Extracellular glycosyl hydrolase activity of the Clostridium strains producing acetone, butanol, and ethanol

Production of acetone, butanol, ethanol, acetic acid, and butyric acid by three strains of anaerobic bacteria, which we identified as Clostridium acetobutylicum, was studied. The yield of acetone and alcohols in 6% wheat flour medium amounted to 12.7–15 g/l with butanol constituting 51.0–55.6%. Activities of these strains towards xylan, β-glucan, carboxymethylcellulose, and crystalline and amorphous celluloses were studied. C. acetobutylicum 6, C. acetobutylicum 7, and C. acetobutylicum VKPM B-4786 produced larger amounts of acetone and alcohols and displayed higher cellulase and hemicellulase activities than the type strain C. acetobutylicum ATCC 824 in lab-scale butch cultures. It was demonstrated that starch in the medium could be partially substituted with plant biomass.     

电子载体对丁醇发酵的影响

研究在培养基中加入不同电子载体对丁醇发酵的影响。结果表明:添加微量的苄基紫精可以促进丁醇的产生,同时可强烈抑制丙酮的合成,丁醇体积分数由66.92%提高到82.35%。苄基紫精可促进菌株快速进入产溶剂期,发酵周期明显缩短,丁醇生产强度显著提高。7%玉米培养基中加入40 mg/L苄基紫精,丁醇产量最高达16.10 g/L,生产强度为0.37 g/(L.h),分别较对照提高10.96%和60.87%。在初始丁醇体积分数较低的条件下,苄基紫精对丁醇合成的促进作用更明显。     

限制葡萄糖、葡萄糖/乙酸双底物条件下自由控制丙丁梭菌ABE发酵丙酮浓度和丙酮/丁醇比

提出一种可以提高和自由控制丙丁梭菌ABE发酵丙酮浓度与丙酮/丁醇比的方法。（1）通过控制糖化酶用量、反应时间和温度调节玉米培养基初始葡萄糖浓度,使发酵进入到产溶剂期后,残留葡萄糖浓度降至接近于0 g/L的水平;（2）在葡萄糖受限的条件下,诱导丙丁梭菌合成分泌糖化酶,分解寡糖,将葡萄糖维持于低浓度,进而限制梭菌胞内糖酵解途径的碳代谢和NADH生成速度。与此同时,外添乙酸形成葡萄糖/乙酸双底物环境。在能量代谢基本不受破坏、丁醇未达到抑制浓度的条件下,适度抑制丁醇生产,有效地利用外添乙酸强化丙酮合成;（3）在外添乙酸的基础上,添加适量酿酒酵母,形成丙丁梭菌/酿酒酵母混合发酵体系,提高梭菌对高丁醇浓度的耐受能力。整个发酵体系可以将丙酮浓度和丙酮/丁醇比自由控制在5~12 g/L和0.5~1.0的水平,最大丙酮浓度和丙酮/丁醇比达到11.74 g/L和1.02,并可维持丁醇浓度于10~14 g/L的正常水平,充分满足工业ABE发酵对于丙酮和丁醇产品的不同需求。     

Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation

ABSTRACT: BACKGROUND: Previously we have developed a butanol tolerant mutant of Clostridium acetobutylicum, Rh8, from the wild type strain DSM 1731. Strain Rh8 can tolerate up to 19 g/L butanol, with solvent titer improved accordingly, thus exhibiting industrial application potential. To test if strain Rh8 can be used for production of high level mixed alcohols, a single secondary alcohol dehydrogenase from Clostridium beijerinckii NRRL B593 was overexpressed in strain Rh8 under the control of constitutive thl promoter. RESULTS: The heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach led to the complete conversion of acetone into isopropanol, achieving a total alcohol titer of 23.88 g/l (7.6 g/l isopropanol, 15 g/l butanol, and 1.28 g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased. CONCLUSIONS: The improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 thus can be considered as a good host for further engineering of solvent/alcohol production.     

丙酮丁醇发酵菌的分子遗传改造

丙酮丁醇梭菌及拜氏梭菌是重要的ABE（丙酮、丁醇和乙醇）工业生产菌株,其发酵产物中的丙酮和丁醇均为重要的化工原料,汽车发动机试验证明丁醇还是一种性能优于乙醇的极具潜力的生物燃料和燃料添加剂。随着新生物技术的不断发展及工业生产的需求,遗传工程改造不断应用于丙酮丁醇生产菌株。在前人研究及工业实践的基础上,对丙酮丁醇生产菌株的遗传特性及其分子遗传改造取得的进展进行了详细概述。     

Mechanism of the acetone formation by Clostridium acetobutylicum

Abstract The production of acetone by Clostridium acetobutylicum was favoured when acetate was added to the culture medium. In the presence of acetate an increase of the acetone concentration from 4.2 to 10.1 g per liter was noted without any change of the butanol concentration. The coenzyme A transferase and the acetoacetate carboxylase were not limiting factors since the addition of acetic acid allowed the biosynthesis of acetone to increase. The control of acetate production by the cells, which is not coupled to the butanol formation, is the key point of the acetone biosynthesis.     

添加有机酸对Clostridium acetobutylicum合成丙酮和丁醇的影响

为提高丙酮-丁醇梭菌厌氧发酵生产丙酮和丁醇的能力,在发酵过程中添加有机酸（乙酸和丁酸）,考察其对菌体生长、溶剂合成影响。实验表明：当添加1.5 g/L乙酸时能够促进菌体的生长,促进丙酮的合成,在600 nm处的最大OD值比参照值高出18.4%,丙酮的最终质量分数提高了21.05%,但不能促进丁醇的合成;当添加1.0g/L丁酸时能够促进菌体生长,促进丁醇的合成,在600 nm处的最大OD比参照值高22.29%,丁醇的最终质量分数比对照组提高了24.32%,但不能促进丙酮的合成。     

Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance

The primary alcohol/aldehyde dehydrogenase (coded by the aad gene) is responsible for butanol formation in Clostridium acetobutylicum. We complemented the non-sporulating, non-solvent-producing C. acetobutylicum M5 strain (which has lost the pSOL1 megaplasmid containing aad and the acetone-formation genes) with aad expressed from the phosphotransbutyrylase promoter and restored butanol production to wild type levels. Because no acetone was produced, no acids (acetate or butyrate) were re-assimilated leading to high butyrate but especially acetate levels. To counter acetate production, we examined thiolase overexpression in order reduce the acetyl-CoA pool and enhance the butyryl-CoA pool. We combined thiolase overexpression with aad overexpression aiming to also enhance butanol formation. While limiting the formation of acetate and ethanol, the butanol titers were not improved. We also generated acetate kinase (AK) and butyrate kinase (BK) knockout (KO) mutants of M5 using a modified protocol to increase the antibiotic-resistance gene expression. These strains exhibited greater than 60% reduction in acetate or butyrate formation, respectively. We complemented the AKKO M5 strain with aad overexpression, but could not successfully transform the BKKO M5 strain. The AKKO M5 strain overexpressing aad produced less acetate, but also less butanol compared to the M5 aad overexpression strain. These data suggest that loss of the pSOL1 megaplasmid renders cells resistant to changes in the two acid-formation pathways, and especially so for butyrate formation. We argue that the difficulty in generating high butanol producers without acetone and acid production is hindered by the inability to control the electron flow, which appears to be affected by unknown pSOL1 genes.     

Genome-scale modeling using flux ratio constraints to enable metabolic engineering of clostridial metabolism in silico

ABSTRACT: BACKGROUND: Genome-scale metabolic networks and flux models are an effective platform for linking an organism genotype to its phenotype. However, few modeling approaches offer predictive capabilities to evaluate potential metabolic engineering strategies in silico. METHODS: A new method called "flux balance analysis with flux ratios (FBrAtio)" was developed in this research and applied to a new genome-scale model of Clostridium acetobutylicum ATCC 824 (iCAC794) that contains 707 metabolites and 794 reactions. FBrAtio was used to model wild-type metabolism and metabolically engineered strains of C. acetobutylicum where only flux ratio constraints and thermodynamic reversibility of reactions were required. The FBrAtio approach allows solutions to be found through standard linear programming. RESULTS: Five flux ratio constraints were required to achieve a qualitative picture of wild-type metabolism for C. acetobutylicum for the production of: (i) acetate, (ii) lactate, (iii) butyrate, (iv) acetone, (v) butanol, (vi) ethanol, (vii) CO2 and (viii) H2. Results of this simulation study coincide with published experimental results and show the knockdown of the acetylacetyl-CoA transferase increases butanol to acetone selectivity, while the simultaneous over-expression of the aldehyde/alcohol dehydrogenase greatly increases ethanol production. CONCLUSIONS: FBrAtio is a promising new method for constraining genome-scale models using internal flux ratios. The method was effective for modeling wild-type and engineered strains of C. acetobutylicum.     

Reaction engineering studies of acetone-butanol-ethanol fermentation with Clostridium acetobutylicum

Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum has been extensively studied in recent years because the organism is recognized as an excellent butanol producer. A parallel bioreactor system with 48 stirred-tank bioreactors on a 12 mL scale was evaluated for batch cultivations of the strictly anaerobic, butanol-producing C. acetobutylicum ATCC 824. Continuous gassing with nitrogen gas was applied to control anaerobic conditions. Process performances of ABE batch fermentations on a milliliter scale were identical to the liter-scale stirred-tank reactor if reaction conditions were identical on the different scales (e.g., initial medium, pH, temperature, specific evaporation rates, specific power input by the stirrers). The effects of varying initial ammonia concentrations (0.1-4.4 g L(-1) ) were studied in parallel with respect to glucose consumption and butanol production of C. acetobutylicum ATCC 824 as a first application example. The highest butanol yield of 33% (mol mol(-1) ) was observed at initial ammonia concentrations of 0.5 and 1.1 g L(-1) . This is the first report on the successful application of a 48 parallel stirred-tank bioreactor system for reaction engineering studies of strictly anaerobic microorganisms at the milliliter scale.     

Disruption of the acetate kinase (<Emphasis Type="Italic">ack</Emphasis>) gene of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> results in delayed acetate production

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.     

高丁醇比丙酮丁醇梭菌的选育与应用

设计了专一性分离方法,从土样中分离了多株能产生溶剂的梭苗,经多次单细胞分离、纯化,再经亚硝基胍和甲基磺酸乙酯诱变和抗性筛选,获得几株高丁醇的丙酮丁醇梭菌。对高产菌株的性状稳定性、发酵过程、混合原料应用、温度的影响进行了研究。结果证明菌株性状稳定,丁醇产量为总溶剂的７０％;过程为典型的丙酮丁醇发酵,对温度可耐受到３９－４０℃;能利用玉米和薯干,玉米和高梁进行正常发酵。菌株已在百吨生产罐,连续应用一年     

丙酮丁醇梭菌发酵菊芋汁生产丁醇

对丙酮丁醇梭菌Clostridium acetobutylicum L7发酵菊芋汁酸水解液生产丁醇进行了初步研究。实验结果表明,以该水解液为底物生产丁醇,不需要添加氮源和生长因子。当水解液初始糖浓度为48.36 g/L时,其发酵性能与以果糖为碳源的对照组基本相同,发酵终点丁醇浓度为8.67 g/L,丁醇、丙酮和乙醇的比例为0.58∶0.36∶0.06,但与以葡萄糖为碳源的对照组相比,发酵时间明显延长,表明该菌株葡萄糖转运能力强于果糖。当水解液初始糖浓度提高到62.87 g/L时,发酵终点残糖浓度从3.09 g/L增加到3.26 g/L,但丁醇浓度却提高到11.21 g/L,丁醇、丙酮和乙醇的比例相应为0.64∶0.29∶0.05,表明适量糖过剩有助于C.acetobutylicum L7胞内代谢从丙酮合成向丁醇合成途径调节;继续提高水解液初始糖浓度,发酵终点残糖浓度迅速升高,丁醇生产的技术经济指标受到明显影响。     

The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain.

Degeneration is the process whereby Clostridium acetobutylicum ATCC 824 loses the capacity to produce acetone and butanol after repeated vegetative transfers or in continuous culture. Two degenerate mutants (M5 and DG1) of C. acetobutylicum ATCC 824 do not contain the four genes (ctfA, ctfB, adc, and aad) for acetone and butanol formation. Strain ATCC 824 contains a 210-kb plasmid (pSOL1) which is absent in M5 and DG1. pSOL1 carries the four acetone and butanol formation genes. A restriction map of pSOL1 was constructed by using ApaI, SmaI, SstII, and NarI digestions. M5 and DG1 could be complemented for acetone and butanol formation by expressing the corresponding genes (ctfA, ctfB, and adc for acetone; aad for butanol) on the plasmid. Degeneration of this strain thus appears to be the result of pSOL1 loss.     

Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition?

Two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of Clostridium acetobutylicum. Inactivation of the butyrate kinase gene (buk) was examined using strain PJC4BK, while the combined effect of buk inactivation and overexpression of the aad gene-encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formation-was examined using strain PJC4BK(pTAAD). The two strains were characterized in controlled pH > or = 5.0 fermentations, and by a recently enhanced method of metabolic flux analysis. Strain PJC4BK was previously genetically characterized, and fermentation experiments at pH > or = 5.5 demonstrated good, but not exceptional, solvent-production capabilities. Here, we show that this strain is a solvent superproducer in pH > or = 5.0 fermentations producing 225 mM (16.7 g/L) of butanol, 76 mM of acetone (4.4 g/L), and 57 mM (2.6 g/L) of ethanol. Strain PJC4BK(pTAAD) produced similar amounts of butanol and acetone but 98 mM (4.5 g/L) of ethanol. Both strains overcame the 180 mM (13 g/L) butanol toxicity limit, without any selection for butanol tolerance. Work with strain PJC4BK(pTAAD) is the first reported use of dual antibiotic selection in C. acetobutylicum. One antibiotic was used for selection of strain PJC4BK while the second antibiotic selected for the pTAAD presence. Overexpression of aad from pTAAD resulted in increased ethanol production but did not increase butanol titers, thus indicating that AAD did not limit butanol production under these fermentation conditions. Metabolic flux analysis showed a decrease in butyrate formation fluxes by up to 75% and an increase in acetate formation fluxes of up to 100% during early growth. The mean specific butanol and ethanol formation fluxes increased significantly in these recombinant strains, up to 300% and 400%, respectively. Onset of solvent production occurred during the exponential-growth phase when the culture optical density was very low and when total and undissociated butyric acid levels were <1 mM. Butyrate levels were low throughout all fermentations, never exceeding 20 mM. Thus, threshold butyrate concentrations are not necessary for solvent production in these stains, suggesting the need for a new phenomenological model to explain solvent formation.     

Use of immobilized microbial membrane fragments to remove oxygen and favor the acetone-butanol fermentation.

Oxygen-reducing membrane fragments obtained from Escherichia coli were used with Clostridium acetobutylicum (C. acetobutylicum) to provide an oxygen-free microenvironment for the conversion of glucose to acetone, butanol, and ethanol (ABE). The batch fermentation of suspended C. acetobutylicum NRRL-B-643 and its ability to produce solvents in the presence of membranes as the oxygen-elimination agent are described and compared with the conventional sparging technique used to maintain anaerobiosis. The use of membrane fragments to remove oxygen for fermentation by C. acetobutylicum was successful and gave slightly improved results over the use of sparing with regard to lag, biomass, and solvent production (e.g., final butanol concentration of 3.25 and 2.7 g/L, respectively). Solvent production is also reported for a continuous columnar reactor with coimmobilized cells and membranes in kappa-carrageenan gel beads and air-saturated liquid feed.     

玉米黄浆水在丙酮-丁醇厌氧梭菌发酵中的应用

为降低丙酮-丁醇厌氧梭菌发酵生产丁醇的成本,研究了不同添加量玉米黄浆水对发酵的影响。与葡萄糖培养基相比,在发酵培养基中添加少量玉米黄浆水对发酵产量无显著影响。当添加体积分数为25％的玉米黄浆水时,丙酮、丁醇和乙醇的最终质量浓度分别是0．31、2．70和8．00g/L,总溶剂量为11．01g／L。通过成本核算,每生产1kg溶剂,添加体积分数25％的玉米黄浆水可比葡萄糖培养基节约成本2．11元。     

Nutritional Factors Affecting the Ratio of Solvents Produced by Clostridium acetobutylicum

Fermentation of whey by Clostridium acetobutylicum yielded butanol and acetone in a ratio of approximately 100:1. This ratio amounted to only 2:1 in synthetic media with glucose, lactose, or glucose plus galactose as substrates. Removal of citrate from whey and addition of minerals resulted in an increase in the amount of acetone produced. Experiments carried out in a chemostat with a low-phosphate synthetic medium revealed that the butanol/acetone ratio could be increased from 2:1 to 3.8:1 by cofermentation of l-lactate and from 2:1 to 8:1 by iron limitation. The performance of the fermentation in a low-iron glucose medium above pH 5.1 yielded l-lactate as the main product.