Growth and mycelial strand production of Rigidoporus lignosus with various nitrogen and carbon sources

Growth and differentiation of mycelial strands in Rigidoporus lignosus have been shown to depend on suitable combinations of the pH of the media and the nature of the nitrogen and carbon sources. Amino acids as sole nitrogen sources gave rise to vegetative mycelium. At pH 4.5, growth and mycelial strand differentiation required asparagine, as the fungus failed to grow in the absence of this amino acid. However, at pH 6, differentiation of strands occurred appreciably in asparagine-deficient media, suggesting a close balance between pH and amino acid requirements. Ammonium was required for strand differentiation, while nitrate, as a sole nitrogen source, maintained the fungus undifferentiated. Of the carbohydrates tested, only glucose, fructose and mannose supported strand differentiation. Starch was found to be particularly effective in promoting growth of vegetative mycelium. Strand differentiation required more specific conditions than growth of the vegetative mycelium.     

Growth morphology of Streptomyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients.

Most media in which the growth of shaken submerged cultures of Streptomyces akiyoshiensis was examined did not support the formation of well-dispersed mycelial suspensions. Investigation of the culture conditions promoting dispersed growth showed the pH of the culture medium to be of critical importance; an initial value of 5.5 minimized aggregation of the mycelium while supporting adequate biomass production. In cultures started at this pH, spore inocula gave better mycelial dispersal than did vegetative inocula; with spore inocula, growth morphology was also less affected by inoculum size. The composition of the nutrient solution influenced the extent of mycelial dispersal; slow growth was often associated with clumping but no clear correlation was observed between pellet formation and the ability of carbon or nitrogen sources to support rapid growth. Increasing the phosphate concentration from 0.5 to 15 mM caused a modest decrease in mycelial aggregation. Conditions promoting a well-dispersed mycelium suitable for studying the physiological control of secondary metabolism also supported the formation of 5-hydroxy-4-oxonorvaline by S. akiyoshiensis.     

簇生沿丝伞的生物学特性

文中以对采自山东省的子实体进行组织分离获取的菌株作为试验材料,对其生物学特性进行研究,对温度、pH、碳源、氮源4个因素进行单因素试验。温度组选取15,20,25,30,35℃5个梯度,pH组选取pH5,6,7,8这4个梯度,碳源组选取葡萄糖、蔗糖、麦芽糖、可溶性淀粉、乳糖、果糖6种,氮源组选取酵母膏、蛋白胨、硫酸铵、牛肉膏、黄豆粉、亚硝酸钠6种,进行单因素试验。根据菌丝在培养皿中的日均生长速度和生长势综合比较,分别选取4个因素中最优的3个,温度组选取20,25,30℃;pH组选取pH 5,6,7;碳源组选取葡萄糖、蔗糖和麦芽糖;氮源组选取蛋白胨、牛肉膏、硫酸铵。然后通过4因素3水平的正交试验,结果表明:4种因子对簇生沿丝伞的影响程度为温度>氮源>碳源>pH,各因素之间呈极显著差异,最终得出该菌菌丝生长的最佳培养条件为温度20℃,pH 5,碳源为蔗糖,氮源为牛肉膏。     

Exopolysaccharide production and mycelial growth in an air-lift bioreactor using Fomitopsis pinicola

For effective exopolysaccharide production and mycelial growth by a liquid culture of Fomitopsis pinicola in an air-lift bioreactor, the culture temperature, pH, carbon source, nitrogen source, and mineral source were initially investigated in a flask. The optimal temperature and pH for mycelial growth and exopolysaccharide production were 25degrees C and 6.0, respectively. Among the various carbon sources tested, glucose was found to be the most suitable carbon source. In particular, the maximum mycelial growth and exopolysaccharide production were achieved in 4% glucose. The best nitrogen sources were yeast extract and malt extract. The optimal concentrations of yeast extract and malt extract were 0.5 and 0.1%, respectively. K2HPO4 and MgSO4 x 7H2O were found to be the best mineral sources for mycelial growth and exopolysaccharide production. In order to investigate the effect of aeration on mycelial growth and exopolysaccharide production in an air-lift bioreactor, various aerations were tested for 8 days. The maximum mycelial growth and exopolysaccharide production were 7.9 g/l and 2.6 g/l, respectively, at 1.5 vvm of aeration. In addition, a batch culture in an air-lift bioreactor was carried out for 11 days under the optimal conditions. The maximum mycelial growth was 10.4 g/l, which was approximately 1.7-fold higher than that of basal medium. The exopolysaccharide production was increased with increased culture time. The maximum concentration of exopolysaccharide was 4.4 g/l, which was about 3.3-fold higher than that of basal medium. These results indicate that exopolysaccharide production increased in parallel with the growth of mycelium, and also show that product formation is associated with mycelial growth. The developed model in an air-lift bioreactor showed good agreement with experimental data and simulated results on mycelial growth and exopolysaccharide production in the culture of F pinicola.     

源自不同寄主的灰葡萄孢生物学特性的比较研究

本研究以分离自番茄、辣椒、草莓、葡萄的灰葡萄孢Botrytis cinerea为供试菌株,从生长温度、pH适应性、碳源、氮源营养利用等方面对不同寄主来源的灰葡萄孢菌株的生物学性状进行了比较研究。结果表明,5个不同寄主来源的灰葡萄孢菌株的菌丝生长温度范围相同,均为0-35℃;但它们的最适生长温度和分生孢子致死温度存在差异,来自和县番茄菌株HX12最适生长温度为20℃,分生孢子致死温度为47℃ 10min,其余最适生长温度均为25℃,分生孢子致死温度均为48℃ 10min;不同菌株在相同温度下的生长速率有显著差异。pH对不同寄主来源灰葡萄孢菌株菌丝生长的影响存在差异,来自长丰辣椒的菌株LJ菌丝在pH2-9的范围内均能生长,以在pH3-6.5时生长较快,pH6时最快;其余4个菌株在pH2-12的范围内均能生长,以在pH3-9时生长较快,pH6左右最快。不同碳源、氮源营养对灰葡萄孢菌株菌丝生长和分生孢子产生均有显著影响,不同寄主来源的菌株间在碳源、氮源营养利用差异均极显著。在相同碳源、氮源营养条件下,不同寄主来源的菌株的线性生长、菌丝干重和分生孢子产量均有显著差异。     

三种香蘑属野生食用菌菌株的培养条件优化

【背景】供试菌株分别是分离自山西省宁武县管涔山的肉色香蘑Lepista irina、斑褶香蘑L. panaeolus和山西省蒲县五鹿山的紫丁香蘑L. nuda 3种野生食用菌的子实体。【目的】获得3种野生食用菌的最佳培养条件。【方法】以菌丝生长速度为指标研究不同碳源、氮源、碳氮比、pH和培养温度等各因素对菌丝生长的影响,根据Box-Benhnken中心组合试验设计原理,采用3因素3水平的响应面法确定使菌丝体达到最快生长速度的最佳培养碳源、氮源和pH。【结果】肉色香蘑在葡萄糖20.9 g/L、土豆196.47 g/L、pH 6.0、培养温度21 °C的条件下,菌丝日均生长速度达到最大,为1.13 mm/d;斑褶香蘑在甘露醇17.4 g/L、酵母膏8.1 g/L、B族维生素0.1 g/L、K2HPO4 2.5 g/L、MgSO4 2.5 g/L、pH 7.9、培养温度25 °C的条件下,菌丝日均生长速度达到最大,为0.73 mm/d;紫丁香蘑在土豆200 g/L、可溶性淀粉20.5 g/L、KNO3 2.1 g/L、K2HPO4 2.5 g/L、MgSO4 2.5 g/L、B族维生素0.1 g/L、pH 7.0、培养温度25 °C的条件下,菌丝日均生长速度达到最大,为2.38 mm/d。【结论】获得了3种香蘑属菌株的最佳培养条件,为后续优质野生食用菌的引种驯化积累了相关数据和资源。     

Semi-defined Medium for in vitro Cultivation of the Fastidious Insect Pathogenic Fungus Cordyceps unilateralis

Cordyceps unilateralis is a fastidious fungal pathogen affecting ants. Up to now, only the complex and expensive Grace’s insect cell culture medium has been used for in vitro cultivation (as blastospores and mycelium) of this fungus. To obtain an inexpensive and less complicated medium, the effects of carbon and nitrogen sources, salt solution and carbon-to-nitrogen (C:N) ratio on the growth of this fungus were examined. Glucose was the most important factor for blastospore formation, and yeast extract could be used as a nitrogen source for blastospore formation and mycelial growth. A suitable C:N ratio (glucose: yeast extract) was 33.3:1. As a result, a new semi-defined medium was achieved, composed of 26.68 g L⁻¹ glucose, 3.3 g L⁻¹ yeast extract and salt solution. This medium supported blastospore formation and mycelial growth of all tested C. unilateralis isolates.     

Effect of culture conditions on antifouling compound production of a sponge-associated fungus

Microorganisms associated with invertebrate hosts have long been suggested to be a source for bioactive metabolites. In this study, we reported that a sponge-associated fungus, Letendraea helminthicola, produced two antifouling compounds: 3-methyl-N-(2-phenylethyl) butanamide and cyclo(D-Pro-D-Phe). To optimize the production of these antifouling compounds, we then examined the production of compounds under different culture conditions (temperature, salinity, pH, and carbon and nitrogen sources). This fungus grew well and produced more compounds at temperatures between 18 and 30°C; the fungus grew well at 75 parts per thousand (ppt) salinity but produced the highest amount of antifouling compounds at 30 and 45 ppt. The optimal initial pH value for mycelial growth was 5.5 to 6.5, whereas the production of the antifouling compounds was maximized at pH 3.5 and 4.5. Glucose and xylose (as carbon sources) increased the production of antifouling compounds. Yeast extract and peptone (as nitrogen sources) maximized the production of mycelial biomass and antifouling compounds. Our results indicate that culture conditions greatly affect the production of bioactive compounds from mycelial fungal cultures as exemplified by strain L. helminthicola and that the conditions favorable for fungal growth may not be the best conditions for bioactive compound production.     

薄皮干酪菌生物学特性及驯化栽培

大型真菌种质资源是国家种质资源库建设重要组成部分,为了充分开发利用大型真菌资源,对采自吉林省寒葱岭的一株野生菌进行分离纯化,并将获取的纯菌株作为实验材料。通过形态学和ITS序列分析,将其鉴定为薄皮干酪菌Tyromyces chioneus。本实验研究了固体培养条件下不同碳源、氮源、pH和温度对其菌丝生长的影响,并从4个单因素实验中选出3个最优水平进行正交实验。同时,在对其菌丝体最适培养条件研究结果的基础上,对其进行驯化栽培研究。结果表明,薄皮干酪菌的最适培养条件为：果糖(20 g/L)、酵母浸粉(2 g/L)、pH 4.0、温度30 ℃。正交实验结果表明,氮源对其菌丝体生长的影响最大,其次是碳源、pH和温度。驯化栽培结果表明,菌丝发菌时间为21 d;空气湿度为85%-95%,适量的散射光,降低温度至18-20 ℃刺激后原基逐步变成菇蕾,将温度、湿度分别提高至24-25 ℃、90%-98%,30 d后子实体成熟。通过本实验的研究为薄皮干酪菌的后续开发利用提供了基础实验数据。     

Use of response surface methodology to examine chitinase regulation in the basidiomycete Moniliophthora perniciosa

We report here the first analysis of chitinase regulation in Moniliophthora perniciosa, the causal agent of the witches' broom disease of cacao. A multivariate statistical approach was employed to evaluate the effect of several variables, including carbon and nitrogen sources and cultivation time, on M. perniciosa non-secreted (detected in mycelium, i.e. in symplasm and cell wall) and secreted (detected in the culture medium) chitinase activities. Non-secreted chitinase activity was enhanced by peptone and chitin and repressed by glucose. Chitinase secretion was increased by yeast extract alone or in combination with other nitrogen sources, and by N-acetylglucosamine, and repressed in presence of chitin. The best cultivation times for non-secreted and secreted chitinase activities were 30 and 20 d, respectively. However, chitinase activity was always higher in the mycelium than in the culture medium, suggesting a relatively poor chitinase secretion activity. Conversely, higher mycelial growth was observed when the activity of the non-secreted chitinase was at its lowest, i.e. when the fungus was grown on glucose and yeast extract as sources of carbon and nitrogen, respectively. Conversely, the induction of non-secreted chitinase activity by chitin decreased the mycelium growth. These results suggest that the culture medium, by the induction or repression of chitinases, affected the hyphal growth. Thus, as an essential component of M. perniciosa growth, chitinases may be a potential target for strategies to control disease.     

Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.     

绒毛栓孔菌液体培养过程中胞外酶活性的研究

本研究以绒毛栓孔菌为材料,采用液体培养的方法分析其在发酵过程中胞外酶的活性变化,并对其菌丝体生物量和发酵液pH值进行了测定。结果表明:胞外酶活性与菌丝体生长状况密切相关。菌丝体生物量增长呈"S"型,6～8d增长最快,第12天达到最大值,在此过程中漆酶、锰过氧化物酶、淀粉酶、羧甲基纤维素酶、果胶酶和蛋白酶活性均出现高峰。酶活性的变化表明,在液体培养过程中绒毛栓孔菌首先分解木质素,其次利用淀粉和纤维素作为碳源,蛋白质作为氮源。若要获得最大菌丝体生物量,缩短培养时间,就必须在培养过程中保证碳氮源的均衡供给。本试验说明不同的酶其分泌高峰期可以作为判断菌丝体营养利用情况和培养周期的依据,以此获取最大菌丝体生物量,为工业生产利用奠定基础。     

Comparative mycelial and spore yield by Trichoderma viride in batch and fed-batch cultures

The effects of cultural parameters such as carbon and nitrogen source and environmental factors including temperature and pH were investigated on spore and mycelial yield of Trichoderma viride, which has potential as a biocontrol agent against species of Fusarium in batch culture and fed-batch culture where there was limiting nutrient. The results obtained indicated that growth and sporulation of T. viride were greatly influenced by various carbon and nitrogen sources, and by environmental factors such as pH and temperature. Mannitol, wheat bran and rice bran as sole carbon sources appear to stimulate high mycelial growth and spore yield in fed-batch culture. Growth and sporulation were also favoured by NaNO₃, peptone and NH₄SO₄ as the nitrogen sources in fed-batch and batch cultures_. Maximum growth and sporulation was between pH 4.5 and 6.0. Temperatures between 30 and 37 °C were good for mycelium growth of T. viride while temperatures between 30 to 45 °C were good for sporulation. The amount of spore and mycelium produced and the time required for attainment of maximum spore yield increased with increasing carbon and nitrogen source in batch culture. The final spore yield obtained in fed-batch culture was two times higher than the apparent spore-carrying capacity of batch culture. These results show that T. viride is capable of growing and sporulating with varied nutritional and environmental conditions, and, therefore, this strain of T. viride may be useful as a biocontrol agent under diverse physiological and environmental conditions.     

菌核侧耳液体培养条件优化研究

运用液体深层培养法,采用不同pH值、不同碳源和氮源对菌核侧耳菌丝体生长的影响进行了探讨。结果表明:菌核侧耳菌丝体生长的培养基适宜pH值为5.0～6.5,葡萄糖、蔗糖、乳糖和纤维素是菌核侧耳菌丝体生长的适用碳源;黄豆、麦麸、酵母膏和蛋白胨等为适宜氮源;其中以黄豆浆作为氮源,蔗糖作为碳源,初始pH5.0为最佳条件组合。     

Growth of and trehalase activity in the thermophilic fungusThermomyces lanuginosus

The thermophilic fungus,Thermomyces lanuginosus, was grown in a glucose-asparagine liquid medium. Optimal mycelial growth occurred at 50°C. The conditions for sporulation were different from those required for vegetative growth. the former being favoured by lower nitrogen level and temperature. Trehalase (α, α-glu coside-l-glucohydrolase, EC 3.2.1.28) was one of the most active glycosidases at 50°C. Non-sporulating mycelium had higher levels of this enzyme than the sporulating mycelium. Trehalase was synthesized constitutively and its activity appears to be controlled by catabolite repression.     

Nitrogen amendments reduce the growth of extramatrical ectomycorrhizal mycelium

The effect of three different nitrogen sources on the growth of external ectomycorrhizal mycelium was studied in Perspex micorocosms. Nonsterile peat was used as substrate. Five different fungal isolates growing in symbiosis with pine seedlings were investigated: two isolates of Paxillus involutus, one of Suillus bovinus and two unidentified ectomycorrhizal fungi isolated from ectomycorrhizal root tips. Three different nitrogen sources were used: ammonium as (NH₄)₂SO₄, nitrate as NaNO₃ and a complete nutrient solution (Ingestad 1979), and three different nitrogen concentrations, 1, 2 or 4 mg N/g dry wt. of peat. The mycelial growth of all fungi was found to be negatively affected by the nitrogen amendments, although the sensitivity to nitrogen varied between the isolates. One of the unidentified isolates was extremely sensitive and growth was completely inhibited by all nitrogen treatments. In contrast, the growth of one of the P. involutus isolates was only slightly reduced by the nitrogen amendments. The different nitrogen sources all reduced growth, and since no significant difference was found between the nitrogen sources or between the different nitrogen concentrations the results were pooled to give one value that summarized the effect of nitrogen on mycelial growth. Thus, the mycelial growth of one of the two P. involutus isolates was reduced to approximately 80% of the growth in the control, the other P. involutus and one of the unidentified fungi, vgk 2 89.10, were reduced to 40–50% of the control growth, S. bovinus to 30% of the control and the most sensitive fungus, the unidentified isolate vg 1 87.10, was reduced to 3% of the growth in the control treatment. In all experiments, the shoot to root ratio generally increased, mainly as a result of increased shoot growth.     

Factors Influencing the Production of Stromata and Microconidia by Sclerotinia fructicola (Wint.) Rehm

Physiological studies were made to determine the effect of differenttemperatures of incubation, hydrogen-ion concentration of theculture medium, and the type and concentration of carbon andnitrogen sources in the substrate on the development of microconidiaand stromata by three isolates of Sclerotinia fructicola (Wint.)Rehm. The optimum initial pH for stroma formation was 4.0 for oneof the isolates and 4.8 for the other two, and these were thepHs at which the most abundant mycelial growth was recorded.Stromata developed most abundantly and in the shortest timeat 25? C, which was the optimum temperature for the growth ofthe mycelium. Glucose, fructose, and sucrose were suitable sourcesof carbon for the growth of S. fructicola and an increase inthe concentration of glucose resulted in an increased rate ofmycelial growth, and of macroconidial and stromatal formation,provided an optimal amount of a suitable nitrogen source wasused. Peptone was the only source of nitrogen of those testedin these experiments that produced normal growth and an increasein concentration of peptone up to 5 per cent resulted in increasednumbers of macroconidia, density of mycelium, and areas of stromaformed. Temperature and initial hydrogen-ion concentration with in theranges permitting vegetative growth did not directly affectmicroconidial formation, but microconidia were initiated whenthe substrate was low in nutrients or when staling of the culturesbecame apparent.     

Effect of Extracellular Factors on Growth and Dimorphism of Rhizopus oryzae with Multiple Enzyme Synthesizing Ability

Rhizopus oryzae PR7 MTCC 9642 was a dimorphic fungus that showed a regular 90 days cycle of filament (mycelium) to pellet (yeast) transformation through a distinct bottom dwelling intermediate state and the pellets never revert back to filamentous form. Apart from the normal cycle, high temperature (37°C and above) and extreme pH also induced the yeast formation. Among the ions tested, calcium and chloride ions were found to restore the filamentous morphology, even in extreme pH and temperature. Cysteine HCl also played noteworthy role in maintaining mycelial growth even at adverse condition. Immobilized spores showed the appearance of intermediate form instead of typical yeast form even at high temperature. The strain could produce a number of extracellular hydrolytic enzymes like cellulolytic, xylanolytic, pectinolytic and amylolytic enzymes. The pellet and mycelial forms were found to be a better producer of cellulase–lignocellulase enzymes and amylolytic enzymes respectively, which might be correlated with their infectivity. Increase in inoculum size, agitation during cultivation, change in carbon and nitrogen source failed to induce mycelial growth in extreme conditions, which might be explained as irreversible change of configuration of protein responsible for mycelial development.     

毛榆孔菌的固体培养、驯化栽培及其纤维素酶活性研究

毛榆孔菌Elmerina hispida在生长过程中分泌出一种很重要的酶——纤维素酶,纤维素酶在工业废水处理、有机颜料脱色等方面具有重要的利用价值。为充分开发利用毛榆孔菌这一野生资源,本研究采用十字画线法测定不同碳源、氮源、pH和温度对毛榆孔菌菌丝生长的影响,从4个单因素试验中选取3个最优水平进行正交试验。研究结果表明,毛榆孔菌菌丝的最适生长碳源是可溶性淀粉,最适生长氮源是酵母膏,最适温度是25℃,最适pH 7.0。驯化栽培的栽培基质为78%木屑,20%麦麸,1%石灰,1%石膏（质量比）,30d左右出现原基,40d以后原基分化,形成子实体。通过测定毛榆孔菌在栽培过程中4个不同时期的纤维素酶,发现酶活力最高可达805.63U/g。本试验成功对毛榆孔菌进行了人工驯化栽培,为该菌进一步的研究和开发提供参考。     

絮缘蘑菇生物学特性及驯化栽培

对采自祁连山地区的一株野生食用菌进行分离,通过形态学及其ITS序列的分子系统发育分析鉴定为絮缘蘑菇Agaricus subfloccosus。以分离纯化获得的菌株作为实验材料,对该种的生物学特性和驯化栽培进行研究。检测了不同碳源、氮源、温度、pH、无机盐、维生素在固体培养条件下对絮缘蘑菇菌丝生长的影响,对以上6个因素进行单因素试验,从中挑选出3个较优因素的最优水平进行正交试验。结果表明：实验范围内,絮缘蘑菇菌丝生长最佳碳源为蔗糖,最佳氮源为硝酸钾,最佳无机盐为硫酸镁,最佳维生素为VB₁₂,最佳温度为16℃,最佳pH为6。影响较大的3个因素为碳源、无机盐和pH,絮缘蘑菇最适的营养因子最佳组合配方为：蔗糖25g/L,硝酸钾2g/L,硫酸镁2g/L,pH为5.0。驯化栽培过程中,栽培配方为：木屑79%、高粱籽5%、玉米面5%、石膏1%、麸皮10%。温度16℃使絮缘蘑菇菌丝在90d左右满袋,覆土可培育出子实体。