产油微藻贴壁培养条件优化与CO2传递及介质材料评价研究
文献类型：学位论文
| 作者 | 季春丽 |
| 学位类别 | 博士 |
| 答辩日期 | 2015 |
| 授予单位 | 中国科学院大学 |
| 授予地点 | 北京 |
| 导师 | 刘天中 |
| 关键词 | 贴壁培养 培养条件 CO2传质 贴壁材料 反应器 |
| 其他题名 | 无 |
| 学位专业 | 化学工程 |
| 中文摘要 | 微藻与传统的生物质原料相比具有更高的光合作用效率和固碳能力，其生长快速、生长周期短、含油量较高而且不与人争粮，不与粮争地，从微藻中提取的生物燃料被认为是唯一有潜力替代化石柴油的生物柴油来源。但大规模微藻生物质资源获得困难和微藻生物能源产品成本过高是目前微藻生物能源技术面临的两大瓶颈。传统液体培养方式产量低、成本高，难以支撑能源微藻产业化发展。贴壁培养是微藻培养的新方法，藻细胞以湿藻生物膜的形式生长，光能利用率高，培养基消耗少，且有克服传统培养方式耗水量大、能耗高、放大困难等潜力，有望实现微藻培养技术的重要突破。但目前对微藻贴壁培养的研究还处于初步阶段，有关微藻贴壁培养的工艺、环境影响因子、生物膜中的营养与碳传递及生长动力学行为、贴壁介质对生物膜的形成与生长的影响等还缺乏深入的了解，而这些问题直接影响到该技术的发展和实际应用。因此本论文以产油栅藻 (Scenedesmus obliquus) 为对象，首先考察了主要培养基组成及补碳条件对栅藻贴壁培养生长的影响，并进行了多因素优化。在此基础上，建立了基于碳传递的栅藻贴壁培养动力学模型，并通过对培养过程pH值的全局拟合实现了动力学过程模拟。首次建立了流室法和T-型刮膜法来评价细胞在贴壁介质表面的粘附强度，进而考察和评价了贴壁培养介质材料。最后对微藻在几种结构的贴壁反应器中的培养进行了实验评价。获得的主要结果如下：(1) 明确了氮、磷、碳三种营养元素和通气速率对栅藻贴壁培养的影响，并通过响应面实验获得了优化的培养条件。实验结果表明氮、磷、碳三种元素是影响栅藻生长和油脂积累的重要因素。生物量积累随培养基中氮、磷浓度的升高而升高，而油脂和TAG积累却相反，但是在某一特定氮、磷浓度下油脂和TAG产率会达到最大值；在培养基循环的方式下，60 L氮浓度为1.76mM (为正常BG11中氮浓度的1/10) 的BG11可使1 m2接种量为10 g m-2的栅藻的总脂和TAG产率达到最大，同时生物量产率也可达到一个较高值，从而实现栅藻生长与油脂的同步积累。CO2浓度过低和通气速率偏小会影响栅藻正常生长，但当CO2浓度和通气速率超出一定范围后，继续增大CO2浓度和通气速率对培养没有意义；对氮、磷、碳浓度以及通气速率的响应面实验结果表明，这四者之间对栅藻生长和油脂积累有协同作用。为获得较高的贴壁培养生物量产率需要较丰富的氮源，其最佳营养条件为：氮浓度3.52 mM、磷浓度0.04 mM、CO2浓度0.7% 和通气速率0.06 vvm。而为获得较高油脂产率，则需要适当降低初始氮和磷浓度，并适当提高通气速率，其最佳营养条件为：氮浓度2.21 mM、磷浓度0.02 mM、CO2浓度0.7% 和通气速率0.07 vvm。(2) 建立了基于CO2传递的栅藻贴壁培养动力学模型，通过对培养过程中培养液的pH变化曲线的全局拟合实现了全过程的模拟。模拟结果表明，CO2浓度对CO2的表观传质系数 (KL) 有重要影响，CO2浓度越低，其表观传质系数越大；通气速率对CO2的表观传质系数影响不明显，但提高培养基循环速率有助于提高CO2的表观传质系数。模型其他参数却基本不受培养条件的影响。当培养 (4天) 结束后，培养液中碳的各种组元组成浓度在各条件下基本相同，且碳源主要以碳酸氢根的形式存在。除了在低浓度CO2条件下，不同条件下的生物膜生长速度基本相当，表明在栅藻贴壁培养时，当通入CO2达到适当浓度，碳的供给不会成为生长的限制性环节。 (3) 对贴壁培养的膜材料和可重复利用合成纤维贴壁材料进行了筛选。实验研究了三种细胞大小的微藻Nannochloropsis sp. (1-3 μm)、Scenedesmus obliquus (3-8 μm) 和Tribonema minus (0.5-3 mm) 在7种膜材料和13种可重复利用的合成纤维材料上的生长以及粘附情况，建立了基于Flow chamber 流室法和T-shape 刮膜法来测定藻细胞生物膜在介质材料表面粘附强度的方法，考察了接种密度、生长期、培养基pH值等对粘附强度的影响。结果显示，细胞对材料的粘附性分别随接种量的增大以及生长期的增长而增大，但是藻液的pH值对细胞的粘附性无明显影响；藻类在亲水性的以及粗糙度较高的膜材料上的粘附性相对较好，但粗糙度太大又不利于生物膜的刮除；相对于单细胞藻Nannochloropsis和Scenedesmus，丝状藻Tribonema对膜材料的粘附作用更显著； Tribonema由于其丝状特性，在所有的合成纤维材料上的生长都较好，而Scenedesmus和Nannochloropsis 由于细胞大小的原因在纤维材料上的接种情况较差，生物量产率较低；减少接种时细胞损失、接种均匀以及使用收获后残留在材料上的藻细胞作为再培养的藻种可以提高微藻在合成纤维上生长的生物量产率，还可以降低细胞在培养过程中被培养基冲刷下来的比率。(4) 研究了Scenedesmus在转鼓、转盘和多层插板反应器中以及Scenedesmus和Tribonema在薄多层反应器中的生长情况。与单层贴壁培养反应器相比，微藻在应用了光稀释原理的反应器中贴壁培养时生物量产率都有不同程度较大幅度的提高，而且生物量产率均随光稀释率的增加而增大；薄多层反应器不仅可以较大幅度地提高微藻在可重复利用合成纤维材料上贴壁生长的生物量产率，而且也可以减小细胞被冲刷或剥离材料表面的程度。 |
| 英文摘要 | Microalgae is widely recognized as the most promising feedstock for biodiesel industry because of its more rapid growth rate, shorter growth cycle, higher photosynthetic efficiency, higher carbon fixation capacity, higher oil content as well as non-competition with food production and arable land usage compared with conventional biological feedstock. However, the major difficulty that hampers economical biodiesel production from microalgae is the severe shortage of cheap algal biomass. Low productivity and high cost are main drawbacks for the conventional liquid cultivation methods. The semi-dry attached cultivation has been proposed and it has demonstrated for several advantages over conventional suspended cultivation of microalgae including higher efficiency for solar energy utilization, easier harvest, decreased water consumption and energy requirement etc. However, many biotechnological aspects including the optimization of the nutrient supply tactic, environmental impact factors, the mechanism of CO2 and nutrients mass transfer in biofilm, the effect of substrata on the formation and growth of microalgae biofilm etc. are needed to be further explored before this technology is pushed into industrial application of microalgae mass cultivation. In this study, the oleaginous microalgae mainly Scenedesmus obliquus was chosen as research object, the effects of primary culture medium compositions and carbon supplement ways on the growth and lipid accumulation of Scenedesmus obliquus were investigated firstly. Then a kinetic model based on carbon dioxide mass transfer and algal biofilm growth was constructed and solved via pH profile fitting. In order to evaluate the adhesive strength of algal biofilm on substrata to provide information for substrata choice for attached cultivation, two methods of flow chamber and T-shape scraper were setup for the first time to measure the adhesive strength between cells and membranous substrata. Finally, cultivation of Scenedesmus obliquus was conducted in several photobioreactors and the cultivation efficiency was evaluated. The achieved main results were as follows:(1) The optimization of Scenedesmus obliquus growth and lipid accumulation in attached cultivation were studied. It was demonstrated that all the concentrations of NaNO3, K2HPO4, aerated CO2 and aeration flow rate had significant influences on the growth and lipid accumulation of Scenedesmus obliquus. Higher N and P concentrations were favorable for biomass yield improvement, however, N and P limitation stimulated the lipid and TAG accumulation. The lipid and TAG productivities reached the highest values when the N and P were at appropriate concentrations. An optimized nitrogen supply strategy for high efficiency production of biomass and total lipids simultaneously was proposed as circulating ca. 60 L of BG-11 medium containing 1/10 of nitrate (1.76 mM) per 1 m2 of cultivation surface with an initial biomass concentration of 10 g m-2. The biomass, lipid and TAG productivity were increased as the CO2 concentration and flow rate increase within certain extent, but further increase of CO2 concentration or flow rete might not be effective to boost biomass and lipid accumulation. The interrelationship of the four parameters were studied and optimized by response surface methodology, 3.52 mM NaNO3, 0.04 mM K2HPO4, 0.7% CO2 and 0.06 vvm flow rate led to the maximum biomass productivity of 9.04 g m-2 d-1, while the maximum total lipid and TAG (1) The optimization of Scenedesmus obliquus growth and lipid accumulation in attached cultivation were studied. It was demonstrated that all the concentrations of NaNO3, K2HPO4, aerated CO2 and aeration flow rate had significant influences on the growth and lipid accumulation of Scenedesmus obliquus. Higher N and P concentrations were favorable for biomass yield improvement, however, N and P limitation stimulated the lipid and TAG accumulation. The lipid and TAG productivities reached the highest values when the N and P were at appropriate concentrations. An optimized nitrogen supply strategy for high efficiency production of biomass and total lipids simultaneously was proposed as circulating ca. 60 L of BG-11 medium containing 1/10 of nitrate (1.76 mM) per 1 m2 of cultivation surface with an initial biomass concentration of 10 g m-2. The biomass, lipid and TAG productivity were increased as the CO2 concentration and flow rate increase within certain extent, but further increase of CO2 concentration or flow rete might not be effective to boost biomass and lipid accumulation. The interrelationship of the four parameters were studied and optimized by response surface methodology, 3.52 mM NaNO3, 0.04 mM K2HPO4, 0.7% CO2 and 0.06 vvm flow rate led to the maximum biomass productivity of 9.04 g m-2 d-1, while the maximum total lipid and TAG productivities of 3.21 and 2.46 g m-2 d-1, respectively were obtained under the conditions of 2.21 mM NaNO3, 0.02 mM K2HPO4, 0.7% CO2 and 0.07 vvm flow rate. (2) A kinetic model based on CO2 mass transfer and algal biofilm growth was developed and the simulation of biofilm cultivation process in attached cultivator was conducted using the analysis of the pH profiles. It elucidated the CO2 mass transfer behavior and the variations of inorganic carbon components in culture medium, and all the kinetic parameters were determined. Apart from the overall mass transfer coefficient KL, the other kinetic parameters were independent from the cultivation conditions including aerated CO2 concentration, aeration rate and medium circulation speed. The concentration of aerated CO2 has significant effect on KL, lower concentration of aerated CO2 resulted higher KL. It indicated that for the attached cultivation of Scenedesmus obliquus, CO2 with lower concentration was to the benefit of higher carbon fixation efficiency. The increase of aeration flow rate had no obvious effect on KL but higher medium circulation speed could improve the KL. The final compositions and concentrations of inorganic carbon compounds in medium under different cultivation conditions were approximately the same and HCO3- was the main component. When the concentration of aerated CO2 was in certain range, there was no obvious difference between algal biofilm growth rates under different cultivation conditions which indicated the carbon supplement was not a limiting factor then.(3) Methodology and characterization of substratum for attached cultivation microalgae were explored. 7 membrane materials and 13 synthetic fiber substrata were tested with three species of microalgae (Scenedesmus obliquus, Nannochloropsis sp. and Tribonema minus). Flow chamber and T-shape scraper were used to evaluate the adhesive strength between cells and membranous substrata, and the effects of inoculation concentration, cultivation time and pH of culture medium etc. on the adhesive strength were studied. The results indicated that microalgae preferred to grow on hydrophilic and rough membranes, but high ratio of surface roughness was unfavourable for clear biofilm scraping. Filamentous algae Tribonema had higher cohesive strength between cells and adhesive strength between cells and substrata. Moreover, adhesive strength increased with higher inoculation concentration and longer cultivation time. pH of medium culture affected adhesive strength insignificantly. The algal biomass accumulation on the synthetic fiber substrata were not as well as that on membranes substrata especially for unicellular microalgae (Scenedesmus and Nannochloropsis). And cells loss when inoculation was the main reason resulted in lower biomass yields. Reduction of cells loss, homogeneous inoculation and usage of residual cells remaining after first harvest as inoculation seeds were effective solutions for better growth and lower wash out ration of the unicellular microalgae on these substrata. Tirbonema showed potential in attached cultivation on synthetic fiber substrata for its wide selectivity of substrata, relative satisfied biomass productivity and lower cells loss ratio when flushed by culture medium in vertically placed photobioreactor.(4) Three photobioreactors including rotary drum, rotation disc and multi-plates bench reactors were used to cultivate microalgae Scenedesmus, moreover, a novel photobioreactor named multi-thin layer phobioreactor was designed to reduce the washout ration of Scenedesmus cells on synthetic fiber substrata. The results revealed that the biomass productivity of Scenedesmus all enhanced in the four reactors compared with single layer cultivation reactor. And higher light dilution rate contributed to higher biomass productivity. Multi-thin layer attached photobioreactor could reduce the cell washout ration of Scenedesmus. |
| 学科主题 | 工学 |
| 语种 | 中文 |
| 公开日期 | 2019-12-31 |
| 源URL | [http://ir.qibebt.ac.cn/handle/337004/8089]  |
| 专题 | 青岛生物能源与过程研究所_微藻生物技术团队 |
| 作者单位 | 中国科学院青岛生物能源与过程研究所 |
| 推荐引用方式 GB/T 7714 | 季春丽. 产油微藻贴壁培养条件优化与CO2传递及介质材料评价研究[D]. 北京. 中国科学院大学. 2015. |
入库方式： OAI收割
来源：青岛生物能源与过程研究所
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