Variation in Gymnoascus reessii Baranetzky

Summary A taxonomic study was made of morphological variability in several cultures ofGymnoascus reessii isolated from dung and soil, mostly in California. There is considerable variation among isolates of this species with regard to cultural and ascocarp characters such as the nature of the free apices of the peridial hyphae. Ascospore characters are quite stable. Although certain isolates possibly could be grouped into varieties, such groupings merge into one another. Hence, at this time it appears that varieties ofG. reessii cannot readily be distinguished. The single heretofore described variety,G. reessii var.deilephilae Hennings, is reduced to synonymy withG. reessii Baran.Supported in part by Botany Department Research Grant # 1344, University of California, Los Angeles, California.     

A nutritional study of eight strains of Gymnoascus reessii

Summary Eight strains ofGymnoascus reessii representing several morphological variants were grown in media which contained a variety of carbon and nitrogen sources in order to determine whether there was a correlation between morphological variation and physiological characteristics. Seven strains were similar in their assimilatory abilities, while one strain, 0-309 (NRRL 3612), was consistently dissimilar from the others. The defined medium which permitted the most growth of all eight isolates contained glycine as the nitrogen source and glucose as the carbon source. Other good, defined sources of carbon and nitrogen included soluble starch, maltose, KNO₃ and NaNO₃. Peptone and casamino acids were effective nitrogen sources also. Seven strains grew better with added growth factors although they did not have an absolute requirement for such factors. The other strain, 0-309, appeared to have a growth factor deficiency. Seven of the eight strains were basically similar in their nutritional characteristics. Only strain 0-309 (NRRL 3612) consistently demonstrated sufficient differences so that it could possibly be considered to be a variety ofG. reessii.     

In-vitro liquid culture of the entomopathogenic nematode,Steinernema colombiense,in orbitally shaken flasks

Steinernema colombiense, an entomopathogenic nematode species (EPN) was grown in two types of orbitally shaken flasks at 130?rpm and 28°C, containing 10 or 20?mL, respectively of a complex culture medium with an initial EPN-concentration of 1,000 Infective Juveniles (IJ)/mL. At the 10th day, the EPN-concentration was 58,771 individuals/mL with 87% of them in the IJ stage. No significant differences were found between the EPN growth kinetics in both types of flasks. The nematode-population growth was modelled by a re-parameterized Gompertz equation of three-parameters with best-fit values of 3.8 days for the lag time, 33.8 day^-1 for the maximum growth rate, and 57.3 (dimensionless) for the maximum asymptotic growth.     

Determination of shear stress thresholds in toxic dinoflagellates cultured in shaken flasks: Implications in bioprocess engineering

Marine dinoflagellates are potentially important innovative sources of high-value toxins in biomedical, toxicological and chemical research programs. However, little is known about the difficulties related to dinoflagellate cultures. In this article, we demonstrate that the shear sensitivity of cells may be one of the main causes. The red-tide Protoceratium reticulatum, a producer of yessotoxins, was used to examine the effect of hydromechanical shear stress associated with intermittent fluid agitation on cell growth. Shaken flasks, widely used in biotechnological process research, were used as model bioreactors, as hydrodynamic shear stress is relatively easy to quantify in them. Intermittent turbulence regime was characterized by three key operating variables: shear stress, cycle time or shaking frequency, and fraction of time shaken per agitation cycle. The light/dark cycle was also used as another variable. Cell damage depended on the combination of the above-mentioned variables. A damage threshold was observed at an average shear stress of approximately 0.16 mN m⁻² (equivalent shear rate of 0.12 s⁻¹). Cell damage from exposure to average detrimental shear stress was also shown to be greater in the dark than in the light period. Preliminary experiments demonstrated that dinoflagellates are also much more sensitive to bubbling than the majority of common fragile microalgae. Although slight toxicity of Pluronic F-68 was observed at a concentration of 0.05% (w/v), this protective medium additive considerably reduced cell breakage. On the other hand, no cell adaptation to stronger shear stress was observed. Finally, the implications of the proposed approach for the hypothetical mass culture of dinoflagellates in bioreactors were also thoroughly assessed.     

Quorum sensing involvement in response surface methodology for optimisation of sclerotiorin production by Penicillium sclerotiorum in shaken flasks and bioreactors

Sclerotiorin, an azaphilone produced by some filamentous fungi including Penicillium sclerotiorum, is a pigment with variety of biological activities including lipoxygenase inhibition, reduction of cholesterol levels, and anti-cancer properties. Sclerotiorin has potential use in pharmaceutical as well as food industries. In this context, the purpose of this study was to provide a simple and robust procedure for optimised production of sclerotiorin by P. sclerotiorum using a central composite design developed through response surface methodology (RSM) and to identify the molecule(s) involved in the signalling mechanism in P. sclerotiorum. The optimisation of sclerotiorin production was carried out using RSM in shaken flasks and the obtained results were then replicated using a 2-L stirred tank bioreactor. Penicillium sclerotiorum ethyl acetate culture extract was analysed using thin layer chromatography (TLC) and potential signalling molecules were identified using Gas chromatography-mass spectrometry (GC-MS). The experimental studies suggested an increase in the sclerotiorin production by 2.1-fold and 2.2-fold in shaken flasks and stirred tank bioreactors respectively. Further analysis of P. sclerotiorum ethyl acetate culture extract reported the presence of ricinoleic acid, an oxylipin, belonging to a family of signalling molecules tentatively involved in the enhancement of sclerotiorin production. This paper has highlighted the positive effect of the optimal supplementation of P. sclerotiorum culture extracts for enhanced production of sclerotiorin. It has also examined potential molecules involved in the signalling mechanism in P. sclerotiorum culture extract for the overproduction of sclerotiorin.     

Prevention of formation of acid drainage from high-sulfur coal refuse by inhibition of iron- and sulfur-oxidizing microorganisms. I. Preliminary experiments in controlled shaken flasks

Changes of pH and sulfate concentration in high-sulfur coal refuse slurries are used as measurements of microbial pyrite oxidation in the laboratory. Sodium lauryl sulfate (SLS), alkylbenzene sulfonate (ABS), benzoic acid (BZ) and combinations of SLS plus BZ and ABS plus BZ effectively inhibited formation of sulfate and acid when added in concentrations greater than 50 mg/L to inoculated 20 or 30% coal refuse slurries. Here 25 mg/L concentrations of SLS, ABS, and ABS + BZ stimulated acid production. Formic, hexanoic, oxalic, propionic, and pyruvic acids at 0.1% concentrations were also effective inhibitors. Four different lignin sulfonates were only slightly effective inhibitors at 0.1% concentrations. It was concluded that acid formation resulting from microbial oxidation in high-sulfur coal refuse can be inhibited.     

Introducing substrate limitations to overcome catabolite repression in a protease producing Bacillus licheniformis strain using membrane-based fed-batch shake flasks

To overcome catabolite repression, industrial fermentation processes are usually operated in substrate-limited fed-batch mode. Therefore, the implementation of such an operating mode at small scale is crucial to maintain comparable process conditions. In this study, Bacillus licheniformis, a well-known producer of proteases, was cultivated with carbon (glucose)- and nitrogen (ammonium)-limited fed-batch conditions using the previously introduced membrane-based fed-batch shake flasks. A repression of protease production by glucose and ammonium was thus avoided and yields increased 1.5- and 2.1-fold relative to batch, respectively. An elevated feeding rate of glucose caused depletion of ammonium, which was recognizable within the oxygen transfer rate (OTR) signal measured with the Respiration Activity MOnitoring System (RAMOS). Ammonium limitation was prevented by feeding ammonium simultaneously with glucose. The OTR signal clearly indicated the initiation of the fed-batch phase and gave direct feedback on the nutrient release kinetics. Increased feeding rates of glucose and ammonium led to an elevated protease activity without affecting the protease yield (Y_P/Glu). In addition to Y_P/Glu, protease yields were determined based on the metabolized amount of oxygen . The results showed that the protease production correlated with the amount of consumed glucose as well as with the amount of consumed oxygen. The membrane-based fed-batch shake flask in combination with the RAMOS device is a powerful combination to investigate the effect of substrate-limited fed-batch conditions.     

Growth of a water strain ofLeptospira in synthetic media

Pertinent parameters of growth of a water strain ofLeptospira in synthetic media are described. Growth rates as a function of aeration, temperature, and substrate concentration were determined. Evidence is presented for the production of acetate and CO₂ from long-chain fatty acids. A marked drop in pH was shown to result from the accumulation of acetate during growth in synthetic medium. Growth was initiated at pH values from 5.2 to 7.7. An absolute CO₂ requirement for initiation of growth was demonstrated. Growth is also described with acetate as the sole carbon and energy source, demonstrating that leptospires are capable of fatty acid synthesis.This investigation was supported by Public Health Service grant AI-05440 from the National Institute of Allergy and Infectious Diseases, and by Public Health Service predoctoral fellowship (1-Fl-GM-40, 062-01) to the senior author from the National Institute of General Medical Sciences. Technical assistance of Mr. Howard Young is gratefully acknowledged.     

Growth and alkaloid production of a cell suspension culture of Thalictrum rugosum in shake flasks and membrane-stirrer reactors with bubble free aeration

Summary A novel membrane-stirrer reactor allowing bubble-free aeration was used for the cultivation of a cell suspension culture of Thalictrum rugosum. The immediate advantages were the absence of any foaming or flotation and the gentle mixing of the suspension by the tumbling movement of a coil or basket of membranes. In a 211 reactor under these conditions the suspensions were grown to high cell densities of 550 g fresh mass or 50 g dry mass/l. A pO₂ of 30% was maintained up to 400 g/l without air bubble formation at the gas-exchange membranes. Provided that the physiological states of the initial suspensions were comparable, growth rates and alkaloid production of shake-flask and fermenter-grown cells were rather similar.Abbreviations NAA -naphthylacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

In situ pH maintenance for mammalian cell cultures in shake flasks and tissue culture flasks

pH in animal cell cultures decreases due to production of metabolites like lactate. pH control via measurement and base addition is not easily possible in small‐scale culture formats like tissue‐culture flasks and shake flasks. A hydrogel‐based system is reported for in situ pH maintenance without pH measurement in such formats, and is demonstrated to maintain pH between 6.8 and 7.2 for a suspension CHO cell line in CD CHO medium and between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS. This system for pH maintenance, along with our previous report of hydrogels for controlled nutrient delivery in shake flasks can allow shake flasks to better mimic bioreactor‐based fed batch operation for initial screening during cell line and process development for recombinant protein production in mammalian cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Growth and monoterpene production by transformed shoot cultures of Mentha citrata and Mentha piperita in flasks and fermenters

This paper reports studies on the growth and biosynthesis of monoterpenes by transformed shoot cultures of Mentha citrata and Mentha piperita, originally developed 5 years ago and since maintained by regular subculturing. Throughout this time, the M. citrata culture has stably maintained production of an oil closely resembling that of the parent plant in which linalool and linalyl acetate are the predominant components. However, M. piperita, which initially showed a divergence from the parent plant in producing significant amounts of menthofuran in addition to the characteristic oil components menthol and menthone, has now been found to produce pulegone and menthofuran as the major components. The cultures were subjected to different environmental conditions of varying periods of light and temperature in an attempt to restore menthol and menthone production. Increased illumination reduced the yields of pulegone and menthofuran but did not stimulate the production of either menthol or menthone, which remained only at trace levels (below 0.2 g/g fresh weight). Cultures of M. citrata were, however, stimulated by increased illumination, and produced more linalool and linalyl acetate. Shoot cultures of M. citrata and M. piperita were grown in 14–1 fermenters for up to 60 dys during which the biomass increased from approximately 100 g to 2.5 kg and 3.5 kg respectively. Both cultures rapidly consumed sucrose with a concomitant release of glucose, and the uptake of inorganic ions was similar except that M. citrata consumed far less Na+ during the fermentation. The total yields of monoterpenes from the fermentations were 1.16 g (M. piperita) and 0.18 g (M. citrata). *** DIRECT SUPPORT *** AG903062 00005     

Growth inhibition of dinoflagellate algae in shake flasks: Not due to shear this time!

Large scale algae cultures present interesting challenges in that they exhibit characteristics of typical bacterial and animal cell cultures. One current commercial food additive, docosahexaenoic acid (DHA), is produced using the dinoflagellate algae, Crypthecodinium cohnii. Like animal cell culture, the perceived sensitivity of algae culture to hydrodynamic forces has potentially limited the agitation and aeration applied to these systems. However, the high density cultivation of C. cohnii required for an economically feasible process inevitably results in high oxygen demand. In this study, we demonstrated what first appeared to be a problem with shear sensitivity in shake flasks is most probably a mass transfer limitation. We subsequently demonstrated the limit of chronic and rapid energy dissipation rate, EDR, that C. cohnii cells can experience. This limit was determined using a microfluidic device connected in a recirculation loop to a stirred tank bioreactor, which has been previously used to repeatedly expose animal cells to high levels of EDR. Inhibition of cell growth was observed when C. cohnii cells were subjected to an EDR of 5.9 × 10⁶ W/m³ with an average frequency of 0.2/min or more. This level of EDR is sufficiently high that C. cohnii can withstand typically encountered hydrodynamic forces in bioprocesses. This result suggests that at least one dinoflagellate algae, C. cohnii, is quite robust with respect to hydrodynamic forces and the scale‐up of process using this type of algae should be more concerned with providing sufficient gas transfer given the relatively high oxygen demand. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Influence of nutrition on the morphology of a strain of Bifidobacterium bifidum

A mucoid variant of Bifidobacterium bifidum was converted from its normal curved rod or bifid form to a highly branched form when grown in a chemically defined minimal medium. Branching could be prevented by the addition of a mixture of dl-alanine, dl-aspartic acid, l(+)-glutamic acid, and dl-serine, but not when any one of these four amino acids was omitted. Although sodium chloride induced pleomorphism, calcium ions were ineffective in suppressing the appearance of these pleomorphic forms. None of the cell wall precursors tested, viz., N-acetyl-d-glucosamine, alpha-epsilon-diaminopimelic acid, and muramic acid, inhibited branching.     

Correlation of actinomycin X2 to the lipid profile in static and shaken cultures of Streptomyces nasri strain YG62

Streptomyces nasri strain YG62 produces a broad-spectrum antibiotic designated actinomycin X2. The influence of static and shaken incubation on the production of actinomycin X2 and lipid profiles of S. nasri strain YG62 was investigated. It was found that shaken incubation was superior to the static process for both actinomycin X2 (2-fold) and total lipids (1.6-fold). Triglyceride and phospholipid levels paralleled the actinomycin X2 production with an increase in the triglyceride (2.8-fold) and phospholipid (1.2-fold) concentrations in the shaken culture over the static incubation. Analysis of fatty acid patterns revealed the occurrence of a wide range of fatty acids (C10-C22). The mean percentage of total saturated fatty acids in shaken culture was higher than those of the static culture. The mean percentage of mono-unsaturated fatty acids was almost the same in both cultures. The mean percentage of the total polyunsaturated fatty acids in the static culture was slightly higher than that of the shaken culture. The polyunsaturated/saturated fatty acid ratio (P/S) was higher in the static culture compared with the shaken culture. A positive correlation was recorded between triglycerides, phospholipids and actinomycin X2. A negative correlation on the other hand, was found between fatty acids and actinomycin X2.