Culture Media Options for Growth and Morphological Characterisation of Cercospora coffeicola Affecting Coffee in Zimbabwe

Cercospora leaf spot is fast turning into a critically important disease in Zimbabwe.The disease is caused by Cercospora coffeicola which significantly reduces productivity and quality of coffee.Disturbingly,optimum sporulation of Cercospora coffeicola in culture remains a limiting factor for microbial analysis and quantitative studies of Cercospora leaf spot.Faced with this challenge,an in-vitro study was conducted at Coffee Research Institute,Manicaland,Zimbabwe to examine growth of Cercospora coffeicola in different nutrient media and to determine the best media for Cercospora coffeicola analysis.Six nutrient media were assessed(corn meal agar,oat meal agar,Czapek Dox agar,malt extract agar,yeast extract agar and potato dextrose agar)for the growth of Cercospora coffeicola.The laboratory-based experiment was duplicated,laid out in a Completely Randomized Design,replicated three times and based on Cercospora coffeicola nutrient inoculation.Data were collected on radial growth,colour and texture of mycelium at 3 and 6 days after inoculation.There were significant differences(p<0.05)in the growth of Cercospora coffeicola in media after 3 and 6 days.Malt extract agar had the greatest radial growth(34 mm and 32 mm)of Cercospora coffeicola for trials 1 and 2 respectively,whilst the least growth was in the oat meal agar(14.2 mm and 15.7 mm)for trials 1 and 2 respectively.There were variations in colour and texture of mycelium with malt extract agar,potato dextrose agar and oat meal agar associated with darker colours and rough texture while smooth white mycelia were found in corn meal agar.After considering all nutrient media,malt extract agar was found to be the best media for the growth of Cercospora coffeicola in-vitro.On the basis of our findings,the authors recommend the use of malt extract agar as the primary media for identification and characterisation of Cercospora coffeicola.

Effect of Three Different Growth Media on Yield and Oil Constituents of Rosemary （Rosmarinus officinalis L.） under Protected Agriculture Conditions

Medicinal plants provide an important source of cure since ancient time. Poor soil resources, scarce and saline water and the harsh environment limited the production of plants in the Arabian Gulf countries. This study aimed to investigate the production potential of rosemary （Rosmarinus officinalis L.） grown on different growth media under greenhouse conditions. Three growth media agricultural soil, compost and hydroponic system were used, whereas tuff （inert volcanic material） was used as substrate. The result indicated that the high salinity of the agricultural soil limited growth and oil yield in rosemary. Shoot height increased in 11 weeks, by 62%, 65% and 114% in plants grown in agricultural soil, hydroponic system and compost, respectively. Na content in plants grown in agricultural soil was significantly higher than in plants grown in the other treatment. Essential oil yield in plants grown in compost exceeded those in agricultural soil by 114%. Essential oil content （0.66%-1.5% w/w） and chemical constituents concentrations did not change significantly with growth media. The main constituents, more or less, are comparable to essential oils constituents reported from other countries. In comparison, better yields are obtained for individual components of the oils of plants grown under our green house conditions. This study demonstrated the great potential of commercial production of rosemary in the greenhouse without compromising the oil quality and oil yield.

The Effect of Culture Condition on Type 5 Capsular Polysaccharide Production of Staphylococcus aureus from Diary Cattle

[Objective] The effect of different culture conditions on type 5 capsular polysaccharide production of Staphylococcus aureus from diary cattle was studied to provide simple way for CP production and preparation and laid foundation for carrying out new polysaccharide vaccine research. [Method] Staph-ylococcus aureus was isolated from milk sample of sick dairy cattle and capsular polysaccharide serotypes were identified. Type 5 capsular polysaccharide was cultured on BHI,solid columbia and mod110 culture media. Glucose and lactose were taken as carbon sources for every culture media in solid and liquid state. Therefore 9 different culture conditions were taken to study the effect of culture conditions on capsular polysaccharide production. [Result] Different culture conditions indicated that compared with columbia culture media, BHI culture media could decline capsular polysaccharide production and mod110 culture media could increase capsular polysaccharide production. While for same culture media, solid culture media was better for capsular polysaccharide production,meanwhile,taken lactose as carbon source could increase capsular polysaccharide production.

Effect of Different Culture Conditions on Sporulation Quantity of Phytophthora capsici in Capsicum annuum L. var. dactylus M. in Xunhua

[Objective] The paper was to explore the effect of different culture conditions on sporulation quantity of Phytophthora capsici in Capsicum annuum L.var.dactylus M.in Xunhua.[Method] The effects of light hours,pH,medium and temperature on sporulation quantity during isolation and culture process of P.capsici were studied.[Result] The sporulation quantity of P.capsici under the conditions of 24 h/day light,pH 7.0,potato medium（PSA） and 30 ℃ was the largest,and pH,basal medium and temperature had greater impact on sporulation quantity.[Conclusion]The study laid foundation for the study on natural incidence condition of C.annuum in Xunhua.

Effects of culture conditions on ligninolytic enzymes and protease production by Phanerochaete chrysosporium in air

The production of ligninolytic enzymes and protease by Phanerochaete chrysosporium was investigated under different culture conditions. Different amounts of medium were employed in free and immobilized culture, together with two kinds of medium with different C/N ratios. Little lignin peroxidase （LIP） （〈 2 U/L） was detected in free culture with nitrogen-limited medium （C/N ratio： 56/2.2, in mmol/L）, while manganese peroxidase （MnP） maximum activity was 231 and 240 U/L in 50 and 100 ml medium culture, respectively. Immobilized culture with 50 ml nitrogen-limited medium gave the highest MnP and LiP production with the maximum values of 410 and 721 U/L separately on the day 5; however, flasks containing 100 ml nitrogen-limited medium only produced less MnP with a peak value of 290 U/L. Comparatively, carbon-limited medium （C/N ratio： 28/44, in mmol/L） was adopted in culture but produced little MnP and LiE Medium type had the greatest impact on protease production. Large amount of protease was produced due to glucose limitation. Culture type and medium volume influence protease activity corporately by affecting oxygen supply. The results implied shallow immobilized culture was a possible way to gain high production of ligninolytic enzymes.

Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media： distinct requirements for TGF-β and IGF-Ⅱ

A paracrine regulation was recently proposed in human embryonic stem cells （hESCs） grown in mouse embryonic fibroblast （MEF）-conditioned media （MEF-CM）, where hESCs spontaneously differentiate into autologous fibroblastlike cells to maintain culture homeostasis by producing TGF-β and insulin-like growth factor-lI （IGF-Ⅱ） in response to basic fibroblast growth factor （bFGF）. Although the importance of TGF-β family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-Ⅱ. In order to ease hESC cul- ture conditions and to reduce xenogenic components, we sought （i） to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts （HFFs） and human mesenchymal stem cells （hM- SCs） rather than MEF-CM, and （ii） to analyze whether the cooperation of bFGF with TGF-β and IGF-Ⅱ to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell （MSC）-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors （FGFR1-4） and specifically produce TGF-β in response to bFGF. However, HFFs but not MSCs secrete IGF-Ⅱ. Despite the absence of IGF-Ⅱ in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-Ⅱ may be dispensable for hESC pluripotency. In fact, IGF-Ⅱ blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-Ⅱ-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-β and IGF-Ⅱ in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.

Effects of Culture Conditions on Growth and Docosahexaenoic Acid Production from Schizochytrium limacinum

The effects of temperature, initial pH, salinity of culture medium, and carbon and nitrogen sources on growth and doco- sahexaenoic acid (C22 : 6 n-3, DHA) production from Schizochytrium limacinum OUC88 were investigated in the present study. The results revealed that the optimal temperature, initial pH and salinity level of the medium for DHA production were 23℃, 7.0 and 18, respectively. Glucose was proved the best carbon source for the growth and DHA production from S. limacinum. Among the nitrogen sources tested, soybean cake hydrolysate, a cheap by-product, was found to be effective for the accumulation of DHA in S. lima- cinum cells. In addition, increasing the concentration of carbon sources in the medium caused a significant increase in cell biomass; however, accumulation of DHA in cells was mainly stimulated by the ratio of C/N in the medium. Under the optimal culture condi- tions, the maximum DHA yield achieved in flasks was 4.08 g L-1 after 5 d of cultivation.

Optimization of Culture Conditions and Medium Composition for the Marine Algicidal Bacterium Alteromonas sp. DH46 by Uniform Design

Harmful algal blooms(HABs) have led to extensive ecological and environmental issues and huge economic losses.Various HAB control techniques have been developed,and biological methods have been paid more attention.Algicidal bacteria is a general designation for bacteria which inhibit algal growth in a direct or indirect manner,and kill or damage the algal cells.A metabolite which is strongly toxic to the dinoflagellate Alexandrium tamarense was produced by strain DH46 of the alga-lysing bacterium Alteromonas sp.The culture conditions were optimized using a single-factor test method.Factors including carbon source,nitrogen source,temperature,initial pH value,rotational speed and salinity were studied.The results showed that the cultivation of the bacteria at 28℃ and 180 r min-1with initial pH 7 and 30 salt contcentration favored both the cell growth and the lysing effect of strain DH46.The optimal medium composition for strain DH46 was determined by means of uniform design experimentation,and the most important components influencing the cell density were tryptone,yeast extract,soluble starch,NaNO3 and MgSO4.When the following culture medium was used(tryptone 14.0g,yeast extract 1.63g,soluble starch 5.0 g,NaNO3 1.6 g,MgSO4 2.3 g in 1L),the largest bacterial dry weight(7.36 g L-1) was obtained,which was an enhancement of 107% compared to the initial medium;and the algal lysis rate was as high as 98.4% which increased nearly 10% after optimization.

Biological characteristics of marine bacterium S-9801 strain and its culture conditions of pigment production

Strain of Flavobacterium sp.(S-9801),was screened from 207 strains of marine bacteria isolated from the Bohai Sea continental shelf and the Zhujiang Estuary,for its red pigment production.The biological characteristics of strain S-9801 and culture conditions of pigment production have been checked out in this study. The color of the bacterial colony on 2216E medium was from coccineus to rose bengal. Optimum culture conditions were sodium chloride concentration(g/dm3),10～30;pH,3～8;temperature,25～28℃;tryptone and yeast extract as nitrogen sources and glucose as carbon source. Under optimum conditions,pigment accumulation started after 12 h,reaching a maximum rate of synthesis at 36 h.

STUDIES ON CULTURE CONDITIONS OF BENTHIC DIATOMS FOR FEEDING ABALONE I.EFFECTS OF TEMPERATURE AND LIGHT INTENSITY ON GROWTH RATE

Effects of temperature and light intensity on the groWth rate of four species of benthic diatoms for feeding abalone were studied by using a novel method (in situ optical density measurement)which was simple, convenient, fast, accurate and reliable for determining biomass of benthic algae. The results showed that the favourable temperature and light intensity were as follows: for Amphora coffeaeformis, 30℃, 18 W/m2 for Cocconeis scutellum var. parv. 18℃, 20 W/m2; for Navicula Corymbosa, 18℃. 12.4 W/m2;for Navicula mollis, 24℃, 6 W/m2.

Are Early Embryo Cleavage Kinetics Affected by Energy Substrates in Different Culture Media?

Objective This study aimed to investigate the influence of different culture media on early embryonic cleavage kinetics using time-lapse analysis and to determine the possible relationships between energy substrates in culture media and the cleavage kinetics.Methods A total of 10021 embryos from 1310 couples were cultured in time-lapse incubators.Embryos cultured in Vitrolife media were allocated to group I,and those in COOK media to group II.Embryo cleavage time points up to the 8-cell stage(t2–t8)were observed after pronuclei fading.Results The baseline demographic features,in vitro fertilization indications,ovarian stimulation protocol,oocyte-cumulus complexes,fertilization rate,together with pregnancy and perinatal outcomes were similar(P>0.05)between groups I and II.According to the time-lapse analysis,all embryos in group I showed significantly faster cleavage speed than those in group II(P<0.05).Furthermore,there was better synchrony in division(s3)and a longer length of the third cell cycle duration(cc3)in group II.Interestingly,implanted embryos in group II showed faster cleavage speed than those in group I,especially at t4 and t7.The glucose contents and multiple major amino acids were similar between the two groups.Lactic and pyruvic acid contents were generally higher in group I than those in group II.Conclusion Because different commercial culture media may influence cleavage kinetics of embryos,it is essential for embryologists to take culture media into consideration in selecting a potential embryo when using a time-lapse system before implantation.

Optimization of Culture Conditions for Production of Cellulase by a Thermophilic Bacillus Strain

The production of cellulase in Bacillus amyloliquefaciens UNPDV-22 was optimized using response surface methodology （RSM）. Central composite design （CCD） was used to study the interactive effect of culture conditions （temperature, pH, and inoculum） on cellulase activity. Results suggested that temperature and pH all have significant impact on cellulase production. The use of RSM resulted in a 96% increase in the cellulase activity over the control of non-optimized basal medium. Optimum cellulase production of 13 U/mL was obtained at a temperature of 42.24 ℃, pH of 5.25, and inoculum size of 4.95% （v/v） in a fermentation medium containing wheat bran, soybean meal and malt dextrin as major nutritional factors.

A Sensitive Competitive ELISA for Determination of Biotin in Transformed Yeast Culture Media

Aim To develop a sensitive competitive ELBA for the determination of biotinin transformed yeast culture media. Methods The ELBA plate was firstly coated with Mycoplasmahyopneumoniae, and then successively incubated with rabbit anti-Mycoplasma hyopneumoniae serum andgoat anti-rabbit IgG-biotin to form the solid biotin, which competed with the biotin in the solution(standard or sample) for the limited streptavidin-horse radish peroxidase conjugate. The standardcalibration curve for biotin analysis was constructed in the range of 50 - 2000 ng·L^(-1). ResultsThe detection limit for biotin was found to be 83 ng·L^(-1), which was about 1000 times lower thanthe lowest determination concentration in the reported ELISA for biotin analysis. The relativestandard deviations for the spiked samples at biotin concentrations of 200 ng·L^(-1), 500ng·L^(-1), and 1000 ng·L^(-1) were 24.87%, 6.15% , and 7.86% , respectively, with the averagerecovery of 101.13% . The wild yeast and its sixty-three transformed yeast culture media wereapplied to the developed ELBA for the determination of biotin. It was found that the biotinconcentrations in more than 85% of the tested samples were enhanced with different increase factorsafter transformation. Conclusion Utilization of Mycoplasma hyopneumoniae as the coating proteinimproves the precision and accuracy of the ELBA assay, which might be used for the biotin assay inother media.

Potential of Natural Killer Cell Enriched Conditioned Media for Skin Care and Anti-Aging

Natural killer (NK) cell is a type of immune cell and is known to be particularly responsible for innate immunity such as anti-cancer immunity, defense mechanisms against infections, and secretion of various cytokines and chemokines for increasing recruitment of other immune cells. In this study, we investigated the potentials of NK-enriched lymphocytes (NKEL) conditioned media (CM) on skin care for cosmeceutical compositions. Various cytokines of NKEL CM can improve wound healing through epithelial-mesenchymal transition (EMT) by increasing KLKs (kallikreins) and reduce metalloproteinase (MMP)-1 and MMP-2 to inhibit wrinkle formation. Our results suggest that NKEL CM which has various cytokines promotes up-regulation of cell migration and KLKs and down-regulation of MMP-1 and MMP-2 by stimulating HaCaT keratinocytes migration. Therefore, NKEL CM can be used as a cosmetic composition that can play a role in skin regeneration and anti-aging.

Experimental Investigation on Media Condition of Leaching Scandium from Silt of Yangtze

The minerals of the silt of the end of Sanxia reservoir of Yangtze River were studied. Through the spectral analysis of the samples, the analysis of chemistry components and the content mensuration of main minerals, chemistry components and composition of the main minerals were tested. The results show that: (1) The silt of Sanxia of the Yangtze in Chongqing is mainly composed of silicolites and quartz, containing Fe and Ti in small quantity and Yt, Yb and Sc in minim quantity. The content of Yt, Yb and Sc is 25.8, 2.06, and 6.60 g·t -1 respectively. (2) Thereinto, the content of Sc is 101 g·t -1 in ilmenite and 121 g·t -1 in titanium-picrite. On the basis of these results, Sc leaching conditions and Sc leaching media are investigated by leaching media of HCl, H_2SO_4 and HNO_3. The results show that HCl can be used as the leaching media and its best leaching conditions are as follows: The leaching concentration is 2280%; the leaching ration on liquid-solid is 2∶1; the leaching temperature is 90 ℃; the leaching time is 10 h and the leaching granularity of the test sample is -0.040 mm. The best condition of HCl adding assiting solvent is that assisting solvent is No.7 and its dosage is 40 g·t -1, which leaching ratio is 81.31%.

ComprehensiveanalysesofseismicsourcelayerinXingtaiandTangshanseismicregionsandtheconditionsofmediaab

Statistic analyses of sectional focal depth frequency relation for 1739 M S≥4.0 events in North China, 541 M S≥4.0 events in Xingtai seismic region and 999 M S≥4.0 events in Tangshan seismic region have been carried out for the first step of the study. Then the sectional focal depth frequency diagrams (H N diagram) for each region were obtained. The study shows that: (1) The intraplate earthquakes in North China occur within the continental crust, and are mainly concentrated within a certain depth range of the middle and upper crust, making up a seismic source layer; (2) The seismic source layer consists of a main layer and an auxiliary layer adjacent to it. The tops and bottoms of the main and auxiliary layers correspond to the G and C, as well as to B and G geologic boundaries respectively; (3) The H N diagrams for Xingtai and Tangshan seismic regions are significantly different from each other, indicating the regionality and localization of the seismic source layers; (4) DSS and MTS observations show that the regionality and localization of the seismic source layer depend mainly on the environment of the deep structures of the region; (5) The seismic source layer is a substance of medium layer, which can be related to the brittle ductile transition zone within the crust. The seismic source layer is also a stress conducting layer. As the media above and below this layer are subjected to failure or flowage, then the stress will concentrate in the stress conducting layer, in which the earthquake occurs.

Isolation Technology of Single Spore and Optimization of Conidia Culture Condition for Ustilaginoidea virens

The single spores were isolated from chlamydospores of Ustilaginoidea virens with three different maturities by PSA. The isolated single spores were cul- tured on different media at different temperatures under natural light for inducing conidia to explore the optimum isolation technique of single spore and optimum cul- ture condition of conidia. The results showed that the successful isolating rate of single spore from yellow rice false smut balls reached 90.00%. The sporulatina quantities of isolated single spores cultured on PSD and PDB media at 22 -29 ~C （variable temperature under natural light） or 28 ℃ （constant temperature under dark condition） for 12 d were up to 6.3× 107 and 1.1× 106 spore/mL, respectively. PSA was the most effective method to isolate single spores from yellow rice false smut balls of U. virens. The optimum conidia culture condition included PSD or PDB medium, 22 -29℃ or 28℃, natural light and vibration culture. Key words Ustilaginoidea virens; Single spore isolation; Conidia; Culture condition

Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli

Anti-lipopolysaccharide factors （ALFs） are important antimicrobial peptides that are isolated from some aquatic species. In a previous study, we isolated ALF genes from Chinese mitten crab, Eriocheir sinensis. In this study, we optimized the production of a recombinant ALF by expressing E. sinensis ALF genes in Eseherichia coli maintained in shake-flasks. In particular, we focused on optimization of both the medium composition and the culture condition. Various medium components were analyzed by the Plackett-Burman design, and two significant screened factors, （NH4）2SO4 and KH2PO4, were further optimized via the central composite design （CCD）. Based on the CCD analysis, we investigated the induction start-up time, the isopropylthio-D-galactoside （IPTG） concentration, the post-induction time, and the temperature by response surface methodology. We found that the highest level of ALF fusion protein was achieved in the medium containing 1.89 g/L （NH4）2SO4 and 3.18 g/L KH2PO4, with a cell optical density of 0.8 at 600 nm before induction, an IPTG concentration of 0.5 mmol/L, a post-induction temperature of 32.7~C, and a post-induction time of 4 h. Applying the whole optimization strategy using all optimal factors improved the target protein content from 6.1% （without optimization） to 13.2%. We further applied the optimized medium and conditions in high cell density cultivation, and determined that the soluble target protein constituted 105% of the total protein. Our identification of the economic medium composition, optimal culture conditions, and details of the fermentation process should facilitate the potential application of ALF for further research.

STUDIES ON CULTURE CONDITIONS OF BENTHIC DIATOMS FOR FEEDING ABALONE Ⅱ. EFFECTS OF SALINITY pH, NITROGENOUS AND PHOSPHATE NUTRIENTS ON GROWTH RATE

Effects of salinity , pH, nitrogenous and phosphate nutrients on the growth rate of four species of benthic diatoms were studied by using the method of in situ optical density measurement . The optimal culture conditions for the four species of diatoms are as follows: for Amphora coffeaeformis , s 35 ; pH 8.5; N03 -N 1.54, NH4-N, 7.0; CO (NH2)2-N, 2.5 (mg/L); PO43--P 1.13 (mg/L) ; for Cocconeis scutellum var parva , s 30 ; pH 8.5 ; NO3-N 3.08, NH4+-N 3.5, CO(NH2)2-N 5.0 (mg/L); pO43-－P0.283 (mg/L), for Navicula corymbosa s 25; pH 8.0; N03- －N 1.54, NH4+ -N 3.5, CO(NH2)2-N 5.0(mg/L); PO43-－P0.565 (mg/L), for Navicula mollis , s 25 ; pH 8.0; NO3- － 1.54 NH4+ - N 1.75, CO(NH2)2 -N 1.25 (mg/L), P043-－P 0.141(mg/L).

Optimization of Culture Conditions for Production of Bioactive Metabolites by <i>Streptomyces</i>spp. Isolated from Soil

The current work was carried out under a screening program targeted at isolation of bioactive Streptomyces species from soil samples. A total of 54 Streptomyces species were isolated from soil samples, out of which 4 isolates were found to be promising. These isolates were identified as Streptomyces spectabilis, Streptomyces purpurascens, Streptomyces coeruleorubidus and Streptomyces lavendofoliae and their sequences have been deposited in the GenBank. The influence of culture conditions including, incubation time, incubation temperature, initial pH and different carbon and nitrogen sources on growth and bioactive compound formation was investigated. Isolate R1, identified as Streptomyces spectabilis, showed maximum bioactive metabolite production with cellobiose and peptone as the carbon and nitrogen sources, on the 5th day at pH 5 at 30℃. The optimum conditions for production by isolate R3, identified as Streptomyces purpurascens, were observed to be starch and casein as the carbon and nitrogen sources, pH 7, temperature 30℃ and an incubation period of eight days. For isolate R5, identified as Streptomyces coeruleorubidus, maximal production resulted on the sixth day at pH 6 and temperature of 35℃ with mannitol and JBM. Isolate Y8, identified as Streptomyces lavendofoliae, was found to produce high levels of bioactive metabolites in the medium supplemented with starch and peptone on the 10th day at pH 7 and at an incubation temperature of 30℃. The four strains tested here behaved differently, each one requiring specific conditions for maximum growth as well as bioactive metabolite production.