Estudio de la extracción de compuestos fenólicos mediante diferentes técnicas de extracción a partir del residuo generado en la obtención de agar-agar

El residuo algal generado en la industria de producción del agar-agar presenta componentes bioactivos de carácter antioxidante con un gran potencial de recuperación y reutilización. Los métodos de extracción comúnmente utilizados para el aislamiento de antioxidantes se basan en extracciones con agua o disolventes orgánicos. En el presente trabajo se ha propuesto el empleo de diversas enzimas para conseguir la extracción de los componentes fenólicos en el que se concluyó que el efecto del tipo de enzima, su concentración y pH del medio afectan en gran medida a la extracción de los componentes de interés. Así mismo se llevó a cabo la caracterización de los hidrolizados enzimáticos por HPLC en el que se ha podido cuantificar la cantidad de azúcares y ácidos formados debido a la acción hidrolítica de las enzimas empleadas.proyecto de Investigación VALORIZACIÓN INTEGRAL DE SUBPRODUCTOS DE LA INDUSTRIA AGROALIMENTARIA MEDIANTE TECNOLOGÍAS EMERGENTES (ALVALOR) financiado por la Junta de Castilla y León y el Fondo Europeo de Desarrollo Regional (BU301P18) y desarrollado en el Grupo de investigación Biotecnología Industrial y Medioambiental, reconocido por la UBU (GIR-UBU BIOIND) y por la Junta de Castilla y León como Unidad de Investigación Consolidada UIC-12

Assisted-enzymatic hydrolysis vs chemical hydrolysis for fractional valorization of microalgae biomass

Producción CientíficaDespite the interest in the utilization of photobioreactors as an alternative wastewater treatment, the research about posterior recovery and valorization of nutrients accumulated in the biomass is still limited. This work compared several hydrolysis methods for the recovery of proteins and carbohydrates from the biomass grown in a photobioreactor treating swine wastewater. Ultrasound-assisted and microwave-assisted enzymatic hydrolysis at mild conditions and chemical methods at different temperatures (40, 60, 120ºC) were applied to the microalgae and bacteria biomass. Alkaline hydrolysis provided the greatest peptide recoveries, increasing with temperature up to a maximum of 81%, but with very small peptide sizes in all temperature range. Acid hydrolysis provided the highest carbohydrate recoveries (60.7% at 120ºC) but degraded proteins, even at mild temperatures. Protein degradation did not vary with temperature in each chemical hydrolysis, obtaining similar peptide sizes in all temperatures, while carbohydrate losses were higher at lower temperatures. Ultrasound-assisted enzymatic extraction recovered 43.6% of the initial proteins as large peptides (up to 135 kDa) with the highest peptide purity (46.7%). Microwave-assistance increased the carbohydrate solubilization of enzymatic hydrolysis, achieving yields of 73% of xylose, but with significant losses.Ministerio de Ciencia e Innovación y Ministerio de Universidades (PID2020-113544RB-I00 y PDC2021-121861-C22)Junta de Castilla y León (UIC 338, CLU 2017-09)UE-FEDER (CLU 2017-09

Agricultural products from algal biomass grown in piggery wastewater: A techno-economic analysis

Producción CientíficaThe intensification of livestock activities lead to an increase in waste generation with high content of nutrients, as is the case of piggery wastewater. However, this type of residue can be used as culture media for algae cultivation in thin-layer cascade photobioreactors to reduce its environment impact and produce a valorizable algal biomass. Biostimulants were produced by enzymatic hydrolysis and ultrasonication of microalgal biomass, using membranes (Scenario 1) or centrifugation (Scenario 2) as harvesting methods. The co-production of biopesticides by solvent extraction was also evaluated using membranes (Scenario 3) or centrifugation (Scenario 4). The four scenarios were analyzed by a technoeconomic assessment estimating the total annualized equivalent cost and the production cost, i.e., the minimum selling price. Centrifugation provided biostimulants approximately 4 times more concentrated than membranes, but with higher expense due to the cost of the centrifuge (contribution of 62.2 % in scenario 2) and the electricity requirements. The biopesticide production resulted the highest contribution to investment cost in scenarios 3 and 4 (34 % and 43 % respectively). The use of membranes was also more advantageous to produce biopesticides, although it was 5 times more diluted than using centrifuge. The biostimulant production cost was 65.5 €/m3 with membranes and 342.6 €/m3 by centrifugation and the biopesticide production cost was 353.7 €/m3 in scenario 3 and 2,122.1 €/m3 in scenario 4. Comparing the treatment of 1 ha of land, the cost of the biostimulant produced in the four scenarios was lower than the commercial one (48.1 %, 22.1 %, 45.1 % and 24.2 % respectively). Finally, using membranes for biomass harvesting allowed economically viable plants with lower capacity and longer distance for biostimulant distribution (up to 300 km) than centrifuge (188 km). The algal biomass valorization for agricultural products production is an environmentally and economically feasible process with the adequate capacity of the plant and distribution distance.Ministerio de Ciencia e Innovación y Ministerio de Universidades (PDC2021-121861-C22, PID2020-113544RB-I00)Ministerio de Ciencia e Innovación y Ministerio de Universidades Doctorate scholarship (PRE2018-083845 and PRE2021-100176)Junta de Castilla y León (UIC 338, CLU 2017-09, CL-EI-2021-07

Valoración de un consorcio de microalgas y bacterias: comparación de métodos convencionales y nuevos métodos de extracción asistida

The microalgae biomass grown in wastewater becomes an interesting renewable raw material able to metabolize the nutrients of waste streams and transform them into high added products such as proteins and carbohydrates. Despite the actual interest in the use of this biomass to improve de viability of conventional wastewater treatment processes, the research carried out on the fractional valorization of these macrocompounds is still very scarce. In the present work, a comparison of several conventional methodologies is developed (chemical hydrolysis, enzymatic hydrolysis and pretreatments coupled with enzymatic hydrolysis) against novel methodologies of enzymatic hydrolysis assisted by ultrasound and microwaves for the solubilization and recovery of macrocompounds, such as proteins and carbohydrates, from a consortium of Scenedesmus almeriensis-bacteria cultivated in a thin-film photobioreactor fed with manure wastewater. The acid hydrolysis with HCl 2M at 120ºC enhanced the carbohydrates solubility (86,6%) with high fermentable monosaccharide recoveries (60.7%). The alkaline hydrolysis with NaOH 2M at 120ºC achieved almost complete solubilization of the biomass components with high solubilization (88.3%) and recovery yields (61.5%) of proteins with peptide size less than 11 kDa, but with high carbohydrates losses. The enzymatic hydrolysis with proteases gave raise to moderate carbohydrates recoveries (14.9%) and peptides (17.6%), while the addition of cellulases to the enzymatic cocktail achieved an improvement in the yields of proteins, although, in both cases, the sizes of the recovered peptides were found in the range of 20-135 kDa. The secuencial application of pretreatments (bead mills or ultrasound) coupled with enzymatic hydrolysis with proteases improved the solubilization of carbohydrates (35%) and proteins (28.2%) of the biomass but, the recovery yields obtained were within the range obtained only with enzymatic hydrolysis with proteases in a single stage (15% and 24%). Finally, regarding the novel enzymatic hydrolysis methods with proteases and the cocktail of proteases and cellulases, the use of microwaves enhanced the release of carbohydrates with a maximum yield of 57.9% and 56.2% respectively, while ultrasounds enhanced the release of proteins up to 49.6% and 44.5% with recovered peptides sizes greater than 48 kDa. In both cases, the yields were much higher than those of the individual enzymatic hydrolysis using a single stage, despite the fact that it was operated with treatment times of only 1h. The presence of bacteria in this biomass could be related to the high losses obtained.La biomasa de microalgas cultivada en aguas residuales resulta ser un interesante material renovable capaz de metabolizar los nutrientes de los residuos y transformarlos en productos con valor añadido como son las proteínas y los carbohidratos. A pesar del reciente interés en el empleo de esta biomasa para mejorar la viabilidad de los procesos convencionales del tratamiento de aguas residuales, la investigación desarrollada sobre la valorización fraccionada de sus macrocomponentes es aún muy escasa. En el presente trabajo se desarrolla una comparación de diferentes metodologías convencionales (hidrólisis química, enzimática y pretratamientos acoplados a hidrólisis enzimática) frente a nuevas metodologías de hidrólisis enzimática asistida por ultrasonidos y microondas en la solubilización y recuperación de macrocomponentes, proteínas y carbohidratos, a partir de un consorcio de Scenedesmus almeriensis-bacterias cultivado en un fotobiorreactor de capa fina alimentado con aguas residuales de purines de cerdo. La hidrólisis ácida con HCl 2M a 120ºC potenció la solubilización de carbohidratos (86.6%) con altas recuperaciones de monosacáridos fermentables (60.7%). La hidrólisis alcalina con NaOH 2M a 120ºC consiguió una solubilización casi completa de los componentes de la biomasa con una alta solubilización (88.3%) y recuperación (61.5%) de proteínas con tamaños de péptidos inferiores a 11 kDa, pero con elevadas pérdidas de carbohidratos. La hidrólisis enzimática con proteasas dio lugar a recuperaciones moderadas de carbohidratos (14.9%) y péptidos (17.6%) mientras que la adición de celulasas al cóctel enzimático consiguió una mejora en los rendimientos de proteínas aunque los tamaños de los péptidos recuperados se encontraron en el rango de 20 – 135 kDa. La aplicación secuencial de pretratamientos (molienda o ultrasonidos) acoplados a la hidrólisis enzimática con proteasas dio lugar a un incremento en la solubilización de carbohidratos (35%) y proteínas (28.2%) de la biomasa pero finalmente las recuperaciones obtenidas se encontraron dentro del rango obtenido únicamente con hidrólisis enzimática con proteasas en una única etapa (15% y 24%) Finalmente, en relación a los novedosos métodos de hidrólisis enzimática con proteasas y combinación de proteasas con celulasas asistidos, el uso de microondas potenció la liberación de carbohidratos con un rendimiento máximo de 57.9% y 56.2% a mientras que la asistencia con ultrasonidos potenció la liberación de proteínas en un 49.6% y 44.5% con péptidos recuperados con tamaños superiores a los 48 kDa. En ambos casos, los rendimientos fueron muy superiores a los de las hidrólisis enzimáticas individuales empleando una única etapa, a pesar de que se operó con tiempos de tratamiento de tan solo 1h. La presencia de bacterias en esta biomasa podría estar relacionada con las elevadas pérdidas registradas.Departamento de Ingeniería Química y Tecnología del Medio AmbienteMáster en Técnicas Avanzadas en Química. Análisis y Control de Calidad Químico

Enzymatic hydrolysis of the industrial solid residue of red seaweed after agar extraction: Extracts characterization and modelling

The efficiency of enzymatic hydrolysis for the release of different biocompounds such as total phenolic compounds (TPC), sugars and proteins from the industrial solid residue of G. sesquipedale after agar extraction has been evaluated. Cellulase (0.25–8%, w/w, enzyme: solid residue, pH = 5) has been proved to be an efficient enzyme to degrade the algae cell wall improving the release of the bound TPC with values up to 7.5 mg gallic acid equivalent/g dry macroalgae residue. Monomer and oligomer carbohydrates were released to the reaction medium, namely glucose, galactose and arabinose with higher yields by increasing cellulase concentration. Enzyme combinations with other hydrolytic enzymes, such as xylanase and protease, did not bring any improvement of the TPC and sugar yields. Protein was also released to the enzymatic medium with protein extraction yields around 30%. The use of protease under basic conditions led to an increase in the release of the protein fraction and of the free amino acids content with a hydrophobic ratio higher than in the raw material. The kinetics of TPC and protein hydrolysis have been fitted to the power law and the Weibull models yielding the Weibull model the best fitting quality.Agencia Estatal de Investigación [grant number PID2019-104950RB-I00/AEI/10.13039/501100011033] and the Junta de Castilla y León (JCyL) and the European Regional Development Fund (ERDF) [grant numbers BU301P18 and BU050P20