Real-Time Monitoring of the Yeast Intracellular State During Bioprocesses With a Toolbox of Biosensors

Industrial fermentation processes strive for high robustness to ensure optimal and consistent performance. Medium components, fermentation products, and physical perturbations may cause stress and lower performance. Cellular stress elicits a range of responses, whose extracellular manifestations have been extensively studied; whereas intracellular aspects remain poorly known due to lack of tools for real-time monitoring. Genetically encoded biosensors have emerged as promising tools and have been used to improve microbial productivity and tolerance toward industrially relevant stresses. Here, fluorescent biosensors able to sense the yeast intracellular environment (pH, ATP levels, oxidative stress, glycolytic flux, and ribosome production) were implemented into a versatile and easy-to-use toolbox. Marker-free and efficient genome integration at a conserved site on chromosome X of Saccharomyces cerevisiae strains and a commercial Saccharomyces boulardii strain was developed. Moreover, multiple biosensors were used to simultaneously monitor different intracellular parameters in a single cell. Even when combined together, the biosensors did not significantly affect key physiological parameters, such as specific growth rate and product yields. Activation and response of each biosensor and their interconnection were assessed using an advanced micro-cultivation system. Finally, the toolbox was used to screen cell behavior in a synthetic lignocellulosic hydrolysate that mimicked harsh industrial substrates, revealing differences in the oxidative stress response between laboratory (CEN.PK113-7D) and industrial (Ethanol Red) S. cerevisiae strains. In summary, the toolbox will allow both the exploration of yeast diversity and physiological responses in natural and complex industrial conditions, as well as the possibility to monitor production processes.

Design, construction and implementation of relative humidity and temperature climatic chamber for metrology laboratory

For the calibration of thermohygrometers it is necessary to have a means of generating temperature and relative humidity for this reason in this development we will build a climatic chamber which will generate and control these two magnitudes. For the generation of temperature a thermoresistance was used and the decrease of relative humidity was done through a silica gel trap through which the air inside the chamber is recirculated with the use of a vacuum pump. On the other hand, an ultrasonic humidity generator also known as fogger was used to increase the relative humidity. For the construction of the chamber, acrylic was used as the main material, since it is translucent and facilitates the visualization of the thermohygrometers’ indications. For the control of the process variables such as temperature and relative humidity, an Arduino card was used, which through PWM pulse width modulation, the control was performed achieving a variation of 0.3 °C and 2 %RH, which will be evaluated by an accredited laboratory in the characterization of isothermal media certified by ONAC, since it is in this way that the inhomogeneity, stability and thermal load of the medium, components that affect the uncertainty of the instruments under calibration, are evaluated. It should be remembered that this development was carried out in order to create an air medium with which temperature and relative humidity measuring instruments can be calibrated in the metrology laboratory VALIDACIONES Y METROLOGIA LM S.A.S., since it is in the process of growth and one of its main objectives is to be accredited in the magnitudes of temperature and relative humidity before the accreditation body in Colombia (ONAC), which is the entity that will finally give the technical approval to release the chamber and put it into operation.

Improvement of clonal micropropagation technique of promising Lonicera caerulea L. cultivars

The work is focused on enhancement of in vitro propagation technique of valuable Lonicera caerulea L. cultivars. The impact of some nutrient medium components at main cultivation stages of the genus Lonicera L. representatives was studied. Some cultivars showed the positive effect of substituting sucrose for glucose in the medium at the multiplication stage (Diana (30 or 40 g L−1), Moskovskaya 23 (20 g L−1) and Yugana (40 g L−1)). When studying the influence of different auxin types on honeysuckle rhizogenesis, Solovey and Yugana preferred addition of 1.0 mg L−1 IBA into the medium. During the research of influence of different auxin concentrations on L. caerulea rooting the increased number of rooted microshoots of Goluboy Desert was demonstrated with the increasing of IAA concentration (from 0.5 to 3.0 mg L−1). Meanwhile, Diana and Yugana better rooted on the medium containing 1.0 mg L−1 IAA but Zolushka reached its highest rooting percentage on the medium supplemented with 0.5 mg L−1 IAA. The assessment of second-year plants undergone the whole cycle of clonal micropropagation was carried out 85% of these plants began to bear fruits.

Stem Cell Induction and Plant Regeneration Are Affected By Medium Components in Maca (Lepidium Meyenii Walp)

Selection, propagation and conservation of important genotypes are important in medicinal-industrial plants. Nowadays, using tissue culture and regeneration techniques of medicinal plants under in vitro conditions has been able to proliferate medicinal plants widely, which is much higher than traditional methods of vegetative propagation. Maca (Lepidium meyenii), is an industrial plant whose root is the usable part. Maca has valuable medicinal effects such as sexual enhancement and reproductive power, infertility treatment, improved sperm count and quality, anti-stress, osteoporosis prevention and more. This study was conducted to induce callus and regeneration of Maca. First, MS medium supplemented with different concentrations of Kinetin, NAA and 2,4-D (0.5, 1 and 2 μM respectively) and control were compared for callus induction from root and leaves. After 38 days of incubation, the first callus appeared, after 50 days of callus induction and after 79 days regeneration occurred. The callus induction experiment was performed for the study of the effect of three explants (leaf, stem and root) and seven hormone levels. The regeneration experiment was carried out by studying the effect of three explants (leaf, stem and root) on eight levels of the hormone. The results of data analysis on callus induction showed that the effects of explants, hormones and their interactions on callus induction percentage were highly significant but not significant on callus growth rate. The results of regression analysis showed that explants, hormones and their interactions had no significant effect on regeneration percentage.

Tofu processing wastewater as a low-cost substrate for high activity nattokinase production using Bacillus subtilis

Background Even though tofu is a traditional Chinese food loved by Asian people the wastewater generated during the production of tofu can pollute the environment, and the treatment of this generated wastewater can increase the operating cost of the plant. In this study, the production of nattokinase could be achieved by using the nitrogen source in tofu processing wastewater (TPW) instead of using the traditional nattokinase medium. This meets the need for the low-cost fermentation of nattokinase and at the same time addresses the environmental pollution concerns caused by the wastewater. Bacillus subtilis 13,932 is, a high yielding strain of nattokinase, which is stored in our laboratory. To increase the activity of nattokinase in the tofu process wastewater fermentation medium, the medium components and culture parameters were optimized. Nattokinase with high enzymatic activity was obtained in 7 L and 100 L bioreactors when TPW was used as the sole nitrogen source catalyzed by Bacillus subtilis. Such a result demonstrates that the production of nattokinase from TPW fermentation using B. subtilis can be implemented at an industrial level. Results The peptide component in TPW is a crucial factor in the production of nattokinase. Box–Behnken design (BBD) experiments were designed to optimize various critical components, i.e., Glucose, TPW, MgSO4·7H2O, CaCl2, in nattokinase fermentation media. A maximum nattokinase activity was recorded at 37 °C, pH 7.0, 70 mL liquid medium, and 200 rpm. The highest nattokinase activities obtained from 7 to 100 L bioreactors were 8628.35 ± 113.87 IU/mL and 10,661.97 ± 72.47 IU/mL, respectively. Conclusions By replacing the nitrogen source in the original medium with TPW, there was an increase in the enzyme activity by 19.25% after optimizing the medium and culture parameters. According to the scale-up experiment from conical flasks to 100 L bioreactors, there was an increase in the activity of nattokinase by 47.89%.

ARTP Mutagenesis of Schizochytrium sp. PKU#Mn4 and Clethodim-Based Mutant Screening for Enhanced Docosahexaenoic Acid Accumulation

Schizochytrium species are one of the best oleaginous thraustochytrids for high-yield production of docosahexaenoic acid (DHA, 22:6). However, the DHA yields from most wild-type (WT) strains of Schizochytrium are unsatisfactory for large-scale production. In this study, we applied the atmospheric and room-temperature plasma (ARTP) tool to obtain the mutant library of a previously isolated strain of Schizochytrium (i.e., PKU#Mn4). Two rounds of ARTP mutagenesis coupled with the acetyl-CoA carboxylase (ACCase) inhibitor (clethodim)-based screening yielded the mutant A78 that not only displayed better growth, glucose uptake and ACCase activity, but also increased (54.1%) DHA content than that of the WT strain. Subsequent optimization of medium components and supplementation improved the DHA content by 75.5% and 37.2%, respectively, compared with that of mutant A78 cultivated in the unoptimized medium. Interestingly, the ACCase activity of mutant A78 in a medium supplemented with biotin, citric acid or sodium citrate was significantly greater than that in a medium without supplementation. This study provides an effective bioengineering approach for improving the DHA accumulation in oleaginous microbes.

Optimisation of Culture media through Response surface methodology to improve Antioxidant activity of E. coli

Antioxidants play a significant role in oxidative stress management and health protection. One variable at a time response surface methodology (RSM) was used to formulate different composition of media to increase the antioxidant property of selected microorganism (Escherichia coli MTCC no. 40). 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assays was used to measure antioxidant activity of selected microorganism. The reduction potentiality of DPPH radical was determined by the DPPH% antioxidant or scavenging activity of the solution. In the present study, we have measured antioxidant activity of both intracellular and extracellular metabolites in dry and wet extracellular metabolites in supernatant respectively. In DPPH assay, extracellular metabolites showed better antioxidant potential in comparison with the reference compound. The comparison was based on antioxidant activity in different responses (number 1-16) for both dry and wet biomass. According to the results maximum DPPH % antioxidant or scavenging activity was showed by response 9 (wet biomass) and response 6 (dry biomass) that is 24.8447 and 35.0142 respectively in comparison to standard (4.4636). Hence, response surface methodology is used in bioprocess technology to optimize the medium components which is the primary step involved to enhance the antioxidant activity of particular microorganism.

Chemical Characterization of Atlantic Cod (Gadusmorhua) Collagen Hydrolyzed Using Enzyme Preparation Derived from Red King Crab (Paralithodes Camtschaticus) and Its Potential as a Core Component of Bacterial Culture Medium

The Atlantic cod (Gadusmorhua) and red king crab (Paralithodescamtschaticus) processing wastes are massive and unutilized in the Murmansk region of Russia. The samples of skin-containing waste of Atlantic cod fillets production were hydrolyzed using enzyme preparations derived from red king crab hepatopancreases, porcine pancreases, and Bacillus subtilis bacteria. The activity of enzymes from crab hepatopancreases was significantly higher than the activity of enzymes derived from other sources. The optimal conditions of the hydrolysis process have been figured out. The samples of cod processing waste hydrolysate were analyzed for amino acid composition and molecular weight distribution. The samples of hydrolysate were used as core components for bacterial culture medium samples. The efficiency of the medium samples was tested for Escherichia coli growth rate; the most efficient sample had an efficiency of 95.3% of that of a commercially available medium based on fish meal. Substitution of medium components with those derived from industrial by-products is one of the ways to decrease a cost of a culture medium in biopharmaceutical drug production.