Development of an agar-plug cultivation system for bioactivity assays of actinomycete strain collections

Natural products are an important source of lead compounds for the development of drug substances. Actinomycetes have been valuable especially for the discovery of antibiotics. Increasing occurrence of antibiotic resistance among bacterial pathogens has revived the interest in actinomycete natural product research. Actinobacteria produce a different set of natural products when cultivated on solid growth media compared with submersed culture. Bioactivity assays involving solid media (e.g. agar-plug assays) require manual manipulation of the strains and agar plugs. This is less convenient for the screening of larger strain collections of several hundred or thousand strains. Thus, the aim of this study was to develop a 96-well microplate-based system suitable for the screening of actinomycete strain collections in agar-plug assays. We developed a medium-throughput cultivation and agar-plug assay workflow that allows the convenient inoculation of solid agar plugs with actinomycete spore suspensions from a strain collection, and the transfer of the agar plugs to petri dishes to conduct agar-plug bioactivity assays. The development steps as well as the challenges that were overcome during the development (e.g. system sterility, handling of the agar plugs) are described. We present the results from one exemplary screening campaign targeted to identify compounds inhibiting Agr-based quorum sensing where the workflow was used successfully. We present a novel and convenient workflow to combine agar diffusion assays with microtiter-plate-based cultivation systems in which strains can grow on a solid surface. This workflow facilitates and speeds up the initial medium throughput screening of natural product-producing actinomycete strain collections against monitor strains in agar-plug assays.

Effect of biosurfactant sophorolipids on Rhizomucor miehei lipase fermentation by Aspergillus oryzae

In this study, the effect of biosurfactant sophorolipids (SLs) on Rhizomucor miehei lipase (RML) fermentation by Aspergillus oryzae was investigated. With the exogenous addition of 0.3% (w/v) SLs in the initial medium, the RML activity reached 430.0 U/mL, an increase of 25.0% compared to the control group. Subsequently, the physiological metabolic responses of A. oryzae to the addition of SLs were further explored. The results showed that though SLs had almost no effect on the RML secretion, it would affect the morphology of the cells. During the late phase of the fermentation, the proportion of middle pellets, which was generally considered as an energetic and stable state for enzyme production was increased with the addition of SLs. Simultaneously, the viscosity of fermentation broth was reduced, which facilitated the increase of oxygen transfer, thereby improving the RML production. Finally, it could be found that the addition of SLs significantly increased the contents of precursor amino acids, especially for those rank first and second of the RML composition, and it could promote the synthesis of RML.

Effect of biosurfactant sophorolipids on Rhizomucor miehei lipase fermentation by Aspergillus oryzae

In this study, the effect of biosurfactant sophorolipids on Rhizomucor miehei lipase (RML) fermentation by Aspergillus oryzae was investigated. With the exogenous addition of 0.3% (w/v) sophorolipids in the initial medium, the RML activity reached 430.0 U/mL, an increase of 25.0% compared to the control group. Subsequently, the physiological metabolic responses of A. oryzae to the addition of sophorolipids were further explored. The results showed that although sophorolipids had almost no effect on the RML secretion, it would affect the morphology of the cells. During the late phase of the fermentation, the proportion of middle pellets, which were generally considered as an energetic and stable state for enzyme production was increased with the addition of sophorolipids. Simultaneously, the viscosity of fermentation broth was reduced, thereby contributing to the oxygen transfer and RML production. Finally, it could be found that the addition of sophorolipids significantly increased the content of precursor amino acids, especially for those rank first and second of the RML composition, and it could promote the synthesis of RML.

Optimum Medium for Lipase Production by Lipolytic Filamentous Fungi Isolated from Kendari Landfill Soil

Lipase produced by Aspergillus is widely known and used in many industrial sectors. Three lipolytic filamentous fungi had been isolated from Kendari (Southeast Sulawesi, Indonesia) landfill soil in previous study and identified as Aspergillus niger KE1, Aspergillus terreus KC1, and Aspergillus fumigatus KE6. However, the optimization of these isolate has not been reported. In this study, statistical optimization is selected because of more effective, efficient, economical, robust in giving the results and the possibility of analysing the interaction effects among factors. Three lipolytic isolates were screened in initial medium to obtain the highest lipolytic isolate and it was used in medium optimization process. Optimization was done using the series experimental design of Taguchi and RSM. Optimization was sucessfully obtain the optimum medium with the reduction of medium component from previously medium reported. Aspergillus niger KE1 is the selected isolate with the highest lipase productivity after 72 h in initial medium. The significant factors affected lipase production are peptone, olive oil, glucose and MgSO4.7H2O. The model equation obtained is Y = 1043 – 228 A + 300 B – 19803 C + 99 A*A + 5720 B*B + 292855 C*C – 979 A*B + 6563 A*C – 56338 B*C. This model predicts the lipase productivity succesfully with R2 of 96.9%. The optimized medium composes of peptone 2%, olive oil 0.1%, glucose 0.5% and MgSO4.7H2O 0.075%. Using the medium, the lipase productivity increases 4.7-folds compared with before optimization. Our results suggest that Aspergillus niger KE1 is a potential lipase source which catalyse the esterification reaction. Further research is needed to purify and characterize the lipase enzyme of this isolate.

Statistical optimization of amylase production using grape fruit peels in submerged fermentation

Amylase production by Bacillus licheniformis isolated from fish gut was statistically optimized using grape fruit peels in submerged fermentation. Nutritional parameters were optimized through one factor at a time approach. The selected parameters were grape fruit peels as carbon source, ammonium nitrate as nitrogen source, magnesium sulphate as mineral salt. These parameters along with initial medium pH was optimized through central composite design of response surface methodology. Maximum amylase production was observed at 5% grape fruit peels, 0.9% ammonium nitrate, 0.6% magnesium sulphate when initial pH of medium was adjusted 7. The amylase was optimally active at pH 9 at 80°C. The highest enzyme activity at 80ºC depicted the potential use of the strain especially in textiles and paper industrial processes which are performed at high temperatures.

THE FORMATION OF BASICS HEALTH AND SAFETY LIFE COMPETENCIES FROM GENERAL EDUCATION SCHOOLSTUDENTS

The article is devoted to the problems of formulating health care and safety of life competence. The article presents the history of the formation and development of health care in pedagogy. The article identifies the main competencies of the 8th-grade courses “Fundamentals of Health” and “Human Biology”. Ascertaining experiment, which was conducted among 8‑form students indicates that the majority consider the course “Fundamentals of Health” — 57.2% necessary to study, but 34.5% of students want a more practical orientation of learning. The formation of students’ competencies of the basics of health and safety of life is considered in the article as a step-by-step process based on the theory of knowledge and should take place in the following stages: empirical, conceptual, and creative, corresponding to the levels of basic concepts: initial, medium, high. In the formative experiment the study of these natural disciplines is carried out using modular learning, which involves the transition of the student to the position of a real subject of educational activity, the basic concepts are highlighted. The use of the educational module involves the structuring of the material, the organization of independent work of students and the implementation of educational tasks, practical work with the application of active teaching methods (problem situations, business games). The structuring of educational material involves its division into blocks: information, practical, integration, control, and testing of knowledge. The humanistic approach is carried out by supplementing the course programs with topics of International Humanitarian Law. The content and activity component of the process, as well as the pedagogical conditions for the formation of students’ competencies in the basics of health and safety of life, have been developed. The methods which it is expedient to use in the course of formation of the given competence at students of comprehensive schools are defined.

The development of digital models of the soil cover in the western part of Bol’shezemel’skaya tundra

The methods of digital mapping are promising for creating soil maps on difficultly accessible territories. This study was aimed at searching of optimal approaches for digital mapping of the soil cover in poorly studied western part of the Bol’shezemel’skaya tundra on different scales. Medium-scale (1 : 200 000) and small-scale (1 : 1 M) soil maps served as the source of initial information about soils of this region; actual information of the state of the territory was obtained from remote sensing data (Landsat 8 scenes, Aug. 14, 2013) and digital elevation model ASTER GDEM v.2. After extraction of information and the choice of predictors, the analysis of digital soil cover models obtained with the use of different algorithms – Random Forest (RF), Multinomial Logistic Regression (MLR) and Linear Discriminant Analysis (LDA) – was performed. The coefficient of agreement between the newly developed digital models and the initial paper-based soil maps (kappa) was calculated. This test demonstrated that the RF algorithm ensures the best results, so the final digital maps were obtained using it. Averaged kappa values for the compared small- and medium-scale models were as follows: RF – 0.39 and 0.36; MLR – 0.31 and 0.31; and LDA – 0.28 and 0.18, respectively. After the preliminary correction of the initial medium-scale map, the kappa values somewhat increased (RF – 0.39, MLR – 0.35, LDA – 0.30). At the stage of evaluation of digital soil maps obtained with the use of RF algorithm, these maps and the initial soil maps were compared with independent point-size terrain data. The degree of agreement between these data and the new digital soil maps proved to be no less than that for the initial maps. For the initial and digital small-scale maps, it reached 24 and 26 %, respectively; for the initial and digital medium-scale maps, 54 and 43 %, respectively. After the preliminary correction of the initial medium-scale map, the degree of agreement between the digital model and terrain data improved considerably and reached 61 %. This method of digital soil mapping on the basis of analogous data seems to be optimal.

Effect of Media Strength and pH on the Growth of Hairy Roots and Production of Gymnemic Acid from Gymnema Sylvestre

Gymnema sylvestre (Madhunashini) is one of the most important medicinal plants used as a crude drug for its preventive and therapeutic properties. Among other constituents of Gymnema, gymnemic acid is a major component responsible for biological and pharmacological actions. The present study deals with the influence of different media strength and initial medium pH on the growth of hairy roots and gymnemic acid production from Gymnema sylvestre. Higher strength of the media (1.5X) favoured the biomass production (114.64 g/L FW and 12.63 g/L DW) and gymnemic acid content (11.7 mg/g DW) in the tested range of 0.25, 0.50, 0.75, 1.0, 1.5 and 2.0 X strength. Among the different hydrogen ion concentration (pH) of 4.0, 4.5, 5.0, 5.5, 5.8, 6.0 and 6.5, initial medium pH of 6.0 favoured the biomass production (102.41 g/L FW and 11.52 g/L DW) and medium pH of 5.8 favoured the gymnemic acid production (11.30 mg/g DW).

Mathematical modeling of frontal polymerization

Frontal polymerization is way to convert liquid resin into a solid material with a self-propagating reaction. The reaction spreads like a flame from the heat of the reaction that diffuses into neighboring regions, starting more reaction. The frontal velocity has been accurately modeled for free-radical polymerization systems. The dynamics of fronts have been studied theoretically and experimentally. If the viscosity of the initial medium is low, then fronts can become unstable due to buoyancy-driven convection. A fascinating aspect of frontal polymerization is that fronts often do not propagate as a plane waves but exhibit complex modes such as “spin modes” and chaos. The kinetics of the polymerization significantly affects the onset of these modes. Multifunctional acrylates exhibit more complex dynamics than monoacrylates. Using multifunctional acrylates and inorganic fillers, 3P LLC created “cure-on demand” systems that do not require mixing before use, have a long shelf life and can be hardened in seconds to minutes. We consider two commercial products using frontal polymerization. The first is a wood filler that can be applied to a damaged section of wood and hardened in a few seconds by the application of heat to the surface. The second product is QuickCure Clay (QCC). QCC has an unlimited working time during which it can be sculpted. QCC is then cured by heating part of the object to 100 °C, setting off the propagating curing front. The modeling of frontal polymerization helped guide the development of these products.