Enhanced butyric acid tolerance and bioproduction by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor

Repeated fed‐batch fermentation of glucose by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor (FBB) was successfully employed to produce butyric acid at a high final concentration as well as to adapt a butyric‐acid‐tolerant strain. At the end of the eighth fed‐batch fermentation, the butyric acid concentration reached 86.9 ± 2.17 g/L, which to our knowledge is the highest butyric acid concentration ever produced in the traditional fermentation process. To understand the mechanism and factors contributing to the improved butyric acid production and enhanced acid tolerance, adapted strains were harvested from the FBB and characterized for their physiological properties, including specific growth rate, acid‐forming enzymes, intracellular pH, membrane‐bound ATPase and cell morphology. Compared with the original culture used to seed the bioreactor, the adapted culture showed significantly reduced inhibition effects of butyric acid on specific growth rate, cellular activities of butyric‐acid‐forming enzyme phosphotransbutyrylase (PTB) and ATPase, together with elevated intracellular pH, and elongated rod morphology. Biotechnol. Bioeng. 2011; 108:31–40. © 2010 Wiley Periodicals, Inc.     

Efficient production of butyric acid from Jerusalem artichoke by immobilized Clostridium tyrobutyricum in a fibrous-bed bioreactor

Butyric acid is an important specialty chemical with wide industrial applications. The feasible large-scale fermentation for the economical production of butyric acid requires low-cost substrate and efficient process. In the present study, butyric acid production by immobilized Clostridium tyrobutyricum was successfully performed in a fibrous-bed bioreactor using Jerusalem artichoke as the substrate. Repeated-batch fermentation was carried out to produce butyric acid with a high butyrate yield (0.44 g/g), high productivity (2.75 g/L/h) and a butyrate concentration of 27.5 g/L. Furthermore, fed-batch fermentation using sulfuric acid pretreated Jerusalem artichoke hydrolysate resulted in a high butyric acid concentration of 60.4 g/L, with the yield of 0.38 g/g and the selectivity of ∼85.1 (85.1 g butyric acid/g acetic acid). Thus, the production of butyric acid from Jerusalem artichoke on a commercial scale could be achieved based on the system developed in this work.     

抗A型产气荚膜梭菌α毒素单链抗体基因的克隆、表达及其生物学活性(英文)

应用RT PCR技术 ,从分泌具有中和活性的抗A型产气荚膜梭菌α毒素单克隆抗体 (McAb)的杂交瘤细胞株 1A8中 ,分别扩增出抗体VH 和VL 基因 ,用linker (Gly4Ser) 3 基因 ,将VH 和VL 基因连接成单链抗体 (ScFv)基因 ,并将其克隆至pGEM T载体中 .经核苷酸序列分析证实 ,VH 和VL 基因及linker基因拼接正确 ,ScFv 1A8基因全长为 72 6bp ,编码2 4 2个氨基酸 ,VH 和VL 基因符合功能性重排的鼠抗体可变区基因特征 ,分别属于鼠免疫球蛋白重链Ⅱ (A)和轻链κⅣ家簇 .将ScFv 1A8基因克隆至表达载体pHOG2 1中 ,构建了重组质粒pHOG 1A8,然后转化至受体菌XL1 BLUE中 ,得到重组菌株XL1 BLUE (pHOG 1A8) .ELISA检测和SDS PAGE分析表明 :经IPTG诱导后所表达的目的蛋白存在于重组菌株XL1 BLUE (pHOG 1A8)的胞周质中 .经薄层扫描分析 :重组菌株XL1 BLUE(pHOG 1A8)的蛋白表达产物占菌体可溶性蛋白的 1 2 % ,其相对分子量约为 31kD .ScFv的生物学活性研究表明 ,ScFv蛋白不但具有中和磷脂酶C的活性 ,而且还能够对致死性腹腔攻击的小鼠产生良好的被动保护作用     

A型产气荚膜梭菌α毒素基因的高效表达

用NooI/EcoRI酶切含α毒素基因质粒pXCPA02,回收1.2kb的α毒素基因片段,通过T4 DNA连接酶,将回收的α毒素基因片段与经NcoI/Eco RI酶切的表达载体pET-28c连接,转化至受体菌BI21(DE3)中,经NcoI/EcoRI,BamHI/Eco RI和NcoI/BamHI/Eco RI酶切反应鉴定和核苷酸序列分析证实,获得的表达质粒aXETA02含有α毒素基因,而且阅读框架是正确的.重组菌株BL21(DE3),(pXETA02)经IPTG诱导后,其表达产物经ELISA检测和SDS-PAGE分析,结果表明重组菌株可以高效表达α毒素蛋白,该蛋白占菌体总蛋白相对含量的36.83%.     

Vacuolization of HeLa cells by a partially purified Clostridium histolyticum cytotoxin

Clostridium histolyticum vacuolating cytotoxin was partially purified from culture broth using ammonium sulfate precipitation, gel filtration and hydrophobic interaction chromatography. The toxin caused vacuolization of HeLa cells visible under a light microscope after 2 h and distinct after 8 h. Transmission electron microscopy revealed the presence of numerous vacuoles, condensation of the mitochondrial matrix, increased cytoplasm density and increased amounts of heterochromatin. Apoptosis was not detected either by electron microscopy or by an apoptosis/necrosis discrimination assay with fluorescein-labeled annexin V and propidium iodide, or DNA fragmentation assay. Calcium ion influx was detected by flow cytometry after labeling cells with Fluo-4 AM. Vacuolation of HeLa cells by C. histolyticum cytotoxin was inhibited by bafilomycin A1, suggesting involvement of H+ -ATPase in the formation of vacuoles.     

Development of a small-scale bioreactor: application to in vivo NMR measurement

A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells. The bioreactor was made of a 10-mm NMR tube. NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter. Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model. Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min. Residence time distribution assays at 0.8-2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed. Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion. In vivo NMR was performed for (31)P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP(gamma) and ADP(beta), ATP(alpha) and ADP(alpha), NADP and NDPG, NDPG and ATP(beta). Cell viability was shown to be maintained as (31)P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH.     

Impact of anaerobiosis strategy and bioreactor geometry on the biochemical response of Clostridium butyricum VPI 1718 during 1,3-propanediol fermentation

The impact of anaerobiosis strategy on 1,3-propanediol production during cultivation of Clostridium butyricum VPI 1718 in different size bioreactors was studied. In batch trials with N2 gas infusion, the fermentation was successfully accomplished, regardless of initial glycerol concentration imposed and bioreactor geometry. However, in the absence of N2 continual sparging, significant variations concerning the biochemical response of the strain were observed. Specifically, at 1-L bioreactor, the absence of N2 infusion at high initial glycerol concentration induced lactate dehydrogenase activity and thus lactic acid synthesis, probably due to partial blockage of phosphoroclastic reaction caused by insufficient self-generated anaerobiosis environment. During fed-batch cultivation with continual N2 sparging, the strain produced ～71 g L(-1) of 1,3-propanediol, whereas under self-generated anaerobiosis, 1,3-propanediol pathway was evidently restricted, as only 30.5 g L(-1) of 1,3-propanediol were finally produced. Apparently, N2 infusion strategy paired with bioreactor geometry can alter the biochemical behavior of the particular strain.     

重组A型产气荚膜梭菌α毒素C端基因在大肠杆菌中的高效表达

在成功克隆A型产气荚膜梭菌α毒素C端基因(cpa408基因)后,发现基因N端ATG后密码子在大肠杆菌中的使用频率普遍偏低,在不改变氨基酸的前提下,对N端基因进行修饰,构建重组表达质粒pBV220cpa408,导入大肠杆菌DH5α中,经温度诱导,cpa408基因获得了高效表达,表达量达菌体总蛋白的4375%,表达产物以可溶形式存在。经Westernblot分析,表达产物能被标准抗α毒素血清识别。     

Solvent-free lipase-catalysed synthesis of saccharide-fatty acid esters: closed-loop bioreactor system with in situ formation of metastable suspensions

Abstract

Saccharide-fatty acid esters – biodegradable, biocompatible and non-ionic bio-based surfactants derived from inexpensive renewable agricultural sources, utilized in foods, cosmetics and pharmaceuticals, have been synthesized under solvent-free conditions using a closed-loop system operated under continuous recirculation consisting of a reservoir, a peristaltic pump and a packed-bed bioreactor containing immobilized Rhizomucor miehei lipase (RML). Metastable suspensions of saccharide crystals were formed through continuous stirring in the reservoir with an in-line filter of 180 µm normal size preventing larger crystals from clogging the tubing. The initial reaction medium consisted of oleic acid/fructose-oleic acid esters 95/5 w/w, with saccharide added periodically to the reservoir to replace consumed acyl acceptor substrate. The liquid-phase water concentration was retained at a previously determined optimal value of ～0.4 wt % through free evaporation of the co-product, water, in the reservoir during the initial phase of the reaction, and N₂(g) bubbling and vacuum pressure after 40 h of reaction. After 200 h, the reaction mixture contained 84 wt % ester, of which 90% of the ester consisted of monoester. Equivalently, the productivity was 0.195 mmol_Ester h^??1 g_RML^??1. The resultant technical grade product can potentially be used directly, without further purification. A mathematical model based on mass balances and a zeroth-order kinetic model was successfully developed to predict the concentration of substrates (oleic acid and saccharide) in the reservoir during the time course of reaction.     

C型产气荚膜梭菌β2毒素基因的克隆与表达

利用PCR技术,从C型产气荚膜梭菌染色体基因组中扩增了0．72kb的β2毒素基因,将纯化的PER产物与载体pGEM—T连接,转化至受体菌JM109中,经NcoI／Bam HI和Bam HI／Eco RI酶切鉴定及核苷酸序列测定证实,重组质粒pXCPB2中含有陡毒素基因。随后用Nco I／Bam HI酶切质粒pXCPB2,回收β2毒素基因片段,插入到事先经同样酶切处理的载体pET-28c中相应酶切位点,构建了表达质粒pETXB2,经Nco I／Bam HI和Nco I／Hind Ⅲ／Bam HI酶切鉴定及核苷酸序列测定证实,表达质粒含有陡毒素基因且基因序列和阅读框架正确。重组菌株BL21（DE3）（pETXB2）表达产物经ELISA检测和SDS—PAGE分析,重组菌株表达的β2毒素蛋白能够被如毒素抗体识别,其表达量占菌体总蛋白相对含量的13．26％。     

Efficient expression and purification of methyltransferase in acetyl-coenzyme a synthesis pathway of the human pathogen Clostridium difficile

The Wood-Ljungdahl pathway is responsible for acetyl-CoA biosynthesis and used as a major mean of generating energy for growth in some anaerobic microbes. Series of genes, from the anaerobic human pathogen Clostridium difficile, have been identified that show striking similarity to the genes involved in this pathway including methyltetrahydrofolate- and corrinoid-dependent methyltransferase. This methyltransferase plays a central role in this pathway that transfers the methyl group from methyltetrahydrofolate to a cob(I)amide center in the corrinoid iron-sulfur protein. In this study, we developed two efficient expression and purification methods for methyltransferase from C. difficile for the first time with two expression vectors MBPHT-mCherry2 and pETDuet-1, respectively. Using the latter vector, more than 50mg MeTr was produced per liter Luria-Bertani broth media. The recombinant methyltransferase was well characterized by SDS-PAGE, gel filtration chromatography, enzyme assay and far-UV circular dichroism (CD). Furthermore, a highly effective approach was established for determining the methyl transfer activity of the methyltetrahydrofolate- and cobalamin-dependent methyltransferase using exogenous cobalamin as a substrate by stopped-flow method. These results will provide a solid basis for further study of the methyltransferase and the Wood-Ljungdahl pathway.     

C型产气荚膜梭菌α毒素基因的克隆与表达

利用PCR技术,从C型产气荚膜梭菌染色体基因组中扩增了1．2kb的α毒素基因,将纯化的PCR产物与载体pGEM-T连接,转化至受体菌JM109中,经NcoI／EcoRI和BamHI／EcoRI酶切鉴定及核苷酸序列测定证实,重组质粒pXCPAl中含有α毒素全基因。随后用NcoI／EcoRI酶切质粒pXCPAl,回收α毒素基因片段,插入到事先经同样酶切处理的载体pET-28c中相应酶切位点,构建了表达质粒pETXAl,经NcoI／EcoRI和BamHI／EcoRI酶切鉴定及核苷酸序列测定证实,表达质粒含有α毒素基因且基因序列和阅读框架正确。重组菌株BL21(DE3)(pETXAl)表达产物经ELISA检测和SDS-PAGE分析,重组菌株表达的α毒素蛋白能够被α毒素单抗识别,其表达量占菌体总蛋白相对含量的16．28％。     

Biodegradation of paint stripper solvents in a modified gas lift loop bioreactor

Paint stripping wastes generated during the decontamination and decommissioning of former nuclear facilities contain paint stripping organics (dichloromethane, 2-propanol, and methanol) and bulk materials containing paint pigments. It is desirable to degrade the organic residues as part of an integrated chemical-biological treatment system. We have developed a modified gas lift loop bioreactor employing a defined consortium of Rhodococcus rhodochrous strain OFS and Hyphomicrobium sp. DM-2 that degrades paint stripper organics. Mass transfer coefficients and kinetic constants for biodegradation in the system were determined. It was found that transfer of organic substrates from surrogate waste into the air and further into the liquid medium in the bioreactor were rapid processes, occurring within minutes. Monod kinetics was employed to model the biodegradation of paint stripping organics. Analysis of the bioreactor process was accomplished with BIOLAB, a mathematical code that simulates coupled mass transfer and biodegradation processes. This code was used to fit experimental data to Monod kinetics and to determine kinetic parameters. The BIOLAB code was also employed to compare activities in the bioreactor of individual microbial cultures to the activities of combined cultures in the bioreactor. This code is of benefit for further optimization and scale-up of the bioreactor for treatment of paint stripping and other volatile organic wastes in bulk materials. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 163-169, 1997.     

Plasmid localization of a type E botulinal neurotoxin gene homologue in toxigenic Clostridium butyricum strains, and absence of this gene in non-toxigenic C. butyricum strains

It has been shown recently that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755) contain a botulinal neurotoxin type E (BoNT/E) gene closely related to that of C. botulinum type E. In this study, we show that this gene is located on a large plasmid in the two toxigenic C. butyricum strains and is absent in 18 non-toxigenic C. butyricum and C. beijerinckii strains. Interestingly, the 230 bp upstream and the 1260 bp downstream of the neurotoxin coding sequence are not present in either the non-toxigenic C. butyricum or C. beijerinckii strains. Our data suggest a BoNT/E gene transfer from C. botulinum E to originally non-toxigenic C. butyricum strains.     

The profile of enzymes relevant to solvent production during direct fermentation of sago starch by <Emphasis Type="Italic">Clostridium saccharobutylicum</Emphasis> P262 utilizing different pH control strategies

The profile of enzymes relevant to solvent production during direct fermentation of sago starch by Clostridium saccharobutylicum P262 in a 2 L stirred tank fermenter was determined utilizing different pH control strategies. During fermentation without pH control (initial pH of 6), the specific activity of crotonase, thiolase, and β-hydroxybutyryl-CoA dehydrogenase increased proportionally with solvent production. The highest crotonase (3,450.7 kat) and phosphotransbutyrylase activity (1,475.6 kat) was observed in fermentation where pH was maintained at 5 during the acidogenic phase and corresponded to a fairly high acid accumulation but low solvent production. During fermentation with a controlled pH of 5.25 during the sol-ventogenic phase, the highest thiolase specific activity (255.7 kat) was obtained and corresponded to the highest production of acetone. On the other hand, the highest specific activities of crotonase, β-hydroxybutyryl-CoA dehydrogenase, and phosphotransbutyrylase were observed at pH 5.5 and corresponded to the highest production of ethanol and butanol. Butyryl-CoA dehydrogenase had no significance role in solvent fermentation. These results suggested that pH control strategies were important for improvement of solvent production during direct fermentation of sago starch by C. saccharobutylicum.     

Effect of sparger design on hydrodynamics of a gas recirculation anaerobic bioreactor

The effects of sparger design and gas flow rate on, gas holdup distribution and liquid (slurry) recirculation velocity have been studied in a surrogate anaerobic bioreactor used for treating bovine waste with a conical bottom mixed by gas recirculation. A single orifice sparger (SOS) and a multi-orifice ring sparger (MORS) with the same orifice open area and gas flow rates (hence the same process power input) are compared in this study. The advanced non-invasive techniques of computer automated tomography (CT) and computer automated radioactive particle tracking (CARPT) were employed to determine gas holdup, liquid recirculation velocity, and the poorly mixed zones. Gas flows (Q(g)) ranging of 0.017 x 10(-3) m(3)/s to 0.083 x 10(-3) m(3)/s were used which correspond to draft tube superficial gas velocities ranging from 1.46 x 10(-2) m/s to 7.35 x 10(-2) m/s (based on draft tube diameter). Air was used for the gas, as the molecular weights of air and biogas (consisting mainly of CH(4) and CO(2)) are in the same range (biogas: 28.32-26.08 kg/kmol and air: 28.58 kg/kmol). When compared to the SOS for a given gas flow rate, the MORS gave better gas holdup distribution in the draft tube, enhanced the liquid (slurry) recirculation, and reduced the fraction of the poorly mixed zones. The improved gas holdup distribution in the draft tube was found to have increased the overall liquid velocity. Hence, for the same process power input the MORS system performed better by enhancing the liquid recirculation and reducing the poorly mixed zones.     

Germination-specific cortex-lytic enzymes from Clostridium perfringens S40 spores: time of synthesis, precursor structure and regulation of enzymatic activity

Germination-specific enzymes, an amidase and a muramidase, of Clostridium perfringens S40 were synthesized at the time of forespore formation during sporulation. The amidase had a unique precursor structure consisting of four domains: the N-terminal pre-sequence, the N-terminal pro-sequence, mature enzyme and the C-terminal pro-sequence. The N-terminal pre-sequence and the C-terminal pro-sequence were sequentially processed at the time of development of phase-bright spores, and the resulting inactive pro-enzyme was activated by cleavage of the N-terminal pro-sequence with a specific protease during germination. A possible mechanism for the regulation of activity of muramidase, which is produced as a mature form and does not need processing for activation, is presented.