【作者】 徐晟；

【导师】 韦中； 梁晓辉；

【作者基本信息】 南京农业大学 ， 农业硕士（专业学位）， 2023， 硕士

【摘要】 微生物发酵技术是功能性微生物利用底物合成某种或某类化合物的重要手段之一,在医学、食品以及农业等诸多领域有着广泛的应用。随着社会的进步和人们对高生活质量的迫切需求,微生物发酵技术开始逐渐取代化工合成并成为研究热点。秸秆的构成使其成为一种很好的固体发酵介质,而其中的可溶性碳水化合物、植物蛋白以及微量元素又可为微生物生长繁殖提供营养。以秸秆为底物构建发酵系统进行微生物发酵具有较大的应用潜力。目前,用于微生物发酵的秸秆多以小麦、玉米等主要粮食作物秸秆为主,缺乏对药用、观赏用作物秸秆的资源化利用。秸秆发酵产物利用多以饲料化、肥料化等为主,缺乏发酵产物多用途、多附加值利用。秸秆发酵菌种多以强降解菌、产酵母菌为主,缺乏可调控土壤微生物环境、促进植物生长发育、抑病等功能菌的参与。本文以实验室保存的一株秸秆降解菌哈茨木霉(Trichoderma harzianum)NJAU4742、两株有益假单胞菌(Pseudomonas moorei)PSE9和(P.vancouverensis)PSE13为材料开展实验。首先通过实验检测菌株PSE9和PSE13的生物学特性,然后通过微生物发酵的方式,对不同秸秆进行发酵,筛选出较好的的微生物-秸秆组合,获得具有一定生物量、功能物质的产品。通过盆栽试验,验证发酵浸提液对植物的促生效果,研究结果如下:1、通过研究两株有益假单胞菌的生物学特性,测定两株菌利用秸秆的能力、产氨、产铁载体、产吲哚乙酸(IAA)和解磷能力。测定两株有益假单胞菌利用秸秆的基本情况,以及是否产促生物质,并拥有一定植物促生的潜力。室内试验结果表明,两株有益假单胞菌(P.moorei)PSE9和(P,vancouverensis)PSE13具有产促生物质IAA(分别为34 nmol/L、40 nmol/L)、铁载体能力(分别为0.28 U、0.23 U),具有一定产氨及解磷能力。两株有益假单胞菌有一定分解纤维素能力,但分解能力较弱,难以充分利用秸秆。2、秸秆本身结构复杂难以被降解利用,通过引入降解菌哈茨木霉NJAU4742可较好地降解秸秆,并为其他有益菌株提供营养物质,因此,有益菌加降解菌可更好地进行发酵过程。通过测定加入降解菌前后不同降解率,试验结果表明,在引入降解菌哈茨木霉NJAU4742后,对比单菌发酵,各处理秸秆降解率均有所增加,尤其菊花秸秆在加入哈茨木霉NJAU4742后降解率提升了20%。进一步通过对六种不同秸秆、两株有益菌和一株降解菌进行组合发酵,共计48种发酵组合进行筛选,以生物量为指标得到一种较优的发酵组合方式。研究表明,六种秸秆中菊花秸秆发酵效果较好,假单胞菌PSE13发酵效果优于假单胞菌PSE9,最终确定哈茨木霉NJAU4742与假单胞菌PSE13接力发酵菊花秸秆作为较优的发酵组合,假单胞菌PSE13生物量达2.7×10~9CFU/g。3、在得到较好的发酵组合(A4)后,测定发酵液处理下的番茄种子萌发情况、发酵液产促生物质IAA、铁载体情况,并进行后续盆栽试验,验证发酵液对番茄植株的促生效果,以及发酵液存放不同时间、不同温度对番茄植株生长的影响。经筛选的发酵组合(A4)发酵后,获得发酵浸提液,处理后的番茄种子发芽率达90%、发酵浸提液含促生物质IAA、铁载体含量分别为34.3 nmol/L、0.38 U。后续盆栽试验结果表明,发酵组合(A4)的发酵浸提液灌根施用,对番茄植株的生长显示有促进作用,番茄植株根长、茎粗、株高较对照组植株增长分别为180%、21%、93%,地上部干鲜重增长分别为109%、74%,地下部干鲜重增长分别为124%、109%。和其他各发酵方式相比,发酵组合(A4)的促生效果最好,植株生长旺盛,干重增长显著。发酵液存放7-14天,在4℃条件下的促生效果优于存放在常温25℃条件。综上所述,通过研究可以发现接力发酵方式(以菊花秸秆为底物,哈茨木霉发酵7天后加入假单胞菌PSE13继续发酵7天)发酵液对番茄有着显著的促生效果,施加发酵浸提液之后,番茄植株的叶绿素含量、植株生物量均有所提高。此外发酵液低温存放7-14天下仍具有一定促生效果,该组合的发酵液在产品化方面有着一定潜力。更多还原

【Abstract】 Microbial fermentation technology is one of the important methods for functional microorganisms to synthesize certain compounds or classes of compounds using substrates,and it has broad applications in many fields such as medicine,food,and agriculture.With the progress of society and people’s urgent need for high-quality life,microbial fermentation technology has gradually replaced chemical synthesis and become a research trend.The composition of straw makes it a good solid fermentation medium,and the soluble carbohydrates,plant proteins,and trace elements in it can provide nutrients for the growth and reproduction of surrounding microorganisms.Constructing a fermentation system for microbial fermentation using straw as a substrate has great application potential.Currently,straw used for microbial fermentation is mainly wheat,corn and other major cereal crop straws,and there is a lack of resource utilization of medicinal and ornamental crop straws.The utilization of straw fermentation products is mainly focused on feed and fertilizer,lacking multi-purpose and high-value-added utilization of fermentation products.The straw fermentation strains are mainly composed of strong degrading bacteria and fermentation yeasts,and there is a lack of participation of bacteria that can regulate the soil microbial environment,promote plant growth and development,and inhibit disease.In this study,an experiment was conducted using a strong degrading fungus Trichoderma harzianum NJAU4742 and two beneficial Pseudomonas moorei PSE9 and Pseudomonas vancouverensis PSE13 preserved in the laboratory.First,the biological characteristics that can distinguish the strains PSE9 and PSE13 were determined through experiments,and the production of growth-promoting substances by these two microbial strains was measured.Then,different straws were fermented by microbial fermentation to screen for better microbial-straw combinations and obtain products with certain biomass and functional substances.The growth-promoting effect of the fermentation extract on plants was determined through pot experiments,and the research results are as follows:1.The biological characteristics of two beneficial Pseudomonas strains were studied,and their ability to use straw,produce ammonia,produce iron carriers,indole-3-acetic acid(IAA),and phosphorus dissolution ability were measured.The basic situation of the two beneficial Pseudomonas strains using straw and whether they produce growth-promoting substances with certain plant growth-promoting potential were determined.The results of indoor experiments showed that the two beneficial Pseudomonas strains PSE9 and PSE13have the ability to produce growth-promoting substance IAA(34 nmol/L and 40 nmol/L)and iron carriers(0.28 U and 0.23 U),and they have a certain ability to produce ammonia and dissolve phosphorus.The two beneficial Pseudomonas strains have a certain ability to decompose cellulose,but the decomposition ability is weak,making it difficult to fully utilize straw.2.Due to the special structure of straw,it is difficult to degrade and utilize.However,by introducing the degradation bacterium Trichoderma NJAU4742,straw can be effectively degraded and provide nutrients for other beneficial strains.Therefore,combining beneficial bacteria with degradation bacteria can better carry out the fermentation process.By measuring the different degradation rates before and after adding the degradation bacteria,it was found that the addition of Trichoderma NJAU4742 increased the straw degradation rate compared to single-strain fermentation(the degradation rate of chrysanthemum straw increased by 20%after adding Trichoderma NJAU4742).Further,by combining six different straws,two beneficial bacteria strains,and one degradation bacteria strain for fermentation,a total of 48 fermentation combinations were screened based on biomass as the core screening criteria,and an optimal fermentation combination was obtained.The study showed that chrysanthemum straw had better fermentation effect among the six types of straws,and the beneficial bacterium PSE13 had better fermentation effect among the two strains of beneficial bacteria.Finally,Trichoderma NJAU4742 and PSE13 were determined as the optimal fermentation combination for chrysanthemum straw,and the biomass of beneficial bacteria reached 2.7×10~9CFU/g.3.After obtaining a good fermentation combination(A4),the germination of tomato seeds,the production of plant growth-promoting substance IAA,iron carrier content in the fermentation solution were measured,and subsequent pot experiments were conducted to verify the growth-promoting effect of the fermentation solution on tomato plants,as well as the effect of storing the fermentation solution at different times and temperatures on tomato plant growth.After fermentation with the selected fermentation combination(A4),the germination rate of tomato seeds treated with the fermented extract reached 90%,and the fermented extract contained plant growth-promoting substance IAA and iron carrier content of 34.3 nmol/L and 0.38 U,respectively.Subsequent pot experiments showed that the fermented extract of the fermentation combination(A4)had a promoting effect on the growth of tomato plants,with increases in root length,stem thickness,and plant height of180%,21%,and 93%compared to the control group,increases in above-ground dry and fresh weight of 109%and 74%,and increases in underground dry and fresh weight of 124%and 109%,respectively.Compared with other fermentation methods,the fermented extract of the fermentation combination(A4)had the best growth-promoting effect,with strong plant growth and significant increases in dry weight.The growth-promoting effect of the fermented extract stored at 4°C was better than that stored at room temperature(25°C).Storing the fermentation solution at different times and temperatures had certain effects on tomato plant growth.In summary,the study found that the relay fermentation method fermentation combination(A4)had a significant growth-promoting effect on tomatoes,and after applying the fermented extract,the Chlorophyll content and plant biomass of tomato plants were both increased.Moreover,the fermented extract still had a certain growth-promoting effect after being stored for a certain period of time and at a certain temperature.Applying this fermented extract can significantly promote the growth of tomato plants and has potential in product development.更多还原