摘要
葡萄修剪枝条是葡萄园管理中产生的一种有机副产物。近年来,随着我国葡萄栽培面积的不断扩大,产生了大量的葡萄修剪枝条,尤其是葡萄冬剪枝条。由于葡萄冬剪枝条木质化较为严重,难于被降解,传统上这些枝条被随意堆放或焚烧,造成资源的严重浪费和环境污染。自然界中存在一些木质素降解微生物,可以实现葡萄枝条木质素的高效降解及转化,如白腐真菌。因此,筛选出高效的枝条木质素降解菌,为提高葡萄枝条木质素的降解效率,促进葡萄枝条的综合利用,具有重要的应用研究价值。
本研究从葡萄园中腐烂的葡萄枝条上,进行分离纯化出木质素降解真菌,再通过愈创木酚平板显色法和苯胺蓝平板退色法初步筛选出产木质素降解酶类(漆酶、锰过氧化物酶等)较高的菌株,并对此筛选出的菌株进行液体产酶试验和固态降解实验,以进一步筛选出产木质素降解酶活高和枝条木质素降解能力强的高效菌株,然后分别研究培养温度和pH值对高效菌株固态降解作用的影响,以对高效菌株的固态降解条件进行初步优化,最后从形态学上对高效菌株进行菌种鉴定。研究结果表明:
1.直接从腐烂的葡萄枝条上,分离纯化到24株在愈创木酚培养基平板上产生红棕色变色圈的木质素降解真菌。以此24菌株分别进行PDA-愈创木酚显色反应和PDA-苯胺蓝退色反应试验,初步筛选出产漆酶和锰过氧化物酶活较高的5个菌株,即A-11、A-02、A-40-1、A-21和A-51-1。
2.以初筛到的5个菌株进行液态产酶试验,结果表明,5个菌株均能在第6 d和8 d分别达到漆酶和锰过氧化物酶的最大产酶高峰,其中菌株A-51-1表现出最强的产漆酶和锰过氧化物酶能力,其最大酶活分别为9.20 U·ml-1和21.60 U·ml-1。
3.以初筛到的5个菌株进行固态降解试验,结果表明,菌株A-51-1的木质素降解能力最强,在30 d和50 d后的木质素降解率分别高达32.53%和63.51%。此外,A-51-1还表现出较强的纤维素和半纤维素的降解能力,以及在固态发酵过程中表现出对木质素、纤维素和半纤维素降解的阶段性和选择性。在发酵前期30 d,菌株A-51-1表现出半纤维素和纤维素的降解速度较快,但在发酵后期木质素的降解速率增强,表现出对木质素降解的较强选择性。
4.酶能力和枝条木质素降解能力,为枝条木质素降解的高效菌株。
5.分别在不同的pH(5.0、6.0、7.0、8.0、9.0)和温度(20℃、25℃、30℃、35℃、40℃)条件下,研究了高效菌株A-51-1对葡萄枝条木质纤维素的降解作用,结果表明,在pH为7.0时,温度为30℃时A-51-1对枝条木质素、纤维素和半纤维素的降解效果最佳。
6.依据《真菌鉴定手册》(魏景超,1979),从菌落、菌丝和分生孢子的形态特征上,将菌株A-51-1鉴别为半知菌类(Fungi Imperfecti)丛梗孢目(Moniliales)丛梗孢科(Moniliaceae)卵形孢霉族(Oosporeae)地霉属(Geotrichum LK.)菌。
Vineyard pruning residues are organic by-products from vineyards management. In recent years, with gradually enlargement of the grape cultivation area in our country, a great quantity vineyard pruning residues are produced, especially vineyard winter pruning residues. Since the greater lignification of vineyard pruning and hardly to be degraded, the vineyard pruning residues were stacked in disorder and burned traditionally, which caused serious waste of resources and environment pollution. There are some lignin-degrading microorganisms which can achieve bio-degradation and microbial conversion efficiently, such as white-rot fungi. Therefore, screening of excellent lignin-degrading fungi, is helpful to improve degradation rate of vineyard pruning lignin and to promote the comprehensive utilization of the vineyard prunings, which has very important application research values.
In this study, lignin-degrading fungi were isolated and purified from the decayed prunings collected in a vineyard. The strains which showed higher lignin-degrading enzymes activities, including laccase and manganese peroxidase enzymes activities, were selected by both colorful reactions of guaiacol plates and decolorizations of Aniline bule plates. By fermentation test in liquid producing enzymes activities and solid pruning degradation test, the excellent strain were selected, with the highest lignin-degrading enzymes activities and the strongest ability of pruning lignin-degradation. And then, the effect of pruning degradation by the excellent strain in different cultivated pH and temperatures of solid fermentation respectively were studied, in order to get a preliminary optimization of solid fermentation conditions. Finally, the excellent strain was identified in morphology. The results show that:
1, Twenty-four lignin-degrading fungi strains which showed color zones on the guaiacol culture plates were obtained directly from decayed prunings by isolation and purification. Through both the colorful reactions test of PDA-guaiacol plates and the decolorizations test of PDA-Aniline bule plates respectively, among the twenty-four strains, five strains with higher Lac and MnP enzymes activities were screened, that were A-11、A-02、A-40-1、A-21 and A-51-1.
2, The liquid fermentation test were carried out by five strains, the results showed that five strains reached the highest peak of producing Lac and MnP enzymes respectively at 6 d and 8 d. Among them, A-51-1 strain showed the strongest ability to produce laccase and manganese peroxidase enzymes, of which the largest enzymes activities were 9.20 U·ml-1 and 21.60 U·ml-1 respectively.
3, The solid fermentation test were carried out by five strains, the results showed that A-51-1 strain had the strongest lignin-degrading ability, and its lignin-degradation rates after 30 d and 50 d were up to 32.53% and 63.51% respectively.In addition, strong cellulose and hemicellulose degrading abilities were showed to A-51-1, and in the process of solid fermentation, the process of lignin, cellulose and hemicellulose degradation by strain were staged and selective. In the prophase 30 d of solid fermentation, A-51-1 strain was showed with higher degradation rate of cellulose and hemicellulose than lignin, while the degradation rate of lignin increased in the late fermentation, which showed strong lignin-degradation selectivity.
4, According to the results of liquid fermentation test and solid fermentation test, it showed that A-51-1 strain was viewed as the excellent lignin-degrading strain, with the strongest abilities of both producing lignin-degrading enzymes activities and vineyard pruning lignin-degradation.
5, In different cultivated pH values including 5.0、6.0、7.0、8.0、9.0 and temperatures of solid fermentation from 20℃to 40℃respectively, the lignocellulose degradation of vineyard prunings by the excellent strain A-51-1 were studied, the results showed that in pH 7.0 or 30℃, the degradations of lignin, cellulose and hemicellulose by A-51-1 strain showed the highest efficiency.
6, Based on the identification of Fungus Handbook by Wei Jingchao, by morphological characteristics of colony, mycelium and conidia,A-51-1 strain was identified as Geotrichum LK., Oosporeae, Moniliaceae, Moniliales, Fungi Imperfecti.
引文
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