响应面法优化自养小球藻产生物柴油油脂

利用响应面法对小球藻(Chlorella vulgaris)在2L气升式生物反应器中对自养产生物柴油油脂的培养条件进行了优化。首先用Plackett-Burman方法对10个相关影响因素的效应进行评价并筛选出对产油有显著影响的3个因素:KNO3浓度、温度和CO2浓度;再用最陡爬坡实验逼近最大产油区域;最后由中心组合实验及响应面分析确定了影响产油主要因素的最佳条件为:KNO3浓度0.31g/L,温度26.5℃,CO2浓度6.80%,最高产油量达到0.42g/L,比优化前提高了近2倍。优化后,在10L气升式生物反应器中进行了扩大培养。     

产油微藻的分离、筛选及自养培养氮源、碳源的优化

从云南滇池的水样中分离筛选得到一株自养产油小球藻(Chlorella vulgaris,C.vulgaris),其油脂产率可达28.6mg/(L·d),进一步考察了不同氮源、氮源浓度和添加无机碳源对其自养生长和油脂积累的影响。结果表明,硝酸钠为优化氮源,氮元素的优化浓度为123mg/L,油脂含量随氮元素浓度升高而降低;添加NaHCO3显著提高了C.vulgaris生物量产率和油脂产率,其优化浓度为800mg/L。在氮源和碳源的优化浓度下,C.vulgaris的最大生物量产率和油脂产率可达332.8mg/(L·d)和100.2mg/(L·d),分别是对照组的3.6和3.4倍。     

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。     

自养与异养小球藻脂溶性化合物对比研究

应用细胞培养技术获绿色自养小球藻与乳黄色异养小球藻细胞。异养藻细胞脂溶性化合物占细胞干重的７２％,是自养藻的４倍多。两类细胞烃类化合物的分布特征发生了显变化,自养藻以正十七烷占绝对优势;异养藻富含长链正烷烃,以正二十五烷烃占优势。两类细胞脂溶性色素化合物和苯洗脱组分化合物以及Ｃ、Ｈ、Ｏ、Ｎ１４种有机元素相对含量也差异显。对比实验结果对于异养小球藻的代谢研究和油脂化合物的开发利用具有重要意义。     

高产油小球藻的筛选及其油脂分析

小球藻广泛分布于各种生境,特别是淡水环境中,适应性强。其同化产物主要是淀粉,但在环境胁迫条件下可显著积累中性脂,其脂肪酸类型主要为C16和C18,适合作为生物柴油的原料。我们从中国部分地区水体中分离纯化到若干株小球藻,通过薄层层析比较分析了21株产油小球藻的油脂含量,筛选到一株三酰基甘油含量较高的藻株Chlorella sp.NMX37N。其适宜生长温区为15—35℃,在25℃时生长速率最快,比生长速率为0.53/d,生长的最适光强为250μmol photons/(m2.s)。批量培养实验显示,藻细胞的三酰基甘油含量随培养时间延长而增加,并在培养的稳定期达到最大值,此时培养液中氮基本被耗尽。在批量培养条件下培养Chlorella sp.NMX37N约40d,藻细胞中总脂含量可达到33%左右,与此相比通过两步培养方式,将培养至对数后期(约20d)的藻细胞缺氮处理48h后,得到的总脂产率相当。通过两步培养方式可以大大缩短培养时间,使得该藻细胞快速有效积累油脂。另外,气相色谱分析显示,该藻的总脂和三酰基甘油的脂肪酸均以C16∶0和C18∶2为主,占总脂肪酸的70%以上,且不含C20以上的长链脂肪酸,可以作为优质的生物柴油原料。     

自养小球藻多糖对人肝癌细胞SMMC-7721凋亡的诱导

应用超声破碎碱提法制备自养小球藻多糖。在体外培养条件下,用不同浓度的小球藻多糖处理肝癌细胞,并通过MTT,双苯并咪唑(Hoechst33258)染色,免疫细胞化学等方法检测。结果表明,1g/L的小球藻多糖作用肝癌细胞SMMC-77211 2h,对其增殖有显著抑制作用,且能通过下调抗凋亡蛋白Bcl-2及上调凋亡执行蛋白Capase-3的表达来诱导其发生凋亡。     

自养条件下小球藻净化氮磷能力的研究

研究了在自养条件下,小球藻净化氮、磷的能力。实验结果表明,小球藻对氮、磷的吸收在开始的前12h内速度比较快,利用率达70％和60％左右;吸收氮、磷2d,利用率可分别达到75％和62％。低浓度的氮、磷组合有利于小球藻对氮的吸收,在磷浓度50～100mg／l范围内可以有效地吸收磷,吸收率接近60％。升高温度有利于小球藻对磷、氮的吸收。     

Fe~(3+)对小球藻的生长及油脂含量的影响

传统化石能源储量日益减少,生物柴油因其环保可再生性成为优质的石化柴油替代品。利用小球藻生产生物柴油速度快、油脂含量高,受到了广泛关注。为进一步提高小球藻生产生物柴油效率,分别探究了Fe3+的浓度及添加时间对自养和异养小球藻生长及产油的影响,获得最优Fe3+培养条件为:自养小球藻延滞期添加10-3 g/L Fe3+,生物量及油脂含量达2.80 g/L及30.90%;异养小球藻指数期添加10-5 g/L Fe3+,生物量及油脂含量达3.30 g/L及29.05%。经脂肪酸分析,以上条件获得的微藻油脂均可作为生物柴油生产原料。     

不同光质对小球藻光自养培养积累油脂的影响

研究了5种光质对小球藻(Chlorella vulgaris)M209256生长和产油的影响。结果表明:蓝光为小球藻的最适生长和产油光质;与其他光质相比,蓝光培养的小球藻生物量和油脂含量均较高,为2.40×107个/mL和28%;红光培养的小球藻生长最慢且油脂含量最低,为1.32×107个/mL和15.13%,表现出明显的"红降"现象。在GCMS分析的基础上,对油脂甲酯化后的十六烷值进行评估,结果发现:蓝光的十六烷值最高;5种光质培养的小球藻所产油脂,甲酯十六烷值均在47以上。因此,小球藻油脂所制备的生物柴油具有较好的燃烧性能。     

Effect of nanoparticle on cellular growth and lipid production in Chlorella vulgaris culture

Magnetic cobalt ferrite/silica nanoparticles (MSNs) and methyl functionalized MSNs (methyl‐MSNs) were used to enhance lipid production in Chlorella vulgaris culture through enhancement of gas‐water mass transfer and increased dissolved concentration of CO₂. Methyl‐MSNs enhanced CO₂–water mass transfer rate better than MSNs, and 0.3 wt% methyl‐MSNs are more effective than 0.1 wt% MSNs. In the cultivation experiment, 0.3 wt% methyl‐MSNs yielded the highest dry cell weight and subsequently, the highest mass transfer rate. However, enhancement of mass transfer rate did not increase lipid content. The volumetric lipid productivity in C. vulgaris culture depends not only on intracellular lipid content but also on the cell mass concentration. Consequently, 0.1 wt% methyl‐MSNs yielded the highest volumetric lipid productivity in C. vulgaris cultivation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:929–933, 2018     

利用市政污水培养Chlorella vulgaris生产生物柴油

为了考察利用南昌市政污水规模化培养富油微藻生产生物柴油,同时达到净化污水的目的,取南昌市青山湖污水处理厂未经任何处理的市政污水作为普通小球藻(Chlorella vulgaris)生长的培养液。监测了C.vulgaris在市政污水中连续培养10 d的特定生长率、生物质产量以及与之相关的市政污水中氨氮(NH4+-N)、总磷(TP)、化学需氧量(COD)、总悬浮固体(TSS)和挥发性悬浮固体(VSS)的清除情况。实验表明:营养物质的水平显著地影响了C.vulgaris的生长。C.vulgaris的生长率在培养8 d后达到最大,OD680为2.856,总的生物质产量日均最大积累速率为0.01 g/L,油脂含量为干质量的18%,油脂的平均日产量为0.001 g/L。培养10 d内NH4+-N、TP和COD的去除率分别为50.0%、32.1%和26.0%,TSS和VSS的日平均去除速率分别为0.01 g/L和0.006 1 g/L。     

pH值对沼液培养的普通小球藻生长及油含量积累的影响

以50％的沼液为普通小球藻的全营养培养基,考察培养基的起始pH值对小球藻生长及油脂含量的影响,普通小球藻对不同初始pH的沼液中氮、磷的去除情况。设定了2组实验,一组只调节初始接种培养液的pH,分别为6.0、6.5、7.0、7.5、8.0、8.5;另一组将培养液pH分别固定在6.0、6.5、7.0、7.5、8.0、8.5,pH用稀HCl和NaOH进行调节。研究发现在pH 6.5和pH 7.0的偏酸环境有利于小球藻生长,而pH在7.0~8.5的偏碱性条件下有利于小球藻油脂的积累,因此综合小球藻生长和油脂积累2个因素,得到最适合小球藻生长和油脂积累的pH为7.0。培养结束后沼液中氮磷的去除率分别达到了95％和97％,沼液中的总氮由原来的134.91 mg/L降至4.86 mg/L,总磷由10.19 mg/L降到0.32 mg/L。     

谷氨酸对异养培养小球藻生长的影响

在不添加其他氮源的小球藻异养培养基中,谷氨酸可促进小球藻的生长,但基本不增加叶绿素的合成;在以铵盐为氮源时,谷氨酸可明显促进小球藻对铵盐的利用,促进小球藻生物量增加和叶绿素合成;在以硝酸盐为氮源时,谷氨酸可增加小球藻的生物量,对叶绿素含量无明显影响。     

UVA‐induced reset of hydroxyl radical ultradian rhythm improves temporal lipid production in Chlorella vulgaris

We report for the first time that the endogenous, pseudo‐steady‐state, specific intracellular levels of the hydroxyl radical (si‐OH) oscillate in an ultradian fashion (model system: the microalga, Chlorella vulgaris), and also characterize the various rhythm parameters. The ultradian rhythm in the endogenous levels of the si‐OH occurred with an approximately 6 h period in the daily cycle of light and darkness. Further, we expected that the rhythm reset to a shorter period could rapidly switch the cellular redox states that could favor lipid accumulation. We reset the endogenous rhythm through entrainment with UVA radiation, and generated two new ultradian rhythms with periods of approximately 2.97 h and 3.8 h in the light phase and dark phase, respectively. The reset increased the window of maximum lipid accumulation from 6 h to 12 h concomitant with the onset of the ultradian rhythms. Further, the saturated fatty acid content increased approximately to 80% of total lipid content, corresponding to the peak maxima of the hydroxyl radical levels in the reset rhythm. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:673–680, 2014     

Electrotransformation of Chlorella vulgaris

Using hygromycin B resistance as a marker for selection, we have established the conditions required for the transformation of Chlorella vulgaris. The exponentially grown C. vulgaris cells were transformed by electroporation with plasmid pIG121-Hm, and transformants were selected with hygromycin B at a concentration of 50 μg/ml. Cell extracts prepared from the late-log cultures of the transformants exhibited glucuronidase activities as conferred by the gus gene on pIG121-Hm. The maintenance of plasmid in the algal cells seemed to be transient as many cultures derived from the hygromycin B-resistant colonies gradually lost the hygromycin resistance upon prolonged growth. The result of Southern blotting of the genomic DNAs prepared from transformant cultures exhibiting persistent hygromycin resistance showed that integration of part of the plasmid DNA into the host chromosome had taken place. Received: 19 December 1997 / Revision received: 5 October 1998 / Accepted: 27 October 1998     

磁处理对小球藻超微结构的影响

用透射电镜研究在不同强度磁场处理下,小球藻超微结构的变化。结果显示:磁处理强度不同,小球藻细胞亚显微结构变化程度不同。细胞壁、叶绿体、线粒体和液泡等部位是受磁处理影响的主要部位。研究发现在较高强度磁处理下,出现质壁分离,类囊体结构轻微破坏,液泡和线粒体增加,能量物质积累等现象,影响小球藻的正常代谢。     

Effect of light supply and carbon source on cell growth and cellular composition of a newly isolated microalga Chlorella vulgaris ESP‐31

Light supply is one of the most important factors affecting autotrophic growth of microalgae. This study investigated the effect of the type and light intensity of artificial light sources on the cell growth of an indigenous microalga Chlorella vulgaris ESP‐31 obtained from southern Taiwan. In addition, a dissolved inorganic carbon source (i.e. sodium bicarbonate) was used to improve the biomass production of strain ESP‐31. The results show that a new fluorescent light source (TL5) was effective in indoor cultivation of microalgae. Better overall productivity of 0.029 g dry cell weight/L‐d was obtained when using TL5 lamps as the light source with a light intensity of 9 W/m². A carbon source (sodium bicarbonate) concentration of 1000 mg/L was found to be optimal for the growth of strain ESP‐31 in terms of both biomass production and carbon source utilization. Under the optimal growth conditions, the resulting microalgal biomass consisted of 25–30% protein, 6–10% carbohydrate, and 30–40% lipid.