High Butanol Production Research Articles

Process engineering of cellulosic n-butanol production from corn-based biomass using Clostridium cellulovorans

The cellulolytic Clostridium cellulovorans has been engineered to produce n-butanol from low-value lignocellulosic biomass by consolidated bioprocessing (CBP). The objective of this study was to establish a robust cellulosic biobutanol production process using a metabolically engineered C. cellulovorans. First, various methods for the pretreatment of four different corn-based residues, including corn cob, corn husk, corn fiber, and corn bran, were investigated. The results showed that better cell growth and a higher concentration of n-butanol were produced from corn cob that was pretreated with sodium hydroxide. Second, the effects of different carbon sources (glucose, cellulose and corn cob), basal media and culture pH values on butanol production were evaluated in the fermentations performed in 2-L bioreactors to identify the optimal CBP conditions. Finally, the engineered C. cellulovorans produced butanol with final concentration >3 g/L, yield >0.14 g/g, and selectivity >3 g/g from pretreated corn cob at pH 6.5 in CBP. This study showed that the fermentation process engineering of C. cellulovorans enabled a high butanol production directly from agricultural residues.

Quantitative proteome profiles help reveal efficient xylose utilization mechanisms in solventogenic Clostridium sp. strain BOH3.

Development of sustainable biobutanol production platforms from lignocellulosic materials is impeded by inefficient five carbon sugar uptake by solventogenic bacteria. The recently isolated Clostridium sp. strain BOH3 is particularly advantaged in this regard as it serves as a model organism which can simultaneously utilize both glucose and xylose for high butanol (>15 g/L) production. Strain BOH3 was, therefore, investigated for its metabolic mechanisms for efficient five carbon sugar uptake using a quantitative proteomics based approach. The proteomics data show that proteins within the CAC1341-1349 operon play a pivotal role for efficient xylose uptake within the cells to produce butanol. Furthermore, up-regulation of key enzymes within the riboflavin synthesis pathway explained that xylose could induce higher riboflavin production capability of the bacteria (e.g., ∼80 mg/L from glucose vs. ∼120 mg/L from xylose). Overall results from the present experimental approach indicated that xylose-fed BOH3 cultures are subjected to high levels of redox stress which coupled with the solvent stress-trigger a sporulation response within the cells earlier than the glucose-fed cultures. The study lays the platform for metabolic engineering strategies in designing organisms for efficient butanol and other value-added chemicals such as riboflavin production. Biotechnol. Bioeng. 2017;114: 1959-1969. © 2017 Wiley Periodicals, Inc.

Impact of pH and butyric acid on butanol production during batch fermentation using a new local isolate of Clostridium acetobutylicum YM1.

The effect of pH and butyric acid supplementation on the production of butanol by a new local isolate of Clostridium acetobutylicum YM1 during batch culture fermentation was investigated. The results showed that pH had a significant effect on bacterial growth and butanol yield and productivity. The optimal initial pH that maximized butanol production was pH 6.0 ± 0.2. Controlled pH was found to be unsuitable for butanol production in strain YM1, while the uncontrolled pH condition with an initial pH of 6.0 ± 0.2 was suitable for bacterial growth, butanol yield and productivity. The maximum butanol concentration of 13.5 ± 1.42 g/L was obtained from cultures grown under the uncontrolled pH condition, resulting in a butanol yield (YP/S) and productivity of 0.27 g/g and 0.188 g/L h, respectively. Supplementation of the pH-controlled cultures with 4.0 g/L butyric acid did not improve butanol production; however, supplementation of the uncontrolled pH cultures resulted in high butanol concentrations, yield and productivity (16.50 ± 0.8 g/L, 0.345 g/g and 0.163 g/L h, respectively). pH influenced the activity of NADH-dependent butanol dehydrogenase, with the highest activity obtained under the uncontrolled pH condition. This study revealed that pH is a very important factor in butanol fermentation by C. acetobutylicum YM1.

Metabolic engineering of Clostridium tyrobutyricum for n-butanol production from sugarcane juice.

Clostridium tyrobutyricum is a promising organism for butyrate and n-butanol production, but cannot grow on sucrose. Three genes (scrA, scrB, and scrK) involved in the sucrose catabolic pathway, along with an aldehyde/alcohol dehydrogenase gene, were cloned from Clostridium acetobutylicum and introduced into C. tyrobutyricum (Δack) with acetate kinase knockout. In batch fermentation, the engineered strain Ct(Δack)-pscrBAK produced 14.8-18.8g/L butanol, with a high butanol/total solvent ratio of ∼0.94 (w/w), from sucrose and sugarcane juice. Moreover, stable high butanol production with a high butanol yield of 0.25g/g and productivity of 0.28g/L∙h was obtained in batch fermentation without using antibiotics for selection pressure, suggesting that Ct(Δack)-pscrBAK is genetically stable. Furthermore, sucrose utilization by Ct(Δack)-pscrBAK was not inhibited by glucose, which would usually cause carbon catabolite repression on solventogenic clostridia. Ct(Δack)-pscrBAK is thus advantageous for use in biobutanol production from sugarcane juice and other sucrose-rich feedstocks.

Comprehensive investigations of biobutanol production by a non-acetone and 1,3-propanediol generating Clostridium strain from glycerol and polysaccharides.

BackgroundLow-cost feedstocks, a single product (butanol), and a high butanol titer are three key points for establishing a sustainable and economically viable process for biological butanol production. Here, we comprehensively investigated the butanol production from mono-substrates, mainly glycerol and polysaccharides, mainly starch and xylan by a newly identified wild-type Clostridium pasteurianum GL11.ResultsStrain GL11 produced 14.7 g/L of butanol with a yield of 0.41 g/g from glycerol in the batch mode without formation of by-products of acetone and 1,3-propanediol (1,3-PDO). With in situ extraction with biodiesel, the amount of butanol was finally improved to 28.8 g/L in the fed-batch mode. Genomic and enzymatic analysis showed that the deficiency of key enzymes involved in acetone and 1,3-PDO pathway within strain GL11 led to the elimination of these by-products, which may also greatly simplify downstream separation. The elimination of acetone and 1,3-PDO and high butanol tolerance contributed to its high butanol production yield from glycerol. More importantly, strain GL11 could directly convert polysaccharides, such as xylan and starch to butanol with secretion of xylanase and amylase via consolidated bioprocessing.ConclusionsThe wild-type strain GL11 was found to be particularly advantageous due to its capability of efficient butanol production from glycerol and polysaccharides with elimination of acetone and 1,3-PDO formation. And the high butanol production with in situ extraction by using biodiesel would significantly enhance the economic feasibility of fermentative production of butanol from glycerol. These unique features of C. pasteurianum GL11 open the door to the possibility of cost-effective biofuels production in large scale.

Bio-butanol production by Clostridium saccharoperbutylacetonicum N1-4 using selected species of brown and red macroalgae.

Macroalgae are a promising option because they can be propagated easily along the seaside thus eliminating the need for land and nutrient resources. Hence, different macroalgae were assessed for their potential in butanol fermentation. In this study, four species of brown macroalgae (Undaria pinnatifida, Laminaria japonica, Ecklonia stolonifera, Hizikia fusiforme, and Sargassum fulvellum) and two species of red macroalgae (Porphyra tenera and Gelidium amansii) were investigated for the production of butanol by Clostridium saccharoperbutylacetonicum N1-4. To hydrolyze the polymeric materials of the algal biomass, dilute acid hydrolysis was carried out using 0.15 M H2SO4 followed by thermal pretreatment at 121°C for 1 h. Using 100 g/L of hydrolyzed brown alga, the highest butanol production (5.51 g/L) was observed for L. japonica. Other brown and red macroalgae did not exceed the butanol production by L. japonica. Moreover, the detoxification of the thermo-chemically pretreated hydrolysate of L. japonica using the activated carbon and overliming method, increased the butanol production by 24.14 and 12.16%, respectively. These results showed that macroalgae could be a promising substrate for butanol fermentation that is cheap, easily propagated, and non-terrestrial and non-food competing.

Enhanced butanol fermentation using metabolically engineered Clostridium acetobutylicum with ex situ recovery of butanol

In this study, metabolic target reactions for strain engineering were searched via intracellular coenzyme A (CoA) metabolite analysis. The metabolic reactions catalyzed by thiolase (AtoB) and aldehyde-alcohol dehydrogenase (AdhE1) were considered potential rate-limiting steps. In addition, CoA transferase (CtfAB) was highlighted as being important for the assimilation of organic acids, in order to achieve high butanol production. Based on this quantitative analysis, the BEKW_E1AB-atoB strain was constructed by overexpressing the thl (atoB), adhE1, and ctfAB genes in Clostridium acetobutylicum strain BEKW, which has the phosphotransacetylase (pta) and butyrate kinase (buk) genes knocked out. After 100h of continuous fermentation coupled with adsorptive ex situ butanol recovery, the concentrations found after considering desorption, yield, and productivity for the BEKW_E1AB-atoB strain were 55.7g/L, 0.38g/g, and 2.64g/L/h, respectively. The level of butanol production achieved (2.64g/L/h) represents the highest reported value obtained after adsorptive, long-term fermentation.

Enhanced Butanol Production Through Adding Organic Acids and Neutral Red by Newly Isolated Butanol-Tolerant Bacteria.

As alternative microorganisms for butanol production with high butanol tolerant and productivity are in high demand, one excellent butanol-tolerant bacterium, S10, was isolated and identified as Clostridium acetobutylicum S10. In order to enhance the performance of butanol production, organic acids and neutral red were added during butanol fermentation. Synergistic effects were exhibited in the combinations of organic acids and neutral red to promote butanol production. Consequently, the optimal concentrations of combined acetate, butyrate, and neutral red were determined at sodium acetate 1.61g/L, sodium butyrate 1.88g/L, and neutral red 0.79g/L, respectively, with the butanol yield of 6.09g/L which was 20.89% higher than that in control. These results indicated that combination of adding organic acid and neutral red is a potential effective measure to improve butanol production.

A Review of Process-Design Challenges for Industrial Fermentation of Butanol from Crude Glycerol by Non-Biphasic Clostridium pasteurianum

Butanol, produced via traditional acetone-butanol-ethanol (ABE) fermentation, suffers from low yield and productivity. In this article, a non-ABE butanol production process is reviewed. Clostridium pasteurianum has a non-biphasic metabolism, alternatively producing 1,3-propanediol (PDO)-butanol-ethanol, referred to as PBE fermentation. This review discusses the advantages of PBE fermentation with an emphasis on applications using biodiesel-derived crude glycerol, currently an inexpensive and readily available feedstock. To address the process design challenges, various strategies have been employed and are examined and reviewed; genetic engineering and mutagenesis of C. pasteurianum, characterization and pretreatment of crude glycerol and various fermentation strategies such as bioreactor design and configuration, increasing cell density and in-situ product removal. Where research deficiencies exist for PBE fermentation, the process solutions as employed for ABE fermentation are reviewed and their suitability for PBE is discussed. Each of the obstacles against high butanol production has multiple solutions, which are reviewed with the end-goal of an integrated process for continuous high level butanol production and recovery using C. pasteurianum and biodiesel-derived crude glycerol.

Effect of Nutrient Supplementation on Biobutanol Production from Cheese Whey by Abe (acetone–butanol–ethanol) Fermentation

Cheese whey is a liquid effluent obtained from the cheese manufacturing process, presenting a high volumetric production and high organic loads related to its lactose content. The aim of this research was to increase the productivity of ABE fermentation with the bacterial strain Clostridium beijerinckii CECT 508, when using nanofiltered sheep cheese whey as a substrate. Thermal sterilization of the whey was essential to avoid the proliferation of lactic acid bacteria. An experimental design including Plackett-Burman and Response Surface Methodology (RSM) was applied to figure out which additional nutrients were necessary for the fermentation process and their optimum concentrations. For this specific whey, it was established that 1 g/L yeast extract, 5 g/L CaCO3, 0.019 g/L FeSO4·7H2O, 0.2 g/L MgSO4·7H2O and 2.1 g/L NH4Cl needed to be added. Under optimal nutrient conditions, the highest butanol production (9.11 g/L) was obtained with an initial lactose concentration of 57 g/L; achieving 49% lactose consumption and 0.311 g/g YB/L yield. However, the best lactose consumption rate (87%) was recorded for lactose initial concentrations of 30 g/L, which also allowed satisfactory fermentation values (8.51 g/L butanol and 0.328 g/g YB/L yield). On the other hand, the best YB/L yields (0.428 g/g; which is close to the theoretical value) were obtained for initial lactose concentrations of 40 g/L, attaining a value of 52% lactose consumption and 8.91 g/L butanol. Therefore, the ABE fermentation could be a feasible solution to treat cheese whey and remove lactose, obtaining acceptable amounts of butanol.

Direct in situ butanol recovery inside the packed bed during continuous acetone-butanol-ethanol (ABE) fermentation.

In this study, the integrated in situ extraction-gas stripping process was coupled with continuous ABE fermentation using immobilized Clostridium acetobutylicum. At the same time, oleyl alcohol was cocurrently flowed into the packed bed reactor with the fresh medium and then recycled back to the packed bed reactor after removing butanol in the stripper. A high glucose consumption of 52g/L and a high butanol productivity of 11g/L/h were achieved, resulting in a high butanol yield of 0.21g-butanol/g-glucose. This can be attributed to both the high bacterial activity for solvent production as well as a threefold increase in the bacterial density inside the packed bed reactor. Also reported is that 64% of the butanol produced can be recovered by the integrated in situ extraction-gas stripping process. A high butanol productivity and a high glucose consumption were simultaneously achieved.

High butanol production by regulating carbon, redox and energy in Clostridia

Butanol is a promising biofuel with high energy intensity and can be used as gasoline substitute. It can be produced as a sustainable energy by microorganisms (such as Clostridia) from low-value biomass. However, the low productivity, yield and selectivity in butanol fermentation are still big challenges due to the lack of an efficient butanol-producing host strain. In this article, we systematically review the host cell engineering of Clostridia, focusing on (1) various strategies to rebalance metabolic flux to achieve a high butanol production by regulating the metabolism of carbon, redox or energy, (2) the challenges in pathway manipulation, and (3) the application of proteomics technology to understand the intracellular metabolism. In addition, the process engineering is also briefly described. The objective of this review is to summarize the previous research achievements in the metabolic engineering of Clostridium and provide guidance for future novel strain construction to effectively produce butanol. Open image in new window

A novel in situ gas stripping-pervaporation process integrated with acetone-butanol-ethanol fermentation for hyper n-butanol production.

Butanol is considered as an advanced biofuel, the development of which is restricted by the intensive energy consumption of product recovery. A novel two-stage gas stripping-pervaporation process integrated with acetone-butanol-ethanol (ABE) fermentation was developed for butanol recovery, with gas stripping as the first-stage and pervaporation as the second-stage using the carbon nanotubes (CNTs) filled polydimethylsiloxane (PDMS) mixed matrix membrane (MMM). Compared to batch fermentation without butanol recovery, more ABE (27.5 g/L acetone, 75.5 g/L butanol, 7.0 g/L ethanol vs. 7.9 g/L acetone, 16.2 g/L butanol, 1.4 g/L ethanol) were produced in the fed-batch fermentation, with a higher butanol productivity (0.34 g/L · h vs. 0.30 g/L · h) due to reduced butanol inhibition by butanol recovery. The first-stage gas stripping produced a condensate containing 155.6 g/L butanol (199.9 g/L ABE), which after phase separation formed an organic phase containing 610.8 g/L butanol (656.1 g/L ABE) and an aqueous phase containing 85.6 g/L butanol (129.7 g/L ABE). Fed with the aqueous phase of the condensate from first-stage gas stripping, the second-stage pervaporation using the CNTs-PDMS MMM produced a condensate containing 441.7 g/L butanol (593.2 g/L ABE), which after mixing with the organic phase from gas stripping gave a highly concentrated product containing 521.3 g/L butanol (622.9 g/L ABE). The outstanding performance of CNTs-PDMS MMM can be attributed to the hydrophobic CNTs giving an alternative route for mass transport through the inner tubes or along the smooth surface of CNTs. This gas stripping-pervaporation process with less contaminated risk is thus effective in increasing butanol production and reducing energy consumption.

Metabolic engineering of Clostridium tyrobutyricum for n-butanol production from maltose and soluble starch by overexpressing α-glucosidase.

Clostridium tyrobutyricum does not have the enzymes needed for using maltose or starch. Two extracellular α-glucosidases encoded by agluI and agluII from Clostridium acetobutylicum ATCC 824 catalyzing the hydrolysis of α-1,4-glycosidic bonds in maltose and starch from the non-reducing end were cloned and expressed in C. tyrobutyricum (Δack, adhE2), and their effects on n-butanol production from maltose and soluble starch in batch fermentations were studied. Compared to the parental strain grown on glucose, mutants expressing agluI showed robust activity in breaking down maltose and produced more butanol (17.2 vs. 9.5 g/L) with a higher butanol yield (0.20 vs. 0.10 g/g) and productivity (0.29 vs. 0.16 g/L h). The mutant was also able to use soluble starch as substrate, although at a slower rate compared to maltose. Compared to C. acetobutylicum ATCC 824, the mutant produced more butanol from maltose (17.2 vs. 11.2 g/L) and soluble starch (16.2 vs. 8.8 g/L) in batch fermentations. The mutant was stable in batch fermentation without adding antibiotics, achieving a high butanol productivity of 0.40 g/L h. This mutant strain thus can be used in industrial production of n-butanol from maltose and soluble starch.

Comparative proteomics analysis of high n-butanol producing metabolically engineered Clostridium tyrobutyricum

The acidogenic Clostridium tyrobutyricum has recently been metabolically engineered to produce n-butanol. The objective of this study was to obtain a comprehensive understanding as to how butanol production was regulated in C. tyrobutyricum to guide the engineering of next-generation strains. We performed a comparative proteomics analysis, covering 78.1% of open reading frames and 95% of core enzymes, using wild type, ACKKO mutant (Δack) producing 37.30g/L of butyrate and ACKKO-adhE2 mutant (Δack-adhE2) producing 16.68g/L of butanol. In ACKKO-adhE2, the expression of most glycolytic enzymes was decreased, the thiolase (thl), acetyl-CoA acetyltransferase (ato), 3-hydroxybutyryl-CoA dehydrogenase (hbd) and crotonase (crt) that convert acetyl-CoA to butyryl-CoA were increased, and the heterologous bifunctional acetaldehyde/alcohol dehydrogenase (adhE2) catalyzing butanol formation was highly expressed. The apparent imbalance of energy and redox was observed due to the downregulation of acids production and the addition of butanol synthesis pathway, which also resulted in increased expression of chaperone proteins and glycerol-3-phosphate dehydrogenase (glpA) and the silence of sporulation transcription factor Spo0A (spo0A) as the cellular responses to butanol production. This study revealed the mechanism of carbon redistribution, and limiting factors and rational metabolic cell and process engineering strategies to achieve high butanol production in C. tyrobutyricum.

Biobutanol Production from Palm Kernel Cake (PKC) using Clostridium saccharoperbutylacetonicum N1-4 in Batch Culture Fermentation

Palm kernel cake (PKC), a by-product of palm oil industry, contains glucose and mannose as hexose sugars. This study was performed to determine the feasibility of using PKC as a lignocellulosic substrate for biobutanol production by Clostridium saccharoperbutylacetonicum N1-4 in an acetone-butanol-ethanol (ABE) fermentation process. Moreover, the effect of tryptone-yeast extract-acetate (TYA) medium and P2 medium on biobutanol production was evaluated. Experimental results showed that butanol production of 3.05 g/L was obtained using mannose sugar, which was comparable to 3.61 g/L butanol production measured using glucose. Moreover, the maximum production of biobutanol (0.38 g/L) was obtained at a PKC concentration of 30%, indicating the possibility of PKC utilization in butanol production. ABE fermentation of PKC using distilled water, TYA medium, and P2 medium showed that the highest butanol production (0.26 g/L) with ABE production of 0.38 g/L was obtained when ABE fermentation was conducted in P2 medium.

Biofuel Fermentation by Hyperbutanol-Producing Mutants in Sweet Corn Stalk Juice Medium

Clostridium acetobutylicum parent strain was treated by ultraviolet irradiation at different minutes. The UV treated strains were cultivated in cornmeal medium with butanol added to the medium prior to cultivation. By increasing butanol content gradually in the medium, high butanol tolerance mutants were screened. Results show that mutants by UV treated for 4 minutes display best butanol tolerance and high butanol productivity. With 12g/L butanol added in the medium, the mutants still grow rapidly and accumulated 18.0g/L butanol at the end of 69 hr cultivation. Using sweet corn stalk juice as raw material, the maximum butanol produced by fermentation with the mutants reached 15.3g/L and 18.3g/L in batch and fed-batch fermentation respectively.

Recent advances and future prospects for increased butanol production by acetone‐butanol‐ethanol fermentation

Presently, several researchers are increasingly focusing on producing butanol as the next‐generation fuel by acetone‐butanol‐ethanol (ABE) fermentation. Butanol has many superior characteristics compared to other biofuels, such as ethanol. However, its production by ABE fermentation faces the challenges of low productivity and yield because of product inhibition and heterofermentation, respectively, and thereby, high costs. Until date, molecular biological techniques and fermentation engineering methods have been applied for high butanol production. Although glucose remains the substrate of choice since traditional research, it is now necessary to substitute glucose derived from edible starch to other substrates from low‐cost feedstock, such as agricultural residue. In addition, ABE‐producing clostridia cannot directly produce butanol from lignocelluloses. Therefore, recent research is focusing on pretreatment and enzymatic saccharification of the complex molecules derived from agricultural residue for use as feedstock in butanol production. This article reviews traditional research, including the metabolism patterns and characteristics of ABE‐producing clostridia. Furthermore, this article describes developments in ABE fermentation with respect to the establishment of highly efficient butanol production processes, such as batch, fed‐batch, and continuous cultures, with the introduction of butanol removal, as well as butanol production from lignocellulosic biomasses or alternative substrates to sugars.

Isolation, characterization and evaluation of hyper 2-propanol producing bacteria from Singapore environment

Three hyper 2-propanol producing strains were isolated from Singapore environment using an enrichment step and a high through-put screening step. The analysis of the amplified 16S rDNA revealed that the isolates belonged to Clostridium species and they were named as Clostridium sp. BT10-1, Clostridium sp. M10-1 and Clostridium sp. PU31-4. At 1L scale, the 2-propanol titer of these positive strains was 1.6-2.1 times of that of Clostridium beijerinckii NRRL B593, which is so far the most efficient natural 2-propanol producer. The highest 2-propanol titer was achieved by isolate BT10-6 and it was 5.26g/L (87.5mM). These three positive strains BT10-6, M10-1 and PU31-4 consumed glucose almost completely in 40-48h and gave 2-propanol productivity at 0.132, 0.118 and 0.087g/L/h, respectively, which is 3.0-4.6 times of 0.029g/L/h given by C. beijerinckii NRRL B593. Butanol was also produced by these positive strains with a slightly lower butanol titer and higher butanol productivity, compared to butanol control strain C. beijerinckii NCIMB 8052.

Decreased hydrogen production leads to selective butanol production in co-cultures of Clostridium thermocellum and Clostridium saccharoperbutylacetonicum strain N1-4

When Clostridium thermocellum and Clostridium saccharoperbutylacetonicum strain N1-4 were co-cultured hydrogen production decreased and butanol was selectively produced with extremely low level of acetone. Since the high butanol production correlates with low hydrogen production, the molecular selection of hydrogenase gene activity is expected to yield strains exhibiting a higher butanol ratio.