氮饥饿胁迫下有机废水培养富油藻-菌生物颗粒的机制与调控

There are 3 neck-bottle problems in the up-stream of microalgae biomass energy production process: high cost of cultivation, contamination of bacteria and difficulty in harvesting. Depressed bacteria overgrowth, enhanced bacterial aggregation and stimulated microalgal produced lipid could be achieved by N starvation stress, when microalgae and bacteria are co-cultivated. This provides a new clue to solve the neck-bottle problems of microalgae heterotrophic cultivation in organic wastewater. Taking N starvation as the main strategy, organic wastewater treatment and lipid transformation by synergistic action of microalgae-bacteria as the subject clue, and rich lipid microalgae-bacteria biogranules cultivation as the critical scientific issue, this proposal aims to study the stress responses of microalgae and bacteria, cell to cell communication and response between microalgae and bacteria, as well as inducted lipid synthesis conditions and pathways, under N starvation stress and microalgae-bacteria synergetic actions. The proposal targets to reveal the response mechanisms and synergetic mechanisms of microalgae and bacteria, and inducted lipid synthesis mechanisms under N starvation stress, with the expectation of efficient production and harvesting of rich lipid biogranules by organic wastewater cultivation. Meanwhile, the process control project of lipid production by organic wastewater with strategies of nitrogen starvation and microalgae-bacterial synergetic action will be set up, by means of designing and scaling up of the systems based on above theory, combined with process operation condition optimization and performances characterization studies.

微藻生物质能源上游生产工艺有3个关键瓶颈问题：培养费用高、细菌污染和微藻收获困难。藻菌共培养时，氮饥饿条件理论上可有效抑制细菌生长、强化细菌聚集功能和刺激藻细胞油脂合成，为解决有机废水异养培养微藻的瓶颈问题提供新的思路。本项目拟以氮饥饿胁迫为主要手段，以微藻和细菌协同作用下有机废水处理与油脂转化为主线，以培养富油藻-菌生物颗粒为关键科学问题，通过解析氮饥饿胁迫下、藻-菌协同作用时，微藻和细菌的应激反应行为、微生物胞间通讯与相互响应、以及油脂诱导合成条件与途径，揭示氮饥饿条件下微藻和细菌的响应机制、藻菌协同作用机制和脂肪酸合成诱导机制，以期奠定有机废水高效生产和收获富油生物颗粒的理论基础。同时，基于上述理论基础设计和构建工艺放大体系，结合关键工艺运行条件解析和行为表征，建立基于氮饥饿胁迫的藻-菌协同作用下有机废水油脂资源转化工艺调控方案。

微藻作为一种可替代传统化石燃料的可再生能源，在废水处理与能源转化方面引起越来越多的关注。本项目针对目前该领域存在的3个关键瓶颈问题，即，培养费用高、微藻收获困难和细菌污染，开展了以下三个方面的研究工作。.（1）微藻生物质高效生产策略。建立了氮限制磷补给的微藻培养技术，生物量和油脂含量分别提高了1.9倍和1.7倍，实现了生物量和油脂含量的同步增长，显著提高了微藻油脂产量和品质。适当过量的磷在胞内形成聚磷，线粒体活性提高25.0%，但大量过量的磷导致细胞中毒，微藻生长降低38.8%。采取分批补给的方式添加过量的磷，微藻生长激素基因和油脂合成基因表达上调，从而充分利用磷过量的优势，同时避免其毒性作用。.（2）微藻采收及生物质利用。建立了内循环生物絮凝剂的生产应用方法，Citrobacter sp细菌分泌的生物聚合物通过吸附架桥絮凝微藻细胞，絮凝后的微藻生物质还可作为该细菌的基质，从而降低产絮菌培养的底物成本。为了进一步提高生物絮凝剂的絮凝效率，建立了AlCl3与生物絮凝剂联合的微藻采收方法，通过絮凝剂的电中和、架桥等作用实现藻细胞的絮凝，收获效率为96.8%，与使用单一絮凝剂相比，生物絮凝剂、AlCl3、助凝剂用量均减少约50%左右，且残余Al3+很低，满足后续微藻利用的工艺要求。微藻生物质作为微生物燃料电池的唯一电子供体，库伦效率为61.5%，产电量可达1.07W/m2，产电效能与乙酸钠作为底物的微生物燃料电池接近，为微藻的资源化利用提供了新方向。.（3）藻菌相互作用及胞间通讯机制。微藻可以感知细菌的信号分子AHLs，分泌芳香族蛋白质与AHLs相互作用，藻细胞聚集成200 μm的生物絮凝体，沉降率最高为41%，而无AHLs的情况下微藻为悬浮态。细菌信号分子还可诱导脂肪酸合成酶基因表达上调，油脂含量增加84%。证明了微菌共存体系存在真核微藻与原核细菌的跨界胞间通讯，促进微藻生物能源生产。另外，我们首次发现微藻可以分泌苯甲酸和水杨酸作为内源性信号物质，刺激DNA复制、TCA循环、糖酵解过程，从而调节微藻生长以应对外界不良环境。