自絮凝颗粒酵母BHL01的絮凝特性及表观生长动力学
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摘要
发酵动力学的提出有助于自絮凝颗粒酵母乙醇发酵工艺的设计与放大。该乙醇发酵体系中除了常规的乙醇、葡萄糖、生物量三项指标外,絮凝颗粒的粒径分布也是重要的影响因素。本文以具有絮凝性状的基因工程酵母菌株BHL01为研究对象,基于聚焦式激光反射测量系统(Focused beam reflectance measurement, FBRM)在线检测絮凝颗粒的粒径分布,进行了以下三方面的研究：
1.对比了BHL01与游离亲本株ATCC4126在连续乙醇发酵过程中的性能
在相同的发酵条件下,分析两者稳态时的各发酵参数和发酵液组分,发现两者单位生物量的发酵效果相似。具有絮凝性状的BHL01受反应器的截留作用,生物量积累高,相应的其乙醇生产能力强；而絮凝形成带来的传质阻力影响,使得BHL01的比生长速率和比乙醇生产速率要明显低于游离酵母ATCC4126。
2.考察了各种物理化学因素对BHL01絮凝性状的影响
采用FBRM对特定物理化学条件下的BHL01絮凝颗粒粒径分布进行实时检测,发现大部分实验结果与其絮凝亲本株SPSC01相似。同时观察到了新的实验现象：BHL01絮凝的最佳pH范围拓宽为3-7.5；乙醇浓度影响上限提高到了30%(v/v)；BHL01的絮凝仅对甘露糖敏感,受其他糖类的影响较弱。综合分析,BHL01属于Flo1型絮凝,其絮凝机理符合类外源凝集素假说。
3.建立了BHL01乙醇发酵体系的表观生长动力学模型
通过两步法研究了自絮凝颗粒酵母BHL01的表观生长动力学。首先通过提高发酵罐搅拌速率来减小颗粒粒径,消除内扩散影响,获得了本征生长动力学模型。进而结合经典的传质理论,基于颗粒内部薄壳物料衡算,建立了表观生长动力学模型。通过不同底物浓度和粒径分布条件下的发酵实验数据拟合,获得了表观生长动力学模型参数,并验证了其可靠性。在此基础上,研究了内扩散效应导致颗粒内部可能出现死区的影响,绘制了发酵初始糖和粒径影响的操作图,并分析了这一发酵体系的性能。结果表明,死区的出现对比生长速率影响显著,且葡萄糖传质是影响表观生长动力学最主要的因素。最后,基于此模型提出了多级串联系统各发酵罐中,自絮凝颗粒酵母粒径分布相应调控的过程工程策略：主发酵罐适宜于较大粒径自絮凝颗粒酵母体系,以利于固定化,提高发酵罐中细胞密度和生产强度；而后发酵罐适宜于较小粒径自絮凝颗粒酵母体系,以减轻底物内扩散影响,保持所需的发酵活性。
综上所述,自絮凝颗粒酵母BHL01应用于乙醇连续发酵工艺优势明显。结合经典的本征生长动力学与传质理论,建立包含粒径分布在内的表观生长动力学模型,为乙醇工艺过程优化提供了有益的参考。
Ethanol fermentation with self-flocculating yeast immobilized within bioreactor is considered as a prospective method to improve the performance of fuel ethanol production. The process development is necessary for industrial application. Process design and amplification is based on the dynamics to quantitatively analyze and predicate the parameters during the ethanol fermentation. Besides ethanol, residual sugar and biomass, the size distribution of flocs is also concerned in the fermentation system. The genetically modified self-flocculating yeast BHL01 was selected as a model strain in this study, and the focused beam reflectance measurement system (FBRM) was used for on-line monitoring size distribution of flocs. In this work, we
1. Compared the performances of continuous ethanol fermentation systems using flocculating yeast BHL01 and free yeast ATCC4126
Under the same conditions, the fermentation parameters and main by-products were analyzed. It was observed that both strains showed similar fermentation performance. Meanwhile, due to the rector's retention, the biomass of BHL01 was higher and the ethanol production capacity of BHL01 was more efficient. However, the specific growth rate of BHL01 was significantly lower because of the mass transfer resistance caused by flocs.
2. Discussed the effects of physical and chemical factors on the floculation of BHL01
The impacts of stirring speed, biomass, temperature, pH, monovalent and bivalent cations, saccharides and ethanol concentration on flocculation of BHL01 was investigated. It was found that most of the experimental results were very similar with its parent strain SPSC01. Moreover, new experiment phenomenons was identified, such as pH range extended to 3-7.5, no influence on the flocs below 30% (v/v) ethanol concentration, only sensitive to mannose. The strain BHL01 is classified as Flol flocculation and the mechanism corresponds with zymolectin hypothesis.
3. Developed observed growth kinetic model for BHL01 continuous ethanol fermentation
An observed growth kinetic model for flocculating yeast BHL01 was developed through two parameter estimating strategies. First, the impact of mass transfer resistance was eliminated by increasing stirring speed of the fermentor to reduce the size of yeast flocs, and an intrinsic growth kinetic model was established. Based on the shell mass balance theory, an observed growth kinetic model was then proposed and model parameters which were verified reliability were obtained through experimental data collecting under the condition of different substrate concentrations and size distribution of yeast flocs. The effects of dead zone which caused by shortfall of glucose and toxicity of ethanol were analyzed, and the operating diagrams were mapped based on the data which generated from the growth model simulation.
The analytical results of the yeast flocs kinetic behavior indicate that the size distribution in each tank should be controlled separately in the tanks-in-series system for continuous ethanol fermentation. Large yeast flocs are preferred for the main tanks where higher sugar concentrations could prevent the occurrence of mass transfer resistance, while small yeast flocs should be applied for the rear tanks to maintain yeast flocs' viability and fermentability in which the sugar depletion significantly increases the risk of mass transfer resistance on yeast flocs. Therefore, comprehensive consideration of all the engineering aspects such as the separation of yeast flocs from the fermentation broth by sedimentation, the effective immobilization of yeast flocs within the tanks by retention and their size distribution are necessary.
In conclusion, self-flocculating yeast BHL01 is adopted competitively in the ethanol industry. The observed growth kinetics which is established by combining with the intrinsic growth kinetics and classic mass transfer theory provides beneficial reference for the ethanol process optimization.
1.对比了BHL01与游离亲本株ATCC4126在连续乙醇发酵过程中的性能
在相同的发酵条件下,分析两者稳态时的各发酵参数和发酵液组分,发现两者单位生物量的发酵效果相似。具有絮凝性状的BHL01受反应器的截留作用,生物量积累高,相应的其乙醇生产能力强；而絮凝形成带来的传质阻力影响,使得BHL01的比生长速率和比乙醇生产速率要明显低于游离酵母ATCC4126。
2.考察了各种物理化学因素对BHL01絮凝性状的影响
采用FBRM对特定物理化学条件下的BHL01絮凝颗粒粒径分布进行实时检测,发现大部分实验结果与其絮凝亲本株SPSC01相似。同时观察到了新的实验现象：BHL01絮凝的最佳pH范围拓宽为3-7.5；乙醇浓度影响上限提高到了30%(v/v)；BHL01的絮凝仅对甘露糖敏感,受其他糖类的影响较弱。综合分析,BHL01属于Flo1型絮凝,其絮凝机理符合类外源凝集素假说。
3.建立了BHL01乙醇发酵体系的表观生长动力学模型
通过两步法研究了自絮凝颗粒酵母BHL01的表观生长动力学。首先通过提高发酵罐搅拌速率来减小颗粒粒径,消除内扩散影响,获得了本征生长动力学模型。进而结合经典的传质理论,基于颗粒内部薄壳物料衡算,建立了表观生长动力学模型。通过不同底物浓度和粒径分布条件下的发酵实验数据拟合,获得了表观生长动力学模型参数,并验证了其可靠性。在此基础上,研究了内扩散效应导致颗粒内部可能出现死区的影响,绘制了发酵初始糖和粒径影响的操作图,并分析了这一发酵体系的性能。结果表明,死区的出现对比生长速率影响显著,且葡萄糖传质是影响表观生长动力学最主要的因素。最后,基于此模型提出了多级串联系统各发酵罐中,自絮凝颗粒酵母粒径分布相应调控的过程工程策略：主发酵罐适宜于较大粒径自絮凝颗粒酵母体系,以利于固定化,提高发酵罐中细胞密度和生产强度；而后发酵罐适宜于较小粒径自絮凝颗粒酵母体系,以减轻底物内扩散影响,保持所需的发酵活性。
综上所述,自絮凝颗粒酵母BHL01应用于乙醇连续发酵工艺优势明显。结合经典的本征生长动力学与传质理论,建立包含粒径分布在内的表观生长动力学模型,为乙醇工艺过程优化提供了有益的参考。
Ethanol fermentation with self-flocculating yeast immobilized within bioreactor is considered as a prospective method to improve the performance of fuel ethanol production. The process development is necessary for industrial application. Process design and amplification is based on the dynamics to quantitatively analyze and predicate the parameters during the ethanol fermentation. Besides ethanol, residual sugar and biomass, the size distribution of flocs is also concerned in the fermentation system. The genetically modified self-flocculating yeast BHL01 was selected as a model strain in this study, and the focused beam reflectance measurement system (FBRM) was used for on-line monitoring size distribution of flocs. In this work, we
1. Compared the performances of continuous ethanol fermentation systems using flocculating yeast BHL01 and free yeast ATCC4126
Under the same conditions, the fermentation parameters and main by-products were analyzed. It was observed that both strains showed similar fermentation performance. Meanwhile, due to the rector's retention, the biomass of BHL01 was higher and the ethanol production capacity of BHL01 was more efficient. However, the specific growth rate of BHL01 was significantly lower because of the mass transfer resistance caused by flocs.
2. Discussed the effects of physical and chemical factors on the floculation of BHL01
The impacts of stirring speed, biomass, temperature, pH, monovalent and bivalent cations, saccharides and ethanol concentration on flocculation of BHL01 was investigated. It was found that most of the experimental results were very similar with its parent strain SPSC01. Moreover, new experiment phenomenons was identified, such as pH range extended to 3-7.5, no influence on the flocs below 30% (v/v) ethanol concentration, only sensitive to mannose. The strain BHL01 is classified as Flol flocculation and the mechanism corresponds with zymolectin hypothesis.
3. Developed observed growth kinetic model for BHL01 continuous ethanol fermentation
An observed growth kinetic model for flocculating yeast BHL01 was developed through two parameter estimating strategies. First, the impact of mass transfer resistance was eliminated by increasing stirring speed of the fermentor to reduce the size of yeast flocs, and an intrinsic growth kinetic model was established. Based on the shell mass balance theory, an observed growth kinetic model was then proposed and model parameters which were verified reliability were obtained through experimental data collecting under the condition of different substrate concentrations and size distribution of yeast flocs. The effects of dead zone which caused by shortfall of glucose and toxicity of ethanol were analyzed, and the operating diagrams were mapped based on the data which generated from the growth model simulation.
The analytical results of the yeast flocs kinetic behavior indicate that the size distribution in each tank should be controlled separately in the tanks-in-series system for continuous ethanol fermentation. Large yeast flocs are preferred for the main tanks where higher sugar concentrations could prevent the occurrence of mass transfer resistance, while small yeast flocs should be applied for the rear tanks to maintain yeast flocs' viability and fermentability in which the sugar depletion significantly increases the risk of mass transfer resistance on yeast flocs. Therefore, comprehensive consideration of all the engineering aspects such as the separation of yeast flocs from the fermentation broth by sedimentation, the effective immobilization of yeast flocs within the tanks by retention and their size distribution are necessary.
In conclusion, self-flocculating yeast BHL01 is adopted competitively in the ethanol industry. The observed growth kinetics which is established by combining with the intrinsic growth kinetics and classic mass transfer theory provides beneficial reference for the ethanol process optimization.
引文
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