Cultivation of Aspergillus niger in a pilot plant external-loop air-lift bioreactor

Abstract: Changes of the main hydrodynamic and oxygen transfer parameters during Aspergillus niger cultivation in an external-loop air-lift bioreactor of 200 dm³ operating capacity were investigated. The final average concentrations of biomass and citric acid obtained in batch fermentations were about 17 g 1^-1 and 90 g 1^-1, respectively. Significant influence of the increasing biomass concentration on the rheological properties of the broth and operating parameters was found. Volumetric oxygen transfer coefficient. k ^L a , was found to be dependent on the apparent viscosity of the broth with an exponent of -0.984.     

Identification of 4-chloroindole-3-acetic acid and indole-3-aldehyde in seeds of Pinus sylvestris

4-Chloroindole-3-acetic acid (4-Cl-IAA) and indole-3-aldehyde (IAId) have been characterized as endogenous constituents in seeds of Pinus sylvestris L. by gas chromatography-mass spectrometry. Quantitative estimates indicate that immature seeds contained 640 pg 4-Cl-IAA (g fresh weight)^-1 while mature seeds contained 340 pg (g dry weight)^-1. 4-Cl-IAA could not be detected in seeds five days after germination. The content of IAld increased from 127 pg (g dry weight)^-1 in mature seeds to 315 pg (g dry weight)^-1 after five days of germination.     

Kinetic and metabolic effects of nitrogen, magnesium and sulphur restriction in Xanthomonas campestris batch cultures

By reducing the concentration of nitrogen (from 5.0 to 2.5 mmol 1^-1), batch cultures of Xanthomonas campestris induced the enzyme UDP-glucose dehydrogenase and stimulated the Entner-Doudoroff pathway enzyme glucose-6-P dehydrogenase. The surplus energy generation was directed to xanthan biosynthesis resulting in a 10% polysaccharide increase. The nitrogen restriction led to a higher consumption of nitrogen (93%) whereas glucose consumption did not surpass 75% utilization. Low concentrations of both magnesium and sulphur exerted a negative effect on xanthan formation. Both restrictions reduced the phosphomannose isomerase enzyme activity by 10-fold turning the mannose transference presumably into the rate-limiting step for xanthan biosynthesis. Conversely, the rate of synthesis of glucuronic acid residues did not affect the rate of xanthan biosynthesis. Polysaccharide synthesis in magnesium and sulphur cultures was negatively affected in comparison with cell formation as the cell volumetric production rate increased from 0.037 to 0.091 g 1^-1 h^-1 and the xanthan volumetric production rate dropped from 0.133 g 1^-1 h^-1 to the minimum obtained at 0.083 g 1^-1 h^-1. The efficiency of the carbon substrate conversion was also greatly changed.     

Nutrient reserves of Salicornia europaea seeds

Seeds of Salicornia europaea L. were analyzed for their nutrient reserves. The content of potassium and sodium was 216 and 39 mmol (kg dry seeds)^-1, respectively. Calcium and magnesium accounted for 30 and 138 mmol (kg dry seeds)^-1, respectively. Whereas most of the alkali metals were water soluble, the alkaline earth metals were mostly acid soluble. The acid-soluble calcium plus magnesium corresponded well with the acid-soluble phosphate. Chloride was accumulated to a level equivalent to that of sodium. Carbonate was present at a concentration of 9 mmol (kg dry seeds)^-1. Carbohydrates accounted for 93 g (kg dry seeds)^-1, nearly half of which was derived from sucrose. Fructose and glucose were present only in traces. Total nitrogen was determined to be 55 g (kg dry seeds)^-1, 16% of which was diethylether soluble. The remaining nitrogen was separated into 39 g (kg dry seeds)^-1 ethanol-insoluble and 8 g (kg dry seeds)^-1 ethanol-soluble nitrogen. About 10% of the ethanol-soluble nitrogen were derived from amino acids. Total lipid content was about 280 g (kg dry seeds)^-1. The alcoholic component of the storage lipids was glycerol and the glycerides were calculated from gas chromatography to be 66% of the total lipids. About 90% of the fatty acids consisted of unsaturated acids, linoleic and oleic acid, the majority (77%) of which was linoleic acid.     

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.     

Effect of some factors on determination of viable microbial cells by peroxyoxalate chemiluminescence method

The effects of physical and chemical factors on the production of H₂O₂ from Escherichia coli cells were studied. When 20 mmol 1^-1 Tris-HCl buffer was used for this purpose the electron transport system (ETS) showed the highest activity at pH 7.6-8.2. KCN promoted the production of H₂O₂ from E. coli cells, and the optimum concentration was changed in different reaction times and pH values. Glucose, 5 mg ml^-1, increased the ETS activity about twofold. The other substrates and surfactants did not increase the chemiluminescence intensity. NaNO₂ and Na₂SO₄ in inorganic salts significantly reduced the ETS activity above 70%. In addition, the optimum temperature for the production of H₂O₂ was 30°C in this study. When glucose (5 mg ml^-1) and KCN (0.2 mmol 1^-1) were added to the reaction buffer containing 0.5 mmol 1^-1 menadione, the detectable minimum cell densities (averages of triplicate assay) of E. coli, Enterobacter cloacae and Serratia marcescens were 5 times 10³ cells ml^-1, 10⁴ cells ml^-1 and 10⁴ cells ml^-1 respectively.     

Nickel and rubidium uptake by whole oat plants in solution culture

Nickel and rubidium uptake by oat plants ( Avena sativa L. cv. Victory) were examined in relation to solution temperature, solution concentrations, metabolic inhibitors, anaerobic root conditions, transpiration and time. Over a 4-h period, uptake rates for both Ni²⁺ and Rb⁺ remained constant at 23°C. Decreasing temperatures to 2°C, 20 μ M concentrations of 2,4-dinitrophenol (DNP), or anaerobic root conditions decreased Ni²⁺ and Rb⁺ uptake rates by 97 to 86% in whole plants. Treatment of excised roots with 20 μ M DNP decreased Ni²⁺ uptake by 93%. Nickel and Rb⁺ uptake rates measured as a function of the external solution concentration followed a typical parabolic curve. K_m (0.012 m M ) and V_max [2.72 μmol (g dry weight)^-1 h^-1] values for Ni²⁺ were nearly 7 times lower than those for Rb⁺ [0.09 m M and 19.2 μmol (g dry weight)^-1 h^-1]. In all experiments, Ni²⁺ and Rb⁺ showed qualitatively similar uptake patterns, but Rb⁺ uptake was quantitatively more sensitive than Ni²⁺ to experimental manipulations.     

Batch and fed batch cultivations for the temperature induced production of a recombinant protein inEscherichia coli

Summary The temperature induced intracellular production of the fused protein SpA-gal (protein A/-galactosidase) withE. coli was compared in batch and fed batch culture. By introducing fed batch cultivation a final cell dry weight of 77.0 g/l was achieved, as compared to 16.4 g/l in batch cultivation. The concentration of SpA-gal in the fed batch cultivation was very high, 19.2 g/l. This corresponded to 25% of the cell dry weight.     

Sensitization of Escherichia coli to nisin by maltol and ethyl maltol

F. SCHVED, M.D. PIERSON AND B.J. JUVEN. 1996. When used separately, 20 mmol 1^-1 maltol or 1600 AU ml^-1 nisin resulted in a 0–0.6 log₁₀ reduction in viable counts of Escherichia coli in a buffer system. However, when added in combination they yielded a 1.8–5. 5–log-cycle reduction in viable counts of E. coli at pH 5.0 and 6.8 respectively. It is postulated that maltol (and ethyl maltol) destabilizes the cell outer membrane by chelation of Mg²⁺ and/or Ca²⁺, thus permeabilizing the E. coli cell to nisin.     

Localization of newly synthesized indoIe-3-methylglucosinolate (= glucobrassicin) in vacuoles from horseradish (Armoracia rusticana)

The presence and concentration of indole-3-methylglucosinolate [= glucobrassicin; 0.49 μmol (g dry weight)-1] and its 1-methoxy derivative [0.38 umol (g dry weight)⁻¹] in Armoracia rusticana P. Gärtner, B. Meyer and Scherb, storage roots were measured. The storage tissue contains L-tryptophan [1.63 μmol (g dry weight)^-1], which is the common precursor amino acid of the indoleglucosinolates. Tissue cylinders convert [¹⁴C]- l -tryptophan efficiently to the indoleglucosinolates (25%) in vivo. The conversion of [¹⁴C]- l -tryptophan to indole-3-methylgluco-sinolate shows biphasic kinetics.
A fraction rich in vacuoles was prepared from tissue sections to which [¹⁴C]- l -tryp-tophan had been fed and allowed to be metabolized. The predominantly vacuolar location of both L-tryptophan and the indoleglucosinolates is demonstrated by correlation with the vacuolar marker acid phosphatase.
The significance for the regulation of the indoleglucosinolate biosynthesis and the role of indole-3-methylglucosinolate as a potential auxin precursor are discussed.     

Elucidation of the DNA Synthetic Cycle of Entamoeba Spp. Using Flow Cytometry and Mathematical Modeling

ABSTRACT. We developed a method to study the DNA synthetic cycles of Entamoeba histolytica and Entamoeba invadens by flow cytometry (FCM) based on a preparative procedure to reduce both high levels of natural fluorescence and non-specific adsorption of fluorochromes. We modeled G₁, S, and G₂ phases as a series of overlapping Gaussian curves. Both E. histolytica and E. invadens displayed G₁, S, and G₂ proportions that are consistent with eukaryotic cell populations in exponential or stationary growth phase. Exponential phase E. histolytica populations contained a hypodiploid subset with a mass of about 20% less than the diploid value which we estimate by FCM to be 24 × 10^-14 g DNA/cell. Exponential phase E. invadens populations contained a hypodiploid subset with a mass of about 6% less than the diploid value which we estimate by FCM to be 30 × 10^-14 g DNA/cell.     

Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch-operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1^-1 yeast extract, 1.0 g 1^-1 glucose, 1.0 g 1^-1 MgSO₄ or 1.0 g 1^-1 K₂HPO₄ to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1^-1 (NH₄)₂SO₄ increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1^-1 (NH₄)₂SO₄ only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.     

Cultivation of Thermoanaerobium brockii in continuous culture with complete cell recycling

The growth behaviour of the thermophilic anaerobic bacterium Thermoanaerobium brockii for the production of its intracellular secondary alcohol dehydrogenase (sADH) has been studied in batch cultures as well as in continuous cultivation with complete cell recycling. In batch culture the maximum specific growth rate, μ_MAX, was 0·5 h⁻¹, resulting in a cell density of 1·2 g l⁻¹ and an sADH activity of 1·3 units ml⁻¹. Higher glucose concentrations resulted in a decrease in ep cf7 max rs, enzyme productivity as well as biomass yield although an increase in total biomass was achieved. To improve cell density and productivity, continuous culture with complete cell recycling was used, resulting in an increase in cell density by 5 times and in productivity of the sADH by 3 times in comparison to those obtained in batch culture.     

High-level production of bacillus subtilis glycine oxidase by fed-batch cultivation of recombinant Escherichia coli Rosetta (DE3)

A fed-batch process for the high cell density cultivation of Escherichia coli Rosetta (DE3) and the production of the recombinant protein glycine oxidase (GOX) from Bacillus subtilis was developed. GOX is a deaminating enzyme that shares substrate specificity with d-amino acid oxidase and sarcosine oxidase and has great biotechnological potential. The B. subtilis gene coding for GOX was expressed in E. coli Rosetta under the strong inducible T7 promotor of the pET28a vector. Exponential feeding based on the specific growth rate and a starvation period for acetate utilization was used to control cell growth, acetate production, and reconsumption and glucose consumption during fed-batch cultivation. Expression of GOX was induced at three different cell densities (20, 40, and 60 g . L(-1)). When cells were induced at intermediate cell density, the amount of GOX produced was 20 U . g(-1) cell dry weight and 1154 U . L(-1) with a final intracellular protein concentration corresponding to approximately 37% of the total cell protein concentration. These values were higher than those previously published for GOX expression and also represent a drastic decrease of 26-fold in the cost of the culture medium.     

High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: Effects of sucrose concentration and inoculum size

High-density cultivation of Perilla frutescens cells for anthocyanin production was carried out in both batch and fed-batch modes in a 500-ml shake flask. In fed-batch cultures, a high cell density of 27.7 g dry cells l⁻¹ and a total anthocyanin production of 3.87 g l⁻¹ by intermittent feeding of all medium components except hormones were obtained. In batch cultures, both initial sucrose concentration and inoculum size showed a conspicuous effect on the kinetics of cell growth, sugar consumption, and secondary metabolite (anthocyanins) production by suspended P. frutescens cells. At an inoculum size of 50 g wet cells l⁻¹, the maximum cell density of 38.3 g dry cells l⁻¹ was obtained after 11 days of cultivation at an initial sucrose concentration of 60 g l⁻¹, the highest pigment production of>5.8 g l⁻¹ was attained after 10 days of cultivation at an initial sucrose concentration of 45 g l⁻¹. These amounts of cell mass and anthocyanin pigments were 3.3 and 24 times higher than those at an initial sucrose concentration of 15 g l⁻¹ and inoculum size of 15 g wet cells l⁻¹, respectively.     

溶氧反馈分批补料高密度培养人骨形成蛋白-2工程菌

对表达人骨形成蛋白－2成熟肽的基因工程大肠杆菌E.coli DH5α/pDH-B2m在500mL摇瓶中进行了培养条件的摸索实验,并在此基础上扩大至NBS Bioflo IV20L发酵罐,利用溶氧反馈－分批补料培养技术：在培养过程中保持适当的溶解氧（40％）,以溶氧值在线反馈控制搅拌速度及流加补料培养基,使细菌保持适当的比生长率,成功地进行了工程菌的高密度培养,最终菌体密度达OD600＝57,每升干菌量22.8g,目的蛋白的表达量占细菌总蛋白的30％,人骨形成蛋白－2成熟肽的理论产率达到3.59g/L。     

生物转化合成苯乳酸工程菌的培养条件优化

本研究旨在优化重组大肠杆菌Escherichia coli BL21 (DE3) harboring pRSF-aad-ldh10-fdh菌株的培养条件,获得高密的供生物转化苯丙氨酸为苯乳酸的细胞。实验考察了摇瓶发酵培养基碳源、氮源种类和浓度,3 L发酵罐中转速和通气量及恒速补料、DO-stat和pH-stat等不同分批补料策略对菌体密度的影响。结果表明,当碳源为4 g/L葡萄糖,氮源为24 g/L安琪酵母浸粉FM802,细胞干重最大可达9.24 g/L;当转速为400 r/min和通气量为1.5 vvm时,细胞干重最大可达10.18 g/L;以4 g/(L·h)恒速流加葡萄糖时,细胞干重最大可达13.71 g/L。本研究还对工程菌酶表达的诱导条件进行了优化,菌体培养2 h后,添加终浓度为0.08 mmol/L IPTG诱导剂,在25℃下诱导培养14 h所得细胞有利于生物转化。底物苯丙氨酸浓度为60 g/L,转化为苯丙酮酸的转化率为50.2%,转化为苯乳酸的转化率为35.2%。     

The effect of calcium concentration on the toxicity of copper, lead and zinc to yolk-sac fry of brown trout, Salmo trutta L., in soft, acid water

Yolk-sac fry of brown trout were exposed to three levels of single trace metals (Cu, 20,40,80 nmol 1^-1; Pb, 12·5,25,50 nmol 1^-1; Zn, 75,150,300 nmol 1^-1) typical of concentrations reported for acid soft waters, in flowing, artificial, soft water media maintained at pH 4·5 and [Ca] of 20 or 200 μmol 1^-1for 30 days.
Mortalities were high in fry subjected to all levels of the three trace metals at [Ca] 20 μmol 1^-1, with 80% of the total deaths occurring between days 11 and 15 of the experiment. 25% mortality occurred at low [Ca] and pH 4·5 in the absence of trace metals, with only one death recorded at [Ca] 200 μmol1^-1'(Cu, 80 nmol 1^-1). At high [Ca] all three levels of Cu and Pb impaired net Na and K uptake; Cu was the only metal to reduce the uptake of Ca. The Zn treatments had no significant effect on mineral uptake. Calcification of centra was reduced by all three Cu treatments at [Ca] 200 μmol 1^-1. The lowest Zn concentration (75 nmol 1^-1) was the only other treatment to impair skeletal development. In the absence of trace metals, low [Ca] significantly reduced Ca, Na and K uptake, skeletal calcification and dry mass at pH 4·5.
The deleterious effects of low Cu, Pb and Zn concentrations at low pH and low [Ca], and the ameliorative effect of higher ambient [Ca], are discussed in relation to fishery status in soft, acid waters.     

高密度发酵制备重组人OCIF活性域多肽融合蛋白

以补料-分批发酵方式在3．7L发酵罐上实现了人破骨细胞形成抑制因子活性域多肽(OCIF AD)的高密度发酵融合表达。通过参数控制。发酵液最终OD600达12．5(相当于35g湿菌体,L),表达量占菌体总蛋白40％左右,含量超过0．6g/L,并且90％,呈可溶性而直接具有生物学活性。直链淀粉树脂亲和层析法一步纯化,纯度达90％以上。     

Fermentation of extremophilic microorganisms

Abstract: The cultivation of the hyperthermophile Pyrococcus furiosus , the thermoacidophile Sulfolobus shibatae and the halophile Marinococcus M52 in dialysis membrane reactors resulted in cell yields of 2.6 g 1⁻¹, 114 g 1⁻¹ and 132 g 1⁻¹ (cell dry weight), respectively. In the case of P. furiosus neither hydrogen (up to 160 μmol 1⁻¹) nor the metabolic products were found to be responsible for growth cessation at a cultivation temperature of 90°C. The low cell yield at an agitation speed of above 1800 rpm demonstrates the sensitivity of P. furiosus to hydrodynamic stress. The oxygen transfer rate into culture medium at extreme temperatures was shown to be equal or even greater than that under mesophilic condition.