Production, Purification, and Characterization of a Xylooligosaccharides-forming Xylanase from High-butanol-producing Strain Clostridium sp. BOH3

An endo-acting xylanase is isolated from the culture medium of Clostridium sp. BOH3 when xylan, glucose, xylose, or sugarcane bagasse hydrolysate (SBH) is used as a carbon source. Crude xylanase is purified by using an anionic Q-column with a yield of 39 %. The pure xylanase has a molecular weight of 35.8 kDa, and it shows optimal activity at pH 5 and 60 °C. When beechwood xylan is used as a substrate, this xylanase liberates short oligosaccharides (XOS) predominantly, ranging from xylobiose (X2) to xylopentaose (X5). However, no xylose can be detected, suggesting that this is an endo-β-1,4-xylanase. Kinetic study of this xylanase reveals that K _m and V _max are 1.36 mg/ml and 212 μmol/(min. mg protein), respectively. On the basis of amino acid sequence, this enzyme shows homology to xylanase (xynb) from Clostridium acetobutylicum ATCC 824, but this enzyme has several distinctive characteristics. For example, its activity can be enhanced with the addition of divalent metal ions, and it produces XOS exclusively when xylan is used as a substrate. These unique characteristics suggest that this is a new enzyme.     

Butanol production from thin stillage using Clostridium pasteurianum

The production of butanol from thin stillage by Clostridium pasteurianum DSM 525 was evaluated in the paper. At initial pH values ranging from 5.0 to 7.0 C. pasteurianum DSM 525 produced 6.2-7.2 g/L of butanol utilizing glycerol in thin stillage as the main carbon source, with yields of 0.32-0.44 g butanol produced/g glycerol consumed, which are higher than previously reported yields (e.g., 0.14-0.31 g butanol/g glycerol, Biebl, 2001). Lactic acid in the thin stillage acted as a buffering agent, maintaining the pH of the medium within a range of 5.7-6.1. Lactic acid was also utilized along with glycerol, enhancing butanol production (6.5 g/L butanol vs. 8.7 g/L butanol with 0 and 16 g/L lactic acid, respectively). These results demonstrate the feasibility of cost-effective butanol production using thin stillage as a nutrient-containing medium with a pH buffering capacity.     

Butanol production from corn fiber xylan using Clostridium acetobutylicum

Acetone, butanol, and ethanol (ABE) were produced from corn fiber arabinoxylan (CFAX) and CFAX sugars (glucose, xylose, galactose, and arabinose) using Clostridium acetobutylicum P260. In mixed sugar (glucose, xylose, galactose, and arabinose) fermentation, the culture preferred glucose and arabinose over galactose and xylose. Under the experimental conditions, CFAX (60 g/L) was not fermented until either 5 g/L xylose or glucose plus xylanase enzyme were added to support initial growth and fermentation. In this system, C. acetobutylicum produced 9.60 g/L ABE from CFAX and xylose. This experiment resulted in a yield and productivity of 0.41 and 0.20 g/L x h, respectively. In the integrated hydrolysis, fermentation, and recovery process, 60 g/L CFAX and 5 g/L xylose produced 24.67 g/L ABE and resulted in a higher yield (0.44) and a higher productivity (0.47 g/L x h). CFAX was hydrolyzed by xylan-hydrolyzing enzymes, and ABE were recovered by gas stripping. This investigation demonstrated that integration of hydrolysis of CFAX, fermentation to ABE, and recovery of ABE in a single system is an economically attractive process. It is suggested that the culture be further developed to hydrolyze CFAX and utilize all xylan sugars simultaneously. This would further increase productivity of the reactor.     

Butanol production from corncob residue using Clostridium beijerinckii NCIMB 8052

Aims: Not all lactic acid bacteria possess the ability to confer health benefits for the host. Thus, it becomes necessary to screen and characterize numerous strains to obtain ideal probiotics. Here, two Lactobacillus plantarum strains (CECT 7315 and CECT 7316) were isolated and characterized. Methods and Results: In vitro and in vivo tests were carried out for demonstrating the abilities as probiotics of CECT 7315/CECT 7316 Lact. plantarum strains. Both strains showed high ability to survive at gastro‐intestinal tract conditions and to adhere to intestinal epithelial cells, as well as great inhibitory activity against a wide range of enteropathogens and ability to induce the production of anti‐inflammatory cytokine IL‐10. Conclusions: Lactobacillus plantarum CECT 7315/CECT 7316 because of their potential probiotic properties could be excellent candidates for being tested in clinical trials aimed to demonstrate beneficial effects on human health. Significance and Impact of the Study: Probiotics are live micro‐organisms that confer a health benefit for the host. However, not all the lactic acid bacteria possess the ability to confer health benefits for the host. In this study, two Lact. plantarum strains (CECT 7315 and CECT 7316) were isolated and characterized to demonstrate their excellent qualities as potential probiotic strains.     

Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran

Wheat bran, a by-product of the wheat milling industry, consists mainly of hemicellulose, starch and protein. In this study, the hydrolysate of wheat bran pretreated with dilute sulfuric acid was used as a substrate to produce ABE (acetone, butanol and ethanol) using Clostridium beijerinckii ATCC 55025. The wheat bran hydrolysate contained 53.1 g/l total reducing sugars, including 21.3 g/l of glucose, 17.4 g/l of xylose and 10.6 g/l of arabinose. C. beijerinckii ATCC 55025 can utilize hexose and pentose simultaneously in the hydrolysate to produce ABE. After 72 h of fermentation, the total ABE in the system was 11.8 g/l, of which acetone, butanol and ethanol were 2.2, 8.8 and 0.8 g/l, respectively. The fermentation resulted in an ABE yield of 0.32 and productivity of 0.16 g l⁻¹ h⁻¹. This study suggests that wheat bran can be a potential renewable resource for ABE fermentation.     

拜氏梭菌Clostridium beijerinckii NCIMB 8052发酵玉米皮生产丁醇

玉米皮作为玉米淀粉加工的副产物,是一种可用于生产液体燃料的潜在廉价优质的生物质资源。本文以玉米皮为原料,对拜氏梭菌发酵生产丁醇进行了研究。实验结果表明,玉米皮首先在最优的预处理温度140℃下使用0.5%硫酸水溶液以固液比1∶8处理20 min,再添加200 IU/g底物糖化酶、1.0 IU/g底物木聚糖酶进行酶解,可以使原料中的淀粉和半纤维素转化为可发酵糖,此时水解液中的总糖浓度为50.46 g/L。然后使用1.0%的活性炭对水解液进行脱毒处理以去除发酵抑制物,再进行丁醇发酵,丁醇产量为9.72 g/L,总溶剂产量可达14.09 g/L,糖醇转化率为35.1%。上述研究结果证明玉米皮作为一种粮食加工废弃物用于液体燃料丁醇的生产在技术上是完全可行的。     

Purification and characterization of the NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum

An NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum was purified 15-fold with a yield of 46%. It was homogeneous by gel electrophoresis after three chromatographic steps. The apparent molecular mass was estimated by column chromatography to be 240 kDa. SDS-gel electrophoresis revealed the presence of 33 kDa subunits. Substrates of the enzyme were ethyl and methyl 3-oxobutyrate, 3-oxobutyryl-N-acetylcysteamine thioester, and 3-oxobutyryl coenzyme A. The specific activities were 340 and 10 U (mg protein)^-1 for the reduction of 3-oxobutyryl coenzyme A and ethyl 3-oxobutyrate, respectively; the Michaelis constants were 300 M and 300 mM, respectively. The identity of 12 N-terminal amino acid residues was determined. The ezmyme was used in a preparative reduction of substrate, yielding ethyl (S)-3-hydroxybutyrate (>99% enantiomeric excess).     

Two cellulolytic Clostridium species: Clostridium cellulosi sp. nov. and Clostridium cellulofermentans sp. nov.

Two cellulolytic clostridia, one thermophilic and the other mesophilic, were isolated and characterized. Cells of the thermophile are gram-negative rods that are motile with lophotrichous flagella and spherical terminal endospores which swell the cells. The optimum growth temperature is 55 to 60 degrees C, with a range of 40 to 65 degrees C. The deoxyribonucleic acid composition is 35 mol% G + C. The name Clostridium cellulosi sp. nov. is proposed. The type strain is AS 1.1777. Cells of the mesophile are gram negative and motile with peritrichous flagella and terminal oval or spherical spores which swell the cells. The deoxyribonucleic acid composition is 34 mol% G + C. The name Clostridium cellulofermentans sp. nov. is proposed. The type strain is AS 1.1775. Both C. cellulosi AS 1.1777 and C. cellulofermentans AS 1.1775 are deposited in the China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Academia Sinica, Beijing, People's Republic of China.     

A rapid method for the screening of plasmids from Clostridium acetobutylicum and other Clostridium sp.

Summary A rapid method was developed for plasmid DNA screening from micro volume cultures of Clostridium acetobutylicum. It involves protoplastization by lysozyme, nuclease inhibition by incorporating EDTA or DEP, followed by lysis with high concentration of SDS. The whole lysate is applied directly to electrophoretic analysis.     

Crystallization and Properties of Amine Dehydrogenase from Pseudomonas sp.

An amine dehydrogenase was purified and crystallized from the cell free extract of a Pseudomonas sp., isolated from soil by means of the enrichment technique. The crystalline enzyme gave a single band on polyacrylamide gel electrophoresis and the molecular weight of the enzyme was estimated to be 100,000 by gel filtration on a Sephadex column. Upon SDS-gel electrophoresis, the enzyme was dissociated into two nonidentical subunits having molecular weights of 60,000 (dehydrogenase) and 39,000 (cytochrome c). The absorption spectrum of the enzyme showed absorption maxima at 550 nm, 524 nm, 411 nm and 280 nm, and a broad shoulder at around 350 nm, indicating that the enzyme was purified as a dehydrogenase-cytochrome c complex. The prosthetic group of the dehydrogenase was identified as covalently bound pyrroloquinoline quinone. The enzyme showed a broad substrate specificity toward various amines including aliphatic monoamines, aliphatic diamines, aromatic amines and polyamines.     

Clostridium hathewayi sp. nov., from human faeces

A strictly anoxic, Gram-positive, sporeforming, rod-shaped bacterium was isolated from a chemostat inoculated with human faeces. The bacterium used carbohydrate as fermentable substrates, producing acetate, ethanol, carbon dioxide and hydrogen as the major products of glucose metabolism, and possessed a G + C content of 50.7 to 50.9 mol%. Comparative 16S rRNA gene sequencing showed that the unidentified bacterium represents a previously unrecognised sub-line within the Clostridium coccoides rRNA group of organisms. The nearest relatives of the unknown bacterium corresponded to Clostridium algidixylanolyticum, C. aerotolerans, C. celerecrescens, C. indolis, C. sphenoides, C. methoxybenzovorans and C. xylanolyticum but 16S rRNA sequence divergence values of >4% demonstrated that it represents a novel species. Based on the presented findings a new species, Clostridium hathewayi, is described. The type strain of Clostridium hathewayi is DSM = 13479T (= CCUG 43506 T).     

Characterization of a d-psicose-producing enzyme, d-psicose 3-epimerase, from Clostridium sp.

The gene coding for d-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co²⁺ as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was d-psicose, and the K _m, turnover number (k _cat), and catalytic efficiency (k _cat/K _m) for d-psicose were 227 mM, 32,185 min^?1, and 141 min^?1 mM^?1, respectively. At pH 8.0 and 55 °C, 120 g d-psicose l^?1 was produced from 500 g d-fructose l^?1 after 5 h.     

Clostridium barkeri sp. n

Clostridium barkeri sp. n. has been described, and its relationship to other clostridia is discussed.     

Potential Applications in Sewage Bioremediation of the Highly Efficient Pyridine-Transforming Paenochrobactrum sp.

Applied Biochemistry and Microbiology - A pyridine-transforming strain P2 was isolated from sewage collected from Guangzhou oil stain field(China).According to the system analysis, it was...     

Butanol from whey ultrafiltrate: batch experiments with Clostridium beyerinckii LMD 27.6

Summary For batch fermentations by Clostridium beyerinckii LMD 27.7 (formerly known as Clostridium butylicum) whey ultrafiltrate, glucose, lactose, and galactose were used as substrates. The aims of the experiments were to find the conditions for butanol production from whey ultrafiltrate and to compare the results with those of other substrates. The conditions necessary for butanol production were established. The mean solvent productivity found on whey ultrafiltrate fermentation was two to three times lower than that found on glucose; the overall solvent yields were comparable. Butanol production from galactose and mixtures of glucose and galactose was also possible.     

Acetone, Isopropanol, and Butanol Production by Clostridium beijerinckii (syn. Clostridium butylicum) and Clostridium aurantibutyricum

Thirty-four strains representing 15 species of anaerobic bacteria were screened for acetone, isopropanol, and n-butanol (solvent) production. Under our culture conditions, several strains of Clostridium beijerinckii and C. aurantibutyricum produced at least 40 mM n-butanol (C. acetobutylicum strains produced up to 41 mM n-butanol under similar conditions). Both solvent-producing and non-solvent-producing strains of C. beijerinckii have high DNA homology with a reference strain of C. beijerinckii. Strains labeled “Clostridium butylicum” are phenotypically similar to C. beijerinckii and showed at least 78% DNA homology to a reference strain of C. beijerinckii. Therefore, these “C. butylicum” strains are members of C. beijerinckii. An earlier DNA homology study has shown that C. beijerinckii, C. aurantibutyricum, and C. acetobutylicum are distinct species.     

Purification and Characterization of 6-Phosphogluconate Dehydrogenase from Phormidium sp.

the native enzyme was 104,000 by gel filtration, and SDS-polyacrylamide gel electrophoresis showed that the enzyme consisted of two subunits with an identical molecular weight of 52,000. The optimum pH of the reaction was 8.0. The Km values for 6-phosphogluconate and NADP were 3.6×10^?5m and 1.3 × 10^?5m, respectively. The enzyme showed no Mg^2𠀫 requirement for the activity, but was activated by Mn^2𠀫 and Ca^2𠀫. The enzyme was inhibited by sulfhydryl reagents, indicating that a sulfhydryl group may be involved in the active site of the enzyme. The enzyme was also inhibited by NADPH₂, ATP, and the intermediates formed during photosynthesis. The substrate 6-phosphogluconate and cofactor NADP partially protected the enzyme from inactivation. The enzyme had enzymological and physicochemical properties similar to enzymes isolated from other sources.