生防菌Ⅲ-61抗真菌活性产物的摇瓶发酵

对生防链霉菌Ⅲ-61产生抗真菌活性物质的摇瓶发酵工艺进行了研究。利用正交试验设计优化了发酵培养基组分,其最适配方为黄豆粉1．5％,蛋白胨0．3％,蔗糖1．0％,淀粉1．3％,磷酸二氢钾0．02％,硫酸镁0．025％,氯化钠0．5％,配咸水溶液,调pH至7～7．4,加碳酸钙1％。通过单因素试验,筛选获得了最优培养条件组合：液体种龄24h,接种量5％～10％,500mL摇瓶培养基装量为80mL,摇床转速240r／min,培养温度31℃,发酵周期96～120h。此优化的发酵培养基与发酵条件的组合昕得菌株Ⅲ-61发酵液对主要靶标黄瓜灰霉病菌的抑菌圈直径达49．5mm,较优化前提高了45．59％。     

Formation of broad-spectrum antibiotics by cultures of the genus Micromonospora]

Data on the study of antibiotic production by the representatives of Micromonospora and the use of ion exchange resins for intensification of screening antibiotic-producing organisms among Micromonospora are presented. It was found that out of 172 strains of Micromonospora tested 92 (53.5 per cent) cultures produced antibiotics, 18 of which were active against gramnegative bacteria. The use of carboxylic ion exchange resins at early microbiological stages of the screening provided an increase in the frequency of finding broad spectrum antibiotics from 10.4 to 19.7 per cent.     

Method of determining the sensitivity of Staphylococcus aureus to methicillin]

The effect of the incubation temperature, i.e. 37 and 30 degrees and the effect of addition of 5 per cent of sodium chloride to the nutrient medium was studied with respect to the results of sensitivity determination of staphylococci to methicillin by 3 methods, such as serial dilutions in liquid and solid media and replica method applied to separate colonies. With the use of all 3 methods incubation of the samples at a temperature of 30 degrees increased the rate of staphylococcal cultures resistant to methicillin. Addition of sodium chloride to the solid nutrient medium increased the level of detecting methicillin resistant cultures in the samples incubated at a temperature of 37 degrees and decreased it in the samples incubated at 30 degrees.     

Growth and development of extreme-thermophilic bacteria at 70 degrees]

Nine cultures of non-sporeforming gram-negative extreme-thermophilic bacterium Thermus flavus have been isolated from hot springs of Kamchatka. Their optimal growth temperature on a solid potato medium was 70 to 76 degrees C, and on a liquid medium (20 per cent potato broth containing 0.5 per cent peptone and 0.1 per cent yeast extract) 70 degrees C. The minimum time of generation of the bacterium, strain 71, growing on the liquid medium at 70 degrees C, was 52 minutes. Other extreme-thermophilic cultures differ from the strain 71 by a lower growth rate. The maximum biomass yield was 1.96 g per litre of the medium after 6 to 9 hours of growth. The extreme-thermophilic bacteria of Kamchatka are similar to those from the hot springs of Japan.     

Potential of solid state fermentation for production of L(+)-lactic acid by Rhizopus oryzae

Production of l(+)-lactic acid by Rhizopus oryzae NRRL 395 was studied in solid medium on sugar-cane bagasse impregnated with a nutrient solution containing glucose and CaCO₃. A comparative study was undertaken in submerged and solid-state cultures. The optimal concentrations in glucose were 120 g/l in liquid culture and 180 g/l in solid-state fermentation corresponding to production of l(+)-lactic acid of 93.8 and 137.0 g/l, respectively. The productivity was 1.38 g/l per hour in liquid medium and 1.43 g/l per hour in solid medium. However, the fermentation yield was about 77% whatever the medium. These figures are significant for l(+)-lactic acid production.     

Action of polyene antibiotics on actinomycetes with a varying lipid makeup]

The effect of polyenic antibiotics, such as nystatin, levorin, amphotericin B and mycoheptin on 93 representatives of various species of the ray fungi was studied. It was shown that resistance of the actinomycetes to the polyens was connected with the absence or insufficient content of sterols (0.001--0.008 per cent in the dry mycelium). On addition of cholesterol to the nutrient media (100 microgram/ml) it was included into the membranes of some cultures and their sensitivity increased 2--60 times. Resistance of Actinomyces sp. LIA 0775 grown on the media with fats differing in their composition decreased 2--4 times. In these cases the culture lipids were characterized by lower content of phospholipids (35--45 per cent from the total lipids as compared to 70--80 per cent when grown on the control medium without fats) and significantly increased content of unsaturated fatty acids (3--4 times).     

Hydrolysis of pectin: an enzymatic approach and its application in banana fiber processing

Sediment samples were collected from different estuarine and marine areas along the West coast of India. Eighteen actinomycete cultures were isolated using starch casein agar and were screened for polygalacturonase activity by growing them on pectin-agar plates. Clear zones were visualized using 1% cetrimide. Out of the 18 strains screened ten cultures could effect hydrolysis of pectin. The above cultures were subjected to secondary screening under submerged fermentation. The actinomycete strain of Streptomyces lydicus was found to be a potent producer of polygalacturonase. Different growth media were screened for enzyme production and the best medium was selected for further studies. The crude enzyme was used for the treatment of raw banana fibers.     

Directed isolation of Micromonospora generic cultures on a selective medium with gentamycin

The results of using selective media with gentamicin for directed isolation of Micromonospora are presented. It was shown that the use of the selective media with gentamicin for isolation of actinomycetes from soils of usual humidity levels markedly increased the frequency of Micronomonospora detection. The use of the selective media with gentamicin for plating out silt substrates containing mainly Micromonospora had practically no effect on the increase in the number of the Micromonospora cultures grown. The number of antibiotic-producing Micromonospora isolated on the media with gentamicin was 3 times higher than that on the control media. The use of the selective media with gentamicin provided directed isolation of Micromonospora.     

seed rot and root rot complex of beans (Phaseolus vulgaris L.)

Fusarium solani (76%),Phyllosticta phaseolina (12%) andAspergillus flavus (10%) were isolated from bean(Phaseolus vulgaris) seeds showing lesions. Out of these the first two caused 30 per cent and 20 per cent seed rotting respectively after artificial seed inoculations. When inoculum was added into the soil before sowing the seed, 35 per cent and 10 per cent pre-emergence rot ocurred and there was 35 per cent and 15 per cent post-emergence blight when soil with healthy seedlings was inoculated withF. solani andP. phaseolina respectively.F. solani caused similar lesions on bean seeds when inoculated after an injury.F. oxysporum was isolated from the roots of dried bean plants showing necrosis.     

Isolation of Legionella from water samples using various culture methods

The efficacy of a non-selective medium and two selective media were compared for the isolation of legionellas from water samples. The effect of acid wash treatment for decontamination of the water samples on the isolation frequency of legionellas was also studied. The 236 samples were taken from cooling, humidifying and drinking water systems; 21% were legionella-positive when inoculated directly on modified Wadowsky-Yee (MWY) medium and 26% were positive when concentrated (x 200) before cultivation on MWY or CCVC media. Inoculation on MWY medium after concentration followed by decontamination by the acid-wash technique gave the highest isolation frequency (31%). The lowest frequency (8%) was found with the non-selective BCYEα medium. An isolation frequency of 28% was achieved with the BCYEα medium after concentration and acid-wash treatment of the samples. Forty per cent of the samples were positive for legionellas when the results from all the culture methods were combined. Not all the legionella-positive samples were identified by a single culture method. Ninety-three of the 95 positive samples were detected with the two best combinations of three culture methods. The best culture method for detecting legionellas depended on the source of the water sample. Some water quality characteristics, like temperature and organic matter content, affected the isolation frequency of Legionella spp.     

Experiment to study some suspension media for the lyophilization of actinomycetes]

Viability and cultural properties of 59 actinomycetes and 17 bacteria lyophilized in polyvinylpyrrolidone (PVP), sodium glutamate, their combinations and horse serum were studied after storage for 2 years at a temperature of 4-10 degrees. A 5 per cent solution of sodium glutamate had a high protective effect on viability of the above organisms. The solution containing 3 per cent of sodium glutamate and 3 per cent of PVP was somewhat less effective. The cultures lyophilized in 5 per cent solution of sodium glutamate had the same viability levels as those lyophilized in horse serum, while the latter had better growth rates on their plating out on nutrient media. A 5 per cent solution of PVP had no advantages over sodium glutamate or horse serum with respect to preservation of the organism viability. No significant differences in the cultural properties: colour of the aerial and substrate mycelium and pigment production were noted in the actinomycetes lyophilized in various protective media and the analogous control cultures maintained by means of passages on fresh nutrient media.     

Selection of the seeding media and the age and quantity of mycelia providing simultaneous biosynthesis of an antibiotic and pigment by the fungus Hypomyces rosellus

The authors observed maximum simultaneous biosynthesis of antibiotic and pigment in the microphilic fungus with using 48-hour seed mycelium having the specific growth rate of 0.008-0.011 h-1 in an amount of 5-7 per cent (v). The Balling 4 degrees wart and Chapek medium with 1 per cent of soybean flower and 1 per cent of corn steep liquor may be used for growing the seed mycelium. No significant effect of the seed medium composition and seed mycelium age on the pigment production was observed.     

Seed culture affects the completion time of L-lactate fermentation using electrodialysis culture

Electrodialysis culture (ED-C) and control culture of Lactococcus lactis IO-1 were inoculated with 3 and 12 h seed cultures. Regardless of seed culture age, control cultures needed 30 h for the completion of L-lactate fermentation. By contrast, the fermentation times for ED-Cs inoculated with 3 and 12 h seed cultures were reduced by 38 and 27% when compared to those of control cultures. Seed culture age can be varied to reduce the fermentation time for ED-C.     

16种茯苓菌株液体发酵产胞内多糖与三萜的比较研究

筛选茯苓高产胞内多糖和胞内三萜的优良液体发酵出发菌株。采用PDA富集固体平板培养与液体发酵培养测定菌丝体生长速率;采用液体发酵策略分析16种茯苓菌株产胞内多糖与胞内三萜的潜能。实验结果表明菌株生长于固体培养基与种子培养基的生长速率之间没有关联性;降低一级种子培养基初始pH值到4.0时能有效缓解茯苓菌株培养物褐化现象;AS5.137胞内多糖含量最高,达377.60±0.10 mg/g,而DB菌株显示出最高的胞内多糖产量,达1.01±0.13 g/L;Y1菌株胞内三萜含量最高,达83.89±4.28 mg/g,而Jingzhou28菌株胞内三萜产量最高,达136.63±26.66 mg/L。就生产茯苓胞内多糖与胞内三萜而言,AS5.137与DB菌株适合作为液体发酵产胞内多糖的出发菌株;Y1,Jingzhou28,Z(z)与Xingpinzhong菌株均较适合作为液体发酵产胞内三萜的出发菌株。     

Morphogenic studies in tissue cultures of the parasiteSantalum album L.

Whole seeds, excised embryos, and excised endosperm ofSantalum album were aseptically cultured with a view to studying seed germination in isolation from the host species, and to establishing callus cultures from both embryo and endosperm for comparative studies et their morphogenesis. Seed germination and seedling formation occurred normally only on modified White's medium supplemented with casein hydrolysate or coconut milk, or with both substances. Neither the excised embryo nor the endosperm grew on any of the culture media tested. However in about 17 per cent seed cultures on White's medium supplemented with 2,4-D, kinetin, and yeast extract, the endosperm degenerated, whereas the embryo callused and subsequently differentiated into innumerable embryoids; eventually the embryoids developed into normal plantlets. Callusing of the endosperm occurred also in seed cultures on four media supplemented variously with 2,4-D, kinetin, and yeast extract. Although the endosperm tissue grew through several passages no organ fornation was observed.     

Various principles of detecting the producers of beta-lactamase inhibitors among soil Actinomycetes

Principles of detecting organisms producing beta-lactamase inhibitors among soil actinomycetes were developed. For detecting such cultures it was recommended to use the Gauze agarized medium No. 1 supplemented with beta-lactam antibiotics. Benzylpenicillin proved to be the most efficient. Various liquid fermentation media for detecting the inhibitory activity of soil actinomycetes were compared. Two media were the most favourable i.e. the glucose-yeast medium No. 18/3 and the soybean-glucose medium with Na2SO4 and CoCl2 No. 20/3. The use of test cultures with relatively low resistance to benzylpenicillin was shown expedient in screening cultures producing beta-lactamase inhibitors. Test cultures with high resistance should be used in more detailed characterization of the selected cultures.     

Directed isolation of Micromonospora generic cultures from moist soils and silts

The data on the study of various soils and substrates for isolation of Micromonospora from them are presented. It was shown that Micromonospora predominated in moist soils and especially in such substrates as silts where their content with respect to the all actinomycetous isolates amounted to 88.9 per cent. In the silts the content of Micromonospora amounted to 66.6--83 per cent, in the sopromely its content was 66 per cent, while in the ordinary non-moist soil its content was from 6 to 11 per cent. Predominance of Micromonospora in silts and moist soils makes its directed search possible.     

Effects of microorganisms on effective oxygen diffusion coefficients and solubilities in fermentation media

Effective oxygen diffusion coefficients and solubilities were measured for submerged cultures of Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. Both effective oxygen diffusion coefficients and solubilities were found to decrease with increasing cell concentrations in the fermentation media. Comparison of the experimental results of effective oxygen diffusion coefficients in fermentation media with values theoretically predicted on the assumption of unpenetrable microbial cells indicates that oxygen molecules diffuse through the cells during the diffusion process. Within the cell concentration range of typical submerged fermentations, the effective oxygen diffusion coefficient of the fermentation media can be described as D(e) = A(1)f + A(2)f(2). In this equation, fis the cell volume fraction and both A(1) and A(2) are functions of the shape of the cells and the ratio of effective oxygen diffusion coefficient in microbial cells to that in the medium.     

The role of succinic acid in the biosynthesis of levorin

The influence of succinic acid as a component of media for biosynthesis of levorin, a polyenic antibiotic was studied. It was shown that with the use of the soybean-corn medium supplemented with succinic acid (0.05-0.4 per cent) the antibiotic content in the fermentation broth was higher than that in the control. The highest stimulating effect (135 per cent) was observed with addition of 0.1 per cent of succinic acid. For providing optimal antibiotic production in the synthetic medium supplemented with succinic acid (0.4 per cent) addition of acetic acid (0.05 per cent) was required. Studies with the soybean-corn medium with and without succinic acid revealed differences in the level of p-aminoacetophenone, an aromatic fragment of the levorin molecule. Under the conditions of the medium with succinic acid the content of p-aminoacetophenone in the mycelium was higher by 10 to 18 per cent as compared to that in the control and depended on the fermentation period. The role of succinic acid in biosynthesis of levorin is discussed.     

Essentiality of Boron for Dinitrogen Fixation in Anabaena sp. PCC 7119

The relationship between the requirement for boron and the form of N supplied in nutrient media to cyanobacterium Anabaena sp. PCC 7119 was investigated. When cells were grown in a medium which contained nitrate or ammonium-N, boron deficiency in the nutrient media did not inhibit growth or change cell composition. However, when cells were dependent on N₂ fixation, the lack of boron inhibited growth (i.e. growth ceased after 96 hours under these conditions). Additionally, boron-deficient cells showed a significant decrease in their content of phycobiliproteins and chlorophyll and accumulated carbohydrates within 24 hours of removing boron from the nutrient media. Inhibition of photosynthetic O₂ evolution accompanied the decrease in photosynthetic pigments. Boron deficiency symptoms were relieved when either boron or combined N was added to boron-deficient cultures. The degree of recovery depended upon the age of the cultures. Assays of nitrogenase activity showed that, after 2 hours of growth, nitrogenase activity of boron-deficient cells was inhibited by 40%. After 24 hours a total inactivation of nitrogenase activity was observed in boron-deficient cells. These results strongly suggest an involvement of boron in N₂ fixation in cyanobacteria.