高温菌共培养发酵木质纤维原料产氢过程加强机制的蛋白质组学研究

The main drawbacks of hydrogen fermentation of lignocellulosic biomass are its low biodegradability and hydrogen yield. Thermophilic fermentation using co-culture of Clostridium thermocellum (C. thermocellum) and other thermophiles has provided a promising avenue for enhanced hydrogen production from cellulose. However, interactions of thermophiles, carbon and hydrogen production metabolisms haven't yet been well understood and improved performance is desired. In this project, the metabolic mechanisms of hydrogen fermentation with co-culture of C. thermocellum and other thermophiles was investigated by using proteomic approach. Relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis will be traced. We seek to elucidate the interactions of microorganisms during cellulosic hydrolysis and hydrogen production metabolisms by observing microstructure of substrate and formation of biofilm, investigating accumulation and metabolism of intermediates of carbohydrate hydrolysis, profiles of thermophiles' density as well as synthesis of products. Moreover, proteomic analysis will be done during fermentation with co-culture under stress of substrate and final products to indentify the key metabolic proteins of lignocellulosic hydrolysis and hydrogen metabolism. Finally, an enhanced hydrogen fermentation of lignocellulosic with the co-culture will be developed based on the above results. This study will not only enhance our understanding to thermophilic hydrogen fermentation with co-culture, but also facilitate the future research on metabolic engineering of thermophilic bacteria capable of processing lignocellulosic biomass to hydrogen.

底物降解难、氢气产率低是木质纤维原料发酵产氢的突出问题，采用有强纤维降解能力的热纤梭菌与其它嗜热菌共培养可以强化纤维素发酵产氢，但高温共培养发酵过程菌菌互作、碳代谢和产氢代谢机制尚不清晰，过程效率有待提高。本课题采用蛋白质组学技术，就包含热纤梭菌的嗜热菌共培养发酵木质纤维原料产氢的过程加强机制进行研究，分析发酵过程与纤维降解、能量平衡和终产物合成的相关关键酶蛋白组分，结合底物结构及生物膜观察、纤维小体在纤维降解中的作用机制、过程糖代谢中间产物积累和分解、菌群动态变化和发酵终产物合成，探索共培养中纤维水解和产氢代谢过程的菌菌协作机制。进一步，通过研究底物和产物抑制下共培养过程蛋白响应，分析影响过程的关键功能蛋白，建立高效的高温共培养发酵产氢过程。该项目不但有利于加强高温共培养发酵产氢过程理论基础，而且对后续高温菌代谢工程研究具有重要意义。

底物降解难、氢气产率低是木质纤维原料生物转化过程的突出问题。本项目围绕纤维原料转化生物燃气过程展开研究，取得主要结果如下：（1）纤维原料生物转化过程中的纤维水解机制：纤维原料直接转化过程底物降解率低，只有37.9%，预处理过程能加强纤维水解，与酸处理相比，碱预处理将更多的纤维组分转化为可溶性组分，脱除更多木质素，提高纤维可及度，最终，其对纤维底物的水解促进作用更为明显，底物降解率提高至59.8%，生物气产率也获得加强。微波可以辅助加强普通酸碱预处理，氢气产率提高到对照的150%。上述结果表明，酸、碱、微波等物化手段能破坏纤维结构，提高纤维可及度，进而提高后续生物转化过程纤维水解和发酵效率。（2）纤维原料共培养发酵产氢过程：尽管其较强的纤维降解能力，但热纤梭菌发酵产氢过程氢气产率仍然不高，将热纤梭菌和热解糖梭菌按照1:0.1的比例接种进行共培养，氢气产率达到59 mL/g秸秆，是单培养过程的160%。共培养发酵过程中热纤梭菌将纤维水解成糖，而热解糖梭菌快速消耗糖产氢，消除了中间产物糖积累，并降低了不利于氢气生成的乙醇、乳酸的合成，乙酸丁酸合成增加，最终氢气产率得到提高。（3）产氢过程机制解析和过程优化控制：共培养产氢过程中，热解糖梭菌接种前24h接种热纤梭菌有利于提高共培养过程纤维水解，纤维降解率提高，产氢水平达到65 mL/g秸秆；碱处理能进一步加强共培养过程纤维降解，采用0.1 g NaOH/g秸秆、120度碱处理20min，最终共培养氢气产率达到102 mL/g秸秆，达到原料秸秆单培养过程的276%、共培养过程的157%，过程加强与化学和生物水解的双重作用有关。发酵过程中，与前期相比，进入稳定期，涉及丙酮酸合成的蛋白降低；而与丙酮酸代谢、终产物合成的蛋白增加。最后进行了共培养过程反应器放大，底物降解和产氢比厌氧瓶提高8%。.综上，项目执行不但有利于加强生物产氢过程理论基础，而且对木质纤维原料转化生物燃料工业化应用提高了技术支撑，具有重要的意义。

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