Poly(3-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> in fed-batch culture

High poly(3-hydroxybutyrate) (PHB) content and volumetric productivity were achieved by fed-batch culture of Halomonas boliviensis using a defined medium. Initial shake flask cultivations in a minimal medium revealed that the growth of H. boliviensis was supported only when the medium was supplemented with aspartic acid, glycine, or glutamine. Addition of 0.1% (w/v) glutamine in the medium resulted in the highest cell dry weight (CDW; 3.9 g l⁻¹). Glutamine was replaced by the less expensive monosodium glutamate (MSG) in the medium without any notable change in the final cell density. Effect of initial concentrations of NH₄Cl and K₂HPO₄ on cell growth and PHB accumulation by H. boliviensis was then analyzed using a fed-batch fermentation system. The best conditions for PHB production by H. boliviensis were attained using 0.4% (w/v) NH₄Cl and 0.22% (w/v) K₂HPO₄ and adding MSG intermittently to the fermentor. Poly(3-hydroxybutyrate) content and CDW reached 90 wt.% and 23 g l⁻¹, respectively, after 18 h of cultivation. In order to increase CDW and PHB content, MSG, NH₄Cl, and K₂HPO₄ were initially fed to the fermentor to maintain their concentrations at 2%, 0.4%, and 0.22% (w/v), respectively, and subsequently their feed was suppressed. This resulted in a CDW of 44 g l⁻¹, PHB content of 81 wt.%, and PHB volumetric productivity of 1.1 g l⁻¹ h⁻¹.     

Nutritional requirements of Arthroderma tuberculatum

Summary Certain nutritional requirements of one strain ofArthroderma tuberculatum were established in shaken flask culture. Growth was measured on a dry weight basis. The optimum medium for growth consisted of 6 % mono sodium glutamate (or glutamic acid), 4 % glucose, 0.1 % K₂HPO₄ and 0.05 % MgSO₄.7H₂O, with an initial pH range of 7.0–8.0. Maximum growth was attained in this medium after about 4 1/2 days incubation at 28 °C on a rotary shaker.     

Optimization of cultivation medium composition for isoamylase production

Summary The medium composition for production of isoamylase by Pseudomonas amyloderamosa JD210 was optimized using response surface methodology. The factors chosen for optimization were maltose, soybean protein hydrolysate (SPH), isoleucine, proline, KH₂PO₄ and MgSO₄. Fractional factorial designs (FFD) and the path of steepest ascent were effective in searching for the major factors and optimum medium composition. By a 2^6–1 FFD, supplementary isoleucine was shown to have a negative effect on enzyme production. The effects of the other five factors were further investigated by a central composite design and the optimum composition was found to be 1.10% maltose, 0.13% SPH, 0.15% proline, 0.38% KH₂PO₄, and 0.05% MgSO₄. When the strain was cultivated in the optimum medium, enzyme production increased 60% compared with ordinary medium. Proline was verified as being a significant factor in promoting enzyme production.     

Growth and β-Glucan 10C3K Production by a Mutant of Alcaligenes faecalis var. myxogenes in Defined Medium

A defined medium was developed in which Alcaligenes faecalis var. myxogenes 10C3 mutant K produced a large quantity of β-glucan 10C3K. The medium contained 4% glucose together with 0.1% citrate, succinate or fumarate as the carbon source, 0.15% (NH₄)₂HPO₄ as the nitrogen source and mineral salts. When NaNH₄HPO₄, KNO₃ or urea was used at a concentration of 0.03% nitrogen as the sole nitrogen source, salts of organic acid were not needed in addition to glucose.

In culture medium containing phosphate buffer (M/15, pH 6.5~8.0) large amounts of polysaccharide were formed and its yield from the 4% glucose added was about 50%. Thus, it was shown that polysaccharide production is enhanced greatly if a suitable pH for polysaccharide production is maintained during incubation.     

Isolation of Myrosinase Producing Microorganism

Screening tests were undertaken to obtain microorganisms which produce myrosinase. One strain of microorganism indicated a strong capasity for producing myrosinase. The morphological and physiological characteristics of this strain were studied. The organism was identified as Enterobacter cloacae, no. 506.

Enzyme production was induced by the addition of 0.01% sinigrin and 6% mustard extract*² to the culture medium. The highest production was obtained after 36~40 hr cultivation when the strain was cultivated at 28°C in a medium containing 0.01% sinigrin, 6% Mustard ext., 0.1% KH₂PO₄, 0.1% NH₄Cl, 0.1% NaCl and 0.1% MgSO₄·7aq, (pH 7.0).     

Corynecin (chloramphenicol analogs) fermentation studies: Selective production of Corynecin I by Corynebacterium hydrocarboclastus grown on acetate

Corynebacterium hydrocarboclastus KY 8835 grew in the acetate medium and accumulated 28mM Corynecins which was the highest production yield among the processes using various carbon sources. Selective production of Corynecin I (over 90% of all Corynecins), which had been desired for increase of the product yield, was achieved in this acetate medium. To keep the concentration of acetate, ammonium, and phosphate ions in the optimum range throughout the fermentation, a solution containing CH₃COOH (50%), CH₃COONH₄ (9%), and KH₂PO₄(0.2%) was fed continuously to the culture medium as the pH controlling agent. The addition of KCl (1%) and NaCl (1%) to the medium at 12 hr after inoculation stimulated the production of Corynecins.     

Isolation of Amylase Inhibitor-producing Microorganism

An amylase inhibitor-producing microorganism was identified as a subspecies of Strepto- myces diastaticus from morphological and physiological studies and was named Streptomyces diastaticus subsp. amylostaticus No. 2476.

When this strain was aerobically cultured in a shaking flask containing 100 ml of medium consisting of 4% corn starch, 2% soy bean flake extract, 0.3 % NaCl, 0.1 % K₂HPO₄, 0.05% MgSO₄·7H₂O, 0.001% FeS0₄ · 7H₂O, 0.0001% CuSO₄-5H₂O, 0.0001% ZnSO₄·7H₂O, and 0.0001% MnS0₄ nH₂O (pH 7.0) at 30°C, the highest inhibitory activity was obtained after 70 ~ 80 hr of cultivation.

This amylase inhibitor (S-AI) had inhibitory activity on α-amylases and glucoamylase, but not on β-amylases and pullulanase.     

Stimulation of heterotrophic and autotrophic growth ofSphaerotilus discophorus by manganous ions

Growth ofS. discophorus in a casamino acids-mineral salts medium was stimulated 3-fold on addition of 0.05% MnSO₄·H₂O to the medium. Growth was measured by determinations of total nitrogen, protein and DNA on the washed cellular material.Autotrophic growth ofS. discophorus strain 43-R was obtained in an inorganic mineral salts medium supplemented with trace amounts of the essential vitamins, thiamin, biotin and cyanocobalamin and with Mn⁺⁺ as the sole available source of energy. A gas mixture of 5% CO₂-95% air was bubbled continuously through the cultures during incubation. Concomitant with growth, Mn⁺⁺ disappeared from the cultures and MnO₂ was formed.     

The effect of headspace renewal in a Temporary Immersion Bioreactor on plantain (Musa AAB) shoot proliferation and quality

The positive effect of ventilation of the culture container on in vitro shoot proliferation and quality was already proven for different species. Hereafter we report on the evolution of the headspace during in vitro culture of plantain in a Temporary Immersion Bioreactor (TIB) on the one hand, and culture on semi-solid medium on the other hand. The CO₂ and C₂H₄ concentration reached a maximum of 12% and 0.45 μl l⁻¹, respectively in the control treatment on semi-solid medium, compared to 5.7% CO₂ and 0.06 μl l⁻¹ C₂H₄ in TIB. The minimal O₂-concentration on semi-solid medium was 15.1%, compared to 19.3% in TIB. The multiplication rate was best in TIB, 6.4 compared to 4.3 in semi-solid conditions, and this was also the case for shoot height (4.3 cm compared to 3.3 cm), and leaf number (2.6 compared to 1.6). Moreover shoots produced on semi-solid medium showed distorted leaves. A typical day-night pattern in CO₂ and O₂ concentration was observed in TIB, as well as on semi-solid medium; this is illustrative for the photosynthetic capacity of the plant material produced in both systems.     

A preliminary study of the nutritional requirements of Myxotrichum uncinatum

Summary Certain nutritional requirements of one strain ofMyxotrichum uncinatum were established. Growth was measured on a dry weight basis. The approximate optimum medium for growth consisted of 5% KNO₃, 4% sucrose, 0.1% K₂HPO₄, 0.05% MgSO₄. 7 H₂O, 0.2 mg % biotin and 1.0 mg % uracil. The best initial pH range was 6.0–7.0. Approximately three to five days incubation on a rotary shaker permitted excellent growth.     

Influence of the phosphorus and nitrogen source on nisin production in Lactococcus lactis subsp. lactis batch fermentations using a complex medium

The influence of different phosphorus and nitrogen sources on Lactococcus lactis subsp. lactis NIZO 22186 growth and nisin production was studied in batch fermentations using a complex medium. KH₂PO₄ was found to be the best phosphorus source for nisin production. Increasing initial phosphate concentrations from 0 to 5% KH₂PO₄ exerted a double effect, creating favourable pH conditions and particularly stimulating the nisin production levels, which were highest at 5% KH₂PO₄. Up to now, no such high initial phosphate concentrations have been reported for the production of other antibiotics or bacteriocins. Nisin, a lanthionine-containing peptide antibiotic with bacteriocin properties, clearly behaved as a primary metabolite, since its formation was linked with active growth and was not suppressed by phosphate concentrations up to 5%. A complex medium supplemented with cotton seed meal as nitrogen source also gave very high nisin yields. Correspondence to: L. De Vuyst     

Phytase production by fermentation of recombinant Pichia pastoris in monosodium glutamate wastewater

A novel method is proposed to produce both phytase and single-cell protein in recombinant Pichia pastoris fermentation using monosodium glutamate wastewater (MSGW) as the basal medium. Recombinant P. pastoris MR33 transformed with a phytase gene (AppA-m) from Escherichia coli was constructed and showed capability to utilize ammonium as the only nitrogen source. The fermentation medium was optimized in shake flasks by single-factor test and response surface methodology. A fed-batch system containing 30% MSGW, 50 g/l glucose, 1.58 g/l CaSO₄, 5.18 g/l MgSO₄ and 6.67 g/l KH₂PO₄ was developed in a 3.7-l bioreactor. The maximum phytase activity in the MSGW medium reached 3,380 U/ml, 84.2% of that in chemically defined medium, and the dry cell weight was 136 g/l. The single-cell protein (SCP; 46.66% dry cell weight) contains a variety of amino acids and is low in fat, which is ideal for utilization in animal feed. Thus, it is feasible to use MSGW medium for the production of enzymes that can be expressed in P. pastoris.     

Increasing CO2 concentration impact upon nutrient absorption and removal efficiency of supra intensive shrimp pond wastewater by marine microalgae Tetraselmis chui

Abstract

The objective of this study was to investigate the effect of increasing CO₂ concentration on the growth and the capability of Tetraselmis chui. in removal of nitrate, ammonium and phosphate from shrimp pond wastewater (SPWW). The factorial experimental design was used with the treatment of SPWW percentage in culture medium, namely: 100% SPWW, 75% SPWW + 25% Sea Water (SW) and 75% SW + 25% SPWW coupled with three CO₂ concentration treatments: 390?ppm, 550?ppm and 1000?ppm using CO₂ system. Growth of T. chui. for lengh of cultivation period tended to be higher at treatments of 390?ppm CO₂ and 100% SPWW, however there was a declining growth over period of cultivation for both treatments. The growth rate of T. chui was higher for all percentage of SPWW treatments in culture medium at 390?ppm CO₂ concentration compared to other percentage of SPWW treatments and CO₂ concentration treatments. There was a decreasing of growth rate with increasing CO₂ concentration at 100% SPWW and 75% SPWW + 25% SW in culture medium. Nitrogen removal efficiency and removal rate by T. chui. were strongly affected by CO₂ concentration. However, there was no significant effect of increasing CO₂ concentration to removal efficiency and rate of PO₄ by T. chui.     

Production of Alcaligenes xylosoxydans EMS33 in a Bench-scale Fermenter

Summary Optimization of medium composition and pH for chitinase production by the Alcaligenes xylosoxydans mutant EMS33 was carried out in the present study and the optimized medium composition and conditions were evaluated in a fermenter. The medium components screened initially using Plackett–Burman design were (NH₄)₂SO₄, MgSO₄ 7H₂O, KH₂PO₄, yeast extract, Tween 20 and chitin in shake flask experiments. The significant medium components identified by the Plackett–Burman method were MgSO₄ 7H₂O, Tween 20 and chitin. Central composite response surface methodology was applied to further optimize chitinase production. The optimized values of MgSO₄ 7H₂O, Tween 20, chitin and pH were found to be 0.6 g/l, 0.05 g/l, 11.5 g/l and 8.0, respectively. Chitinase and biomass production of Alcaligenes xylosoxydans EMS33, was studied in a 2-l fermenter containing (g/l): chitin, 11.5; yeast extract, 0.5; (NH₄)₂SO₄, 1; MgSO₄ 7H₂O, 0.6; KH₂PO₄, 1.36 and Tween 20, 0.05. The highest chitinase production was 54 units/ml at 60 h and pH 8.0 when the dissolved O₂ concentration was 60%, whereas the highest biomass production was achieved at 36 h and pH 7.5 without any dissolved O₂ control.     

Synthesis of (R)-2-phenylpropanoic acid from its racemate through an isomerase-involving reaction by Nocardia diaphanozonaria

(R)-2-Phenylpropanoic acid was synthesized from the racemic acid through an isomerization reaction involving resting cells of Nocardia diaphanozonaria JCM3208. The isomerization activity of the cells was enhanced 25-fold by adding 5.5 mM racemic 2-phenylpropanoic acid to the culture medium. When 5 mM racemic 2-phenylpropanoic acid was included in the reaction mixture (4 ml) containing resting cells (100 mg dry cell wt) in 25 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.0) at 30 °C for 8 h, 4.56 mM (R)-2-phenylpropanoic acid (95.8% e.e.) was formed with a 91% molar conversion yield.     

Determination of an economical medium for growth of Lactobacillus fermentum using response surface methodology

Aim: Lactobacillus fermentum is a widely utilized probiotic compound fed as an alternative to antibiotics for growth promotion in a wide variety of livestock species. The objective of this research is to develop an economical and practical fermentation medium for the growth of Lact. fermentum using response surface methodology. Methods and Results: A two‐level Plackett–Burman design was used to determine which factors in the fermentation medium influence the growth of Lact. fermentum. Under our experimental conditions, peptone, urea and yeast extract were found to be major factors. Then, the steepest ascent method and the central composite design were applied to optimize the culture of Lact. fermentum. The following composition of the fermentation medium was estimated to be the most economical formula (per litre): 30 g corn syrup, 15 g glucose, 14·4 g peptone, 7 g (NH₄)₂SO₄, 0·5 g urea, 3 g sodium acetate, 4 g sodium citrate, 0·1 g MnSO₄·4H₂O, 0·5 g MgSO₄·7H₂O, 7·3 g yeast extract, 0·5 g K₂HPO₄. Conclusion: Based on 10 side‐by‐side comparisons, we found that the yield of Lact. fermentum using our fermentation medium was 64% greater than those using modified de Man, Rogosa and Sharp broth (MRS) medium (1·8 × 10⁹ CFU ml^?1vs 1·1 × 10⁹ CFU ml^?1, respectively), while the cost was 89% lower than MRS. This research indicates that it is possible to increase bacterial yield by using inexpensive materials. Significance and Impact of the Study: It is more likely that the use of Lact. fermentum as a probiotic will increase. The low cost medium developed in this research can be used for large‐scale, commercial application where economics are quite likely to be important.     

Biosynthesis of protein by microscopic fungi in solid state fermentation. II. Optimization of aspergillus oryzae A. or. 11 cultivation for protein enrichment of starchy raw materials

Study was made of the effect of medium humidity, source and dose of nitrogen, dose of phosphorus, dose of inoculum and aeration on protein biosynthesis by strain Aspergillus oryzae A. or. 11 in solid state fermentation. It was found that for a maximal protein yield the medium ought to contain about: 60% of water, 36% of starchy raw materials d. m. (e.g. 28.8% of coarse rye meal and 7.2% of beet pulp), 0.85% of nitrogen sources (1.6% of (NH₄)₂SO₄ and 1.1% of urea), and 0.35% of phosphorus source (1.3% of KH₂PO₄). pH of medium should be near 6.5. The dose of inoculum should not be lower than 10⁸ spores/100 g medium. The duration of culture ought to be 20–22 h at 30–35°C, with aeration at least 40 dm³ of air/h × 1000 g medium. Under these conditions the protein yield is about 6.0–6.3 g/100 g of starting medium d.m. at the cost of utilization of about 25 g total carbohydrates.     

Optimization of medium components for high-molecular-weight hyaluronic acid production by <Emphasis Type="Italic">Streptococcus</Emphasis> sp. ID9102 via a statistical approach

Hyaluronic acid (HA), linear high-molecular-weight glycosaminoglycan produced from Streptococcus sp., has raised interest in the medical and cosmetics industries because of the various biological functions of HA. In this paper, we report on the optimization of medium components for HA production in Streptococcus sp. ID9102 (KCTC 11935BP) by two-step optimization (one-factor-at-a-time and taguchi orthogonal array design). In the first step, medium components, such as carbon, nitrogen, phosphate, and mineral sources, were selected for HA production in Streptococcus sp. ID9102 (KCTC 11935BP) using the one-factor-at-a-time method. In the second step, the concentration of the selected medium components was optimized using taguchi orthogonal array design. The design for medium optimization was developed and analyzed using MINITAB 14 software. In addition, the effect of amino acid and organic acid, such as glutamine, glutamate, and oxalic acid, was studied for HA production in Streptococcus sp. ID9102 (KCTC 11935BP). Through these processes, the optimum medium comprising 4% glucose, 0.75% yeast extract, 1.0% casein peptone, 0.25% K₂HPO₄, 0.05% MgCl₂, 0.5% NaCl, 0.04% glutamine, 0.06% glutamate, and 0.02% oxalic acid was determined. We were able to produce HA with a molecular weight of 5.9 × 10⁶ at a productivity of 6.94 g/l on pilot scale fermentation.     

Biological Activities of Siolipin (Ester of Lipoamino Acid)

Siolipin has shown three biological activities; (1) hemolytic action for rabbit erythrocyte, (2) acceleration of fibrin clot formation, (3) antibacterial activity on Bacillus subtilis PCI-219 grown on a synthetic medium (1% glucose, 01% KH₂PO₄, 0.1% (NH₄)₂HPO₄, 0.5% NaCl, 0.04% MgSO₄·7H₂O, 1.5% agar). The antibacterial activity of siolipin was reversed by l-histidine, l-cysteine etc.     

Optimization of the production of bacterial cellulose using multivariable linear regression analysis

The biosynthesis of bacterial cellulose by Acetobacter xylinum was optimized by numerically finding the maximum of an arbitrarily chosen second order polynomial model function of several variables (describing the dependence of the cellulose production on the concentrations of the medium components), using multivariable linear regression analysis. The chosen function appeared to describe the analyzed correlation sufficiently well. Consequently, three to six stages of optimization made the determination of the optimum medium compositions possible for 16 days of fermentation at 30°C in a medium based on fructose (wt%: fructose, 3.68; yeast extract, 5.02; (NH₄)₂NO₃, 0.001; KH₂PO₄, 0.3; MgSO₄ × 7 H₂O, 0.05; resulting in a cellulose production equal to 0.505 wt.% – namely 5.6 times higher than before the optimization) and for 7 days fermentations at 30°C in a medium based on sucrose and ethanol (wt.%: sucrose, 5.0; ethanol, 1.36; yeast extract, 1.27; (NH₄)₂SO₄, 0.5; KH₂PO₄, 0.3; MgSO₄ × 7 H₂O, 0.05; resulting in a cellulose production equal to 0.251 wt.% – namely 1.5 times higher than before the optimization).