Effect of glycerin on the biosynthesis of heliomycin by Streptomyces olivocinereus

Glycerol as the sole carbon source was added to the medium or biosynthesis of heliomycin by Streptomyces olivocinereus and the effect of its concentration on the culture growth and antibiotic production was studied. The culture growth and the amount of the antibiotic synthesized per 1 unit of the fermentation broth were limited by glycerol added in quantities of 0.05 to 1 per cent. Further increasing of the glycerol concentration had no significant effect on the culture growth and antibiotic biosynthesis. The amount of the antibiotic synthesized per 1 unit of the mycelial mass relatively slightly depended on the glycerol concentration. The rate of glycerol consumption by the young 24-hour culture in batch fermentations markedly exceeded that of glycerol consumption by the 48-hour culture. The younger mycelium significantly increased its rate of glycerol consumption when the initial concentration was increased whereas the rate of glycerol consumption by the more mature mycelium did not depend on the initial concentration of the carbon source (within 0.5-2 per cent). The rate of heliomycin biosynthesis practically slightly depended on the initial concentration of glycerol.     

Characteristics of the intrinsic luminescence of Actinomyces olivocinereus--producer of heliomycin]

Some characteristics of UV-induced luminescence were studied with Actinomyces olivocinereus producing the antibiotic heliomycin. The luminescence of the growth medium was found to be caused not by heliomycin, but by some other factors. The luminescence of heliomycin in the colonies was quenched as a result of its screening with melanin pigments located in a layer between the aerial and substrate mycelium.     

L-arabinose control of the formation of heliomycin complex synthesized by Streptomyces olivocinereus

The unfavourable effect of arabinose on biosynthesis of heliomycin resembled by its outer appearance the "glucose effect", a well known phenomenon relevant to glucose inhibition of the synthesis of catabolic enzymes of other sugars. Arabinose inhibited glycerol utilization by the cells of S. olivocinereus preadapted to it. The effect of decreased glycerol consumption by the mycelium of S. olivocinereus in the presence of arabinose resembled the effect of the inductor exclusion or/and catabolic inhibition described for glucose. Arabinose inhibited the synthesis of the enzymatic heliomycin-synthesizing complex.     

Effect of L-arabinose and sucrose on the biosynthesis of heliomycin by its producer Streptomyces olivocinereus 11-98

When Streptomyces olivocinereus 11-98 MFU was grown in media containing L-arabinose or sucrose there was observed a converse relation between the culture growth and heliomycin biosynthesis. In media with two carbon sources: L-arabinose and glycerol or sucrose and glycerol at first L-arabinose or sucrose was consumed while the level of glycerol consumption remained low as compared to the control. After exhaustion of the first carbon source there was observed increased consumption of the second one i.e. glycerol. While the medium contained L-arabinose or sucrose the culture growth was mainly provided by these carbon sources and biosynthesis of heliomycin was inhibited. The culture started biosynthesis of heliomycin when L-arabinose or sucrose in the medium was exhausted. Probably control of heliomycin biosynthesis by L-arabinose or sucrose is achieved by catabolic type carbon regulation known as the general mechanism regulating biosynthesis of various antibiotics.     

Kinetic aspects of the relation of the growth of the producer and the biosynthesis of heliomycin depending on the carbon source in the medium

Kinetic parameters of Streptomyces olivocinereus 11-98 growth and biosynthesis of heliomycin were studied. It was shown that carbon sources such as glycerol, mannitol and ramnose were the most favourable for the antibiotic biosynthesis. These carbon sources belonged to the group of substances providing high growth rates of the culture. Ranging of the culture growth rates and antibiotic production levels revealed a set of carbon sources providing a converse relationship between the growth rate and antibiotic biosynthesis i.e. L-arabinose, potassium gluconate, raffinose and sucrose. It was suggested that these compounds were catabolic type regulators of heliomycin biosynthesis.     

Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa

The feasibility of the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids, as a novel approach to reduce their production costs, was demonstrated by the cultivation of Pseudomonas aeruginosa IFO3924. Fairly large amounts of PHAs and rhamnolipids were obtained from the bacterial cells and the culture supernatant, respectively. Decanoate was a more suitable carbon source than ethanol and glucose for the simultaneous production, although glucose was suitable for cell growth without an induction period under pH control. The kind of carbon source affected PHA monomer composition markedly and PHA molecular weight slightly. Monorhamnolipids and dirhamnolipids were included in the rhamnolipids extracted from the culture supernatant using decanoate, glucose, or ethanol as the carbon source. Both PHAs and rhamnolipids were synthesized after the growth phase. PHA content in the cell reached a maximum when the carbon source was exhausted. After exhaustion of the carbon source, PHA content decreased rapidly, but rhamnolipid synthesis, which followed PHA synthesis, continued. This resulted in a time lag for the attainment of maximum levels of PHAs and rhamnolipids. The reusability of the cells used in rhamnolipid production was evaluated in the repeated batch culture of P. aeruginosa IFO3924 for the simultaneous production of PHAs and rhamnolipids. High concentrations of rhamnolipids in the culture supernatant were attained at the end of both the first and second batch cultures. High PHA content was achieved in the resting cells that were finally harvested after the second batch. Simultaneous production of PHAs and rhamnolipids will enhance the availability of valuable biocatalysts of bacterial cells, and dispel the common belief that the production cost of PHAs accumulated intracellularly is almost impossible to become lower than that of cells themselves.     

Immobilized Aspergillus sydowii produces xylanase

Summary Spores ofAspergillus sydowii, immobilized in 2.5% caleium alginate was used as inoculum in batch cultures for production of xylanase enzyme using xylan as the sole carbon source. Partially germinated mycelium from these entrapped spores produced significant amount of the enzyme in a short period of 24 hours and the same inoculum could be used repeatedly for at least 5 cycles with less than 10% loss of enzyme activity.     

Stable sulfur isotope fractionation during the reduction of thiosulfate by Dethiosulfovibrio russensis

Stable sulfur isotope fractionation was investigated during reduction of thiosulfate by growing batch cultures of Dethiosulfovibrio russensis at a cell-specific reduction rate of 2.4 +/- 0.72 fmol cell(-1) d(-1) (28 degrees C). Citrate was used as carbon and energy source. The hydrogen sulfide produced by this sulfur- and thiosulfate-reducing bacterium was depleted in 34S by 11% compared to total thiosulfate sulfur, in agreement with previous results observed for sulfate-reducing bacteria. This indicates the operation of a similar pathway for thiosulfate reduction in these phylogenetically different bacteria.     

Carbon source regulation of cephem antibiotic production by resting cells of Streptomyces clavuligerus and its reversal by protein synthesis inhibitors

The effect of utilizable carbon sources on the production of cephem antibiotics by Streptomyces clavuligerus has been studied. The pattern of utilizable carbon sources was found to be very restricted. Glycerol, maltose and starch supported the most extensive growth. Increasing the initial concentrations of carbon sources decreased both the volumetric and the specific production of cephems. A resting cell system was adopted for this study. Linear production of cephems continued for 4–7 h. The production rate of the resting cell system was higher with mycelia harvested at an early exponential stage than with those harvested at a late stage of growth. Addition of carbon source to the resting cell system decreased the production rate of cephems. This suppressive effect was prevented by the addition of chloramphenicol (or streptomycin) although uptake of carbon source was not inhibited by such a protein synthesis inhibitor.     

Experience in scale-up of homogeneous perfusion culture for hybridomas

A perfusion system for production of monoclonal antibodies was developed using an externally-mounted, hollow-fibre cartridge. The experimental apparatus was operated for 420 h and demonstrated increased steady-state viable cell concentration with increase in perfusion rate. Antibody titres were up to three times those measured for batch cultures and specific antibody productivity was doubled.The procedure was successfully scaled to a 10 dm³ system which produced antibody under conditions of Good Manufacturing Practice (GMP). A calculation of productivity between the scaled perfusion system and 260 dm³ batch cultures resulted in comparable antibody production, whereas the perfusion allowed a halving in medium utilisation. Reactivity assays conducted on the purified antibody from both batch and perfusion cultures showed no evidence of proteolysis or altered antibody activity in the final perfusion product. This study provides additional support for the use of homogeneous perfusion cultures in production of monoclonal antibodies under GMP conditions.     

Biosynthesis of cephamycin by resting cells of Streptomyces lactamdurans L 2/6

In order to investigate the nutritional conditions of cephamycin biosynthesis independently of the biomass growth process, the nutrient limited-resting cell system was used. A replacement medium eliminating cell multiplication was developed. The presence of Mg2+, carbon source and nitrogen source was necessary for cephamycin production by resting cells of Streptomyces lactamdurans L 2/6. Maximum antibiotic production was obtained when maltose, saccharose, and fructose were used as carbon source, and L-asparagine as nitrogen source. An inhibitory effect on the process was exerted by the calcium ions. There was no visible inhibition of cephamycin biosynthesis by inorganic phosphate ions in concentration up to 100 mM.     

Exploring low-cost carbon sources for microbial lipids production by fed-batch cultivation of <Emphasis Type="Italic">Cryptococcus albidus</Emphasis>

Volatile fatty acids (VFAs), acetic acid, acetates, and ethanol were used as carbon sources for the production of microbial lipids using Cryptococcus albidus in batch cultures. C. albidus utilized organic acids less than glucose in the production of lipids, resulting in a lipid yield coefficient on VFAs of 0.125 g/g. In a two-stage batch culture, the lipid content increased to 43.8% (w/w) when VFAs were used as the sole carbon source in the second stage, which was two times higher than that of the batch culture. Furthermore, a 192 h, two-stage fed-batch cultivation of C. albidus produced a dry cell weight, lipid concentration, and lipid content of 26.4 g/L, 14.5 g/L, and 55.1% (w/w), respectively. The fed-batch culture model used in this study featured pure VFA solutions, with intermittent feeding, under oxygen-enriched air supply conditions. This study investigated several alternative carbon sources to reduce the cost of microbial lipids production and proved the feasibility of using VFAs as the carbon source for the provision of a high lipid content and productivity.     

Bioenergetics and end-product regulation of Clostridium thermosaccharolyticum in response to nutrient limitation

Fermentation of xylose by Clostridium thermosaccharolyticum was studied in batch and continuous culture in which the limiting nutrient was either xylose, phosphate, or ammonia. Transient results obtained in continuous cultures with batch grown inoculum and progressively higher feed substrate concentrations exhibited ethanol selectivities (moles ethanol/moles other products) in excess of 11. The hypothesis that this high ethanol selectivity was a general response to mineral nutrient limitation was tested but could not be supported. Growth and substrate consumption were related by the equation q(s)(1 - Y(x) (c))G(ATP) = (mu/Y(ATP) (max)) + m, with q(s) the specific rate of xylose consumption (moles xylose/hour . g cells), Y(x) (c) the carbon based cell yield (g cell carbon/g substrate carbon), G(ATP) the ATP gain (moles ATP produces/mol substrate catabolized), mu the specific growth rate (1/h), Y(ATP) (max) the ATP-based cell yield (g cells/mol ATP), and m the maintenance coefficient (moles ATP/hour . g cells). Y(ATP) (max) was found to be 11.6 g cells/mol ATP, and m 9.3 mol ATP/hour . g cells for growth on defined medium. Different responses to nutrient limitation were observed depending on the mode of cultivation. Batch and immobilized cell continuous cultures decreased G(ATP) by initiating production of the secondary metabolites, propanediol, and in some cases, D-lactate; in addition, batch cultures increased the fractional allocation of ATP to maintenance and/or wastage. Nitrogen-limited continuous free-cell cultures maintained a constant cell yield, whereas phosphate-limited continuous free-cell cultures did not. In the case of phosphate limitation, the decreased ATP demand associated with the lowered cell yield was accompanied by an increased rate of ATP consumption for maintenance and/or wastage. Neither nitrogen or phosphorus-limited continuous free-cell cultures exhibited an altered G(ATP) in response to mineral nutrient limitation, and neither produced secondary metabolites. (c) 1993 John Wiley & Sons, Inc.     

Production of Chitinases and beta-1,3-Glucanases by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani

The in vitro production of chitinases and beta-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO(3). The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for beta-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and beta-1,3-glucanases were detected. S. elegans culture filtrates, possessing beta-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium.     

Polyhydroxybutyrate production by Saccharophagus degradans using raw starch as carbon source

Biosynthesis of poly(3‐hydroxybutyrate) (PHB) from raw starch as the carbon source by the polysaccharide‐digesting bacteria Saccharophagus degradans was investigated in a fed‐batch culture. The production and properties of the PHB synthesized from starch were compared to those obtained using glucose as carbon source. In fed‐batch cultures, S. degradans accumulated 21.35 and 17.46% of PHB, using glucose or starch as carbon source, respectively. The physical properties of the biopolymer produced from each carbon source were similar between them. Molecular mass, melting temperature and heat of fusion were 54.23 kDa, 165.61°C and 59.59 J/g, respectively, using glucose; and 57.07 kDa, 174.31°C and 67.66 J/g, respectively, using starch. This is the first work describing the capability of S. degradans to utilize raw starch as the sole carbon source for the production of PHB.     

Improved podophyllotoxin production by transformed cultures of Linum album

Various cell and hairy root cultures of L. album were developed and analyzed for podophyllotoxin content. Transformed callus and hairy root cultures developed from infection of stem portions of in vitro-germinated L. album plant with Agrobacterium rhizogenes NCIM 5140 strain were selected on the basis of high podophyllotoxin content and growth. Based on the integration of Ri T(L)-DNA and T(R)-DNA, integration of only the ags and not the rol gene in transformed cell culture indicated fragmented integration pattern. The effect of different cultivation media and carbon source on growth and podophyllotoxin production were studied in shake-flask suspension cultures. Detailed batch growth and production kinetics with sugar consumption profile were also established. Maximum volumetric productivity of 4.40 and 2.75 mg/L per day was obtained in cell suspension and hairy root cultures, respectively.     

Improvement of acid phosphatase production by immobilization of Humicola lutea mycelium in polyurethane sponge

Acid phosphatase production by the fungus Humicola lutea 120-5, immobilized in polyurethane sponge, was studied under semicontinuous shake flask fermentation and compared to the enzyme secretion by free cells. The effect of parameters such as the carrier content and the duration of the batch in repeated batch experiments on the phosphatase production half-life was investigated. The best results were obtained with 1.0 g of sponge cubes (about 1.0 cm per side) per culture flask using 72 h runs. In these conditions the half-life of enzyme production by immobilized biocatalyst was 15 sequential cycles (45 days) compared to three cycles (9 days) for the free mycelium. The maximal phosphatase titre registered in free cell fermentation was 2500 U/l (i.e. 100%), while the relative enzyme activity of the optimal immobilized system was over 100% during the whole half-life time of 45 days. Significant improvement (200–215%) in the yield was observed in one-third of this period or 15 days. The supernatant medium obtained at any stage of the repeated batch cultures did not contain free cells and, due to the low pH (3.0–3.5), the whole process was carried out without any bacterial contamination. In comparison with free cell fermentation, the significant improvement of the acid phosphatase production by polyurethane sponge-immobilized H. lutea mycelium as well as its operation stability was confirmed by scanning electron microscopy.     

Cell growth and alpha-amylase production characteristics of Bacillus amyloliquefaciens

Growth and alpha-amylase production characteristics of Bacillus amyloliquefaciens strain F (ATCC 23350) in batch cultures are examined using glucose or maltose as the carbon source. While the cell growth is rapid when glucose is used as the carbon source, higher cell mass, higher total and specific enzyme activities, and higher enzyme production rates are obtained when maltose is used as the carbon source. The overall specific enzyme activity decreases with an increase in the initial concentration of carbon source. The oxygen requirement and carbon dioxide generation vary linearly with the maximum amount of cell mass produced. For experiments conducted using glucose as the carbon source, the kinetics of cell growth and glucose consumption are described using a special form of the Vavilin equation. For a given amount of initial carbon source, the enzyme synthesis capability is retained by the microorganism, although at a substantially reduced level, under severe oxygen limitation.     

海藻酸钙固定赤霉菌菌丝产素的研究

将赤霉菌丝固定在海藻酸钙微球中进行连续发酵,考察产赤霉素情况。对海藻酸钠和钙盐浓度固定赤霉菌菌丝的微球稳定性进行初步研究,讨论了固定不同菌龄的赤霉菌微球在不同葡萄糖浓度下的产素能力及菌丝生长能力。实验表明:菌丝微球较稳定的固定条件是菌丝8 g/L、海藻酸钠浓度3 g/L和钙离子浓度3 mol/L;摇瓶发酵72 h,90 h的菌丝微球中菌丝营养生长基本停止,当培养液葡萄糖浓度为2 g/L时,赤霉素终浓度为1 145.5 μg/ml,比生产速率为4.61×10^-3/h;在该条件下固定菌丝球的床层式连续发酵,赤霉素比生产速率为4.82×10^-3/h,是相应分批发酵过程中最大赤霉素生产速率的1.87倍。     

Continuous culture with complete cell recycle: cultivation of Pseudomonas cepacia ATCC 29351 on salicylate for production of salicylate hydroxylase

Summary Pseudomonas cepacia ATCC 29351 was cultivated on salicylate as sole carbon source in a system with complete cell recycling by means of a hollow fiber microfilter. Comparisons with batch cultures and continuous cultures are made with respect to cell densities, yield coefficients, specific enzyme activities and volumetric productivities. In batch cultures the toxicity of the salicylate limits the cell densities to below 1 g/l. In a recycling cultivation, a cell concentration of 15.1 g/l was obtained and the volumetric productivity of the cell mass was increased by a factor of 3. No serious effects on the cells were noted.