微藻生物质制备燃料乙醇关键技术研究进展

燃料乙醇作为一种优良的可再生液体燃料,其开发利用受到了人们的广泛关注。微藻是一种高光合、高产生物量的生物质资源,很多的藻体细胞中含有大量的淀粉、纤维素（Iα型）等多糖物质,是制备燃料乙醇的优良原料。发展利用微藻制备燃料乙醇技术工艺,对于缓解我国目前日益短缺的能源问题,减少温室气体排放和环境污染等具有很好的应用前景。综述了国内外利用微藻生物质制备燃料乙醇中所用到的关键技术、存在的问题以及今后的发展前景等。     

白色LED复合光谱对4种淡水微藻的影响

利用光效高、耗能小的LED光谱作为光源培养微藻能够降低微藻培养的成本,促进微藻培养实现工业化。比较了6种已市场化的,具有不同光强、不同光谱组成的白色LED复合光谱(1号,光强2 162 lx;2号,光强2 227lx;3号,光强2 794 lx;4号,光强4 587 lx;5号,光强5 356 lx;6号,光强6 244 lx)对4种淡水微藻生长情况和叶绿素含量的影响。结果发现:四尾栅藻在5号光源下,有最大生物质质量浓度和比生长速率,分别为2.89 g/L和0.32g/(L·d)(以细胞干质量计);钝顶螺旋藻在4号光源下,有最大生物质质量浓度和比生长速率,分别为5.05 g/L和0.33 g/(L·d);布朗葡萄藻在6号光源下,有最大生物质质量浓度和比生长速率,分别为1.22 g/L和0.25g/(L·d);而集胞藻在光强较小的光源下生长较好,当光强为2 162 lx时,生物质质量浓度和比生长速率分别为3.05 g/L和0.22 g/(L·d)。在光强较低的情况下,光质的红蓝比对四尾栅藻和布朗葡萄藻的生长没有显著影响(p0.05);与蓝光相比,红光更利于集胞藻和钝顶螺旋藻的生长,分别在红蓝比(R/B)为11.7的1号光源和4号光源下有最大藻细胞密度3.05和5.05 g/L。四尾栅藻、钝顶螺旋藻和布朗葡萄藻的单位水体内叶绿素含量与比生长速率成正比,而单位质量干藻细胞内的叶绿素含量随光强的增大而有所降低。     

一株富含碳水化合物微藻的筛选和分子鉴定

微藻生长快,单位体积碳水化合物产率高,是发酵生产生物乙醇的理想原料。本研究采用通气培养系统,对初筛得到的10株微藻进行分批培养,以单位体积碳水化合物产率为主要指标,筛选富含碳水化合物的优良藻种。研究结果显示：10株微藻的生物质干重、可溶性糖含量、碳水化合物含量和碳水化合物产率变化范围分别在0.922~1.965 g/L、4.42%~19.23%、26.8%~60.9% 和36.17~149.67 mg·L-1·d-1之间,其中藻株GZ-57的碳水化合物产率和可溶糖含量最高,分别为149.67 mg·L-1·d-1 和19.23%,表明藻株GZ-57是一株具有培养潜力的高产碳水化合物微藻。进一步对其进行形态特征及基于18S rDNA、ITS序列的分子系统学分析,发现藻株GZ-57与栅藻科（Scenedesmaceae）链带藻属（Desmodesmus）的极大链带藻（Desmodesmus maximus）亲缘关系较近,因此将其鉴定为极大链带藻（Desmodesmus maximus）。     

用解淀粉酵母和酿酒酵母的互补混合物进行淀粉酒精发酵

将一种分解淀粉的酵母(Saccharomycopsis fibuligera)和不分解淀粉的酿酒酵母(S-accharomyces cerevisiae)一起培养,发酵未经水解的淀粉生成乙醇。转化率为理论上最大值的90％以上。发酵最适pH为5.0～6.0。使用的淀粉浓度为10％(w/v),S.fibuligera     

微藻生物能源的产业化进程及其在CO2减排中的应用进展

基于微藻能源的第三代生物燃料,是一种通过微藻的光合作用积累生物量和油脂而获得的新型清洁生物能源。微藻是由阳光驱动的细胞工厂,它可以在常温常压下实现对CO2的高效吸收,通过微藻细胞高效的光合作用,将光能转化为脂肪或淀粉等碳水化合物的化学能,并释放出O2。将就生物能源、微藻生物能源及其在CO2减排中的应用和产业化进程进行总结和展望。     

乙醇-黄原胶耦联两步发酵

初步探索乙醇-黄原胶耦联两步发酵的可行性和生产工艺,即控制乙醇发酵,发酵液去除固形物后蒸馏回收酒精,然后利用酒精糟液中剩余营养物质进行黄原胶发酵。摇瓶和小罐试验证实方案是可行的,耦联发酵工艺优化条件:选择木薯淀粉进行乙醇发酵60 h后的酒精糟液,添加2 g/L KH2PO4,不添加无机N源。在优化条件下,7 L发酵罐中耦联发酵的黄原胶产量达到20 g/L,转化率约为50%。     

蛋白核小球藻高效同化硝态氮联产微藻蛋白

本研究旨在建立利用微藻去除高浓度废水中硝酸根并转化为藻蛋白的创新技术。先在摇瓶中研究了培养模式和光照模式对于混养蛋白核小球藻的生物量产量、硝酸根同化速率和藻蛋白产量的影响,随后在5 L光发酵罐中成功进行了放大验证。结果表明,在摇瓶培养中,不回流培养基的补料分批培养是最佳培养模式,可获得最高生物量产量为35.95 g/L,硝酸根平均同化速率为2.06 g/(L·d),藻蛋白含量可高达42.44%干重;采用阶梯式增加光强的光照模式,能显著提高细胞比生长速率,最高达到0.65 d–1。在5 L光发酵罐中连续培养128 h,最高生物量产量和硝酸根平均同化速率分别达到66.22 g/L和4.38 g/(L·d),最高藻蛋白含量可达干重的47.13%。本研究能为高效处理工业废硝酸或高浓度硝酸盐废水提供微藻光发酵技术,基于微藻的生物转化过程可联产高蛋白微藻生物质,有利于实现这类废水资源化利用、变废为宝。     

微藻藻种的筛选和育种及基因工程改造

正利用微藻油脂、烷烃或微藻淀粉生产生物柴油或生物酒精已成为国际生物能源研究领域的前沿和各个国家尤其是西方发达国家能源科技竞争的热点。然而,微藻生物燃料面临用于大规模工业化培养的微藻品种较少、生产成本高而难于商业化应用的问题。文章深入研究微藻生物技术的发展,对目前微藻藻种筛选、育种和基因工程技术改造进行分析,为进一步发掘筛选新的微藻生物资源、获得富含生物燃料原料成分及多种生物活性成分的优良藻种提供指导,加快微藻的生物产品和生物燃料商业化生产。     

pH对小球藻Chlorella sp. XQ-200419光合作用、生长和产油的影响

以一种生长快、油脂含量高的小球藻(Chlorella sp. XQ-200419)为实验材料, 利用测定净光合放氧速率的方法研究了pH对其光合作用的影响; 使用改良的BG-11培养基在微藻环形培养池模拟系统中进行分批培养, 培养周期为8d, 培养过程中使用 pH控制仪在线监测藻液的pH, 根据pH变化, 自动接通、关闭CO2通气管道, 将藻液pH分别控制在5.06.0, 7.08.0, 8.09.0, 9.010.0, 10.011.0内, 研究pH对生长速率、生物质面积产率、总脂含量和总脂面积产率的影响。主要结果如下: 藻液pH对小球藻Chlorella sp. XQ-200419光合放氧、生长速率、生物质产率、总脂含量和产率都有显著影响, 适宜的pH范围是7.09.0, 在此范围内, 光合放氧、生长速率、生物质产率、总脂含量和产率均保持较高水平, 且pH的影响不显著; pH低于7.0, 高于9.0, 其光合放氧、生长速率、生物质产率、总脂含量和产率都显著降低。这表明pH对小球藻Chlorella sp. XQ-200419光合作用的影响和对生长、产油的影响是一致的。pH 7.08.0, 小球藻的生物质平均面积产率和总脂平均面积产率都达到最大值, 分别是8.9 g/(m2d)和2269.5 mg/(m2d); 当藻液pH超过10.0, 生物质平均面积产率和总脂平均面积产率分别降低42.1%和60.0%。适合于小球藻生长的pH也有利于其积累油脂, 所以, pH对小球藻产油的影响是一种适宜模式, 而非胁迫模式。规模化培养小球藻Chlorella sp. XQ-200419, 通过补充CO2将藻液pH控制在7.09.0内, 可以获得高生物质产率和总脂产率。研究结果反映出pH对小球藻光合作用、生长和产油影响的规律, 也为规模化培养小球藻生产微藻油脂过程中合理控制藻液pH提供了依据。       

利用外源γ-氨基丁酸调控海洋绿藻亚心形四爿藻淀粉积累

微藻被看作第三代生物质能源的来源。微藻淀粉结构与高等植物的高度相似性使其可以作为粮食作物的替代,在生物能源领域有广泛的应用。γ-氨基丁酸(GABA)被认为是一种信号分子,可以调节植物细胞的生长代谢。本研究在缺氮培养条件下添加外源GABA调控海洋绿藻亚心形四爿藻生理代谢和淀粉积累。结果表明,添加外源GABA可以抑制细胞生长,降低光合作用效率;OJIP实验显示,GABA的添加增强了光合器官能量耗散,降低了光能利用效率,阻碍了电子传递,造成额外胁迫,从而促使细胞将碳流更多地分配到淀粉积累,导致藻细胞的淀粉含量、淀粉产量和淀粉产率提高。添加10 mmol/L GABA获得最大淀粉含量39%DW,比未添加GABA的对照组淀粉含量提高39%;同时获得最大淀粉产量和产率为1.72 g·L^-1和0.36 g·L^-1·d^-1,分别比未添加GABA的对照组提高39%和50%。以上结果表明在缺氮条件下添加外源GABA是一种调控亚心形四爿藻细胞代谢并提高其淀粉生产的有效方法。     

Micro and macroalgal biomass: A renewable source for bioethanol

Population outburst together with increased motorization has led to an overwhelming increase in the demand for fuel. In the milieu of economical and environmental concern, algae capable of accumulating high starch/cellulose can serve as an excellent alternative to food crops for bioethanol production, a green fuel for sustainable future. Certain species of algae can produce ethanol during dark-anaerobic fermentation and thus serve as a direct source for ethanol production. Of late, oleaginous microalgae generate high starch/cellulose biomass waste after oil extraction, which can be hydrolyzed to generate sugary syrup to be used as substrate for ethanol production. Macroalgae are also harnessed as renewable source of biomass intended for ethanol production. Currently there are very few studies on this issue, and intense research is required in future in this area for efficient utilization of algal biomass and their industrial wastes to produce environmentally friendly fuel bioethanol.     

Tracing carbon monoxide uptake by Clostridium ljungdahlii during ethanol fermentation using 13C-enrichment technique

Conversion of synthesis gas (CO and H₂) to ethanol can be an alternative, promising technology to produce biofuels from renewable biomass. To distinguish microbial utilization of carbon source between fructose and synthesis gas CO and to evaluate biological production of ethanol from CO, we adopted the ¹³C-enrichment of the CO substrate and hypothesized that the residual increase in δ¹³C of the cell biomass would reflect the increased contribution of ¹³C-enriched CO. Addition of synthesis gas to live culture medium for ethanol fermentation by Clostridum ljungdahlii increased the microbial growth and ethanol production. Despite the high ¹³C-enrichment in CO (99 atom % ¹³C), however, microbial δ¹³C increased relatively small compared to the microbial growth. The uptake efficiency of CO estimated using the isotope mass balance equation was also very low: 0.0014 % for the low CO and 0.0016 % for the high CO treatment. Furthermore, the fast production of ethanol in the early stage indicated that the presence of sugar in fermentation medium would limit the utilization of CO as a carbon source by C. ljungdahlii.     

Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity

Alcohol fermentation of starch was investigated using a direct starch fermenting yeast, Saccharomyces cerevisiae SR93, constructed by integrating a glucoamylase-producing gene (STA1) into the chromosome of Saccharomyces cerevisiae SH1089. The glucoamylase was constitutively produced by the recombinant yeast. The ethanol concentration produced by the recombinant yeast was 14.3 g/L which was about 1.5-fold higher than by the conventional mixed culture using an amylolytic microorganism and a fermenting microorganism. About 60% of the starch was converted into ethanol by the recombinant yeast, and the ethanol yield reached its maximum value of 0.48 at the initial starch concentration of 50 g/L. The fed-batch culture, which maintains the starch concentration in the range of 30 to 50 g/L, was used to produce a large amount of ethanol from starch. The amount of ethanol produced in the fed-batch culture increased about 20% compared to the batch culture. (c) 1997 John Wiley & Sons, Inc.     

Fermentation of starch by Klebsiella oxytoca p2, containing plasmids with alpha-amylase and pullulanase genes.

Klebsiella oxytoca P2(pC46), an ethanol-producing recombinant, has been evaluated in fermentation of maltose and starch. The maximum ethanol produced by P2(pC46) was 0.34 g ethanol/g maltose and 0.38, 0.40, or 0.36 g ethanol/g starch in fermentation of 1, 2, or 4% starch, representing 68, 71, and 64% the theoretical yield. The pC46 plasmid transformed to cells of K. oxytoca P2 reduced the ethanol production from maltose and starch. In fermentation of starch after its digestion at 60 degrees C for 24 h, in two-step fermentation, the time for maximum ethanol production was reduced to 12-24 h and the theoretical yield was around 90%. The increase in starch concentration resulted in lower alpha-amylase activity but in higher pullulanase activity. The high activity and thermostability of the amylolytic enzymes from this transformant suggest that it has a potential for amylolytic enzymes source.     

Fermentation of biomass-generated producer gas to ethanol

The development of low-cost, sustainable, and renewable energy sources has been a major focus since the 1970s. Fuel-grade ethanol is one energy source that has great potential for being generated from biomass. The demonstration of the fermentation of biomass-generated producer gas to ethanol is the major focus of this article in addition to assessing the effects of producer gas on the fermentation process. In this work, producer gas (primarily CO, CO(2), CH(4), H(2), and N(2)) was generated from switchgrass via gasification. The fluidized-bed gasifier generated gas with a composition of 56.8% N(2), 14.7% CO, 16.5% CO(2), 4.4% H(2), and 4.2% CH(4). The producer gas was utilized in a 4-L bioreactor to generate ethanol and other products via fermentation using a novel clostridial bacterium. The effects of biomass-generated producer gas on cell concentration, hydrogen uptake, and acid/alcohol production are shown in comparison with "clean" bottled gases of similar compositions for CO, CO(2), and H(2). The successful implementation of generating producer gas from biomass and then fermenting the producer gas to ethanol was demonstrated. Several key findings following the introduction of producer gas included: (1) the cells stopped growing but were still viable, (2) ethanol was primarily produced once the cells stopped growing (ethanol is nongrowth associated), (3) H(2) utilization stopped, and (4) cells began growing again if "clean" bottled gases were introduced following exposure to the producer gas.     

Anaerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture: involvement of the pentose phosphate pathway.

Under anaerobic 2-ketogluconate-limited growth conditions (D = 0.1 h-1), Klebsiella pneumoniae NCTC 418 was found to convert this carbon source to biomass, acetate, formate, CO2, ethanol and succinate. The observed fermentation pattern is in agreement with the simultaneous functioning of the pentose phosphate pathway and the Entner-Doudoroff pathway in 2-ketogluconate catabolism. When cultured at pH 8.0 apparent YATP values were lower than those found at culture pH 6.5. This difference can be explained by assuming that at high culture pH values approximately 0.5 mol ATP was invested in the uptake of 1 mol 2-ketogluconate. Sudden relief of 2-ketogluconate-limited conditions led to lowering of the intracellular NADPH/NADP ratio and (possibly as a result of this) to inhibition of biosynthesis. Whereas production of ethanol stopped, lactate was produced at high rate. This product was formed, at least partly, via the methylglyoxal bypass.     

The CO2-concentrating mechanism in the physiological context: lowering the CO2 supply diminishes culture growth and economises starch utilisation in Chlamydomonas reinhardtii

In a synchronously grown Chlamydomonas reinhardtii (Chlorophyceae) culture the CO2-concentrating mechanism (CCM) was induced by lowering the CO2 level from 4% to 0.036% CO2 (culture HL). The effects of the reduced carbon supply on starch levels were studied over a period of up to 100 h and compared with control cultures kept either at 4% CO2 (culture H) or continuously at ambient air (0.036% CO2, culture L). Lowering the CO2 supply reduced culture growth as estimated by chlorophyll, protein and cell density. The starch level continued to show diurnal variations with an initially reduced rate of starch synthesis at reduced or abolished culture growth. Subsequently, starch maxima and minima increased. After 4 days the resulting pattern for culture HL was similar to that of culture L, which possessed higher minima but identical maxima to culture H. The intracellular starch localisation was examined on electron micrographs. Cell extracts were assayed for ADP-glucose pyrophosphorylase (EC 2.7.7.27) and starch phosphorylase (EC 2.4.1.1) activities. Over the assayed period of 2 days, there was a good correlation between the observed changes in the starch levels and the measured enzyme activities. The rate of CO2-dependent oxygen evolution of culture HL declined from 100% to 60% of the control over the day. This indicates that the diminished or abolished growth and the impairment of starch accumulation upon CO2 depletion are not simply consequences of the lowered level of the substrate CO2. The diminished growth and the peculiar starch accumulation pattern with higher positions of the starch minima in low-CO2 cells are interpreted as economised starch utilisation as long-term aspects of induction of the CCM.     

Flocculation of microalgae using cationic starch

Due to their small size and low concentration in the culture medium, cost-efficient harvesting of microalgae is a major challenge. We evaluated the potential of cationic starch as a flocculant for harvesting microalgae using jar test experiments. Cationic starch was an efficient flocculant for freshwater (Parachlorella, Scenedesmus) but not for marine microalgae (Phaeodactylum, Nannochloropsis). At high cationic starch doses, dispersion restabilization was observed. The required cationic starch dose to induce flocculation increased linearly with the initial algal biomass concentration. Of the two commercial cationic starch flocculants tested, Greenfloc 120 (used in wastewater treatment) was more efficient than Cargill C*Bond HR 35.849 (used in paper manufacturing). For flocculation of Parachlorella using Greenfloc 120, the cationic starch to algal biomass ratio required to flocculate 80% of algal biomass was 0.1. For Scenedesmus, a lower dose was required (ratio 0.03). Flocculation of Parachlorella using Greenfloc 120 was independent of pH in the pH range of 5 to 10. Measurements of the maximum quantum yield of PSII suggest that Greenfloc 120 cationic starch was not toxic to Parachlorella. Cationic starch may be used as an efficient, nontoxic, cost-effective, and widely available flocculant for harvesting microalgal biomass.     

Direct and efficient ethanol production from high-yielding rice using a Saccharomyces cerevisiae strain that express amylases

Efficient ethanol producing yeast Saccharomyces cerevisiae cannot produce ethanol from raw starch directly. Thus the conventional ethanol production required expensive and complex process. In this study, we developed a direct and efficient ethanol production process from high-yielding rice harvested in Japan by using amylase expressing yeast without any pretreatment or addition of enzymes or nutrients. Ethanol productivity from high-yielding brown rice (1.1g/L/h) was about 5-fold higher than that obtained from purified raw corn starch (0.2g/L/h) when nutrients were added. Using an inoculum volume equivalent to 10% of the fermentation volume without any nutrient supplementation resulted in ethanol productivity and yield reaching 1.2g/L/h and 101%, respectively, in a 24-h period. High-yielding rice was demonstrated to be a suitable feedstock for bioethanol production. In addition, our polyploid amylase-expressing yeast was sufficiently robust to produce ethanol efficiently from real biomass. This is first report of direct ethanol production on real biomass using an amylase-expressing yeast strain without any pretreatment or commercial enzyme addition.