小球藻高密度培养及油脂提取条件的优化

【目的】高密度培养小球藻及优化油脂提取条件。【方法】通过进行单因素实验研究不同培养基组成及环境因子对其细胞生长影响,并采用超声波提取法进行正交实验对藻粉油脂提取条件进行研究。【结果】对椭圆小球藻Y4进行异养培养,最适培养条件为:葡萄糖50 g/L,硝酸钾2 g/L,适宜的培养温度、摇床转速和接种量分别为29°C、180 r/min和20%。在此基础上,进行了1 L发酵罐培养实验,获得了干重18.25 g/L的生物量。通过对油脂提取条件进行优化,Y4的油脂提取率由优化前的25.0%提高到60.2%,提高了35.2%。【结论】优化了小球藻的培养条件及油脂提取条件,促进了小球藻的开发和利用。     

自养和兼养条件下蛋白核小球藻昼夜节律的响应

【目的】研究自养和兼养两种培养方式对蛋白核小球藻(Chlorella pyrenoidosa)生长、细胞分裂和生化组分积累的影响,探讨人工培养蛋白核小球藻的昼夜节律响应机制和优化技术。【方法】小球藻自养培养采用BG11培养基,兼养培养基在BG11培养基中添加4种不同浓度(1、5、10、20 g/L)的葡萄糖,培养周期为10 d。血球板计数法测定藻细胞浓度,干重法测定藻细胞生物量。显微观察藻细胞大小和分裂情况。脂染色法测定小球藻总脂的含量,藻细胞的叶绿素、蛋白和淀粉分别采用甲醇、氢氧化钠、硝酸钙浸提后通过紫外分光光度法定量测定。【结果】葡萄糖兼养培养对蛋白核小球藻具有显著的促生长效应,最适浓度为10 g/L。10 d收获时,兼养组(10 g/L葡萄糖)藻细胞浓度和干重分别是自养组的2.57倍和6.73倍。分析一昼夜中的藻细胞增殖规律可知,第2天和第5天时自养组中增殖的新生子细胞约有76.00%在黑暗期分裂产生,而兼养组中第2天和第5天光照期的新细胞增殖量占比分别达到40.90%和67.50%。一昼夜内藻细胞大小的迁移动态监测表明,第2天自养组藻细胞的体积变化静息期为8 h,兼养组只有4 h;第5天两组藻细胞大小迁移动态的昼夜节律明显,但兼养组黑暗结束后较大细胞(D6μm)占比显著高于自养组。第8天时,兼养组藻细胞已处于稳定期,总脂和蛋白含量均显著高于自养组,藻细胞总脂和色素含量在一昼夜中相对稳定,但蛋白和淀粉含量分别在光照8 h和12 h左右达到峰值。从第2天开始,对兼养组细胞每天进行2 h光延长,收获时藻细胞浓度和干重分别比对照组提高13%和11%。【结论】葡萄糖兼养培养能大幅提高蛋白核小球藻的生物量。蛋白核小球藻生长增殖与生化组分积累均受昼夜节律调控,自养条件下藻细胞以光照期生长黑暗期增殖为主。兼养培养提高藻细胞生物量的机制在于缩短藻细胞生长静息期,在昼夜节律中加速藻细胞生长并显著提高通过细胞周期检查点的细胞比例,光照期效应尤其明显。藻细胞蛋白和淀粉含量昼夜节律明显,最佳收获时间分别在光照8 h和12 h后。     

热带普通小球藻培养模式的筛选及其培养基的优化

【背景】从海南热带海区中分离得到一株微藻,其生长速度快、适应力强,经鉴定该微藻为普通小球藻。【目的】提高热带普通小球藻的生长速率。【方法】以"宁波大学3#微藻培养液配方"为基础培养液,分别添加有机碳(C6H12O6和CH3COONa)对热带普通小球藻进行自养、兼养及异养培养,获得促进热带普通小球藻快速生长的培养方式。在"宁波大学3#微藻培养液配方"的基础上对热带普通小球藻的兼养培养基配方进行优化,并用优化兼养培养基与"宁波大学3#微藻培养基"对比培养热带普通小球藻。【结果】添加6 g/L CH3COONa的兼养模式促进热带普通小球藻生长效果最好;优化的兼养培养基配方为:6 g/L CH3COONa,20 mg/L(NH4)2SO4-N,5 mg/L Na H2PO4-P,3 mg/L Fe SO4-Fe,1 mg/L Vitamin B1和0.000 5 mg/L Vitamin B12。对比培养实验结果显示,培养第6天,兼养培养液收获的生物量(细胞密度)达4.20×107 cells/m L,是"宁波大学3#配方微藻培养液"的2.30倍。【结论】兼养培养模式为热带普通小球藻的最佳培养模式,优化的兼养培养基极显著地提高了热带普通小球藻的生物量(P0.01)。     

葡萄糖对小球藻(Chloralla sp. HN08)光合作用和生长的影响

【目的】探讨葡萄糖作为外加碳源对热带海洋小球藻(Chloralla sp.HN08)生物质生产和脂、光合色素、碳水化合物及可溶性蛋白等细胞主要成份含量的影响。【方法】分析比较小球藻HN08在光合自养和兼养(添加10 g/L葡萄糖)2种营养方式下的生长速率、细胞密度、光合放氧速率、油脂相对含量,以及可溶性总糖、淀粉和可溶性蛋白的含量。【结果】结果表明,在光照条件下葡萄糖(10 g/L)能促进小球藻(Chloralla sp.HN08)生长,提高细胞终密度,而异养条件下藻细胞逐渐衰亡。兼养条件下,细胞相对生长速率及细胞终密度分别是自养条件下的6.8倍和1.3倍。兼养藻细胞中可溶性糖、淀粉、油脂含量显著高于(P0.05)光合自养细胞,然而可溶性蛋白质和光合色素含量显著低于(P0.05)光合自养细胞。添加葡萄糖的小球藻液的光饱和点和呼吸速率均高于光自养条件下的细胞,但2种培养条件下藻液的净光合速率无显著差异(P0.05)。【结论】光照条件下,添加葡萄糖可显著提高小球藻HN08相对生长速率和细胞终密度,促进油脂与淀粉的积累。     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoidosa No.2。研究了培养基组成及培养条件对其细胞生长和油脂积累的影响。结果表明, 最适培养基组成为(g/L)：葡萄糖 20, 甘氨酸 0.08, MgSO4·7H2O 0.4, K2HPO4 1.0, FeSO4·7H2O 0.004; 适宜的培养温度、初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和 650 Lux。在上述优化条件下培养7 d, Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L 和 40.15%提高到6.56 g/L和59.90%, 油脂产量提高了162%。Chlorella pyrenoidosa No.2能以木糖为碳源产油脂, 可望用于以木质纤维素等可再生生物质资源为原料生产油脂。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 不饱和脂肪酸含量达71％左右, 可作为生产生物柴油的原料。     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoi-dosa No.2.研究了培养基组成及培养条件对其细胞生长和油脂积累的影响.结果表明,最适培养基组成为(g/L):葡萄糖20,甘氨酸0.08,MgSO4·7H2O 0.4,K2HPO4 1.0,FeSO4·7H2O 0.004;适宜的培养温度,初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和650 Lux.在上述优化条件下培养7 d,Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L和40.15%提高到6.56 g/L和59.90%,油脂产量提高了162%.Chlorella pyrenoidosa No.2能以木糖为碳源产油脂,可望用于以木质纤维素等可再生生物质资源为原料生产油脂.气相色谱分析表明该油脂的脂肪酸组成与植物油相似,不饱和脂肪酸含量达71%左右,可作为生产生物柴油的原料.     

一株高蛋白含量小球藻TX的分离鉴定及其诱变育种

【背景】小球藻由于蛋白含量高、营养丰富,在水产养殖上可直接作为鱼、虾、贝类的优质饵料。【目的】对从养殖环境中分离的小球藻进行诱变,选育生长快、蛋白含量高的突变株,为水产养殖天然饵料生产提供优良藻种资源。【方法】以从养殖环境中筛选的生长相对较快且蛋白含量较高的TX作为出发藻株,对该藻株进行分子鉴定,并对该藻株进行紫外诱变、甲基磺酸乙脂(ethyl methyl sulfonate,EMS)诱变和复合诱变,采用96孔板高通量筛选技术和递进式重复筛选方法选育高生物量、高蛋白突变株。【结果】经18SrRNA基因序列分析,TX鉴定为Chlorella sorokiniana,从540个可能的突变株中筛选到8个遗传稳定且生长较快的突变株,其中H10的总蛋白含量达64.2%,可溶性蛋白含量达0.44g/L,干重达0.72g/L,分别较出发藻株提高3.4%、15.8%和26.2%。【结论】突变株H10蛋白含量高且生长较快,可用于天然饵料生产。     

葡萄糖对单针藻异养、兼养生长及油脂合成的影响

利用Andrew方程考查了异养和兼养培养模式下葡萄糖对单针藻Monoraphidium sp.FXY-10生长的影响,拟合出葡萄糖消耗动力学模型;同时研究了不同培养模式下葡萄糖对单针藻油脂合成的影响,拟合出最佳葡萄糖浓度时油脂合成动力学模型。结果表明,葡萄糖浓度为10.0g/L时,异养、兼养培养条件下藻细胞生物量和油脂含量达到最大值;异养、兼养培养条件下葡萄糖的半饱和常数KS分别为17.82g/L和1.89g/L,说明兼养培养时单针藻对葡萄糖的吸收能力相对较高。     

不同营养方式对普通小球藻生长代谢及生化组分的影响

摘要:【目的】系统研究自养、混养和异养3种营养方式对真核模式微藻———普通小球藻(Chlorella vulgaris)生长特性、细胞生化组分和碳代谢途径关键酶活性的影响。【方法】以C．vulgaris为研究对象,通过设置光合自养、混养和异养3 种营养方式,采用光谱学、色谱学方法,研究不同营养方式对C．vulgaris从生长特性、细胞组分合成和碳代谢等方面的影响。【结果】C．vulgaris依次经自养至混养和异养的培养方式转变中,藻细胞的可溶性糖和油脂含量显著提高,油脂中C16、C18不饱和脂肪酸的相对含量降低,而饱和脂肪酸的含量升高;蛋白质含量、光合色素含量显著下降,18种氨基酸的相对含量也呈下降趋势;葡萄糖的添加可抑制藻细胞吸收和积累除碳元素以外的其他测试元素。在添加葡萄糖的前提下,光照可促进藻细胞的生长量、不饱和脂肪酸和氨基酸,以及除碳元素以外的其他测试参数增加。对微藻胞外碳酸酐酶和核酮糖-1,5-二磷酸 羧化酶的活性分析结果表明,异养和混养直接影响C．vulgaris的碳代谢途径。【结论】光源和葡萄糖的供给与否直接影响C．vulgaris的生长代谢和生化组分合成,葡萄糖的添加在显著促进藻细胞生物量积累的同时,刺激碳素(糖类和油脂)生化成分的合成,而抑制氮素成分(蛋白质和光合色素)的合成;在光照条件下培养基质中葡萄糖的浓度和消耗水平直接决定藻细胞主营自养或异养生长。添加有机碳源葡萄糖的混养(光照)和异养(暗处理)培养可促进藻细胞的生长,异养和自养的生物量之和接近于混养,表明混养是最佳的藻细胞营养生长方式。     

富油微藻的筛选及其产油能力的评价

【目的】筛选具有较快生长速率及较强产油能力的微藻,探究所获得微藻的生理生化性能及不同培养方式对其生物量、产油能力、碳消耗等生长特性的影响与藻种对pH的适应能力。【方法】通过磷酸香草醛测定法及尼罗红染色对微藻进行初筛复筛,通过设置光合自养、异养和混养等3种培养方式,并采用气质联用等方法,研究不同培养方式对所获微藻生长特性、所产油脂脂肪酸组成以及碳代谢等方面的影响。【结果】筛选出两株产油能力较强的藻株H、Z_8,其油脂产量分别可达1.14±0.05 g/L和1.33±0.10 g/L,经形态观察和分子生物学鉴定初步表明藻株H属布朗单针藻(Chlorolobion braunii)、藻株Z_8属链带藻(Desmodesmus intermedius)。构建了不同培养方式下微藻动力学模型,H、Z_8属于生长偶联型。当培养环境的pH处于6.0–9.0,对藻株H、Z_8的总脂量与生物量无明显差异(P0.05)。【结论】筛选获得的藻株H、Z_8中C16与C18脂肪酸占总脂肪酸的比率能达到90%以上。藻种在混养条件下生物量积累优于异养,但异养条件下更加有利于油脂的积累,且H、Z_8均具有较为宽泛的pH适应能力,是具有一定产业化应用潜力的优良产油藻株。     

Heterotrophic production of lutein by selected Chlorella strains

Seven Chlorella strains representing three species obtained from culture collections and research laboratories were screened for their potential of heterotrophic production of lutein on two different media (Basal and Kuhl) containing glucose. While both media supported good growth and lutein formation of the seven strains in darkness, higher biomass concentrations and lutein content were achieved on Basal medium. Chlorella protothecoides CS-41 was chosen from the seven strains for further investigation due to its higher productivities of both biomass and lutein. The maximal biomass concentration and lutein content of C. protothecoides cultivated heterotrophically with 9 g L^-1 glucose in a 3.7-L fermentor were respectively 4.6 g dry cells L^-1 and 4.60 mg lutein g^-1 dry cells on Basal medium, and 4.0 g dry cells L^-1 and 4.36 mg lutein g^-1 dry cells on Kuhl medium. The heterotrophic cultivation process was scaled up successfully to 30 L using a fermentor, in which the Basal medium containing 36 g L^-1 glucose was used; the maximal biomass concentration of 16.4 g dry cells L^-1, specific growth rate of 0.92 d^-1,lutein content of 4.85 mg lutein g^-1 dry cells,growth yield of 0.47 g dry cells g^-1 glucose and lutein yield of 1.93 mg lutein g^-1 glucose were respectively achieved. This revised version was published online in August 2006 with corrections to the Cover Date.     

以异养细胞作为种子的椭圆小球藻产油脂光自养培养优化

异养细胞种子/光自养培养方法是一种可异养培养的能源微藻培养的有效方法,但已有文献尚未从工艺优化角度考察其发展潜力。为了获得较高细胞密度的用于光自养培养的种子和提高光自养培养的细胞密度与油脂产率,对异养细胞种子/光自养培养的培养基和培养条件进行了优化。结果表明,采用优化后的培养基,椭圆小球藻在摇瓶中异养培养的最高藻细胞密度可达11.04 g/L,比在初始培养基条件下提高了28.0%,在5 L发酵罐中异养培养的藻细胞密度达到73.89 g/L;在2 L柱式光生物反应器中光自养培养的藻细胞密度、油脂含量和油脂产率分别达1.62 g/L、36.34%和6.1 mg/(L·h),油脂成分主要为含C16-C18碳链的脂肪酸,是制备生物柴油的理想原料。经过优化,异养细胞种子/光自养培养这一方法能够显著地提高椭圆小球藻产油脂的能力,这进一步表明异养细胞种子/光自养培养方法有望成为可异养的能源微藻的高效培养方式。     

光合自养缺陷型小球藻的筛选及生物能源应用

小球藻Chlorella protothecoides(C.protothecoides)是潜在的、可用于工业生产生物柴油的高产油微藻.本研究通过体外诱变的手段,获得了一株完全不能进行光合自养生长的突变体Al64.利用尼罗红染色和叶绿素自发荧光分析和电子显微镜分析细胞的亚显微结构,结果显示该突变体中叶绿体严重退化,其中类囊体膜结构缺失,导致该突变体缺乏叶绿素,无法进行光合自养生长.在富糖富氮的培养条件下,该光合自养缺陷型突变体的细胞密度和油脂含量比野生型细胞分别高5.54%和6.76%,分析还发现,该突变体产油能力为0.158 g L?1 h?1,比野生型提高12.8%.本文通过缺失光合作用突变体的构建,在异养高氮条件下实现了生物量及细胞内油脂含量的同步提高,为进一步提高微藻生产生物柴油的产量提供了新的研究平台.     

Sequential heterotrophic/autotrophic cultivation – An efficient method of producing Chlorella biomass for health food and animal feed

Sequential heterotrophic/autotrophic cultivation method was investigated for production of high concentration of Chlorella biomass with high cellular protein and chlorophyll contents. By using autotrophic growth medium, which contains glucose as organic carbon source, for heterotrophic culture, the protein and chlorophyll contents of the cells could be increased by simply illuminating the culture broth and aerating with CO₂-enriched air at the end of the heterotrophic culture. A system was then constructed for continuous sequential heterotrophic/autotrophic production of algal biomass. The system was composed of the conventional mini-jar fermentor for the heterotrophic phase and a tubular photobioreactor for the autotrophic phase. The exhaust gas from the heterotrophic phase was used for aeration of the autotrophic phase in order to reduce the CO₂ emission into the atmosphere. With this system, it was possible to produce high Chlorella biomass concentration (14 g L-¹) containing 60.1% protein and 3.6% chlorophyll continuously for more than 640 h. During the steady state, about 27% of the CO₂ produced in the heterotrophic phase was re-utilized in the autotrophic phase. When the tubular photobioreactor was replaced with a 3.5-L internally illuminated photobioreactor, the productivity increased from 2 g L-¹ d-¹ to 4 g L-¹ d-¹. However, the chlorophyll content of the cells was lower due to the lower light supply coefficient of the photobioreactor. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of Trophic Modes to Maximize Biomass and Lipid Productivity of <Emphasis Type="Italic">Micractinium inermum</Emphasis> NLP-F014

An optimum trophic mode condition was investigated to maximize biomass and lipid productivity of Micractinium inermum NLP-F014, which grown successfully in blended wastewater medium. In this study, four trophic modes were used, including photoautotrophic, photoheterotrophic, heterotrophic and mixotrophic modes. Mixotrophic mode showed the highest biomass and lipid productivity. However, a high concentration of organics resulted the negative effect on the growth of M. inermum NLP-F014. Mixotrophic cultivation using glucose below 500 mg/L was able to produce maximum biomass productivity up to 0.90 ± 0.03 g/L/day as well as maximum lipid productivity up to 129.31 ± 0.10 mg/L/day. From lipid analysis on mixotrophic mode using glucose, the major fatty acids are oleic acid (C18:1), linoleic acid (C18:2) and palmitic acid (C16:0). These results suggest that mixotrophic mode cultivation with wastewater containing chemical oxygen demand (COD) below 500 mg/L could be applicable for biodiesel production of M. inermum NLP-F014.     

2种小球藻在鱼糜加工废水处理过程中的生长和油脂积累规律

以小球藻Chlorella pyrenoidsa F-9和C.vulgaris HYS-2为材料,研究小球藻在鱼糜加工废水中的生长速率、脂类积累规律和对氮磷营养的消除效果。结果表明,2种小球藻在鱼糜加工废水中快速生长,最高生物量达到0.293 g/L和0.276 g/L,分别是f/2对照培养基的2.4和1.6倍。小球藻HYS-2在鱼糜加工废水中油脂百分含量最高达到36.22%,为对照培养基的1.27倍,小球藻F-9在鱼糜加工废水中的油脂含量达到17.89%,为f/2培养基中的0.89倍。培养15 d后,2种小球藻对鱼糜加工废水中氮磷去除率均达90%以上。以上研究表明,用未经任何化学处理的鱼糜加工废水养殖小球藻在生长速率上远远大于f/2培养基,说明其具有较好地发展潜力,可以实现微藻的低成本养殖。     

花生四烯酸产生菌高山被孢霉的高糖驯化研究

【目的】提高花生四烯酸(Arachidonic acid,ARA)产量,克服ARA产生菌高山被孢霉(Mortierella alpina)在长期的保存及使用过程中易受到外界条件影响发生退化,从而导致菌种耗糖量降低、影响菌种摄入营养的能力和不利于工业化生产的缺点。【方法】首先采用固体培养基驯化,将菌种逐级涂布于梯度高糖PDA平板(含糖量分别为2%、5%、7%、10%和15%)培养,挑选经固体驯化后能耐受10%高糖浓度平板的菌种,转接到两种含不同氮源的梯度高糖(含糖量分别为3%、4%、5%和6%)液体培养基中进行驯化,最后对驯化后的菌种进行2 L发酵罐放大实验。【结果】当培养基中以酵母粉为氮源时,驯化后菌体的最高耗糖量由3 g/(L.d)提高到12 g/(L.d);当培养基中以玉米浆为氮源时,驯化后菌体的最高耗糖量由7 g/(L.d)提高到12 g/(L.d)。摇瓶驯化实验结果表明以玉米浆为氮源驯化的菌种发酵效果较好,发酵罐实验结果显示菌体生物量为50 g/L,总油脂为18 g/L,目的产物ARA产量为8 g/L。相比未驯化之前的发酵结果,生物量和总油脂含量提高了近3倍,ARA产量提高了近4倍。【结论】经过高糖驯化,菌种的耗糖能力得到提高,生物量、总油脂及ARA的产量也都有所增加,从而可以使菌种在保存和使用过程中不易退化,保持稳定。     

Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources

The effects of nitrate, ammonium, and urea as nitrogen sources on the heterotrophic growth of Chlorella protothecoides were investigated using flask cultures. No appreciable inhibitory effect on the algal growth was observed over a nitrogen concentration range of 0.85-1.7 g l(-)(1). In contrast, differences in specific growth rate and biomass production were found among the cultures with the various nitrogen compounds. The influence of different nitrogen sources at a concentration equivalent to 1.7 g l(-)(1) nitrogen on the heterotrophic production of biomass and lutein by C. protothecoides was investigated using the culture medium containing 40 g l(-)(1) glucose as the sole carbon and energy source in fermentors. The maximum biomass concentrations in the three cultures with nitrate, ammonium, and urea were 18.4, 18.9, and 19.6 g l(-)(1) dry cells, respectively. The maximum lutein yields in these cultures were between 68.42 and 83.81 mg l(-)(1). The highest yields of both biomass and lutein were achieved in the culture with urea. It was therefore concluded that urea was the best nitrogen source for the production of biomass and lutein. Based on the experimental results, a group of kinetic models describing cell growth, lutein production, and glucose and nitrogen consumption were proposed and a satisfactory fit was found between the experimental results and predicted values. Dynamic analysis of models demonstrated that enhancing initial nitrogen concentration in fermentor cultures, which correspondingly enhances cell growth and lutein formation, may shorten the fermentation cycle by 25-46%.