Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

Phenol biodegradation by the thermoacidophilic archaeon <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> 98/2 in a fed-batch bioreactor

Toxic at low concentrations, phenol is one of the most common organic pollutants in air and water. In this work, phenol biodegradation was studied in extreme conditions (80°C, pH = 3.2) in a 2.7 l bioreactor with the thermoacidophilic archaeon Sulfolobus solfataricus 98/2. The strain was first acclimatized to phenol on a mixture of glucose (2000 mg l⁻¹) and phenol (94 mg l⁻¹) at a constant dissolved oxygen concentration of 1.5 mg l⁻¹. After a short lag-phase, only glucose was consumed. Phenol degradation then began while glucose was still present in the reactor. When glucose was exhausted, phenol was used for respiration and then for biomass build-up. After several batch runs (phenol < 365 mg l⁻¹), specific growth rate (μ_X) was 0.034 ± 0.001 h⁻¹, specific phenol degradation rate (q_P) was 57.5 ± 2 mg g⁻¹ h⁻¹, biomass yield (Y_X/P) was 52.2 ± 1.1 g mol⁻¹, and oxygen yield factor ( \textY_{\textX/\textO 2} ) \left( {{\text{Y}}_{{{\text{X}}/{\text{O}}_{ 2} }} } \right) was 9.2 ± 0.2 g mol⁻¹. A carbon recovery close to 100% suggested that phenol was exclusively transformed into biomass (35%) and CO₂ (65%). Molar phenol oxidation constant ( \textY_{\textO 2 /\textP} ) \left( {{\text{Y}}_{{{\text{O}}_{ 2} /{\text{P}}}} } \right) was calculated from stoichiometry of phenol oxidation and introducing experimental biomass and CO₂ conversion yields on phenol, leading to values varying between 4.78 and 5.22 mol mol⁻¹. Respiratory quotient was about 0.84 mol mol⁻¹, very close to theoretical value (0.87 mol mol⁻¹). Carbon dioxide production, oxygen demand and redox potential, monitored on-line, were good indicators of growth, substrate consumption and exhaustion, and can therefore be usefully employed for industrial phenol bioremediation in extreme environments.     

Effects of Corn Steep Liquor on Growth Rate and Pyrene Degradation by Pseudomonas strains

The growth rates and pyrene degradation rates of Pseudomonas sp. LP1 and Pseudomonas aeruginosa LP5 were increased in corn steep liquor (CSL) supplemented. On pyrene alone the highest specific growth rate of LP1 was 0.018 h⁻¹, while on CSL-supplemented pyrene MSM, the value was 0.026 h⁻1. For LP5 the highest growth rate on CSL-supplemented pyrene-MSM was 0.034 h⁻¹. Conversely, on pyrene alone the highest rate was 0.024 h⁻¹. CSL led to marked reduction in residual pyrene. In the case of Pseudomonas sp. LP1 values of residual pyrene were 58.54 and 45.47%, respectively, for the unsupplemented and supplemented broth cultures, showing a difference of 13.09%. For LP5 the corresponding values were 64.01 and 26.96%, respectively, showing a difference of 37.05%. The rate of pyrene utilization by LP1 were 0.08 and 0.11 mg l⁻¹ h⁻¹ on unsupplemented and supplemented media, respectively. The corresponding values for LP5 were 0.07 and 0.015 mg l⁻¹ h⁻¹, respectively. These results suggest that CSL, a cheap and readily available waste product, could be very useful in the bioremediation of environments contaminated with pyrene.     

Decalactone Production by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> under increased O<Subscript>2</Subscript> Transfer Rates

Yarrowia lipolytica converts methyl ricinoleate to γ-decalactone, a high-value fruity aroma compound. The highest amount of 3-hydroxy-γ-decalactone produced by the yeast (263 mg l^-1) occurred by increasing the k_La up to 120 h⁻¹ at atmospheric pressure; above it, its concentration decreased, suggesting a predominance of the activity of 3-hydroxyacyl-CoA dehydrogenase. Cultures were grown under high-pressure, i.e., under increased O₂ solubility, but, although growth was accelerated, γ-decalactone production decreased. However, by applying 0.5 MPa during growth and biotransformation gave increased concentrations of dec−2-en-4-olide and dec-3-en-4-olide (70 mg l⁻¹).     

Oxygen consumption rate and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in early developmental stages of the sea urchin <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> Lam.

Changes in oxygen consumption rate and Na⁺/K⁺-ATPase activity during early development were studied in the sea urchin Paracentrotus lividus Lam. The oxygen consumption rate increased from 0.12 μmol O₂ mg protein⁻¹ h⁻¹ in unfertilized eggs to 0.38 μmol O₂ mg protein⁻¹ h⁻¹ 25 min after fertilization. Specific activity of the Na⁺/K⁺-ATPase was significantly stimulated after fertilization, ranging up to 1.07 μmol P_i h⁻¹ mg protein⁻¹ in the late blastula stage and slightly lower values in the early and late pluteus stages.     

Kinetic limitations during the simultaneous removal of <Emphasis Type="Italic">p</Emphasis>-cresol and sulfide in a denitrifying process

The aim of this study was to evaluate the capacity of a denitrifying consortium to achieve the simultaneous removal of nitrate, sulfide and p-cresol and elucidate the rate-limiting steps in the mixotrophic process. Nitrite reduction appeared as the most evident rate-limiting step in the denitrifying respiratory process. The nitrite reduction rate achieved was up to 57 times lower than the nitrate reduction rate during the simultaneous removal of sulfide and p-cresol. Negligible accumulation of N₂O occurred in the denitrifying cultures corroborating that nitrite reduction was the main rate-limiting step of the respiratory process. A synergistic effect of nitrate and sulfide is proposed to explain the accumulation of nitrite. The study also points at the oxidation of S⁰ as another rate-limiting step in the denitrifying process. Different respiratory rates were achieved with the distinct electron donors provided (p-cresol and sulfide). The oxidation rate of p-cresol (q_CRES) was generally higher (up to 2.6-fold in terms of reducing equivalents) than the sulfide oxidation rate (q_S2−), except for the experiments performed at 100 mg S²⁻ L⁻¹ in which q_S2− was slightly (~1.4-fold in terms of reducing equivalents) higher than q_CRES. The present study provides kinetic information, which should be considered when designing and operating denitrifying reactors to treat industrial wastewaters containing large amounts of sulfurous, nitrogenous and phenolic contaminants such as those generated from petrochemical refineries.     

Fed-batch versus batch cultures of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> for γ-decalactone production from methyl ricinoleate

Constant medium feeding rate and intermittent fed-batch fermentation strategies were investigated aiming to increase the yields of γ-decalactone production by Yarrowia lipolytica, using methyl ricinoleate as substrate and ricinoleic acid source. The accumulation of another compound, 3-hydroxy-γ-decalactone, was also analyzed since it derives from the direct precursor of γ-decalactone thereby providing information about the enzymatic activities of the pathway. Both strategies were compared with the traditional batch mode in terms of overall productivity and yield in respect to the substrate. Although the productivity of γ-decalactone was considerably higher in the batch mode (168 mg l⁻¹ h⁻¹), substrate conversion to lactone (73 mg γ-decalactone g⁻¹) was greater in the intermittent fed-batch giving 6.8 g γ-decalactone l⁻¹. This last strategy therefore has potential for γ-decalactone production at an industrial level.     

Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Recombinant human interferon-gamma (rhIFN-γ) is a protein of great potential for clinical therapy due to its multiple biological activities. However, overexpressing rhIFN-γ in Escherichia coli was found to accumulate as cytoplasmic inclusion bodies. In this work, a system for soluble and active expression of rhIFN-γ was constructed by coexpressing chaperonin GroEL/GroES in E. coli. The rhIFN-γ gene was fused to a pET-28a expression vector, and rhIFN-γ was partially expressed as the soluble form following coexpression with a second vector producing chaperonin GroEL/GroES. The fermentation of recombinant E. coli harboring rhIFN-γ and GroEL/GroES plasmids was investigated, and the optimized conditions were as follows: culture temperature of 25°C, incubation time of 8 h, isopropyl-β-d-thio-galactoside concentration of 0.2 mM, and l-arabinose concentration of 0.5 g/L. As a result, the expression level of rhIFN-γ was improved accordingly by 2.2-fold than the control, while a significantly positive correlation was also found between the ratio of supernatant to precipitate of rhIFN-γ and the amount of chaperonin. Circular dichroism spectra, fluorescence spectra, size exclusion chromatography, and chemical cross-linking method were applied to characterize rhIFN-γ, indicating that the three-dimensional structure of rhIFN-γ was identical to that of the native rhIFN-γ. The enzyme-linked immunosorbent assay for active rhIFN-γ quantification showed that coexpression yielded 72.91 mg rhIFN-γ per liter fermentation broth. Finally, protein–protein interactions between rhIFN-γ and chaperonin were analyzed using the yeast two-hybrid system, which provided the direct evidence that chaperonin GroEL/GroES interacted with rhIFN-γ to increase the soluble expression and presented the potential in producing efficiently recombinant proteins.     

Role of extracellular protease in nitrogen substrate management during antibiotic fermentation: a process model and experimental validation

The production of compound K and aglycon protopanaxadiol (APPD) from ginsenoside Rd and ginseng root extract was performed using a recombinant β-glycosidase from Pyrococcus furiosus. The activity for Rd was optimal at pH 5.5 and 95°C with a half-life of 68 h at 95°C. β-Glycosidase converted Rb₁, Rb₂, Rc, and Rd to APPD via compound K. With increases in the enzyme activity, the productivities of compound K and APPD increased. The substrate concentration was optimal at 4.0 mM Rd or 10% (w/v) ginseng root extract; 4 mM of Rd was converted to 3.3 mM compound K with a yield of 82.5% (mol/mol) and a productivity of 2,010 mg l⁻¹ h⁻¹ at 1 h and was hydrolyzed completely to APPD with 364 mg l⁻¹ h⁻¹ after 5 h. Rb₁, Rb₂, Rc, and Rd at 3.9 mM in 10% ginseng root extract were converted to 3.1 mM compound K with 79.5% and 1,610 mg l⁻¹ h⁻¹ at 1.2 h and were hydrolyzed completely to APPD with 300 mg l⁻¹ h⁻¹ after 6 h. The concentrations and productivities of compound K and APPD in the present study are the highest ever reported.     

Growth-associated production of poly-3-hydroxybutyrate byBacillus mycoides

Bacillus mycoides strain RIJ B-017, a growth-associated poly-3-hydroxybutyrate (PHB) producer was grown on sucrose-containing media. PHB accumulated in cells up to 72% of dry cell mass. The overall maximum value of PHB yield (Y _p/s) and productivities (Q _p andq _p) 250 mg_p/g_s, 120 mg_p L⁻¹ h⁻¹ and 30 mg_p g_x ⁻¹ h⁻¹, respectively, were obtained at 15 g/L sucrose. Differential scanning calorimeter heating curve showed two peaks, one at 95.9 °C and another at 165.4°C with a shoulder around 154.6 °C. The viscosity-average molar mass in chloroform at 27°C was 505 kDa. The carbon content of PHB was 55.4% of the mass.     

Autotrophic and heterotrophic respiration in needle fir and Quercus-dominated stands in a cool-temperate forest, central Korea

To investigate annual variation in soil respiration (R _S) and its components [autotrophic (R _A) and heterotrophic (R _H)] in relation to seasonal changes in soil temperature (ST) and soil water content (SWC) in an Abies holophylla stand (stand A) and a Quercus-dominated stand (stand Q), we set up trenched plots and measured R _S, ST and SWC for 2 years. The mean annual rate of R _S was 436 mg CO₂ m⁻² h⁻¹, ranging from 76 to 1,170 mg CO₂ m⁻² h⁻¹, in stand A and 376 mg CO₂ m⁻² h⁻¹, ranging from 82 to 1,133 mg CO₂ m⁻² h⁻¹, in stand Q. A significant relationship between R _S and its components and ST was observed over the 2 years in both stands, whereas a significant correlation between R _A and SWC was detected only in stand Q. On average over the 2 years, R _A accounted for approximately 34% (range 17–67%) and 31% (15–82%) of the variation in R _S in stands A and Q, respectively. Our results suggested that vegetation type did not significantly affect the annual mean contributions of R _A or R _H, but did affect the pattern of seasonal change in the contribution of R _A to R _S.     

Enhancement of oxytetracycline production after gamma irradiation-induced mutagenesis of <Emphasis Type="Italic">Streptomyces rimosus</Emphasis> CN08 strain

Streptomyces rimosus CN08 isolated from Tunisian soil produced 8.6 mg l⁻¹ of oxytetracycline (OTC) under submerged fermentation (SmF). Attempts were made for enhancing OTC production after irradiation-induced mutagenesis of Streptomyces rimosus CN08 with Co⁶⁰-γ rays. 125 OTC-producing colonies were obtained after screening on kanamycin containing medium. One mutant called Streptomyces rimosus γ-45 whose OTC production increased 19-fold (165 mg l⁻¹) versus wild-type strain was selected. γ-45 mutant was used for OTC production under solid-state fermentation (SSF). Wheat bran (WB) was used as solid substrate and process parameters influencing OTC production were optimized. Solid-state fermentation increased the yield of antibiotic production (257 mg g⁻¹) when compared with submerged fermentation. Ammonium sulphate as additional nitrogen source enhanced OTC level to 298 mg g⁻¹. Interestingly, OTC production by γ-45 mutant was insensitive to phosphate which opens the way to high OTC production even in medium containing phosphate necessary for optimal mycelia growth.     

Bioethanol production in batch mode by a native strain of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

Two wild strains of Zymomonas mobilis were isolated (named as ML1 and ML2) from sugar cane molasses obtained from different farms of Santander, Colombia. Initially, selection of the best ethanol-producer strains was carried out using ethanol production parameters obtained with a commercial strain Z. mobilis DSM 3580. Three isolated strains were cultivated in a culture medium containing yeast extract, peptone, glucose and salts, at pH 6 and 32°C with stirring rate of 65 rpm during 62 h. The best results of ethanol production were obtained with the native strain ML1, reaching a maximum ethanol concentration of 79.78 g l⁻¹. ML1 and ML2 strains were identified as Z. mobilis, according to the morphology, biochemical tests and molecular characterization by PCR of specific DNA sequences from Z. mobilis. Subsequently, the effect of different nitrogen sources on production of ethanol was evaluated. The best results were obtained using urea at a 0.73 g/l. In this case, maximum concentration of ethanol was 83.81 g l⁻¹, with kinetic parameters of yield of ethanol on biomass (Y_P/X) = 69.01(g g⁻¹), maximum volumetric productivity of ethanol (Qp_max) = 2.28 (g l⁻¹ h⁻¹), specific productivity of ethanol (q_P) = 3.54 (h⁻¹) and specific growth rate (μ) = 0.12 h⁻¹. Finally, we studied the effect of different culture conditions (pH, temperature, stirring, C/N ratio) with a Placket-Burman′s experimental design. This optimization indicated that the most significant variables were temperature and stirring. In the best culture conditions a significant increase in all variables of response was achieved, reaching a maximum ethanol concentration of 93.55 g l⁻¹.     

Quasi steady state growth of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in glucose-limited acceleration stat (A-stat) cultures

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h⁻² after initial stabilization of the cultures in chemostat at D = 0.2–0.3 h⁻¹. It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used—at an acceleration rate 0.003 h⁻² the quasi steady state growth was observed until μ _crit = 0.59 h⁻¹, which is also the μ _max value for the culture. Lower values of μ _crit were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h⁻¹ was immediately disrupted after initiating acceleration at the highest acceleration rate studied—0.06 h⁻². Observation was made that differences [Δ(μ − D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h⁻² (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate—it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S ₀ = 5 g L⁻¹ glucose instead of S ₀ = 10 g L⁻¹. It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility—Δ(μ − D) was less than 5%, standard deviations of biomass yields per ATP produced (Y _ATP), and biomass yields per glucose consumed (Y _XS) were less than 15%.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

High-frequency adventitious shoots regeneration from leaf of non-heading Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>) cultured in vitro

The objective of the present work was selection of cultivar and suitable medium for regenerating shoots from leaf segments of non-heading Chinese cabbage. We evaluated six types of supplemented media with 2.0, 5.0 and 10.0 mg l⁻¹ 6-BA; 1.0 and 2.0 mg l⁻¹ TDZ; 0.1, 0.3, 0.5, 0.8 and 1.0 mg l⁻¹NAA; 3.0, 5.0 and 7.5 mg l⁻¹AgNO₃; 0.01 mg l⁻¹ 2–4, D and 4.0 mg l⁻¹ KT for shoot regeneration and six cultivars “Sanchidaye”, “Liuchuandasuomian”, “Qingyou 4”, “Liangbaiye”, “AiKang 5” and “Hanxiao F3”, furthermore for root formation three types of supplemented media with 0.2, 0.3, 0.5 mg l⁻¹ NAA, and for survival rate two types of base media: turf + vermiculite + manure (1:2:0.2) and soil + vermiculite (1:2). Culturing leaf segments on MS medium supplemented with 2 mg l⁻¹ TDZ; 0.5 mg l⁻¹ NAA and 7.5 mg l⁻¹ AgNO₃ gave the highest number of shoots per leaf segment (66) while roots were best formed on the medium supplemented with 0.2 mg l⁻¹ NAA. Survival rate was highest (61.6%) in the turf: vermiculite: manure (1:2:0.2) medium. The highest percentage of responding leaf segments, number of shoots per leaf segment, rooting percentage and survival rate were observed in “Liuchuandasuomian”. The plantlets were transferred to the soil and grown into mature plants in pots. These results could be used for preliminary selections of cultivars to transfer disease resistance (Bt) gene through agrobacterium in non-heading Chinese cabbage.     

Enhanced degradation of phenol by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. CP4 entrapped in agar and calcium alginate beads in batch and continuous processes

Phenol is one of the major toxic pollutants in the wastes generated by a number of industries and needs to be eliminated before their discharge. Although microbial degradation is a preferred method of waste treatment for phenol removal, the general inability of the degrading strains to tolerate higher substrate concentrations has been a bottleneck. Immobilization of the microorganism in suitable matrices has been shown to circumvent this problem to some extent. In this study, cells of Pseudomonas sp. CP4, a laboratory isolate that degrades phenol, cresols, and other aromatics, were immobilized by entrapment in Ca-alginate and agar gel beads, separately and their performance in a fluidized bed bioreactor was compared. In batch runs, with an aeration rate of 1 vol⁻¹ vol⁻¹ min⁻¹, at 30°C and pH 7.0 ± 0.2, agar-encapsulated cells degraded up to 3000 mg l⁻¹ of phenol as compared to 1500 mg l⁻¹ by Ca-alginate-entrapped cells whereas free cells could tolerate only 1000 mg l⁻¹. In a continuous process with Ca-alginate entrapped cells a degradation rate of 200 mg phenol l⁻¹ h⁻¹ was obtained while agar-entrapped cells were far superior and could withstand and degrade up to 4000 mg phenol l⁻¹ in the feed with a maximum degradation rate of 400 mg phenol l⁻¹ h⁻¹. The results indicate a clear possibility of development of an efficient treatment technology for phenol containing waste waters with the agar-entrapped bacterial strain, Pseudomonas sp. CP4.     

The production of xylitol from d-xylose by fermentation with Hansenula polymorpha

We have analysed the influence of the initial pH of the medium and the quantity of aeration provided during the batch fermentation of solutions of d-xylose by the yeast Hansenula polymorpha (34438 ATCC). The initial pH was altered between 3.5 and 6.5 whilst aeration varied between 0.0 and 0.3 vvm. The temperature was kept at 30 °C during all the experiments. Hansenula polymorpha is known to produce high quantities of xylitol and low quantities of ethanol. The most favourable conditions for the growth of xylitol turned out to be: an initial pH of between 4.5 and 5.5 and the aeration provided by the stirring vortex alone. Thus, at an initial pH of 5.5, the maximum specific production rate (μ_m) was 0.41 h⁻¹, the overall biomass yield (Y _x/s ^G) was 0.12 g g⁻¹, the specific d-xylose-consumption rate (q _s ) was 0.075 g g⁻¹ h⁻¹ (for t = 75 h), the specific xylitol-production rate (q _Xy ) was 0.31 g g⁻¹ h⁻¹ (for t = 30 h) and the overall yields of ethanol (Y _E/s ^G) and xylitol (Y _Xy/s ^G) were 0.017 and 0.61 g g⁻¹ respectively. Both q _s and q _Xy decreased during the course of the experiments once the exponential growth phase had finished. Received: 26 March 1998 / Received revision: 30 June 1998 / Accepted: 2 July 1998     

Enhanced regeneration of haploid plantlets from microspores of <Emphasis Type="Italic">Brassica napus</Emphasis> L. using bleomycin,PCIB, and phytohormones

The effects of three periods of incubation (10, 20 and 30 min) at different levels of bleomycin (0, 0.1, 0.2, 0.3, 0.4 and 0.5 μg ml⁻¹), as well as three periods of exposure (12, 24 and 48 h) to different levels of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), including 1, 2, 3, 4 and 5 mg l⁻¹, on microspore embryogenesis of rapeseed cv. ‘Amica’ were investigated. Microspore embryogenesis was significantly enhanced following 20 min treatment with 0.2 μg ml⁻¹ bleomycin compared with untreated cultures. Highest embryo yield (163 embryos Petri dish⁻¹) was observed with 24 h treatment of 4 mg l⁻¹ PCIB. The highest percentage of secondary embryogenesis was observed on B₅ medium containing 0.15 mg l⁻¹ of gibberellic acid (GA₃) and 0.2 mg l⁻¹ 6-benzyladenine (BA) in 4–6 mm microspore-derived embryos (MDEs). Most callus formed on B₅ medium containing 0.15 mg l⁻¹ GA₃, 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ indole-3-acetic acid (IAA) when 4–6 mm embryos were used. Regeneration was highest on B₅ medium containing 0.05 mg l⁻¹ GA₃ or 0.1 mg l⁻¹ BA and 0.2 mg l⁻¹ IAA with 2–4 mm embryos. Microspore embryogenesis and plant regeneration could be improved by both bleomycin and PCIB when the appropriate MDE length and phytohormone level were selected.     

Effect of growth rate on the production of<Emphasis Type="SmallCaps">l</Emphasis>-proline in the fed-batch culture of<Emphasis Type="Italic">Corynebacterium acetoacidophilum</Emphasis>

Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06, 0.08, and 0.1 h⁻¹. The results show that the highest production ofl-proline was obtained at μ=0.04 h⁻¹. The specificl-proline production rate (Q_p) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at μ=0.08 h⁻¹. Thus, the highest productivity ofl-proline was 1.66 g L⁻¹ h⁻¹ at μ=0.08 h⁻¹. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics.