Production ofPaecilomyces Fumosoroseus conidia in submerged culture

Mexican isolates ofPaecilomyces fumosoroseus (Wize) Brown & Smith virulent to nymphs and adults ofBemisia tabaci Gennadius (Homoptera: Aleyrodidae) were screened in terms of spore production in submerged culture. Effects of light, temperature stress and yeast extract on sporulation were studied. Cycles of 12 hours light/12 hours dark increased spore production as well as an incubation for 24 hours at 37°C prior to incubation at 30°C. In absence of organic nitrogen both fungal growth and sporulation were very low. Spore production in fermentors with a culture media of a C:N ratio of 25 was doubled as compared to a media with a C:N ratio of 11. Both conidia and blastospores were produced. Production of conidia directly from blastospores through microcyclic sporulation was observed. The proportion of conidia obtained under optimal conditions was 88.8%. Submerged culture ofP. fumosoroseus seemed advantageous compared to ricefilled plastic bags production method because of shorter fermentation times, higher spore yields and substantially higher volumetric spore productivity. Results indicated that careful manipulation of nutritional and environmental conditions allowed for production of conidia during submerged growth ofP. fumosoroseus, microcyclic sporulation being induced under a set of environmental conditions including temperature stress and nutrients limitation.     

Effect of culture conditions on growth and sporulation ofBacillus thuringiensis subsp.israelensis and development of media for production of the protein crystal endotoxin

Summary The influence of medium composition on the inoculum and production stages of theBacillus thuringiensis subsp.israelensis bioinsecticide fermentation was investigated. Media which inhibited sporulation were selected for inoculum development stages. Bioinsecticide production media were designed to produce high cell counts and >90% sporulation in a 48h fermentation. Maximum insecticidal activity occurred at the point of maximum bacterial cell lysis/spore release. A process involving two inoculum stages and a 48h production stage in a 40 l fermenter yielded a viable cell count of 6.5 x 10⁹/ml with greater than 95% sporulation. Good correlation existed between spore counts and bioinsecticide activity.     

Conidiation ofTrichoderma atroviride isolate during submerged cultivation in a laboratory stirred-tank fermenter

Conditions for conidiation of a natural isolate of Trichoderma atroviride during submerged cultivation in Erlenmeyer flasks and in a laboratory stirred-tank fermenter were optimized. From the simple sugars tested, cellobiose was the best substrate for conidia production while cellulose fines from paper mill waste proved to be a suitable cheap complex carbon source. Optimum temperature for conidiation was 24-26 degrees C, and the required dissolved oxygen level was > 40% saturation. After initial slight decrease during the 1st d after inoculation, the pH of the culture medium constantly increased throughout the sporulation period. Attempts to regulate the pH during fermentation did not improve the spore yields. The most intense formation of conidia took place between 2nd and 3rd d of growth and the overall volumetric productivity of conidia was 4.1-8.2 x 10(9) conidia per L/h.     

Sporulation of Streptomyces griseus in submerged culture.

A wild-type strain of Streptomyces griseus forms spores both on solid media (aerial spores) and in liquid culture (submerged spores). Both spore types are highly resistant to sonication, but only aerial spores are resistant to lysozyme digestion. Electron micrographs suggest that lysozyme sensitivity may result from the thinner walls of the submerged spores. Studies of the life cycle indicate that neither streptomycin excretion nor extracellular protease activity is required for sporulation: the analysis of mutants, however, suggests that antibiotic production may be correlated with the ability to sporulate. A method was devised to induce the rapid sporulation of S. griseus in a submerged culture. This method, which depends on nutrient deprivation, was used to determine that either ammonia or phosphate starvation can trigger sporulation and that the enzyme glutamine synthetase may be useful as a sporulation marker after phosphate deprivation.     

A new two-phase kinetic model of sporulation of Clonostachys rosea in a new solid-state fermentation reactor

A new two-phase kinetic model of sporulation of Clonostachys rosea in a new solid-state fermentation (SSF) reactor was proposed. The model including exponential and logistic models was applied to study the simultaneous effect of temperature, initial moisture content, medium thickness and surface porosity of the plastic membrane on C. rosea sporulation. The model fits experimental data very well and allows accurate predictions of spore production. The maximum spore production achieved 3.360 × 10¹⁰ (spores/gDM), about 10 times greater than that in traditional SSF reactor(data not shown). The new reactor can provide two times sporulation surface area. Moisture content can be adjusted by changing the surface porosity to meet the spore production. Two mixings carried out during fermentation makes medium loose and results in a mass of new sporulation surface area. Therefore, the new SSF reactor would have great potential for application in bulk spore production of fungal biocontrol agents.     

Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus

The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L⁻¹. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m⁻² s⁻¹ and a spore concentration of 1,320 spore cm⁻² produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible.     

The Sporulation of Penicillium notatum Westling in Submerged Liquid Culture: I. THE EFFECT OF CALCIUM AND NUTRIENTS ON SPORULATION INTENSITY

The growth and sporulation of Penicillium notatum Westling inshaken submerged culture has been studied. Sporulation occurredonly when calcium ions were added to the basal sucrose-mineralsalts medium. Concentrations of 5 p.p.m. or less of calciumwere ineffective and at least 35 p.p.m was necessary for themaximum degree of sporulation. The replacement of calcium inthe medium by strontium or barium enabled sporulation to occur,although these metal ions were less effective than calcium inthe order. The calcium requirement for sporulation diminishedas the total nutrient concentration was lowered and was similarlyreduced if the concentration of the nitrogen source alone (sodiumnitrate) was lowered, whereas reductions in the levels of othersingle nutrients caused relatively small alterations in thecalcium requirement. Lowering the concentration of the nitrogensource caused increased sporulation in the presence of bothlimiting and non-limiting levels of calcium.     

Characterization study of the sporulation kinetics of Bacillus thuringiensis

A wild-type and an rDNA strain of Bacillus thuringiensis were cultured in a net-draft-tube modified 20-L airlift bioreactor. A comparison of the sporulation patterns suggests that the early sporulation strain has a lower final spore count. Results from off-gas analysis suggests that the CO(2) profile could be an alternative indication to spore counts for the examination of fermentation performance or even the mortality in bioassay of the cultivation product. The difference in mortality tests exhibited by the microorganism was attributed to different patterns of sporulation as well as different levels of gene control inside the cell itself. The sporulation kinetics of B. thuringiensis was simulated by a simple modified Hill equation, where the initial glucose concentration could affect the timing of the onset of sporulation. The equation matches well with the experimental sporulation data for B. thuringiensis in both wild-type and rDNA strains.     

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.     

Production of Calcium Gluconate by Penicillium chrysogenum in Submerged Culture

The waste mycelium of Penicillium chrysogenum HA-10 (obtained at the end of penicillin fermentation), or a 24-hr-old freshly grown vegetative inoculum of this organism, was found to utilize glucose for the production of calcium gluconate by submerged fermentation in shake flasks. After 72 to 96 hr of fermentation at 24 C, 90 to 95% of the reducing sugar from the 15% glucose medium was converted to calcium gluconate. Reuse of the mycelium for successive experiments reduced the fermentation period to 72 hr or less because of an enhancement of glucose utilization. Ten successive batches of 15% glucose medium were fermented by the reuse method. Fermentation media containing up to 30% glucose could be used, provided boric acid was added to prevent the precipitation of calcium gluconate formed. We found that 30% hydrol (a by-product of glucose manufacture containing 50 to 55% reducing sugar), when used in place of glucose in the fermentation medium, inhibited the rate of glucose utilization. However, this effect was partially reversed by pretreatment of hydrol with 2 to 4% activated charcoal before addition to the fermentation medium.     

Efficient Induction of Sporulation of Dictyostelium Prespore Cells by 8-Bromocyclic AMP under Both Submerged- and Shaken-Culture Conditions and Involvement of Protein Kinase(s) in Its Action

It is important to establish an experimental system in which sporulation of Dictyostelium can be induced at high cell densities to obtain sufficient amounts of materials for analysis of the molecular events leading to sporulation. 8-Bromo cAMP (Br-cAMP) was found to be effective for inducing sporulation by prespore cells of Dictyostelium discoideum NC4 at high cell densities under both submerged- and shaken-culture conditions. Ultrastructural studies revealed that the morphological changes associated with this sporulation proceeded normally in vitro. The effect of Br-cAMP was inhibited by two protein kinase inhibitors, K252a and staurosporine. Protein-phosphorylation experiments showed that Br-cAMP induced increased phosphorylations of a 96 kDa spore coat protein (SP96) and a protein with a mobility corresponding to a molecular weight of 50 kDa (p50-4). The protein kinase inhibitor K252a blocked the phosphorylations of both proteins. These proteins may be targets of particular protein kinase(s) that is activated by Br-cAMP. These findings indicate that the present experimental system should be useful for elucidating the molecular events involved in normal sporulation and the mechanism by which Br-cAMP induces sporulation in vitro.     

Antigenic characterization of Penicillium camemberti and related common cheese contaminants

Twenty-four isolates of Penicillium (including a green-spored mutant from a French Brie cheese, Penicillium camemberti) with a proposed relationship to the white cheese mold P. camemberti were investigated by immunological procedures. These penicillia, which are representative of species that have caused considerable taxonomic confusion, had common micromorphology (terverticillate penicilli with rough and smooth stipes and smooth ellipsoidal to subglobose [(3 to 5) X 2 1/2 to 4 1/2 microns] conidia); growth rates; good growth on creatine sucrose agar, cheese, and other products with a high amount of protein and lipid as a primary habitat; production (with the exception of Penicillium solitum) of cyclopiazonic acid; and the ability to grow at low temperatures and water activities. The isolates that were investigated proved to be strictly antigenically related. Absorbed antiserum of the green-spored mutant of P. camemberti showed a specific precipitin band when tested by immunodiffusion either with its homologous reference antigen or with the exoantigens obtained from different isolates. The precipitin band was not present in any P. camemberti starter culture but in many unwanted cheese contaminants. The precipitin band can be used in the purity control of P. camemberti starter culture spore preparations. Analysis of the exoantigens of all the cultures by reversed phase high-performance liquid chromatography allowed us to subdivide these penicillia into nine groups below the species level. The results indicate that P. commune Thom is the wild-type ancestor of P. camemberti.     

Cortex content of asporogenous mutants of Bacillus subtilis.

A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content. During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation. Spore cortex contents of various spo mutants of B. subitils were surveyed. Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation. Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores. One spo III mutant had a low amount of cortex, but four others had none.     

Effect of fermentation system on the production and properties of tannase of Aspergillus niger van Tieghem MTCC 2425

The tannase-producing efficiency of liquid-surface fermentation (LSF) and solid-state fermentation (SSF) vis-à-vis submerged fermentation (SmF) was investigated in a strain of Aspergillus niger, besides finding out if there was a change in the activity pattern of tannase in these fermentation processes. The studies on the physicochemical properties were confined to intracellular tannase as only this form of enzyme was produced by A. niger in all three fermentation processes. In LSF and SmF, the maximum production of tannase was observed by 120 h, whereas in SSF its activity peaked at 96 h of growth. SSF had the maximum efficiency of enzyme production. Tannase produced by the SmF, LSF and SSF processes had similar properties except that the one produced during SSF had a broader pH stability of 4.5-6.5 and thermostability of 20 degrees-60 degrees C.     

Beauveria bassiana submerged conidia production in a defined medium containing chitin,two hexosamines or glucose

Summary Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH₄Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 10⁵/ ml after two days in GlcNAc medium as compared to 8.9 × 10⁵/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 10⁶/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.     

Tryptophan supplementation and pH adjustment for optimizing the sporulation of <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

To determine the effect of tryptophan and pH on sporulation of Coniothyrium minitans, the fungus was cultivated using a two-stage, agar plate method in which addition of tryptophan and pH were controlled at the sporulation stage. The spore yield was enhanced by 4 times with 0.1 g tryptophan/l addition after 72 h. The optimal pH values were 4 for mycelia growth and 5.8–6 for sporulation. Mycelia grown at pH 6 had a higher productivity of spore production than did those grown at pH 4.     

Proteolytic processing of the protease which initiates degradation of small, acid-soluble proteins during germination of Bacillus subtilis spores.

Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.