Enzymelektrode zur Bestimmung von L-Lysin in Aminosäuregemischen

A specific enzyme electrode with L-Lysine decarboxylase (E.C. 4.1.1.18) from Klebsiella pneumoniae and a CO₂-sensor are described. The electrode can be used for assay of L-Lysine in amino acid mixtures as protein hydrolysates or culture filtrates from fermentation processes. The accuracy and reproducibility are those of an amino acid analyzer. The electrode works well for several weeks.     

Herstellung von L-Lysindecarboxylase aus Klebsiella pneumoniae

L-Lysine decarboxylase (LL-Lysine carboxylase E.C. 4.1.1.18) is produced by a strain of the genus Klebsiella. The bacterial cells are cultivated at a temperature of 37°C with and without shaking. The production of LL-Lysine decarboxylase is increased by adding of LL-Lysine to the medium. At the end of cultivation the cells are killed by thymol, washed in water and dried by means of acetone and ether. Compared with specimen derived from other bacteria the LL-Lysine decarboxylase in the shape of dried cells shows a high specific activity. The preparations are free of disturbing enzyme activities, have a high level of stability and may be used for determining of LL-Lysine by means of enzyme electrodes.     

Improving probiotic spore yield using rice straw hydrolysate

Spore-forming Bacillus sp. has been extensively studied for their probiotic properties. In this study, an acid-treated rice straw hydrolysate was used as carbon source to produce the spores of Bacillus coagulans. The results showed that this hydrolysate significantly improved the spore yield compared with other carbon sources such as glucose. Three significant medium components including rice straw hydrolysate, MnSO₄ and yeast extract were screened by Plackett–Burman design. These significant variables were further optimized by response surface methodology (RSM). The optimal values of the medium components were rice straw hydolysate of 27% (v/v), MnSO₄ of 0·78 g l⁻¹ and yeast extract of 1·2 g l⁻¹. The optimized medium and RSM model for spore production were validated in a 5 l bioreactor. Overall, this sporulation medium containing acid-treated rice straw hydrolysate has a potential to be used in the production of B. coagulans spores.     

Cultivation of yeasts on hydrolysate nutrient medium obtained from the production of microcrystalline cellulose

Studies of the biomass production during a continuous cultivation of yeasts on a nutrient medium, prepared from a hydrolysate from the production of microcrystalline cellulose, have been carried out. A new strain of yeasts has been used. Its cultivation has been achieved without addition of biostimulators to the nutrient medium in spite of their absence in the initial hydrolysate. Practically a complete assimilation of sugars has been achieved at high dilution rates (D = 0.25 to 0.50 h⁺¹). The yield of biomass achieved is above 50% compared to the initial sugars and it contains 48.89% true protein. The results obtained offer the possibility of a complex utilization of the products of cellulose hydrolysis in the production of microcrystalline cellulose with a realization of a waste free technology.     

Sequential Primary and Secondary Shunt Metabolism in Penicillium chrysogenum

Penicillium chrysogenum was grown on a rich medium and on a more sparse medium which favored penicillin production. Mycelia grown on both media were examined for changes in lipid, mannitol, erythritol, glycerol, pentitol, trehalose, and residual mycelium, and the filtrates were examined for penicillin. Penicillin production took place after the bulk of trehalose, polyol, and lipid had accumulated, and hence the sequential pattern of primary and secondary shunt metabolism, as observed in the case of ergot alkaloid production by Claviceps purpurea, was demonstrated in this example of penicillin production.     

Einfluß von Tricyclazol auf die Entwicklung und Melaningehalte der Sklerotien von Botrytis cinerea

Effect of tricyclazole on production and melanin contents of sclerotia of Botrytis cinerea Tricyclazole retarded production of sclerotia of Botrytis cinerea on agar medium more severely than mycelium growth. At a concentration range (50–200 mg/l) that did not control Botrytis on grape leaves, sclerotia production was significantly reduced. There was a negative relation between the bleaching duration of sclerotia and the tricyclazole concentrations in the medium on which they were formed. Light microscopical studies showed that sclerotia from tricyclazole-containing medium contained a significantly poorer developed outer melanin layer than that from the control medium. Ultrastructural studies with 5 days old untreated sclerotia revealed intense electron impermeable deposits in the intercellular spaces and a small electron dense layer in the outer cell walls, on the other hand treated sclerotia of the same age showed only sporadic small pigmented deposits between the cells and the pigmentation of the outer cell wall was absent.     

Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis

Aim: Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae. Methods and Results: Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small‐scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1 : 2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l^?1 (0·22 mol l^?1)]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1 : 5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1 : 10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0·42 ± 0·01 g ethanol (g glucose)^?1 were observed for both yeasts in 1 : 10 hydrolysate. In small‐scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1 : 5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1 : 2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate. Conclusions: Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate. Significance and Impact of the study: This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates.     

Effect of nitrogen source concentration on curdlan production by Agrobacterium sp. ATCC 31749 grown on prairie cordgrass hydrolysates

The effect of nitrogen source concentration on the production of the polysaccharide curdlan by the bacterium Agrobacterium sp. ATCC 31749 from hydrolysates of prairie cordgrass was examined. The highest curdlan concentrations were produced by ATCC 31749 when grown on a medium containing a solids-only hydrolysate and the nitrogen source ammonium phosphate (2.2 mM) or on a medium containing a complete hydrolysate and 3.3 mM ammonium phosphate. The latter medium sustained a higher level of bacterial curdlan production than the former medium after 144 hr. Biomass production by ATCC 31749 was highest after 144 hr when grown on a medium containing a solids-only hydrolysate and 2.2 or 8.7 mM ammonium phosphate. On the medium containing the complete hydrolysate, biomass production by ATCC 31749 was highest after 144 hr when 3.3 mM ammonium phosphate was present. Bacterial biomass production after 144 hr was greater on the complete hydrolysate medium compared to the solids-only hydrolysate medium. Curdlan yield produced by ATCC 31749 after 144 hr from the complete hydrolysate medium containing 3.3 mM ammonium phosphate was higher than from the solids-only hydrolysate medium containing 2.2 mM ammonium phosphate.     

Penicillin G production by immobilized whole cells of Penicillium chrysogenum.

Penicillium chrysogenum was immobilized in polyacrylamide gel prepared from 5% acrylamide monomers (85% acrylamide and 15% N,N'-methylene bisacrylamide). Penicillin produced from glucose by the immobilized mycelium was 17% of that produced by washed mycelium. However, the activity of penicillin production of the washed mycelium decreased with repeated use. On the other hand, the activity of the immobilized mycelium increased initially and decreased gradually with repeated use. The rate of oxygen uptake of the immobilized mycelium was about 30% of that of the washed mycelium. The immobilized mycelium required oxygen for the production of penicillin.     

Ethylene Production and Mycelium Growth of the Tulip Strain of Fusarium oxysporum as Influenced by Shaking of and Oxygen Supply to the Culture Medium

The fungus Fusarium oxysporum Schlecht f. sp. tulipae Apt. can produce ethylene abundantly in vitro when grown in Pratt's liquid medium with glucose as the only organic substrate. This production starts after a lag phase of about 4 days, and peak production occurs when mycelium weight has reached its maximum value. For several days the rate of production is more or less linearly dependent on pO₂. The total production is also dependent on the oxygen concentration, but pure oxygen inhibits the total production by about 50% as compared with 21% oxygen. The high production in shake cultures, as compared with the low production in stagnant cultures, is probably the result of a better oxygen supply in the culture medium. The mycelium weight proved not to be a valid referential basis for the production of ethylene.     

Effect of Pamamycin-607 on Secondary Metabolite Production by Streptomyces spp.

The effect of the aerial mycelium-inducing compound, pamamycin-607, on antibiotic production by several Streptomyces spp. was examined. Exposure to 6.6 μM pamamycin-607 stimulated by 2.7 fold the puromycin production by Streptomyces alboniger NBRC 12738, in which pamamycin-607 had first been isolated, and restored aerial mycelium formation. Pamamycin-607 also stimulated the respective production of streptomycin by S. griseus NBRC 12875 and that of cinerubins A and B by S. tauricus JCM 4837 by approximately 1.5, 1.7 and 1.9 fold. The antibiotic produced by Streptomyces sp. 91-a was identified as virginiamycin M₁, and its synthesis was enhanced 2.6 fold by pamamycin-607. These results demonstrate that pamamycin-607 not only restored or stimulated aerial mycelium formation, but also stimulated secondary metabolite production.     

Citric Acid Production from Hydrolysate of Pretreated Straw Cellulose by Yarrowia lipolytica SWJ-1b Using Batch and Fed-Batch Cultivation

In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH₄)₂SO₄, KH₂PO₄, or Mg²⁺ were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.     

Use of elephant grass (Pennisetum purpureum) acid hydrolysate for microbial oil production by Trichosporon cutaneum

Elephant grass (Pennisetum purpureum) dilute acid hydrolysate contains 34.6?g/L total sugars. The potential of lipid production by oleaginous yeast Trichosporon cutaneum grown on elephant grass acid hydrolysate was investigated for the first time. During the fermentation process on the elephant grass acid hydrolysate, glucose, xylose, and arabinose could be well utilized as carbon sources by T. cutaneum. Interestingly, xylose was almost no use before glucose was consumed completely. This illustrated that simultaneous saccharification of xylose and glucose by T. cutaneum did not occur on elephant grass acid hydrolysate. The highest biomass, lipid content, lipid yield, and lipid coefficient of T. cutaneum were measured after the sixth day of fermentation and were 22.76?g/L, 24.0%, 5.46?g/L, and 16.1%, respectively. Therefore, elephant grass is a promising raw material for microbial oil production by T. cutaneum.     

Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates

The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA‐barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4‐fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate‐derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance.     

促进黄花蒿发根青蒿素合成的内生真菌诱导子的制备

应用酸解法对黄花蒿(ArtemisiaannuaL.)内生胶孢炭疽菌(Colletotrichumgloeosporioides)菌丝体进行提取,在黄花蒿发根培养系统中比较了各制备提取物的青蒿素诱导活性。活性提取物经过SephadexG25层析后,部分纯化的内生菌寡糖提取物(MW<2500)可显著促进发根青蒿素的合成,培养23d的发根经诱导子(0.4mg/mL)处理4d后,青蒿素产量可达13.51mg/L,比同期对照产量提高51.63%,诱导作用与诱导子浓度、作用时间相关。内生菌寡糖诱导子的制备和使用,在青蒿素生物技术生产研究中为首次应用。     

Beneficial Effect of Corncob Acid Hydrolysate on the Lipid Production by Oleaginous Yeast Trichosporon dermatis

In this work, corncob acid hydrolysate and its simulated medium whose sugar composition was the same as the corncob acid hydrolysate were used as fermentation substrate for lipid production by oleaginous yeast Trichosporon dermatis. On the corncob acid hydrolysate, after 7 days of fermentation, the biomass, lipid content, lipid yield, and lipid coefficient of T. dermatis were 17.3 g/L, 40.2%, 7.0 g/L, and 16.5%, respectively. Interestingly, during the lipid fermentation on the corncob acid hydrolysate, glucose, xylose, arabinose, and even acetic acid could be well utilized as carbon sources by T. dermatis. Surprisingly, the lipid yield (7.0 g/L) of T. dermatis on the corncob acid hydrolysate was much higher than that (3.8 g/L) on the simulated medium, in spite of the fact that the lipid coefficient (17.4%) on the simulated medium was a little higher. This phenomenon further showed that lignocellulosic acid hydrolysate was a suitable substrate for lipid fermentation by T. dermatis. This work would help the comprehensive utilization of lignocellulosic biomass for lipid production.     

Das Wirkungsspektrum der lichtabhängigen Zonierung der Koremien von zwei Mutanten von Penicillium claviforme Bainier

Summary An action spectrum of light induced coremia-zonation was obtained for the fungus Penicillium claviforme mut. olivicolor Abe et Ura. Zonation is induced only by light of wavelengths shorter than 510 nm. The action spectrum has maxima at 370 nm and at 450–460 nm and a definite shoulder at 470–480 nm. Penicillium claviforme mut. album is somewhat less sensitive to light but possesses the same spectral sensitivity.Measurable amounts of carotenoids are not found in the mycelium. The presence of diphenylamine in the nutrition medium has no effect on the fungal sensitivity to light. It is therefore assumed that the photoreceptor pigment involved is a flavoprotein.

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Die Arbeit wurde während eines Studienaufenthaltes im Zentralinstitut für Genetik und Kulturpflanzenforschung der Deutschen Akademie der Wissenschaften zu Berlin in Gatersleben angefertigt.     

In vitro-Untersuchungen und mikroskopische Studien zur Entwicklung von Phoma chrysanthemicola Hollos f. sp. chrysanthemicola, dem Erreger einer Wurzel- und Stengelgrundfäule der Chrysantheme

In vitro investigations and microscopic studies about the development of Phoma chrysanthemicola Hollos f. sp. chrysanthemicola, the causal pathogen of a collar and root rot of chrysanthemums Different media were tested for cultivation of Phoma chrysanthemicola. The pathogen grew best on PDA and carrot-Agar at a temperature of 25°C. The three strains used showed many variations, concerning the structure and colour of the mycelium. Fructification has been promoted by irradiation (ultra-violer ray). The morphogenesis, especially the formation of pycnidia, conidia and sclerotial structures has been examined with help of the scanning electrone microscope (SEM), results are presented., So far unknown organs probably belonging to the fungus were observed, their identity is still not clear.     

Enhanced development of somatic embryos of Plantago ovata Forsk. By additives

Summary Plantago ovata Forsk (commonly known as Isabgul) is an economically important medicinal plant. In the present investigation, in vitro plant regeneration of P. ovata was attempted through somatic embryogenesis. Casein hydrolysate and coconut water were used in different concentrations in Murashige and Skoog medium along with 1-naphthaleneacetic acid and N⁶-benzyladenine to increase the amount of callus and number of somatic embryos. Light and scanning electron microscopic studies followed the developmental stages of embryo formation. Results indicated that optimum concentrations of casein hydrolysate and coconut water are useful for promoting the growth of embryogenic cultures. However, a supra-optimal dose of casein hydrolysate and coconut water induced polyphenol synthesis and caused browning of callus and also eventual death of embryos. The use of additives such as coconut water and casein hydrolysate promotes large-scale production of P. ovata through in vitro somatic embryogenesis.     

Combining chemical flocculation and bacterial co-culture of Cupriavidus taiwanensis and Ureibacillus thermosphaericus to detoxify a hardwood hemicelluloses hydrolysate and enable acetone–butanol–ethanol fermentation leading to butanol

Butanol, a fuel with better characteristics than ethanol, can be produced via acetone–butanol–ethanol (ABE) fermentation using lignocellulosic biomass as a carbon source. However, many inhibitors present in the hydrolysate limit the yield of the fermentation process. In this work, a detoxification technology combining flocculation and biodetoxification within a bacterial co-culture composed of Ureibacillus thermosphaericus and Cupriavidus taiwanensis is presented for the first time. Co-culture-based strategies to detoxify filtered and unfiltered hydrolysates have been investigated. The best results of detoxification were obtained for a two-step approach combining flocculation to biodetoxification. This sequential process led to a final phenolic compounds concentration of 1.4 g/L, a value close to the minimum inhibitory level observed for flocculated hydrolysate (1.1 g/L). The generated hydrolysate was then fermented with Clostridium acetobutylicum ATCC 824 for 120 h. A final butanol production of 8 g/L was obtained, although the detoxified hydrolysate was diluted to reach 0.3 g/L of phenolics to ensure noninhibitory conditions. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2753, 2019.