Improve Butanol Production Research Articles

Transcriptomic characterization of recombinant Clostridium beijerinckii NCIMB 8052 expressing methylglyoxal synthase and glyoxal reductase from Clostridium pasteurianum ATCC 6013.

Biological production of commodity chemicals from abundant waste streams such as whey permeate represents an opportunity for decarbonizing chemical production. Whey permeate remains a vastly underutilized feedstock for bioproduction purposes. Thus, enhanced understanding of the cellular and metabolic repertoires of lactose-mediated production of chemicals such as butanol promises to identify new targets that can be fine tuned in recombinant and native microbial strains to engender stronger coupling of whey permeate-borne lactose to value-added chemicals. Our results highlight new genetic targets for future engineering of C. beijerinckii for improved butanol production on lactose and ultimately in whey permeate.

Decoupling acidogenic cascade via bio-electrogenic induced ABE pathway for enhanced n-butanol synthesis in Clostridium acetobutylicum ATCC-824: Bioenergetics and gene expression analysis

This investigation scrutinized the quantitative aspects of biobutanol production in conventional and bioelectrogenic induced ABE fermentation using Clostridium acetobutylicum ATCC-824. The primary objective of the study was to investigate how alterations in the extracellular redox potential (-0.4 V vs. Ag/AgCl (3.5 M KCl)) affect the hetero-fermentation towards the production of acetone, butanol, and ethanol during the bi-phasic fermentation, utilizing different glucose concentrations from 20 g/L − 60 g/L. The study showed significant results with 68.86 % increased butanol titer (6.67 g/L) compared to conventional fermentation (3.95 g/L butanol). Additionally, the acetone production also increased to 2.34 g/L as compared to conventional fermentation (1.96 g/L). Furthermore, gene expression and network analysis showed that various ABE pathway genes were significantly up or down regulated during the bi-phasic fermentation, indicating a difference between the conventional and EF reactors. The external potential influenced the overexpression of genes like pta, crt, bcd, ask, adh (CA_C1574), and hbd (CA_C3375), while simultaneously downregulation of buk gene, suggesting the acidogenic decoupling and transition from cold channel (butyric acid) towards hot channel pathway (directly from acetyl CoA through butyryl-CoA) for improved butanol production. Moreover, the study also revealed that entropy and enthalpy changes played a significant role in butanol production and ABE fermentation. The EF showed more negative free energy (-3.33 × 104 kJ/mol) when compared with conventional fermentation (-2.8 × 104 kJ/mol) suggesting the mechanism to be more spontaneous and viable. Overall, this study provided evidence of metabolic rerouting with non-genetic approach by regulating electron flow, culminating in an impact on the bi-phasic fermentation towards specific metabolites.

Using nitrogen starvation and excess phosphorus for two-stage algae cultivation to improve butanol production of lipid-extracted algae

Lipid-extracted algae (LEA) can be processed via acetone, butanol and ethanol fermentation to produce butanol as an additive to algal biofuel, but direct fermentation was ineffective. In this study, nitrogen and phosphorus regulation for algae cultivation was applied to modify the biochemical composition of algal biomass and hence improve butanol production from LEA. The results show that when nitrogen starvation and excess phosphorus were used during algae cultivation, a significant rise of starch productivity and decline of protein productivity for algal biomass were obtained, which resulted in the highest butanol productivity of 20.16 mg L−1 d−1, a 5.48-fold increase over traditional one-stage cultivation. Mechanism analysis suggested that nitrogen starvation enhanced lipid and starch contents and reduced protein content, which modified C/N ratio of LEA, prevented acid crash and improved butanol production; excess phosphorus modified amino acid contents of phenylalanine and arginine, which strengthened fluxes directing to butanol generation. Through this investigation, the approach of using nitrogen starvation and excess phosphorus to modify biochemical composition of algal biomass and subsequently improve butanol production of LEA was demonstrated to be applicable.

A dynamic kinetic model captures cell-free metabolism for improved butanol production

Cell-free systems are useful tools for prototyping metabolic pathways and optimizing the production of various bioproducts. Mechanistically-based kinetic models are uniquely suited to analyze dynamic experimental data collected from cell-free systems and provide vital qualitative insight. However, to date, dynamic kinetic models have not been applied with rigorous biological constraints or trained on adequate experimental data to the degree that they would give high confidence in predictions and broadly demonstrate the potential for widespread use of such kinetic models. In this work, we construct a large-scale dynamic model of cell-free metabolism with the goal of understanding and optimizing butanol production in a cell-free system. Using a combination of parameterization methods, the resultant model captures experimental metabolite measurements across two experimental conditions for nine metabolites at timepoints between 0 and 24 h. We present analysis of the model predictions, provide recommendations for butanol optimization, and identify the aldehyde/alcohol dehydrogenase as the primary bottleneck in butanol production. Sensitivity analysis further reveals the extent to which various parameters are constrained, and our approach for probing valid parameter ranges can be applied to other modeling efforts.

Formation and multiple catalysis of copper-cysteine complex enhance butanol fermentation from rice straw

Butanol production from rice straw via Acetone-Butanol-Ethanol (ABE) fermentation has been considered a sustainable and cost-effective approach to cope with energy crisis and environmental issues. However, a number of factors, particularly low butanol production and energy-intensive separation, impede its industrial application. In this study, Cu2+, Zn2+, Fe2+, and Ni2+ were individually induced into the ABE fermentation system of Clostridium beijerinckii NCIMB 8052 with butyric acid and sugars both from rice straw as co-substrates to increase the specific butanol yield. The results demonstrated that the additional 0.20 g/L Cu2+ (i.e. 1.25 mmol/L) significantly increased SBY and butanol/acetone to 0.194 ± 0.006 g/g rice straw and 3.90 ± 0.21, respectively, compared to those of 0.126 ± 0.004 g/g rice straw and 2.61 ± 0.17 in control system. The improved butanol production was attributed to the formation of Cu2+-cysteine/Cu+-cysteine complex, which enhanced the adsorption of butyrate from rice straw by upgrading the activity of key enzyme (ctfAB) and NADH/NAD+. The study provides a reinforcement strategy for efficient lignocellulosic butanol production.

Effectiveness of Low-Cost Bioreactors Integrated with a Gas Stripping System for Butanol Fermentation from Sugarcane Molasses by Clostridium beijerinckii

The effectiveness of column bioreactors for butanol fermentation from sugarcane molasses by Clostridium beijerinckii TISTR 1461 was investigated. This fermentation was operated at an initial pH of 6.5 and temperature of 37 °C under anaerobic conditions. A 1-L bubble column bioreactor was used with various gas circulation rates ranging from 0.2 to 1.0 L/min. The highest butanol concentration (PB, 8.72 g/L), productivity (QB, 0.24 g/L∙h) and yield (YB/S, 0.21 g/g) were obtained with a gas circulation of 0.2 L/min. To improve butanol production efficiency, gas-lift column bioreactors with internal and external loops at 0.2 L/min of circulating gas were used. Higher PB (10.50–10.58 g/L), QB (0.29 g/L∙h) and YB/S (0.22–0.23 g/g) values were obtained in gas-lift column bioreactors. These values were similar to those using a more complex 2-L stirred-tank bioreactor (PB, 10.10 g/L; QB, 0.28 g/L h and YB/S, 0.22 g/g). Hence, gas-lift column bioreactors have potential for use as low-cost fermenters instead of stirred-tank bioreactors for butanol fermentation. When the gas-lift column bioreactor with an internal loop was coupled with a gas stripping system, it yielded an enhanced PB and sugar consumption of approximately 9% and 7%, respectively, compared to a system with no gas stripping.

Enhanced butanol production in Clostridium acetobutylicum by manipulating metabolic pathway genes

Biobutanol is naturally produced by solventogenic clostridia during acetone-butanol-ethanol (ABE) fermentation, which is hampered by low butanol titer, yield and productivity. Here, Clostridium acetobutylicum ATCC 55025 and its robust mutant HKKO with histidine kinase genes cac3319/cac0323 knockout were metabolically engineered to eliminate acetone formation and improve butanol production. By knocking out acetoacetate decarboxylase gene adc in ATCC 55025 and HKKO, the acetone titer decreased to 0.1 g/L and the butanol/ABE ratio increased by 39.0 % and 40.9 %, respectively. However, the butanol production was limited to ∼11 g/L. To improve butanol production, an alternative strategy was used by overexpressing acetaldehyde/alcohol dehydrogenase gene adhE2 in HKKO, and the mutant produced butanol of 19.7 g/L than that of 18.2 g/L from HKKO with high butanol productivity of 0.41 g/L/h and yield of 0.26 g/g. This study provides novel insights into the roles of adc and adhE2 on butanol biosynthesis and generates a hyper-butanol-producing strain.

Fed-batch simultaneous saccharification and fermentation including in-situ recovery for enhanced butanol production from rice straw

This paper describes a study of fed-batch SSFR (simultaneous saccharification, fermentation and recovery) for butanol production from alkaline-pretreated rice straw (RS) in a 2-L stirred tank reactor. The initial solid (9.2% w/v) and enzyme (19.9 FPU g-dw-1) loadings were previously optimized by 50-mL batch SSF assays. Maximum butanol concentration of 24.80 g L-1 was obtained after three biomass feedings that doubled the RS load (18.4% w/v). Butanol productivity (0.344 g L-1h−1) also increased two-fold in comparison with batch SSF without recovery (0.170 g L-1h−1). Although fed-batch SSFR was able to operate with a single initial enzyme dosage, an extra dosage of nutrients was required with the biomass additions to achieve this high productivity. The study showed that SSFR can efficiently improve butanol production from a lignocellulosic biomass accompanied by the efficient use of the enzyme.

Biobutanol Production Using High Cell Density Fermentation in a Large Extractant Volume

Biobutanol is well known as a suitable substitute for gasoline, which can be applied without engine modification. Butanol toxicity to the producer strain causes difficulties to grow strain of higher than 4 g/L dry cell weight and to produce butanol higher than 20 g/L. Fermentation using high initial cell density has been reported to enhance butanol productivity. In addition, oleyl alcohol has been recognized for effective extraction of butanol because of its selectivity and biocompatibility with reduced the effect of toxicity. Butanol fermentation with high cell density and large extractant volume has not been reported and is expected to improve butanol production in a minimum medium volume setting. Clostridium saccharoperbutylacetonicum N1-4, C. beijerinckii NCIMB 8052 (8052), and C. acetobutylicum ATCC 824 (824) were used in this study. Three kinds of media, TYA, TY, and TY-CaCO3, were used in this conventional extractive fermentation. Then, in situ extractive fermentation with Ve/Vb ratios at 0.1, 0.5, 1.0, and 10 were used. Total butanol concentration was defined as the broth-based total butanol, which is the total amount of butanol produced in broth and extractant per the volume of broth. TYA medium yielded the highest total butanol concentrations at N1-4 (12 g/L), 8052 (11 g/L), and 824 (15 g/L), and the highest partition coefficient (3.7) among the three media with similar Ve/Vb ratio at 0.5. N1-4 yielded the highest increment of total butanol production (22 g/L) in the extractive fermentation with high cell density. Low butanol concentration of 0.8 g/L in the broth was maintained using the extractant at a broth volume ratio (Ve/Vb) much lower than 4.4 g/L with a ratio of 0.5. Ve/Vb ratio of 10 which provided 2-fold higher total butanol concentration (28 g/L) than that of 11 g/L obtained using a Ve/Vb ratio of 0.5. These results indicated that a larger volume of extractant to broth improved total butanol concentration by reducing butanol toxicity and led to high medium based butanol yield in fermentation using high cell density.

Acetone, butanol, and ethanol production from puerariae slag hydrolysate through ultrasound-assisted dilute acid by Clostridium beijerinckii YBS3

In this study, puerariae slag (PS) was evaluated as a renewable raw material for acetone–butanol–ethanol (ABE) fermentation. To accelerate the hydrolysis of PS, the method of ultrasound-assisted dilute acid hydrolysis (UAAH) was used. With this effort, 0.69 g reducing sugar was obtained from 1 g raw material under the optimal pretreatment condition. Subsequently, the butanol and total solvent production of 8.79 ± 0.16 g/L and 12.32 ± 0.26 g/L were obtained from the non-detoxified diluted hydrolysate, and the yield and productivity of butanol were 0.19 g/g and 0.12 g/L/h, respectively. Additionally, the changes in the structure of PS after different pretreatment methods were observed using SEM and FT-IR. UAAH resulted in more severe and distinct damage to the dense structure of PS. This study suggests that the UAAH is an attainable but effective pretreatment method, thereby is a promising technique for lignocellulose hydrolysis and improve butanol production.

Metabolic Engineering of Clostridium cellulovorans to Improve Butanol Production by Consolidated Bioprocessing.

Clostridium cellulovorans DSM 743B can produce butyrate when grown on lignocellulose, but it can hardly synthesize butanol. In a previous study, C. cellulovorans was successfully engineered to switch the metabolism from butyryl-CoA to butanol by overexpressing an alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824; however, its full potential in butanol production is still unexplored. In the study, a metabolic engineering approach based on a push-pull strategy was developed to further enhance cellulosic butanol production. In order to accomplish this, the carbon flux from acetyl-CoA to butyryl-CoA was pulled by overexpressing a trans-enoyl-coenzyme A reductase gene (ter), which can irreversibly catalyze crotonyl-CoA to butyryl-CoA. Then an acid reassimilation pathway uncoupled with acetone production was introduced to redirect the carbon flow from butyrate and acetate toward butyryl-CoA. Finally, xylose metabolism engineering was implemented by inactivating xylR (Clocel_0594) and araR (Clocel_1253), as well as overexpressing xylT (CA_C1345), which is expected to supply additional carbon and reducing power for CoA and butanol synthesis pathways. The final engineered strain produced 4.96 g/L of n-butanol from alkali extracted corn cobs (AECC), increasing by 235-fold compared to that of the wild type. It serves as a promising butanol producer by consolidated bioprocessing.

Intracellular metabolism analysis of Clostridium cellulovorans via modeling integrating proteomics, metabolomics and fermentation

Abstract A consolidated bioprocess for cellulosic n-butanol production has been developed by engineering Clostridium cellulovorans to overexpress a bifunctional aldehyde/alcohol dehydrogenase. Rational metabolic engineering is important to further improve butanol production. This study aimed to investigate intracellular metabolism and identify the key regulators of cellulosic butanol formation in C. cellulovorans via integrated Omics and fermentation kinetics data analysis. First, comparative proteomics and metabolomics analyses of wild type and n-butanol producing mutant strain were conducted, which quantified 624 host cell proteins and 474 primary and secondary metabolites. Compared to wild type, most cellulases in cellulolysis were up-regulated, but three glycolysis enzymes and three enzymes in central pathway were down-regulated in the n-butanol producing strain. Second, a dynamic model integrating Omics and fermentation data was developed to identify key regulators in butanol biosynthesis, which were ranked by further metabolic control analysis. Finally, rational metabolic engineering was performed in C. cellulovorans by overexpressing two genes (thl and hbd) identified as important factors limiting butanol biosynthesis, which improved butanol yield and C4/C2 ratio. This study demonstrated a research approach to integrate multi-Omics and fermentation data of C. cellulovorans and guide its rational metabolic engineering, which can also be applied to other microorganisms.

Kinetic modeling of butyric acid effects on butanol fermentation by Clostridium saccharoperbutylacetonicum

A kinetic model of acetone-butanol-ethanol (ABE) fermentation taking into account butyric acid effects was developed and implemented in COPASI. The model was validated by comparing the simulation results with experimental data in batch cultures of Clostridium saccharoperbutylacetonicum under various concentrations of initial glucose (97.1 to 152.6 mM) and butyric acid (90.7 to 153.2 mM). The modeling results suggested that increasing the conversion rates from butyryl-CoA (BCoA) to butanol, from butyrate to BCoA, or from pyruvate to lactate would increase butanol synthesis. Similarly, reducing glucose uptake rate or the reaction rates from pyruvate to acetyl CoA (ACoA), from acetoacetyl CoA (AACoA) to BCoA, or from BCoA to butyrate would improve butanol production. Overall, the kinetic model developed can accurately predict the dynamic behavior of metabolites in ABE fermentation with butyric acid addition, which may subsequently be used to identify genetic manipulation strategies for higher bio-butanol production.

Recent Advances in Microbial Production of Butanol as a Biofuel

In lieu of rising crude oil prices, exhaustion of petroleum feed stocks and environmental challenges, only renewable fuels have the potential to match the energy requirements of the future. Among the various renewable fuels, butanol has recently gained a lot of attention because of its advantages over other biofuels. Its microbial production by clostridia through ABE fermentation is being explored for improved yield and cost effectiveness. Using lignocellulosic wastes successfully for butanol production through ABE fermentation is a major breakthrough to deal with the future energy crisis. Genetic engineering of microbes to increase the carbon and redox balance, cell recycling, media optimization, mathematical modelling and tolerance improvement strategies are being attempted to overcome the hurdles of high production cost, by products formation leading to low yield and product toxicity. Along with genetic engineering major research is cantered on heterologous host engineering for improved butanol production and tolerance. This review highlights the recent advances in improving yield and tolerance to butanol in both Clostridial and heterologous hosts from genetic engineering and fermentation methodology aspects.
 Int. J. Appl. Sci. Biotechnol. Vol 7(2): 130-152

Enhanced Butanol Production Using Non-ionic Surfactant-Based Extractive Fermentation: Effect of Substrates and Immobilization of Cell.

The foremost aim of the present study was to enhance butanol production in an extractive fermentation study in presence of non-ionic surfactant using immobilized cells. Earlier studies had shown improved butanol production with non-ionic surfactant and immobilized cells independently. Therefore, in the present work, the combined effect of extractive fermentation and immobilized cells on butanol production was studied. Different matrices (brick, bamboo, cotton fiber, flannel cloth, and polyurethane foam) were tested for immobilization of Clostridium sporogenes. Immobilized biomass thus obtained was used in an extractive fermentation study with non-ionic surfactant L62. Biomass immobilized on polyurethane foam (PF) doubled the butanol production in presence of 6% (v/v) L62 with respect to control (free cells without surfactant). Further, the effect of different carbon sources was also studied to check the suitability of different industrial wastes containing different carbon sources. Glucose was found to be the best carbon source.

Hybrid dilute sulfuric acid and aqueous ammonia pretreatment for improving butanol production from corn stover with reduced wastewater generation

An efficient hybrid pretreatment method was developed for butanol production from corn stover using dilute sulfuric acid (DA) and aqueous ammonia (AA). With the optimized AA concentration, treatment temperature and time of 10% AA, 80 °C and 24 h, the hybrid pretreatment could effectively dissolve hemicellulose and lignin with solid recovery rate of 37.45% and lignin reduction rate of 86.77% compared to those of 57.75% and 45.84% from single DA pretreatment. By washing 1 time after each step treatment, sugar yield and butanol production were increased to 401.76 mg/g-CS and 10.89 g/L from 346.04 mg/g-CS and 9.33 g/L obtained without washing. Compared with conventional single DA and AA pretreatment methods, wastewater generation was reduced to 0.83 L/g-butanol from 2.11 and 3.46 L/g-butanol, indicating this hybrid pretreatment could be an effective approach for improving butanol production from lignocellulosic feedstocks.

Nutrient modulation based process engineering strategy for improved butanol production from Clostridium acetobutylicum.

The present study demonstrates a process engineering strategy to achieve high butanol titer and productivity from wild type Clostridium acetobutylicum MTCC 11274. In the first step, two different media were optimized with the objectives of maximizing the biomass and butanol productivity, respectively. In the next step, attributes of these two media compositions were integrated to design a two-stage fed-batch process which resulted in maximal butanol productivity of 0.55 g L-1 h-1 with titer of 13.1 g L-1 . Further, two-stage fed-batch process along with combinatorial use of magnesium limitation and calcium supplementation resulted in the highest butanol titer and productivity of 16.5 g L-1 and 0.59 g L-1 h-1 , respectively. Finally, integration of the process with gas stripping and modulation of feeding duration resulted in a cumulative butanol titer of 54.3 g L-1 and productivity of 0.58 g L-1 h-1 . The strategy opens up possibility of developing a viable butanol bioprocess. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2771, 2019.

Semi-hydrolysate of paper pulp without pretreatment enables a consolidated fermentation system with in situ product recovery for the production of butanol

Utilization of lignocellulosic biomasses for biobutanol fermentation usually requires costly processes of pretreatment and enzymatic hydrolysis. In this study, paper pulp (93.2% glucan) was used as a starting biomass material to produce biobutanol. We conducted enzymatic semi-hydrolysis of paper pulp without pretreatment and with low enzyme loading, which produced high concentrations of cellobiose (13.9 g L−1) and glucose (21.3 g L−1). In addition, efficient fermentation of the semi-hydrolysate was achieved similar to that with the use of commercial sugars without inhibitors. Finally, we designed a novel non-isothermal simultaneous saccharification and fermentation with in situ butanol recovery, which was composed of a repeated semi-hydrolysis process and successive butanol-extractive fermentation process under the respective optimal conditions. The consolidated system improved butanol production, butanol yields, and butanol productivities and enabled repeated use of medium when compared with other integrated hydrolysis and fermentation processes.

Features of a Staged Acidogenic/Solventogenic Fermentation Process To Improve Butanol Production from Rice Straw

This study introduced an innovative two-stage fermentation process to maximize sugar utilization and biobutanol production from alkaline-pretreated rice straw. The new bioconversion process was composed of an acidogenic fermentation process followed by an acetone–butanol–ethanol (ABE) fermentation process. A sugar-rich hydrolysate (90.4 g/L reducing sugar) and a high acid content fermentation broth (33.9 g/L butyric acid), both produced from rice straw, were mixed together to increase the yield of the biofuels in the ABE fermentation process. Butyric acid and acetic acid generated from the acidogenic fermentation process play a critical role in the ABE fermentation process, which was confirmed by gene expression analysis of five messenger RNAs. Compared with the conventional process, this unique strategy increased the final butanol concentration from 6.2 to 15.9 g/L with 3-fold lower cellulase loading. Furthermore, an enhanced production of 149 g butanol and 36 L hydrogen gas from 1 kg rice straw was achi...

Improving Fructose Utilization and Butanol Production by Clostridium acetobutylicum via Extracellular Redox Potential Regulation and Intracellular Metabolite Analysis.

Jerusalem artichoke (JA) can grow well in marginal lands with high biomass yield, and thus is a potential energy crop for biorefinery. The major biomass of JA is from tubers, which contain inulin that can be easily hydrolyzed into a mixture of fructose and glucose, but fructose utilization for producing butanol as an advanced biofuel is poor compared to glucose-based ABE fermentation by Clostridium acetobutylicum. In this article, the impact of extracellular redox potential (ORP) on the process is studied using a mixture of fructose and glucose to simulate the hydrolysate of JA tubers. When the extracellular ORP is controlled above -460 mV, 13.2 g L-1 butanol is produced from 51.0 g L-1 total sugars (40.1 g L-1 fructose and 10.9 g L-1 glucose), leading to dramatically increased butanol yield and butanol/ABE ratio of 0.26 g g-1 and 0.67, respectively. Intracellular metabolite and q-PCR analysis further indicate that intracellular ATP and NADH availabilities are significantly improved together with the fructose-specific PTS expression at the lag phase, which consequently facilitate fructose transport, metabolic shift toward solventogenesis and carbon flux redistribution for butanol biosynthesis. Therefore, the extracellular ORP control can be an effective strategy to improve butanol production from fructose-based feedstock.