Production of Bacillus thuringiensis Berliner var. kurstaki Grown in Alternative Media

Agro - industrial residues and by - products available in southeastern Brazil were used as ingredients for low - cost culture media for liquid fermentation of Bacillus thuringiensis var. kurstaki. Highest spore yield was obtained with a medium containing cheese whey , soya bean milk and molasses (WSM) . Crystals and spores were produced in all media and potency of the final product was highest for nutrient broth + yeast extract medium (NBY) . There was no correlation between the number of spores in the fermented media and the potency of the preparations . Considering all three factors , the potencies , costs and yields of the final products , lowest relative cost was obtained with BMM medium ( Bombyx mori pupae + molasses) . NBY and WSM had intermediate relative cost approximately nine times higher than BMM . The cost analysis suggests that BMM medium should be preferred for local production of B. thuringiensis var . kurstaki in comparison to other media tested . The results also demonstrate the importance of considering yields , cost and potency of the B. thuringiensis preparations in selecting the production medium .     

On the reaction of 2-aminopropionitrile in aqueous media

The reactions of 2- and 3-aminopropionitrile (APN), and 2,2-iminodipropionitrile (IDPN) were carried out in aqueous ammoniacal media. 2-APN was found to give IDPN, N-(1-cyanoethyl)alanine amide, N-(1-cyanoethyl)alanine, N-(1-carbamoylethyl)alanine, 2,2-iminodipropionic acid, alanine amide, and alanine. Compounds of biological significance such as peptides and amino acids other than alanine were not formed. The results were well consistent with those obtained for aminoacetonitrile. IDPN which can be formed easily from 2-APN in aqueous media, also yielded the same products as with 2-APN. On the other hand, 3-APN gave 3-alanine via 3-alanine amide under similar conditions. 3-APN was found to be more stable than 2-APN in aqueous media.     

Thermoanaerobacter ethanolicus Growth and Product Yield from Elevated Levels of Xylose or Glucose in Continuous Cultures

The performance of Thermoanaerobacter ethanolicus was evaluated in continuous culture with media containing concentrations of xylose (8 to 20 g/liter) greater than those previously reported. The ethanol yield declined from to 0.42 to 0.29 g of ethanol per g of xylose consumed when input xylose was increased from 4 to 20 g/liter. Yields of both total C₂ and C₃ products from consumed xylose and of cell biomass from ATP produced declined as the input xylose concentration was increased, which was not the case when glucose was the substrate. This suggested that yeast extract functioned as a significant energy and carbon source for cells in fermentations of xylose but not of glucose. The feasibility of this interpretation was confirmed by (i) the calculation of the products theoretically obtainable from yeast extract and (ii) the observation of significant quantities of fermentation products in inoculated sugar-free media. Markedly different patterns of metabolism for the two sugar substrates were also evidenced by the cell yield for glucose being twice that of xylose at elevated sugar concentrations. It was noted that caution must be exerted when results obtained at low xylose concentrations are extrapolated to predict those which can be obtained at higher concentrations.     

Microbial transformation of androst-4-ene-3,17-dione by Beauveria bassiana

The microbial transformation of androst-4-ene-3,17-dione (I) by the fungus Beauveria bassiana CCTCC AF206001 has been investigated using pH 6.0 and 7.0 media. Two hydroxylated metabolites were obtained with the pH 6.0 medium. The major product was 11alpha-hydroxyandrost-4-ene-3,17-dione (II) whereas the minor product was 6beta,11alpha-dihydroxyandrost-4-ene-3,17-dione (III). On the other hand, four hydroxylated and/or reduced metabolites were obtained with the pH 7.0 medium. The major product was 11alpha,17beta-dihydroxyandrost-ene-3-one (V) and the minor products were 17beta-hydroxyandrost-ene-3-one (IV), 6beta,11alpha,17beta-trihydroxyandrost-ene-3-one (VI) and 3alpha,11alpha,17beta-trihydroxy-5alpha-androstane (VII). The products were purified by chromatographic methods, and were identified on the basis of spectroscopic methods. This fungus strain is clearly an efficient biocatalyst for 11alpha-hydroxylation and reduction of the 17-carbonyl group.     

N-(2-Carboxyethyl)chitosans: regioselective synthesis,characterisation and protolytic equilibria

N-(2-Carboxyethyl)chitosans were obtained by reaction of low molecular weight chitosan with a low degree of acetylation and 3-halopropionic acids under mild alkaline media (pH 8-9, NaHCO3) at 60 degrees C. The chemical structure of the derivatives obtained was determined by 1H and 13C NMR spectroscopies. It was found that alkylation of chitosan by 3-halopropionic acids proceeds exclusively at the amino groups. The products obtained are described in terms of their degrees of carboxyethylation and ratio of mono-, di-substitution and free amine content. The protonation constants of amino and carboxylate groups of a series of N-(2-carboxyethyl)chitosans were determined by pH-titration at ionic strength 0.1 M KNO3 and 25 degrees C.     

Comparison of fluorescent gentamicin-thallous-carbonate and KF streptococcal agars to enumerate enterococci and fecal streptococci in meats.

Two selective and differential media were compared for their abilities to enumerate enterococci and fecal streptococci in pork, beef, and poultry products. Counts obtained on KF streptococcal (KF) agar were compared with counts obtained on fluorescent gentamicin-thallous-carbonate (fGTC) agar. Reactions of 13 known enterococcal species were also observed. All 13 species of enterococci as well as Streptococcus bovis and Streptococcus equinus grew equally well on fGTC agar. KF streptococcal medium allowed growth of most species of enterococci but not S. bovis and S. equinus. Quantitative comparisons between the two media inoculated with pure cultures of known species of enterococci revealed equivalent plate counts following incubation. However, when meat samples were plated, counts on fGTC agar were consistently and significantly higher than counts on KF agar for all sample sources.     

Influence of the reaction medium on the product distribution of peroxidase-catalysed oxidation of p-cresol

Summary p-Cresol was oxidized by hydrogen peroxide in a reaction catalysed by horseradish peroxidase and the low molecular weight products were investigated. In aqueous media Pummerer's ketone (I) was the dominating product but in organic media the product distribution was quite different; 2,2'-dihydroxy-5,5'-dimethyldiphenyl (II) was the main low molecular weight product. Similar product distributions were obtained with peroxidase adsorbed on a solid support and suspended in toluene and with peroxidase solubilized in a microemulsion containing the same solvent. The best selectivity for the formation of (II) was obtained when the enzyme was adsorbed on Celite and suspended in water-saturated chloroform with 0.5% (v/v) extra water added. The yield of low molecular weight products in this case was 28%; of this fraction, 95% was (II). Offprint requests to: P. Adlercreutz     

Morphogenetic responses obtained from a variety of somatic explant tissues of data palm

Shoot tip, bud, leaf, stem and root explants from bearing trees, offshoots, seedlings, and asexual plantlets ofPhoenix dactylifera L. were cultured on modified Murashige and Skoog nutrient media containing 3 g/l activated charcoal, 100 mg/l 2,4-dichlorophenoxyacetic acid, 3 mg/l N ⁶-(Δ²-isopentyl)adenine to obtain callus. Differential morphogenetic responses were obtained from calli dependent on the explant type and parent source. Subcultured shoot tips and leafy lateral buds callus on nutrient media devoid of charcoal and supplemented with 0.1 mg/l α-naphthaleneacetic acid (NAA) produced adventitious plantlets. Subcultured leaf calli produced roots only. Root callus failed to exhibit any morphogenetic response upon subculturing. Undifferentiated non-leafy buds and stem tissues did not give rise to callus, regardless of the parent source. Generally, the best callus and embryogenetic responses from explants were obtained from seedling and plantlet parent sources. Similarly, organogenetic responses such as root formation and shoot development from shoot tips cultured on media containing 10 mg/l NAA were also related to the parent explant source. Mention of a trademark or proprietary product in the paper does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be available.     

Function of Growth Factors for Rumen Microorganisms I. Nutritional Characteristics of Selenomonas ruminantium

Nutritional characteristics of Selenomonas ruminantium var. lactilytica isolated from a sheep rumen were studied. The organism required for growth the addition of a clarified rumen fluid to a Trypticase-yeast extract medium with either lactate or glucose as an energy source. The requirement for rumen fluid was found to be satisfied by volatile fatty acids in glucose media and by biotin in lactate media. Straight-chain saturated fatty acids with C(3) to C(10) carbon skeleton had been found to be effective. Among them, n-valerate was most effective at the lowest concentration. An abnormal morphology was observed with n-valerate-deficient glucose media. n-Valerate was essential in glucose media, and it was stimulatory in lactate media. Fermentation products from glucose were lactate, propionate, and acetate, and fermentation products from lactate were propionate and acetate. When cells were grown in a glucose medium containing n-valerate-C(14), the label was present in cell fractions. Almost all of the activity was found in lipid materials.     

Effect of particle size on the rate of enzymatic hydrolysis of cellulose

The effect of particle size on enzymatic hydrolysis of cellulose has been investigated. The average size of microcrystalline cotton cellulose has been reduced to submicron scale by using a media mill. The milled products were further subjected to hydrolysis using cellulase. High cellulose concentration (7%) appeared to retard the size reduction and resulted in greater particles and smaller specific surface areas than those at low concentration (3%) with the same milling time. Initial rate method was employed to explore the rate of enzymatic hydrolysis of cellulose. The production rate of cellobiose was increased at least 5-folds due to the size reduction. The yield of glucose was also significantly increased depending upon the ratio of enzyme to substrate. A high glucose yield (60%) was obtained in 10-h hydrolysis when the average particle size was in submicron scale.     

Development of novel Alicyclobacillus spp. isolation medium

AIM: To develop a new isolation medium with higher recovery rates of Alicyclobacillus spp. METHODS AND RESULTS: SK agar was developed with optimized incubation temperature, pH, acidulant, Tween 80 concentration and divalent cation addition. Results indicate that detection of Alicyclobacillus spp. by SK agar was significantly higher (P > 0.05) than those obtained by K agar, orange serum agar, and potato dextrose agar. CONCLUSIONS: Current media used for Alicyclobacillus spp. isolation still resulted in high numbers of false negative products. The sensitivity of SK agar to Alicyclobacillus spp. allows detection of low numbers of Alicyclobacillus spp. and also provides a more higher isolation results compared with currently used media. SIGNIFICANCE AND IMPACT OF THE STUDY: SK agar will be useful to the fruit juice industry to obtain more accurate numbers of contaminant Alicyclobacillus spp. With this media, false negative samples can be reduced, and the likelihood of exported products being rejected can be greatly reduced.     

Bioactive metabolites from the endophytic fungus Ampelomyces sp. isolated from the medicinal plant Urospermum picroides

Extracts of cultures grown in liquid or on solid rice media of the fungal endophyte Ampelomyces sp. isolated from the medicinal plant Urospermum picroides exhibited considerable cytotoxic activity when tested in vitro against L5178Y cells. Chromatographic separation yielded 14 natural products that were unequivocally identified based on their ¹H and ¹³C NMR as well as mass spectra and comparison with previously published data. Six compounds (2, 4, 5, 7, 9 and 11) were natural products. Both fungal extracts differed considerably in their secondary metabolites. The extract obtained from liquid cultures afforded a pyrone (2) and sulfated anthraquinones (7 and 9) along with the known compounds 1, 3, 6 and 8. When grown on solid rice medium the fungus yielded three compounds 4, 5 and 11 in addition to several known metabolites including 6, 8, 10, 12, 13 and 14. Compounds 4, 8 and 10 showed the strongest cytotoxic activity against L5178Y cells with EC₅₀ values ranging from 0.2–7.3 μg/ml. Furthermore, 8 and 10 displayed antimicrobial activity against the Gram-positive pathogens, Staphylococcus aureus, S. epidermidis and Enterococcus faecalis at minimal inhibitory concentrations (MIC) of 12.5 μg/ml and 12.5–25 μg/ml, respectively. Interestingly, 6 and 8 were also identified as constituents of an extract derived from a healthy plant sample of the host plant U. picroides thereby indicating that the production of bioactive natural products by the endophyte proceeds also under in situ conditions within the host plant.     

Stability of T-2 mycotoxin in aqueous media

3H-labeled T-2 mycotoxin was dissolved in various aqueous media and stored for up to 3 weeks at 4, 22, and 37 degrees C. At periods ranging from 1 to 21 days, aliquots were assayed by thin-layer chromatography. Thin-layer chromatography plates were scanned for breakdown products by use of a radioisotope scanner, and breakdown products were identified based on their comigration with known standards. Results indicated that T-2 toxin was more stable in tissue culture medium with or without serum, than in Hanks balanced salt solution (HBSS), at all temperatures. The metabolites HT-2, T-2 triol, and T-2 tetraol were detected as early as 1 day (HBSS; 37 degrees C) and as late as 3 weeks (HBSS; 4 degrees C) after storage. Stability of the toxin in aqueous media decreased with increasing temperature.     

In vitro effect of larval stages of Ascaris lumbricoides on human blood clotting

The effects of larval stages of Ascaris lumbricoides on human blood clotting was studied in vitro. Extracts and excretory/secretory products of third-stage larvae (L3) and late third-stage larvae (LL3) cultured from ova obtained from infected patients were analysed for anti-coagulant activity. Prothrombin time (PT) was prolonged by the addition of either whole extract of L3/LL3 or ES products of L3/LL3 as compared to controls. Partial thromboplastin time with kaolin (PTTK) was also prolonged on the addition of either extracts of ES products of L3/LL3. The prolongation of PTTK was significantly higher with extracts/ES products of L3 when compared to the extracts/ES products of LL3 (p less than 0.005). Thrombin time (TT) was prolonged by extracts of L3/LL3 and their ES products.     

Stability of T-2 mycotoxin in aqueous media.

3H-labeled T-2 mycotoxin was dissolved in various aqueous media and stored for up to 3 weeks at 4, 22, and 37 degrees C. At periods ranging from 1 to 21 days, aliquots were assayed by thin-layer chromatography. Thin-layer chromatography plates were scanned for breakdown products by use of a radioisotope scanner, and breakdown products were identified based on their comigration with known standards. Results indicated that T-2 toxin was more stable in tissue culture medium with or without serum, than in Hanks balanced salt solution (HBSS), at all temperatures. The metabolites HT-2, T-2 triol, and T-2 tetraol were detected as early as 1 day (HBSS; 37 degrees C) and as late as 3 weeks (HBSS; 4 degrees C) after storage. Stability of the toxin in aqueous media decreased with increasing temperature.     

Hormonal control of glycogenolysis in parenchymal liver cells by Kupffer and endothelial liver cells

Conditioned media of isolated Kupffer and endothelial liver cells were added to incubations of parenchymal liver cells, in order to test whether secretory products of Kupffer and endothelial liver cells could influence parenchymal liver cell metabolism. With Kupffer cell medium an average stimulation of glucose production by parenchymal liver cells of 140% was obtained, while endothelial liver cell medium stimulated with an average of 127%. The separation of the secretory products of Kupffer and endothelial liver cells in a low and a high molecular weight fraction indicated that the active factor(s) had a low molecular weight. Media, obtained from aspirin-pretreated Kupffer and endothelial liver cells, had no effect on the glucose production by parenchymal liver cells. Because aspirin blocks prostaglandin synthesis, it was tested if prostaglandins could be responsible for the effect of media on parenchymal liver cells. It was found that prostaglandin (PG) E1, E2, and D2 all stimulated the glucose production by parenchymal liver cells, PGD2 being the most potent. Kupffer and endothelial liver cell media as well as prostaglandins E1, E2, and D2 stimulated the activity of phosphorylase, the regulatory enzyme in glycogenolysis. The data indicate that prostaglandins, present in media from Kupffer and endothelial liver cells, may stimulate glycogenolysis in parenchymal liver cells. This implies that products of Kupffer and endothelial liver cells may play a role in the regulation of glucose homeostasis by the liver.     

Biochar successfully replaces activated charcoal for in vitro culture of two white poplar clones reducing ethylene concentration

Biochar (BC) is a carbon rich product resulting from the biomass pyrolysis process and there have been no reports until now on BC effects in tissue cultures as a suitable substitute for activated charcoal (AC). The results of an experiment on two clones of white poplar (Populus alba L.) grown in culture media with different amounts of BC (0, 0.5 and 1.5?g/dm³) showed that its addition did not damage the plants and there were no significant differences comparing the data obtained for the same concentrations of AC. Both BC and AC addition was shown to increase root dry biomass and number of roots per shoot and these effects appeared to be independent of genotype and concentrations of the added products. A greater elongation was also recorded for shoots grown on a substrate containing BC than those grown on media without BC. These effects did not seem to be caused by darkening due to the addition of BC as there are no significant differences between the temperatures of the different culture media, but are probably due to the adsorption of molecules such as ethylene. Indeed, during the experiment, the hormone concentration in the atmosphere was lower in vials containing the media with BC than the BC-free ones after 14 and 21?days: the lower amount of ethylene in the medium with BC could explain the difference in shoot elongation and the abundant root biomass since high ethylene concentration could inhibit organogenesis.     

Heat shock modulates the ability of A-549 cell line from human lung adenocarcinoma to regulate the intensity of DNA and protein biosynthesis by an autocrine mechanism]

A-549 cells of human lung adenocarcinoma were subjected to heat shock (30 min, 44 degrees C) which caused substantial decreases in the rates of biosynthesis of the great bulk of cellular proteins with simultaneous increases in the synthesis rates of the 70 kDa protein predominantly localized in cell cytosol. By the 6th hour after the heat shock cessation this protein synthesis reached its maximum; by the 18th hour it was no longer detectable, while the protein itself was not denatured. During the recovery after the heat shock the ability of the serum-free culture medium conditioned by A-549 cells in autocrine regulation of [3H]thymidine incorporation into DNA and [3H]leucine incorporation into proteins changed also. The conditioned medium obtained within 1-3 hours after the heat shock did not influence the intensity of DNA synthesis, while the medium obtained 4-48 hours after the heat shock stimulated this process, the maximal effect (3.3-fold stimulation) being observed in the case of the 48-hour conditioned medium. Temporary (1 hour) acidification of the conditioned media down to pH 2.0 resulted in complete inhibition of the stimulating activity. Besides, these media acquired an ability to inhibit [3H]thymidine incorporation into the DNA of tracer cells. Study of effects of conditioned media on the rate of [3H]leucine incorporation into A-549 cell proteins revealed that the media obtained 1-4 hours after the heat shock inhibited this process, while the media obtained 6-18 hours thereafter stimulated it 1.2-2.1-fold. In the test systems under study temporary acidification of the media increased their stimulating influence on [3H]leucine incorporation into cellular proteins.     

Somatic embryogenesis and plant regeneration in carob (Ceratonia siliqua L.)

Summary Somatic embryos of carob (Ceratonia siliqua L.) were induced from cotyledonary segments excised from immature seeds when cultured on Murashige and Skoog media supplemented with several combinations of 6-benzylaminopurine (BA) and indole-3-butyric acid (IBA). The best frequencies of induction (33.8%) were obtained when 4.4 μM BA and 0.5 μM IBA were used. Shoots were also sporadically formed in the same media. When IBA was replaced by other auxins in the induction media, only α-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) could induce somatic embryogenesis, although at lower rates than IBA. 2,4-Dichlorophenoxyacetic acid and 4-amino-3,5,6-trichloropicolinic acid were completely ineffective. Besides culture media composition, the developmental stage of the explants at the time of culture showed a strong influence on somatic embryogenesis induction, with cotyledons from stage II pods providing the highest levels of induction. By contrast, the genotype of the explant did not determine a significant role in the induction process. Attempts to achieve somatic embryo germination were mostly unsuccessful, since only shoot development was observed; the highest frequencies of development occurred on media containing only gibberellic acid (3.0 μM). For plant regeneration, the developed shoots were further rooted on IBA-supplemented media, and the plantlets obtained were transferred to soil, where c. 88% of them survived. Histological observations showed the presence of morphologically normal and abnormal somatic embryos, the latter displaying an abnormal pattern of vascular bundles. Ultrastructural analysis showed that the cells of the globular embryos had a dense cytoplasm, whereas those not involved in somatic embryo formation showed signs of senescence. Histological studies were also used to distinguish between somatic embryos and shoots originated in the same media.     

Cause analysis of the effects of acid-catalyzed steam-exploded corn stover prehydrolyzate on ethanol fermentation by Pichia stipitis CBS 5776

The prehydrolyzate obtained from acid-catalyzed steam-exploded corn stover (ASC) mainly contains xylose and a number of inhibitory compounds that inhibit ethanol fermentation by Pichia stipitis. In this study, the effects of the ASC prehydrolyzate, specifically those of the carbohydrate-degradation products, lignin-degradation products (which were extracted from ASC prehydrolyzate using ethyl acetate), and six major phenolic compounds (added to pure-sugar media individually or in combination), on ethanol fermentation were investigated. Results indicate that the effects of the carbohydrate-degradation products were negligible (10 h delayed) compared with those of pure-sugar fermentation, whereas the effects of the lignin-degradation products were significant (52 h delayed). Meanwhile, the inhibitory effects of the major phenolic compounds were not caused by certain types of inhibitors, but were due to the synergistic effects of various inhibitors.