供液固态发酵基质水分特性演化规律及灵芝对水分演化的应答

Environmental concern and energy security have aroused renewed interests in solid-state fermentation (SSF) owing to its special advantages of water and energy saving. Severe evaporation caused by metabolic heat removal results in liquid water losing. Hence, it requires available water supply in most SSF. The newly constructed water-supply SSF technology can effectively promote fermentation efficiency. However, how does the feed water migrate and transform in the fermentation for microbial utilization? How does the microorganism respond to the liquid? The process remains unclear and needs exploration. In this project, a water-supply SSF bioreactor, negative-pressure auto-controlled irrigation (NPACI) coupled with super absorbent polymers (SAPs), was constructed based on the principle of water states. Ganoderma lucidum ACCC53264, a kind of edible and medicinal mushroom, was chosen as the reference strain with pretreated sawdust and wheat straw as the fermentation substrate. Low-field nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI), and heavy water labeling are used to analyze the evolutionary laws of the water occurrence characteristics including the transfer, mobility, distribution and transform of three forms of water, i.e, hygroscopic, capillary and free water. Accordingly, the model of changes in matrix physical structure during the fermentation process will be established. Then, the response of ACCC53264 to water evolution will be revealed. The results will provide theoretical basis and practical guidance for moisture controlling in SSF, and supply technical support for industrialization of modern water-supply SSF and promotion of edible and medicinal mushroom industry.

环境污染和能源危机加剧使天然具有节水、节能优势的固态发酵重新成为研究的热点。发酵中强烈的水分蒸散使多数微生物提出供液要求。新构建的供液固态发酵技术能够有效促进微生物固态发酵。然而供液水分在发酵中如何迁移、演化并为微生物利用？微生物又如何响应这些演化过程？这些关键问题亟待解决。本项目拟以食药用真菌灵芝Ganoderma lucidum ACCC53264为研究对象，以木屑、小麦秸秆为发酵基质，利用基于水分能态原理构建的负水势供液耦合保水剂固态发酵装置，采用低场核磁共振技术、重水标记等方法研究发酵中基质水分(吸湿水、毛管水和自由水)迁移和分布状态演化规律，建立发酵过程中基质物理结构变化模型。并解析菌株ACCC53264对基质水分演化的响应机制。预期研究结果为固态发酵水分可控化研究提供理论依据和实践指导，为实现供液固态发酵规模化和推动食药用菌产业升级提供技术支持。

环境污染和能源危机加剧使天然具有节水、节能优势的固态发酵重新成为研究热点。固态发酵虽然前景广阔，但发酵中强烈的水分蒸散使多数微生物提出供液要求，存在基质有效水分调控难题。新构建的供液固态发酵技术能够为微生物提供有效水分，促进固态发酵。项目研究了供液固态发酵基质水分特性演化规律并以食药用真菌灵芝Ganoderma lucidum ACCC53264为研究对象研究其对水分演化的应答，取得以下结果：.1）研究灵芝固态发酵体系中基质物性参数确定灵芝固态发酵最佳条件：湿料最适堆料密度550 kg/m3，颗粒度20目，堆料高度4 cm，起始含水量60%；分析固态发酵体系三相组成，证明了水分变异在三相变化中的最显著性作用；构建的保水剂等供液方式能够促进灵芝固态发酵产生物量和关键酶活：7%的水饱和保水剂添加量最佳，生物量增加44%，FPA、CMC、β-葡聚糖、GS和GR酶酶活分别增加14.98%、15.67%、11.7%、19.05%和32%；.2）利用低场核磁共振等技术准确解析水分在发酵中的形态变化和特性演化过程。结果表明：随着基质含水量的增加，低场核磁共振图谱最高峰保留时间增加，基质水分自由度也在逐渐增加。基质中水的存在和散失主要由毛管水控制。发酵结束，供液固态发酵的毛管水含量分别比对照和冷模固态发酵中高20.48%和17.20%，弛豫时间减少量最高，达56.53%。保水剂能通过持续不断释放毛管水缓解灵芝固态发酵过程中的水分散失；.3）利用比较转录组技术分析、筛选并验证了灵芝供液固态发酵体系关键转录差异表达基因和表达基因的生理功能；揭示了筛选的差异表达的编码MnP的基因通过ROS信号参与水分影响灵芝固态发酵生理功能；.4）解析了纤维素酶基因转录激活因子ClrB-1、ClrB-2以及水通道蛋白AQP响应水分供液的机制：供液通过促进ClrB-1、ClrB-2表达，提高纤维素酶酶活，促进生长和次级代谢产物积累；GlAQP通过ROS调控干旱胁迫下灵芝三萜合成，干旱胁迫前期和后期GlAQP对ROS水平和灵芝三萜合成具有相反影响且GlAQP与NOX之间存在相互调控作用。研究从微生物生理角度揭示了灵芝对供液固态发酵中水分演化的应答机制。

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