Non- Formal Education Efforts to Improve Human Resources in Facing The Challenges of Global Digitalization Through Online Courses

The rapid changes in world civilization in various countries are marked by the unlimited use of information technology. The rapid development of information technology in the digital era makes people need more than just literacy; they need a learning perspective that is able to educate them about the benefits of effective and critical filtering of valid information. To develop abilities and skills, not only can they be achieved through formal education. One of them is a digital-based non-formal learning program that facilitates the community's development in the digitalization era, namely online courses. The research method used is the System Literature Reviewer, or SLR, method. Where in the process do researchers collect, enter, process, and store data? This method aims to solve the problems that researchers have through a literature review of previous journals. Nonformal education provides many options for meeting needs in a digital era; this iS certainly tailored to the needs and circumstances of the community to be addressed. Social society will be studied as the fulfillment of needs occurs so that adjustments to nonformal education methods will increasingly develop. Online learning outcomes are as good or better than conventional learning outcomes. Although it has been found that there are positive outcomes from online learning, it is not certain that this is applicable to all courses. There is no evidence that online learning is superior as a learning medium, which is in line with previous literature

Integration of enzymatic data in <i>Bacillus subtilis</i> genome-scale metabolic model improves phenotype predictions and enables in silico design of poly-γ-glutamic acid production strains

Abstract Background Genome-scale metabolic models (GEMs) allow predicting metabolic phenotypes from limited data on uptake and secretion fluxes by defining the space of all the feasible solutions and excluding physio-chemically and biologically unfeasible behaviors. The integration of additional biological information in genome-scale models, e.g., transcriptomic or proteomic profiles, has the potential to improve phenotype prediction accuracy. This is particularly important for metabolic engineering applications where more accurate model predictions can translate to more reliable model-based strain design. Results Here we present a GEM with Enzymatic Constraints using Kinetic and Omics data (GECKO) model of Bacillus subtilis, which uses publicly available proteomic data and enzyme kinetic parameters for central carbon (CC) metabolic reactions to constrain the flux solution space. This model allows more accurate prediction of the flux distribution and growth rate of wild-type and single-gene/operon deletion strains compared to a standard genome-scale metabolic model. The flux prediction error decreased by 43% and 36% for wild-type and mutants respectively. The model additionally increased the number of correctly predicted essential genes in CC pathways by 2.5-fold and significantly decreased flux variability in more than 80% of the reactions with variable flux. Finally, the model was used to find new gene deletion targets to optimize the flux toward the biosynthesis of poly-γ-glutamic acid (γ-PGA) polymer in engineered B. subtilis. We implemented the single-reaction deletion targets identified by the model experimentally and showed that the new strains have a twofold higher γ-PGA concentration and production rate compared to the ancestral strain. Conclusions This work confirms that integration of enzyme constraints is a powerful tool to improve existing genome-scale models, and demonstrates the successful use of enzyme-constrained models in B. subtilis metabolic engineering. We expect that the new model can be used to guide future metabolic engineering efforts in the important industrial production host B. subtilis

Design and application of microbial consortia for enhanced biomanufacturing

The global energy demand is experiencing rapid growth and fossil reserves cannot anymore meet this demand. Moreover, environmental issues such as global warming and glacier melting, underscore the need for alternative renewable energy sources. To address these challenges and align with the long-term goals of a sustainable circular economy, lignocellulosic biomass (LCB), an abundant and renewable feedstock, has garnered significant attention as resource that can fulfill energy needs while reducing dependence on fossil fuels and mitigating environmental concerns.Establishing biorefineries for microbial fermentation of LCB for value-added products and energy production is a promising way to convert such materials efficiently and economically. To be used in fermentation processes, LCB requires a previous step of pretreatment to disassemble lignin, breakdown hemicellulose, diminish cellulose crystallinity and liberate monomeric sugars for fermentation. However, despite releasing sugars, pretreatment also generate distinct toxic compounds including furans, phenolic compounds, and weak acids, which inhibit the microbial metabolism during fermentation. Therefore, to have a successful fermentation, the concentration of inhibitory compounds needs to be reduced to a minimum that do not affect the microbial performance. Detoxification methods to reduce the concentration of inhibitors in hemicellulosic hydrolysates are currently challenged by low efficiency, prolonged operational time, high costs, undesired sugar loss, and environmental concerns. Alternatively, microbial detoxification with bacteria able to degrade specific inhibitors could be a valuable option to avoid such challenges.In this thesis, the ability of six bacteria to consume inhibitory compounds present in brewer’s spent grain (BSG - a scarcely valorized and abundant by-product of beer industry) hemicellulosic hydrolysate was assessed. Two out of the six bacteria, namely P. putida and Rhodococcus sp., were further studied for their ability to metabolize some of the most frequent lignocellulose-derived inhibitors as sole carbon source. Then, BSG hemicellulosic hydrolysate was used to produce 2,3-butanediol (2,3-BDO) by Paenibacillus polymyxa. 2,3-BDO is a versatile platform chemical with diverse applications across industries, and with a current global market value of US$ 270.4 Mn (million), projected to grow 3.5% in the next 8 years. Presently, the industrial production of 2,3-BDO primarily relies on petroleum-derived hydrocarbons but high production yield has been shown by the bacterium Paenibacillus polymyxa. Different strategies have been studied to enhance the production of 2,3-BDO and the development of a more sustainable bioprocess for its production, including: 1) reducing by-products formation; 2) improving bacterial productivity by genetic engineering; 3) optimizing the fermentation parameters. Utilization of cheap and abundant agro-industrial biomasses remains the best alternative for the development of biomanufacturing in-line with the principle of circular economy. In this thesis, simultaneous saccharification and fermentation (SSF), and simultaneous saccharification and co-fermentation (SSCF) processes were used in monoculture of P. polymyxa and also in co-culture of this strain with an engineered P. putida (unable to use sugars), utilizing BSG as a feedstock. Co-culture was proposed for detoxification of lignocellulosic-derived inhibitory compounds. An enhanced production of 2,3-BDO (20.94 g/L) was achieved using SSCF, when the microbial detoxification of inhibitory compounds was operated. This approach represents a significant progress for the establishment of an efficient biomanufacturing of 2,3-BDO from lignocellulosic biomass. This study was concluded with a techno-economic assessment of the SSCF process using a co-culture system of P. polymyxa with Pseudomonas putida for 2,3-BDO production from BSG

Does SwrA affect DegU-mediated transcriptional activation of aprE?

Bacillus subtilis subtilisin E (AprE) is one of main industrial proteases. Expression of aprE is regulated by a complex network of activators and repressors that comprises AbrB, SinR, ScoC, CodY and DegU. The latter is a global regulator activated via phosphorylation mediated by its cognate sensor kinase DegS. It has been shown that DegU~P can physically interact with SwrA, a small protein essential for swarming motility. The cooperative effect of DegU~P and SwrA has been demonstrated on two promoters: PA(fla/che), which codes for the major structural components of the flagellum; and Ppgs, that encodes the genes for γ-PGA biosynthesis. On those promoters, SwrA appears to act as a modulator of DegU activity, promoting transcriptional activation. To date, no studies addressed the role of SwrA on the regulation exerted by DegU~P on PaprE. The aim of this work is to investigate whether SwrA is involved in modifying DegU activity also at the subtilisin promoter. To this end, the construct containing a GFP reporter gene under the control of PaprE, kindly provided by Jan-Willem Veening, was used to carry out quantitative and qualitative analyses of aprE expression. The construct was inserted in isogenic Bacillus strains, differing for the status of both the swrA allele (swrA+, swrA- and ΔswrA) and the degUS TCS (degUSwt, degU32Hy, degS200Hy). The PaprE transcriptional profile will be presented together with fluorescent microscopy data

Silver Doped Magnesium Ferrite Nanoparticles: Physico-Chemical Characterization and Antibacterial Activity

Spinel phases, with unique and outstanding physical properties, are attracting a great deal of interest in many fields. In particular, MgFe2O4, a partially inverted spinel phase, could find applications in medicine thanks to the remarkable antibacterial properties attributed to the generation of reactive oxygen species. In this paper, undoped and Ag-doped MgFe2-xAgxO4 (x = 0.1 and 0.3) nanoparticles were prepared using microwave-assisted combustion and sol–gel methods. X-ray powder diffraction, with Rietveld structural refinements combined with micro-Raman spectroscopy, allowed to determine sample purity and the inversion degree of the spinel, passing from about 0.4 to 0.7 when Ag was introduced as dopant. The results are discussed in view of the antibacterial activity towards Escherichia coli and Staphylococcus aureus, representative strains of Gram-negative and Gram-positive bacteria. The sol–gel particles were more efficient towards the chosen bacteria, possibly thanks to the nanometric sizes of metallic silver, which were well distributed in the powders and in the spinel phase, with respect to microwave ones, that, however, acquired antibacterial activity after thermal treatment, probably due to the nucleation of hematite, itself displaying well-known antibacterial properties and which could synergistically act with silver and spinel

An overall framework for the E. coli γ-glutamyltransferase-catalyzed transpeptidation reactions

γ-Glutamyl derivatives of proteinogenic or modified amino acids raise considerable interest as flavor enhancers or biologically active compounds. However, their supply, on a large scale and at reasonable costs, remains challenging. Enzymatic synthesis has been recognized as a possible affordable alternative with respect to both isolation procedures from natural sources, burdened by low-yield and by the requirement of massive amount of starting material, and chemical synthesis, inconvenient because of the need of protection/deprotection steps. The E. coli γ-glutamyltransferase (Ec-GGT) has already been proposed as a biocatalyst for the synthesis of various γ-glutamyl derivatives. However, enzymatic syntheses using this enzyme usually provide the desired products in limited yield. Hydrolysis and autotranspeptidation of the donor substrate have been identified as the side reactions affecting the final yield of the catalytic process. In addition, experimental conditions need to be specifically adjusted for each acceptor substrate. Substrate specificity and the fine characterization of the activities exerted by the enzyme over time has so far escaped rationalization. In this work, reactions catalyzed by Ec-GGT between the γ-glutamyl donor glutamine and several representative acceptor amino acids have been finely analyzed with the identification of single reaction products over time. This approach allowed to rationalize the effect of donor/acceptor molar ratio on the outcome of the transpeptidation reaction and on the distribution of the different byproducts, inferring a general scheme for Ec-GGT-catalyzed reactions. The propensity to react of the different acceptor substrates is in agreement with recent findings obtained using model substrates and further supported by x-ray crystallography and will contribute to characterize the still elusive acceptor binding site of the enzyme

OPTIMIZATION OF GAMMA-PGA BIOSYNTHESIS SUPPORTED BY SYNTHETIC BIOLOGY AND METABOLIC ENGINEERING STRATEGIES

Poly-γ-glutamate (γ-PGA) is a natural polymer made of glutamic acid residues, synthesized by the pgs operon of Bacillus subtilis. γ-PGA has a wide range of applications as food, cosmetics and pharmaceutical additive. However, to increase its industrial attractiveness, it is necessary to cut production costs utilizing cost-competitive feedstocks for fermentation. Raw glycerol is a low-cost by-product of biodiesel plants (it accounts for 10% of the final product) that can be used as feedstock. To achieve cost-competitive γ-PGA production from glycerol a multifaceted approach has been set up that includes: 1) Characterization and optimization of pgs operon regulation: the strength of the pgs operon regulatory elements has been analysed both by a synthetic biology approach, exploiting the well-characterized expression operating unit (EOU) inserted in amyE, and by a classical in-locus transcriptional fusion. Results from the two settings will be compared. These data will now be used to finely tune pgs expression through an inducible promoter to optimize γ-PGA yield. 2) Accumulation of γ-PGA precursors by metabolic engineering: a genome-scale metabolic model was used to identify suitable targets for enhancing central carbon pathway flux toward γ-PGA synthesis. The first two B. subtilis strains, engineered according to this analysis, showed enhanced polymer production. Other target genes are under investigation. 3) Enhancement of glycerol metabolism: B. subtilis tolerance to raw glycerol obtained from a biodiesel plant (from both vegetable and animal origin) was verified. Further investigations are underway to improve glycerol uptake and consumption

An In Vitro Study on the Role of Cellulases and Xylanases of <i>Bacillus subtilis</i> in Dairy Cattle Nutrition

The administration of Bacilli to dairy cows exerts beneficial effects on dry matter intake, lactation performance, and milk composition, but the rationale behind their efficacy is still poorly understood. In this work, we sought to establish whether cellulases and xylanases, among the enzymes secreted by B. subtilis, are involved in the positive effect exerted by Bacilli on ruminal performance. We took advantage of two isogenic B. subtilis strains, only differing in the secretion levels of those two enzymes. A multi-factorial study was conducted in which eight feed ingredients were treated in vitro, using ruminal fluid from cannulated cows, with cultures of the two strains conveniently grown in a growth medium based on inexpensive waste. Feed degradability and gas production were assessed. Fiber degradability was 10% higher (p p B. subtilis cellulases and xylanases effectively contribute to improving forage quality, justifying the use of Bacilli as direct-fed microbials to increase animal productivity