Bruno Rodríguez-Morgado

Proteomic analysis of enzyme production by Bacillus licheniformis using different feather wastes as the sole fermentation media.

This study evaluates the use of different types of feathers as fermentation media for enzyme production. Bacillus licheniformis was grown on the feathers, which lead to total biodegradation due to bacterial enzymatic hydrolytic excretion. B. licheniformis excretes protease and lipase activity, with feather concentration being the main parameter controlling their generation. Using a proteomic approach, the proteins excreted during fermentation were identified, and the influence of the chemical composition of the feathers on protein secretion was tested. The identified proteins are hydrolytic enzymes such as keratinase, gamma-glutamyltranspeptidase, chitosanases, and glicosidases. The diversity of proteins is related to the chemical complexity of the feathers. Understanding the composition of a hydrolytic system, when B. licheniformis is cultured on different feathers, may assist in utilizing such a system for producing different hydrolytic enzymes. The data indicate that proteomics can be a valuable tool for describing the physiological state of B. licheniformis cell populations growing on different wastes.

Behavior of oxyfluorfen in soils amended with different sources of organic matter. Effects on soil biology

We performed a laboratory study on the effect of oxyfluorfen at a rate of 4lha(-1) on biological properties of a soil amended with four organic wastes (two biostimulants/biofertilizers, obtained from rice bran, RB1 and RB2; municipal solid waste, MSW; and sheep manure, SM). Soil was mixed with SM at a rate of 1%, MSW at a rate of 0.52%, RB1 at a rate of 0.39% and RB2 at a rate of 0.30%, in order to apply the same amount of organic matter to the soil. The enzymatic activities and microbial community in the soil were determined during the incubation times. The application of RB1 and RB2 to soil without oxyfluorfen increased the enzymatic activities and biodiversity, peaking at day 10 of the incubation period. This stimulation was higher in the soil amended with RB2 than in that amended with RB1. In SM and CF-amended soils, the stimulation of enzymatic activities and soil biodiversity increased during the experiment. The application of herbicide in organic-amended soils decreased the inhibition of soil enzymatic activities and soil biodiversity. Possibly the low molecular weight protein content easily assimilated by soil microorganisms and the higher fat content in the biostimulants/biofertilizers are responsible for the lower inhibition of these soil biological properties.

Enzymatic Vegetable Organic Extracts as Soil Biochemical Biostimulants and Atrazine Extenders

The purpose of this study was to gather information on the potential effects of organic biostimulants on soil activity and atrazine biodegradation. Carob germ enzymatic extract (CGEE) and wheat condensed distiller solubles enzymatic extract (WCDS-EE) have been obtained using an enzymatic process; their main organic components are soluble carbohydrates and proteins in the form of peptides and free amino acids. Their application to soil results in high biostimulation, rapidly increased dehydrogenase, phosphatase and glucosidase activities, and an observed atrazine extender capacity due to inhibition of its mineralization. The extender capacity of both extracts is proportional to the protein/carbohydrate ratio content. As a result, these enzymatic extracts are highly microbially available, leading to two independent phenomena, fertility and an atrazine persistence that is linked to increased soil activity.

Antiproliferative and immunoactivatory ability of an enzymatic extract from rice bran.

The validation of natural products as source of functional foods or nutraceuticals has become an important issue in current health research. Thus, the present work has tested on MOLT-4 cells (human T cell acute lymphoblastic leukemic) the antiproliferative effect of a water-soluble enzymatic extract from rice bran (EERB). Present work shows that EERB induces cellular death in MOLT-4 cells in a dose-dependent way (0-10mg/mL) but not in non-tumoral lymphocytes. Flow cytometric analysis of MOLT-4 cells treated with EERB showed the presence of death cells by apoptosis rather than necrosis. Additionally, EERB also exerts an immunoactivatory effect on N13 microglia cells, by inducing TNF-alpha (tumour necrosis factor-α) expression, which plays a key role in the innate immune response to infection. Accordingly, we can propose EERB as a useful natural standardized extract with antiproliferative and immunoactivatory ability that would be beneficial to apply in the functional food field.

Pre-fermentative addition of an enzymatic grape seed hydrolysate in warm climate winemaking. Effect on the differential colorimetry, copigmentation and polyphenolic profiles.

The effect of adding an enzymatic hydrolysate of grape seeds (EH-GS) during Syrah wine fermentation in a warm climate has been evaluated. We focused on the polyphenolic composition as well as the application of differential and tristimulus colorimetry to colour data. This is the first attempt at using this oenological alternative to avoid common colour losses of red wines elaborated in a warm climate. The addition of 250g (simple dose, SW) of EH-GS to 120kg of fermentation material promoted a significant (p<0.05) increase in the polyphenolic content of stored wines, especially in benzoic acid, hydroxycinnamic acid derivative, flavonol and anthocyanin levels. This increase could promote a higher copigmentation percentage and maximum colour stabilization (C(∗)ab) without significantly changing the wine tonality. Unexpectedly, the use of a double quantity (DW) of EH-GS resulted in significantly less chroma than for control wines (CW), demonstrating visually perceptible colour changes (ΔE(∗)ab>3 CIELAB units).

Accelerated degradation of PAHs using edaphic biostimulants obtained from sewage sludge and chicken feathers

We studied in the laboratory the bioremediation effects over a 100-day period of three edaphic biostimulants (BS) obtained from sewage sludge (SS) and from two different types of chicken feathers (CF1 and CF2), in a soil polluted with three polycyclic aromatic hydrocarbons (PAH) (phenanthrene, Phe; pyrene, Py; and benzo(a)pyrene, BaP), at a concentration of 100 mg kg(-1) soil. We determined their effects on enzymatic activities and on soil microbial community. Those BS with larger amounts of proteins and a higher proportion of peptides (<300 daltons), exerted a greater stimulation on the soil biochemical properties and microbial community, possibly because low molecular weight proteins can be easily assimilated by soil microorganisms. The soil dehydrogenase, urease, β-glucosidase and phosphatase activities and microbial community decreased in PAH-polluted soil. This decrease was more pronounced in soils contaminated with BaP than with Py and Phe. The application of the BS to PAH-polluted soils decreased the inhibition of the soil biological properties, principally at 7 days into the experiment. This decrease was more pronounced in soils contaminated with BaP than with Py and Phe and was higher in polluted soils amended with CF2, followed by SS and CF1, respectively.

Obtaining edaphic biostimulants/biofertilizers from different sewage sludges. Effects on soil biological properties

We studied the influence of six edaphic biostimulants/biofertilizers (BSs) manufactured by the pH-stat method from different sewage sludge (SS): SS1 (an anaerobic mature sludge, one year old), SS2 (an aerobic young sludge, without maturation) and SS3 (an aerobic mature sludge, four months old), not previously autoclaved (A) and autoclaved (B), by analysing their effects on soil biological properties. Soil enzymatic activities were measured at 1, 3, 5, 7, 15, 30 and 60 days of the incubation period, whereas the 16S rDNA-DGGE profiles were determined at 0, 5 and 60 days. The enzymatic activities were significantly stimulated. The highest stimulation was found in the B2 treatment followed by B3, A2, A3, B1 and A1 treatments. Increasing the number of lower molecular weight proteins in the BS enhances the stimulation of soil enzymatic activities. The application of BS caused at 5 days of the incubation period temporal variations in the soil bacterial community structure.

Obtaining edaphic biostimulants/biofertilizers from sewage sludge using fermentative processes. Short-time effects on soil biochemical properties

ABSTRACT In this manuscript, we study the manufacture and effect on soils of different edaphic biostimulants/biofertilizers (BS) obtained from sewage sludge using Bacillus licheniformis as biological tool. These BS consist of different combinations of organic matter, bacteria and enzymes that were subjected to several treatments. These BS were applied in soil in order to observe their influence on the biochemical properties (enzymatic activities and ergosterol content). Dehydrogenase, urease, β-glucosidase, phosphatase activities and ergosterol content were measured at different incubation days. Only dehydrogenase activity and ergosterol content were significantly stimulated after the application of BS1 and BS4. Rest of the extracellular activities were not stimulated probably because B. licheniformis practically has digested all organic substrates during fermentation process.

Effect of l-lactic acid from whey wastes on enzyme activities and bacterial diversity of soil

The l-lactic acid obtained from a whey waste valorization process decreased pH and increased phosphate solubilization of a Plagic Antrosol soil. This behavior was reversible due to the complete and rapid degradation of lactic acid by soil microorganisms, leading to a strong soil biological stimulation. Both dehydrogenase (DHA) and acid phosphomonoesterase (APA) activities were also significantly stimulated, due to the stimulation of microbial activity, whereas the β-glucosidase activity was unaffected. There was also a shift in bacterial biodiversity with growth of bacterial strains using lactic acid as a C source. The bacteria enriched belonged to the plant growth-promoting bacteria (PGPb), such as Rhizobium and Azotobacter genus, involved in N2 fixation, and Pseudomonas and Bacillus genera, involved in phosphorous bioavailability. We conclude that lactic acid application improves fertility parameters on soils. Further research at field scale is needed to confirm these results.