【作者】 苏小军；

【导师】 熊兴耀； 谭兴和；

【作者基本信息】 湖南农业大学 ， 园艺学， 2009， 博士

【摘要】 我国是世界上最大的马铃薯生产国,种植面积达8000万亩,产量超过8000万吨。尽管生产占据第一位,但加工业却很薄弱,严重制约了整个产业的发展。以马铃薯为原料生产乙醇可为我国马铃薯的深加工开辟一条新途径。乙醇通过进一步的纯化,即可成为食用乙醇,也可用作工业乙醇,还可成为燃料乙醇。随着全球性能源安全问题的出现,以马铃薯为原料生产燃料乙醇是最有发展前景的。目前,能耗较大和成本偏高是制约乙醇产业发展的主要问题,因此,有关乙醇生产的研究也主要集中在这两方面。以淀粉质为原料发酵生产乙醇,传统工艺必须先通过高温高压蒸煮工序,而该蒸煮工序消耗的能量占到了整个生产过程总能耗的30%-40%。生料发酵技术省去了高温蒸煮糊化工艺,避免了高温条件下可发酵性糖的损失,因而具有降低能耗、简化操作工序、降低生产成本等优点,但目前其工业化应用受到了菌株产酶活力低、酶活力不稳定、发酵周期长、效率低、容易污染等因素的限制。生料发酵技术的关键在于生淀粉的水解糖化,因此通过选育生淀粉酶高产菌株和提高淀粉对酶解的敏感性等途径来增加生淀粉的糖化效率,是当前的研究重点和热点。本文以马铃薯为对象,开展生料糖化发酵技术转化乙醇的研究。一方面通过选育马铃薯生淀粉酶高产菌株,并通过产酶条件的优化,获得高活力生淀粉酶,以达到实现生料糖化发酵转化乙醇的目的。另一方面,采用辐照技术对马铃薯原料进行预处理,以提高糖化效率,并通过进一步的条件优化,实现马铃薯生料发酵转化乙醇。本研究旨在建立起以马铃薯为原料转化乙醇新技术体系,为解决我国马铃薯加工产业问题提供新思路,为乙醇产业发展开辟原料新途径提供技术支持,对于我国马铃薯和乙醇产业乃至能源的可持续发展都有重要意义。取得研究结果如下:1、通过从马铃薯加工基地、酒精厂和淀粉厂附近的腐殖土壤及腐烂马铃薯中广泛取样,初步筛选出了10株具有较强马铃薯生淀粉糖化能力的菌株,并通过进一步筛选,获得了1株对马铃薯生淀粉糖化能力相对较强的菌株,其液体培养所产的生淀粉酶活力达30.5 U/ml,固体培养所产生淀粉酶的活力达19.3U/ml。对菌株的形态学研究和ITS rDNA序列同源性比较分析表明,该菌株属于黑曲霉（Aspergillus niger）。2、采用紫外辐照和硝基胍处理相结合的方法对上述菌株进行了诱变,得到1株产酶能力大大提高的突变株,其固体发酵所产生淀粉酶的酶活力为43.8U/ml,比出发菌株提高了127%。经连续6代传代试验验证,该突变株的遗传性能稳定。为方便起见,将该突变菌株命名为Aspergillus niger AF-1。3、对菌株AF-1固态发酵所产生淀粉酶的酶学性质分析表明,酶的最适反应温度为55℃,在60℃以下稳定,具有一定的耐热能力;最适反应pH为4.0,在pH 3.5-5.0范围内稳定,为一种酸性酶;Mn²⁺、Fe²⁺、Zn²⁺、Mg²⁺、Ca²⁺、Ba²⁺对酶有一定的激活作用,而Cu²⁺、K⁺、Na⁺则抑制酶的活性。酶对底物的作用方式表现出多样性,以表面扩散侵蚀的方式作用于马铃薯和木薯生淀粉,以表面扩散侵蚀和形成大侵蚀孔洞的方式同时作用于红薯淀粉。4、利用响应面法对菌株AF-1固态发酵产生淀粉酶的条件进行了优化。首先采用单因素试验筛选出马铃薯粉、豆粕粉、FeSO₄为最适碳源、氮源和无机盐。然后通过Plackett-Burman设计对影响产酶条件的12个相关因素进行效应评价,筛选出具有显著效应的豆粕粉、温度和麸皮三个因素。再利用最陡爬坡试验逼近以上三个因素的最大响应区域。最后采用响应面中心组合设计对显著因素进行优化,得出豆粕粉含量、温度和麸皮添加量的最佳值分别为11.46%、26.26℃和17.41g。优化后的酶活提高到204 u/ml,比初始酶活提高了3.85倍。5、采用γ射线对马铃薯进行辐照处理,发现马铃薯淀粉经50-400kGy剂量照射后原有颗粒形貌并没有改变,但经200kGy剂量处理后溶于水6h颗粒表面出现明显裂痕,经400kGy剂量处理后溶于水3h颗粒表面出现大量裂痕,6h颗粒失去原有形貌。进一步的分析表明,马铃薯粉经不同剂量辐照处理后,溶解度增加,其中以400kGy剂量处理增加效果最显著,溶解度达61%,比对照的提高了4倍;辐照处理具有直接糖化效果,还原糖的含量随着辐照剂量的加大而增加,其中以400kGy剂量辐照处理的糖化效果最明显,DE值达5.1%;马铃薯经辐照处理后,对酶的作用变得敏感,且随着辐照剂量的增加而加强;200kGy剂量范围内辐照处理对糖化效率的提高不如95℃蒸煮显著,但400kGy剂量辐照处理的要远远高于蒸煮的;离子色谱法分析检测表明,马铃薯粉经200、400kGy剂量辐照处理后,降解产物主要为葡萄糖和麦芽糖;经400kGy辐照处理后酶解产物主要为葡萄糖,酶解的主要产物单一。6、以生马铃薯粉为原料,采用酵母菌和菌株Aspergillus niger AF-1所产生淀粉酶进行同步糖化发酵转化乙醇时,合适的酶添加量为150U/g,料水比为1:2.5,酵母添加量为5×10⁷个/ml。另外,添加外源氮源硫酸铵能够提高发酵液的乙醇浓度。Ca²⁺、Mg²⁺对发酵有一定的促进作用,分别在8mmol/L和4mmol/L浓度条件下表现出最大效应。50kGy剂量辐照处理对生淀粉酶和酵母菌同步糖化发酵产乙醇的影响不大;100kGy剂量辐照处理对发酵有一定的促进效果;200kGy、400kGy剂量辐照处理的促进效果最显著,发酵液最终的乙醇浓度均达到11%。7、50kGy、100kGy剂量辐照处理对商品糖化酶和酵母菌同步糖化发酵产乙醇的影响不大;200kGy剂量辐照处理对发酵有一定的促进效果,但不是很显著;400kGy剂量辐照处理的促进效果最强烈,发酵液最终的乙醇浓度可达10.1%。采用单因素试验、Plackett-Burman试验、最陡爬坡试验和响应面分析相结合的试验方法对经400kGy剂量辐照处理后的马铃薯生料商品酶和酵母菌同步糖化发酵产乙醇的工艺条件进行了优化,优化条件下发酵液的乙醇浓度可达12.4%,较优化前提高了22.8%。通过条件优化研究,将马铃薯生料发酵生产乙醇的最佳条件确定为:温度35.27℃、料水比1:2.07、pH4.0、装瓶量125ml、（NH₄）₂SO₄ 0.2%、接种量2.5×10⁷个/ml、糖化酶200U/g、淀粉酶15U/g、纤维素酶10U/g、发酵周期48h。更多还原

【Abstract】 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.更多还原