Pyricularia's Capability of Infecting Different Grasses in Two Regions of Mexico.

The genus Pyricularia includes species that are phytopathogenic fungi, which infect different species of Poaceae, such as rice and sorghum. However, few isolates have been genetically characterized in North America. The current study addresses this lack of information by characterizing an additional 57 strains of three grasses (Stenotaphrum secundatum, Cenchrus ciliaris and Digitaria ciliaris) from two distant regions of Mexico. A Pyricularia dataset with ITS sequences retrieved from GenBank and the studied sequences were used to build a haplotype network that allowed us to identify a few redundant haplotypes highly related to P. oryzae species. An analysis considering only the Mexican sequences allowed us to identify non-redundant haplotypes in the isolates of C. ciliaris and D. ciliaris, with a high identity with P. pennisetigena. The Pot2-TIR genomic fingerprinting technique resulted in high variability and allowed for the isolates to be grouped according to their host grass, whilst the ERIC-PCR technique was able to separate the isolates according to their host grass and their region of collection. Representative isolates from different host grasses were chosen to explore the pathogenic potential of these isolates. The selected isolates showed a differential pathogenic profile. Cross-infection with representative isolates from S. secundatum and C. ciliaris showed that these were unable to infect D. ciliaris grass and that the DY1 isolate from D. ciliaris was only able to infect its host grass. The results support the identification of pathogenic strains of Pyricularia isolates and their cross-infection potential in different grasses surrounding important crops in Mexico.

Two-methods approach to follow up biomass by impedance spectroscopy: Bacillus thuringiensis fermentations as a study model.

Impedance spectroscopy is used for the characterization of electrochemical systems as well as for the monitoring of bioprocesses. However, the data obtained using this technique allow multiple interpretations, depending on the methodology implemented. Hence, it is necessary to establish a robust methodology to reliably follow-up biomass in fermentations. In the present work, two methodological approaches, mainly used for the characterization of electrochemical systems, were employed to characterize and determine a frequency that allows the monitoring of biomass in Bacillus thuringiensis fermentations by impedance spectroscopy. The first approach, based on a conventional analysis, revealed a single distribution with a characteristic frequency of around 2 kHz. In contrast, the second approach, based on the distribution of relaxation times, gave three distributions (A, B, and C). The C distribution, found near 9 kHz, was more related to the microbial biomass than the distribution at 2 kHz using the equivalent circuits. The time course of the B. thuringiensis fermentation was followed; bacilli, spores, glucose, and acid and base consumption for pH were determined out of line; and capacitance at 9 kHz was monitored. The correlation between the time course data and the capacitance profile indicated that the monitoring of B. thuringiensis at 9 kHz mainly corresponds to extracellular activity and, in a second instance, to the cellular concentration. These results show that it is necessary to establish a robust and reliable methodology to monitor fermentation processes by impedance spectroscopy, and the distribution of relaxation times was more appropriate. KEY POINTS: • Application of impedance spectroscopy for bioprocess monitoring • Low-frequency monitoring of biomass in fermentations • Analysis of impedance data by two methodological approaches.

Use of equivalent circuit analysis and Cole-Cole model in evaluation of bioreactor operating conditions for biomass monitoring by impedance spectroscopy.

The most important parameter in bioprocesses is biomass, where not only the quantity produced in a culture but also the behavior that is presented are important concerns. It is clear that conditions of operation in a bioreactor affect biomass production, but how operation conditions affect the measurement of biomass on-line is of special interest. We studied the effect of bioreactor operating condition variations on model parameters using impedance spectroscopy for biomass monitoring. The model parameters analyzed were capacitance, resistance, alpha (α), conductivity delta (∆σ) and critical frequency (fc). These model parameters were obtained by fitting data from impedance measurements to an equivalent circuit model and Cole-Cole conductivity model. The effect of operating conditions on the medium with no cells was estimated by the percentage of change in each model parameter. The operating conditions with the most significant percentage of change were determined, by comparing to the percentage of change of the same model parameters obtained, during a fermentation of Bacillus thuringiensis as a cellular model. Equivalent circuit parameters were mainly affected by variation in pH, temperature and aeration, whereas Cole-Cole parameters were affected by variation in agitation, aeration, temperature and pH. Therefore, any variation in these operating conditions (within the test interval) during a fermentation may generate changes in monitoring parameters, which will not be a direct consequence of any change in the properties of the biomass.

Flocculation and Expression of FLO Genes of a Saccharomyces cerevisiae Mezcal Strain with High Stress Tolerance.

Mezcal is a distillate produced by spontaneous fermentation of the must obtained from stalks of Agave spp. plants that are cooked and pressed. Agave must contains a high amount of fructose and phenolic compounds, and fermentation usually occurs under stressful (and uncontrolled) environmental conditions. Yeasts capable of growing under such conditions usually display advantageous biological and industrial traits for stress tolerance such as flocculation. In this study, seven Saccharomyces cerevisiae strains isolated from mezcal must were exposed to temperatures ranging between 10 and 40 °C, and to different sugar sources (fructose or glucose). Yeasts grown in fructose increased their stress tolerance, determined by colony count in a microdrop assay, under low temperature (10 °C) compared to the growth at 40 °C on solid cultures. The most stress-tolerant mezcal strain (Sc3Y8) and a commercial wine (Fermichamp) strain, used as control, were grown under fermentation conditions and exposed to long-term temperature stress to determine their performance and their potential for flocculation. Compared to glucose, fermentation on fructose increased the metabolite accumulation at the end of culture, particularly at 40 °C, with 2.3, 1.3 and 3.4 times more glycerol (8.6 g/L), ethanol (43.6 g/L) and acetic acid (7.3 g/L), respectively. Using confocal microscopy analysis, we detected morphological changes such as aggregation and wall recognition at the level of budding scars in yeast, particularly in the Sc3Y8 strain when it was exposed to 40 °C. The analysis confirmed that this mezcal strain was positive for flocculation in the presence of Ca2+ ions. Analysis of FLO1, FLO5 and FLO11 gene expression implicated in flocculation in both Saccharomyces strains showed a strong transcriptional induction, mainly of the FLO5 gene in the mezcal Sc3Y8 strain.

Reactance and resistance: main properties to follow the cell differentiation process in Bacillus thuringiensis by dielectric spectroscopy in real time.

During growth, Bacillus thuringiensis presents three phases: exponential phase (EP), transition state (TS), and sporulation phase (SP). In order to form a dormant spore and to synthesize delta-endotoxins during SP, bacteria must undergo a cellular differentiation process initiated during the TS. Dielectric spectroscopy is a technique that can be utilized for continuous and in situ monitoring of the cellular state. In order to study on-line cell behavior in B. thuringiensis cultures, we conducted a number of batch cultures under different conditions, by scanning 200 frequencies from 42 Hz to 5 MHz and applying fixed current and voltage of 20 mA and 5 V DC, respectively. The resulting signals included Impedance (Z), Angle phase (Deg), Voltage (V), Current (I), Conductance (G), Reactance (X), and Resistance (R). Individual raw data relating to observed dielectric property profiles were correlated with the different growth phases established using data from cellular growth, cry1Ac gene expression, and free spores obtained with conventional techniques and fermentation parameters. Based on these correlations, frequencies of 0.1, 0.5, and 1.225 MHz were selected for the purpose of measuring dielectric properties in independent batch cultures, at a fixed frequency. X and R manifest more propitious behavior in relation to EP, TS, SP, and spore release, due to particular changes in their signals. Interestingly, these profiles underwent pronounced changes during EP and TS that were not noticed when using conventional methods, but were indicative of the beginning of the B. thuringiensis cell differentiation process.

The effect of hexose ratios on metabolite production in Saccharomyces cerevisiae strains obtained from the spontaneous fermentation of mezcal.

Mezcal from Tamaulipas (México) is produced by spontaneous alcoholic fermentation using Agave spp. musts, which are rich in fructose. In this study eight Saccharomyces cerevisiae isolates obtained at the final stage of fermentation from a traditional mezcal winery were analysed in three semi-synthetic media. Medium M1 had a sugar content of 100 g l(-1) and a glucose/fructose (G/F) of 9:1. Medium M2 had a sugar content of 100 g l(-1) and a G/F of 1:9. Medium M3 had a sugar content of 200 g l(-1) and a G/F of 1:1. In the three types of media tested, the highest ethanol yield was obtained from the glucophilic strain LCBG-3Y5, while strain LCBG-3Y8 was highly resistant to ethanol and the most fructophilic of the mezcal strains. Strain LCBG-3Y5 produced more glycerol (4.4 g l(-1)) and acetic acid (1 g l(-1)) in M2 than in M1 (1.7 and 0.5 g l(-1), respectively), and the ethanol yields were higher for all strains in M1 except for LCBG-3Y5, -3Y8 and the Fermichamp strain. In medium M3, only the Fermichamp strain was able to fully consume the 100 g of fructose l(-1) but left a residual 32 g of glucose l(-1). Regarding the hexose transporters, a high number of amino acid polymorphisms were found in the Hxt1p sequences. Strain LCBG-3Y8 exhibited eight unique amino acid changes, followed by the Fermichamp strain with three changes. In Hxt3p, we observed nine amino acid polymorphisms unique for the Fermichamp strain and five unique changes for the mezcal strains.

Oil and air dispersion in a simulated fermentation broth as a function of mycelial morphology.

The culture conditions of a multiphase fermentation involving morphologically complex mycelia were simulated in order to investigate the influence of mycelial morphology (Trichoderma harzianum) on castor oil and air dispersion. Measurements of oil drops and air bubbles were obtained using an image analysis system coupled to a mixing tank. Complex interactions of the phases involved could be clearly observed. The Sauter diameter and the size distributions of drops and bubbles were affected by the morphological type of biomass (pellets or dispersed mycelia) added to the system. Larger oil drop sizes were obtained with dispersed mycelia than with pellets, as a result of the high apparent viscosity of the broth, which caused a drop in the power drawn, reducing oil drop break-up. Unexpectedly, bubble sizes observed with dispersed mycelia were smaller than with pellets, a phenomenon which can be explained by the segregation occurring at high biomass concentrations with the dispersed mycelia. Very complex oil drops were produced, containing air bubbles and a high number of structures likely consisting of small water droplets. Bubble location was influenced by biomass morphology. The percentage (in volume) of oil-trapped bubbles increased (from 32 to 80%) as dispersed mycelia concentration increased. A practically constant (32%) percentage of oil-trapped bubbles was observed with pelleted morphology at all biomass concentrations. The results evidenced the high complexity of phases interactions and the importance of mycelial morphology in such processes.