以高油脂产率为目标的小球藻光自养培养工艺优化与初步放大
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摘要
随着化石燃料的短缺和二氧化碳减排的迫切性,微藻生物质能源产业化的发展备受关注。微藻培养是微藻生物质能源产业化中的重要环节,且高油脂产率是微藻培养的核心目标。高油脂产率微藻培养工艺优化与放大的研究成为近些年的研究热点,但文献结果大多以室内研究为主,即使涉及户外培养但其规模也很小。小球藻是淡水能源微藻中的主要藻种,但对其高产油脂光自养培养工艺优化和放大方面的研究很少。
本文主要以小球藻(Chlorella sp.)为研究对象,考察了营养方式、昼夜温度、pH值、营养盐浓度、培养模式等条件对油脂产率的影响,建立了“异养种子-光自养培养”新模式,获得了高产油脂蛋白核小球藻的光自养培养基,并建立了室内高油脂产率小球藻的培养工艺。结合室内培养工艺,建立了户外0.35m2敞开式反应器中的小球藻光自养培养工艺并对其进行了优化。将种子制备工艺从5L摇瓶放大到500L发酵罐,为户外大规模光自养培养提供了充足的种子。此外,还将户外光自养培养规模从0.35m2反应器成功放大到80m2水泥跑道池。本文主要研究结论如下：
(1)建立了小球藻“异养种子-光自养培养”新模式(以异养细胞作为种子进行光自养培养)。考虑到能源微藻光自养接种时,由于种子自养培养存在耗时长、易被杂藻和原生动物污染等缺点从而难以提供充足且稳定的藻种,本文首次建立了小球藻“异养种子-光自养培养”的新模式。对于蛋白核小球藻而言,藻种制备过程中异养培养的细胞产率是自养培养的20.9倍；“异养种子-光自养培养”模式下的油脂产率是“自养种子-光自养培养”(以自养细胞作为种子进行光自养培养)的1.59倍；且该模式下的脂肪酸组成主要以2-3个双键且碳链长度在16-18之间的脂肪酸为主,十分适合作为生产生物燃料的原料。此外,该模式还适用于椭圆小球藻和普通小球藻：该模式下椭圆小球藻和普通小球藻的油脂产率分别是“自养种子-光自养培养”模式下的1.52倍和1.58倍。
(2)通过筛选及优化获得了适合室内和户外生长的小球藻光自养培养基。结果表明,在室内及户外3L平板式反应器中,小球藻在优化后培养基中的油脂产率分别是初始培养基中的1.61和1.67倍；在户外0.35m2敞开式反应器中,小球藻在优化后培养基中的油脂产率是初始培养基中的1.33倍。
(3)白昼温度对油脂产率影响显著。由于户外培养时存在昼夜的变化,夜晚藻细胞密度和油脂含量的下降导致了油脂产率的降低。因此,在一定光暗周期下考察温度与藻细胞密度、油脂含量和脂肪酸组成的变化规律是十分重要的。研究结果表明,30℃是小球藻光自养培养的最适温度。夜晚藻细胞损失会随着白天温度的升高或夜晚温度的降低而下降；夜晚油脂含量的增幅则会随着白天温度的升高或夜晚温度的降低而增加。此外,在任何温度下,随着培养时间的延长(生物量增加),夜晚细胞损失也会逐渐减小：在6种主要的脂肪酸中C16：0含量最高且最易受温度的影响,其值随着温度的升高而增加。
(4)在室内建立了基于CO2控制最佳pH及氮胁迫的半连续光自养培养工艺。在分批培养过程中通CO2调节藻液pH值在7.0±0.2时可以提高细胞的油脂产率：在该条件下的油脂产率是分批培养过程中不控pH处理的2.64倍。细胞在适度氮胁迫条件下培养时有利于提高油脂产率。初始硝酸钠浓度为200mg/L处理的油脂含量高达38.7%,是初始硝酸钠浓度为500mg/L处理的1.25倍。此外,在相同的培养条件下,半连续培养方式下的油脂产率是分批培养的1.47倍。因此,在半连续培养过程中结合通CO2进行pH调控及氮胁迫的培养策略可以明显提高油脂产率；而且在每隔24小时带放1/3体积藻液的半连续培养操作时的油脂产率最高,其值是分批培养中不控pH和不限氮条件下的3.64倍。
(5)在户外0.35m2敞开式反应器中建立了蛋白核小球藻光自养培养工艺。结果表明,培养过程中的最佳pH值为7.0±0.2且发酵尾气可代替纯CO2进行pH调控以及作为碳源：白天时最佳通气量为0.5vvm,夜晚则不通气最好；最佳的液位深度和初始接种密度分别为14cm和0.3g/L。结合室内培养工艺,进一步优化户外培养工艺：当户外温度较低时(最高温度小于30℃),提高白天培养温度使其值在30-36℃,小球藻的油脂产率可提高81%；在户外相同培养条件下,小球藻在半连续培养方式下的油脂产率是分批培养时的1.85倍。
(6)将种子制备工艺从5L摇瓶成功放大到50L和500L发酵罐,为户外放大提供了充足的藻种。首次将“异养种子-光自养培养”模式应用于蛋白核小球藻户外大规模培养,并将培养工艺逐步放大到了80m2水泥跑道池,该培养规模下的油脂产率高达46.4mg/L/d (5.22g/m2/d),是目前文献报道的最大规模及最高油脂产率。
上述研究结果不仅为高油脂产率小球藻培养的产业化奠定了基础,而且还对其他能源微藻的培养研究具有重要的参考价值。
Due to the lack of fossil energy and the urgency of CO2emission reduction, the development for industrilization of biofuels from microalgae has been paid much more attention. Microalage cultivation is the key to biofuels from microalgae, and high lipid productivity is the central aim of microalgae cultivation. Therefore, optimizaition and scale up of microalgae photoautotrophic cultivation for high lipid productivity has been a research focus in recent years. However, many researches in the literature mainly focused on indoor cultivation; some results refered to outdoor culture but the scales of them were not large. Chlorella spp. are main species of fresh oleaginous algae, however, few research has done into the optimization and scale up of Chlorella photoautotrophic cultivation for high lipid productivity.
Chlorella sp. has been used as the research object. The effects of trophic modes, temperature, nutrient concentrations and culture modes on the lipid productivity have been investigated; the novel model of "heterotrophic seed-photoautotrophic cultivaton" of Chlorella has been found; photoautotrophic medium of C. pyrenoidosa for high lipid productivity has been selected and optimized; and the C. pyrenoidosa indoor photoautotrophic cultivation for high lipid productivity has been established. Based on the indoor results, the Chlorella outdoor photoautotrophic culture has been established and optimized in0.35m2open basins. Moreover, for outdoor photoautotrophic mass culture, the seed preparation scale has been magnified from5L flasks to50L and500L fermenters, and the photoautotrophic cultivation scale has been enlarged from0.35m2open basins to80m2raceway ponds. The main results are as follows:
(1) Establish a novel Chlorella culture model of "heterotrophic seed-photoautotrophic cultivation"("HS-PC", the photoautotrophic culture with heterotrophic cells as seed). For overcoming the long period of seed cultured photoautotrophically, difficulty for algal species control and inadequate cells supply for the inoculation of microalgae photoautotrophic cultivation, a novel model of "HS-PC" has been firstly established. For C. pyrenoidosa, the biomass productivity of heterotrophy was20.9-fold higher than that of photoautotrophy during the seed preparation process; the lipid productivity of "HS-PC" was1.59times higher than that of "PS-PC"(the photoautotrophic culture with photoautotrophic cells as seed); The fatty acids of "HS-PC" model were in medium length (16-18carbons) with2or3unsaturation degree, which were suitable for biofuels-making. Furthermore, the "HS-PC" model also can be applied to C. ellipsoidea and C. vulgaris; the lipid productivity of C. ellipsoidea and C. vulgaris in "HS-PC" model were1.52-fold and1.58-fold higher than those in "PS-PC" model, respectively.
(2) Obtain the optimum C. pyrenoidosa photoautotrophic medium for both indoor and outdoor culture through the selection and optimization. The results showed that the lipid productivity of the cells cultured with the optimized medium were1.61-fold and1.67-fold higher than those with the original one in the3L panel bioreactor for indoor and outdoor culture; the lipid productivity with the optimized medium was1.33-fold higher than that with the original one in the outdoor0.35m2open basins.
(3) Temperatures during the daytime and night have great effects on lipid productivity. For outdoor culture with light-dark cycle, the losing of biomass and lipid at night decreased the biomass and lipid productivity. Hence, studies on the effects of temperature on the variations of biomass concentration, lipid content and fatty acids composition under a light-dark cycle culture were absolutely important. The results showed that30℃was the optimal temperature for achieving high biomass and lipid productivity. The night biomass loss decreased with the increasing daytime temperature or the decreasing night temperature; the extent to the lipid content at night increased with the increasing daytime temperature or the decreasing night temperature. In addition, the night biomass loss decreased with the increasing biomass concentration at any temperature; among the six main fatty acids, C16:0was the most abundant fatty acid and apparently influenced by the temperature, which was increased with the increasing temperature.
(4) Establish an indoor photoautotrophic cultivation strategy of semi-continuous culture with pH-regulation by CO2and nitrogen-limitation. In the batch culture, the lipid productivity can be enhanced with pH-regulation at7±0.2by CO2, which was2.64-fold higher than that without pH control. The lipid productivity of the cells can be increased under the nitrogen-limitation conditions; the lipid content of Chlorella with the initial NaNO3concentration at200mg/L was38.7%, which was1.25times higher than that at500mg/L. Additionally, under the same culture conditions, the lipid productivity of the cells in the semi-continuous culture was1.47-fold higher than that in the batch culture. Hence, the culture strategy of integrating both nitrogen-limitation and pH-regulation by CO2in a semi-continuous cultivation was investigated for enhancing the lipid productivity. The results showed that the lipid productivity of the above semi-continuous system with one-third replacement and24h's interval was3.64times higher than that in the batch culture without pH control and nitrogen limitation.
(5) Establish an outdoor photoautotrophic cultivation in0.35m2open basins. The results showed that the optimal pH was7±0.2and the fermenter exhaust-gas can take place of pure CO2as carbon source and for pH-regulation; the optimal ventilation volumn, broth depth and inoculation concentration were0.5vvm,14cm and0.3g/L as well as no mixing during the night. Additionally, the Chlorella outdoor photoautotrophic culture has been optimized by indoor culture results:when the natural temperature was low (maximium temperature<30℃), the lipid productivity can be enhanced by81%through controlling the daytime temperature between30℃and36℃; Under the same culture conditions, the lipid productivity of Chlorella in the semi-continuous culture with nitrogen-limitation was1.85-fold higher than that in the batch culture.
(6) The seed preparation process has been enlarged from5L flasks to500L fermenters for adequate cells supply for the inoculation of the photoautotrophic culture. The "HS-PC" model was firstly carried out for outdoor mass culture:the photoautotrophic culture scale was magnified to80m2raceway ponds. The maximum lipid productivity of Chlorella cultured in80m2raceway ponds reached46.4mg/L/d (5.22g/m2/d), which was the biggest scale and highest lipid productivity in the literature.
Our results can not only load a good foundation for industrialization of Chlorella cultivation with high lipid productivity, but also provide a valuable reference for other oleaginous microalgae cultivation.
本文主要以小球藻(Chlorella sp.)为研究对象,考察了营养方式、昼夜温度、pH值、营养盐浓度、培养模式等条件对油脂产率的影响,建立了“异养种子-光自养培养”新模式,获得了高产油脂蛋白核小球藻的光自养培养基,并建立了室内高油脂产率小球藻的培养工艺。结合室内培养工艺,建立了户外0.35m2敞开式反应器中的小球藻光自养培养工艺并对其进行了优化。将种子制备工艺从5L摇瓶放大到500L发酵罐,为户外大规模光自养培养提供了充足的种子。此外,还将户外光自养培养规模从0.35m2反应器成功放大到80m2水泥跑道池。本文主要研究结论如下：
(1)建立了小球藻“异养种子-光自养培养”新模式(以异养细胞作为种子进行光自养培养)。考虑到能源微藻光自养接种时,由于种子自养培养存在耗时长、易被杂藻和原生动物污染等缺点从而难以提供充足且稳定的藻种,本文首次建立了小球藻“异养种子-光自养培养”的新模式。对于蛋白核小球藻而言,藻种制备过程中异养培养的细胞产率是自养培养的20.9倍；“异养种子-光自养培养”模式下的油脂产率是“自养种子-光自养培养”(以自养细胞作为种子进行光自养培养)的1.59倍；且该模式下的脂肪酸组成主要以2-3个双键且碳链长度在16-18之间的脂肪酸为主,十分适合作为生产生物燃料的原料。此外,该模式还适用于椭圆小球藻和普通小球藻：该模式下椭圆小球藻和普通小球藻的油脂产率分别是“自养种子-光自养培养”模式下的1.52倍和1.58倍。
(2)通过筛选及优化获得了适合室内和户外生长的小球藻光自养培养基。结果表明,在室内及户外3L平板式反应器中,小球藻在优化后培养基中的油脂产率分别是初始培养基中的1.61和1.67倍；在户外0.35m2敞开式反应器中,小球藻在优化后培养基中的油脂产率是初始培养基中的1.33倍。
(3)白昼温度对油脂产率影响显著。由于户外培养时存在昼夜的变化,夜晚藻细胞密度和油脂含量的下降导致了油脂产率的降低。因此,在一定光暗周期下考察温度与藻细胞密度、油脂含量和脂肪酸组成的变化规律是十分重要的。研究结果表明,30℃是小球藻光自养培养的最适温度。夜晚藻细胞损失会随着白天温度的升高或夜晚温度的降低而下降；夜晚油脂含量的增幅则会随着白天温度的升高或夜晚温度的降低而增加。此外,在任何温度下,随着培养时间的延长(生物量增加),夜晚细胞损失也会逐渐减小：在6种主要的脂肪酸中C16：0含量最高且最易受温度的影响,其值随着温度的升高而增加。
(4)在室内建立了基于CO2控制最佳pH及氮胁迫的半连续光自养培养工艺。在分批培养过程中通CO2调节藻液pH值在7.0±0.2时可以提高细胞的油脂产率：在该条件下的油脂产率是分批培养过程中不控pH处理的2.64倍。细胞在适度氮胁迫条件下培养时有利于提高油脂产率。初始硝酸钠浓度为200mg/L处理的油脂含量高达38.7%,是初始硝酸钠浓度为500mg/L处理的1.25倍。此外,在相同的培养条件下,半连续培养方式下的油脂产率是分批培养的1.47倍。因此,在半连续培养过程中结合通CO2进行pH调控及氮胁迫的培养策略可以明显提高油脂产率；而且在每隔24小时带放1/3体积藻液的半连续培养操作时的油脂产率最高,其值是分批培养中不控pH和不限氮条件下的3.64倍。
(5)在户外0.35m2敞开式反应器中建立了蛋白核小球藻光自养培养工艺。结果表明,培养过程中的最佳pH值为7.0±0.2且发酵尾气可代替纯CO2进行pH调控以及作为碳源：白天时最佳通气量为0.5vvm,夜晚则不通气最好；最佳的液位深度和初始接种密度分别为14cm和0.3g/L。结合室内培养工艺,进一步优化户外培养工艺：当户外温度较低时(最高温度小于30℃),提高白天培养温度使其值在30-36℃,小球藻的油脂产率可提高81%；在户外相同培养条件下,小球藻在半连续培养方式下的油脂产率是分批培养时的1.85倍。
(6)将种子制备工艺从5L摇瓶成功放大到50L和500L发酵罐,为户外放大提供了充足的藻种。首次将“异养种子-光自养培养”模式应用于蛋白核小球藻户外大规模培养,并将培养工艺逐步放大到了80m2水泥跑道池,该培养规模下的油脂产率高达46.4mg/L/d (5.22g/m2/d),是目前文献报道的最大规模及最高油脂产率。
上述研究结果不仅为高油脂产率小球藻培养的产业化奠定了基础,而且还对其他能源微藻的培养研究具有重要的参考价值。
Due to the lack of fossil energy and the urgency of CO2emission reduction, the development for industrilization of biofuels from microalgae has been paid much more attention. Microalage cultivation is the key to biofuels from microalgae, and high lipid productivity is the central aim of microalgae cultivation. Therefore, optimizaition and scale up of microalgae photoautotrophic cultivation for high lipid productivity has been a research focus in recent years. However, many researches in the literature mainly focused on indoor cultivation; some results refered to outdoor culture but the scales of them were not large. Chlorella spp. are main species of fresh oleaginous algae, however, few research has done into the optimization and scale up of Chlorella photoautotrophic cultivation for high lipid productivity.
Chlorella sp. has been used as the research object. The effects of trophic modes, temperature, nutrient concentrations and culture modes on the lipid productivity have been investigated; the novel model of "heterotrophic seed-photoautotrophic cultivaton" of Chlorella has been found; photoautotrophic medium of C. pyrenoidosa for high lipid productivity has been selected and optimized; and the C. pyrenoidosa indoor photoautotrophic cultivation for high lipid productivity has been established. Based on the indoor results, the Chlorella outdoor photoautotrophic culture has been established and optimized in0.35m2open basins. Moreover, for outdoor photoautotrophic mass culture, the seed preparation scale has been magnified from5L flasks to50L and500L fermenters, and the photoautotrophic cultivation scale has been enlarged from0.35m2open basins to80m2raceway ponds. The main results are as follows:
(1) Establish a novel Chlorella culture model of "heterotrophic seed-photoautotrophic cultivation"("HS-PC", the photoautotrophic culture with heterotrophic cells as seed). For overcoming the long period of seed cultured photoautotrophically, difficulty for algal species control and inadequate cells supply for the inoculation of microalgae photoautotrophic cultivation, a novel model of "HS-PC" has been firstly established. For C. pyrenoidosa, the biomass productivity of heterotrophy was20.9-fold higher than that of photoautotrophy during the seed preparation process; the lipid productivity of "HS-PC" was1.59times higher than that of "PS-PC"(the photoautotrophic culture with photoautotrophic cells as seed); The fatty acids of "HS-PC" model were in medium length (16-18carbons) with2or3unsaturation degree, which were suitable for biofuels-making. Furthermore, the "HS-PC" model also can be applied to C. ellipsoidea and C. vulgaris; the lipid productivity of C. ellipsoidea and C. vulgaris in "HS-PC" model were1.52-fold and1.58-fold higher than those in "PS-PC" model, respectively.
(2) Obtain the optimum C. pyrenoidosa photoautotrophic medium for both indoor and outdoor culture through the selection and optimization. The results showed that the lipid productivity of the cells cultured with the optimized medium were1.61-fold and1.67-fold higher than those with the original one in the3L panel bioreactor for indoor and outdoor culture; the lipid productivity with the optimized medium was1.33-fold higher than that with the original one in the outdoor0.35m2open basins.
(3) Temperatures during the daytime and night have great effects on lipid productivity. For outdoor culture with light-dark cycle, the losing of biomass and lipid at night decreased the biomass and lipid productivity. Hence, studies on the effects of temperature on the variations of biomass concentration, lipid content and fatty acids composition under a light-dark cycle culture were absolutely important. The results showed that30℃was the optimal temperature for achieving high biomass and lipid productivity. The night biomass loss decreased with the increasing daytime temperature or the decreasing night temperature; the extent to the lipid content at night increased with the increasing daytime temperature or the decreasing night temperature. In addition, the night biomass loss decreased with the increasing biomass concentration at any temperature; among the six main fatty acids, C16:0was the most abundant fatty acid and apparently influenced by the temperature, which was increased with the increasing temperature.
(4) Establish an indoor photoautotrophic cultivation strategy of semi-continuous culture with pH-regulation by CO2and nitrogen-limitation. In the batch culture, the lipid productivity can be enhanced with pH-regulation at7±0.2by CO2, which was2.64-fold higher than that without pH control. The lipid productivity of the cells can be increased under the nitrogen-limitation conditions; the lipid content of Chlorella with the initial NaNO3concentration at200mg/L was38.7%, which was1.25times higher than that at500mg/L. Additionally, under the same culture conditions, the lipid productivity of the cells in the semi-continuous culture was1.47-fold higher than that in the batch culture. Hence, the culture strategy of integrating both nitrogen-limitation and pH-regulation by CO2in a semi-continuous cultivation was investigated for enhancing the lipid productivity. The results showed that the lipid productivity of the above semi-continuous system with one-third replacement and24h's interval was3.64times higher than that in the batch culture without pH control and nitrogen limitation.
(5) Establish an outdoor photoautotrophic cultivation in0.35m2open basins. The results showed that the optimal pH was7±0.2and the fermenter exhaust-gas can take place of pure CO2as carbon source and for pH-regulation; the optimal ventilation volumn, broth depth and inoculation concentration were0.5vvm,14cm and0.3g/L as well as no mixing during the night. Additionally, the Chlorella outdoor photoautotrophic culture has been optimized by indoor culture results:when the natural temperature was low (maximium temperature<30℃), the lipid productivity can be enhanced by81%through controlling the daytime temperature between30℃and36℃; Under the same culture conditions, the lipid productivity of Chlorella in the semi-continuous culture with nitrogen-limitation was1.85-fold higher than that in the batch culture.
(6) The seed preparation process has been enlarged from5L flasks to500L fermenters for adequate cells supply for the inoculation of the photoautotrophic culture. The "HS-PC" model was firstly carried out for outdoor mass culture:the photoautotrophic culture scale was magnified to80m2raceway ponds. The maximum lipid productivity of Chlorella cultured in80m2raceway ponds reached46.4mg/L/d (5.22g/m2/d), which was the biggest scale and highest lipid productivity in the literature.
Our results can not only load a good foundation for industrialization of Chlorella cultivation with high lipid productivity, but also provide a valuable reference for other oleaginous microalgae cultivation.
引文
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