Effect of the culture medium composition on bacitracin synthesis and sporulation in Bacillus lichenformis 28 KA

The effect of the culture medium composition on the bacitracin synthesis and sporulation in Bacillus licheniformis 28 KA was being studied. During the producer's growth on a medium with tripton a biphasic pattern of the process was observed, i.e. a rapid growth of the bacterial biomass was accompanied by the production of insignificant amounts of the antibiotic. The maximum efficiency of the antibiotic synthesis coincides with the end of the exponential growth phase and the onset of sporification. The efficiency of sporification amounts up to 95% when the above mentioned medium is used. The substitution of glutamate for trypton results in a dramatic deceleration of the bacterial growth and biomass accumulation, and the process of the antibiotic biosynthesis ceases to be biphasic, i. e. the antibiotic is synthesized during the trophophase. Under the conditions when the antibiotic production drops to zero by the middle of the exponential growth phase, sporulation is virtually suppressed.     

Effect of inoculum parameters on the specificity of secondary synthesis in cultures producing novobiocin and mycoheptin]

The impact of the vegetative inoculum parameters on specificity of the secondary synthesis in the cultures producing novobiocin and mycoheptin was studied. During the study the fermentation conditions were varied by using the vegetative inoculum differing in the respiration rate after its transfer to the fermentation medium. To show the decisive role of the inoculum parameters in regulation of the specificity of the secondary synthesis, the dynamics of accumulation of certain metabolites forming from glucose along with the main antibiotic and the activity of the key enzymes of the carbohydrate metabolism during the culture growth in the fermentation media were studied. It was found that the specificity of the secondary synthesis with respect to certain metabolites was defined by the intensity of carbohydrate metabolism, i. e. the ratio of the activity of enzymes of glycolysis and the pentosephosphate pathway. In this regard, the inoculum with the maximum respiration rate in an amount of 10 to 20 per cent promoted the highest productivity of the mycelium by the synthesis of novobiocin and mycoheptin while the rate of accumulation of fatty acids, carbohydrates and phenol compounds (for Streptomyces spheroides) and mycopentene (for Streptoverticillium mycoheptinicum) decreased.     

Formation of antibiotic 2562 by a culture of the actinomycete, Streptomyces griseovariabilis]

An actinomycetous culture 2562 inhibiting the growth of gramnegative bacteria was isolated from a soil sample. The culture was classified as Streptomyces griseovariabilis. It was found that culture 2562 produced an antibiotic belonging to the group of novobiocin. It consists of 2 components. One of them is identical to chlorobiocin and the other is a minor component of this group. Some parameters of the antibiotic complex production by strain 2562 under submerged conditions were studied. Nutrient media providing the predominant biosynthesis of the first (main) or the second component of the antibiotic were developed.     

Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris

A Pichia pastoris system was used to express a single-chain antibody (scFv) targeted against Mamestra configurata (bertha armyworm) serpins. To improve scFv production we examined parameters such as proteinase activity, temperature, cell density, osmotic stress, medium composition, pH, and reiterative induction. P. pastoris was found to express several proteases; however, adjustment of medium pH to limit their activity did not correlate with increased scFv recovery. Induction medium pH values of 6.5-8.0 were most conducive to scFv production, despite significant differences in cell growth rates. Increasing inoculum density limited growth potential but gave rise to higher levels of scFv production. Three factors, medium composition, pre-induction osmotic stress, and temperature, had the greatest effects on protein production. Supplementation of the induction medium with arganine, casamino acids, or EDTA increased scFv production several fold, as did cultivation under osmotic stress conditions during pre-induction biomass accumulation. Incubation at 15 versus 30 degrees C extended the period whereby cells were capable of producing scFv from 1 to 7 days. Under optimal conditions, yeast cultures yielded 25 mg/L of functional scFv and could be subject to five reiterative inductions.     

Selective action of novobiocin on Act. levoris, the producer of levorin and levoristatin]

The effect of sodium novobiocin on Act. levoris, strain LIA 0868 producing levorin and levoristatin was studied. High lethal effect of the antibiotic on Act. levoris was found. The effect increased with a rise in the antibiotic concentration in the agar from 2 to 6 lambda/ml. The inhibitory effect of novobiocin on Act, levoris was evident from a marked increase in the number of morphologically changed colonies with a low level of levorin production. The selective effect of novobiocin on the organism producing levorin and levoristatin was evident from selection of variants with high levels of levoristatin production on media containing novobiocin.     

S-Adenosylmethionine (SAM) and antibiotic biosynthesis: effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces

The production of antibiotics in different Streptomyces strains has been reported to be stimulated by the external addition of S-adenosylmethionine (SAM) and by overexpression of the SAM synthetase gene metK. We investigated the influence of SAM addition, and of the expression of SAM biosynthetic genes, on the production of the aminocoumarin antibiotic novobiocin in the heterologous producer strain Streptomyces coelicolor M512 (nov-BG1). External addition of SAM did not influence novobiocin accumulation. However, overexpression of a SAM synthase gene stimulated novobiocin formation, concomitant with an increase of the intracellular SAM concentration. Streptomyces genomes contain orthologs of all genes required for the SAM cycle known from mammals. In contrast, most other bacteria use a different cycle for SAM regeneration. Three secondary metabolic gene clusters, coding for the biosynthesis of structurally very different antibiotics in different Streptomyces strains, were found to contain an operon comprising all five putative genes of the SAM cycle. We cloned one of these operons into an expression plasmid, under control of a strong constitutive promoter. However, transformation of the heterologous novobiocin producer strain with this plasmid did not stimulate novobiocin production, but rather showed a detrimental effect on cell viability in the stationary phase and strongly reduced novobiocin accumulation.     

Effect of light emitting diodes on the cultivation of Spirulina platensis using NPK‐10:26:26 complex fertilizer

NPK‐10:26:26 complex fertilizer based culture medium was studied for the mass production of Spirulina platensis using different light emitting diodes (LEDs). First, cultivation was carried out under white LED to formulate the optimum fertilizer loading for which Spirulina growth was maximized. Optimum composition for newly formulated fertilizer medium was NPK fertilizer ?0.76 g L^?1 and sodium bicarbonate ?10.0 g L^?1 and corresponding biomass productivity was found to be 76.67 mg L^?1 day^?1. The effect of different LEDs (for example, blue, white, red, green and yellow) on Spirulina growth kinetics and the accumulation of chlorophyll, protein and lipid content was determined using the optimum NPK fertilizer medium. Kinetic parameters (i.e., biomass productivity, maximum specific growth rate, maximum biomass concentration, nitrogen‐to‐biomass conversion factor and phosphorus‐to‐biomass conversion factor) and chlorophyll accumulation were affected by the use of different LEDs and follow the following trend: blue > white > red > green > yellow, whereas protein and lipid accumulation was almost independent of LEDs used. Elemental C, N, P and K concentrations were measured to find the effects of nutrients for the growth of Spirulina platensis. Physicochemical parameters (pH and conductivity) were also monitored during biomass growth under different LEDs. Finally, biomass growth using NPK‐10:26:26 fertilizer under different LEDs was compared with standard Zarrouk medium and better growth results were obtained using optimally formulated NPK‐10:26:26 fertilizer medium.     

Theoretical Design of Continuous Antibiotic Fermentation Units

A graphic method of predicting antibiotic yields in continuous flow reactors is presented and discussed using the novobiocin fermentation as a model process. Extension to other antibiotic fermentations and steroid bioconversions is emphasized. In the case of the novobiocin fermentation it was concluded that a combination of one growth stage and one or two antibiotic production stages would be the most economic reactor system to conduct such a fermentation on a continuous basis.     

Production of an antifungal antibiotic by Streptomyces aburaviensis 1DA-28

A broad-spectrum antifungal Streptomyces isolate, 1DA-28, from Indian soil has been characterized and identified as Streptomyces aburaviensis var. ablastmyceticus (MTCC 2469). Nutritional and cultural conditions for the production of antibiotic by this organism under shake-flask conditions have been determined. Antibiotic production in synthetic medium reached the maximum on the 5th day of incubation at 30 degreesC. Glucose and starch were found to be the best carbon sources while NH4NO3 was preferred as nitrogen source. Optimum temperature and pH for antibiotic production were 32 degreesC and 7.4, respectively. Phosphate at a concentration sub-optimal for growth enhanced antibiotic production. Supplementation of medium with casein hydrolysate improved both growth and antibiotic titre but yeast extract exhibited marked inhibition.     

Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.)

In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30?months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87?mg?l^?1) and hyoscyamine (107.7?mg 1^?1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60?g/l sucrose or Gamborg??s B5 with 40?g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine.     

Action of migillin on its own producer, Asp. fumigatus]

Addition of migillin to the nutrient medium at early stages of the fungus development induced at one hand inhibition of the fungus growth during the first 24-48 hours and stimulation of the biomass accumulation during the following hours of the development and on the other hand it induced inactivation of migillin added and inhibition of production of the "own" antibiotic. The migillin effect was accompanied by a delay in liberation of the substance with adsorption at 260nm from the mycelium.     

Developmental cycle and the cytomorphological characteristics of the oleandomycin producer Streptomyces antibioticus

The developmental cycle and cytomorphological features of the industrial strain OL-1 and its variant 0968 of the oleandomycin-producing organism were studied. Variant 0968 was obtained as a result of exposure of the spores of strain OL-1 to UV light. When grown under submerged conditions in flasks with the rich medium, the strains were characterized by a complete developmental cycle consisting of three generations of the hyphae. Every generation had a tendency for formation of submerged spores. The UV-induced variant differed from the industrial strain by higher levels of the antibiotic accumulation which correlated with higher rates of the spore germination. The strains were characterized by polymorphism of the mycelium and formation of submerged spores during their cultivation which is likely to prolong the antibiotic synthesis from 120 to 216 hours from the inoculation moment. The long-term selection of the oleandomycin-producing organism on the rich medium markedly changed the culture genotype and resulted in significant changes in the developmental cycle under submerged cultivation conditions. The data may be used for the microscopic control of the process of oleandomycin production.     

Effect of iron and aeration on superoxide dismutase and catalase activity of PHB-producing Azotobacter chroococcum

The effect of aeration level and iron concentration on Azotobacter chroococcum 23 growth, PHB accumulation and antioxidative enzyme activities was investigated in shake flask experiments. Biomass yield and carbon source conversation coefficients increased in the presence of iron in the growth medium and under decreased aeration. The highest biomass production was observed for the culture grown in a medium with 36 μM of initial iron concentration and moderate aeration level. The highest PHB accumulation level (70–72% from cell dry weight) under our experimental conditions was observed at decreased aeration in the growth medium with 180 μM of initial iron concentration. Results obtained prove that both aeration level and iron supply have a marked influence on the activity of SOD and catalase. Bearing in mind the necessity of iron for the synthesis of both enzymes, only catalase showed a specific dependence on the intracellular iron accumulation level.     

Beta-lactam and aminoglycoside production from streptomycetes

Streptomyces cattleya, S. fradiae and S. griseus produced different amounts of growth when cultured sequentially through sporulation, vegetative and antibiotic production media. Only S. griseus grew well on all three types of medium. Streptomyces cattleya grew poorly on both sporulation and vegetative media. Growth was 1.6 and 8.0 mg/1/h respectively. For all three species, biomass yield in the final antibiotic production medium was dependent on amount of inoculum. Antibiotic yields were obtained only from production media. Under slow growth conditions L-cysteine and L-valine supplementation stimulated S. cattleya beta-lactam production, giving 1000 micrograms/ml beta-lactam equivalents compared with 45 micrograms/ml beta-lactam equivalents for no supplementation. For aminoglycosides the agar well diffusion bioassay was more sensitive towards the hydrochloride than the neutral salt. Paper chromatography confirmed the main antibiotic classes. RF values for replicate samples indicated aminoglycoside homogeneity and beta-lactam heterogeneity.     

Effects of Temperature and pH on Cell Growth of a Flocculating Microalga (Chlorella vulgaris) JSC-7

微藻是可广泛用于健康食品及水产养殖的饵料,同时,微藻细胞内积累的油脂可作为可再生生物燃料,因此微藻的生长和代谢受到广泛关注。温度和pH对微藻的生物量积累有很大影响,考察不同温度和pH条件下微藻细胞的生长有助于寻找最佳的条件进行微藻的培养。自絮凝小球藻JSC-7（Chlorella vulgaris JSC-7）可实现自沉降采收,有利于降低微藻生产成本,优化其生长条件对更好地利用该微藻具有重要意义。考察了温度（22∽40℃）及pH（6.0∽10.0）对其细胞生长、叶绿素含量和油脂产量的影响。在所选取的温度及pH范围内,JSC-7细胞均可生长,显示该藻种可以适应广泛的温度和pH条件。适合细胞生长的温度依次为31℃〉28℃〉35℃〉25℃,pH依次为7.0〉8.0〉6.0。pH 8.0时生物量和油脂的积累量最多,说明该藻株在弱碱条件下更适合生长和产油。当温度为31℃、pH为7.0时,可获得最高的生长量（OD690=0.941）、叶绿素含量（19 mg/L）及油脂产量（39.07%/克干重）。     

Effect of amino acids on the growth of a B. polymyza 1538 culture and the biosynthesis of polymyxin B]

The effect of a number of the most important amino acids on the growth of B. polymyxa 1538 and polymyxin B biosynthesis was studied. It was found that all of the amino acids tested except D-L-threonin and L-alpha-gamma-DABA inhibited the initial growth of the organism. D-L-threonine and L-alpha-gamma-DABA had some stimulating effect on the culture growth without affecting the final accumulation of the biomass. The antibiotic synthesis proceeded more vigorously when the medium contained D-L-threonine, L-alpha-gamma-DABA, L-asparagine, L-proline, L-glutamine, D-L-asparaginic acid and L-glutamic acid in the concentrations tested. Neither of the tested amino acids used alone or in combination provided the levels of polymyxin B biosynthesis observed on the media containing rich sources of organic nitrogen.     

Optimization of culturing conditions for the production of biomass and phenolics from adventitious roots ofEchinacea angustifolia

We investigated different concentrations of auxins (1AA, IBA, NAA), the strength of the MS medium, sucrose and ammonium/nitrate contents, initial medium pH, and inoculum size to determine their effects on biomass increase and the accumulation of total phenols and flavonoids in adventitious roots ofEchinacea angustifolia. These roots were cultured under darkness in shake flasks for 4 weeks. IBA proved the best auxin for inducing root proliferation. Root growth was inhibited when the initial pH was maintained below 5.0 or above 6.0. Nitrate, rather than ammonium, was more necessary for root growth and phenolics accumulations. Overall, biomass increased and total phenol and flavonoid contents were maximized under the following conditions: half-strength MS medium supplemented with 2 mg L^-1 IBA, 5% (w/v) sucrose, 5:25 (mM) ammonium/nitrate ratio, pH adjusted to 6.0 before autoclaving, and an inoculum size of 10 g L^-1 FW. These results indicate that the type ofin vitro environment strongly affects growth and the accumulation of phenolics from adventitious root cultures ofE. angustifolia. Such optimization is beneficial to large-scale production of biomass and secondary metabolites in that species.     

Use of Epifluorescence Microscopy in Studies of the Germination and Recovery of Thermoactinomycetes

Spore germination and growth of thermoactinomycetes were observed by epifluorescence microscopy. Each of the principal stages in germination was recognized and found to correspond to changes in phase-contrast appearance commonly monitored during endospore germination. The effects of novobiocin and nalidixic acid on germination of Thermoactinomyces vulgaris and T. thalpophilus were studied by using epifluorescence microscopy. Outgrowth but not initiation was inhibited by 100 μg of nalidixic acid per ml and 50 μg of novobiocin per ml. When each of the compounds, at various concentrations, was incorporated into a medium for thermoactinomycete recovery, the antibiotics were found to inhibit colony development. Samples of water and sediment incubated in a growth medium containing novobiocin and selective for thermoactinomycete species were examined by epifluorescence microscopy for total numbers of outgrown spores. Direct viable counts of outgrown spores indicated that the standard plate count enumerated less than 10% of the viable population of thermoactinomycetes.     

Degeneration of Streptomyces niveus with Repeated Transfers

Novobiocin production by Streptomyces niveus decreased drastically as the culture was transferred at regular intervals under both sporulating and nonsporulating conditions. Addition of degenerated live mycelium as second inoculum to shake flask fermentations already inoculated with a high-producing strain resulted in sharply depressed novobiocin formation. Fractionated medium of low-producing strain containing either no cells or dead cells had no adverse effect on the antibiotic yield of the high-producing mycelium. It appears that the low-producing mycelium was outgrowing the high-producing mycelium. A study of the growth rates of the two types of mycelium in a clear broth medium indicated no differences in generation time. However, the low-producing strain proved to have a higher efficiency of carbohydrate utilization, thus overgrowing the high-producing strain. It was speculated that culture instability of S. niveus is due to heterocaryosis.     

Heterologous expression of Vitreoscilla hemoglobin (VHb) and cultivation conditions affect the alkaloid profile of Hyoscyamus muticus hairy roots

Fast-growing hairy root cultures of Hyoscyamus muticus induced by Agrobacterium rhizogenes offer a potential production system for tropane alkaloids. Oxygen deficiency has been shown to limit growth and biomass accumulation of hairy roots, whereas little experimental data is available on the effect of oxygen on alkaloid production. We have investigated the effect of Vitreoscilla hemoglobin (VHb) expression and cultivation conditions on the complete alkaloid profile of H. muticus hairy roots in shake flasks and in a laboratory scale bioreactor. We optimized the growth medium composition and studied the effects of sucrose, ammonium, nitrate, and phosphate on growth and alkaloid production. Maximum biomass accumulation was achieved with the highest and maximum hyoscyamine content with the lowest sucrose concentration. The optimum nitrate concentration for growth was higher for the VHb line than the control. Neither VHb expression nor aeration improved the hyoscyamine content significantly, thus suggesting that hyoscyamine biosynthesis is not limited by oxygen availability. Interestingly, the effect of VHb expression on the alkaloid profile was slightly different from that of aeration. VHb expression did not affect the concentrations of cuscohygrine, which was increased by aeration. Therefore, the effect of VHb is probably not related only to its ability to increase the intracellular effective oxygen concentration.