A kinetic model for lutein production by the green microalga Chlorella protothecoides in heterotrophic culture

Production of lutein by the green microalga Chlorella protothecoides grown heterotrophically in a fermentor using glucose as the carbon source and urea as the nitrogen source was investigated. An unstructured kinetic model was proposed to describe the microalgal culture system including cell growth, lutein formation, as well as glucose and nitrogen consumption. The inhibition potentials of biomass, product and substrates on growth and lutein formation were examined and incorporated into the kinetic model. Values of the kinetic model parameters were estimated. The resulting model predictions were in good agreement with the experimental results. The model can be helpful in scale-up, optimization and control of the C. protothecoides culture process, and can also be used as a guideline for similar microalgal cultivation systems. Received 28 January 1999/ Accepted in revised form 27 August 1999     

Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress

The fast growing unicellular green microalgae Chlorella protothecoides has attracted interest as a promising organism for commercial production of a high-value carotenoid, lutein, by heterotrophic fermentation. Effects of two oxidant-forming reactive oxygen species (ROS) on the biomass concen-tration, and yield and content of lutein in batch culture of heterotrophic Chlorella protothecoides were investigated in this study. The addition of 0.1 mmol/L H2O2 and 0.01 mmol/L NaClO plus 0.5 mmol/L Fe2 to the culture led to the generation of ·OH and enhanced the lutein content from 1.75 to 1.90 and 1.95 mg/g, respectively. The lutein content further increased to 1.98 mg/g when 0.01 mmol/L H2O2 and 0.5 mmol/L NaClO were added to generate 1O2. The maximum yield of lutein (28.5, 29.8 and 31.4 mg/L) and a high biomass concentration (15.0, 15.3 and 15.9 g/L) were also achieved through the above treatments. The results indicated that 1O2 could promote lutein formation and enhance lutein production in hetero-trophic Chlorella protothecoides. Moreover, 1O2 produced from the reaction of H2O2 and NaClO was more effective in enhancing lutein production and reducing biomass loss than ·OH from the reaction of H2O2 or NaClO plus Fe2 .     

Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress

The fast growing unicellular green microalgae Chlorella protothecoides has attracted interest as a promising organism for commercial production of a high-value carotenoid, lutein, by heterotrophic fermentation. Effects of two oxidant-forming reactive oxygen species (ROS) on the biomass concentration, and yield and content of lutein in batch culture of heterotrophic Chlorella protothecoides were investigated in this study. The addition of 0.1 mmol/L H₂O₂ and 0.01 mmol/L NaClO plus 0.5 mmol/L Fe²⁺ to the culture led to the generation of ^·OH and enhanced the lutein content from 1.75 to 1.90 and 1.95 mg/g, respectively. The lutein content further increased to 1.98 mg/g when 0.01 mmol/L H₂O₂ and 0.5 mmol/L NaClO were added to generate ¹O₂. The maximum yield of lutein (28.5, 29.8 and 31.4 mg/L) and a high biomass concentration (15.0, 15.3 and 15.9 g/L) were also achieved through the above treatments. The results indicated that 1O2 could promote lutein formation and enhance lutein production in heterotrophic Chlorella protothecoides. Moreover, ¹O₂ produced from the reaction of H₂O₂ and NaClO was more effective in enhancing lutein production and reducing biomass loss than ^·OH from the reaction of H₂O₂ or NaClO plus Fe²⁺. Supported by the National Key Project of Sci & Tech Supporting Programs Funded by Ministry of Science & Technology of China (Grant No. 2006BAD27B03), Sci & Tech Project of Guangzhou (Grant No. 2005Z3-E0331) and Sci & Tech Project of Guangdong (Grant No. 20052050166)     

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.     

植物激素IBA与6-BA对摇瓶分批流加异养培养小球藻的生长及化学组成的影响

研究了植物激素IBA与6-BA对摇瓶分批流加异养培养小球藻的生长及化学组成的影响。结果表明,IBA与6-BA处理摇瓶分批流加异养培养的小球藻,促进了小球藻的生长,提高了小球藻的生物量(15．0％)与对糖转化率(14．8％),同时也提高了小球藻的蛋白质含量(16．3％)。氨基酸分析结果表明,IBA与6-BA能提高摇瓶分批流加异养培养的小球藻的α-氨基酸含量(含量达31．2％,而对照的仅26．0％),增加必需氨基酸的含量及比例,尤其两种重要α-氨基酸Arg和Lys的含量分别提高达22．9％和30．1％,强化了异养小球藻的营养价值。IBA与6-BA能提高摇瓶分批流加异养培养的小球藻叶绿素及类胡萝卜素含量。培养工艺研究与化学组成分析将为植物激素应用于小球藻的异养培养奠定基础。     

Fatty acid production by heterotrophic Chlorella saccharophila

The fatty acid composition of the alga Chlorella saccharophila was investigated under different growth conditions. Using glucose as the sole carbon source, heterotrophically-grown Chlorella saccharophila produced a greater proportion of the polyunsaturated fatty acids (C18: 2 and C18: 3) than photosynthetic cultures, with linoleic acid (C18: 2) predominating. An unexpected discovery was the observation that at the lowest glucose concentration (2.5 gl^–1) the lipid content of the algae increased to between 36–47% of the cell weight, depending on the temperature. At glucose concentrations of 5 g l^–1 or more, the lipid content fell to 10–12% of the cell, although total fatty acid yield was higher due to higher biomass concentrations. Aeration of heterotrophic cultures promoted the production of unsaturated fatty acids compared to non-aerated cultures.     

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)。     

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。     

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     

Optimization for high-density cultivation of heterotrophic Chlorella based on a hybrid neural network model

AIMS: The purpose of this study was to develop a reliable hybrid neural network (HNN) model for heterotrophic growth of Chlorella, based on which optimization for fed-batch (FB) cultivation of Chlorella may be successfully realized. METHODS AND RESULTS: Deterministic kinetic model was preliminarily developed for the optimization of FB cultivation of Chlorella. The highest biomass concentration and the maximum productivity were obtained as: 104.9 g l(-1) dry cell weight and 0.613 g l(-1) h(-1), respectively. After several cultivations had been performed, an HNN model was developed. The efficiency of biomass production was further increased by the optimization using this model. The highest biomass concentration and the maximum productivity attained was: 116.2 g l(-1) dry cell weight and 1.020 g l(-1) h(-1), respectively. CONCLUSION: The HNN model agreed well with experimental results in different cultivations. Comparison between the HNN model and the deterministic model showed that the former had better generalization ability, which made it a reliable tool in modelling and optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: The high cell density and productivity of biomass obtained in this study is of significance for the commercial cultivation of Chlorella. The simple and efficient optimization strategy proposed in this paper may be employed in heterotrophic mass culture of Chlorella as well as other similar organisms.     

Pyrolytic characteristics of heterotrophic Chlorella protothecoides for renewable bio-fuel production

Heterotrophic Chlorella protothecoides cells were pyrolyzed in athermogravimetric analyzer to investigate the pyrolytic characteristics anddetermine the kinetic parameters. Heating rates of 15, 40, 60 and 80 ^°C^-1 up to a final temperature of 800 ^°C wereused. The pyrolysis reactions mainly took place between 160–520^°C with a volatile yield of about 80%. The devolatilization stageconsisted of two main temperature zones (I and II) with a transition at300–320 ^°C. Crude lipid in cells decomposed at Zone II whileother main components at Zone I. The increase of heating rate caused alateral shift to higher temperatures in the thermograms, a decrease ofactivation energies for the devolatilization stage and an increase of both theinstantaneous maximum and average reaction rates. The difference ofactivation energies between two zones implied that more energy input forlipid pyrolysis seems needed in comparison with other main components.These data are useful for the design, operation, and modeling of thepyrolysis systems for microalgae.     

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     

无机碳源对小球藻自养产油脂的影响

旨在研究小球藻利用无机碳自养产油脂,考察了3种无机碳源 (Na2CO3、NaHCO3和CO2) 及其初始浓度对小球藻产油特性的影响。结果表明,小球藻能利用Na2CO3、NaHCO3和CO2产油;经Na2CO3、NaHCO3和CO2培养10 d后,随着每种无机碳源浓度的增加,小球藻产量均先增加后减少。小球藻经3种无机碳源培养后,其培养液pH值上升。最适宜的Na2CO3和NaHCO3添加量均为40 mmol/L,其生物量分别达到0.52 g/L和0.67 g/L,产油量分别达到0.19 g/L和0.22 g/L。在3种无机碳源中,CO2是最佳无机碳源,当CO2浓度为6％时,小球藻生长最快,生物量达2.42 g/L,产油量最高达0.72 g/L;当CO2浓度过低时,无机碳供应不足,油脂产量低;当CO2浓度过高时,培养液pH偏低,小球藻油脂积累受到抑制。Na2CO3和NaHCO3较CO2更有利于小球藻积累不饱和脂肪酸。     

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。     

响应面法优化普通小球藻混合营养培养基组成生产生物质

利用响应面法优化了混合营养培养普通小球藻生产生物质的培养基组成.首先采用Plackett-Burman设计对11个相关营养因素的效应进行了评价,并筛选出影响小球藻细胞生长的3个主要因素为KNO3、葡萄糖和NaC1;然后结合Box-Behnken设计建立了以小球藻浓度为响应值的二次回归方程模型,获得优化的培养基组成为KNO31.64g/L、葡萄糖45g/L、NaC1 1.57g/L;模型预测的最大浓度为5.28g/L,验证值为5.68g/L;验证结果表明,所建立模型预测精度较好,可用于优化小球藻的混养培养基组成.优化条件下混养小球藻细胞的蛋白质和色素含量较优化前降低,而可溶性糖和油脂含量提高,脂肪酸以棕榈酸和油酸为主;细胞组分分析结果显示,混养培养所得小球藻生物质具有作为生产微藻生物能源原料的潜力.     

Strategies to enhance cell growth and achieve high‐level oil production of a Chlorella vulgaris isolate

The autotrophic growth of an oil‐rich indigenous microalgal isolate, identified as Chlorella vulgaris C? C, was promoted by using engineering strategies to obtain the microalgal oil for biodiesel synthesis. Illumination with a light/dark cycle of 14/10 (i.e., 14 h light‐on and 10 h light‐off) resulted in a high overall oil production rate (v_oil) of 9.78 mg/L/day and a high electricity conversion efficiency (E_c) of 23.7 mg cell/kw h. When using a NaHCO₃ concentration of 1,500 mg/L as carbon source, the v_oil and E_c were maximal at 100 mg/L/day and 128 mg/kw h, respectively. A Monod type model was used to describe the microalgal growth kinetics with an estimated maximum specific growth rate (μ_max) of 0.605 day^?1 and a half saturation coefficient (K_s) of 124.9 mg/L. An optimal nitrogen source (KNO₃) concentration of 625 mg/L could further enhance the microalgal biomass and oil production, leading to a nearly 6.19 fold increase in v_oil value. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

粗甘油优化污泥脱水液培养小球藻过程的研究

污泥脱水液为污泥压缩过程产生的污水,因其含有N、P等营养物,可用于微藻的培养。但污泥脱水液碳氮比低,可利用碳源有限,影响微藻生长。本研究考察外加不同浓度(1 g/L,2 g/L,4 g/L,6 g/L)生物柴油副产物-粗甘油对污泥脱水液培养小球藻过程的影响。结果表明:1 g/L、2 g/L粗甘油浓度能促进小球藻生长,藻生物量为1.29 g/L、1.45 g/L;2 g/L粗甘油浓度下氨氮、总氮去除率达99.32%和97.52%。粗甘油被分解后易使培养体系pH降至7以下,使总磷去除率比对照组略低。外加1 g/L、2 g/L粗甘油组的COD去除量分别为553.00 mg/L和405.00 mg/L。藻细胞元素和傅里叶红外光谱分析表明补加粗甘油后藻细胞中C元素和H元素相对含量均明显增加,C元素含量约为对照组的1.5倍;2 g/L粗甘油组的蛋白质与脂类物质含量均高于对照组。MPBR半连续培养小球藻过程中,HRT为5 d时藻生物量维持在1.99~2.21 g/L,大约为批次生物量的1.50倍;氨氮、总氮、总磷、COD的去除率分别在96.26%~99.20%、92.44%~94.04%、53.63%~58.58%、59.44%~65.57%。     

Optimization of a culture medium for xylanase production by Bacillus sp. using statistical experimental designs

The concentrations of oat spelt xylan, casein hydrolysate and NH4Cl in the culture medium for production of xylanase from Bacillus sp. I-1018 were optimized by means of response surface methods. The path of steepest ascent was used to approach the optimal region of the medium composition. The optimum composition of the nutrient medium was then easily determined by using a central composite design and was found to be 3.16g/l of xylan, 1.94g/l casein hydrolysate, 0.8g/l of NH4Cl. The xylanase production was increased by 135% when the strain was grown in the optimized medium compared to initial medium.     

High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture

The green microalga Chlorella protothecoides was grown heterotrophically in batch mode in a 3.7-L fermenter containing 40 g/L glucose and 3.6 g/L urea. In the late exponential phase, concentrated nutrients containing glucose and urea were fed into the culture, in which the nitrogen source was sufficient compared to carbon source. As a result, a maximum cell dry weight concentration of 48 g/L was achieved. This cell dry weight concentration was 28.4 g/L higher than that obtained in batch culture under the same growth conditions. In another cultivation run, the culture was provided with the same initial concentrations of glucose (40 g/L) and urea (3.6 g/L) as in the batch mode, followed by a relatively reduced supply of nitrogen source in the fed-batch mode to establish a nitrogen-limited culture. Such a modification resulted in an enhanced lutein production without significantly lowering biomass production. The cellular lutein content was 0.27 mg/g higher than that obtained in the N-sufficient culture. The improvements were also reflected by higher maximum lutein yield, lutein productivity, and lutein yield coefficient on glucose. This N-limited fed-batch culture was successfully scaled up from 3.7 L to 30 L, and a three-step cultivation process was developed for the high-yield production of lutein. The maximum cell dry weight concentration (45.8 g/L) achieved in the large fermenter (30 L) was comparable to that in the small one (3.7 L). The maintenance of the culture at a higher temperature (i.e., 32 degrees C) for 84 h resulted in a 19.9% increase in lutein content but a 13.6% decrease in cell dry weight concentration as compared to the fed-batch culture (30 L) without such a treatment. The enhancement of lutein production resulted from the combination of nitrogen limitation and high-temperature stress.