Medium optimization for the production of penicillin V acylase fromBacillus sphaericus

Summary Bacillus sphaericus (NCIM 2478) produced high levels of penicillin V acylase (100 U / g dry cells) when grown on cornsteep liquor — minerals medium at 25°C for 20 h. Supplementation of the medium either with 1 % (w/v) whole wheat bran or its aqueous extract brought about more than 70 % increase in the total enzyme activity. Moreover, deletion of Na₂HPO₄, MgSO₄, CaCl₂, FeSO₄, CuSO₄ and KCl from the medium affected neither growth nor enzyme production.NCL Communication No; 4964.     

Acetone and Butanol Production by Clostridium acetobutylicum in a Synthetic Medium

The effect of the component concentrations of a synthetic medium on acetone and butanol fermentation by Clostridium acetobutylicum ATCC 824 was investigated. Cell growth was dependent on the presence of Mg, Fe, and K in the medium. Mg and Mn had deleterious effects when in excess. Ammonium acetate in excess caused acid fermentation. The metabolism was composed of two phases: an acid phase and a solvent one. Low concentrations of glucose allowed the first phase only. The theoretical ratio of the conversion of glucose to solvents, which was 28 to 33%, was obtained with the following medium: MgSO₄, 50 to 200 mg/liter; MnSO₄, 0 to 20 mg/liter; KCl, 0.015 to 8 g/liter (an equivalent concentration of K⁺ was supplied in the form of KH₂PO₄ and K₂HPO₄); FeSO₄, 1 to 50 mg/liter; ammonium acetate, 1.1 to 2.2 g/liter; para-aminobenzoic acid, 1 mg/liter; biotin, 0.01 mg/liter; glucose, 20 to 60 g/liter.     

THE GROWTH AND MORPHOLOGY OF THE SILICOFLAGELLATE DICTYOCHA FIBULA EHRENBERG IN CULTURE1,2

Dictyocha fibula was cultured in an enriched seawater medium from collections taken at Edmonds, Washington. The optimum temperature for growth is 10 C at salinity 24% and 160 ft-c illumination in Provasoli enriched seawater at the concentration of 7.5 ml/liter seawater. Average generation time is 49 hr. In addition to swimming cells with skeletons, swimming cells without skeletons and nonswimming coenocytes were observed in clonal cultures at 15 C.     

Puccinellia festucaeformis (Host) Parl.: Germinazione e crescita iniziale in funzione della salinità del substrato

Abstract

Puccinellia festucaeformis (Host) Parl.: germination and early growth on different salt substrates. Germination behaviour of Puccinellia festucaeformis seeds and early growth of seedlings at different experimental conditions was analysed. The following growth substrates were utilized: NaCl, KCl, KNO₃, MgCl₂, MgSO₄, Na₂SO₄, NaNO₃, CaCl₂ at the decreasing concentrations of 0.50, 0.25, 0.12, 0.06M. Caryopses were allowed to imbibe and grow at alternating temperatures (10°-20°C or 20°-30°C) in the dark for 3 days. Seedling were grown for 15 days, at controlled light and temperature conditions, in the same nutrient substrates as those used for the germination experiments.

The germination experiments showed a high tolerance to salts up to 0.25M solution and for the whole range of MgSO₄ concentrations. High growth temperatures increased the depressive effects of salt concentrations. Seedling growth was highly reduced when salt concentration was higher than 0.12M. High salt tolerance - maximum shoot and root growth - was showed by seedling allowed to grow on 0.50M MgSO₄.

Germination and growth condition of Puccinellia festucaeformis is discussed in relation to the ecological features of this species and to its possible importance as bioindicator of MgSO₄ rich natural substrates.     

CULTURE AND PHYSIOLOGICAL REQUIREMENTS OF A BACTERIZED CLADOPHORA GLOMERATA (L.) KUTZ FROM LAKE MICHIGAN

A defined medium was developed for a bacterized Gladophora glomerata from Lake Michigan yielding 0.8 g dry weight/m²/20 days and a doubling time of 1.2 days. Best growth was at 25 C under continuous aeration and 200–500 ft-c light. Aeration with 2% CO₂, with or without THIS buffer, inhibited growth. Calcium nitrate, urea, and NaNO₃ were all utilized as nitrogen sources; however, more NO₃ nitrogen was required than urea or Ca nitrate N for maximum yields. Germination of zoospores through zoosporangial walls was observed.     

Production of Candida utilis protein from peat extracts

Sphagnum peat extracts or hydrolysates have been obtained and used as a culture medium for the production of Candida utilis biomass as single cell proteins. Acid hydrolysis of ground peat (4–60 mesh) in an autoclave operated under a set of conditions for acid strength (0.3-1.5 (v/v) H₂SO₄), holding time (1–4 hr), temperature (100–165°C), and weight ratio of dry peat to solution (3.3–16.7 g dry peat/100 g solution) yielded carbohydrate-rich extracts of different concentrations (1–34g/liter). The best yield (mg total carbohydrate/g dry peat) was obtained for a holding time of I hr and a temperature of 152°C. Low peat concentratio (4.1 g dry peat/100 g solution)resulted in high yield(280mg total carbohydrate/gdry peat) with a corresponding low carbohydrate content in hydrolysate (13 g/liter), while a lower yield with a higher carbohydrate content (34 g/liter)in hydrolysate were found when increasing peat concentration (16.7 g dry peat/100 g solution). Shake-fladk experiments using peat hydrolysates as the culture medium together with NH₄OH (～4.8 g/liter) and K₂HPO₄(5 g/liter) as nitrogen and phosphate supplement, respectively, gave a maximum biomass concentration of 7.5 g/liter after 60 hr at 30°C and 200rpm. Batch cultivation in a fermentor under controlled conditions for aeration (4.2 liter/min), agitation (500rpm), temperature (30°C), and pH (5.0) produced a maximum biomass of 10 g/liter after 20 hr with a specific growth rate of 0.13 hr^?1. For the continuous cultivation, a maximal biomass productivity of 1.24 g/gliter-he was obtained at a dilution rate of 0.125 hr ^?1. Monod's equation's equation has been used for the estimation of the coefficients μ_Max, K_s, and Y. It was found that the yield coefficient Y is not constant during the progress of batch cultivation.     

Halophilic and salts tolerant protoplast cultures of mangrove plants, Sonneratia alba and Avicennia alba

The effects of sea salts, NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂, on the growth of protoplast cultures of two mangrove species, Sonneratia alba and Avicennia alba, were investigated using 96-well culture plates. Plants of these two species naturally grow at the seaward side of a mangrove forest. Cotyledon protoplasts of S. alba showed halophilic nature to NaCl, KCl, and MgCl₂ at low concentrations (10–50 mM) when cultured in Murashige and Skoog’s (MS) medium containing 0.6 M mannitol. CaCl₂ at a concentration higher than 25 mM was inhibitory to cell growth. On the other hand, in protoplast culture of A. alba suspension cells, which were induced from cotyledon tissues, in the modified amino acid (mAA) medium containing 1.2 M sorbitol, tolerance to NaCl, MgCl₂ and MgSO₄ were observed at a wide range of concentrations up to 400 mM. CaCl₂ was always inhibitory for cell divisions in A. alba, but stimulatory for spherical enlargement of cells. However, no difference in cell enlargement was observed among other salts. Similarity and difference in reactivity to salts between protoplasts and suspension cells from our previous studies were discussed in relation to the site of salt tolerance or halophilic adaptation within mangrove cells. For protoplast cultures, the site(s) for response of S. alba and A. alba are located in the cytoplasm and/or the cell membrane.     

THE UPTAKE OF GLUCOSE BY CHLAMYDOMONAS SP.12

The glucose uptake of a species of Chlamydomonas was studied at various concentrations of d -glucose plus glucose-1-¹⁴C (0.003–10.0 mg/liter) and at various light levels (0–220 ft-c). The alga grows at 4 C either in the light or in the dark with added glucose, cellobiose, maltose, or fructose. Uptake of glucose could be described by the Michaelis-Menten equation, and both the maximum velocity of uptake and the half-saturation constant increased when the cells were exposed to glucose in the dark. However, the high value of the half-saturation constant (5 mg glucose/liter) compared with the low levels of glucose in nature (5–10 μg/liter) makes it unlikely that a transport system is effective under natural conditions. Even if a total of 10.0 mg/liter of glucose plus other organic compounds were available as substrate, the rate of photosynthesis would still be more than 10 times higher (at 220 ft-c) than the rate of organic substrate uptake. Light had no effect on the total uptake of glucose but did reduce the percentage of ¹⁴CO₂ evolved from 61% of the total ¹⁴CO taken up in the dark to 0% at 220 ft-c. This decrease could be due to either preferential use of the ¹⁴CO₂ in photosynthesis or of the photosynthate in respiration.     

Determination of L-Cysteine with L-Methionine γ-Lyase

A chemically defined medium was devised to examine the growth, production and biochemical pathway of tetrocarcin A. The production of tetrocarcin A was greatly stimulated by l-feucine and its corresponding keto acid, α-ketoisocaproate, suggesting that l-leucine is involved in the biosynthesis of tetrocarcin A. About 10–12 μg/ml of tetrocarcin A was produced in a chemically defined medium consisting of 20 g sucrose, 2.5 g KNO₃, 5 g MgSO₄·7H₂O, 5 g KH₂PO₄ and 1 g l-leucine per liter of water (pH 7.0).     

NUTRITIONAL STUDIES OF ASCOBOLUS IMMERSUS

A synthetic medium for vegetative growth and apothecial formation of 2 compatible strains of A immersus has been formulated as follows: KH₂PO₄, 0.5 g; K₂HPO₄, 0.6 g; MgSO₄·7H₂O, 0.5 g; micronutrients, 0.1 ml (Vogel's); asparagine, 5 g (for vegetative growth), or urea, 1 g (for apothecial formation); dextrin (Merck), 20 g; biotin, 5 μg; thiamine, 100 μg; and distilled water, 1 liter. This medium had a pH of 6.2 to 6.7 without adjustment and was satisfactory. For apothecial formation, 20 g Noble agar (Difco) and 2 g cellulose powder (Whatman) were added to the above medium. Apothecial formation was better at 23–24 than at 25–26 C. Light was necessary for apothecial formation. Dextrin, soluble starch, glucose and mannose were satisfactory carbon sources for both vegetative growth and apothecial formation. This fungus could not use lactose, sucrose, sorbose, mannitol, sorbitol or insulin as carbon sources. It could assimilate nitrate in the form of KNO₃. Optimum yields were obtained with asparagine, aspartic acid, or glutamic acid as the source of nitrogen. The optimum nitrogen-carbon ratio for apothecial formation was about 2% dextrin and 0.1% urea. This ratio was given the highest apothecial rating 10. Ascobolus immersus was deficient in the ability to synthesize biotin and thiamine.     

The Effects of Inorganic Elements on the Reduction of Phytophthora Stem Rot Disease of Soybean, the Growth Rate and Zoospore Release of Phytophthora sojae

The effects of several inorganic elements contained in B5 medium on Phytophthora stem rot disease reduction of Glycine max (L.) Merr. cv. Chusei‐Hikarikuro, fungal growth of Phytophthora sojae isolate and zoospore release were investigated. Application of B5 solution and macro inorganic nutrients in the B5 medium prior to inoculation significantly inhibited infection, compared with controls. Various concentrations of KNO₃, (NH₄)₂SO₄, MgSO₄, CaCl₂ and NaH₂PO₄ in the presence of macro inorganic nutrients were investigated in an effort to determine the elements most effective in suppressing the incidence of disease. A concentration of 2.47–24.7 mm KNO₃ and 0.1–10.2 mm CaCl₂ greatly inhibited infection. Although mycelium growth of the isolate was affected by the potassium and calcium concentration, no significant relationship was observed between inhibition of the growth rate and disease reduction at 2.47 mm KNO₃ and 0.1–5.1 mm CaCl₂ application. Disease suppression recorded in laboratory experiments using pathogen mycelium was due to the response of plant tissues rather than a direct inhibition of pathogen fungal growth by the application of potassium or calcium. The extent of disease reduction was related to an increased potassium and calcium uptake by plants, suggesting that the effective elements in reducing Phytophthora stem rot were potassium and calcium. The presence of 2.47–247 mm KNO₃ and 5.1–10.2 mm CaCl₂ decreased the release of zoospores, although 0.1–2.5 mm CaCl₂ significantly induced zoospore release. These results suggest that applying a solution containing more than 2.47 mm of potassium and 5.1 mm of calcium can decrease the incidence of disease in agricultural fields by the inhibition of zoospore release.     

THE HETEROTROPHIC CAPABILITIES OF CYCLOTELLA MENEGHINIANA1

Cyclotella meneghiniana grew heterotrophically in darkness when glucose in concentrations from 5 mg/liter to 10 g/liter was provided. The other compounds tested did not support growth. However, in continuous light (300 ft-c) growth wax not enhanced if glucose wax provided. Under diurnal conditions of light (300 ft-c) approximately 12–14 hr of darkness were required to observe the enhancement effects of glucose. Uptake studies with labeled glucose indicated that uptake is not dependent on glucose, but that it occurs only at low light intensities. Cells required 12–14 hr of darkness to develop the uptake system.     

An inexpensive inorganic medium for the mass cultivation of freshwater microalgae

A new and inexpensive inorganic medium (‘DS medium’) for the mass cultivation of freshwater blue-green algae (Cyanobacteria) and green algae has been developed. It consists basically of distilled or demineralized water (90%) and seawater (10%) and requires only little addition of pure purchasable chemicals (phosphate, trace elements, if necessary nitrate). No addition of macronutrients (NaCl, MgCl₂ or MgSO₄, KCl or K₂SO₄, CaCl₂) and of boron is required because they are sufficiently provided by the seawater. Decalcified water may also be suitable instead of demineralized water. For the cultivation of green algae, a higher trace element concentration is recommended than for blue-green algae. Because of its low total salt concentration the DS medium is freshwater-like. It is easy to prepare and effects rapid algal growth. It may be of special value for algal mass culture in regions which are close to the sea.     

Effects of mineral salts on the growth,sporulation and virulence of Esteya vermicola,an endoparasitic fungus of the pinewood nematode,Bursaphelenchus xylophilus

Esteya vermicola, an endoparasitic fungus of pinewood nematode, exhibits great potential as a biological agent against nematodes. In this study, various mineral supplements, such as chloride salts (KCl, CaCl₂, MgCl₂, FeCl₂, and FeCl₃) and calcium salts (CaCl₂, CaCO₃, and CaSO₄) were evaluated for their ability to enhance the growth, sporulation and virulence of E. vermicola. Of the cations tested, CaCl₂ provided the greatest enhancement of growth speed and sporulation. Of the anions tested, CaCO₃ produced the highest proportion of lunate conidia, and CaCl₂ produced the highest adhesive rate and mortality against the nematode, Bursaphelenchus xylophilus. The optimum concentration of CaCl₂ for optimization of sporulation and virulence was 0.4–0.6%. In conclusion, CaCl₂ is highly effective in enhancing growth, sporulation and virulence of Esteya vermicola.     

Induction of somatic embryos by macrosalt stress from mature zygotic embryos of Panax ginseng

Mature zygotic embryos of ginseng (Panax ginseng C. A. Meyer) were germinated on a Murashige and Skoog medium lacking growth regulators. However, when the zygotic embryos were cultured on MS medium containing increased levels of macrosalts (NH₄NO₃, KNO₃, KH₂PO₄, MgSO₄, or CaCl₂) to result is a mild salt stress, growth of zygotic embryos was strongly suppressed and eventually browning occurred. Somatic embryos or embryogenic calli were formed directly from these abnormal stressed zygotic embryos. Cotyledons were the most competent tissue for somatic embryo production. The highest frequency of somatic embryo formation (56.3%) was observed on medium containing 61.8 mM of NH₄NO₃. The highest frequency of somatic embryo formation by five different macrosalt treatments occurred in the following order: NH₄NO₃> KNO₃> KH₂PO₄> MgSO₄> CaCl₂. Somatic embryos were regenerated into plants with a shoot and root, and the plants survived on soil in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimal conditions for the production of exopolysaccharide by marine microorganismHahella chejuensis

A marine microorganism, strain 96CJ10356 produced exopolysaccharide, designated as EPS-R. To optimize culture conditions for the production of EPS-R, carbon and nitrogen sources, mineral salts, temperature, and pH were examined. From this study, STN medium for the production of EPS-R was suggested as follows; sucrose 20 g, tryptone 10 g, NaCl 10 g, MgSO₄ 5 g, CaCl₂ 1 g, KH₂PO₄ 76 mg, K₂HPO₄ 83 mg, FeCl₂ 5 mg, MnCl₂ 1 mg, NaMoO₄ 1 mg, and ZnCl₂ 1 mg per liter at pH 7.0. About 9.23 g/L of EPS-R was obtained from STN medium after cultivation for 120 h at 25°C in a 5-liter jar fermentor with an aeration rate of 0.17 vvm. Apparent viscosity and flocculation activity of the culture broth were increased with the production of EPS-R and the maximal values were 415 cP and 1400 unit/mL against 0.5% activated carbon, respectively.     

In vitro salt and thermal tolerance of fungal endophytes of Nicotiana spp. growing in arid regions of north-western Australia

Abstract

Endophytic fungal strains isolated from indigenous Nicotiana plants naturally growing in dry and hot regions of north-western Australia were characterised based on their tolerance to salinity and temperature. Sixty-eight fungal isolates were tested on eight levels (0.5 M, 1.0 M, 1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M and 4.0 M) of five different of salts solutions NaCl, KCl, MgCl₂, CaCl₂ and MgSO₄ and at various temperatures (25–50?°C). The salt adaptation test indicated that the fungal strains namely Aspergillus niger (E-202), A. ochraceous-A (E-134), Aurantiporus sp. (E-135), Cladosporium halotolerance (E-128), Pleurostomophora richardsiae (E-13) and Trichoderma sp. (E-185.1) were tolerant to higher concentrations of various salts. The most growth-limiting salt turned out to be MgCl₂ followed by the chaotrope CaCl₂. Responses to temperature tolerance revealed that most fungi tested could grow at 30?°C. About 50% all the fungi did not show any growth when the temperature was raised above 30?°C. When the temperature was raised up to 50?°C all the fungi failed to grow but the fungus Rasamsonia piperina (E-172). Endophyte strains identified could be promising candidates for future research in investigating the fungus–plant interactions and their roles in plant adaptation to inhospitable environments.     

EFFECTS OF pH AND OF VARIOUS CONCENTRATIONS OF SODIUM,POTASSIUM, AND CALCIUM CHLORIDE ON MUSCULAR ACTIVITY OF THE ISOLATED CROP OF PERIPLANETA AMERICANA (ORTHOPTERA)

1. Twenty-five solutions which contained KCl (0.0, 0.2, 0.4, 0.6, and 0.8 gm. per liter), in combination with CaCl₂ (0.0, 0.2, 0.4, 0.6, and 0.8 gm. per liter), 10.0 gm. of NaCl, and 0.2 gm. of NaHCO₃ per liter of solution were tested in order to determine satisfactory KCl/CaCl₂ ratios in an insect physiological salt mixture for the maintenance of muscular activity by the isolated crop of the American roach. Satisfactory activity products (0.390 to 0.549) were obtained in seven mixtures with KCl/CaCl₂ ratios of 0.2/0.2, 0.4/0.4, 0.6/0.6, 0.8/0.8, 0.2/0.4, 0.4/0.6, and 0.6/0.8, expressed as gram per liter. These ratios lie between 0.50 and 1.00. In solutions which contained calcium, but no potassium, approximately 50 per cent of the crops exhibited an initial tone increase and were arrested in rigor. See Fig. 2. In solutions which contained potassium, but no calcium, all crops showed an initial loss of tone and arrest in relaxation. See Fig. 2. 2. Seven KCl/CaCl₂ ratios (see paragraph 1 above) were tested with eight NaCl concentrations (1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, and 1.8 per cent) at a pH of 8.0. In these mixtures, the ones with KCl/CaCl₂ ratios of less than 1.0 produced higher activity products than those with ratios equal to 1.00. The highest average activity product (0.849) was obtained in the solutions with 0.2 gm. of KCl and 0.4 gm. of CaCl₂ per liter. 3. Four KCl/CaCl₂ ratios (0.2/0.2, 0.4/0.4, 0.2/0.4, and 0.4/0.6 gm. per liter) were tested with 1.4, 1.5, and 1.6 per cent NaCl at a pH of 7.5. When analyzed with data from comparable solutions at a pH of 8.0, it was found that 1.4 per cent NaCl afforded an optimum environment for isolated crop activity. 4. Effects of hydrogen and hydroxyl ion concentrations were studied at pH values of 6.8, 7.5, 8.0, and 8.9. The highest average activity product, 1.011, was produced at a pH of about 8.0. 5. A satisfactory physiological salt solution for the isolated foregut of the American roach, Periplaneta americana, would contain 14.0 gm. of NaCl, 0.4 gm. of CaCl₂, 0.2 gm. of KCl, and 0.2 gm. of NaHCO₃ per liter of solution. This mixture should have a pH value between 7.8 and 8.2. 6. Durations of crop activity extending over periods as long as 25 hours were quite common, and several crops maintained contractions for more than 30 hours. The greatest longevity was for crop 814, from a female, which continued activity for slightly more than 47 hours. 7. A significant difference between the activity products of the crops from males and the crops from females was recorded. Although there was not a significant difference in the amount of food ingested by males and females, 12 hours after feeding there was more food in the females'' crops, and the food progressed more rapidly through the males'' crops than through the females''. In addition, crops from the two sexes reacted differently to the effects of day old solutions. This sex difference is apparently related to an inherently increased activity of the crop from the male roach.     

Optimization of Culture Medium for Sporulation and Biomass Production of a Nematophagous Fungus: Consideration of Nutritional and Environmental Conditions

This study reports the effects of various nutritional and environmental factors on sporulation and biomass of Paecilomyces lilacinus IPC‐P. These factors included carbon and nitrogen sources, carbon‐to‐nitrogen ratios, mineral elements and vitamins together with water potentials, temperatures, dark/light cycles and pH. On the basis of these results, together with a ‘two‐step’ cultivation and orthogonal method, the culture conditions for sporulation of this fungus were optimized. The spore suspension was inoculated on a basal medium (sucrose 19.00 g/l, soy peptone 4.06 g/l, K₂HPO₄ 1.00 g/l, KCl 0.50 g/l, MgSO₄ 0.50 g/l, FeSO₄ 0.01 g/l, agar 13.00 g/l) for 4 days, before being transferred to a sporulation medium (dextrin 2.27 g/l, urea 2.13 g/l, CaCl₂ 3.00 g/l, ZnSO₄·7H₂O 0.01 g/l, agar 13.00 g/l) for a further 4 days under the following environmental conditions: ?3.9 MPa/pH 7/light 24 h/temperature 29°C; these conditions were altered to ?0.3 MPa/pH 6/light 24 h/temperature 23°C in order to obtain better biomass yields. The data presented provide information on the nutrient and environmental requirements of this fungus, which will be essential for its commercial production.     

Reduced major minerals and increased minor nutrients improve micropropagation in three apple cultivars

Mineral nutrient medium requirements for propagation of in vitro shoots of apple (Malus domestica Borkh) ‘Golden Delicious’, ‘Maksat’, and ‘Voskhod’ were studied using response surface methodology (RSM). The mineral nutritional factors evaluated were based on Murashige and Skoog (MS) mineral nutrients (NH₄NO₃, KNO₃, CaCl₂, KH₂PO₄, MgSO₄, and minor nutrients), with concentrations ranging from 0.5 to 3.0× the MS concentrations. Nine plant growth qualities were evaluated. The most significant factors were NH₄NO₃ at 0.5 to 1.0× MS, and minor nutrients at 2.0× MS. Most of the other factors were optimal at 0.5×. The quality rating was highest when minor nutrients were 2.0× MS, and most other nutrients were standard concentrations or lower. Increased KH₂PO₄ and minor nutrients were the most significant for improved multiplication, and higher KNO₃ for shoot length. Optimized media were developed for each cultivar based on these models. The cultivars were grown on the three individual optimized media, a general medium based on the three optimizations, and MS. The optimized medium for each cultivar was significantly better for shoot quality and shoot length of each cultivar than MS, but the generalized medium of minors at 2.0× and NH₄NO₃, CaCl₂, and MgSO₄ at 0.5× MS, was significantly better for two of the three cultivars and not significantly different for the third. The next step to develop a final optimized medium will require the evaluation of the minor nutrients, determination of optimal concentrations of each, and screening a wide range of Malus germplasm on the finalized medium.