Studies On Saccharification And Fermentation Of Raw Potato Flour For Ethanol Production

China is the largest potato producing country in the world. However the industrial process of potato is very weak, it has been holding up the development of the whole potato industry. Ethanol production from potato provides a new way for potato process industry. Ethanol can be edible, industrial and fuel use, in which fuel ethanol has good prospects.However, main issues with fuel ethanol are high production cost and high energy consumption. Hence researches have been focused on these two aspects. The process of producing fuel ethanol from starch, traditionally involves high temperature cooking, this step consumes about 30 - 40% of energy of the whole process. While the technology of raw material fermentation removes the step of high temperature cooking, avoids the loss of fermentable sugar under high temperature, hence it has the advantages of lower energy consumption, simplying process, and lower costs. However, it's industrialization is still inhibited by issues like low activity of enzyme, unstable enzyme activity, long fermentation cycle, low efficiency, easy to be contaminated etc. The critical point of the technology of raw material fermentation lies in the saccharification of raw starch, hence screening microbes producing raw-starch-hydrolysing enzyme and improving the sensitivity of enzyme would be the critical and hot subject of current researches.This article uses potato as the material, focusing on the raw material fermenting technology to produce fuel ethanol. From one aspect, try to obtain high activity raw-starch-hydrolysing enzyme by screening microbes, and optimizing conditions to produce enzymes; on the other hand, utilize radiation technology to pretreat potato to increase enzyme degradation efficiency, at the same time, optimize the process conditions to produce fuel ethanol by fermenting potatoes. The purpose of this research is to set up a fuel ethanol production system by fermenting potato, hence provide a new idea of industrial process of potato, provide a new path of exploring fuel ethanol industry production. Here are the research results:1,Screening microbes from soil around potato industry bases, ethanol producing plant, starch producing plant and spoiled potatoes, preliminarily screened out 10 species of fungi that has the ability of producing raw potato starch hydrolysing enzymes. By further screening, obtained one species of fungi that has strong hydrolysing ability. It's amylase activity gets high of 30.5 U/ml in liquid fermentation and 19.3 U/ml in solid state fermentation. Morphology of this fungi and ITS rDNA sequence same origin comparison indicate that this species belongs to Aspergillus niger.2,Tried to mutate the species by UV radiation combined with NTG treatment, obtained one mutated species that has much higher enzyme producing ability. It's raw-potato-starch-hydrolysing amylase activity is high of 43.8U/ml in solid state fermentation, which was improved 127% compared to the original species. This mutated species has genetic stability, named as Aspergillus niger AF-1.3,The enzymology characteristic analysis of the amylase produced by the mutant indicates that the optimal temperature of reaction is 55℃, it's stable under 60℃, the enzyme is heat resistant. The optimal pH is at 4.0, it's stable between pH 3.5-5.0. Mn²⁺, Fe²⁺,Zn²⁺, Mg²⁺, Ca²⁺, Ba²⁺ can activate the enzyme, while Cu²⁺, K⁺, Na⁺ can actually inhibit the activity of the enzyme. The enzyme reacted to difference substrates in two ways, showed centrifugal hydrolysis on potato and cassava granules, and showed both centrifugal and centripetal hydrolysis on sweet potato starch with deep holes into the granules.4,Response surface methodology was used to optimize solid state fermentation for raw-starch-hydrolysing amylase production by Aspergillus niger AF-1. Potato flour, soybean powder, FeSO₄ were screened out as the most suitable carbon source, nitrogen source and inorganic salt source, respectively. Based on these, screening methodology Plackett-Burman design was used to evaluate the effects of twelve factors related to amylase production and three statistically significant factors soybean powder, temperature, bran were selected. The path of steepest ascent was used to approach the optimal region of above three factors subsequently. These optimal factors were further optimized using central composite designs and response surface methodology and determined as follows: soybean powder 11.46%, temperature 26.26℃and bran 17.41g. Amylase activity after optimization of the fermentation medium increased to 204 u/ml, increased by 3.85 times. 5,After treating the potatoes withγray, the granule morphology of potato starch did not change under the doses of 50-400kGy. However, under dose of 200kGy treatment, the surface of the granule cracked obviously 6 hours after being dissolved in water, under the dose of 400kGy , granual cracked 3 hrs after being dissolved in water, lost it's original morphology 6 hrs after being dissolved in water. Further analysis showed that the dissolving ability increased after being treated at different doses ofγray, among which 400kGy increased the dissolving ability the most to 61%, which is 4 times that of the control,γray irradiation has the effect of dehydrolysating directly, the content of sugars increased with the dose ofγray, among which 400kGy is the best, has DE value of 5.1%. Potatoes became more sensitive to enzyme after exposed to y ray, 400kGy increased saccharification efficiency much greater than that of 95℃cooking, but 200kGy has less saccharification efficiency than that of 95℃cooking. Ion-chromatography analysis showed that after potatoes being treated with y ray at 200 and 400 kGy, it's degraded product are mainly glucose and maltose, while at 400kGy, degraded product was mainly glucose only.6,In ethanol production from raw potato flour using simultaneous saccharification and fermentation technology by yeast and raw-starch-hydrolysing amylase produced by Aspergillus niger AF-1, the suitable enzyme amount is 150U/g, solid-liquid rate 1:2.5, yeast inoculation amount 5×10⁷cell/ml,. It is also discovered that foreign nitrogen source (NH₄)₂SO₄ can increase ethanol concentration in fermentation. Ca²⁺, Mg²⁺ can improve the ferment process, most effective at 8mmol/L and 4 mmol/L. 50kGy dose treatment on potato has little effect on ethanol yield, 100kGy can improve the fermentation, 200kGy and 400 kGy can significantly improve the fermentation, ethanol concentration reached 11% at the end of fermentation.7,50kGy dose treatment on potato has little effect on ethanol production from raw potato flour using simultaneous saccharification and fermentation technology by yeast and glucoamylase , 100kGy and 200kGy can improve the fermentation, 400 kGy can significantly improve the fermentation, ethanol concentration reached 10.1% at the end of fermentation. Used single factor experiment, Plackett-Burman design, the path of steepest ascent design, central composite designs and response surface methodology to optimize the fermentation of potatoes treated at 400 kGy does, the optimized condition are temperature 35,27°C, solid-liquid rate 1:2.07, pH4.0, bottle filled up to 125ml, (NH₄)2SO₄ 0.2%, yeast inoculation amount 2.5×10⁷cell/ml, glucoamylase 200U/g,α-amylase 15U/g, cellulase 10U/g, fermentation cycle 48 h. Under the optimal conditions, ethanol concentration could reach 12.4%, the ethanol concentration increased 22.8% compared with non-optimized conditions.