Jesús Córdova

Improvement of xylanase production by a parasexual cross between Aspergillus niger strains

A diploid strain (D4) isolated via parasexual recombination between two Aspergillus niger xylanase overproducing mutants was characterised in terms of enzyme production and catabolite repression by glucose. This strain increased xylanase production (607 nkat/ml), which was nearly 100% higher than titers achieved by the wild type strain (305 nkat/ml) and 28% higher than the best mutant used to induce parasexual cycle. Diploid D4 was also less sensitive to carbon catabolite repression by glucose, since xylanolytic activity was detected under conditions normally repressing production by the wild type strain. No decrease in maximal xylanase levels was observed in the presence of glucose for diploid D4.

In vitro antagonism of Thielaviopsis paradoxa by Trichoderma longibrachiatum.

Seventy-nine Trichoderma strains were isolated from soil taken from 28 commercial plantations of Agave tequilana cv. ‘Azul’ in the State of Jalisco, Mexico. Nine of these isolates produced nonvolatile metabolites that completely inhibited the growth of Thielaviopsis paradoxa on potato dextrose agar plates. These isolates were identified as Trichoderma longibrachiatum on the basis of their morphology and DNA sequence analysis of two genes (ITS rDNA and translation elongation factor EF-1β). Mycoparasitism of Th. paradoxa by T. longibrachiatum strains in dual cultures was examined by scanning electron microscopy. The Trichoderma hyphae grew alongside the Th. paradoxa hyphae, but penetration of Thielaviopsis hyphae by Trichoderma was no apparent. Aleurioconidia of Th. paradoxa were parasitized by Trichoderma. Both hyphae and aleurioconidia of Th. paradoxa lost turgor pressure, wrinkled, collapsed and finally disintegrated. In liquid cultures, all nine Trichoderma isolates produced proteases, β-1,3-glucanases and chitinases that would be responsible for the degradation of Thielaviopsis hyphae. These results demonstrate that the modes of action of T. longibrachiatum involved against Th. paradoxa in vitro experiments are mycoparasitism and the production of nonvolatile toxic metabolites.

Improvement of growth, fermentative efficiency and ethanol tolerance of Kloeckera africana during the fermentation of Agave tequilana juice by addition of yeast extract

BACKGROUND The aim of this work was to improve the productivity and yield of tequila fermentation and to propose the use of a recently isolated non-Saccharomyces yeast in order to obtain a greater diversity of flavour and aroma of the beverage. For that, the effects of the addition of different nitrogen (N) sources to Agave tequilana juice on the growth, fermentative capacity and ethanol tolerance of Kloeckera africana and Saccharomyces cerevisiae were studied and compared. RESULTS Kloeckera africana K1 and S. cerevisiae S1 were cultured in A. tequilana juice supplemented with ammonium sulfate, diammonium phosphate or yeast extract. Kloeckera africana did not assimilate inorganic N sources, while S. cerevisiae utilised any N source. Yeast extract stimulated the growth, fermentative capacity and alcohol tolerance of K. africana, giving kinetic parameter values similar to those calculated for S. cerevisiae. CONCLUSION This study revealed the importance of supplementing A. tequilana juice with a convenient N source to achieve fast and complete conversion of sugars in ethanol, particularly in the case of K. africana. This yeast exhibited similar growth and fermentative capacity to S. cerevisiae. The utilisation of K. africana in the tequila industry is promising because of its variety of synthesised aromatic compounds, which would enrich the attributes of this beverage.

Polyhydroxybutyrate production by Saccharophagus degradans using raw starch as carbon source

Biosynthesis of poly(3‐hydroxybutyrate) (PHB) from raw starch as the carbon source by the polysaccharide‐digesting bacteria Saccharophagus degradans was investigated in a fed‐batch culture. The production and properties of the PHB synthesized from starch were compared to those obtained using glucose as carbon source. In fed‐batch cultures, S. degradans accumulated 21.35 and 17.46% of PHB, using glucose or starch as carbon source, respectively. The physical properties of the biopolymer produced from each carbon source were similar between them. Molecular mass, melting temperature and heat of fusion were 54.23 kDa, 165.61°C and 59.59 J/g, respectively, using glucose; and 57.07 kDa, 174.31°C and 67.66 J/g, respectively, using starch. This is the first work describing the capability of S. degradans to utilize raw starch as the sole carbon source for the production of PHB.

Fermentation of Agave tequilana juice by Kloeckera africana : influence of amino-acid supplementations

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.

Effect of dilution rate and nutrients addition on the fermentative capability and synthesis of aromatic compounds of two indigenous strains of Saccharomyces cerevisiae in continuous cultures fed with Agave tequilana juice.

Knowledge of physiological behavior of indigenous tequila yeast used in fermentation process is still limited. Yeasts have significant impact on the productivity fermentation process as well as the sensorial characteristics of the alcoholic beverage. For these reasons a better knowledge of the physiological and metabolic features of these yeasts is required. The effects of dilution rate, nitrogen and phosphorus source addition and micro-aeration on growth, fermentation and synthesis of volatile compounds of two native Saccharomyces cerevisiae strains, cultured in continuous fed with Agave tequilana juice were studied. For S1 and S2 strains, maximal concentrations of biomass, ethanol, consumed sugars, alcohols and esters were obtained at 0.04 h⁻¹. Those concentrations quickly decreased as D increased. For S. cerevisiae S1 cultures (at D=0.08 h⁻¹) supplemented with ammonium phosphate (AP) from 1 to 4 g/L, concentrations of residual sugars decreased from 29.42 to 17.60 g/L and ethanol increased from 29.63 to 40.08 g/L, respectively. The S1 culture supplemented with AP was then micro-aerated from 0 to 0.02 vvm, improving all the kinetics parameters: biomass, ethanol and glycerol concentrations increased from 5.66, 40.08 and 3.11 g/L to 8.04, 45.91 and 4.88 g/L; residual sugars decreased from 17.67 g/L to 4.48 g/L; and rates of productions of biomass and ethanol, and consumption of sugars increased from 0.45, 3.21 and 7.33 g/L·h to 0.64, 3.67 and 8.38 g/L·h, respectively. Concentrations of volatile compounds were also influenced by the micro-aeration rate. Ester and alcohol concentrations were higher, in none aerated and in aerated cultures respectively.

Biosynthesis of Extracellular Polymeric Substances by the Marine Bacterium Saccharophagus degradans under Different Nutritional Conditions

The effect of carbon source, carbon to nitrogen (C/N) ratio, and limitation in nutrients (N, P, K, Ca, Mg, and Fe) on extracellular polymeric substances (EPS) synthesis by the marine bacterium Saccharophagus degradans was studied. This strain was able to grow in mineral medium and produce EPS with different efficiency according to the C source used (g EPS/L): glucose or starch (1.5 ± 0.2); galactose, sucrose, or xylose (0.7 ± 0.2); and fructose (0.3 ± 0.1). The C/N ratio (glucose/ammonium) had a significant effect on EPS biosynthesis due to its production rise as the C/N ratio increased from 3 to 100 (0.7 to 2.1 g EPS/L). It was also observed that limitation in nutrients such as N, P, K, Ca, Mg, and Fe also favored EPS biosynthesis. When taking into account both factors (C/N ratio, 100; nutrients limitation, 50%) a positive synergistic effect was noted on EPS production since under these conditions the maximum concentration obtained was 4.12 ± 0.3 g/L after 72 h of culture. The polymer was found to be a polysaccharide of mainly glucose, mannose, and galactose. This is the first report on EPS production by S. degradans which is a new feature of this versatile marine bacterium.

MULTIPLEX GAS SAMPLER FOR MONITORING RESPIROMETRY IN COLUMN-TYPE BIOREACTORS USED IN SOLID-STATE FERMENTATION

ABSTRACT A multiplex gas sampler for monitoring CO2 and O2 for column-type bioreactors used in solid-state fermentation was designed. The sampler mechanically couples up to 24 bioreactors to a central set of CO2 and O2 sensors. The user can set fermentation time, the number of bioreactors to be sampled by the sampling port, the sampling rate and the delay time between sampling (to guarantee the complete replacement of a gas sample). The user has the possibility to enable or disable sampling from bioreactor-ports. Due to its small size and weight, the gas sampler is portable. This device is also quite economical. This gas sampler was validated using two solid-state fermentation experiments. First, the CO2 and O2 measurements were confirmed to be highly reproducible. No significant differences were found for the fermentations of 19 experimental units (bioreactors) simultaneously run with Rhizopus sp. cultured at the same conditions. Second, the versatility of the gas sampler, operating simultaneously with several microorganisms cultured at different conditions, was demonstrated through the simultaneous monitoring of A. awamori and Rhizopus sp. solid-state fermentations, cultured at different temperatures and pH. By using the multiplex gas sampler, the above study was done in only one set of experiments rather than in five sets of experiments that would have been required by using other sampler, representing a great time saving.