优化黑曲霉菌丝球形成条件提高豆制品废水处理效率

豆制品废水的处理是豆制品厂的一个重要环节。利用黑曲霉在培养过程中形成的菌丝球处理豆制品废水具有安全性好、菌体回收简单、成本低等优点。本实验中以察氏培养基为基础研究了黑曲霉菌丝球的形成条件,并评价了对豆制品废水的处理效果。结果表明在黑曲霉孢子浓度为7.23×10~3/L~8.68×10~6/L、初始pH 3.5~pH 7.5、葡萄糖浓度为0 g/L~30 g/L的条件下都可以形成茵丝球。在豆制品废水中黑曲霉孢子浓度为1.45×10~4/L~7.23×10~5/L、pH 3.7~pH 6.6均可以形成茵丝球,而且在初始pH 5.6时经过72 h的培养豆制品废水变得澄清,COD下降了56.34%。本研究表明利用黑曲霉在豆制品废水中形成菌丝球的方式是一种有潜力的处理方式。     

静电作用和疏水作用对黑曲霉孢子凝聚的影响

真菌孢子的凝聚对真菌菌丝球的形成有重要作用,也影响真菌产物的产量。黑曲霉是一种生产多种代谢产物的重要工业微生物。本文以黑曲霉孢子为研究对象,通过磷酸缓冲液处理和溶胀手段改变孢子之间的静电作用和疏水作用,探究了静电作用和疏水作用对黑曲霉孢子凝聚的影响。用磷酸缓冲液处理孢子,去除孢子外层的部分黑色素,减少了孢子的羧基基团以及疏水蛋白,导致孢子间的凝聚增加。孢子的溶胀过程破坏了孢子最外部的疏水蛋白层,减少了孢子间的疏水作用,并使Zeta电位上升,增加孢子的负电荷,使得孢子聚集减少。而未处理的孢子一直展现出较强的凝聚,没有随着pH的升高而显示出显著差异。溶胀孢子的凝聚颗粒随着p H升高而明显变小,表明疏水性降低和电荷的增加导致孢子间凝聚程度减弱,且疏水作用对孢子凝聚的影响大于静电作用。本研究为真菌液态深层发酵过程中菌丝球的形成提供了理论依据。     

pH值对Gigaspora margarita孢子萌发、菌丝生长与聚磷酸盐含量的影响

土壤pH值是影响AM真菌的生理与生态过程的重要因子之一,本试验在培养基上接种Gigasporamargarita的孢子,研究了pH值分别为5.2、6.0和6.8时孢子萌发率、菌丝生长和菌丝中聚磷酸盐(polyP)的含量。结果表明,不同pH条件下的孢子萌发率没有明显差异,培养12d后的萌发率为70%左右;随着pH的升高,菌丝的长度逐渐增加,表明低pH对菌丝的生长有一定的抑制效应;培养12d后,孢子中polyP含量低于菌丝中polyP含量,pH6.0和pH6.8的条件下菌丝中polyP含量明显高于pH5.2的含量,表明低pH也能降低菌丝中的聚磷酸盐含量。认为低pH对菌丝生长和polyP含量的抑制可能是其限制AM真菌功能发挥的重要机制之一。     

pH值对Gigaspora margarita孢子萌发、菌丝生长与聚磷酸盐含量的影响

土壤pH值是影响AM真菌的生理与生态过程的重要因子之一,本试验在培养基上接种Gigasporamargarita的孢子,研究了pH值分别为5.2、6.0和6.8时孢子萌发率、菌丝生长和菌丝中聚磷酸盐(polyP)的含量。结果表明,不同pH条件下的孢子萌发率没有明显差异,培养12d后的萌发率为70%左右;随着pH的升高,菌丝的长度逐渐增加,表明低pH对菌丝的生长有一定的抑制效应;培养12d后,孢子中polyP含量低于菌丝中polyP含量,pH6.0和pH6.8的条件下菌丝中polyP含量明显高于pH5.2的含量,表明低pH也能降低菌丝中的聚磷酸盐含量。认为低pH对菌丝生长和polyP含量的抑制可能是其限制AM真菌功能发挥的重要机制之一。     

潮霉素B抗性为选择标记的整合型表达载体的构建及在米根霉中的遗传转化

为了建立适合米根霉的遗传转化体系,应用重叠延伸PCR的方法构建了以潮霉素B抗性为选择标记的单交换整合型表达载体p BS-hygro-ldh A;分别采用PEG/Ca Cl2介导的原生质体转化、原生质体电转化及萌发孢子电转化的方法将表达载体p BS-hygro-ldh A转化入米根霉AS 3.819菌株中,并研究了菌丝酶解时间、孢子萌发时间以及电转化电场强度对于转化效率的影响;通过荧光定量PCR(q PCR)对米根霉转化子基因组中质粒整合拷贝数进行了检测,并研究了其对米根霉转化子抗性稳定性的影响。实验结果表明成功获得整合了表达载体p BS-hygro-ldh A的米根霉转化子。菌丝酶解140 min产生的原生质体其再生率和转化率最高,原生质体电转化最佳电场强度为13 k V/cm,孢子萌发2.5 h转化率最高,萌发孢子电转化最佳电场强度为14 k V/cm。萌发孢子电转化方法转化率要高于原生质体转化的方法。荧光定量PCR检测结果表明,在一定范围内,高质粒整合拷贝数的米根霉转化子比较稳定。研究建立了用于工业米根霉菌株的遗传转化体系,为米根霉代谢调控研究以及菌种改造工作提供了基础与支持。     

尖顶羊肚菌液体培养基质与条件的研究

通过对尖顶羊肚菌液体培养基质与条件的研究,明确其菌丝生长的最适pH值、最适温度、适宜光照条件、适宜葡萄糖和蛋白胨浓度、适宜培养基,以便应用于尖顶羊肚菌液体菌种的生产和工业发酵。结果表明:菌丝的最适生长温度为2 5℃;最适生长pH值为6 ;葡萄糖和蛋白胨最适浓度分别为2 0 0g/L和10g/L ;菌丝在黑暗环境下生长良好,光照对菌丝生长具有抑制作用;用胡萝卜酵母膏培养基振荡培养形成的菌丝球多,菌丝生长量大;菌丝球在不同培养基中生长,可引起培养液pH值的上升或者下降;菌丝球可利用培养基内的氨基酸,使氨基酸降解,在胡萝卜酵母膏培养基中振荡培养8d的菌液总氨基酸含量较原液减少了36 71% ,亮氨酸、异亮氨酸和甲硫氨酸含量的下降幅度最大     

环境pH诱导出芽短梗霉细胞多形化

出芽短梗霉具有酵母状细胞、膨大细胞、菌丝、厚垣孢子、念珠状菌丝和分生组织状结构。在最适pH条件下,出芽短梗霉生长繁殖以酵母状细胞（CBS100225等4菌株）或膨大细胞（CBS249.65等4菌株）为主。pH 2.2或pH 7.0诱导全部8株出芽短梗霉形成分生组织状结构。酵母状细胞转变成膨大细胞受低pH值诱导的占75%,还受高pH诱导的占50%。膨大细胞是多形性细胞转变的中心环节,可以转变成菌丝、厚垣孢子或分生组织状结构。     

哈茨木霉的培养及其对烟草疫霉生长的抑制研究*

哈茨木霉是一类重要的植病生防因子。哈茨木霉TH-1分别在PDA培养基、麦芽糖培养基、查氏培养基和琼脂培养基上培养均能产孢,其中PDA培养基为最适培养基。PDA培养基上,菌丝生长适宜温度27.5℃~35℃,最适温度32.5℃,产孢最适温度27.5℃。菌丝生长适宜pH值为3~7,产孢适宜pH值为5~9,生长与产孢最适pH值为5。光照对菌丝生长影响不大但明显影响菌株的产孢数量,光照时间越长产孢量越大。对峙培养试验表明TH-1明显抑制疫霉菌的生长速率,其无菌滤液明显抑制烟草疫霉菌游动孢子的萌发,并抑制游动孢子芽管     

真菌菌丝球形成的自组织机理

采用数码显微摄像技术,拍摄了几种丝状真菌存悬液培养下的菌丝生长照片,探讨了真菌自组织形成菌丝球的机理,发现孢子的聚集在菌丝球的形成中起了重要作用,还研究了影响菌丝球形成的某些物化因素,结果表明在接种20日龄的桔青霉孢子,孢子悬液浓度为10^5个/ml,培养液中加入表面活性剂吐温80或曲拉通X-100（浓度0.1%),摇床转速为140r/min的条件下,于25℃培养5d,可形成机械强度较好的菌丝球。     

橘青霉菌丝球形成条件及其处理废水的应用

探讨了橘青霉 (Penicilliumcitrinum)呈球状体生长的条件和形成的机理。发现在培养液初始pH为 7.0 ,孢子年龄为16～ 2 0天 ,孢子悬液浓度为 10 5个·mL-1,表面活性剂Tween80或TritonX - 10 0质量浓度为 1g/L ,摇床转速为 140r/min的条件下 ,于 2 5℃培养 5天 ,可形成大小适中 ,有一定机械强度 ,光滑均匀的菌丝球。孢子的聚集在菌丝球的形成中起了重要作用。用此菌丝球吸附质量浓度为 5 0mg·L-1的 1 氨基 2 萘酚 4 磺酸水溶液模拟处理染料废水 ,吸附率达到 6 2 .4%。     

米根霉ME—F12发酵产富马酸的菌体形态控制

丝状真菌发酵体系中菌体形态对产量有着重要影响。考察富马酸产生菌Rhizopus oryzae ME—F12种子培养过程中不同pH条件、孢子悬浮液密度以及CaCl2添加量对其形态的影响。结果表明,当控制种子培养液pH2．3～2．7、接种孢子的终密度为1．5×10^8～3．0×10^8／L和添加0．5g／LCaCl2时,培养可获得直径约为0．65mm光滑规整茵球,后继的产酸发酵中富马酸量高达58．9g／L。正交实验表明,pH是影响菌球形成的最主要因素,孢子液密度主要影响菌体生物量,而CaCl2则是菌球表面光滑度的主要影响因素。     

离子注入选育高产L-乳酸的米根霉突变株及其发酵特性研究

通过氮离子注入获得米根霉突变株RQ4012,其利用木糖的能力比出发菌株提高了1.6倍;通过多次传代,证明其具有良好的遗传稳定性。试验测定菌株RQ4012发酵木糖生产L-乳酸的最佳发酵条件:木糖10%,生理盐水浸泡孢子9 h,(NH4)2SO43 g/L,接种量4%,CaCO3添加量6%,装液量20%,温度37℃,转速200 r/min,在此条件下,乳酸产量达到79.51 g/L。对混合糖的发酵进行了探索,结果表明该菌能高效利用混合糖生产L-乳酸,在利用植物纤维素水解液生产L-乳酸上有良好的应用前景。     

里氏木霉与米根霉混合固态发酵产纤维素酶

以里氏木霉及米根霉单菌固态发酵为对象,考察不同混合发酵形式对里氏木霉与米根霉混合固态发酵产纤维素酶的影响。结果表明:同时接种里氏木霉与米根霉,试验考察的两菌种接种量比1∶1(以孢子个数计)及5∶1条件下,两菌未产生明显协同产酶作用。米根霉延时(24 h)接种且菌种量比5∶1以及米根霉延时(48 h)接种且菌种量比1∶1,2种发酵形式产酶情况类似,滤纸酶活(FPA)及羧甲基纤维素酶(CMCase)酶活相对米根霉单菌发酵有所提高,而β-葡萄糖苷酶(β-GA)酶活相对里氏木霉单菌固态发酵结束时分别增加4.66及4.40倍,可以发现两菌产生一定协同作用。在米根霉延时(48 h)接种且菌种量比5∶1的发酵形式下,FPA及CMCase在发酵第7天酶活分别达到44.04 IU/g、627.14 U/g(以1 g干曲计),分别是里氏木霉固态单菌发酵产酶达到稳定期时酶活的1.36和1.63倍,两菌产生了有效的协同作用。     

Growth ofRhizopus arrhizus in fermentation media

Summary Rhizopus arrhizus biomass attached itself to fermentor walls, baffles and impellers when grown in casein/ glucose media. In shake flasks, dispersed filamentous growth was produced in media containing certain concentrations of glucose and soya flour. Other media tested produced pelleted or clumpy growth. Medium initial pH did not affect morphology type. Dispersed growth could not be obtained by addition of detergents, oils and polymers to a clear glucose/soya peptone medium. Addition of maize solids to this medium resulted in dispersed growth which occurred even in the presence of calcium, which in most media caused pellet formation. Mycelia appeared to bind to the maize particles and use these as growth centres thereby preventing pellet or clump formation. Mycelial pellets appeared to originate either from a single spore or by interaction of branched hyphae from different spores. Medium composition and macro-morphology type correlate with differences in hyphal structures.     

Effect of individual amino acids and glucose on activation and germination of Rhizopus oligosporus sporangiospores in tempe starter

AIM: To understand the conditions promoting activation and germination of spores, and to contribute to the control of tempe starters. METHODS AND RESULTS: Using microscopic counts of fluorescent labelled spores, the following results were obtained: (1) L-alanine plays an important role (of the same order as that of peptone) in stimulation of germination of dormant spores. Alanine can satisfy the requirements of carbon as well as nitrogen for spore germination; (2) L-proline, on the other hand, inhibits alanine uptake presumably by blocking/congesting transporters of spore cells, resulting in apparent low viability on agar media; (3) L-leucine and L-isoleucine slightly favour spore germination while L-arginine and L-lysine do not have any stimulating effect; (4) The stimulatory role of glucose was only evident in the presence of phosphate (in minimal medium); when glucose is used in the absence of phosphate, either alone or in combination with single amino acids its role is hardly distinguishable; (5) Phosphate plays a facilitating role in spore germination. CONCLUSIONS: Glucose and amino acids play important roles in activation and germination of sporangiospores of Rhizopus oligosporus in tempe starter (stored for 12 months). The ability and rate of germination of dormant/old sporangiospores of R. oligosporus, depend on their ability for uptake of individual amino acids and/or glucose. SIGNIFICANCE AND IMPACT OF STUDY: New light was shed on the counteractive role of proline and the stimulating effect of phosphate. Soybeans subjected to traditional preparation for tempe making are heavily leached; germination of starter spores on such beans is sub-optimal, and bean processing could be optimized.     

The comparative virulence of thermotolerant Mucorales species in mice

The comparative virulence of thermotolerant Mucorales was determined for cortisone-treated and untreated Swiss mice by intravenous administration of spores. The measure of virulence was based on an LD50 value, calculated after the 30-day observation period. Of the known etiological agents of mucormycosis, Mucor meihei, M. pusillus, Rhizopus arrhizus, R. chinensis, R. cohnii, R. microsporus, R. oryzae, R. rhizopodiformis and Cunninghamella elegans were able to produce fatal infections in mice; whereas, Mucor alternans, M. ramosissimus and Syncephalastrum racemosum were avirulent at dosages of up to 10(5) spores. Of those thermotolerant species which have not been reported to cause mucormycosis in human beings, Radiomyces embreei, R. spectabilis, Rhizopus oligosporus and Thermomucor indicae-seudaticae were found to produce fatal infections in mice; whereas, an isolate of Mycotypha africana was avirulent. Cortisone treatment of mice was found to lower their resistance to infection at a given spore dosage as measured by ET50 values.     

Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798.

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

Factors affecting production of mold mycelium and protein in synthetic media.

The effects of certain cultural conditions on the yield of dry mycelium, protein, and total amino acid content of Rhizopus oligosporus Saito (NRRL 2710), Rhizopus rhizopodiformis (Cohn apud Lichtheim) Zopf (NRRL 6246), and Absidia corymbifera (Cohn) Sacc. et Trotter (NRRL 6247) were studied. The yield of mycelium was found to significantly increase as the spore inoculum was increased from 187,500 to 2,250,000 spores. But the total amino acids (grams/liter) did not change significantly, whereas the percentage of crude protein decreased. An inoculum containing approximately 750,000 spores/ml was used in all of the other experiments. Mycelial production was highest at 37 degrees C for all three molds. However, the best temperature for percentage of crude protein and total amino acids varied with the organism. The mycelial yield and total crude protein of R. oligosporus showed some significant changes as the C/N ratio was increased in 3% glucose medium. In a synthetic medium having a 15:1 C/N ratio, the strains of R. oligosporus, R. rhizopodiformis, and A. corymbifera had better yields from falactose than glucose, not only in dry mycelium but also in total crude protein (grams/liter) and total amino acids (grams/liter). R. oligosporus grew very well on several ammonium salts. but the maximum yield of dry mycelium, total crude protein (grams/liter), and total amino acids (grams/liter) occurred with ammonium sulfate. The optimum pH for both Rhizopus species was 4.0, although R. oligosporus grew equally well at pH 3.0 and slightly less at pH 5.0. The highest yield of mycelium for A. corymbifera was obtained in a medium with an initial pH of 8.0. It was calculated that a fermenter chanrged with an adequate medium and 1,000 lb (about 450 kg) of R. oligosporus or A. corymbifera cells could produce 88 or 90 lb of protein (on a dry-weight basis) per h if the product was removed continuously.