The oxygen transfer rate as key parameter for the characterization of<Emphasis Type="Italic"> Hansenula polymorpha</Emphasis> screening cultures

Screening cultures are usually non-monitored and non-controlled due to a lack of appropriate measuring techniques. A new device for online measurement of oxygen transfer rate (OTR) in shaking-flask cultures was used for monitoring the screening of Hansenula polymorpha. A shaking frequency of 300 rpm and a filling volume of 20 ml in 250-ml flasks ensured a sufficient oxygen transfer capacity of 0.032 mol (l h)^–1 and thus a respiration not limited by oxygen. Medium buffered with 0.01 mol phosphate l^–1 (pH 6.0) resulted in pH-inhibited respiration, whereas buffering with 0.12 mol phosphate l^–1 (pH 4.1) resulted in respiration that was not inhibited by pH. The ammonium demand was balanced by establishing fixed relations between oxygen, ammonium, and glycerol consumption with 0.245±0.015 mol ammonium per mol glycerol. Plate precultures with complex glucose medium reduced the specific growth rate coefficient to 0.18 h^–1 in subsequent cultures with minimal glycerol medium. The specific growth rate coefficient increased to 0.26 h^–1 when exponentially growing precultures with minimal glycerol medium were used for inoculation. Changes in biomass, glycerol, ammonium, and pH over time were simulated on the basis of oxygen consumption.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

Fed-batch sorbose fermentation using pulse and multiple feeding strategies for productivity improvement

Microbial oxidation of D-sorbitol tol-sorbose byAcetobacter suboxydans is of commercial importance since it is the only biochemical process in vitamin C synthesis. The main bottleneck in the batch oxidation of sorbitol to sorbose is that the process is severely inhibited by sorbitol. Suitable fed-batch fermentation designs can eliminate the inherent substrate inhibition and improve sorbose productivity. Fed-batch sorbose fermentations were conducted by using two nutrient feeding strategies. For fed-batch fermentation with pulse feeding highly concentrated sorbitol (600 g/L) along with other nutrients were fed intermittently in four pulses of 0.5 liter in response to the increased DO signal. The fed-batch fermentation was over in 24 h with a sorbose productivity of 13.40 g/L/h and a final sorbose concentration of 320.48 g/L. On the other hand, in fed-batch fermentation with multiple feeds, two pulse feeds of 0.5 liter nutrient medium containing 600 g/L sorbitol was followed by the addition of 1.5 liter nutrient medium containing 600 g/L sorbitol at a constant feed rate of 0.36 L/h till the full working capacity of the reactor. The fermentation was completed in 24 h with an enhanced sorbose productivity of 15.09 g/L/h and a sorbose concentration of 332.60 g/L. The sorbose concentration and productivity obtained by multiple feeding of nutrients was found to be higher than that obtained by pulse feeding and was therefore a better strategy for fed-batch sorbose fermentation.     

Physiology of a colony of<Emphasis Type="Italic"> Pleurotus pulmonarius</Emphasis> grown on medium overlaid with a Cellophane membrane

The physiology of a colony of Pleurotus pulmonarius grown on potato dextrose agar overlaid with a Cellophane membrane (PDA-WC) was studied. On PDA-WC, the colony presented higher biomass density and productivity of fruit body formation (4.84±0.23 mg mycelial biomass/cm² and 23.7±3.12 g ml^–1 h^–1, respectively) than that which developed on potato dextrose agar without Cellophane (PDA-OC; 0.26±0.01 mg mycelial biomass/cm² and 10.8±1.57 g ml^–1 h^–1, respectively). In cultures developed on PDA-WC, intracellular laccases and -1,3-glucanases activities were lower [12±0.9 arbitrary units (AU)/g mycelial biomass and 1.33±0.1 international units (IU)/g mycelial biomass, respectively] than those observed on PDA-OC (20.65±1.0 AU/g mycelial biomass and 3.67±0.2 IU/g mycelial biomass, respectively). In cultures developed on PDA-WC, intracellular protein and glycogen concentrations were lower (1.9±0.9 and 117±3.5 mg/g mycelial biomass, respectively) than those observed on PDA-OC (14.3±1.1 and 347±2.9 mg/g mycelial biomass, respectively). The radial growth rate and the content of glucans in the cell wall were not significantly different between cultures developed on PDA-WC and PDA-OC. These results show that the use of the Cellophane as a tool to study in vitro fungal physiology might affect the interpretation of experimental results, since the physiology under otherwise similar conditions was different on medium with and without Cellophane.     

Physiological regulation of competence induction for natural transformation in Acinetobacter calcoaceticus

Acinetobacter calcoaceticus induced competence for natural transformation maximally after dilution of a stationary culture into fresh medium. Competence was gradually lost during prolonged exponential growth and after entrance into the stationary state. Growth cessation and nutrient upshift were involved in the induction of competence. The level of competence of a chemostat culture of A. calcoaceticus was dependent on the nature of the growth limitation. Under potassium limitation a transformation frequency of ±1x10^-4 was obtained. This frequency was independent of the specific growth rate. In phosphate-, nitrogen-, and carbon-limited chemostat cultures, in contrast, the transformation frequency depended on the specific growth rate; the transformation frequency equalled±10^-4 at dilution rates close to µ_max of 0.6h^-1 and decreased to ±10^-7 at a dilution rate of 0.1 h^-1. We conclude that (1) DNA uptake for natural transformation in A. calcoaceticus does not serve a nutrient function and (2) competence induction is regulated via a promoter(s) that resembles the fis promoter from Escherichia coli.     

Fed-batch cultivation ofEscherichia coli YK537 (pAET-8) for production ofphoA promoter-controlled human epidermal growth factor

Secretion of the expressed heterologous proteins can reduce the stress to the host cells and is beneficial to their recovery and purification. In this study, fed-batch cultures ofEscherichia coliYK537 (pAET-8) were conducted in a 5-L fermentor for the secretory production of human epidermal growth factor (hEGF) whose expression, was under the control of alkaline phosphatase promoter. The effects of feeding of glucose and complex nitrogen sources on hEGF production were investigated. When the fed-batch culture was conducted in a chemically defined medium, the cell density was 9.68 g/L and the secreted hEGF was 44.7 mg/L in a period of 60 h. When a complex medium was used and glucose was added in pH-stat mode, the secreted hEGF was improved to 345 mg/L. When the culture was fed with glucose at a constant specific rate of 0.25 gg⁻¹h⁻¹, hEGF reached 514 mg/L. The effects of adding a solution containing yeast extract and tryptone were further studied. Different rate of the nitrogen source feeding resulted in different levels of phosphate and acetic acid formation, thus affected hEGF expression. At the optimal feeding rate, hEGF production achieved 686 mg/L.     

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h⁻¹ and a maximum prodigiosin concentration of 80 ng of prodigiosin · mg of cell protein⁻¹ were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h⁻¹), and maximum prodigiosin concentrations were higher (1,500 ng · mg of protein⁻¹). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na⁺; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na⁺ up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH₂PO₄ above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion · liter⁻¹, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng · mg protein⁻¹) was observed in chemostat cultures at a dilution rate of 0.057 h⁻¹. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h⁻¹, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.     

Callus Growth and Proline Accumulation in Response to Sorbitol and Sucrose-Induced Osmotic Stress in Rice

This study investigated the influence of osmotic stress, induced by sorbitol and sucrose combinations, on growth and proline accumulation in callus cultures of rice (Oryza sativa L.). Dehusked mature seeds, cv. Hassawi, were induced to callus on MS medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.32 µM 6-furfurylaminopurine (kinetin). The medium also contained 29.2, 58.4, 87.6, and 116.8 mM sucrose combined with 0, 54.9, 109.8, and 164.7 mM sorbitol. Callus formation was observed in about 35 % of the cultured seeds irrespective of the sugar treatment. An increase in callus mass was observed as sucrose concentration increased reaching a maximum growth at 87.6 mM. Callus growth was enhanced in response to 54.9 mM sorbitol but at higher concentration it was inhibitory. Best callus growth was obtained on a medium containing 54.9 mM sorbitol combined with 87.6 mM sucrose. Increasing osmotic stress, as a consequence of increasing sucrose and sorbitol concentrations, induced proline accumulation and the highest concentration of proline, 5.8 µmol g^–1(f.m.), was obtained on 164.7 mM sorbitol combined with 116.8 mM sucrose.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

In Vitro Long-Term Storage of Date Palm

A reliable method for the long-term conservation of date palm tissue cultures is described. In vitro shoot bud and callus culture were successfully stored for 12 months at 5°C in the dark. At this conditions high percent of cultures remained viable without serious signs of senescence. However, the growth rate decreased as storage period increased. The role of sorbitol as osmotic agent in storage was examined. Health shoot bud cultures were obtained after 6 months of storage on medium containing 40 g dm^–3 sorbitol. This period extended for 9 months in case of callus cultures.     

Oxidation of amines by yeasts grown on 1-aminoalkanes or putrescine as the sole source of carbon,nitrogen and energy

The maximum growth rate of Trichosporon cutaneum CBS 8111 in chemostat cultures was 0.185 h^-1 on ethylamine and 0.21 h^-1 on butylamine, that of Candida famata CBS 8109 was 0.32 h^-1 on putrescine.The amine oxidation pattern of the ascomycetous strains studied, viz. Candida famata CBS 8109, Stephanoascus ciferrii CBS 4856 and Trichosporon adeninovorans CBS 8244 was independent of the amine that had been used as the growth substrate. It resembled that of benzylamine/putrescine oxidase found in other ascomycetous yeasts. However, differences in pH optimum and substrate specificity were observed between the amine-oxidizing systems of these three species.The amine oxidation pattern of cell-free extracts of Trichosporon cutaneum CBS 8111 varied with the amine that was used as growth substrate. The enzyme system produced by Cryptococcus laurentii CBS 7140 failed to oxidize isobutylamine and benzylamine, and showed a high pH optimum.The synthesis of amine oxidase in the four yeast strains studied was not repressed by ammonium chloride and was weakly repressed by glucose but was strongly repressed if both compounds were present in the growth medium.     

Cultivation of Candida langeronii in sugar cane bagasse hemicellulosic hydrolyzate for the production of single cell protein

Sugar cane bagasse hemicellulosic fraction was hydrolysed by treatment with 70 mg of sulphuric acid per gram of dry mass at 125 °C for 2 h. The hydrolysate was used as the substrate to grow Candida langeronii RLJ Y-019 at 42 °C; initial pH 6.0; stirring at 700 rev/min and aeration at 1.0 and 2.0 v/v/min. The utilization of D-xylose, L-arabinose, and acetic acid were delayed due to the presence of D-glucose, but after D-glucose depletion the other carbon sources were utilized. The kinetic parameters calculated for both cultivations at 1.0 and 2.0 v/v/min included: maximum specific growth rate (_max) of 0.29 ± 0.01 h^–1 and 0.43 ± 0.016 h^–1, yields (Y _x/s) of 0.36 ± 0.012 and 0.40 ± 0.012 g_x/g_s and productivity (Q _x) of 0.81 ± 0.016 and 0.97 ± 0.012 g_x/l/h, respectively, and compared favourably with published results obtained with Candida utilis and Geotrichum candidum. Candida langeronii appeared superior to C. utilis for biomass production from hemicellulose hydrolysate, in that it utilized L-arabinose and was capable of growth at higher temperatures. The biomass contained 48.2, 1.4, 5.8 and 23.4% of total protein, DNA, RNA and carbohydrate, respectively and contained essential amino acids for animal feed.     

Dichloromethane as the sole carbon source forHyphomicrobium sp. strain DM2 under denitrification conditions

Hyphomicrobium sp. strain DM2 was found to grow anaerobically in the presence of nitrate with methanol, formaldehyde, formate or dichloromethane. The estimated growth rate constants with methanol and dichloromethane under denitrification conditions were 0.04 h^–1 and 0.015 h^–1, respectively, which is twofold and fourfold lower than the rates of aerobic growth with these substrates. Slight accumulation of nitrite was observed in all cultures grown anaerobically with nitrate. Dichloromethane dehalogenase, the key enzyme in the utilization of this carbon source, was induced under denitrification conditions to the same specific activity level as under aerobic conditions. In a fed batch culture under denitrification conditionsHyphomicrobium sp. DM2 cumulatively degraded 35 mM dichloromethane within 24 days. This corresponds to a volumetric degradation rate of 5 mg dichloromethane/l·h and demonstrates that denitrificative degradation offers an attractive possibility for the development of anaerobic treatment systems to remove dichloromethane from contaminated groundwater.     

Toxicity of ochratoxin A in aBrevibacillus brevis — Growth inhibition assay

Ochratoxin A (OTA) is a nephrotoxic, carcinogenic and immunosuppressive mycotoxin. It can be detoxified by various microorganisms, e.g. different yeast strains, via metabolisation into ochratoxin α (OTα). Within this study a growth inhibition assay was developed to compare the toxicity of OTA and its degradation product OTα. As an indicator organismBrevibacillus brevis was used. The assay was performed in microtiterplates. Growth inhibition was determined by comparing the optical density values ofBrevibacillus brevis cultures grown in medium supplemented with OTA/OTα and OTA/OTα-free medium, respectively. It could be shown thatB. brevis is sensitive to OTA (EC₁₀₀=0.5 mg/L±0.03 mg/L), which is not the case for its metabolite OTα. Therefore this bioassay is a useful tool to show the detoxification of OTA to OTα by microbial degradation. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Plant regeneration from protoplasts isolated from embryogenic calli of the forage legume<Emphasis Type="Italic"> Astragalus melilotoides</Emphasis> Pall.

An efficient and reproducible protocol is described for the regeneration of Astragalus melilotoides protoplasts isolated from hypocotyl-derived embryogenic calli. Maximum protoplast yield (11.74±0.6×10⁵/g FW) and viability (87.07±2.8%) were achieved using a mixture of 2% (w/v) Cellulase Onozuka R10, 0.5% (w/v) Cellulase Onozuka RS, 0.5% (w/v) Macerozyme R10, 0.5% (w/v) Hemicellulase, and 1% (w/v) Pectinase, all dissolved in a cell protoplast wash (CPW) salt solution with 13% (w/v) sorbitol. First divisions occurred 3–7 days following culture initiation. The highest division frequency (9.86±0.68%) and plating efficiency (1.68±0.05%) were obtained in solid-liquid medium (KM8P) supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 0.5 mg/l 6-benzylaminopurine (BA), 0.2 mg/l kinetin, 0.2 M glucose, 0.3 M mannitol and 500 mg/l casein hydrolysate. Upon transfer to MS medium with 0.5 mg/l -naphthaleneacetic acid and 1-2 mg/l BA, the protoplast-derived calli produced plantlets via somatic embryogenesis (56.3±4.1%) and organogenesis (21.6±0.6%). Somatic embryos or adventitious shoots developed into well-rooted plantlets on MS medium without any plant growth regulators or supplemented with 3.0 mg/l indole-3-butyric acid, respectively. About 81% of the regenerants survived in soil, and all were normal with respect to morphology and growth characters.Abbreviations BA: 6-Benzylaminopurine - CH: Casein hydrolysate - CPW: Cell protoplast wash - 2,4-D: 2,4-Dichlorophenoxyacetic acid - FDA: Fluorescein diacetate - IBA: Indole-3-butyric acid - KIN: Kinetin - MES: 2-(N-morpholino) Ethanesulphonic acid - NAA: -Naphthaleneacetic acidCommunicated by A. Altman     

Growth kinetic parameter estimation of Candida rugopelliculosa using a fish manufacturing effluent

A fourth order Runge–Kutta approximation was used to determine the Monod kinetics of Candida rugopelliculosa by using unsteady state data from only one continuous unsteady state operation at a fixed dilution rate. The maximum microbial growth rates, max, and half saturation coefficient, K _s, were 0.82 ± 0.22 h^–1 and 690 ± 220 mg soluble chemical oxygen demand (SCOD) l^–1, respectively. The microbial yield coefficient, Y, and microbial decay rate coefficient, k _d, were 1.39 ± 0.22 × 10⁴ cells mg^–1 SCOD and 0.06 ± 0.01 h^–1, respectively.     

n-3 Polyunsaturated fatty acid productivity of the marine microalgaIsochrysis galbana. Growth conditions and phenotypic selection

Two set of isolates were obtained in an isolation/selection programme to select eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) rich strains ofIsochrysis galbana. EPA content was improved up to an average of 5.3% (d.wt) for the second set of isolates. On the other hand, with the aeration rate, pH and irradiance kept at low levels, the growth rate was slow and EPA synthesis was enhanced, but productivity increased when growth rates were maximum. A model relating steady-state dilution rates in chemostat cultures ofI. galbana to internal average irradiance is proposed. The greatest productivities were obtained between 0.0295 h^–1 and 0.0355 h^–1 with 300 mg m^–3 h^–1 for EPA and 130 mg m^–3 h^–1 for DHA.     

Mixed-acid fermentation and polysaccharide production by Lactobacillus helveticus in milk cultures

Lactobacillus helveticus grown in milk with pH control at 6.2 had a slower growth rate (=0.27 h^–1) and produced less exopolysaccharide (49 mg l^–1) but increased lactic acid production (425 mM) compared to cultures without pH control (=0.5 h^–1, 380 mg exopolysaccharide l^–1, and 210 mM lactate), respectively. Both cultures displayed a mixed-acid fermentation with formation of acetate, which is linked not only to citrate metabolism, but also to alternative pathways from pyruvate.     

Optimization of some parameters influencingThermoascus aurantiacus growth: Effects of lignin-related compounds

Statistical designs were used to optimize some parameters affecting the growth rate of a Brazilian strain ofThermoascus aurantiacus. The mycelial growth rate was measured using the horizontal tube method. Temperature of incubation and initial pH were the major factors affecting the growth rate. They were optimal at 6.0 and 48°C, respectively. The maximum growth rate was obtained in solid Czapek modified medium containing 1.5% glucose and 38.4 mEq L^–1NaNO₃. Under these conditions, the growth rate ofT. aurantiacus was 5.16±0.10 mm h^–1. Lignin-related compounds such as tannins and extractive substances added at 0.1% (w/v) to the minimal Czapek medium increased growth rate 14% and 29%, respectively.     

Enhanced colchicine production in root cultures of Gloriosa superba by direct and indirect precursors of the biosynthetic pathway

Excised root cultures of Gloriosa superba reached 7.5 g dry wt l^–1 and accumulated 240±40 g colchicine g^–1 cell dry wt after 4 weeks growth. While all precursors (except trans-cinnamic acid) enhanced colchicine content of root cultures without adversely affecting root growth, treatment with p-coumaric acid + tyramine (each at 20 mg l^–1) increased colchicine content to 1.9 mg g^–1 cell dry wt.