scholarly journals Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

2011 ◽  
Vol 77 (18) ◽  
pp. 6470-6475 ◽  
Author(s):  
Shunichi Nakayama ◽  
Keiji Kiyoshi ◽  
Toshimori Kadokura ◽  
Atsumi Nakazato
Keyword(s):  
Clostridium Acetobutylicum ◽  
Clostridium Thermocellum ◽  
Clostridium Beijerinckii ◽  
Crystalline Cellulose ◽  
Butanol Production ◽  
Content Type ◽  
Coculture System

ABSTRACTWe investigated butanol production from crystalline cellulose by cocultured cellulolyticClostridium thermocellumand the butanol-producing strain,Clostridium saccharoperbutylacetonicum(strain N1-4). Butanol was produced from Avicel cellulose after it was incubated withC. thermocellumfor at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by coculturedClostridium acetobutylicumandClostridium beijerinckiithan by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.

scholarly journals Production of Heterologous and Chimeric Scaffoldins by Clostridium acetobutylicum ATCC 824

2004 ◽  
Vol 186 (1) ◽  
pp. 253-257 ◽  
Author(s):  
S. Perret ◽  
L. Casalot ◽  
H.-P. Fierobe ◽  
C. Tardif ◽  
F. Sabathe ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Clostridium Thermocellum ◽  
Crystalline Cellulose ◽  
Truncated Form ◽  
Clostridium Cellulolyticum ◽  
Clostridium Acetobutylicum Atcc 824

ABSTRACT Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C. acetobutylicum. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.

scholarly journals Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation

2011 ◽  
Vol 78 (5) ◽  
pp. 1416-1423 ◽  
Author(s):  
Joungmin Lee ◽  
Yu-Sin Jang ◽  
Sung Jun Choi ◽  
Jung Ae Im ◽  
Hyohak Song ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Clostridium Acetobutylicum ◽  
Secondary Alcohol ◽  
Clostridium Beijerinckii ◽  
Fuel Additive ◽  
Gas Stripping ◽  
Alcohol Fermentation ◽  
Content Type ◽  
Formation Pathway ◽  
Total Alcohol

ABSTRACTClostridium acetobutylicumnaturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineeredC. acetobutylicumcan be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene fromClostridium beijerinckiiNRRL B-593 (i.e.,adhB-593) inC. acetobutylicumATCC 824. To increase the total alcohol titer, a synthetic acetone operon (actoperon;adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in thebukgene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged byin situremoval of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.

scholarly journals d-2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum

2011 ◽  
Vol 77 (8) ◽  
pp. 2582-2588 ◽  
Author(s):  
Marco A. J. Siemerink ◽  
Wouter Kuit ◽  
Ana M. López Contreras ◽  
Gerrit Eggink ◽  
John van der Oost ◽  
...  
Keyword(s):  
Experimental Data ◽  
Heterologous Expression ◽  
Metabolic Network ◽  
Clostridium Acetobutylicum ◽  
Clostridium Beijerinckii ◽  
Racemic Mixture ◽  
Gene Encoding ◽  
Content Type ◽  
Batch Cultures

ABSTRACTAcetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions,Clostridium acetobutylicumATCC 824 (and strains derived from it) generates bothd- andl-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR fromClostridium beijerinckiiNCIMB 8052 was functionally expressed inC. acetobutylicumunder the control of two strong promoters, the constitutivethlpromoter and the late exponentialadcpromoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mMd-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of bothd-2,3-butanediol andmeso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain ofC. acetobutylicum.

scholarly journals Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose, as Revealed byIn VitroReconstitution of the Clostridium thermocellum Cellulosome

2015 ◽  
Vol 81 (14) ◽  
pp. 4756-4766 ◽  
Author(s):  
Katsuaki Hirano ◽  
Satoshi Nihei ◽  
Hiroki Hasegawa ◽  
Mitsuru Haruki ◽  
Nobutaka Hirano
Keyword(s):  
Clostridium Thermocellum ◽  
Full Length ◽  
Size Exclusion ◽  
Molar Ratio ◽  
Crystalline Cellulose ◽  
Content Type ◽  
Crystalline Substrate ◽  
Exclusion Chromatography ◽  
Enzyme Molecules ◽  
Species Specific

ABSTRACTThe cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of theClostridium thermocellumcellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.

scholarly journals Enhanced Biobutanol Production in Batch Simultaneous Saccharification and Fermentation From Agriculture Residues Using Novel Clostridial Fusants

2021 ◽  
Author(s):  
Sarkar Begum
Keyword(s):  
Enzymatic Activity ◽  
Wheat Straw ◽  
Clostridium Acetobutylicum ◽  
Clostridium Thermocellum ◽  
Simultaneous Saccharification And Fermentation ◽  
Incubation Temperature ◽  
Clostridium Beijerinckii ◽  
Raw Material ◽  
Production Cycle ◽  
Simultaneous Saccharification

A novel approach was introduced for the enhancement of biobutanol production in ABE fermenetation. Thermostable Clostridia species were developed through protoplast fusion between mesophilic and thermophilic Clostridial species. Two parental strains of Clostridia species, Clostridium acetobutylicum (ATCC 4259) and Clostridium beijerinckii (ATCC BA101), along with three fused strains, Clostridium acetobutylicum Clostridium thermocellum (CaCt), Clostridium beijerinckii Clostridium thermocellum (CbCt) and Clostridium acetobutylicum Clostridium beijerinckii (CaCb), were examined for biobutanol production using wheat straw as a feed stock in a batch process of simultaneous saccharification and fermentation (SSF). The objective of the study was to use the thermotolerant fused strains to enhance enzymatic activity during the SSF by raising the incubation temperature and thereby eventually increasing biobutanol production. Economic and residence time analysis of SSF found that addition of cellulase increases the cost of fermentation and prolongs biobutanol production cycle. However, results from the current study indicated that the fused strains, at the higher temperature, were able to produce the required enzymes for the saccharification of wheat straw (i.e., endoglucanase, exoglucanase, and b- glucosidase). This will have a major impact on eliminating costs associated with adding enzymes as raw material to the saccharification process. Fused strain CbCt achieved the highest biobutanol production of up to 14.13 g/L (i.e., a yield of 0.29) was reached at 45°C with total sugar consumption of 82%. Enzymatic activity of CbCt was found to be 61.67 FPU (Filter Paper Unit) which was the highest in comparison with other fused strains evaluated in this study. These results indicate a breakthrough for the technological and economical obstacles associated with industrialization of the SSF process.

scholarly journals Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Changhee Cho ◽  
Seungpyo Hong ◽  
Hyeon Gi Moon ◽  
Yu-Sin Jang ◽  
Dongsup Kim ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Protein Design ◽  
Clostridium Acetobutylicum ◽  
Genetic Manipulation ◽  
Random Mutagenesis ◽  
Strain Improvement ◽  
Downstream Processing ◽  
Enzyme Engineering ◽  
Butanol Production ◽  
Content Type

ABSTRACTButanol production byClostridium acetobutylicumis accompanied by coproduction of acetone and ethanol, which reduces the yield of butanol and increases the production cost. Here, we report development of several clostridial aldehyde/alcohol dehydrogenase (AAD) variants showing increased butanol selectivity by a series of design and analysis procedures, including random mutagenesis, substrate specificity feature analysis, and structure-based butanol selectivity design. The butanol/ethanol ratios (B/E ratios) were dramatically increased to 17.47 and 15.91 g butanol/g ethanol for AADF716Land AADN655H, respectively, which are 5.8-fold and 5.3-fold higher than the ratios obtained with the wild-type AAD. The much-increased B/E ratio obtained was due to the dramatic reduction in ethanol production (0.59 ± 0.01 g/liter) that resulted from engineering the substrate binding chamber and the active site of AAD. This protein design strategy can be applied generally for engineering enzymes to alter substrate selectivity.IMPORTANCERenewable biofuel represents one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, clostridial strain improvement has been slower than improvement of other microorganisms. Furthermore, fermentation coproducing various by-products requires costly downstream processing for butanol purification. Here, we report the results of enzyme engineering of aldehyde/alcohol dehydrogenase (AAD) to increase butanol selectivity. A metabolically engineeredClostridium acetobutylicumstrain expressing the engineered aldehyde/alcohol dehydrogenase gene was capable of producing butanol at a high level of selectivity.

scholarly journals Enhanced Butanol Production Obtained by Reinforcing the Direct Butanol-Forming Route in Clostridium acetobutylicum

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Yu-Sin Jang ◽  
Jin Young Lee ◽  
Joungmin Lee ◽  
Jin Hwan Park ◽  
Jung Ae Im ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Clostridium Acetobutylicum ◽  
Metabolic Flux ◽  
Genetic Manipulation ◽  
High Titer ◽  
Strain Improvement ◽  
High Yield ◽  
Butanol Production ◽  
Wild Type ◽  
Content Type

ABSTRACTButanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation byClostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of theptaandbukgenes, encoding phosphotransacetylase and butyrate kinase, while theadhE1D485Ggene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD**) strain. By reinforcing the direct butanol-forming flux inC. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain within siturecovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production inC. acetobutylicumdescribed here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer.IMPORTANCERenewable biofuel is one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered development of engineered clostridia having highly efficient and selective butanol production capability. Here we report for the first time the results of systems metabolic engineering studies of two butanol-forming routes and their relative importances in butanol production. Based on these findings, a metabolically engineeredClostridium acetobutylicumstrain capable of producing butanol to a high titer with high yield and selectivity could be developed by reinforcing the direct butanol-forming flux.

scholarly journals Enhanced Biobutanol Production in Batch Simultaneous Saccharification and Fermentation From Agriculture Residues Using Novel Clostridial Fusants

2021 ◽  
Author(s):  
Sarkar Begum
Keyword(s):  
Enzymatic Activity ◽  
Wheat Straw ◽  
Clostridium Acetobutylicum ◽  
Clostridium Thermocellum ◽  
Simultaneous Saccharification And Fermentation ◽  
Incubation Temperature ◽  
Clostridium Beijerinckii ◽  
Raw Material ◽  
Production Cycle ◽  
Simultaneous Saccharification

A novel approach was introduced for the enhancement of biobutanol production in ABE fermenetation. Thermostable Clostridia species were developed through protoplast fusion between mesophilic and thermophilic Clostridial species. Two parental strains of Clostridia species, Clostridium acetobutylicum (ATCC 4259) and Clostridium beijerinckii (ATCC BA101), along with three fused strains, Clostridium acetobutylicum Clostridium thermocellum (CaCt), Clostridium beijerinckii Clostridium thermocellum (CbCt) and Clostridium acetobutylicum Clostridium beijerinckii (CaCb), were examined for biobutanol production using wheat straw as a feed stock in a batch process of simultaneous saccharification and fermentation (SSF). The objective of the study was to use the thermotolerant fused strains to enhance enzymatic activity during the SSF by raising the incubation temperature and thereby eventually increasing biobutanol production. Economic and residence time analysis of SSF found that addition of cellulase increases the cost of fermentation and prolongs biobutanol production cycle. However, results from the current study indicated that the fused strains, at the higher temperature, were able to produce the required enzymes for the saccharification of wheat straw (i.e., endoglucanase, exoglucanase, and b- glucosidase). This will have a major impact on eliminating costs associated with adding enzymes as raw material to the saccharification process. Fused strain CbCt achieved the highest biobutanol production of up to 14.13 g/L (i.e., a yield of 0.29) was reached at 45°C with total sugar consumption of 82%. Enzymatic activity of CbCt was found to be 61.67 FPU (Filter Paper Unit) which was the highest in comparison with other fused strains evaluated in this study. These results indicate a breakthrough for the technological and economical obstacles associated with industrialization of the SSF process.

scholarly journals Feedback Regulation between Aquatic Microorganisms and the Bloom-Forming Cyanobacterium Microcystis aeruginosa

2019 ◽  
Vol 85 (21) ◽  
Author(s):  
Meng Zhang ◽  
Tao Lu ◽  
Hans W. Paerl ◽  
Yiling Chen ◽  
Zhenyan Zhang ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Microcystis Aeruginosa ◽  
Lake Taihu ◽  
Inorganic Nitrogen ◽  
Microbial Community Composition ◽  
Nitrogen And Phosphorus ◽  
Content Type ◽  
Coculture System ◽  
Eukaryotic Microorganisms ◽  
Aquatic Microorganisms

ABSTRACT The frequency and intensity of cyanobacterial blooms are increasing worldwide. Interactions between toxic cyanobacteria and aquatic microorganisms need to be critically evaluated to understand microbial drivers and modulators of the blooms. In this study, we applied 16S/18S rRNA gene sequencing and metabolomics analyses to measure the microbial community composition and metabolic responses of the cyanobacterium Microcystis aeruginosa in a coculture system receiving dissolved inorganic nitrogen and phosphorus (DIP) close to representative concentrations in Lake Taihu, China. M. aeruginosa secreted alkaline phosphatase using a DIP source produced by moribund and decaying microorganisms when the P source was insufficient. During this process, M. aeruginosa accumulated several intermediates in energy metabolism pathways to provide energy for sustained high growth rates and increased intracellular sugars to enhance its competitive capacity and ability to defend itself against microbial attack. It also produced a variety of toxic substances, including microcystins, to inhibit metabolite formation via energy metabolism pathways of aquatic microorganisms, leading to a negative effect on bacterial and eukaryotic microbial richness and diversity. Overall, compared with the monoculture system, the growth of M. aeruginosa was accelerated in coculture, while the growth of some cooccurring microorganisms was inhibited, with the diversity and richness of eukaryotic microorganisms being more negatively impacted than those of prokaryotic microorganisms. These findings provide valuable information for clarifying how M. aeruginosa can potentially modulate its associations with other microorganisms, with ramifications for its dominance in aquatic ecosystems. IMPORTANCE We measured the microbial community composition and metabolic responses of Microcystis aeruginosa in a microcosm coculture system receiving dissolved inorganic nitrogen and phosphorus (DIP) close to the average concentrations in Lake Taihu. In the coculture system, DIP is depleted and the growth and production of aquatic microorganisms can be stressed by a lack of DIP availability. M. aeruginosa could accelerate its growth via interactions with specific cooccurring microorganisms and the accumulation of several intermediates in energy metabolism-related pathways. Furthermore, M. aeruginosa can decrease the carbohydrate metabolism of cooccurring aquatic microorganisms and thus disrupt microbial activities in the coculture. This also had a negative effect on bacterial and eukaryotic microbial richness and diversity. Microcystin was capable of decreasing the biomass of total phytoplankton in aquatic microcosms. Overall, compared to the monoculture, the growth of total aquatic microorganisms is inhibited, with the diversity and richness of eukaryotic microorganisms being more negatively impacted than those of prokaryotic microorganisms. The only exception is M. aeruginosa in the coculture system, whose growth was accelerated.

Sign in / Sign up

Export Citation Format

Share Document