In vitro differentiation of diakinesis figures in human testis

Summary Fragments of tissue from human testicular biopsies were incubated at 31°C in medium supplemented with 10% w/v foetal calf serum and enriched with 4 mM L-glutamine. The tissue was supported during growth at the gas/medium interface. Autoradiography was used to follow the progress of differentiating spermatogonia. Activity of the testicular epithelium was observed throughout 21 days of culture. Many diakinesis figures were observed between 10–14 days growth in culture.     

Production of extracellular alkaline proteases by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis>

Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH₄NO₃ showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.     

Production of bacterial cellulose from <Emphasis Type="Italic">Enterobacter amnigenus</Emphasis> GH-1 isolated from rotten apple

Bacterial cellulose finds novel applications in biomedical, biosensor, food, textile and other industries. The optimum fermentation conditions for the production of cellulose by newly isolated Enterobacter amnigenus GH-1 were investigated. The strain was able to produce cellulose at temperature 25–35°C with a maximum at 28°C. Cellulose production occurred at pH 4.0–7.0 with a maximum at 6.5. After 14 days of incubation, the strain produced 2.5 g cellulose/l in standard medium whereas cellulose yield in the improved medium was found to be 4.1 g/l. The improved medium consisted of 4% (w/v) fructose, 0.6% (w/v) casein hydrolysate, 0.5% (w/v) yeast extract, 0.4% (w/v) disodium phosphate, and 0.115% (w/v) citrate. Addition of metal ions like zinc, magnesium, and calcium and solvents like methanol and ethanol were found to be stimulatory for cellulose production by the strain. The strain used natural carbon sources like molasses, starch hydrolysate, sugar cane juice, coconut water, coconut milk, pineapple juice, orange juice, and pomegranate juice for growth and cellulose production. Fruit juices can play important role in commercial exploitation of bacterial cellulose by lowering the cost of the production medium.     

Effects of Pluronic F-68 on growth of transformed roots ofSolanum dulcamara

Summary The effects of the non-ionic surfactant, Pluronic F-68, on the growth of transformed roots ofSolanum dulcamara L. have been studied. Growth was stimulated by addition of low concentrations (0.001–0.1% w/v) of freshly-prepared commercial grade Pluronic to liquid culture medium, with maximum increases in root fresh and dry weights at 0.01% (w/v). In contrast, higher concentrations (0.25–1.00% w/v) of freshly-prepared Pluronic inhibited growth. Freshly-prepared purified Pluronic retarded root growth, even at concentrations that were stimulatory with the commercial preparation. Similarly, commercial grade Pluronic solutions stored at 4°C or 22°C for 5 days (aged) were inhibitory to root growth.     

Optimized protocol for the pilot-scale preparation of fungal cellulase

Summary A 25-l scale protocol is devised for the optimal secretion and recovery of fungal cellulase. Using a selected higher yieldingTrichoderma viride SMC strain, a protocol consisted of: a) an optimized production medium rich in microcrystalline cellulose (MCC), fortified with 1% (w/v) ammonium sulphate, 0.5% (w/v) soybean flour, 0.1% (v/v) Tween-80 and other trace nutrients; b) optimized physical parameters of production, such as an inoculum containing a homogeneous suspension of 6×10⁷ conidia per 1,28±1°C, pH 4.0±0.5, 300±20 rpm, 11000±1000 l/h aeration, and 170–220 h duration; c) optimal recovery through a filter press (450 l/h rate of filtration) followed by precipitation with 2.5–3.0 volumes of acetone (15°C and basket centrifugation (27°C, 1700 rpm)); and d) vacuum drying (35°C, 4–6 h). This afforded 70% recovery of cellulase in the form of white fluffy powder containing 20000±2000 carboxy methyl cellulase and 1000±50 units filter paperase per g activities, with raw material cost of US$ 8–10 per million carboxy methyl cellulase units. During storage for 18 months at 4°C, ambient temperature and 37°C, the cellulase preparation was found to retain 100, 75 and 60% of its initial activity, respectively.     

Effects of Pluronic F-68 on Tetrahymena cells: protection against chemical and physical stress and prolongation of survival under toxic conditions

The effects of the non-ionic surfactant Pluronic F-68 (0.01% w/v) on Tetrahymena cells have been studied. A marked protection against chemical and physical stress was observed. The chemical stress effects were studied in cells suspended in buffer (starvation) or in buffers with added ingredients from a chemically defined medium (Ca2+, Mg2+, Na+, K+, trace metal ions). The physical stress was due to mechanical stress or hyperthermia. The data show that Pluronic: (a) prolongs the survival of low concentration cell suspensions during starvation; (b) prevents the cell death caused by low concentrations of Ca2+ (70 microM); (c) prolongs the survival of cells exposed to higher ion concentrations (10 mM Ca2+, or Na+ or K+); (d) postpones the death caused by trace metal ions like Zn2+, Fe3+ and, Cu2+; (e) protects cells from the death caused by shearing forces; and (f) prolongs the survival of cells exposed to hyperthermia (43 degrees C). The cellular survival is increased at reduced temperatures (e.g. 4 degrees C instead of 36 degrees C) and at increased cellular concentrations (e.g. 100 cells ml(-1) instead of 25 or 10 cells ml(-1)). There is no effect of pre-incubation with Pluronic. The protective effect of Pluronic towards Tetrahymena is observed for concentrations in the range from 0.001 to 0.1% w/v.     

Partial characterization of β-glucosidase activity produced by Kluyveromyces marxianus IMB3 during growth on cellobiose-containing media at 45°C

Summary We report- the partial characterization of a -glucosidase produced during growth of the thermotolerant yeast, K. marxianus IMB3 on lactose-containing media at 45°C. The enzyme had Km values of 1.1mM and 14.8mM for the substrates p-nitrophenyl--D-glucoside and cellobiose, respectively. The enzyme had a pH optimum of 5.5 and was optimally active at 50°C. It was stable up to 125 hours at 25°C and 35°, with half-lives of 45 hours and 2 hours at 45°C and 50°C, respectively. The enzyme was inhibited to varying degrees in the presence of metal ions and was completely inactivated by Hg²⁺. Ethanol concentrations [1–10% (v/v)] had little effect on activity. Glucose (20mM) caused inhibition when p-nitrophenyl--D-glucoside was used as substrate, whereas lactose at similar concentrations had no effect.     

Production of α-amylase fromHalobacterium halobium

Maximum production of extracellular -amylase activity inHalobacterium halobium was at 40°C in a medium containing 25% (w/v) NaCl, 1% (w/v) soluble starch and 1% (w/v) peptone, in presence of 0.1mm ZnSO₄ after 5 days in shaking cultures. The amylase had optimal activity at pH 6.5 in the presence of 1 to 3% (w/v) NaCl at 53°C.S. Patel, N. Jain and D. Madamwar are with the Post Graduate Department of Biosciences, Sadar Patel University, Vallabh Vidyanagar-388120, India.     

The effect of cultural conditions on the production of lipase by Acremonium strictum

Summary The optimum cultural conditions for the production of lipase byA. strictum under stationary condition are: period of incubation, 7 days; temperature, 30°C; xylose at a concentration of 2% (w/v) and 3.5% (w/v) soyabean meal as carbon and nitrogen sources respectively. Incorporation of 1% (v/v) of Tween 80 in culture medium enhanced enzyme production while the presence of fatty acids reduced both fungal growth and lipase production. The enzyme showed broad substrate specificity.     

Growth and hyoscyamine production of ‘hairy root’ cultures of Datura stramonium in a modified stirred tank reactor

Summary The growth and hyoscyamine production of transformed roots of Datura stramonium have been examined in a modified 14-1 stirred tank reactor in both batch and continuous fermentations on media containing half or full strength Gamborg's B5 salts and at three different temperatures. Under a range of conditions, roots grown on half strength B5 salts with 3% w/v sucrose had a higher dry matter content (up to 8.3% w/w) and a higher hyoscyamine content (up to 0.52 mg·g^–1 wet weight) than roots grown on full strength B5 salts with the same level of sucrose (up to 4.6% w/w dry matter and up to 0.33 mg hyoscyamine g^–1 wet weight). Growth at 30°C was initially faster than at either 25°C or 35°C and by day 12, the drained weight of roots in the fermentor at 30°C was about fourfold greater than at 25°C and twice that at 35°C. The ultimate hyoscyamine levels attained (approximately 0.5 mg·g^–1 wet weight) were similar at both 25°C and 30°C but some 40% lower at 35°C. Final packing densities of 70% w/v were achieved for roots after 37 days growth at 25°C and the highest production rate of 8.2 mg hyoscyamine l^–1 per day was obtained for roots grown at 30°C. In continuous fermentation at 25°C, the release of hyoscyamine into the culture medium was low (less than 0.5% w/w of the total) but was up to sevenfold higher in fermentors operated at 30°C or 35°C. Offprint requests to: M. G. Hilton     

Effect of fermentation variables on cellulase production by Trichoderma Sp.

Summary Batch cultivation ofTrichodermma reesei QM9414 was carried out in Mandels medium containing(w/v) 1% beech wood cellulose and 0.05% yeast extract at 29°C. Use of 36 hours old inoculum(10% v/v),3.2 1/min aeration rate at 400 rpm(K_La 220/h) and pH cycling strategy produced 4 g/1 cell mass and 21.5 IU/1/h FPA cellulase.     

Production of a cellulase-free xylanase from agricultural waste materials by a thermotolerant Streptomyces sp.

1444 microorganisms were isolated from soil samples from the northern Thai and screened at 55 °C by using basal medium supplemented with 1% carboxymethyl cellulose as a sole carbon source. One isolate, Streptomyces Ab106, had a high activity of a cellulase-free xylanase also without mannanase activity. The maximum cellulase-free xylanase activities of 3.5, 3.3, 3.1 and 2.7 IU were after growth of the organism with 1% (w/v) corn hull, corncob, bagasse and oat spelt xylan, respectively, at 55 °C for 6 days, respectively. The activity was more than 5 times higher than that at 35 °C.     

Regeneration of plants from cryopreserved embryogenic cell suspension and callus cultures of cotton (Gossypium hirsutum L.)

Successful regeneration of cotton (Gossypium hirsutum L.) plants from cryopreserved embryogenic callus and cell suspension cultures is described. The cryoprotectant mixture consisting of a modified Murashige and Skoog (1962) medium with sucrose (5% w/v), DMSO (5% v/v) and glycerol (5% v/v) gave the highest survival rate (70%) from cell suspension cultures cryopreserved in liquid nitrogen after slow cooling (0.5 to 1.0°C/min). A cooling rate of 0.5°C/min provided a satisfactory recovery rate (30%) from cryopreserved embryogenic callus cultures and was superior to a cooling rate of 1°C/min. Regenerated plants from cell suspension and embryogenic callus cultures cryopreserved for more than four years exhibited normal morphology, growth and boll set upon transfer to soil.Abbreviations DMSO dimethylsulfoxide - MS Murashige and Skoog (1962) - MMS modified MS - NAA -naphthaleneacetic acid     

Isolation of thermotolerant,fermentative yeasts growing at 52°C and producing ethanol at 45°C and 50°C

Five thermotolerant, alcohol-producing yeast cultures were isolated from samples obtained from India. Two were identified as ofKluyveromyces marxianus. All five grew on plate-cultures up to 52°C, with maximum growth rates in liquid culture at 40°C. All produced relatively high alcohol concentrations: 5.7 to 7.0% (w/v) at 45°C and 5.0 to 5.5% (w/v) at 50°C when growing on 14.0% (w/v) glucose. All five isolates fermented diluted molasses containing 16.0% (w/v) total sugars, producing 5.6 to 6.0% (w/v) alcohol concentrations. Supplementing the molasses with P, K, Mg and Mn resulted in a 13 to 20% increase in alcohol production at 40°C. The maximum amounts of alcohol produced on supplemented molasses were 7.5 to 8.0 and 6.5 to 7.0% (w/v) at 37°C and 40°C, respectively.     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

Cellulolytic enzymes of a thermotolerantAspergillus terreus strain and their action on cellulosic substrates

A thermotolerant fungal strainAspergillus terreus produced high activities of cellulolytic enzymes when grown in shake flasks for 8 days at 40°C or 14 days at 28°C in medium containing 2.5% (w/v) cellulose powder and 1% (w/v) wheat bran. There was little difference between the final activities of endo-(1,4)--glucanase (ca. 14.4 U/ml); filter paper activity (ca. 1.3 U/ml) and -glucosidase (ca. 10 U/ml). Endoglucanase had maximum activity at 60°C and pH 3.8; the other two enzymes were optimal at 60°C and pH 4.8. The maximum hydrolysis of different cellulosic substrates (about 50%) was obtained within 48 h when 1.1 U/ml of filter paper cellulase activity were employed to saccharify 100 mg alkali-treated cotton, filter paper, bagasse, and rice straw at 50°C and pH 4.8. The major end-product, glucose, was produced from all substrates, with traces of cellobiose and other larger oligosaccharides being present in rice straw hydrolysates.     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me₂SO), is toxic at high concentrations at temperatures >4 °C and has harmful effects on the biological functionality of stem cell as well as on treated patients.Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me₂SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from −16 to −25 °C. The CPAs, beside Me₂SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.     

Interface-mediated death of unconditioned Tetrahymena cells: effect of the medium composition

ABSTRACT We have previously shown that the cell death of Tetrahymena thermophila in low inocula cultures in a chemically-defined medium is not apoptotic. The death is caused by a cell lysis occurring at the medium-air interface and can be prevented by the addition of insulin or Pluronic F-68. Here, we report that cell death can also be caused by the medium. The specific effects of several medium constituents were tested in the presence and absence of an interface. Four of the 19 amino acids (arginine, aspartic acid, glutamic acid, and histidine in millimolar concentration) as well as Ca²⁺ (68 μM) and Mg²⁺ (2 mM) and trace metal ions (micromolar concentrations) are all sufficient to induce the interface-mediated death. The effect of the amino acids and the salt ions Ca²⁺ and Mg²⁺ can be abolished by the addition of insulin (10^-6 M) or Pluronic F-68 (0.01% w/v), whereas insulin/Pluronic F-68 only postpones the death induced by trace metal ions. On the basis of our findings, a new recipe for a chemically-defined medium has been formulated. Single cells can grow in this medium in the presence of medium-air interface without any supplements.     

Bi-phasic production of a-amylase of Bacillus flavothermusin batch fermentation

The facultative thermophile, Bacillus flavothermus, produced highest a-amylase activity (28.6 units/ml) with lactose (4%, w/v) and Yeatex (2%, w/v) and an initial pH 6.0 at 55°C. In batch fermentation, biomass and a-amylase activity peaked twice. In the first of these growth cycles the organism utilised the nitrogen source, Yeatex, and maximum enzyme was associated with cell lysis. In the second growth phase the carbohydrate source, lactose, was utilised and enzyme peaked in the early stages of growth.