Effect of carbon sources on cellulase production by clostridium cellulolyticum

The effect of varying carbon source on cellulase activity in C. cellulolyticum has been investigated using a basal culture medium supplemented with various compounds, including Avicel which gave the best results indicating that both the production of cellulase components (FPAse, CMCase, PNPGase and xylanase) and the bacterial hydrolysis yield increase with the degree of crystallinity. Glucose and cellobiose alternative products of cellulolysis, had contrasting effects. At concentrations from 0.5 to 5 mM when mixed with Avicel glucose enhances, while cellobiose inhibits, the production of cellulase. In contrast, in the absence of Avicel, cellobiose has a greater effect in enhancing cellulase production than glucose. At 7.5 g.l⁻¹ other sugars (fructose, xylose and arabinose) are also more effective than cellobiose in promoting cellulase production. Glucose, in contrast to cellobiose, has a greater effect on derepression of Avicelase and inhibits neither Avicelase activity in solution nor bacterial cellulolysis in solid culture media. Cultures grown in a fermenter, using either sugar, show that growth inhibition can be lowered and production greatly improved. The best results were obtained under conditions of cellobiose limitation using ultrafiltration to the growth inhibiting metabolites. Culture carboxymethylcellulose (CMC), a soluble cellulose derivative, causes both immediate growth and a rapid production of CMCase, PNGase and xylanase activities whereas the capacity to hydrolyse cellulose of filter paper (FPase) is absent. The sugars released during cellulolysis inhibit both cell growth and cellulase production indicating that C. cellulolyticum produces a “true cellulase system” subject to end-product regulation.     

Phototrophic hydrogen production from glucose by pure and co-cultures of Clostridium butyricum and Rhodobacter sphaeroides

Phototrophic hydrogen production from glucose by pure and co-cultures of Clostridium butyricum and Rhodobacter sphaeroides was studied in batch experiments. Results showed that in all batches hydrogen was produced after a lag phase of about 10 h; pure culture of R. sphaeroides produced hydrogen at rates substantially lower than C. butyricum. In co-culture systems, R. sphaeroides even with cell populations 5.9 times higher still could not compete with C. butyricum for glucose. In co-culture systems, R. sphaeroides syntrophically interacted with C. butyricum, using the acetate and butyrate produced by the latter as substrate for hydrogen production. Hydrogen production was ceased in all batches when the pH was lowered to the level of pH 6.5, resulting from the accumulation of fatty acids. It was also demonstrated in this study that fluorescence in situ hybridization (FISH) was an effective means for the quantification of the relative abundance of individual bacteria in a co-culture system.     

Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase gene isolated from Clostridium butyricum

The [FeFe]-hydrogenase (hydA) from Clostridium butyricum TERI BH05-2 strain was isolated to elucidate its molecular characterization. A 1953 bp DNA fragment encompassing the ORF and the putative promoter region of hydA gene was PCR amplified and subcloned into pGEM^®-T-Easy cloning vector (pGEM^®-T-hydA). The hydA DNA sequence revealed the presence of a 1725 bp length ORF (including the stop codon) encoding 574 amino acids with a predicted isoelectric point and molecular mass of 6.8 and 63097.67 Da, respectively. The hydA ORF was PCR amplified from pGEM^®-T-hydA and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-5X-hydA) and transformed into Escherichia coli BL-21. The recombinant E. coli BL-21 was investigated for fermentative hydrogen production under anaerobic condition from glucose. Heterologous expression of the Clostridium butyricum hydA resulted in 1.9 fold increase in hydrogen productivity as compared to that from the wild type strain, C. butyricum TERI BH05-2. The hydrogen yield of the recombinant strain was 3.2 mol H₂/mol glucose, 1.68 fold higher than the wild type parent strain.     

Optimization of biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent using response surface methodology

Clostridium butyricum EB6 successfully produced hydrogen gas from palm oil mill effluent (POME). In this study, central composite design and response surface methodology were applied to determine the optimum conditions for hydrogen production (P_c) and maximum hydrogen production rate (R_max) from POME. Experimental results showed that the pH, temperature and chemical oxygen demand (COD) of POME affected both the hydrogen production and production rate, both individually and interactively. The optimum conditions for hydrogen production (P_c) were pH 5.69, 36 °C, and 92 g COD/l; with an estimated P_c value of 306 ml H₂/g carbohydrate. The optimum conditions for maximum hydrogen production rate (R_max) were pH 6.52, 41 °C and 60 g COD/l; with an estimated R_max value of 914 ml H₂/h. An overlay study was performed to obtain an overall model optimization. The optimized conditions for the overall model were pH 6.05, 36 °C and 94 g COD/l. The hydrogen content in the biogas produced ranged from 60% to 75%.     

纤维素酶固态发酵动力学的研究

以稻壳和麸皮混合物为基质,进行纤维素酶固态发酵动力学的研究.以氨基葡萄糖含量表征生物量,建立了纤维素酶固态发酵过程中细胞生长、产酶和二氧化碳释放速率动力学模型,并得到模型参数.动力学分析结果表明,在不同培养温度和基质配比下,最大菌体量与最大酶活呈正相关;在适宜培养温度下,预期最大酶活与最大CO2释放速率也呈一定的正相关关系.     

Biological hydrogen production from phenol-containing wastewater using Clostridium butyricum

Toxicity renders certain industrial effluents unfit for recovering its bioenergy content. An enriched single strain, Clostridium butyricum, was herein applied to fermentatively produce hydrogen from glucose in the presence of 200–1500 mg L⁻¹ of phenol. The enriched C. butyricum yielded hydrogen at approximately 1.4 mol H₂ mol⁻¹ glucose in the presence of 200–400 mg L⁻¹ phenol. Significant inhibition of cell metabolism was noted at phenol concentration >1000 mg L⁻¹. During glucose fermentation, phenol dosed at 200–400 mg L⁻¹ was partly co-degraded. Ethanol and acetate were the primary metabolites, whose yields increased with increasing phenol concentration. The present results revealed the potential to harvest hydrogen from a toxic (phenol-containing) wastewater.     

微波预处理稻壳对纤维素酶固态发酵的影响

以稻壳为原料,通过微波预处理后用于固态发酵生产纤维素酶,研究了微波处理对后续发酵过程的影响。采用正交试验与单因素试验确定了微波处理的条件,并分析了微波功率与处理时间对发酵过程中纤维素酶活性及戊糖、还原糖含量的影响。试验结果表明,用功率为300W的微波处理稻壳7min后进行发酵,可以得到最高的纤维素酶酶活,其中滤纸酶活（干基质）可达7．09 IU／g,CMC酶活（干基质）可达87．24 IU／g,分别比未经处理的稻壳提高了21％和15％。若以单位能耗产生的酶活增加量计算,微波处理稻壳5min后发酵,可以得到最高的酶活性增加量。     

The preparation and application of crude cellulase for cellulose-hydrogen production by anaerobic fermentation

Strategies were adopted to cost-efficiently produce cellulose-hydrogen by anaerobic fermentation in this paper. First, cellulase used for hydrolyzing cellulose was prepared by solid-state fermentation (SSF) on cheap biomass from Trichoderma viride. Several cultural conditions for cellulase production on cheap biomass such as moisture content, inoculum size and culture time were studied. And the components of solid-state medium were optimized using statistical methods to further improve cellulase capability. Second, the crude cellulase was applied to cellulose-hydrogen process directly. The maximal hydrogen yield of 122 ml/g-TVS was obtained at the substrate concentration of 20 g/L and cultured time of 53 h. The value was about 45-fold than that of raw corn stalk wastes. The hydrogen content in the biogas was 44–57%(v/v) and there was no significant methane gas observed.     

纤维素原料生产燃料酒精的技术现状

综述了纤维素原料的预处理及水解为葡萄糖技术的研究进展，介绍了纤维素原料发酵生产酒精技术的概况，对不同的预处理、水解和发酵方法进行了比较，指出制约以纤维素为原料生产酒精的因素依然是成本问题，如何有效地降低成本是当前亟待解决的一个难题。     

Effects of pH, glucose and iron sulfate concentration on the yield of biohydrogen by Clostridium butyricum EB6

A local bacterial isolate from palm oil mill effluent (POME) sludge, identified as Clostridium butyricum EB6, was used for biohydrogen production. Optimization of biohydrogen production was performed via statistical analysis, namely response surface methodology (RSM), with respect to pH, glucose and iron concentration. The results show that pH, glucose concentration and iron concentration significantly influenced the biohydrogen gas production individually, interactively and quadratically (P < 0.05). The center composite design (CCD) results indicated that pH 5.6, 15.7 g/L glucose and 0.39 g/L FeSO₄ were the optimal conditions for biohydrogen production, yielding 2.2 mol H₂/mol glucose. In confirmation of the experimental model, t-test results showed that curve fitted to the experimental data had a high confidence level, at 95% with t = 2.225. Based on the results of this study, optimization of the culture conditions for C. butyricum EB6 significantly increased the production of biohydrogen.     

Isolation and characterization of high hydrogen-producing strain Clostridium beijerinckii PS-3 from fermented oil palm sap

Felled oil palm trunk (OPT) (25 years old) is an abundant biomass in Southern Thailand. The OPT composition was 31.28–42.85% cellulose, 19.73–25.56% hemicellulose, 10.74–18.47% lignin, 1.63–2.25% protein, 1.60–1.83% fat, 1.12–1.35% ash and trace amount of minerals (0.01–0.40%). Oil palm sap extracted from OPT was found to contain 15.72 g/L glucose, 2.25 g/L xylose, and 0.086 g/L arabinose. A total of twenty samples from hot springs (45–75 °C and pH 6.5–8.4), oil palm sap and palm oil mill effluent were enriched for isolation of hydrogen-producing bacteria. The highest hydrogen-producing strain was isolated from oil palm sap and identified as Clostridium beijerinckii PS-3 using biochemical test and 16S rRNA gene analysis. Among various carbon sources tested, glucose, xylose, starch and cellulose were the preferred substrates for hydrogen production. The strain PS-3 could produce the maximum hydrogen yield of 140.9 ml H₂/g total sugar and the cumulative hydrogen production of 1973  ml/L-oil palm sap. Therefore, C. beijerinckii PS-3 is a potential candidate for fermentative hydrogen production from mixed sugars of the oil palm sap.     

Impact of low lignin containing brown midrib sorghum mutants to harness biohydrogen production using mixed anaerobic consortia

Three low lignin containing bmr 3 derivatives, namely DRT 07K1, DRT 07K6 and DRT 07K15 developed through backcrossing were used along with the parent, bmr 3 source mutant (IS 21888) for evaluation of biohydrogen production. Results demonstrated that biohydrogen production varied amongst bmr derivatives under similar fermentation conditions. Significant negative correlation was observed between lignin content and fermentative biohydrogen production. All bmr derivatives with lower lignin content produced higher levels of biohydrogen compared to source bmr 3 (IS 21888) which has more lignin content. The maximum and a minimum biohydrogen production observed was 72 and 50 ml/g Total Volatile Solids (TVS) for the DRT 07K6 bmr3 derivative and bmr 3 (IS 21888) respectively. Acetate and butyrate were accounted >85% of volatile fatty acids, indicating acid type fermentations. Statistical analysis revealed that all bmr mutant derivatives with respect to source differ significantly in cumulative biohydrogen production, plant height, grain yield and lignin content. Biohydrogen production from biomass associated at least two different levels, one at lignin entanglement another at the polymeric nature of cellulose and hemicellulose. Further studies are necessary to determine the effect of biomass structure associated with different bmr traits on the microbial growth and biohydrogen production rate.     

Improved bio-hydrogen production by overexpression of glucose-6-phosphate dehydrogenase and FeFe hydrogenase in Clostridium acetobutylicum

Clostridium acetobutylicum is an attractive industrial microorganism for biochemical production, but there have been few attempts for bio-hydrogen production based on metabolic engineering. In this study, metabolically engineered C. acetobutylicum carrying glucose-6-phosphate dehydrogenase (zwf) and FeFe hydrogenase (hydA) were constructed as recombinant strains CA-zwf(pIMP-zwf) and CA-hydA(pMTL-hydA), respectively, to improve hydrogen productivity. The results showed that the engineered strains produced 1.15 and 1.39-fold higher hydrogen yield, respectively, than the wild type. Furthermore, when pH and glucose concentration were optimized for the CA-hydA strain, enhanced hydrogen productivity of 25.8% was achieved in 7 L jar scale fermentation. This result provides an insight into the future direction for metabolic engineering of C. acetobutylicum for improved hydrogen production.     

Pretreatment of Miscanthus for hydrogen production by Thermotoga elfii

Pretreatment methods for the production of fermentable substrates from Miscanthus, a lignocellulosic biomass, were investigated. Results demonstrated an inverse relationship between lignin content and the efficiency of enzymatic hydrolysis of polysaccharides. High delignification values were obtained by the combination of mechanical, i.e. extrusion or milling, and chemical pretreatment (sodium hydroxide). An optimized process consisted of a one-step extrusion-NaOH pretreatment at moderate temperature (70°C). A mass balance of this process in combination with enzymatic hydrolysis showed the following: pretreatment resulted in 77% delignification, a cellulose yield of more than 95% and 44% hydrolysis of hemicellulose. After enzymatic hydrolysis 69% and 38% of the initial cellulose and hemicellulose fraction, respectively, was converted into glucose, xylose and arabinose. Of the initial biomass, 33% was converted into monosaccharides. Normal growth of Thermotoga elfii on hydrolysate was observed and high amounts of hydrogen were produced.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.     

Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3

A hydrogen producing strain newly isolated from anaerobic sludge in an anaerobic bioreactor, was identified as Clostridium beijerinckii Fanp3 by 16S rDNA gene sequence analysis and detection by BioMerieux Vitek. The strain could utilize various carbon and nitrogen sources to produce hydrogen, which indicates that it has the potential of converting renewable wastes into hydrogen. In batch cultivations, the optimal initial pH of the culture medium was between 6.47 and 6.98. Using 0.15 M phosphate as buffer could alleviate the medium acidification and improve the overall performance of C. beijerinckii Fanp3 in hydrogen production. Culture temperature of 35 °C was established to be the most favorable for maximum rate of hydrogen production. The distribution of soluble metabolic products (SMP) was also greatly affected by temperature. Considering glucose as a substrate, the activation energy (E_a) for hydrogen production was calculated as 81.01 kcal/mol and 21.4% of substrate energy was recovered in the form of hydrogen. The maximal hydrogen yield and the hydrogen production rate were obtained as 2.52 mol/mol-glucose and 39.0 ml/g-glucose h⁻¹, respectively. These results indicate that C. beijerinckii Fanp3 is an ideal candidate for the fermentative hydrogen production.     

Mesophilic biohydrogen production by Clostridium butyricum CWBI1009 in trickling biofilter reactor

This study investigates the mesophilic biohydrogen production from glucose using a strictly anaerobic strain, Clostridium butyricum CWBI1009, immobilized in a trickling bed sequenced batch reactor (TBSBR) packed with a Lantec HD Q-PAC^® packing material (132 ft²/ft³ specific surface). The reactor was operated for 62 days. The main parameters measured here were hydrogen composition, hydrogen production rate and soluble metabolic products. pH, temperature, recirculation flow rate and inlet glucose concentration at 10 g/L were the controlled parameters. The maximum specific hydrogen production rate and the hydrogen yield found from this study were 146 mmol H₂/L.d and 1.67 mol H₂/mol glucose. The maximum hydrogen composition was 83%. Following a thermal treatment, the culture was active without adding fresh inoculum in the subsequent feeding and both the hydrogen yield and the hydrogen production rate were improved. For all sequences, the soluble metabolites were dominated by the presence of butyric and acetic acids compared to other volatile fatty acids. The results from the standard biohydrogen production (BHP) test which was conducted using samples from TBSBR as inoculum confirmed that the culture generated more biogas and hydrogen compared to the pure strain of C. butyricum CWBI1009. The effect of biofilm activity was studied by completely removing (100%) the mixed liquid and by adding fresh medium with glucose. For three subsequent sequences, similar results were recorded as in the previous sequences with 40% removal of spent medium. The TBSBR biofilm density varied from top to bottom in the packing bed and the highest biofilm density was found at the bottom plates. Moreover, no clogging was evidenced in this packing material, which is characterized by a relatively high specific surface area. Following a PCA test, contaminants of the Bacillus genus were isolated and a standard BHP test was conducted, resulting in no hydrogen production.     

Improved electrochemical activity of LiMnPO4 by high-energy ball-milling

Olivine lithium manganese phosphate (LiMnPO₄) becomes research focus because of its high energy density and improved thermal stability. However, its application in lithium ion batteries suffers severely from poor electrochemical activity due to low conductivity and structural instability upon the charge and discharge process. By applying a high-energy ball-milling method we succeed in improving the capacity delivery and rate capability. LiMnPO₄ materials ball-milled without or with acetylene black are able to deliver a high capacity of 135 and 127 mAh g⁻¹, respectively, more than 50% greater than the pristine one. Particularly, the latter also shows an improved discharge plateau and stable cyclability. High-energy synchrotron radiation X-ray diffraction (XRD), scanning electron microscopy (SEM), Raman spectroscopy, laser particle analysis, and galvanostatic charge and discharge are employed to understand the effect of ball-milling on the LiMnPO₄ material.     

Biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent

A hydrogen producer was successfully isolated from anaerobic digested palm oil mill effluent (POME) sludge. The strain, designated as Clostridium butyricum EB6, efficiently produced hydrogen concurrently with cell growth. A controlled study was done on a synthetic medium at an initial pH value of 6.0 with 10 g/L glucose with the maximum hydrogen production at 948 mL H₂/L-medium and the volumetric hydrogen production rate at 172 mL H₂/L-medium/h. The supplementation of yeast extract was shown to have a significant effect with a maximum hydrogen production of 992 mL H₂/L-medium at 4 g/L of yeast extract added. The effect of pH on hydrogen production from POME was investigated. Experimental results showed that the optimum hydrogen production ability occurred at pH 5.5. The maximum hydrogen production and maximum volumetric hydrogen production rate were at 3195 mL H₂/L-medium and 1034 mL H₂/L-medium/h, respectively. The hydrogen content in the biogas produced was in the range of 60–70%.