD1 + gldA1 strain increased cell growth (> 50%), the rate of furfural detoxification (up to 68%), and ABE production (up to 40%), relative to the plasmid control. Likewise, over-expression of [(dhaD1 + gldA1) dhaK] improved butanol and ABE production by 70% and 50%, respectively, in the presence of 5 and 6 g/L furfural relative to the plasmid control. Overexpression of Cp gldhs and dhaK in Cb significantly enhanced glycerol utilization, ABE production, and furfural tolerance by Cb. Future research will address the inability of recombinant Cb to metabolize glycerol as a sole substrate.

  • Use of Cupriavidus basilensis-aided bioabatement to enhance fermentation of acid-pretreated biomass hydrolysates by Clostridium beijerinckii
    Journal of Industrial Microbiology & Biotechnology, 2016
    Co-Authors: Victor Ujor, Venkat Gopalan, Thaddeus Chukwuemeka Ezeji
    Abstract:

    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. First, we demonstrate that growth of C. basilensis is possible on furfural, 5-hydroxymethyfurfural, cinnamaldehyde, 4-hydroxybenzaldehyde, syringaldehyde, vanillin, and ferulic, p -coumaric, syringic and vanillic acid, as sole carbon sources. Second, we report that C. basilensis detoxified and metabolized ~98 % LDMICs present in dilute acid-pretreated MG hydrolysates. Last, this bioabatement resulted in significant payoffs during acetone-butanol-ethanol (ABE) fermentation by C. beijerinckii : 70, 50 and 73 % improvement in ABE concentration, yield and productivity, respectively. Together, our results show that biological detoxification of acid-pretreated MG hydrolysates prior to fermentation is feasible and beneficial.

  • identification purification and characterization of furfural transforming enzymes from Clostridium beijerinckii ncimb 8052
    Anaerobe, 2015
    Co-Authors: Yan Zhang, Victor Ujor, Macdonald Wick, Thaddeus Chukwuemeka Ezeji
    Abstract:

    Abstract 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. To delineate the mechanism by which C. beijerinckii detoxifies furfural and HMF, an aldo/keto reductase (AKR) and a short-chain dehydrogenase/reductase (SDR) found to be over-expressed in furfural-challenged cultures of C. beijerinckii were cloned and over-expressed in Escherichia coli Rosetta-gami™ B(DE3)pLysS, and purified by histidine tag-assisted immobilized metal affinity chromatography. Protein gel analysis showed that the molecular weights of purified AKR and SDR are close to the predicted values of 37 kDa and 27 kDa, respectively. While AKR has apparent Km and Vmax values of 32.4 mM and 254.2 mM s−1 respectively, using furfural as substrate, SDR showed lower Km (26.4 mM) and Vmax (22.6 mM s−1) values on the same substrate. However, AKR showed 7.1-fold higher specific activity on furfural than SDR. Further, both AKR and SDR were found to be active on HMF, benzaldehyde, and butyraldehyde. Both enzymes require NADPH as a cofactor for aldehydes reduction. Based on these results, it is proposed that AKR and SDR are involved in the biotransformation of furfural and HMF by C. beijerinckii.

  • process integration for simultaneous saccharification fermentation and recovery ssfr production of butanol from corn stover using Clostridium beijerinckii p260
    Bioresource Technology, 2014
    Co-Authors: Nasib Qureshi, Thaddeus Chukwuemeka Ezeji, Badal C. Saha, Vijay Singh, Michael A. Cotta
    Abstract:

    Abstract A simultaneous saccharification, fermentation, and recovery (SSFR) process was developed for the production of acetone–butanol–ethanol (AB or ABE), of which butanol is the main product, from corn stover employing Clostridium beijerinckii P260. Of the 86 g L−1 corn stover provided, over 97% of the sugars were released during hydrolysis and these were fermented completely with an ABE productivity of 0.34 g L−1 h−1 and yield of 0.39. This productivity is higher than 0.31 g L−1 h−1 when using glucose as a substrate demonstrating that AB could be produced efficiently from lignocellulosic biomass. Acetic acid that was released from the biomass during pretreatment and hydrolysis was also used by the culture to produce AB. An average rate of generation of sugars during corn stover hydrolysis was 0.98 g L−1 h−1. In this system AB was recovered using vacuum, and as a result of this (simultaneous product recovery), 100% sugars were used by the culture.