Conversion for Avicel and AFEX pretreated corn stover by Clostridium thermocellum and simultaneous saccharification and fermentation: insights into microbial conversion of pretreated cellulosic biomass

In this study, efforts were taken to compare solubilization of Avicel and AFEX pretreated corn stover (AFEX CS) by SSF and Clostridium thermocellum fermentation, with an aim to gain insights into microbial conversion of pretreated cellulosic biomass. Solubilization rates for AFEX CS are comparable for the two systems while solubilization of Avicel is much faster by C. thermocellum. Initial catalyst loading impacts final cellulose conversion for SSF but not for C. thermocellum. Hydrolysis of the two substrates using cell-free C. thermocellum fermentation broth revealed much smaller difference in cellulose conversion than the difference observed for growing cultures. Tests on hemicellulose removal and particle size reduction for AFEX CS indicated that substrate accessibility is very important for enhanced solubilization by C. thermocellum.     

Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405)

Our lab and most others have not been able to close a carbon balance for fermentation by the thermophilic, cellulolytic anaerobe, Clostridium thermocellum. We undertook a detailed accounting of product formation in C. thermocellum ATCC 27405. Elemental analysis revealed that for both cellulose (Avicel) and cellobiose, >92% of the substrate carbon utilized could be accounted for in the pellet, supernatant and off-gas when including sampling. However, 11.1% of the original substrate carbon was found in the liquid phase and not in the form of commonly-measured fermentation products - ethanol, acetate, lactate, and formate. Further detailed analysis revealed all the products to be <720 da and have not usually been associated with C. thermocellum fermentation, including malate, pyruvate, uracil, soluble glucans, and extracellular free amino acids. By accounting for these products, 92.9% and 93.2% of the final product carbon was identified during growth on cellobiose and Avicel, respectively.     

Application of vitamin B(12)-targeting site on Lactobacillus helveticus B-1 to vitamin B(12) assay by chemiluminescence method

Lactobacillus helveticus B-1 is assumed to have a vitamin B(12)-targeting (or B(12)-binding) site on the cells, since the binding reaction of vitamin B(12) with L. helveticus B-1 cells proceeded instantly and quantitatively. This reaction is specific to complete B(12) compounds, cobalamins, and can be used for a vitamin B(12) assay method by chemiluminescence. The calibration graph was linear from 0.1 to 10.0 ng/mL. The B(12) contents in oyster and sardine were 75.9 and 39.4 microg/100g, respectively. These values were very close to those obtained using a chemilumi-ADVIA Centaur immunoassay system with intrinsic factor and to those obtained by microbiological assays.     

Thermophilic anaerobic fermentation of hemicellulose and hemicellulose-derived aldose sugars by Thermoanaerobacter strain B6A

The thermophilic, anaerobic fermentation of hemicellulosic subtrates by Thermoanaerobacter strain B6A was investigated using a variety of commercially-available hemicelluloses which had been characterized by physical and chemical analysis. Products of hemicellulose fermentation included ethanol, acetic acid, lactic acid, H₂, and CO₂ in ratios which varied depending on the hemicellulose used. Both the rate and extent of substrate utilization (as estimated from product formation) varied in the order: unidentified mannan/xyloglucan > xylan > 4-O-methylglucuronoxylan > arabinoxylan > type II arabinogalactan=0. Rates of product formation were enhanced up to twofold by autoclaving of substrates,which partially depolymerized the substrates and in some cases altered their composition. Total extent of product formation, however, was similar in autoclaved and non-autoclaved substrates. Hemicellulose fermentations were mediated by one or more constitutive extracellular enzyme activities. Component monosaccharides of hemicelluloses in their natural isomeric configurations supported rapid growth (_max = 0.3–1.0 h^–1), while unnatural isomers were not utilized.The wide carbohydrate utilization spectrum of this strain apparently reflects its role as a versatile primary consumer in the hot spring bacterial-algal mat from which it was isolated.Abbreviations GC Gas chromatography - HPLC High-performance liquid chromatography - MS Mass spectroscopy - TLC Thinlayer chromatography - TMS Trimethylsilyl Contribution No. 3696 of the Central Research and Development Department     

Clostridium thermocellum与Bacillus licheniformis共培养分解纤维素的性质

【目的】采取人工构建复合菌系的方法探索微生物协同降解纤维素的机理及菌间关系。【方法】从一组高温发酵木质纤维素原料产沼气的菌群中分离获得若干菌株,其中一株细菌经16S rRNA基因全序列测序比对后鉴定为地衣芽孢杆菌(Bacillus licheniformis),将该菌株与厌氧纤维素分解菌Clostridium thermocellum CTL-6进行共培养,菌株组合表现出很强的滤纸纤维素分解能力。【结果】两菌共培养9 d,累计滤纸分解量为484.6 mg,滤纸相对分解率高达93.2%;pH变化呈先下降后逐步回升,培养3 d后pH由初始时的7.00降到最低值6.57,第9天升至7.73;菌株组合能同时产生纤维素酶和半纤维素酶,培养过程中两种酶活性大小均呈不断上升趋势,最大值分别为0.32 U/m L和0.57 U/m L。利用HPLC监测了乳酸、甲酸、乙酸、丙酸和丁酸5种有机酸含量的变化,其中丁酸、丙酸代谢量最高,分别为1 477.3 mg/L和1 068.8 mg/L;除丙酸外,其他4种有机酸含量变化趋势与滤纸降解的变化均无明显相关性。5种有机酸总含量的变化与p H的变化趋势一致,表明对pH变化起决定性作用的很可能是某种未检测的酸性较强的物质含量变化。【结论】Bacillus licheniformis能有效促进Clostridium thermocellum CTL-6的纤维素分解活性,且该菌株组合可作为后期进一步构建纤维素甲烷转化复合菌系的基础。     

Processing of glutathionylcobalamin by a bovine B12 trafficking chaperone bCblC involved in intracellular B12 metabolism

Glutathionylcobalamin (GSCbl) is a biologically relevant vitamin B₁₂ derivative and contains glutathione as the upper axial ligand thought formation of a cobalt-sulfur bond. GSCbl has been shown to be an effective precursor of enzyme cofactors, however processing of the cobalamin in intracellular B₁₂ metabolism has not been fully elucidated. In this study, we discovered that bCblC, a bovine B₁₂ trafficking chaperone, catalyzes elimination of the glutathione ligand from GSCbl by using the reduced form of glutathione (GSH). Deglutathionylation products are base-off cob(II)alamin and glutathione disulfide, which are generated stoichiometrically to GSH. Although cob(I)alamin was not detected due to its instability, deglutathionylation is likely analogous to dealkylation of alkylcobalamins, which uses the thiolate of GSH for nucleophilic displacement. The catalytic turnover number for the deglutathionylation of GSCbl is ?1.62 ± 0.13 min⁻¹, which is, at least, an order of magnitude higher than that for elimination of upper axial ligands from other cobalamins. Considering the prevalence of GSH at millimolar concentrations in cells, our results explain the previous finding that GSCbl is more effective than other cobalamins for synthesis of enzyme cofactors.     

Influence of initial cellulose concentration on the carbon flow distribution during batch fermentation by Clostridium thermocellum ATCC 27405

The objective of this research was to understand how carbon loading influences hydrogen (H₂) synthesis and metabolic flow patterns in the thermophilic, cellulolytic bacterium, Clostridium thermocellum. C. thermocellum was cultivated in batch cultures with high (5 g L⁻¹) and low (1 g L⁻¹) initial concentrations of α-cellulose at 60°C. The growth rate of C. thermocellum was 22% lower (0.15 h⁻¹) in cultures with low-cellulose concentration compared with cultures with high-cellulose concentrations. Although substrate depletion coincided with the end of log-growth in low-cellulose cultures, the prime reason for growth arrest in high-cellulose cultures was not identified. Ethanol, acetate, and formate were the major soluble end-products with concomitant release of H₂ and CO₂ under both conditions. Lactate appeared during the late log phase in high-carbon cultures when pH dropped below 6.4 and became the major end-product in stationary phase. During the exponential phase of cell growth, significantly higher yields for H₂ and acetate (1.90 ± 0.14 and 1.11 ± 0.04 mol/mol glucose equivalent, respectively) were obtained from low-cellulose cultures compared to those from high-cellulose cultures. The maximum specific rate of H₂ production, 6.41 ± 0.13 mmol H₂/g dry cell/h, obtained during the exponential phase from low-carbon cultures was about 37% higher than that obtained from high-carbon cultures.     

Expression, purification and structural characterization of the scaffoldin hydrophilic X-module from the cellulosome of Clostridium thermocellum

The cellulosome is a membrane-bound, extracellular multi-subunit complex responsible for the degradation of crystalline cellulose by a number of organisms including anaerobic bacteria and fungi. The hydrophilic X-module (CipA-X) from the modular scaffoldin subunit of Clostridium thermocellum cellulosome has been proposed to play various roles in cellulosomal function, including thermal and structural stability. Towards elucidating the function of CipA-X using structural and biophysical studies, the region comprising residues 1692-1785 from the C. thermocellum CipA cDNA encoding CipA-X was cloned into a pET21b expression vector. When expressed in Escherichia coli, the C-terminal His-tagged protein accumulated in the insoluble fraction. Cell fractionation experiments showed that the recombinant protein was localized to inclusion bodies. Refolding and purification involved denaturation of the whole cell lysate by addition of urea, followed by a nickel-Sepharose chromatography step and dialysis into native conditions (25 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 10 mM EDTA). A final gel filtration step purified the protein to homogeneity, yielding 40 mg/L. The two-dimensional 1H-15N correlation spectrum of uniformly 15N-labelled CipA-X showed the characteristics of a well-folded protein comprising significant beta-structure, which is in agreement with the circular dichroism data.     

The AAA motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy

Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and CobT, with molecular masses of 137, 37 and 71 kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA⁺ superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and CobT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and CobT form a chaperone-like complex, characteristic for the AAA⁺ class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.     

丁酸梭菌发酵培养基的优化及发酵产物对黄曲霉毒素B1的降解

【背景】丁酸梭菌是专性厌氧的新一代芽孢益生菌,耐热、耐酸、抗逆性强,极具应用价值和开发前景。【目的】优化丁酸梭菌发酵培养基并初步研究其发酵液对黄曲霉菌的抑制作用和降解黄曲霉毒素B₁ (aflatoxin B₁, AFB₁)的能力。【方法】利用响应面法对发酵培养基进行优化,采用牛津杯法对丁酸梭菌发酵液抑制黄曲霉菌生长进行研究,并通过酶联免疫法测定发酵液对AFB₁的降解能力。【结果】优化后的发酵培养基为：葡萄糖18.1g/L,大豆蛋白胨29.7g/L,磷酸氢二钾3.8 g/L,氯化钠2.0 g/L,乙酸钠4.0 g/L,结晶硫酸镁1.2 g/L,L-半胱氨酸盐酸盐0.3 g/L。优化后的丁酸梭菌生物量由8.99×10⁸个/mL提高至2.28×10⁹个/mL,是优化前的2.54倍。丁酸梭菌发酵液对致病真菌黄曲霉菌的抑菌效果十分显著,其上清液经浓缩后对AFB₁降解72h的降解率达到68.65%,初步分析表明上清液中对AFB₁     

Interconversions of different forms of vitamin B6 in tobacco plants

There are six different vitamin B₆ (VB₆) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5′-phosphate (PLP), pyridoxamine 5′-phosphate (PMP), and pyridoxine 5′-phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB₆ in the human diet, and VB₆ plays an important role in plants, the mechanisms underlying the interconversions of different VB₆ forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100 mg/L of PM, PL or PN and the abundance of the different B₆ vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB₆ was about 3.9 μg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB₆ by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B₆ vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of non-phosphorylated forms.     

Development of a real time PCR Taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum

In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.     

The binding properties of the human receptor for the cellular uptake of vitamin B12

Cellular uptake of vitamin B(12) (cobalamin, Cbl) is mediated by a receptor expressed on the plasma membrane that binds transcobalamin (TC) saturated with Cbl and internalizes the TC-Cbl by endocytosis. A few reports have described the characterization of the receptor protein. However, many discrepancies have emerged in the functional and structural properties of the receptor and therefore, the identity and primary structure of this protein remains unconfirmed. In this report, we provide evidence of a 58 kDa monomeric protein as the likely receptor for the uptake of TC-Cbl and that the functional activity is not associated with a 72/144 kDa monomer/dimer with immunoglobulin Fc structural domain that has been purported to be the receptor in a number of publications.     

An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B12 by Pseudomonas denitrificans

In order to improve the productivity of vitamin B(12) by Pseudomonas denitrificans carried out in a 120-m(3) fermenter, the effect of pH on vitamin B(12) biosynthesis was investigated. Results obtained from shake flask experiments showed that the feeding of carbon source (beet molasses or glucose) and methyl-group donor (betaine or choline chloride) significantly influenced the pH and the biosynthesis of vitamin B(12). In contrast to beet molasses or choline chloride, using glucose as a feed medium and betaine as a methyl-group donor, pH could be maintained at a stable range. As a result, higher vitamin B(12) production was achieved. Accordingly, an effective and simplified pH-stat control strategy was established for the fermentation of vitamin B(12) in a 120-m(3) industrial fermenter. When the new pH control strategy was applied, pH was stably kept in the range of 7.15-7.30 during fermentation. Thus, 214.3 mug/mL of vitamin B(12) was achieved.     

Spirochaeta caldaria sp. nov., a thermophilic bacterium that enhances cellulose degradation by Clostridium thermocellum

Two strains of obligately anaerobic, thermophilic spirochetes were isolated from cyanobacterial mat samples collected at freshwater hot springs in Oregon and Utah, USA. The isolates grew optimally between 48° and 52°C, and did not grow at 25° or 60°C. Both strains fermented various pentoses, hexoses, and disaccharides. Amino acids or cellulose did not serve as fermentable substrates for growth. H₂, CO₂, acetate, and lactate were end products of d-glucose fermentation. On the basis of physiological characteristics, guanine + cytosine content of DNA, and comparisons of 16S ribosomal RNA sequences, it was concluded that the two isolates were representatives of a novel species of Spirochaeta for which the name Spirochaeta caldaria is proposed. One of the two strains was grown in coculture with a thermophilic cellulolytic bacterium (Clostridium thermocellum) in a medium containing cellulose as the only fermentable substrate. In the coculture cellulose was broken down at a faster rate than in the clostridial monoculture. The results are consistent with the suggestion that interactions between cellulolytic bacteria and non-cellulolytic spirochetes enhance cellulose breakdown in natural environments in which cellulose-containing plant material is biodegraded.     

Lessons in biology from patients with inborn errors of vitamin B12 metabolism

Background

Since 1975 cells lines from patients with suspected inborn errors of vitamin B₁₂ metabolism have been referred to our laboratory because of elevations of homocysteine, methylmalonic acid, or both.

Design

Cultured fibroblasts from patients were subjected to a battery of tests: incorporation of labelled propionate and methyltetrahydrofolate into cellular macromolecules, to test the functional integrity of methylmalonyl-CoA mutase and methionine synthase, respectively; uptake of labelled cyanocobalamin and synthesis of adenosylcobalamin and methylcobalamin; and, where applicable, complementation analysis.

Results

This approach has allowed for the discovery of novel steps in the cellular transport and metabolism of vitamin B₁₂, including those involving cellular uptake, the efflux of vitamin B₁₂ from lysosomes, and the synthesis of adenosylcobalamin and methylcobalamin. For all of these disorders, the responsible genes have been discovered.

Conclusion

The study of highly selected patients with suspected inborn errors of metabolism has consistently resulted in the discovery of previously unknown metabolic steps and has provided new lessons in biology.     

Effect of nutrient limitation and two-stage continuous fermentor design on productivities during "Clostridium ragsdalei" syngas fermentation

The effect of three limiting nutrients, calcium pantothenate, vitamin B₁₂ and cobalt chloride (CoCl₂), on syngas fermentation using “Clostridium ragsdalei” was determined using serum bottle fermentation studies. Significant results from the bottle studies were translated into single- and two-stage continuous fermentor designs. Studies indicated that three-way interactions between the three limiting nutrients, and two-way interactions between vitamin B₁₂ and CoCl₂ had a significant positive effect on ethanol and acetic acid formation. In general, ethanol and acetic acid production ceased at the end of 9 days corresponding to the production of 2.01 and 1.95 g L⁻¹ for the above interactions. Reactor studies indicated the three-way nutrient limitation in two-stage fermentor showed improved acetic acid (17.51 g g⁻¹ cells) and ethanol (14.74 g g⁻¹ cells) yield compared to treatments in single-stage fermentors. These results further support the hypothesis that it is possible to modulate the product formation by limiting key nutrients during C. ragsdalei syngas fermentation.     

Glutathione increases the binding affinity of a bovine B12 trafficking chaperone bCblC for vitamin B12

Intracellular B₁₂ metabolism involves a B₁₂ trafficking chaperone CblC that is well conserved in mammals including human. The protein CblC is known to bind cyanocobalamin (CNCbl, vitamin B₁₂) inducing the base-off transition and convert it into an intermediate that can be used in enzyme cofactor synthesis. The binding affinity of human CblC for CNCbl was determined to be K_d = ≈6–16 μM, which is relatively low considering sub-micromolar B₁₂ concentrations (0.03–0.7 μM) in normal cells. In the current study, we discovered that the base-off transition of CNCbl upon binding to bCblC, a bovine homolog of human CblC, is facilitated in the presence of reduced form of glutathione (GSH). In addition, GSH dramatically increases the binding affinity for CNCbl lowering the K_d from 27.1 ± 0.2–0.24 ± 0.09 μM. The effect of GSH is due to conformational change of bCblC upon binding with GSH, which was indicated by limited proteolysis and urea-induced equilibrium denaturation of the protein. The results of this study suggest that GSH positively modulates bCblC by increasing the binding affinity for CNCbl, which would enhance functional efficiency of the protein.     

Comparative genotyping of Clostridium thermocellum strains isolated from biogas plants: Genetic markers and characterization of cellulolytic potential

Clostridium thermocellum is among the most prevalent of known anaerobic cellulolytic bacteria. In this study, genetic and phenotypic variations among C. thermocellum strains isolated from different biogas plants were determined and different genotyping methods were evaluated on these isolates. At least two C. thermocellum strains were isolated independently from each of nine different biogas plants via enrichment on cellulose. Various DNA-based genotyping methods such as ribotyping, RAPD (Random Amplified Polymorphic DNA) and VNTR (Variable Number of Tandem Repeats) were applied to these isolates. One novel approach – the amplification of unknown target sequences between copies of a previously discovered Random Inserted Mobile Element (RIME) – was also tested. The genotyping method with the highest discriminatory power was found to be the amplification of the sequences between the insertion elements, where isolates from each biogas plant yielded a different band pattern. Cellulolytic potentials, optimal growth conditions and substrate spectra of all isolates were characterized to help identify phenotypic variations. Irrespective of the genotyping method used, the isolates from each individual biogas plant always exhibited identical patterns. This is suggestive of a single C. thermocellum strain exhibiting dominance in each biogas plant. The genotypic groups reflect the results of the physiological characterization of the isolates like substrate diversity and cellulase activity. Conversely, strains isolated across a range of biogas plants differed in their genotyping results and physiological properties. Both strains isolated from one biogas plant had the best specific cellulose-degrading properties and might therefore achieve superior substrate utilization yields in biogas fermenters.