松茸菌丝原生质体形成与再生条件的研究

采用多因素实验正交优选法比较不同酶、渗透压稳定剂、菌丝生理状况、菌丝培养基、预处理液和酶解时间等因素对松茸原生质体形成的影响和再生培养基、明胶、酪蛋白对松茸原生质体再生的影响.以0.6MMgSO4作为渗透压稳定剂,2-硫基苏糖醇为预处理液,用2%蜗牛酶在30℃酶解6h,每克菌丝可以得到1×106个原生质体.再生最优条件0.6MMgSO4,PDY再生培养基,2.5%明胶,松茸菌丝再生率最高4.97%.     

松茸利用淀粉的初步研究

目的：研究松茸在以淀粉为碳源的人工培养基上生长的可能性,初步了解共生菌根真菌松茸离体培养下的营养生长习性。方法：采用改良真菌合成培养基和单因子试验方法,比较了葡萄糖、麦芽糖、蔗糖、可溶性淀粉和普通玉米淀粉等不同碳源对松茸纯培养菌丝体生长的影响,描述了菌丝体萌动生长速度与菌落扩展的态势,定期收获并称量菌丝体生物量（干重）,然后进行统计分析。结果：当查氏（Czapek）培养基以KNO3为供试氮源,供试碳源是普通玉米淀粉时,移植的松茸菌块萌发菌丝体相对延迟,长势较弱,松茸菌落扩展速度为0.13～0.2mm·d-1之间,生物量积累较少;以NH4Cl为优化氮源,松茸菌落扩展速度提高至0.37mm·d-1,1个月生物量积累27.3mg;进一步添加1‰（W/V）马铃薯汁,菌落扩展速度达到0.63mm·d-1,生物量积累达73.4mg。结论：共生真菌松茸可以利用淀粉作为单一碳源进行营养生长,但菌丝体扩展速度和生长态势较其它供试碳源条件下弱。而普通淀粉经改性加工成可溶性淀粉后,松茸长势有所改善。优化氮源,补充生长因子,则显著促进了松茸利用普通玉米淀粉的生长水平。     

矿质营养与其他生长物质对荷叶离褶伞菌丝生长的影响

1mg／mL KCl促进荷叶离褶伞菌丝生长;5或10mg／mL NaCl、5或10mg／mL MgSO4、5或10mg／mLKCl、10mg／mL H2PO4和1mg／mL CaSO4抑制菌丝生长;0．8mg／mL的MnSO4和CuSO4以及0．5mg／mL FeSO4、0．2或0．5mg／mL CoCl2和0．2、0．5或0．8mg／mL ZnSO4促进菌丝生长;0．5或0．2mg／mL CuSO4、0．2或0．5mg／mL MnSO4及0．8或0．2mg／mL FeSO4对菌丝生长的影响不显著;维生素B6、维生素C、维生素PP和维生素B1可促进菌丝生长,在含有10μg/L维生素B6的培养基上菌丝生长速度最快,但维生素C试用浓度较低（50μg／L）时对菌丝生长的影响不显著;吲哚丁酸、吲哚乙酸、奈乙酸对菌丝生长具有促进作用,但0．1、0．5或1．0μg／L赤霉素对菌丝生长的影响不显著。     

K88大肠杆菌菌毛蛋白发酵工艺条件优化研究

分别采用LB培养基、牛肉膏蛋白胨培养基、强化营养培养基、玉米浆培养基对大肠杆菌K88进行发酵培养,选出最适于大肠杆菌K88生长的玉米浆培养基;采用正交实验对玉米浆培养基的C／N、K2HPO4／KH2PO4、Mg^2＋的配比进行优化,筛选最适于大肠杆菌K88生长的营养配比;研究生长曲线、接种量以及菌体和菌毛生产量的相关性,根据实验结果优化发酵培养条件,确定菌种的最佳发酵工艺,以收获最多的K88菌毛蛋白。研究表明,K88大肠杆菌在玉米浆培养基C／N、K2HP04／KH2PO4的配比分别为5／11、1／1,Mg^2＋为0．1g/mL,pH值为7．2,转速为200r／min,接种量为4．5％的条件下发酵26个小时,菌体和菌毛生产量均达到高峰,同时得出菌毛蛋白产生量和菌体量成正相关。     

不同培养基对冬虫夏草菌丝生长的影响

不同培养基对3个来源不同的冬虫夏草菌菌丝生长影响的研究结果表明,在不同培养基上,各菌株之间在菌丝萌发时间、40d菌落直径和菌落形态上表现出一定的差异。同时筛选到的最优培养基比已报道培养基的菌落生长速度快2～3倍,且菌丝为白色、粗壮,菌丝质量较优。     

蚊幼虫病原真菌贵阳腐霉的生物学研究

为了开发灭蚊真菌贵阳腐霉Pythiumguiyangense用于蚊虫防治,对贵阳腐霉生物学特性进行了研究。采用菌丝生长率、真菌产孢情况以及对致倦库蚊Culexquinquefaciatus1龄幼虫的毒力作为评价的指标。测试了7种人工培养基、7种单糖、7种氮源、真菌生长所需的pH范围和温度范围、以及4种光-暗比的光照程序对真菌的影响。结果证明该真菌生长的适合温度为5℃～35℃,最佳温度为25℃～30℃;适合pH范围是5～12;在pH9～11范围内菌丝和游动孢子生长最好;测试的人工培养基中,按照菌丝生长速度从高到低排列,依次为Czapek’SFE、PYG、KPYG2、SDAY、CMA和PDA。其中从真菌的游动孢子形成量和对蚊幼虫的毒力来看,最佳培养基为SFE;葡萄糖、果糖、乳糖、蔗糖、麦芽糖、甘露糖和可溶性淀粉都是本真菌的合适碳氢营养源;含有机氮的培养基比含无机氮的培养基好;不同的光照程序没有表现明显的影响,但是观察到紫外光对本真菌有明显的抑制作用。     

松茸液态发酵菌丝生长量因子的研究

研究了液态发酵条件下通气量、转速、pH、可溶性淀粉浓度对松茸菌丝生长量的影响,探讨了可溶性淀粉稳定pH的作用机理。试验结果表明：通气量≤0.4vvm时松茸菌丝产量随通气量的增大而增长,当通气量＞0.5vvm时,菌丝产量增加不明显;转速增大菌丝产量增加,转速＞80r/min,菌丝产量下降;松茸菌丝发酵的最适pH为5.5,初始pH不同,发酵罐中培养基最终的pH趋于6.0;可溶性淀粉是影响松茸菌丝量的重要因子,可溶性淀粉浓度≤4g/100mL时菌丝量随可溶性淀粉浓度的增加而增大,当其浓度为4g/100mL时,菌     

发酵法生产富铁姬松茸菌丝体的产业化研究

对发酵法生产富铁姬松茸菌丝体进行了产业化研究.结果表明,姬松茸的耐铁能力和富铁能力都很强,在铁浓度为0.1～1.5g/L的液体培养基上,菌丝均能生长,当铁浓度超过1.5g/L 时,菌丝生长受到一定影响;采用液体深层培养,最佳的铁源为FeSO4·7H2O ,最佳铁添加量为0.4～0.5g/L.此时,姬松茸菌丝体的生物量得率和富铁率达到最高,同时,菌丝体含铁量和多糖含量也较高.另外,扩大到10t罐进行工业化生产,富铁姬松茸干菌粉得率达到2.82%,菌丝体含铁量可达14.77mg/g.姬松茸菌丝体产品产量高,质量稳定, 已推向市场,提供给一些企业,初步实现了产业化.     

蚊幼虫病原真菌贵阳腐霉的生物学研究

为了开发灭蚊真菌贵阳腐霉Pythium guiyangense用于蚊虫防治,对贵阳腐霉生物学特性进行了研究。采用菌丝生长率、真菌产孢情况以及对致倦库蚊Culex quinquefaciatus1龄幼虫的毒力作为评价的指标。测试了7种人工培养基、7种单糖、7种氮源、真菌生长所需的pH范围和温度范围、以及4种光-暗比的光照程序对真菌的影响。结果证明该真菌生长的适合温度为5℃～35℃,最佳温度为25℃～30℃;适合pH范围是5～12;在pH 9～11范围内菌丝和游动孢子生长最好;测试的人工培养基中,按照菌丝生长速度从高到低排列,依次为Czapek’SFE、PYG、KPYG₂、SDAY、CMA和PDA。其中从真菌的游动孢子形成量和对蚊幼虫的毒力来看,最佳培养基为SFE;葡萄糖、果糖、乳糖、蔗糖、麦芽糖、甘露糖和可溶性淀粉都是本真菌的合适碳氢营养源;含有机氮的培养基比含无机氮的培养基好;不同的光照程序没有表现明显的影响,但是观察到紫外光对本真菌有明显的抑制作用。     

姬松茸摇瓶种子培养初探

对姬松茸(Agaricus bLazei.Murrill)的摇瓶培养作初步探索。结果表明:①适合姬松茸菌丝活化的培养基为:PDA,麸皮100 g/L,pH自然;②摇瓶培养适宜的培养基为:蔗糖2%,黄豆粉1%,玉米粉2%,酵母粉0.2%,(NH4)2SO4 0.1%,MgSO4·7H2O 0.05%,CaCO3 0.1%。     

联合固氮菌Enterobactergergoviae57—7泌铵突变株的分离和特性

经 Tn5转座子诱变从野生型菌株 ( Enterobacter gergoviae 5 7- 7)筛选到抗甲胺 ( 0 .3mol/L )的泌铵突变株 MG61 ,该突变株在固氮生长时能分泌铵 2 .0 mmol/L。由于泌铵 MG61的生长比野生型菌株慢 ,在培养基中含铵 ( 5 mmol/L或 30 mmol/L)条件下 ,MG61的生长速率与对照相同 ,而远比野生型菌株慢 ,表明 MG 61不能很好地利用铵。在含 2 0 mmol/L铵的培养基中 MG 61仍表达 86%的固氮活性 ,而野生型菌株完全丧失了固氮活性。在谷氨酸存在下 MG61的生长速率及固氮酶活性都比对照高。在硝酸盐存在下 MG61的生长速率与对照相同 ,但泌铵量达 7.8mmol/L。     

耐铵型产琥珀酸放线杆菌的选育及铵离子对其生长代谢的影响

经硫酸二乙酯(DES)诱变,在含61～242mmol/LNH4+梯度平板中,筛选到一株耐铵型突变株YZ25,该菌株在含121mmol/LNH4+发酵培养基中,琥珀酸产量达32.68g/L,转化率为65.4%,比出发菌提高了180.5%。进一步考察了不同形态铵盐对YZ25生长的影响,结果表明添加少量铵盐能够提高突变菌的生长速率,但当超过一定量后菌株生长受到抑制,不同铵盐对菌株的抑制程度不同,硫酸铵、碳酸氢铵、氯化铵和硝酸铵对突变株YZ25的半抑制浓度分别为:215mmol/L、265mmol/L、235mmol/L、210mmol/L。为了考察铵离子对YZ25发酵产琥珀酸的影响过程,在3.0L发酵罐以氨水作为pH的调控剂发酵,结果表明在稳定期前菌株生长基本不受铵离子抑制,生物量能够达到正常水平,但是进入稳定期后铵离子抑制作用越来越明显,导致菌株生长提前结束,耗糖不完全,产酸受阻。最后结合产琥珀酸放线杆菌Actinobacillus succinogenes代谢途径分析了铵离子对菌株抑制作用的机理。     

The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots

The growth, the alkaloid production, as well as the scopolamine/hyoscyamine ratio of two clones of belladonna hairy roots were studied. The effects of nitrate and ammonium concentrations on these cultures were investigated. A rise in ammonium concentration caused the decline of the hairy roots, while nitrate had a marked effect on the alkaloid content. The alkaloid production obtained with 15.8 mM of NO3- and 20.5 mM of NH4+ was 1.2-1.4 times higher than that obtained when the roots were grown in the standard Murashige and Skoog medium (MS medium, 39.5 mM of NO3- and 20.5 mM of NH4+). The nitrate and ammonium concentrations in the culture medium also had a strong influence on the scopolamine/hyoscyamine ratio. When nitrate or ammonium concentrations were raised, that ratio also was increased 2-3-fold. The hairy root clones originating from transformations with two distinct strains of Agrobacterium had similar responses.     

产多聚唾液酸的菌种筛选及产酸条件

通过对40株大肠杆菌进行产多聚唾液酸的筛选,得到一株高产多聚唾液酸菌株C-8,对该菌的一系列培养条件进行了研究。最佳培养基为：山梨醇2.5％,硫酸铵0.5％,磷酸氢二钾90mmol／L,胰蛋白陈1.5％,硫酸镁0.04％,pH7．8。在37℃,250r／min摇床培养65h,可使菌体在每毫升培养液中产多聚唾液酸1200μg。     

NUTRITIONAL REQUIREMENTS FOR OVULE FORMATION IN EXCISED PISTILS OF NIGELLA

The nutritional requirements for ovule formation in Nigella saliva L. were investigated by growing excised pistils on defined media. Pistils grown on a medium containing the minerals of Murashige and Skoog produced significantly more ovules than on a medium containing the minerals of Bilderback. When the nitrogen, sulfate, and phosphate of the Bilderback medium were adjusted to levels comparable to those of the Murashige and Skoog medium, a similar number of ovules was formed. The effect of different forms and concentrations of nitrogen on ovule formation and pistil growth was investigated. High concentrations of nitrate (40 mil) favored pistil growth and ovule formation, but comparable levels of ammonium were toxic. When ammonium at concentrations above 10 mM was added to nitrate media, ovule formation was inhibited. A medium containing low concentrations of ammonium (10 mM) and nitrate (5 mM) supported more ovule formation and pistil growth in young pistils than a low-nitrate (5 mM) medium without ammonium. However, ovule formation on a medium containing 10 mM ammonium and 5 mM nitrate was significantly less than on a medium containing only 15 mM nitrate. Low concentrations of organic nitrogen in the form of α-alanine (1 mM) and γ-aminobutyric acid (5 mM) supported ovule formation and pistil growth similar to a high nitrate medium.     

INORGANIC NITROGEN NUTRITION OF THE SEEDLINGS OF THE ORCHID,CATTLEYA

When grown in vitro in a medium containing NH₄NO₃ as the sole source of nitrogen, seeds ro the orchid, Cattleya (C. labiata ‘Wonder’ X C. labiata ‘Treasure'), germinated readily and proceeded to form small plantlets. Development of the embryos was accompanied by an increase in their total nitrogen and a decline in the percent dry weight. Growth responses of the seedlings in other ammonium salts like (NH₄)₂SO₄, (NH₄)₂HPO₄, NH₄Cl, ammonium acetate and ammonium oxalate were similar to that in NH₄NO₃. However, when grown in a medium containing NaNO₃, development of the seedlings was drastically inhibited; KNO₃, Ca(NO₃)₂, KNO₂ and NaNO₂ also were poor nitrogen sources. Attempts to grow the seedlings in NaNO₃ by changing the pH or by addition of kinetin, molybdenum or ascorbic acid as supplements were completely unsuccessful. When seedlings growing in NH₄NO₃ for varying periods were transferred to NaNO₃, it was found that those plants allowed to grow for 60 or more days in NH₄NO₃ could resume normal growth thereafter in NaNO₃. Determination of the nitrate reductase activity in seedlings of different ages grown in NaNO₃, after NH₄NO₃, showed that the ability of the seedlings to assimilate inorganic nitrogen was paralleled by the appearance of the enzyme.     

Effect of calcium, phosphate and nitrogen on cell growth and biosynthesis of cell wall polysaccharides by Silene vulgaris cell culture

Medium nutrients such as calcium, phosphorus, nitrogen and nitrate to ammonium ratio have significant influence on the growth, biosynthetic and biochemical characteristics of polysaccharides produced by Silene vulgaris (M.) G. cell culture. Cell growth and production of polysaccharides was limited by an absence of any of these components in the medium. Optimal growth of the callus and production of arabinogalactan were achieved at 1.5-4.5 microM calcium while the optimal production of pectin named silenan was observed at 3.0-4.5 microM. The phosphate contents in the medium in the range of 0.63-3.75 microM were favorable for callus growth. Production of silenan was maximal at 1.25-3.75 microM phosphate. Optimal growth of the callus was achieved at 30-90 microM nitrogen. Maximal production of silenan was observed at 60 microM nitrogen while the optimal production of arabinogalactan was at 90 microM nitrogen (at ratio of NH(4)(+):NO(3)(-) as 1:2). A presence both of nitrate and ammonium is needed for the silenan biosynthesis (the NH(4)(+):NO(3)(-) ratio as 1:1 and 1:2). Yields and volumetric production of arabinogalactan were maximal at deletion of ammonium from the nutrient medium (ratio 0:1). Absence of calcium or nitrogen in the medium leads to a decrease of the galacturonic acid residues in silenan. The galactose residues contents in arabinogalactan were decreased in the absence of nitrogen and calcium in the medium.     

Isolation and Characterization of NH+4-Excreting Mutants of Enterobacter gergoviae

NH4+-excreting mutants were isolated from Enterobacter gergoviae 57–7 wild type as methylamine resistant strains which were obtained by mutagenesis with a transposable element Tn5. The MG 61 mutants excreted 2 mmol/L of ammonium during a diazotrophic growth. The growth of MG 61 mutants were slower than the growth of wild types because of its excreting ammonium. MG 61 mutants expressed up to 86% of the fully depressed nitrogenase activity when grown in a medium containing 20 mmol/L ammonium. By contrast the ammonium grown cultures of wild type had no nitrogenase activity. In the presence of 5 mmol/L or 30 mmol/L of ammonium in the medium, the growth of MG 61 mutants was as same as CK and much slower than that of the wild types which means that the mutants could not utilize amonium very well in the medium. But MG 61 mutants could utilize glutamate as a sole nitrogen source. In the presence of nitrate (10 mmol/L) in the medium, MG 61 mutants grew slowly but excreted 7.8 mmol/L of ammonium.     

The effects of amino acids and ammonium on the growth of plant cells in suspension culture

A suspension culture of soybean (Glycine max L.) was grown on a defined medium in which the nitrogen sources were nitrate (25 mM) and ammonium (2 mM). The cells did not grow on nitrate unless the medium was supplemented with ammonium or glutamine. The l- and d-isomers of 12 amino acids tested singly could not replace ammonium. Most amino acids (4 mM) inhibited growth when the cells were cultured on nitrate and ammonium. Cells from five other plants (Reseda luteoli L.; Triticum monococcum L.; flax, Linum usitatissimum L.; horseradish, Amoracia lapathifolia Gilib; Haplopappus gracilis L.) grew on the defined medium with nitrate (25 mM) as the sole nitrogen source. Higher cell yields were obtained when ammonium (2 mM) or glutamine also was present. Supplementing the defined medium with high concentrations of ammonium (20 mM) inhibited growth of soybean, Haplopappus, and wheat cells. Addition of citrate (5 mM) relieved the inhibitory effects of ammonium in soybean and wheat cells but not in the Haplopappus cells.     

Nitrogen regulation of erythromycin formation in Streptomyces erythreus

Abstract Erythromycin formation decreased in Streptomyces erythreus as a function of the ammonium concentration present in the medium. Total inhibition of synthesis was obtained with 100 mM NH₄Cl but medium pH and culture growth were not significantly affected. A similar effect was obtained with NH₄NO₃ or (NH₄)₂SO₄ indicating that ammonium ion probably repressed formation of antibiotic.