废糟液全循环对絮凝酵母乙醇连续发酵的影响
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摘要
燃料乙醇作为一种可再生清洁能源,对缓解石油资源短缺、降低环境污染具有重要意义。目前糖质和淀粉质原料发酵生产的燃料乙醇占市场总量的90%以上,但生产成本高一直是这一产业面临的主要问题,使其严重依赖政府补贴。能耗是仅次于原料消耗的燃料乙醇生产第二大成本,主要用于乙醇蒸馏和废糟液处理,特别是粮食类淀粉质原料燃料乙醇生产过程中,废糟液处理普遍采用的多效蒸发系统。显然,废糟液直接循环使用是减少废液排放和降低其处理能耗最具有竞争力的策略。本论文研究开发了废糟液循环条件下絮凝酵母乙醇连续发酵新工艺。由于酵母细胞在发酵罐内实现自固定化而不随发酵液的排出而流失,发酵液乙醇蒸馏过程中酵母细胞自溶产生的有害副产物显著减少,为提高废糟液循环比例,乃至实现全循环,奠定了良好基础。
本文首先研究了废糟液全循环对絮凝酵母生长和乙醇发酵过程的影响。与无废糟液循环发酵过程相比,废糟液全循环对乙醇发酵过程产生了明显的抑制,表现为发酵液中残糖浓度的升高,乙醇和生物量平均浓度分别降低了5.3%和10.9%。发酵液中副产物和金属离子检测结果表明,在废糟液全循环过程中,除琥珀酸和5-羟甲基糠醛外,其余10种副产物和4种金属离子均有明显积累,其积累浓度为对照组的3倍以上。其中Ca2+、甘油和丙酸的积累浓度最高,分别为1126mg/L,15080mg/L和2100mg/L。其余副产物浓度都远小于1g/L。进一步的碳流分布分析表明,废糟液全循环对酵母糖酵解和磷酸戊糖途径的碳流分配没有产生明显的抑制,但使酵母细胞对底物的吸收速率和糖酵解途径三个限速酶己糖激酶、6-磷酸果糖激酶和丙酮酸激酶活性逐渐降低,到整个废糟液全循环结束时,流向乙醇的碳流量降低了33%。
为了找出主要抑制物,进行了添加副产物实验研究。单一副产物添加实验研究结果表明,柠檬酸、α-酮戊二酸、富马酸、乳酸、丙酮酸、琥珀酸、2-苯乙醇、糠醛和5-羟甲基糠醛浓度低于1g/L时,对絮凝酵母生长的抑制率低于5%,对乙醇发酵的抑制率低于4%,且低浓度单一金属离子可促进酵母生长和乙醇发酵。而丙酸浓度低于1g/L时,对絮凝酵母生长的抑制率低于20.3%,2g/L时对生长的抑制率达到30.2%,对乙醇发酵的抑制率低于5%。除丙酸外(MIC10:0.4g/L),其余所有副产物和金属离子在实际发酵液中的积累浓度远小于其最小抑制浓度(MIC10)。混合添加实验结果表明金属离子对酵母生长和乙醇发酵有促进作用,进一步的协同实验证实了丙酸是影响絮凝酵母生长和乙醇发酵的主要抑制物。
系统研究了丙酸对絮凝酵母代谢过程的影响。丙酸对酵母生长的影响非常显著,当发酵培养基中丙酸浓度超过40mmol/L时,对絮凝酵母生长和乙醇发酵、糖酵解途径三个限速酶活性以及酵母絮凝均产生显著抑制。当培养基中丙酸浓度达到60mmol/L时对生长的抑制率达到67%,对乙醇发酵的抑制率达到66%,酵母絮凝颗粒粒径降低至50μm,接近游离酵母,不利于废糟液全循环絮凝酵母乙醇连续发酵的稳定运行。
酵母代谢不生成丙酸,进一步分析检测发现玉米原料中半纤维素在酸性和高温条件下的分解是产生丙酸的主要来源,且其浓度随原料处理温度和时间的增加而升高,但玉米糖化液在60℃长时间储存时,丙酸浓度几乎无变化。据此提出了发酵培养基不经高温灭菌直接用于废糟液全循环乙醇连续发酵的工程策略,并通过实验进行了验证。实验结果表明发酵液各副产物浓度明显低于培养基高温灭菌的废糟液全循环工艺,其中丙酸浓度降低了62%,乙醇和生物量浓度分别提高了7.4%和59.3%,残还原糖浓度降低了40.1%,且优于培养基高温灭菌无废液循环乙醇发酵工艺各主要参数。保证了废糟液全循环絮凝酵母发酵连续发酵的稳定运行。
这些研究结果对生产装置运行中提高废糟液直接循环使用比例,实现节能减排,具有重要的指导意义。
As a renewable and clean fuel, ethanol is an alternative to petroleum-based transportation fuels, and in the meantime alleviates environmental impact caused by the over-consumption of these products. At present, over90%fuel ethanol is produced from sugar-and starch-based feedstocks by microbial fermentations, and high production cost makes the industry heavily depended on governmental subsidies. While feedstock consumption is the major cost in the fuel ethanol production, energy consumption is the second largest, which mainly comes from ethanol distillation and stillage treatment, particularly for fuel ethanol production with grain-based feedstocks in which the multi-evaporation process is employed for stillage treatment. Apparently, stillage backset is an economically competitive strategy for reducing stillage as well as saving energy consumption for stillage treatment. In this work, a novel process has been developed for ethanol fermentation by the flocculating yeast under stillage backset conditions. Due to the self-immobilization of yeast cells within the fermentor through flocculation, harmful byproducts generated with the lysis of yeast cells during ethanol distillation were significantly reduced, making more stillage backset, even without discharge and all stillage backset.
Impact on the fermentation process was investigated under all stillage backset conditions. Compared to ethanol fermentation without stillage backset, inhibition in yeast growth and ethanol fermentation was observed, the average concentration of ethanol and biomass were decreased by5.3%and10.9%respectivily. As a result, less biomass was accumulated within the fermentation system, and consequently more sugars were remained unfermented, with less ethanol produced. The analysis of byproducts indicated that except succinic acid and5-(hydroxymethyl) furfural, all other ten major byproducts and four metal ions accumulated significantly under the stillage backset condition. Compared to the control without stillage backset, their average concentrations increased at least3times. Exception of Ca2+, glycerol and propionic acid (were1126mg/L,15080mg/L and2100mg/L respectivily), concentrations of all other byproducts and metal ions were far less than1g/L. Metabolic flux analysis illustrated that the stillage backset had no significant impact on ratio of carbon flux distributions in the glycolysis and pentose phosphate pathway associated with yeast metabolism, but substrate uptake rate was inhibited, and the activities of rate-limiting enzymes hexokinase,6-phosphofructose and pyruvatekinae in the glycolysis were down-regulated, making the net carbon flow to ethanol production decreased33%at the end of ethanol fermentation with stillage backset.
In order to identify the main inhibitors, effect of individual byproducts on the ethanol fermentation was studied by supplementing them into the medium and feeding into the fermentation system. When the concentration of citric acid, α-ketoglutaric acid, fumaric acid, lactic acid, pyruvic acid, succinic acid,2-phenylethanol, furfural and5-hydroxymethyl furfural were lower than1g/L, experimental results indicated that the growth and ethanol fermentation inhibition rate were lower than5%and4%respectively. And the yeast growth and ethanol fermentation were facilitated when metal ions were in a low concentration. However, the growth inhibition rate were lower than20.3%when1g/L propionic acid was supplemented. Furthermore, exception of propionic acid (MIC10:0.4g/L), concentrations detected within the fermentation system of all other byproducts and metal ions were much lower than their minimum inhibition concentrations (MIC10). When a mixture of these byproducts with concentrations of their averages detected within the fermentation system under the stillage backset condition was supplemented, no synergic effect was observed, and propionic acid was still the dominated inhibitor on yeast growth and ethanol fermentation.
The results of effect of propionic acid on ethanol fermentation by the flocculating yeast indicated that the significant decrease of ehanol production, key enzyme activities and yeast flocculation were observed, When the propionic acid concentration was larger than IC50(40mmol/L). and propionic acid exhibited the strongest inhibition in yeast growth and ethanol fermentation with an inhibition rate of67%and66%respectively, when supplemented with the concentration of60mmol/L. and the size of flocs closes to50μm, its performance likes the free cells, which is absolutely a bad news for the process by using flocculating yeast.
However, propionic acid was produced by the dehydration of pentose sugars liberated from hemicelluloses in the feedstock under high temperature and acidic conditions associated with mash liqufication, hydrolysate sterilization and ethanol distillation, but propionic acid production was negligible during mash saccharification at60℃. and a practical strategy was developed for ethanol fermentation by the flocculating yeast under stillage backset conditions Without fermentation medium was sterilized at high temperature. The result indicated that the propionic acid accumulation within the fermentation system decreased62%, and more sugars were fermented and the reducing sugar was decreased by40.1%, with an increase of7.4%and59.3%in ethanol production and biomass respectivily.
No doubt, the research progress is significant for fuel ethanol production with more stillage backset to reduce the discharge and save energy consumption for stillage treatment.
本文首先研究了废糟液全循环对絮凝酵母生长和乙醇发酵过程的影响。与无废糟液循环发酵过程相比,废糟液全循环对乙醇发酵过程产生了明显的抑制,表现为发酵液中残糖浓度的升高,乙醇和生物量平均浓度分别降低了5.3%和10.9%。发酵液中副产物和金属离子检测结果表明,在废糟液全循环过程中,除琥珀酸和5-羟甲基糠醛外,其余10种副产物和4种金属离子均有明显积累,其积累浓度为对照组的3倍以上。其中Ca2+、甘油和丙酸的积累浓度最高,分别为1126mg/L,15080mg/L和2100mg/L。其余副产物浓度都远小于1g/L。进一步的碳流分布分析表明,废糟液全循环对酵母糖酵解和磷酸戊糖途径的碳流分配没有产生明显的抑制,但使酵母细胞对底物的吸收速率和糖酵解途径三个限速酶己糖激酶、6-磷酸果糖激酶和丙酮酸激酶活性逐渐降低,到整个废糟液全循环结束时,流向乙醇的碳流量降低了33%。
为了找出主要抑制物,进行了添加副产物实验研究。单一副产物添加实验研究结果表明,柠檬酸、α-酮戊二酸、富马酸、乳酸、丙酮酸、琥珀酸、2-苯乙醇、糠醛和5-羟甲基糠醛浓度低于1g/L时,对絮凝酵母生长的抑制率低于5%,对乙醇发酵的抑制率低于4%,且低浓度单一金属离子可促进酵母生长和乙醇发酵。而丙酸浓度低于1g/L时,对絮凝酵母生长的抑制率低于20.3%,2g/L时对生长的抑制率达到30.2%,对乙醇发酵的抑制率低于5%。除丙酸外(MIC10:0.4g/L),其余所有副产物和金属离子在实际发酵液中的积累浓度远小于其最小抑制浓度(MIC10)。混合添加实验结果表明金属离子对酵母生长和乙醇发酵有促进作用,进一步的协同实验证实了丙酸是影响絮凝酵母生长和乙醇发酵的主要抑制物。
系统研究了丙酸对絮凝酵母代谢过程的影响。丙酸对酵母生长的影响非常显著,当发酵培养基中丙酸浓度超过40mmol/L时,对絮凝酵母生长和乙醇发酵、糖酵解途径三个限速酶活性以及酵母絮凝均产生显著抑制。当培养基中丙酸浓度达到60mmol/L时对生长的抑制率达到67%,对乙醇发酵的抑制率达到66%,酵母絮凝颗粒粒径降低至50μm,接近游离酵母,不利于废糟液全循环絮凝酵母乙醇连续发酵的稳定运行。
酵母代谢不生成丙酸,进一步分析检测发现玉米原料中半纤维素在酸性和高温条件下的分解是产生丙酸的主要来源,且其浓度随原料处理温度和时间的增加而升高,但玉米糖化液在60℃长时间储存时,丙酸浓度几乎无变化。据此提出了发酵培养基不经高温灭菌直接用于废糟液全循环乙醇连续发酵的工程策略,并通过实验进行了验证。实验结果表明发酵液各副产物浓度明显低于培养基高温灭菌的废糟液全循环工艺,其中丙酸浓度降低了62%,乙醇和生物量浓度分别提高了7.4%和59.3%,残还原糖浓度降低了40.1%,且优于培养基高温灭菌无废液循环乙醇发酵工艺各主要参数。保证了废糟液全循环絮凝酵母发酵连续发酵的稳定运行。
这些研究结果对生产装置运行中提高废糟液直接循环使用比例,实现节能减排,具有重要的指导意义。
As a renewable and clean fuel, ethanol is an alternative to petroleum-based transportation fuels, and in the meantime alleviates environmental impact caused by the over-consumption of these products. At present, over90%fuel ethanol is produced from sugar-and starch-based feedstocks by microbial fermentations, and high production cost makes the industry heavily depended on governmental subsidies. While feedstock consumption is the major cost in the fuel ethanol production, energy consumption is the second largest, which mainly comes from ethanol distillation and stillage treatment, particularly for fuel ethanol production with grain-based feedstocks in which the multi-evaporation process is employed for stillage treatment. Apparently, stillage backset is an economically competitive strategy for reducing stillage as well as saving energy consumption for stillage treatment. In this work, a novel process has been developed for ethanol fermentation by the flocculating yeast under stillage backset conditions. Due to the self-immobilization of yeast cells within the fermentor through flocculation, harmful byproducts generated with the lysis of yeast cells during ethanol distillation were significantly reduced, making more stillage backset, even without discharge and all stillage backset.
Impact on the fermentation process was investigated under all stillage backset conditions. Compared to ethanol fermentation without stillage backset, inhibition in yeast growth and ethanol fermentation was observed, the average concentration of ethanol and biomass were decreased by5.3%and10.9%respectivily. As a result, less biomass was accumulated within the fermentation system, and consequently more sugars were remained unfermented, with less ethanol produced. The analysis of byproducts indicated that except succinic acid and5-(hydroxymethyl) furfural, all other ten major byproducts and four metal ions accumulated significantly under the stillage backset condition. Compared to the control without stillage backset, their average concentrations increased at least3times. Exception of Ca2+, glycerol and propionic acid (were1126mg/L,15080mg/L and2100mg/L respectivily), concentrations of all other byproducts and metal ions were far less than1g/L. Metabolic flux analysis illustrated that the stillage backset had no significant impact on ratio of carbon flux distributions in the glycolysis and pentose phosphate pathway associated with yeast metabolism, but substrate uptake rate was inhibited, and the activities of rate-limiting enzymes hexokinase,6-phosphofructose and pyruvatekinae in the glycolysis were down-regulated, making the net carbon flow to ethanol production decreased33%at the end of ethanol fermentation with stillage backset.
In order to identify the main inhibitors, effect of individual byproducts on the ethanol fermentation was studied by supplementing them into the medium and feeding into the fermentation system. When the concentration of citric acid, α-ketoglutaric acid, fumaric acid, lactic acid, pyruvic acid, succinic acid,2-phenylethanol, furfural and5-hydroxymethyl furfural were lower than1g/L, experimental results indicated that the growth and ethanol fermentation inhibition rate were lower than5%and4%respectively. And the yeast growth and ethanol fermentation were facilitated when metal ions were in a low concentration. However, the growth inhibition rate were lower than20.3%when1g/L propionic acid was supplemented. Furthermore, exception of propionic acid (MIC10:0.4g/L), concentrations detected within the fermentation system of all other byproducts and metal ions were much lower than their minimum inhibition concentrations (MIC10). When a mixture of these byproducts with concentrations of their averages detected within the fermentation system under the stillage backset condition was supplemented, no synergic effect was observed, and propionic acid was still the dominated inhibitor on yeast growth and ethanol fermentation.
The results of effect of propionic acid on ethanol fermentation by the flocculating yeast indicated that the significant decrease of ehanol production, key enzyme activities and yeast flocculation were observed, When the propionic acid concentration was larger than IC50(40mmol/L). and propionic acid exhibited the strongest inhibition in yeast growth and ethanol fermentation with an inhibition rate of67%and66%respectively, when supplemented with the concentration of60mmol/L. and the size of flocs closes to50μm, its performance likes the free cells, which is absolutely a bad news for the process by using flocculating yeast.
However, propionic acid was produced by the dehydration of pentose sugars liberated from hemicelluloses in the feedstock under high temperature and acidic conditions associated with mash liqufication, hydrolysate sterilization and ethanol distillation, but propionic acid production was negligible during mash saccharification at60℃. and a practical strategy was developed for ethanol fermentation by the flocculating yeast under stillage backset conditions Without fermentation medium was sterilized at high temperature. The result indicated that the propionic acid accumulation within the fermentation system decreased62%, and more sugars were fermented and the reducing sugar was decreased by40.1%, with an increase of7.4%and59.3%in ethanol production and biomass respectivily.
No doubt, the research progress is significant for fuel ethanol production with more stillage backset to reduce the discharge and save energy consumption for stillage treatment.
引文
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