酵母菌PNY2013驱动的同步脱氮除磷新机理及其应用

The theory and technology of denitrification and phosphorus removal is under the sustaining pressure of nitrogen and phosphorus pollution in water treatment. And besides the optimization and reformation the process, it is necessary and urgent to discover new facilitate strains and develop corresponding process for the denitrification and phosphorus removal. Given this, by using a purified strain of yeast PNY2013, which is reported to be able to simultaneously remove the nitrogen and phosphorus for the first time, as the our material, the present project firstly focuses on the key functional gene for metabolic mechanism of nitrogen and phosphorus by genome sequencing, gene knockout and transcriptomics, as well as to completely harness relationship between metabolism of nitrogen, phosphorus and carbon under molecular level; secondly, the present project focuses on the application of PNY2013 to treat wastewater with different carbon source and concentration. Base on the evaluation and certification of the application of PNY2013 on the removal of nitrogen and phosphorus mixtured with active sludge and digested sludge. The present aim of project to harness a feasible application method. The results of the present project will enrich and fulfill the novel mechanism of simultaneous removal of nitrogen and phosphorus by strain PNY2013 in molecular level, supplement the nowadays route for the substance cycling, as well as amplify the current theory and technology of bio-treatment, all of which play important roles in microbiology and realistic engineering of denitrification and phosphorus removal.

现有的生物脱氮除磷理论及技术面临着当前水体氮磷污染治理的持续压力，寻找新菌株并开发相应的脱氮除磷工艺具有现实的必要性及紧迫感。本项目针对首次分离得到的一株好厌氧条件下均具有同步脱氮除磷新功能的酵母菌PNY2013，首先，进行全基因组和转录组分析，结合基因敲除最终明确其氮磷代谢关键基因，揭示其驱动的同步脱氮除磷新机理。其次，聚焦PNY2013同步脱氮除磷应用的碳源问题，进行包括含糖类工业废水在内的不同碳源条件下的连续运行实验，明确不同碳源条件下的实际操作方法。最后，针对PNY2013同步脱氮除磷应用的优势生长问题，进行其与现有的活性污泥及消化污泥共存竞争条件下的连续运行实验，掌握切实可行的操作方法。本项目的研究结果将从分子基因水平揭示出PNY2013驱动的同步脱氮除磷新机理，补充现有的物质循环径路因而具有重大的科学意义，同时还能扩充生物处理理论及技术，在脱氮除磷处理工程领域发挥重要作用。

本项目针对首次分离得到的一株好厌氧条件下均具有同步脱氮除磷新功能的酵母菌PNY2013，分别就其驱动的同步脱氮除磷机理、其同步脱氮除磷应用的碳源及竞争生长问题的研究结果表明：首先，PNY2013好氧和厌氧条件下共用同一条有别于常规的碳代谢通路，以丙酮酸脱氢酶aceE及甲酸脱氢酶FDH主导丙酮酸及乙醛酸的脱氢代谢产生乙酸、乙醇、氢气及二氧化碳，同时，丙酮酸通过转化为2-羟基戊二酸或草酰乙酸酯，为合成谷氨酸提供碳骨架并和氮代谢关联；以氨氮转运蛋白酶、联氨水解酶HH及联氨脱氢酶HD将氨氮转运进微生物体内代谢并产生氮气，同时，以GDHA和abat主导谷氨酸的合成完成氨氮的进一步代谢；以ppk、ppx、pst等主导磷的转运并通过ADP/ATP能量代谢系统完成磷的代谢。其次，PNY2013以葡萄糖、乙醇及乙酸为碳源时其μmax分别为0. 1327、0. 1252及0. 1115h-1, 其脱氮除磷的最佳条件均为：温度30°C, pH=8.0, 溶解氧0-2mg·L-1, C/N=200:5；长期连续运行条件下的实验表明, 与进水氨氮及磷酸根浓度分别达400及80 mg·L-1, 且两者去除率均接近100%的葡萄糖为碳源条件下的能力相比, 乙醇及乙酸为碳源时, 两者的去除率也可达60-80（氨氮）及40%（磷酸根）；在模拟制糖、淀粉、啤酒、味精四种工业废水为碳源条件下, 除淀粉外PNY2013均能有效去除COD、氨氮及磷酸根, 其去除率分别可达40（制糖）, 89（啤酒）和96%（味精）；85（制糖）, 94（啤酒）和76%（味精）；90%（制糖、啤酒及味精）。最后，PNY2013与现有污泥共存系统在不同碳源模式下均显示出良好的处理性能，如对葡萄糖、氨氮、总氮、磷酸盐和总磷的去除率分别可达99、98、94、97及95%，比单独现有污泥的去除率分别提高了48、12、14、45及43%；共存长期竞争条件下的实验结果表明其比COD、比氨氮及比磷酸盐污泥负荷分别可达1.084、0.011及0.0017 kg/[kg(MLSS)·d]，分别是单独现有污泥的2.8、4.4及4.3倍；共存长期竞争条件下的微生物结构分析表明，尽管丰度有所下降PNY2013最终以70%的丰度取得优势地位；同时随着PNY2013的加入，其与原有微生物共生重构了系统的代谢模式，形成了独特有效的碳、氮及磷代谢通路。