Metabolic Analysis Of Ethanol Fermentation By Neurospora Crassa AS3.1602

The inevitable depletion of the world's petroleum supply and the increased problems of greenhouse gas effects have resulted in the worldwide interest in alternative, nonpetroleum-based energy. Lignocellulosics are the most abundant renewable resource in the world. In China, the annual production of corn straw exceeds 600 million tons. Fuels derived from cellulosic biomass offer one such alternative to conventional energy sources that can dramatically impact national economic growth, national energy security, and environmental goals.Ethanol is the most attractive liquid fuel from lignocellulosic materials. At present, most bioethanol is made from corn sugars and starch. With the fast increase of population and severe shortage of food, the development of ethanol production from food will be restricted. In the latest thirty years, under the help of International Energy Agency and the governments of several countries, scientists were engaged in the investigation of technologies to convert renewable lignocellulose into fuel ethanol. Rapid progress has been made in material pretreatment, organisms screening and improvement, cellulase preparation and enzymatic hydrolysis, and ethanol fermentation technology. Part of these technologies has been carried out in pilot-scale application. However, due to some economic and technical reasons, the technology of lignocellulosics conversion is still immature. One of the major difficulties is process simplification and costs reduction.Neurospora crassa has both abilities of producing cellulase and hemicellulase, and fermenting glucose and xylose to ethanol. Therefore, the additional enzyme production and enzymatic lignocellulosic raw material hydrolytic processes are unnecessary, if N. crassa is used for bioethanol production from biomass. It is termed as direct microbial conversion or consolidated bioprocessing, and is regarded to have good prospects in the future. The objectives of this study were to investigate the regulating mechanisms of glycolysis, pentose phosphate pathway and tricarboxylic acis (TCA) cycle, the metabolic status of the cells, and the controlling factors of ethanol fermentation during the direct conversion of lignocellulosics to ethanol by N. crassa AS3.1602, and to provide experimental data and theoretical principle for the whole utilization and efficient bioconversion of lignocellulosics to ethanol. The innovative research results were obtained as following:I. Method establishment for extraction and analysis of the intracellular metabolites of N. crassa AS3.1602Intracellular metabolites were extracted using methanol. Samples were condensed with vacuum evaporation and solid-phase extraction (SPE), respectively, and the sample pretreatment method based on SPE was development to render the cell extracts suitable for analysis of their sugar phosphate content with anion-exchange chromatograph with pulsed amperometric detection (AEC-PAD). The analysis conditions of organic acid and sugar phosphate with AEC were determined. This provided the basis for metabolite investigation of N. crassa in ethanol fermentation.II. Metabolic analyses of glucose fermentation, xylose fermentation, glucose/xylose co-fermentation and cellulose direct conversion of N. crassa AS3.1602The metabolic analysis was carried out from three aspects, including extracellular metabolites, intracellular metabolites and intracellular enzyme activity analyses, complemented with metabolic flux analysis (MFA).1. Glucose fermentation by N. crassa AS3.1602Characteristics of glucose fermentation of Neurospora crassa AS3.1602 was studied using batch cultures. This work was carried out from two aspects: high substrate concentration and ethanol endurance, comparing with industrial Saccharomyces cerevisiae NAN27. Glucose metabolic performance changed little when initial glucose concentration varied from 20 g/L to 100 g/L. Ethanol inhibited glucose fermentation greatly and additional 5 % (V/V) ethanol destroyed mycelia and stopped fermentation totally. Intracellular enzyme assays indicated that the activities of related enzymes decreased sharply by 2 % ethanol. 2. Xylose fermentation by N. crassa AS3.1602The effects of oxygen limitation on xylose fermentation by N. crassa AS3.1602 were investigated, complemented with MFA. With the increase of oxygen limitation, xylose uptake and cell growth rates decreased. Oxygen had great effects on ethanol production. With the increase of oxygen limitation, the metabolic fluxes of ethanol production changed gradually. At OTR of 8.4 mmol/L·h, the productivity and final concentration of ethanol reached the highest values.Intracellular metabolites were determined at various oxygen transfer rates (OTRs). Concentrations of most of the intracellular metabolites decreased with oxygen limitation. Intracellular enzyme activities of xylose reductase (XR), xyltiol dehydrogenase (XDH) and xylulokinase (XK), the first three enzymes in xylose metabolic pathway, decreased with the increase of oxygen limitation, resulting in the decreased xylose uptake rate. When OTR varied from 12.6 to 0 mmol/L·h, the activities of transaldolase (TAL) and transketolase (TKL) were always maintained at low levels, indicating a great control on xylose metabolism. The enzyme of glucose-6-phosphate dehydrogenase (G6PDH) played a major role in NADPH regeneration and its activity decreased remarkably with the increase of oxygen limitation.3. Glucose-xylose co-fermentation by N. crassa AS3.1602Glucose-xylose co-fermentation was carried out under oxygen-limited conditions. When glucose and xylose were co-fermented by N. crassa, there was obvious order in substrates utilization. Glucose inhibited the uptake of xylose. The whole fermentation process could be divided into two stages at 48 h. Before 48 h, glucose was utilized quickly and ethanol accumulated continuously. After 48 h with glucose depletion, the activities of the first three enzymes involved in xylose metabolism increased significantly and the xylose uptake rate increased sharply. With the consumption of xylose, xylitol accumulated continuosly. Flux flew form pentose phosphate pathway into glycolysis, resulting in the increase of fructose-6-phosphate level. Fructose-6-phosphate was converted to glucose-6-phosphate, and then glucose-6-phosphate flew into the oxidative branch of pentose phosphate pathway for NADPH regeneration.Acetoin, as the artificial electron acceptor, could alleviate the xylitol accumulation during glucose-xylose co-fermentation.4. Cellulose fermentation by N. crassa AS3.1602Characteristics of celluose fermentation by Neurospora crassa AS3.1602 was studied using batch cultures. Aerobic fermentation of 3 d with 20 g/L avicel for cellulase production was made, and then the pre-culrures were transferred into 100 ml serum flask with additional 20 g/L avicel for anaerobic fermentation. After 96 h, ethanol concentration reached 6.3 g/L and only small amount of byproducts of lactate and acetate were observed. During the fermentation, the level of intracellular fructose-6-phosphate decreased, indicating an efflux from pentose phosphate pathway into glycolysis, resulting in the ethanol production. Most of intracellular enzyme activities decreased, indicating the decreases of cell activity and fermentation efficiency.III. Principle component analysis of intracellular metabolite concentration and enzyme activitiesVariations of intracellular metabolite concentration and enzyme activities under different fermentation conditions were evaluated using principal component analysis. The key factors, which affected the glucose and xylose ethanol fermentation of N. crassa AS3.1602, were osmotic pressure, aeration and subtrate components. Useful information for future genetic modification or process optimization for improvement of ethanol production of this strain was provided by this research.IV. Direct conversion of lignocellulosic raw materials into ethanol by N. crassa AS3.1602The optimization of medium components for cellulase and hemicellulase production was carried out, using wheat bran and cob powder as the carbon sources and soybean cake powder, ammonium sulphate and urea as the nitrogen sources. The direct conversion of the xylose-production-residues from corn cob by N. crassa AS3.1602 was carried out under oxygen-limited conditions with fermentation volume of 2 L. 6.8 g/L of ethanol was produced.