Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides

For the first time, a fed-batch fermentation process of Tuber melanosporum was developed for the efficient production of bioactive mycelia and Tuber polysaccharides. Each 1.67 g/L of peptone and 8.33 g/L of yeast extract were added on day 3, 6, and 9, respectively, and sucrose was fed to maintain its concentration around 35–5 g/L when its residual level decreased to 10–5 g/L. Then, the maximal biomass, the production of extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) reached 53.72 ± 2.57 g DW/L, 7.09 ± 0.62 and 4.43 ± 0.21 g/L, respectively. Compared with the batch culture conducted in the enriched medium, the biomass, the production of EPS and IPS were enhanced by 55.8%, 222.3% and 103.2%, respectively. Not only the cell density but also the production of EPS and IPS were the highest ever reported in truffle fermentation, and the biomass was also the highest as ever reported in mushroom fermentation.     

Antibacterial and antioxidant activities of four kaempferol methyl ethers isolated from Dodonaea viscosa Jacq. var. angustifolia leaf extracts

Fractionation of dichloromethane and acetone fractions obtained by serial extraction from the leaf powder of Dodonaea viscosa Jacq. var. angustifolia (Sapindaceae) resulted in the isolation of four kaempferol methyl ethers. The compounds were identified by spectral data (¹H NMR, ¹³C NMR and MS) as: 3, 5, 7-trihydroxy-4'-methoxyflavone (1); 5, 7, 4'-trihydroxy-3, 6-dimethoxyflavone (2); 5, 7-dihydroxy-3, 6, 4'-trimethoxyflavone (santin) (3); and 5-hydroxy -3, 7, 4'-trimethoxyflavone (4) together with 3,4',5,7-tetrahydroxy flavone (kaempferol) (5). Antioxidant potential of the compounds was evaluated using a DPPH spectrophotometric assay, while antibacterial activity was determined using a serial dilution microplate technique. The isolates demonstrated varying degrees of antioxidant and antibacterial activities. Of all the compounds investigated, compounds 1 and 5 demonstrated some antioxidant activity (EC₅₀ = 75.49 ± 1.76 µM and 35.06 ± 0.85 respectively) but lower than l-ascorbic acid (EC₅₀ = 13.55 ± 0.28 µM) used as a standard antioxidant agent. The minimum inhibitory concentration (MIC) of isolated compounds against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa varied from 16 µg/ml to more than 250 µg/ml. Some structure activity relationships could be established for these compounds.     

High-level expression of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas diminuta NK703 in Escherichia coli by combined optimization strategies

In this work, a glutaryl-7-aminocephalosporanic acid acylase (GLA) coding gene was cloned from Pseudomonas diminuta NK703 which was screened from oilfield. The concerted effects of the expression system, inducing condition and culture medium on the expression of NK703 GLA in E. coli were firstly investigated. The best combination was the recombinant E. coli strain of pET-28a + GLA/BL21(DE3) with 2.0% (w/v) lactose inducing in YT medium at 25 °C. Then, by optimizing the components of culture medium, a synthetic medium with dextrin and a feeding medium with glycerol as the carbon sources were developed to further enhance the GLA yield and improve the GLA solubility. In the end, the NK703 GLA activity increased about 50-fold, reached 14,470 ± 465 U/L, and the GLA productivity and the proportion of soluble GLA to the total soluble protein attained 206.0 ± 9.033 U L⁻¹ h⁻¹ and 60.13%, respectively. Scaling up the GLA production in 3.7 L fermenter under the optimized conditions identified in shake flask, the GLA activity also reached 12,406 ± 521 U/L, which was the highest report at fermenter level.     

Teladorsagia circumcincta: Survival of adults in vitro is enhanced by the presence of a mammalian cell line

Adult Teladorsagia circumcincta survival and motility in vitro was examined in a range of different cell culture media, supplements and gas mixes. Under optimum conditions, worms survived for 14 days, exhibiting high motility for 9 days and egg production for 72 h. Optimum conditions involved co-culture of worms with a HeLa cell line in a supplemented cell medium (CEM) and an atmosphere containing 10% CO₂, 5% O₂ 85% N₂, 65% humidity at 37 °C. The incubation medium consisted of Minimum Essential Medium with 10% fetal calf serum, 1% non-essential amino acids, 1% glutamax and 1% penicillin-neomycin-streptomycin cocktail mix. Compared with optimum conditions, incubation in CEM alone, cell conditioned CEM, RPMI alone, Medium 199 alone, reduced CO₂ or O₂, or when cells were replaced with Escherichia coli, both survival and motility were reduced. Optimum conditions for adult T. circumcincta maintenance for culture, anthelmintic testing or generation of excretory/secretory products are described.     

Isolation,identification and characterization of a novel Rhodococcus sp. strain in biodegradation of tetrahydrofuran and its medium optimization using sequential statistics-based experimental designs

Statistics-based experimental designs were applied to optimize the culture conditions for tetrahydrofuran (THF) degradation by a newly isolated Rhodococcus sp. YYL that tolerates high THF concentrations. Single factor experiments were undertaken for determining the optimum range of each of four factors (initial pH and concentrations of K₂HPO₄ · 3H₂O, NH₄Cl and yeast extract) and these factors were subsequently optimized using the response surface methodology. The Plackett–Burman design was used to identify three trace elements (Mg²⁺, Zn²⁺and Fe²⁺) that significantly increased the THF degradation rate. The optimum conditions were found to be: 1.80 g/L NH₄Cl, 0.81 g/L K₂HPO₄ · 3H₂O, 0.06 g/L yeast extract, 0.40 g/L MgSO₄ · 7H₂O, 0.006 g/L ZnSO₄ · 7H₂O, 0.024 g/L FeSO₄ · 7H₂O, and an initial pH of 8.26. Under these optimized conditions, the maximum THF degradation rate increased to 137.60 mg THF h⁻¹ g dry weight in Rhodococcus sp. YYL, which was nearly five times of that by the previously described THF degrading Rhodococcus strain.     

Tyrosinase activity of Greyia flanaganii (Bolus) constituents

Hyper-pigmentation of the skin is a common problem that is prevalent in middle aged and elderly people. It is caused by over production of melanin. Tyrosinase is known to be the key enzyme in melanin production. Ethanolic extract of Greyia flanaganii leaves showed significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 32.62 μg/ml. The total extract was further investigated for its toxicity and effect on melanin production by melanocytes cells, and showed significant inhibition (P < 0.05) (20%) of melanin production at 6.25 μg/ml and low levels of cytotoxicity (IC₅₀ < 400 μg/ml). The amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (EC₅₀) by the total ethanolic extract was found to be 22.01 μg/ml. The effect of G. flanaganii against acne causing bacteria, Propionibacterium acnes, was investigated using microdilution assay. The MIC of the extract of G. flanaganii was found to be 250 μg/ml. Bioassay-guided fractionation led to the isolation of (3S)-4-hydroxyphenethyl 3-hydroxy-5-phenylpentanoate (1), 2′,4′,6′-trihydroxydihydrochalcone (2), 2′,6′,4-trihydroxy-4′-methoxydihydrochalcone (3), 2′,6′-dihydroxy-4′-methoxydihydrochalcone (4), 5,7-dihydroxyflavanone [(2S)-pinocembrin] (5), 2′,6′-dihydroxy-4′,4-dimethoxy dihydrochalcone (6) and (2R,3R)-3,5,7-trihydroxy-3-O-acetylflavanone (7). The isolated compounds were tested for their antioxidant, cytotoxicity, tyrosinase inhibition and antibacterial activities. Compound 2 exhibited significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 69.15 μM. The isolated compounds showed low toxicity of the cells with reduction of melanin content of the cells. All compounds tested showed good radical scavenging activity. These data indicates that G. flanaganii extract and its isolated phenolic constituents could be possible skin lightening agents.     

In vitro effects of growth factors and hormones on three Perkinsus species and increased proliferation of P. marinus during cloning

Clonal cultures are essential for the genotypic and phenotypic characterization of Perkinsus species but their cloning, especially of P. marinus, can be tedious. The use of a growth factor and hormone supplement to facilitate cloning was, therefore, investigated. Many of the 16 supplements tested significantly increased P. marinus and P. olseni proliferation but only two significantly increased P. chesapeaki proliferation. The concentration of the most effective supplement for all three Perkinsus species (i.e., endothelial cell growth supplement, ECGS) and medium dilution were then optimized for P. marinus cultured at low densities. Finally, the advantage of using conditioned culture medium, a feeder layer, and ECGS alone and in different combinations to improve cloning of P. marinus were compared. Using conditioned culture medium, a feeder layer and ECGS in combination, each cell (N = 7) seeded singly yielded clonal cultures with 253 ± 167 cells after 21 days. In contrast, only 4 out of 7 cells seeded singly in culture medium yielded clonal cultures with 5 ± 4 cells after 21 days.     

Optimization of exopolysaccharide welan gum production by Alcaligenes sp. CGMCC2428 with Tween-40 using response surface methodology

The effect of additives on welan gum production produced by fermentation with Alcaligenes sp. CGMCC2428 was studied. Tween-40 was the best additive for improving welan gum production and welan gum displayed better rheological properties than that obtained by control fermentation without additives. Response surface methodology was employed to optimize the culture conditions for welan gum production in the shake flask culture, including Tween-40 concentration, pH and culture temperature. The optimal conditions were determined as follows: Tween-40 concentration 0.94 g/l, pH 6.9 and temperature 29.6 °C. The corresponding experimental concentration of welan gum was 23.62 ± 0.60 g/l, which was agreed closely with the predicted value (23.48 g/l). Validation experiments were also carried out to prove the adequacy and the accuracy of the model obtained. The welan gum fermentation in a 7.5 l bioreactor reached 24.90 ± 0.68 g/l.     

Optimized production of Pichia guilliermondii biomass with zinc accumulation by fermentation

Zinc is an essential nutrient that plays an important role in several biological processes of living organisms. When bound to an organic substrate, Zn is more efficiently absorbed by organisms, has a high biological activity and a low toxicity. Due to its ability to incorporate metals, yeast biomass has been used frequently as a delivery vehicle for many mineral supplements. This study describes the screening of strains of yeast for production of biomass enriched with Zn by submerged fermentation. Five strains of yeasts, belonging to the genera Saccharomyces, Kluyveromyces and Pichia, were evaluated. The highest Zn concentration was 6820 mg/kg of dry weight biomass, using Pichia guilliermondii Wickerham LPB 063 after 120 h of cultivation in a medium with 0.5 g/L ZnSO₄. Process conditions were optimized using statistical experimental design methodology. Four parameters were identified in the 2⁸⁻⁴ fractional factorial design as having a significant effect on Zn accumulation: ZnSO₄ and Fe₂(SO₄)₃ concentrations, time of addition of the ZnSO₄ solution and concentration of soybean molasses. In the 3² experimental design, the influence of ZnSO₄ and Fe₂(SO₄)₃ concentrations were studied more closely. The highest Zn concentration (75,090 mg/kg dry weight) in the biomass was reached using the conditions: ZnSO₄, 10.0 g/L; Fe₂(SO₄)₃, 0.1 g/L in Erlenmeyer flasks. A batch liquid fermentation was carried out in a 2 L bioreactor for production of P. guilliermondii Wickerham LPB 063 containing organically bound Zn. The concentration of organically bound Zn after 144 h of fermentation was of 96,030 mg/kg, with a biomass production of 30 g/L. The maximum specific growth rate obtained (μ_max) was 0.0077/h, while the maximum productivity of biomass was at 0.1511 g/L/h.     

Coronatine,a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate

Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received much attention recently for its potential to act as a plant growth regulator and elicitor of plant secondary metabolism. To gain more insight into the mechanism by which elicitors can affect the biosynthesis of paclitaxel (Px) and related taxanes, the effect of coronatine (Cor) and methyl jasmonate (MeJA) on Taxus media cell cultures has been studied. For this study, a two-stage cell culture was established, in which cells were first cultured for 14 days in a medium optimised for growth, after which the cells were transferred to medium optimised for secondary metabolite production. The two elicitors were added to the medium at the beginning of the second stage. Total taxane production in the cell suspension was significantly enhanced by both elicitors, increasing from a maximum level of 8.14 mg/L in control conditions to 21.48 mg/L (day 12) with MeJA and 77.46 mg/L (day 16) with Cor. Expression analysis indicated that the txs, t13oh, t2oh, t7oh, dbat, pam, bata and dbtnbt genes were variably induced by the presence of the elicitors. Genes encoding enzymes involved in the formation of the polihydroxylated hypothetical intermediate (TXS, T13OH, T2OH, T7OH) and the phenylalanoil CoA chain (PAM) were stronger induced than those encoding enzymes catalysing the last steps of the Px biosynthetic pathway (DBAT, BAPT and DBTNBT). Notably, although taxane accumulation differed qualitatively and quantitatively following MeJA- or Cor-elicitation, gene expression induction patterns were similar, inferring that both elicitors may involve distinct but yet uncharacterised regulatory mechanisms.     

Evaporative water loss and energy metabolic in two small mammals,voles (Eothenomys miletus) and mice (Apodemus chevrieri), in Hengduan mountains region

Evaporative water loss (EWL) and energy metabolism were measured at different temperatures in Eothenomys miletus and Apodemus chevrieri in dry air. The thermal neutral zone (TNZ) of E. miletus was 22.5–30 °C and that of A. chevrieri was 20–27.5 °C. Mean body temperatures of the two species were 35.75±0.5 and 36.54±0.61 °C. Basal metabolic rates (BMR) were 1.92±0.17 and 2.7±0.5 ml O₂/g h, respectively. Average minimum thermal conductance (C_m) were 0.23±0.08 and 0.25±0.06 ml O₂/g h °C. EWL in E. miletus and A. chevrieri increased with the increase in temperature; the maximal EWL at 35 °C was 4.78±0.6 mg H₂O/g h in E. miletus, and 5.92±0.43 mg H₂O/g h in A. chevrieri. Percentage of evaporative heat loss to total heat production (EHL/HP) increased with the increase in temperature; the maximal EHL/HP was 22.45% at 30 °C in E. miletus, and in A. chevrieri it was 19.96% at 27.5 °C. The results may reflect features of small rodents in the Hengduan mountains region: both E. miletus and A. chevrieri have high levels of BMR and high levels of total thermal conductance, compared with the predicted values based on their body masses, while their body temperatures are relatively low. EWL plays an important role in temperature regulation.     

Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid

Crude glycerol is a major byproduct of the biodiesel industry; previous research has proved the feasibility of producing docosahexaenoic acid (DHA, 22:6 n − 3) through fermentation of the algae Schizochytrium limacinum on crude glycerol. The objective of this work is to investigate the cell growth kinetics, substrate utilization efficiency, and DHA production of the algae through a continuous culture. Steady-state biomass yield, biomass productivity, growth yield on glycerol, specific glycerol consumption rate, and fatty acid composition were investigated within the range of dilution rate (D) from 0.2 to 0.6 day⁻¹, and the range of feed crude glycerol concentration (S₀) from 15 to 120 g/L. The maximum specific growth rate was determined as 0.692 day⁻¹. The cells had a true growth yield of 0.283 g/g but with a relatively high maintenance coefficient (0.2216 day⁻¹). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day⁻¹ and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day⁻¹ and S₀ = 90 g/L due to the higher DHA content at S₀ = 90 g/L. The biomass and DHA productivity of the continuous culture was comparable to those of batch culture, while lower than the fed-batch culture, mainly because of the lower DHA content obtained by the continuous culture. Overall, the results show that continuous culture is a powerful tool to investigate the cell growth kinetics and physiological behaviors of the algae growing on biodiesel-derived crude glycerol.     

Use of glycerol for producing 1,3-dihydroxyacetone by Gluconobacter oxydans in an airlift bioreactor

1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6 g/l yeast extract, 4.7 g/l glycerol, 42.1 g/l mannitol, 0.5 g/l K₂HPO₄, 0.5 g/l KH₂PO₄, 0.1 g/l MgSO₄·7H₂O, and 2.0 g/l CaCO₃ with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3 ± 7.8 g/l of maximal DHA concentration with 89.8 ± 2.4% of conversion rate of glycerol to DHA was achieved after 72 h of biotransformation using 10 g/l resting cells at 30 °C, pH 5.0 and 1.5 vvm of aeration rate.     

Modulation of ethanol toxicity by Asian ginseng (Panax ginseng) in Japanese ricefish (Oryzias latipes) embryogenesis

Alcohol consumption by women during pregnancy often induces fetal alcohol spectrum disorder (FASD) in children who have serious central nervous system (CNS), cardiovascular, and craniofacial defects. Prevention of FASD, other than women abstaining from alcohol drinking during pregnancy, is not known. A limitation of the use of synthetic anti-alcoholic drugs during pregnancy led us to investigate herbal products. In particular, many plants including Asian ginseng (Panax ginseng) have therapeutic potential for the treatment of alcoholism. We used Japanese ricefish (medaka) (Oryzias latipes), an animal model of FASD, for identifying herbal medicines that can attenuate ethanol toxicity. Fertilized eggs in standard laboratory conditions were exposed to ginseng (PG) root extract (0–2 mg/mL) either 0–2 (group A) or 1–3 (group B) day post fertilization (dpf) followed by maintenance in a clean hatching solution. The calculated IC₅₀ as determined 10 dpf in A and B groups were 355.3 ± 1.12 and 679.7 ± 1.6 μg/mL, respectively. Simultaneous exposure of embryos in sub-lethal concentrations of PG (50–200 μg/mL) and ethanol (300 mM) for 48 h disrupted vessel circulation and enhanced mortality. However, PG (100 μg/mL) may partially protect trabecular cartilage (TC) deformities in the neurocranium in B group embryos induced by ethanol (300 mM). To understand the mechanism, embryonic ethanol concentration was measured at 2 dpf and adh5, adh8, aldh2, aldh9a, catalase, GST, and GR mRNAs were analyzed at 6 dpf. It was observed that although ethanol is able to reduce adh8 and GST mRNA contents, the simultaneous addition of PG was unable to alter ethanol level as well as mRNA contents in these embryos. Therefore, antagonistic effects of PG on ethanol toxicity are mediated by a mechanism which is different from those regulating ethanol metabolism and oxidative stress.     

Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43)

A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5 mg/L 2,4-D and 1.0 mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD₆₀₀ = 0.8) were co-cultured on LS30-AsPC medium for 2 days at 25 ± 2 °C in the dark. Based on GUS expression analysis, 10 min co-cultivation time with 100 μM acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10 g/L PVP, 10% coconut water and 250 mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T₁) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T₀ and T₁).     

Optimization of medium composition for butyric acid production by Clostridium thermobutyricum using response surface methodology

The optimal medium for butyric acid production by Clostridium thermobutyricum in a shake flask culture was studied using statistical experimental design and analysis. The optimal composition of the fermentation medium for maximum butyric acid yield, as determined on the basis of a three-level four-factor Box-Behnken design (BBD), was obtained by response surface methodology (RSM). The high correlation between the predicted and observed values indicated the validity of the model. A maximum butyric acid yield of 12.05 g/l was obtained at K₂HPO₄ 7.2 g/l, 34.9 g/l glucose, 20 g/l yeast extract, and 15 g/l acetate, which compared well to the predicated production of 12.13 g/l.     

Hydrocarbon degradation and bioemulsifier production by thermophilic Geobacillus pallidus strains

Geobacillus pallidus XS2 and XS3 were isolated from oil contaminated soil samples in Yumen oilfield, China, and were able to produce bioemulsifiers on different hydrocarbons. Biodegradation assays exhibited that approximately 70% of PAH (250 mg/L) or 85% of crude oil (500 mg/L) was removed by the thermophilic bacteria after 20 days. The bioemulsifiers of the two strains were isolated and obtained a productive yield of 4.24 ± 0.08 and 3.82 ± 0.11 g/L, respectively. GPC analysis revealed that the number-average molecular weights (M_n) of the two bioemulsifiers were 271,785 Da and 526,369 Da, with PDI values of 1.104 and 1.027, respectively. Chemical composition studies exhibited that the bioemulsifier XS2 consisted of carbohydrates (68.6%), lipids (22.7%) and proteins (8.7%) while the bioemulsifier XS3 was composed by carbohydrates (41.1%), lipids (47.6%) and proteins (11.3%). Emulsification assays approved the effectiveness of bioemulsifiers over a wide range of temperature, pH and salinity.     

Antiproliferative and newly attributed apoptotic activities from an invasive marine alga: Caulerpa racemosa var. cylindracea

Caulerpa racemosa (Forsskål) is a green marine alga which spreads from tropical to warm-water regions. Due to having invasive capacity C. racemosa var. cylindracea is a well-known biological pollution in Mediterranean Sea. One of the most important secondary metabolites of C. racemosa is Caulerpenyne (CPN). In the present study, antiproliferative and apoptotic effects of C. racemosa var. cylindracea extract and purified CPN on two well-known neuroblastoma cell lines, SHSY5Y and Kelly, are investigated. The antiproliferative and, additionally, newly attributed apoptotic effects of both C. racemosa var. cylindracea extract and purified CPN on SHSY5Y and Kelly cell lines have been shown in the present study. IC₅₀ values are 0.59 ± 0.06; 1.06 ± 0.23 g wet alga/methanol and 5.64 ± 0.09; 6.02 ± 0.09 μM CPN for C. racemosa var. cylindracea extract and purified CPN on SHSY5Y and Kelly cell lines, respectively. Percentages of apoptotic cells of SHSY5Y and Kelly in 0, 0.1 and 1 μM CPN conditions were 1.00 ± 0.71, 3.00 ± 0.71 and 49.40 ± 3.78, 39.60 ± 6.19 and 78.00 ± 2.74, 69.40 ± 3.78, respectively. In conclusion, the present study shows the antiproliferative effect of C. racemosa var. cylindracea extract and newly attributed apoptotic effects of C. racemosa var. cylindracea this extract. Compared to other alkylating anticancer drugs, CPN and also C. racemosa var. cylindracea extract might be considered as an alternative native source of antitumor drugs. Inasmuch as both C. racemosa extract and CPN have shown both antiproliferative and apoptotic effects on SHSY5Y and Kelly cell lines, the CPN and CPN derivatives might be considered as multifunctional agents in cell metabolism.