Providing inoculum for Didymella bryoniae studies: the effect of light spectrum and storing at low temperature

The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media (PDA, V8, ML, and PDAB) and, to evaluate fungus’ survivability stored at -20°C over time. The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media. However, there was considerable variation in the amount of each structure between the different culture media. The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover, the culture medium composition influences the type of fungal structure produced, as well as spores’ size and quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months without loss of viability.

Bacterial Succinoglycans: Structure, Physical Properties, and Applications

Succinoglycan is a type of bacterial anionic exopolysaccharide produced from Rhizobium, Agrobacterium, and other soil bacteria. The exact structure of succinoglycan depends in part on the type of bacterial strain, and the final production yield also depends on the medium composition, culture conditions, and genotype of each strain. Various bacterial polysaccharides, such as cellulose, xanthan, gellan, and pullulan, that can be mass-produced for biotechnology are being actively studied. However, in the case of succinoglycan, a bacterial polysaccharide, relatively few reports on production strains or chemical and structural characteristics have been published. Physical properties of succinoglycan, a non-Newtonian and shear thinning fluid, have been reported according to the ratio of substituents (pyruvyl, succinyl, acetyl group), molecular weight (Mw), and measurement conditions (concentration, temperature, pH, metal ion, etc.). Due to its unique rheological properties, succinoglycan has been mainly used as a thickener and emulsifier in the cosmetic and food industries. However, in recent reports, succinoglycan and its derivatives have been used as functional biomaterials, e.g., in stimuli-responsive drug delivery systems, therapeutics, and cell culture scaffolds. This suggests a new and expanded application of succinoglycan as promising biomaterials in biomedical fields, such as tissue engineering, regenerative medicine, and pharmaceuticals using drug delivery.

Primary morphogenesis of Sorbus Torminalis L. (Grantz) into in vitro culture

Nowadays in vitro methods, combined with ex situ and becoming an increasingly important means of preserving and maintaining the level of phytodiversity stability. Sorbus torminalis L. is a tree of the Rosaceae family, which grows on the territory of Ukraine, belongs to rare, valuable aboriginal species and is listed in the Red Book of Ukraine, with its protection status - endangered. The peculiarities of introduction of in vitro culture of perennial representatives of S. torminalis with the use of different types of explants, sterilizing substances, cultivation conditions and nutrient medium composition are presented in the paper. For in vitro culture of S. torminalis, annual shoots with apical and lateral buds 15-25 cm long are optimal. The influence of different sterilization options on the development of primary microshoots has been studied. For sterilization of artificially awakened and young shoots it is most effective to use 0.1% solution of AgNO3 (7 min) and 15% solution of H2O2 (10 min). The developed method of sterilization of S. torminalis explants provided 80-90% yield of aseptic plant material. It was found that the sterilization regime did not significantly affect the primary morphogenesis of explants and was uniform. The optimal components of nutrient media at the stage of introduction into vitro culture and primary morphogenesis of S. torminalis have been established. For the cultivation of different types of explants of S. torminalis, used WPM nutrient media with the addition of synthetic plant growth regulators 6-benzylaminopurine, thidiazuron, kinetin 0,5-1,5 mg mg∙l-1 and α-naphthylacetic acid 0,01-0,05 mg∙l-1 both alone and in combination with each other. In particular, for the regeneration of plants from the lateral and apical buds of explants is effective WPM medium with the addition of BAP 1,5 mg∙l-1 + 0,5 mg∙l-1 NAA and WPM + TDZ medium 0,5 mg∙l-1 with adding PVP 200 mg∙l-1. To induce the laying of additional buds and shoots on the explant from the apical meristems in the medium should be added 4,0 mg∙l-1 BAP + 0,01 mg∙l-1 NAA with the addition of PVP 200 mg∙l-1.

Diagnostic method and device for evaluating and forecasting the technical condition of farm machinery in operation

The paper discusses methods and ways to diagnose the technical condition of agricultural machines and harvesters, existing practices, and approaches to get reliable data on the current health of the machinery used. The device for assessing and predicting machines’ technical condition includes software and technical means developed with virtual technologies to measure diagnostic parameters of the machinery. The main device elements are digital sensors with physical modifiers (pressure, temperature, medium composition and motion sensors, a-d converters with signal amplifiers), software to configure data gathering, and output to conduct analyses and produce recommendations. The core of the present approach is the technology of virtual prediction of breakdowns by changes in the technical condition parameters. It is based on modular devices, software with an interface that collects and processes data and provides a complete set of failure diagnostics and forecasting. The given method based on a device operating in the information and communication network increases farm machinery’s performance. Furthermore, it reduces operating costs due to the prevention of expensive breakdowns, individual forecasting, and scheduled maintenance of machines in operation. The approach under consideration was applied in the laboratory of digital engineering technologies of the Bashkir State Agrarian University Republic of Bashkortostan of the Russian Federation. The given work is aimed to boost the efficiency of the farm machinery diagnostics and maintenance system by applying a virtual breakdown prediction technology to conduct an automated evaluation, registration, and analysis of a machine’s condition. It can be achieved by developing software and technical means to register data and their structure systematization.

Effect of Nutritional Factors and Copper on the Regulation of Laccase Enzyme Production in Pleurotus ostreatus

This research aimed to establish the relationship between carbon–nitrogen nutritional factors and copper sulfate on laccase activity (LA) by Pleurotus ostreatus. Culture media composition was tested to choose the nitrogen source. Yeast extract (YE) was selected as a better nitrogen source than ammonium sulfate. Then, the effect of glucose and YE concentrations on biomass production and LA as response variables was evaluated using central composite experimental designs with and without copper. The results showed that the best culture medium composition was glucose 45 gL−1 and YE 15 gL−1, simultaneously optimizing these two response variables. The fungal transcriptome was obtained in this medium with or without copper, and the differentially expressed genes were found. The main upregulated transcripts included three laccase genes (lacc2, lacc6, and lacc10) regulated by copper, whereas the principal downregulated transcripts included a copper transporter (ctr1) and a regulator of nitrogen metabolism (nmr1). These results suggest that Ctr1, which facilitates the entry of copper into the cell, is regulated by nutrient-sufficiency conditions. Once inside, copper induces transcription of laccase genes. This finding could explain why a 10–20-fold increase in LA occurs with copper compared to cultures without copper when using the optimal concentration of YE as nitrogen sources.

Hypersecretion of vaccine antigen outer membrane lipoprotein A in Corynebacterium glutamicum through high-throughput based development process

Outer membrane lipoprotein A (OmlA) is a vaccine antigen against porcine contagious pleuropneumonia (PCP), a disease severely affecting the swine industry. Here, we aimed to systematically potentiate the secretory production of OmlA in Corynebacterium glutamicum (C. glutamicum), a widely used microorganism in the food industry, by establishing a holistic development process based on our high-throughput culture platform. The expression patterns, expression element combinations, medium composition, and induction conditions were comprehensively screened or optimized in microwell plates (MWPs), followed by fermentation parameter optimization in a 4×1 L parallel fermentation system (CUBER4). An unprecedented yield of 1.01 g/L OmlA was ultimately achieved in a 5-L bioreactor following the scaling-up strategy of fixed oxygen mass transfer coefficient (kLa), and the produced OmlA antigen showed well-protective immunity against Actinobacillus pleuropneumoniae challenge. This result provides a rapid and reliable pipeline to achieve the hyper-production of OmlA, and possibly other recombinant vaccines, in C. glutamicum.

H2O2-Elicitation of Black Carrot Hairy Roots Induces a Controlled Oxidative Burst Leading to Increased Anthocyanin Production

Hairy roots (HRs) grown in vitro are a powerful platform for plant biotechnological advances and for the bio-based production of metabolites of interest. In this work, black carrot HRs able to accumulate anthocyanin as major secondary metabolite were used. Biomass and anthocyanin accumulation were improved by modulating growth medium composition—different Murashige & Skoog (MS)-based media—and H2O2-elicitation, and the level of the main antioxidant enzymes on elicited HRs was measured. Higher growth was obtained on liquid 1/2 MS medium supplemented with 60 g/L sucrose for HRs grown over 20 days. In this medium, 200 µM H2O2 applied on day 12 induced anthocyanin accumulation by 20%. The activity of superoxide dismutase (SOD)—which generates H2O2 from O2•−—increased by over 50%, whereas the activity of H2O2-scavenging enzymes was not enhanced. Elicitation in the HRs can result in a controlled oxidative burst, in which SOD activity increased H2O2 levels, whereas anthocyanins, as effective reactive oxygen species scavengers, could be induced to modulate the oxidative burst generated. Moreover, given the proven stability of the HR lines used and their remarkable productivity, this system appears as suitable for elucidating the interplay between antioxidant and secondary metabolism.

Isolation and Optimization of Culture Conditions of a Bioflocculant-Producing Fungi from Kombucha Tea SCOBY

Biolocculants are gaining attention in research due to their environmental friendliness and innocuousness to human in comparison to the conventional flocculants. The present study aimed to investigate the ability of fungi from Kombucha tea SCOBY to produce effective bioflocculant in bulk. A 16S rRNA gene sequence analysis was utilized to identify the isolate. The medium composition (carbon and nitrogen sources) and culture conditions (inoculum size, temperature, shaking speed, pH, and time) were optimized using one-factor-at-a-time method. The functional groups, morphology, and crystallinity of the bioflocculant were evaluated using Fourier transform infrared (FT-IR), scan electron microscope (SEM) and X-ray diffractometry (XRD). The fungus was found to be Pichia kudriavzevii MH545928.1. It produced a bioflocculant with flocculating activity of 99.1% under optimum conditions; 1% (v/v) inoculum size, glucose and peptone as nutrient sources, 35 °C, pH 7 and the shaking speed of 140 rpm for 60 h. A cumulus-like structure was revealed by SEM; FT-IR displayed the presence of hydroxyl, carboxyl, amine, and thiocynates. The XRD analysis demonstrated the bioflocculant to have big particles with diffraction peaks at 10° and 40° indicating its crystallinity. Based on the obtained results, P. kudriavzevii MH545928.1 has potential industrial applicability as a bioflocculant producer.

Phosphorus and Nitrogen Limitation as a Part of the Strategy to Stimulate Microbial Lipid Biosynthesis

Microbial lipids called a sustainable alternative to traditional vegetable oils invariably capture the attention of researchers. In this study, the effect of limiting inorganic phosphorus (KH2PO4) and nitrogen ((NH4)2SO4) sources in lipid-rich culture medium on the efficiency of cellular lipid biosynthesis by Y. lipolytica yeast has been investigated. In batch cultures, the carbon source was rapeseed waste post-frying oil (50 g/dm3). A significant relationship between the concentration of KH2PO4 and the amount of lipids accumulated has been revealed. In the shake-flask cultures, storage lipid yield was correlated with lower doses of phosphorus source in the medium. In bioreactor culture in mineral medium with (g/dm3) 3.0 KH2PO4 and 3.0 (NH4)2SO4, the cellular lipid yield was 47.5% (w/w). Simultaneous limitation of both phosphorus and nitrogen sources promoted lipid accumulation in cells, but at the same time created unfavorable conditions for biomass growth (0.78 gd.m./dm3). Increased phosphorus availability with limited cellular access to nitrogen resulted in higher biomass yields (7.45 gd.m./dm3) than phosphorus limitation in a nitrogen-rich medium (4.56 gd.m./dm3), with comparable lipid yields (30% and 32%). Regardless of the medium composition, the yeast preferentially accumulated oleic and linoleic acids as well as linolenic acid up to 8.89%. Further, it is crucial to determine the correlation between N/P molar ratios, biomass growth and efficient lipid accumulation. In particular, considering the contribution of phosphorus as a component of coenzymes in many metabolic pathways, including lipid biosynthesis and respiration processes, its importance as a factor in the cultivation of the oleaginous microorganisms was highlighted.