垃圾堆肥高效纤维素分解菌的筛选与培育技术

从堆肥、马粪、果园土、污泥等20个样品中，分离筛选出3株对滤纸分解旺盛的纤维素分解菌：C1、C2、C3，并外购康氏木霉、白腐菌、变色栓菌一起作为出发株，经紫外诱变处理后，在含葡萄糖的产酶培养基平板上筛选到能形成较大透明圈的突变株，并进行CMC酶活、微晶纤维素酶活及天然粗纤维分解能力测定。实验结果证明白腐菌经紫外线照射60s诱变而得的C16不仅透明圈大，CMC酶活高（60.08U/mL）是出发株的2倍，而且其对天然粗纤维分解能力强，10d分解率达35％。     

Impact of a native Streptomyces flavovirens from mushroom compost on green mold control and yield of Agaricus bisporus

ABSTRACT

Thirty-five actinobacterial isolates, obtained from button mushroom (Agaricus bisporus) substrates (i.e., compost in different phases of composting, black peat or casing layer) in Serbia in 2014–2016 were tested in vitro against the causal agents of green mold in cultivated mushroom. Out of six most promising isolates, A06 induced 42.4% in vitro growth inhibition of Trichoderma harzianum T54, and 27.6% inhibition of T. aggressivum f. europaeum T77. The novel strain A06 was identified as Streptomyces flavovirens based on macroscopic and cultural characteristics and 16S rDNA sequence and used in mushroom growing room experiments. Actinobacteria had no negative influence on mycelial growth of the cultivated mushroom in compost in situ. Isolate S. flavovirens A06 enhanced mushroom yield significantly, up to 31.5%. The A06 isolate was more efficient in enhancing yield after inoculation with the compost mold T. aggressivum (26.1%), compared to casing mold T. harzianum (8%). Considering disease incidence, actinobacteria significantly prevented green mold in compost caused by T. aggressivum (6.8%). However, fungicide prochloraz-Mn had a more significant role in reducing symptoms of casing mold, T. harzianum, in comparison with actinobacteria (24.2 and 11.8%, respectively). No significant differences between efficacies of S. flavovirens A06 and the fungicide prochloraz-Mn against T. aggressivum were revealed. These results imply that S. flavovirens A06 can be used to increase mushroom yield and contribute to disease control against the aggressive compost green mold disease caused by Trichoderma aggressivum.     

Exploitation of Trichoderma harzianum mycelial waste for the removal of rhodamine 6G from aqueous solution

The harvested mycelial waste of Trichoderma harzianum was used as an adsorbent for the removal of rhodamine 6G and was studied in batch mode. The effects of agitation time and initial dye concentration, adsorbent dosage and pH were examined. The study revealed that the amount of dye adsorbed (mgg(-1)) increased with increase in agitation time and reached equilibrium after 120 min, for dye concentrations of 10-50 mg L(-1). The adsorbent dosage of 1.0 g/50 mL and pH of 8.0 were found to be optimum for maximum dye removal. The batch mode adsorption data followed both the Langmuir and Freundlich isotherms. The pseudo first- and second-order rate kinetics were applied to the adsorbent system. The adsorption kinetics of rhodamine 6G showed that the pseudo-second-order kinetic model provided the best correlation of the equilibrium data. The study implies that it is possible to develop a dye removal system by using T. harzianum biomass, which occurs as sludge in waste stream of fermentation industries.     

Chemical synthesis of 6-pentyl-2H-pyran-2-one: a natural antifungal biosynthesized by Trichoderma spp.

Trichoderma spp. biosynthesize 6-pentyl-2H-pyran-2-one (6-PP), a natural antifungal pyrone which could be used as biological control agent (BCA). Unfortunately, biotechnical processes are limited by inhibition of biomass at high concentration of 6-PP. We report herein a new easy synthesis of this natural pyrone, using readily available starting materials. This synthesis, compatible with a large production scale, permit to obtain overweight amounts of 6-PP that in biotechnological routes.     

多环芳烃污染土壤的微生物-紫花苜蓿联合修复效应

微生物-植物联合修复技术作为一种低耗高效的新型修复手段已经被广泛应用于有机污染土壤的修复领域并取得了较好的效果,新型生物资源的应用将推动该方法的进一步发展。本研究采用温室盆栽实验,以里氏木霉（Trichodermaressei FS10-C）、根瘤菌（Rhizobium meliloti）和紫花苜蓿（Medicago sativa L.）作为供试生物,设置添加灭活菌剂-无紫花苜蓿（CK）、添加灭活菌剂-种植紫花苜蓿（A）、接种木霉菌剂-种植紫花苜蓿（TA）、接种木霉菌根瘤菌复合菌剂-种植紫花苜蓿（TRA）4种处理,探究微生物-植物联合修复对多环芳烃（PAHs）污染土壤的生物修复效果及其微生态效应。结果表明,经过60 d的培养,微生物不仅促进了紫花苜蓿的生长,而且在紫花苜蓿的协同作用下进一步提高了土壤中PAHs降解率。TA处理中紫花苜蓿生物量增加了5.88%,而TRA处理进一步促进了紫花苜蓿的生长,其生物量增加了11.15%;A、TA和TRA处理下土壤中PAHs的降解率分别为17.02%、25.62%、32.93%,显著（p〈0.05）高于处理CK（5.67%）。此外,接种菌剂处理（TA、TRA）对土壤中高分子量PAHs具有更好的降解效果,A处理土壤中4-、5（＋6）环PAHs的降解率分别为18.13%、24.74%,TA处理为21.41%、28.34%,而TRA处理则为21.29%、30.11%。同时,紫花苜蓿能够通过其根际效应显著促进土壤微生物活性,相较于CK处理,A、TA、TRA处理土壤脱氢酶活性分别提高了33.20%、34.58%、32.65%,A、TA、TRA处理AWCD值和微生物群落多样性指数均显著（p〈0.05）高于CK。通过木霉、根瘤菌与紫花苜蓿联合作用不仅可以有效地降解土壤中的PAHs,而且能够恢复土壤微生物生态功能多样性和稳定性。因此,该方法是一种极具潜力的生物修复手段,具有广阔的市场应用前景。     

一株苯并[a]芘高效降解真菌的筛选与降解特性

从长期受多环芳烃(PAHs)污染的土壤中分离出一株能够降解苯并[a]芘(B[a]P)的真菌,经鉴定为绿色木霉(Trichoderma viride)(命名为BF-1),并对其以B[a]P为唯一碳源进行反复驯化,考察了B[a]P浓度、不同重金属和培养基对其降解能力的影响.结果表明,菌株BF-1在B[a]P浓度为5mg.L-1,32℃振荡培养约6d的条件下,降解速度最快,B[a]P的降解率达68.28%.BF-1在B[a]P浓度分别为10与25mg.L-1,32℃振荡培养6d的条件下,B[a]P的降解率分别为73.29%与87.36%.Cu2+(50mg.L-1)基本不影响BF-1对B[a]P的降解率;Cd2+(100mg.L-1)、Pb2+(300mg.L-1)对BF-1降解B[a]P有一定影响,但仍表现出较高的耐受能力;而Zn2+(200mg.L-1)对BF-1有明显的抑制作用.选用含5mg.L-1B[a]P的土豆葡萄糖液体培养基,6d后B[a]P的降解率为71.31%.对比前述实验结果表明,培养基对B[a]P降解率的影响并不明显.因此,BF-1的应用价值较高.     

纤维素酶产生菌的选育及发酵条件优化

通过紫外线和化学诱变方法对一株纤维素酶产生菌--绿色木霉他进行了菌株选育,提高了产酶能力,变异株T211的CMCase和FPA分别达到19.17 IU/mL和1.94 IU/mL.进一步对其发酵条件进行研究,优化了培养基和培养条件.优化的培养基为:微晶纤维素10 g/L,麸皮10 g/L,蛋白胨0.4 g/L,尿素0.4 g/L,硫酸铵2.0 g/L,吐温-80 2.0 mL/L,其他同Mandel's营养盐液;培养条件包括温度、摇瓶装量、转速和接种量分别为:27～28℃、50 mL/250 mL、180 r/min和4%～6%.在上述条件下,CMCase达到25 IU/mL,FPA达到2.5 IU/mL.     

Construction and functional analysis of Trichoderma harzianum mutants that modulate maize resistance to the pathogen Curvularia lunata

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to generate an insertional mutant library of the mycelial fungus Trichoderma harzianum. From a total of 450 mutants, six mutants that showed significant influence on maize resistance to C. lunata were analyzed in detail. Maize coated with these mutants was more susceptible to C. lunata compared with those coated with a wild-type (WT) strain. Similar to other fungal ATMT libraries, all six mutants were single copy integrations, which occurred preferentially in noncoding regions (except two mutants) and were frequently accompanied by the loss of border sequences. Two mutants (T66 and T312) that were linked to resistance were characterized further. Maize seeds coated with T66 and T312 were more susceptible to C. lunata than those treated with WT. Moreover, the mutants affected the resistance of maize to C. lunata by enhancing jasmonate-responsive gene expression. T66 and T312 induced maize resistance to C. lunata infection through a jasmonic acid-dependent pathway.     

Metabolism of nC11 fatty acid fed to Trichoderma koningii and Penicillium janthinellum II: Production of intracellular and extracellular lipids

Abstract

Little is known about the fungal metabolism of nC₁₀ and nC₁₁ fatty acids and their conversion into lipids. A mixed batch culture of soil fungi, T. koningii and P. janthinellum, was grown on undecanoic acid (UDA), a mixture of UDA and potato dextrose broth (UDA+PDB), and PDB alone to examine their metabolic conversion during growth. We quantified seven intracellular and extracellular lipid classes using Iatroscan thin-layer chromatography with flame ionization detection (TLC-FID). Gas chromatography with flame ionization detection (GC-FID) was used to quantify 42 individual fatty acids. Per 150 mL culture, the mixed fungal culture grown on UDA+PDB produced the highest amount of intracellular (531 mg) and extracellular (14.7 mg) lipids during the exponential phase. The content of total intracellular lipids represented 25% of the total biomass-carbon, or 10% of the total biomass dry weight produced. Fatty acids made up the largest class of intracellular lipids (457 mg/150 mL culture) and they were synthesized at a rate of 2.4 mg/h during the exponential phase, and decomposed at a rate of 1.8 mg/h during the stationary phase, when UDA+PDB was the carbon source. Palmitic acid (C_16:0), stearic acid (C_18:0), oleic acid (C_18:1), linoleic acid (C_18:2) and vaccenic acid (C_18:1) accounted for >80% of the total intracellular fatty acids. During exponential growth on UDA+PDB, hydrocarbons were the largest pool of all extracellular lipids (6.5 mg), and intracellularly they were synthesized at a rate of 64 μg/h. The mixed fungal species culture of T. koningii and P. janthinellum produced many lipids for potential use as industrial feedstocks or bioproducts in biorefineries.     

Isolation of a salt tolerant laccase secreting strain of Trichoderma sp. NFCCI-2745 and optimization of culture conditions and assessing its effectiveness in treating saline phenolic effluents

Most of the hazardous pollutants are phenolic in nature and persists in the environment. The ability of laccases to oxidize phenolic compounds and reduce molecular oxygen to water has led to intensive studies of these enzymes. Therefore the fungal strains with high laccase activity and substrate affinity that can tolerate harsh environmental conditions have a potential for biotechnological applications. Salt tolerant laccase secreting fungi can be utilized in treatment of saline and phenolic rich industrial effluents such as coir effluent and textile effluent that needed to be diluted several fold before microbial treatment. This is the first study describing the isolation and optimization of a salt tolerant strain of Trichoderma sp. potential for industrial applications. The fungus was identified based on morphological characteristics and was subsequently confirmed with molecular techniques and deposited at National Fungal Culture Collections of India （NFCCI） under the Accession No. Trichoderma viride NFCCI 2745. In contrast to other laccase secreting fungi, light conditions did not exert much influence on laccase production of this strain and salinity enhanced its laccase secretion. The fungus effectively removed the phenolic content of the textile effluent, coir-ret liquor and wood processing effluent within 96 hr of incubation. The tolerance of the fungus to high salinity and phenolic compounds makes this strain ideal for treating saline and phenolic rich industrial effluents.