Hydrogen-producing Bacteria And Its Metabolic Characteristics From Corn Stover Bioconversion

With the problem of energy crisis, food shortages, enviroment pollution become more and more serious, how to make the waste harmless and resources has become the urgent issue to solve worldwide. Straw is an aboudant agricultural waste, because of its sufficient supply and low price, if convert it to hydrogen by microorganisms, it can not only reduce the cost of hydrogen production, but can also reclaim waste materials, which has remarkable social and economic benefits on the aspects of energy development and waste ultization. Straw was mainly composed of cellulose, hemicellulose and lignin, cellulose and hemicellulose are fermentable carbohydrates, which can be converted to sugars by microorganisms or enzymes before their use for biohydrogen generation. But take straw as raw materials, biofuels researches in most cases are emphasis on the conversion of cellulose, hemicellulose is disposed as waste, which not only cause environmental pollution but also wastes of resources. Meanwhile, in cellulose conversion process, the major obstacles of cellulase technology are lower cellulase activity and higher production costs. Aiming at current main technical bottleneck of conversion straw to biohydrogen, the pentose fermentation bacteria and cellulose degrading bacteria for hydrogen production were isolated and the performance of hydrogen production from hemicellulose hydrolysate and cellulosic materials were investigated respectively. Simultaneously, associated with pretreatment technology, a combined system for efficient utilization of corn stover for hydrogen production was established. The main achievements obtained by this research were as follows:A moderately thermophilic bacterium, Thermoanaerobacterium thermosaccharolyticum W16, was isolated for efficiently producing hydrogen from pentose. Under the conditions of carbon source 10g/L, temperature 60°C, and initial pH 6.5, the maximum cumulative H2 yield and H2 production rate in glucose and xylose are 2.42mol H2/mol glucose, 12.9mmol H2/L·h and 2.19mol H2/mol xylose, 10.7mmol H2/L·h, respectively. The metabolic characteristics under mixed glucose and xylose are also discussed. It discovered that strain W16 could simultaneously uptake glucose and xylose, although, the consumption rate of glucose was faster than the consumption rate of xylose. The effect of common inhibitors derived from hydrolysate showed that strain W16 had high tolerance to acetate sodium and vanillin, however, it behaved more sensitive to furfural, hydroxymethylfurfural (HMF) and syringaldehyde. By using response surface methodology, the hydrolysis conditions were optimized to produce hemicellulosic hydrolysates. Under the optimal hydrolysis conditions of acid concentration 1.69%, processing time 117min, the removal efficiency of hemicellulose was up to 80.%,and the content of xylose, arobinose and glucose were 9.11g/L,1.85g/L和0.88g/L, respectively. The performance of hydrogen production from such hydrolysates by the strain W16 was also investigated. The results indicated that the strain W16 could effectively utilize hemicellulosic hydrolysates for fermentative hydrogen production; the inhibitors derived from hydrolysate such as acetate sodium, furfural didn't have inhibitory effect on the strain W16.The screening method was constructed to isolate thermophilic anaerobic cellulolytic hydrogen-producing bacteria, and a good cellulytic hydrogen-producing bacteria Thermoanaerobacterium thermosaccharolyticum M18 was obtained. This strain could efficiently uptake microcrystalline cellulose, xylan, filter paper, and sodium carboxymethylcellulose for hydrogen production, as well as unpretreated corn stover , rice straw, and corn cob. While inoculated to the medium containing 5g/L microcrystalline cellulose or corn stover, the hydrogen production yield and the substrate degradation efficiency were 44.55mmol/L (243.7ml/g-microcrystalline cellulose), 8.45mmol/L (80.2ml/g-corn stover) and 81.9% , 47.2%, respectively. The cellulases characteristics analysis indicated that the cellulases produced by the strain M18 were inducible enzymes, which only be induced in the existence of cellulosic materials. The activity assays on different enzymes of respective component show that the distribution of produced cellulases is mainly outside the cell.By virtue of SEM, FTIR, XRD, 13C-NMR, GC-MS, and the detection of cellulase and hemicelllase activities, the cellulolytic characteristics of the strain M18 were systematically analyzed. It can be concluded that the process of induced enzyme production coupled with corn stover degradation. The strain M18 exhibited degradation superiority on cellulose and hemicellulose and the degradation of hemicellulose was priority than the degradation of cellulose. In the degradation process, the intramolecular hydrogen bonds of the cellulose molecule were ruptured and the areas of crystal and amorphous were both destroyed. Cellulose and hemicellulose were decomposed into small molecule substances under the effect of cellulases and hemicellulases, and then fermented into hydrogen. It also suggested that lignin is the main barrier to restrict bioconversion of corn stover.Due to the existence of lignin influenced the bioconversion of cellulose and hemicellulose, the sodium hydroxide pretreatment was used to delignify corn stover. After pretreatment, the cellulosic solid residues and alkali-soluable hemicellulose sugar were obtained. During hydrogen production from corn stover, the bioconversion of cellulose and hemicellulose in NaOH-pretreated corn stover to hydrogen by M18 and alkali-soluable hemicellulose sugar recovery by W16 could be integrated into a biorefinery scheme, thus the maximum bioconversion efficiency of corn stover was acchieved. Compared with unpretreated corn stover, the hydrogen yield increased 36.8%.