Ana Torrado

Submerged Citric Acid Fermentation on Orange Peel Autohydrolysate

The citrus-processing industry generates in the Mediterranean area huge amounts of orange peel as a byproduct from the industrial extraction of citrus juices. To reduce its environmental impact as well as to provide an extra profit, this residue was investigated in this study as an alternative substrate for the fermentative production of citric acid. Orange peel contained 16.9% soluble sugars, 9.21% cellulose, 10.5% hemicellulose, and 42.5% pectin as the most important components. To get solutions rich in soluble and starchy sugars to be used as a carbon source for citric acid fermentation, this raw material was submitted to autohydrolysis, a process that does not make use of any acidic catalyst. Liquors obtained by this process under optimum conditions (temperature of 130 degrees C and a liquid/solid ratio of 8.0 g/g) contained 38.2 g/L free sugars (8.3 g/L sucrose, 13.7 g/L glucose, and 16.2 g/L fructose) and significant amounts of metals, particularly Mg, Ca, Zn, and K. Without additional nutrients, these liquors were employed for citric acid production by Aspergillus niger CECT 2090 (ATCC 9142, NRRL 599). Addition of calcium carbonate enhanced citric acid production because it prevented progressive acidification of the medium. Moreover, the influence of methanol addition on citric acid formation was investigated. Under the best conditions (40 mL of methanol/kg of medium), an effective conversion of sugars into citric acid was ensured (maximum citric acid concentration of 9.2 g/L, volumetric productivity of 0.128 g/(L.h), and yield of product on consumed sugars of 0.53 g/g), hence demonstrating the potential of orange peel wastes as an alternative raw material for citric acid fermentation.

Complete bioconversion of hemicellulosic sugars from agricultural residues into lactic acid by Lactobacillus pentosus

On the basis of previous knowledge, different agroindustrial wastes were submitted to dilute-acid hydrolysis with H2SO4 to obtain hemicellulosic sugars and then employed for lactic acid production by Lactobacillus pentosus. Toxic compounds released from lignin did not affect lactic acid fermentation when hydrolysates from trimming vine shoots, barley bran husks, or comcobs were employed as carbon source, and complete bioconversion of hemicellulosic sugars was achieved. Nevertheless, Eucalyptus globulus hydrolysates had to be submitted to a detoxification process with activated charcoal. Maximum lactic acid concentration (33 g/L) was reached employing barley bran hydrolysates, whereas corncobs, trimming vine shoots, and detoxified E. globulus hydrolysates yielded 26, 24, and 14.5 g/L of lactic acid, respectively. The maximum product yield from pentoses (0.76 g/g) was achieved using hydrolysates from trimming vine shoots, followed by hydrolysates from detoxified E. globulus (0.70 g/g), barley bran (0.57 g/g), and corncob (0.53 g/g). These results confirm that L. pentosus can be employed to ferment hemicellulosic sugars (mainly xylose, glucose, and arabinose) from acid hydrolysates of most agricultural residues without appreciable substrate inhibition.

Citric acid production from orange peel wastes by solid-state fermentation

Valencia orange (Citrus sinensis) peel was employed in this work as raw material for the production of citric acid (CA) by solid-state fermentation (SSF) of Aspergillus niger CECT-2090 (ATCC 9142, NRRL 599) in Erlenmeyer flasks. To investigate the effects of the main operating variables, the inoculum concentration was varied in the range 0.5·103 to 0.7·108 spores/g dry orange peel, the bed loading from 1.0 to 4.8 g of dry orange peel (corresponding to 35-80 % of the total volume), and the moisture content between 50 and 100 % of the maximum water retention capacity (MWRC) of the material. Moreover, additional experiments were done adding methanol or water in different proportions and ways. The optimal conditions for CA production revealed to be an inoculum of 0.5·106 spores/g dry orange peel, a bed loading of 1.0 g of dry orange peel, and a humidification pattern of 70 % MWRC at the beginning of the incubation with posterior addition of 0.12 mL H2O/g dry orange peel (corresponding to 3.3 % of the MWRC) every 12 h starting from 62 h. The addition of methanol was detrimental for the CA production. Under these conditions, the SSF ensured an effective specific production of CA (193 mg CA/g dry orange peel), corresponding to yields of product on total initial and consumed sugars (glucose, fructose and sucrose) of 376 and 383 mg CA/g, respectively. These results, which demonstrate the viability of the CA production by SSF from orange peel without addition of other nutrients, could be of interest to possible, future industrial applications.

Stability and Emulsifying Capacity of Biosurfactants Obtained from Lignocellulosic Sources Using Lactobacillus pentosus

Lactobacillus pentosus grown on sugars from agricultural residues produces biosurfactants with emulsifying properties that could facilitate the bioremediation of hydrocarbon contaminated sites. The biosurfactans obtained after growing L. pentosus cells on distilled grape marc hydrolyzates gave values of relative emulsion volume (EV) close to 50%, being stable after 72 h when gasoline or kerosene were employed. These EV values were higher than those achieved using commercial surfactin (14.1% for gasoline and 27.2% for kerosene). Moreover, assays carried out with kerosene showed that L. pentosus produced biosurfactants from distilled grape marc hydrolyzates with the highest stabilizing capacity value (ES) to maintain the emulsion (99%) followed by biosurfactants produced from hazelnut shell hydrolyzates (97%). These data are comparable with those obtained using sodium dodecyl sulfate, SDS (87.7%), whereas surfactin only gave an ES value of 65.4%. Consequently, this work shows that utilization of low-cost feedstock agricultural residues as substrates for producing biosurfactants/bioemulsifiers is possible thus removing obstacles for the wide-scale industrial application of biosurfactants/bioemulsifiers.

Use of waste materials for Lactococcus lactis development.

BACKGROUND Lactococcus lactis is an interesting microorganism with several industrial applications, particularly in the food industry. As well as being a probiotic species, L. lactis produces several metabolites with interesting properties, such as lactic acid (LA) and biosurfactants. Nevertheless, L. lactis is an especially demanding species since it has strong nutritional requirements, implying the use of complex and expensive culture media. RESULTS The results showed the potential of L. lactis CECT-4434 as a LA and biosurfactant producer. The economical cost of L. lactis cultures can be reduced by replacing the MRS medium by the use of two waste materials: trimming vine shoots as C source, and 20 g L(-1) distilled wine lees (vinasses) as N, P and micronutrient sources. From the hemicellulosic fraction, 14.3 g L(-1) LA and 1.7 mg L(-1) surfactin equivalent were achieved after 74 h (surface tension reduction of 14.4 mN m(-1)); meanwhile, a simultaneous saccharification and fermentation process allowed the generation of 10.8 g L(-1) LA and 1.5 mg L(-1) surfactin equivalent after 72 h, reducing the surface tension by 12.1 units at the end of fermentation. CONCLUSIONS Trimming vine shoots and vinasses can be used as alternative economical media for LA and cell-bound biosurfactant production.

Fermentation kinetics and chemical characterisation of vino tostado, a traditional sweet wine from Galicia (NW Spain).

BACKGROUND Grapes after harvesting are air dried and pressed in order to concentrate sugars, acids and flavour compounds to produce vino tostado (toasted wine), a wine with intense aroma and flavour notes and high residual sugar concentration. In order to get a better knowledge of the difficulties involved, several fermentations were conducted at 12 and 28 degrees C using 0, 15 and 30 g hL(-1) ammonium sulfate and 0, 25 and 50 g hL(-1) exogenous commercial yeast (Saccharomyces cerevisiae var. bayanus) to study the kinetics of sugar consumption and ethanol, acetic acid and glycerol production. RESULTS Fermentation kinetic parameters were calculated and metal concentrations and antioxidant activity were analysed. CONCLUSION The spontaneous fermentation at 12 degrees C and all fermentations conducted with the commercial yeast gave vino tostado of adequate quality, while the spontaneous fermentation at 28 degrees C was sluggish. High-temperature fermentations led to sweeter wines with higher volumetric productivities, although low-temperature fermentations produced better wines in terms of higher glycerol and lower acetic acid levels. Fructose was the only sugar to be consumed during spontaneous fermentations, while both glucose and fructose were consumed during fermentations of the inoculated musts, with preference for each monosaccharide depending on temperature.

Stabilization of kerosene/water emulsions using bioemulsifiers obtained by fermentation of hemicellulosic sugars with Lactobacillus pentosus.

The results of the present study show that Lactobacillus pentosus can produce extracellular bioemulsifiers by utilizing hemicellulosic sugars from grape marc as a source of carbon. The effectiveness of these bioemulsifiers (LPEM) was studied by preparing kerosene/water (K/W) emulsions in the presence and absence of these emulsifiers. Various parameters such as relative emulsion volume (EV), stabilizing capacity (ES), viscosity, and droplet size of K/W emulsions were measured. The EV values for K/W emulsions stabilized by concentrated LPEM were approximately 74.5% after 72 h of emulsion formation, with ES values of 97%. These values were higher than those obtained with dodecyl sodium sulfate as emulsifier (EV=62.3% and ES=87.7%). Additionally, K/W emulsions stabilized by LPEM produced polydisperse emulsions containing droplets of radius between 10 and 40 μm, which were smaller than those obtained for K/W emulsions without LPEM (droplet radius=60-100 μm). Moreover, the viscosity values of the K/W emulsions without and with LPEM were approximately 236 and 495 cP, respectively.

Modelling the Biphasic Growth and Product Formation by Enterococcus faecium CECT 410 in Realkalized Fed-Batch Fermentations in Whey

The influence of initial pH on growth and nutrient (total sugars, nitrogen, and phosphorous) consumption by Enterococcus faecium CECT 410 was studied during batch cultures in whey. With these data, two realkalized fed-batch fermentations were developed using different feeding substrates. The shift from homolactic to mixed acid fermentation, the biphasic kinetics observed for cell growth and nitrogen consumption and the increase in the concentrations of biomass and products (lactic acid, acetic acid, ethanol, and butane-2,3-diol) were the most noteworthy observations of these cultures. Modelling the fed-batch growth of Ent. faecium with the Logistic and bi-Logistic models was not satisfactory. However, biomass production was best mathematically described with the use of a double Monod model, which was expressed in terms of biomass, product accumulation, and nitrogen utilization. Product formation was successfully modelled with a modified form of the Luedeking and Piret model developed in this study.

Modelling the enzymatic activity of two lipases isoenzymes commonly used in the food industry Modelado de la actividad enzimática de dos isoenzimas lipasas comúnmente utilizadas en la industria alimentaria

An in-depth analysis of the kinetics of two lipases isoenzymes (Lip1 and Lip2) in triacetin hydrolysis in absence and in presence of hexane was carried out. The addition of hexane led to an increase in enzymatic activities of both enzymes for all triacetin concentrations, and the kinetic data described a hyperbola which was consistent with the classical Michaelis–Menten model. Without hexane, the time-course of the triacetin hydrolysis by Lip1 and Lip2 did not follow a Michaelian behaviour. In this case, a first phase of low enzymatic activity (at triacetin concentrations from 0 to 250 mM) was followed by a rapid increase in velocity at triacetin concentrations ≥250 mM. The Michaelis–Menten model was unable to describe the first phase due to the linear (nonhyperbolic) relationship between the velocity and the triacetin concentration, meanwhile the logistic model provided a satisfactory description of the experimental data corresponding to the second phase of activity. En este trabajo se llevó a cabo un profundo análisis de la cinética de dos isoenzimas lipasas (Lip1 y Lip2) en la hidrólisis de triacetina, en ausencia y en presencia de hexano. La adición de hexano a la mezcla de reacción incrementó las actividades enzimáticas de ambas enzimas para todas las concentraciones de triacetina, obteniéndose una relación hiperbólica compatible con el modelo clásico de Michaelis-Menten. En ausencia de hexano, la actividad de Lip1 y Lip2 no mostró un comportamiento Michaeliano, observándose una fase inicial de baja velocidad a concentraciones de triacetina entre 0–250 mM, seguida de un rápido incremento en la actividad enzimática ([triacetina] ≥ 250 mM). El modelo de Michaelis-Menten no pudo ser utilizado para describir la primera fase debido al incremento lineal (no hiperbólico) de la velocidad con la concentración de triacetina, mientras el modelo logístico describió adecuadamente la cinética de hidrólisis en la segunda fase.