【作者】 李凡；

【导师】 白凤武；

【作者基本信息】 大连理工大学 ， 生物化工， 2010， 博士

【摘要】 燃料乙醇具有清洁环保、可再生的优点,是目前应用最广泛的生物能源之一。燃料乙醇生产成本分析表明,能耗成本占淀粉质原料燃料乙醇总生产成本的30%左右,仅次于原料成本,采用高浓度(Very high gravity, VHG)发酵技术,提高发酵终点的乙醇浓度,可以节省发酵醪精馏操作的能耗,减少废糟液总量,进而节省采用全蒸发浓缩技术(DDGS)处理废糟液的能耗,是燃料乙醇生产节能技术的发展方向。VHG乙醇发酵技术面临的最大问题是发酵终点高达15%(v／v)以上乙醇浓度对细胞的强烈抑制作用。批次发酵反应器和平推流反应器是产物抑制型反应体系最理想的选择,但传统的批次发酵过程反应器内生物量密度低、发酵周期长；而多级串联发酵虽能在一定程度上减轻返混,生产12-13%(v／v)的乙醇,但仍然难以满足VHG乙醇发酵的要求。酵母絮凝是一种由絮凝基因调控的,并受环境因素影响的,通过酵母细胞互相粘附形成酵母颗粒团的现象。在乙醇发酵工业中,絮凝酵母可以作为细胞固定化的方法来提高反应器中的生物量密度,进而提高发酵效率及设备生产强度。本论文利用过程工程的原理,利用絮凝酵母SPSC01菌株进行VHG乙醇发酵的研究,旨在解决目前VHG乙醇发酵生产效率低的技术难题。论文研究工作主要包括以下内容：1.建立了SPSC01重复批次VHG乙醇发酵工艺利用絮凝酵母SPSC01进行传统的批次发酵,虽然可以达到较高的终点乙醇浓度,但是发酵时间较长、生产效率较低,与游离酵母相比,不具备技术经济优势。利用絮凝酵母SPSC01进行单级和多级连续发酵,在不进行补充接种的条件下,发酵初期菌体活性较好,可以维持高浓度生产,但持续高浓度的乙醇对细胞造成严重毒害,菌体活性很快降低,系统无法稳定运行。因此,基于过程工程的原理,开发了高密度重复批次发酵工艺,减少了高浓度底物和产物对酵母的生长抑制和毒害,并且能够在发酵终点利用菌体沉降特性迅速进行细胞分离。与其他高浓度乙醇发酵工艺相比,利用絮凝酵母进行重复批次发酵,在发酵终点乙醇浓度为15%(v／v)的前提下,设备生产强度达到8.62g/L／h,具有明显的经济技术优势。2.研究了重复批次发酵过程中SPSC01沉降性能下降的原因针对在重复批次发酵过程中随着发酵批次的增加,酵母沉降性能下降的现象,研究了SPSC01沉降性能的变化规律。絮凝酵母经过培养后,呈颗粒形态,以自由沉降为特征,随着重复发酵过程的进行,酵母由颗粒形态转化为絮状形态,沉降表现为干扰沉降和压缩沉降。这种沉降方式速度缓慢,不利于菌体快速沉降分离,导致每批次发酵结束后酵母持续受到乙醇毒害而逐渐丧失活力。考察了酵母密度、表观粘度、颗粒粒径等因素对重复批次过程中SPSC01沉降性能的影响,判断酵母颗粒粒径的减小及发酵液粘度的变化是造成沉降性能下降的主要原因。通过对培养后用钙离子螯合剂处理的不同粒径酵母体系表观粘度、沉降性能的考察,明确了酵母颗粒大小决定表观粘度大小及沉降速率快慢的规律。酵母颗粒大小反映了酵母絮凝性能的强弱,SPSC01絮凝性能的衰退造成了沉降性能的下降。3.重复批次发酵过程SPSC01絮凝性能衰退原因的分析及实验验证课题组前期研究表明,絮凝酵母SPSC01中FLO1基因对其絮凝性能至关重要。本论文研究证明,FLO1基因的的表达会赋予酵母絮凝特性和疏水性。论文对酵母絮凝能力与疏水性的研究结果表明,疏水性不是影响SPSC01絮凝性能的主要因素。高浓度乙醇对于絮凝酵母细胞壁表面的理化作用,以及对于其线粒体功能的影响,都不是导致发酵过程絮凝性能衰退的主要原因。SPSC01菌株在重复批次发酵过程前后,其絮凝基因片段完整性没有发生改变。将重复批次发酵末期的酵母菌体重新摇瓶培养,其絮凝性能可以回复。对比摇瓶和重复批次发酵末期的SPSC01菌体FLO1基因的转录水平可知,该基因在VHG高强度乙醇发酵条件下,转录水平大幅下降,这是导致SPSC01絮凝性能衰退的根本原因。对SPSC01重复批次发酵进行工艺优化,在批次发酵终点采出酵母菌体以降低生产强度,减轻乙醇生产过快产生的抑制,刺激菌体生长,可以使酵母菌体通过自我繁殖更新实现发酵过程的稳定和持续性,不但省去额外菌体培养过程,其酵母絮凝能力和沉降性能也可以满足工艺要求。4.乙醇浓度及其变化速率对SPSC0l絮凝基因FLO1转录及细胞形态的影响发现乙醇对于SPSC01菌体絮凝性能的影响是通过抑制絮凝基因FLO1的转录水平来实现的。乙醇浓度越高、乙醇浓度的变化速率越快,其对SPSC01絮凝基因FLO1的转录抑制越强。较快的乙醇浓度变化,也对SPSC01的形态造成改变,产生了假丝形态。这种形态变化,不是由于氮元素缺乏造成,而可能来自于重复批次发酵过程中周期变化的乙醇浓度对于酵母细胞周期造成的影响。利用组成型表达SPSC01絮凝基因FLO1的重组菌BHL01进行重复批次VHG乙醇发酵,菌体发酵性能及絮凝性能的稳定性均优于SPSC01。综上所述,本论文利用絮凝酵母SPSC01进行VHG乙醇发酵的工艺探索,建立了重复批次发酵的生产工艺,提高了发酵生产强度；从絮凝基因FLO1的转录水平解释了重复批次发酵过程中酵母絮凝性能衰退现象及由此而导致的沉降性能下降问题；分析了乙醇浓度及其变化速率对于FLO1的转录水平及细胞形态的影响；以上述研究为依据,将生化过程工程与基因工程结合,对重复批次发酵工艺进行了工艺优化,并利用基因重组絮凝酵母大幅度提升了VHG重复批次乙醇发酵工艺性能。更多还原

【Abstract】 Fuel ethanol, both environmentally friendly and renewable, is the mostly widely used biofuel in the world. Cost analysis of ethanol production from starch-based feedstocks reveals that energy consumption contributes about 30% of the total cost, the second largest only after that from feedstocks consumption. Very high gravity (VHG) fermentation, aiming at achieving more than 15% (v/v) ethanol, can significantly save energy consumption, not only in the distillation, but also in the treatment of the distillage with the DDGS (distilled dry grain with solubles) process, making it much more promising. However, the biggest challenge of VHG fermentation comes from the strong inhibition of ethanol. Batch and plug flow reactors are two ideal models to alleviate product inhibition, but biomass conconcentration is usually low within a batch reactor, which correspondingly needs longer fermentation time. The multistage fermentation system that can alleviate backmixing to some extent and produce 12-13% (v/v) ethanol is still problematic for VHG ethanol fermentation.Yeast flocculation is a phenomenon that is controlled by FLO genes and affected by bivalent cations, particularly Ca2+, through which yeast cells adhere together and form flocs. In the brewing industry, yeast flocculation benefits the separation of biomass at the end of the fermentation. Recently, ethanol fermentation with the self-flocculating yeast SPSC01 immobilized within bioreactors was developed for fuel ethanol production. In this thesis, VHG ethanol fermentation by SPSC01 was studied, with the following focuses:1. Development of consecutive batch VHG ethanol fermentation strategyConventional batch mode using the flocculating yeast SPSC01 under VHG conditions needs much longer fermentation time, and thus its ethanol productivity is very low, which confers no advantages over free yeast cells. Moreover, without a seed culture system to supplement viable yeast flocs into the fermentation system periodically, continuous ethanol fermentation using SPSC01, whether a single fermentor or tanks in series, produced high ethanol concentration for a while, but was unstable thereafter due to significant viability loss of yeast flocs suffered from severe ethanol inhibition. Consecutive batch fermentation with SPSC01 at high biomass density was then developed to shorten fermentation time. Taking advantage of yeast flocs to separate from fermentation broth by self-sedimentation, the inhibition of ethanol in yeast cell growth was significantly alleviated. Compared to other VHG fermentation technologies, the consecutive batch mode produced more than 15% (v/v) ethanol, with an average productivity as high as 8.62 g/L/h.2. Investigation of the settling performance of SPSC01Within the duration of the consecutive batch fermentation, yeast flocs experienced significant change in their settling performance, from free settling of intact flocs to hindered settling of snowflake-like incompact flocs, followed by compression settling, which was slow and undesirable for separating yeast flocs from fermentation broth, exacerbating ethanol inhibition in the yeast flocs. The impact of the density and size of yeast flocs and the apparent viscosity of fermentation broth on their settling performance was thus investigated. It was found that the decrease of the size of yeast flocs was the main reason for this phenomenon.3. Mechanism of the deflocculation of SPSC01 and process modificationPrevious studies indicated that the gene FLO1 is crucial to the flocculation of SPSC01, and its expression confers both flocculation and hydrophobicity to yeast cells. It was found that the hydrophobicity of yeast flocs was not the main factor affecting their flocculation performance. Neither were the physiochemical effect of ethanol on yeast cell wall and its physiological effect on intracellular mitochondrial functions. Moreover, no significant change in the gene intergrity was detected, since the flocculating ability of the de-flocculating yeast flocs could be restored by the regeneration of them in flask culture. The analysis of the FLO1 transcription indicated that compared with flask culture, its expression level decreased sharply at the end of the consetutive batch fermentation, which was the underlying reason for the deflocculation of SPSC01.The process was modified by purging yeast cells at the end of each batch within the duration of the consecutive batch fermentation, which lowered the density of yeast flocs within the fermentor, and thus simulated their growth. The flocculationd settling performance of yeast flocs were improved, and the fermentatioin system was run reliably.4. Effect of ethanol concentration and production rate on FLO] transcriptionEthanol affects the flocculating ability of yeast flocs by inhibiting FLO1 transcription. It was found that the higher the ethanol concentration and the faster the production rate, the more severe was the gene transcription inhibited. The high ethanol production rate change not only impaired FL01 transcription, but also induced pseudohyphal development, a significant morphology change.In summary, consecutive VHG batch fermentation using SPSC01 that could improve ethanol productivity was established. The deflocculation of the yeast flocs under VHG conditions and subsequent impact in their settling performance were contributed to the decrease in the FLO1 transcription. The effect of ethanol concentration and production rate on the FLO1 transcription and morphology of SPSC01 were observed. With these understandings, the consecutive VHG batch fermentation process was modified and the performance was greatly imporved.更多还原