丙酮丁醇梭菌XY16丁醇发酵特性

以诱变选育的1株突变菌株丙酮丁醇梭菌XY16为对象,对影响该菌发酵特性的相关因素(N源、生长因子、热激)进行研究。结果显示:无机N源乙酸铵比其他N源更有利于丙酮丁醇的发酵,玉米浆或玉米蛋白可以直接替代生长因子进行丙酮丁醇发酵,热激可以提高总溶剂产量,最高可以达到21.28 g/L。该菌还可以同时利用葡萄糖和木糖,当葡萄糖利用完后,木糖才能被有效利用。     

Recognition sequence of a new methyl-specific restriction system from Clostridium acetobutylicum strain ABKn8

A new type II restriction endonuclease, named Cac8I was detected in Clostridium acetobutylicum strain ABKn8. Cac8I cleaved the hexanucleotide sequence [5'-GCN decreases NGC-3'] and generated blunt ends. Up to now no isoschizomer of Cac8I has been described [corrected].     

Induction of acetoacetate decarboxylase in Clostridium acetobutylicum

Abstract Factors that may initiate the biosynthesis of acetoacetate decarboxylase were investigated in resting cells of Clostridium acetobutylicum . Linear acids from C₁ to C₄ were inducers, whereas branched acids and linear acids from C₅ to C₇ were not inducers of acetoacetate decarboxylase biosynthesis. Induction of acetoacetate decarboxylase was maximal at pH 4.8 in the presence of acid concentrations comparable with those found during fermentation. In growth conditions repression of acetoacetate decarboxylase biosynthesis was found. This fact explains that acetone production by Clostridium acetobutylicum occurs when growth slows down.     

Host-plasmid interactions in recombinant strains of Clostridium acetobutylicum ATCC 824

Abstract Plasmid-containing strains of Clostridium acetobutylicum produced higher levels of solvents and lower levels of acids than wild-type cells in controlled pH 4.5 batch fermentations. This effect was observed regardless of whether or not the plasmids contained C. acetobutylicum genes. The effect was less prevalent in higher pH fermentations and apparently independent of the actual DNA sequences contained on these plasmids. The plasmid-containing strains were found to have lower growth-rates and higher solventogenic enzyme activities than wild-type cells. However, similar activity levels were found for both butyrate-pathway enzymes.     

Phenotypic and genotypic differences between certain strains of Clostridium acetobutylicum

Abstract It has become evident that several of the strains of Clostridium acetobutylicum that have been employed in physiological studies of the acetone-butanol fermentation, are heterogeneous. Studies of the phenotypic and genotypic characteristics of several of these strains (involving inter alia both pyrolysis mass spectrometry and 16S rRNA sequence determinations) demonstrated that the type strain obtained from ATCC was not identical with that supplied by NCIMB, and that NCIMB 8052T is in fact Clostridium beijerinckii . We therefore suggest that the name Clostridium acetobutylicum should be restricted to those strains that are genetically closely related to ATCC 824T (which include strains DSM 792 and DSM 1731 but not strain P262).     

Genome analysis of a hyper acetone‐butanol‐ethanol (ABE) producing Clostridium acetobutylicum BKM19

Previously the development of a hyper acetone‐butanol‐ethanol (ABE) producing Clostridium acetobutylicum BKM19 strain capable of producing 30.5% more total solvent by random mutagenesis of its parental strain PJC4BK, which is a buk mutant C. acetobutylicum ATCC 824 strain is reported. Here, BKM19 and PJC4BK strains are re‐sequenced by a high‐throughput sequencing technique to understand the mutations responsible for enhanced solvent production. In comparison with the C. acetobutylicum PJC4BK, 13 single nucleotide variants (SNVs), one deletion and one back mutation SNV are identified in the C. acetobutylicum BKM19 genome. Except for one SNV found in the megaplasmid, all mutations are found in the chromosome of BKM19. Among them, a mutation in the thlA gene encoding thiolase is further studied with respect to enzyme activity and butanol production. The mutant thiolase (thlA^V5A) is showed a 32% higher activity than that of the wild‐type thiolase (thlA^WT). In batch fermentation, butanol production is increased by 26% and 23% when the thlA^V5A gene is overexpressed in the wild‐type C. acetobutylicum ATCC 824 and in its derivative, the thlA‐knockdown TKW‐A strain, respectively. Based on structural analysis, the mutation in thiolase does not have a direct effect on the regulatory determinant region (RDR). However, the mutation at the 5^th residue seems to influence the stability of the RDR, and thus, increases the enzymatic activity and enhances solvent production in the BKM19 strain.     

The improvement of glucose/xylose fermentation by Clostridium acetobutylicum using calcium carbonate

Batch fermentation of 60g/l glucose/xylose mixture by Clostridium acetobutylicum ATCC 824 was investigated on complex culture medium. Different proportions of mixtures, ranged between 10 and 50g of each sugar/l, were fermented during pH control at 4.8 (optimum pH for solventogenesis) or during CaCO₃ addition. Using xylose-pregrown cells and pH control, an important amount of xylose was left over at the end of the fermentation when the glucose concentration was higher than that of xylose. The addition of 10g of CaCO₃/l (to prevent the pH dropping below 4.8) increased xylose uptake: a substantial decrease of residual xylose was observed when xylose-pregrown cells as well as glucose-pregrown cells were used as inoculum for all the mixture proportions studied. MgCO₃ (Mg²⁺-containing compound) and CaCl₂ (Ca²⁺-containing compound) reduced residual xylose only during pH control at 4.8 by NaOH addition. As butanol is the major limiting factor of xylose uptake in C. acetobutylicum, fermentations were carried out with or without CaCO₃ in butanol-containing media or in iron deficient media (under iron limitation, butanol synthesis occurred early and could inhibit xylose uptake). Results showed that an excess of CaCOCaCO₃ could increase butanol tolerance which resulted in an increase in xylose utilization. This positive effect seem to be specific to Ca²⁺- or Mg²⁺-containing compounds, going beyond the buffering effect of carbonate.     

The regulation of thiomethylgalactoside transport in Clostridium acetobutylicum P262 by inducer exclusion and inducer expulsion mechanisms

Abstract Clostridium acetobutylicum P262 had phosphotransferase systems for glucose and lactose, and the lactose system was inducible. When C. acetobutylicum P262 was provided with glucose and lactose, the cultures grew in a diauxic fashion, and glucose was used preferentially. Cells grown on lactose took up thiomethylgalactoside, and retained this non-metabolizable lactose analog for long periods of time. Because glucose inhibited thiomethylgalactoside uptake and caused the efflux of thiomethylgalactoside that had already been taken up, it appeared that C. acetobutylicum P262 had inducer exclusion and inducer expulsion mechanisms similar to those found in lactic acid bacteria.     

Pervaporative butanol fermentation by Clostridium acetobutylicum B18

Extractive acetone-butanol-ethanol (ABE) fermentation was carried out successfully using pervaporation and a low-acid-producing Clostridium acetobutylicum B18. A pervaporation module with 0.17 m(2) of surface area was made of silicone membrane of 240 mum thickness. Pervaporation experiments using make-up solutions showed that butanol and acetone fluxes increased linearly with their concentrations in the aqueous phase. Fickian diffusion coefficients were constants for fixed air flow rates, and increased at higher sweep air flow rates. During batch and fed-batch fermentations, pervaporation at an air flow rate of 8 L/min removed butanol and acetone efficiently. Butanol concentration was maintained below 4.5 g/L even though Clostridium acetobutylicum B18 produced butanol steadily. Pervaporation could not remove organic acids efficiently, but organic acids did not accumulate because strain B18 produced little organic acid and recycled added organic acids efficiently. With pervaporation, glucose consumption rate increased compared to without pervaporation, and up to 160 g/L of glucose was consumed during 80 h. Cell growth was not inhibited by possible salt accumulation or oxygen diffusion through the silicone tubing. The culture volume was maintained relatively constant during fed-batch operation because of an offsetting effect of water and product removal by pervaporation and addition of nutrient supplements. (c) 1994 John Wiley & Sons, Inc.     

In situ butanol recovery from Clostridium acetobutylicum fermentations by expanded bed adsorption

Although butanol is a promising biofuel, its fermentative production suffers from inhibition caused by end product toxicity. The in situ removal of butanol from cultures via expanded bed adsorption offers an effective strategy for mitigating the effects of product toxicity while eliminating the need to clarify cultures via microfiltration. The hydrophobic polymer resin Dowex Optipore L‐493 was found to be both an effective butanol adsorbent and suitable for use in expanded bed adsorption. Recirculation rates through the adsorption column were strongly correlated with and ultimately controlled rates of butanol uptake from the media which, reaching as high as 41.1 g/L h, easily exceed those of its production in a typical fermentation. Vacuum application with vapor collection was found to be an effective means of adsorbent regeneration, with an average of 81% butanol recovery possible, with butanol concentrations in the cold trap reaching as high as 85.8 g/L. Integration of expanded bed adsorption with a fed‐batch Clostridium acetobutylicum ATCC 824 fermentation and its continuous operation for 38.5 h enabled the net production (i.e., in solution and adsorbed) of butanol and total solvent products at up to 27.2 and 40.7 g/L of culture, respectively, representing 2.2‐ and 2.3‐fold improvements over conventional batch culture. While adsorbent biofouling was found to be minimal, further investigation of biofouling in longer‐term studies will provide useful and further insight regarding the robustness of the process strategy. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:68–78, 2014     

Molecular genetics and the initiation of solventogenesis in Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052

Abstract: A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum ) NCIMB 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. The 14 rrn operons together with 40 genes have been assigned positions on the map. Genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. Experiments using the available genetic tools have shown that spo0A plays a cardinal role in controlling several aspects of the transition from exponential growth to stationary phase in C. beijerinckii . These include initiation of sporulation, accumulation of the storage polysaccharide, granulose, and production of acetone and butanol. Several C. beijerinckii and C. acetobutylicum genes concerned with fermentative metabolism, whose expression is modulated at the onset of solventogenesis, contain sequence motifs resembling 0A boxes in their 5' regulatory regions. This invites the speculation that they are under direct control of Spo0A, and additional data are now required to test this prediction.     

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。     

CAC2634-disrupted mutant of Clostridium acetobutylicum can be electrotransformed in air

Aims: To simplify the electrotransformation process of Clostridium acetobutylicum, which currently needs to be performed in an anaerobic chamber, thus laborious and time‐consuming. Methods and Results: The CAC2634 gene encoding PerR is a known peroxide regulon repressor in Cl. acetobutylicum. CAC2634 in a previously constructed Restriction–Modification system deficient Cl. acetobutylicum mutant SMB009 was disrupted using ClosTron method. The resulted mutant SMB012 can be electrotransformed in air with an efficiency of 1·2–3·1 × 10³ transformants μg^?1 DNA. Conclusions: We demonstrated that the disruption of CAC2634 in Cl. acetobutylicum enables its electrotransformation in air. Significance and Impact of the Study: The electrotransformation process of Cl. acetobutylicum could be significantly simplified, especially when operating multiple electrotransformations.     

Anaerobic transformation of 2,4,6-TNT and related nitroaromatic compounds by Clostridium acetobutylicum

The transformation of TNT and related aminated nitrotoluenes by Clostridium acetobutylicum was investigated. 2,4,6-trinitrotoluene (TNT) was rapidly reduced (537 nM min⁻¹ mg protein⁻¹) to undetermined end products via monohydroxylamino derivatives. TNT reduction was more rapid than that of 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene. The metabolic phase of clostridial cultures affected rates and extents of transformation of TNT and its intermediates. Acidogenic cultures showed rapid transformation rates and the ability to transform TNT and its primary reduction products to below detection limits; solventogenic cultures did not transform TNT completely, and showed accumulation of its hydroxylamino derivatives. Carbon monoxide-induced solventogenesis was capable of slowing the transformation of TNT and intermediates. Studies employing [ring-U-¹⁴C]-TNT demonstrated that no significant mineralization occurred and that products of transformation were water-soluble. Received 06 November 1995/ Accepted in revised form 15 August 1996     

Construction ofEscherichia coli-Clostridium acetobutylicum shuttle vectors and transformation ofClostridium acetobutylicum strains

Summary SeveralE. coli-C. acetobutylicum shuttle vectors were constructed and used to transform twoC. acetobutylicum strains ATCC 824 and NCIMB 8052. Other than pSYL2, none of these vectors were able to transform ATCC 824 due to the presence of a restriction system. However, all of them could transform NCIMB 8052 with efficiencies of 8×10²–6×10³ transformants per g DNA.     

Structure and transcription of genes within the β-hbd-adh1 region of Clostridium acetobutylicum P262

Abstract The 1.2-kb DNA fragment upstream of the linked β- hbd (3-hydroxybutyryl-CoA dehydrogenase) and adh1 (NADPH-dependent alcohol dehydrogenase) genes from Clostridium acetobutylicum P262 was sequenced. The upstream region contained an open reading frame (ORFB) which was found to have 44% amino acid identity to the fixB gene products of yRhizobium and Azorhizobium . The β- hbd and ORFB genes were expressed during the acidogenic and solventogenic phases. The β- hbd gene was transcribed on a single mRNA species of 2.0 kb, whereas the ORFB gene was transcribed on two species of mRNA of 2.0 and 3.5 kb, respectively. The adh1 gene was induced or derepressed at the pH breakpoint before the onset of solventogenesis and was transcribed on a single species of mRNA of 2.4 kb.