Integral multi-valorization of agro-industrial wastes: A review.

Agriculture and industries related to the agriculture sector generate a large amount of waste each year. These wastes are usually burned or dumped, causing damage to the environment, the economy and society. Due to their composition, they have great potential for obtaining high value-added products in biorefineries. This fact, added to the growing demand for energy and chemicals from fossil resources, is driving the interest of the scientific community in them. Biorefinery processes are hardly profitable when applied individually, so a better alternative is to develop integrated multi-feedstock and multi-product biorefinery schemes using all biomass fractions in a zero-waste approach. However, for industrial scale application, extensive research, scale-up studies, and techno-economic and environmental feasibility analyses are needed. This review compiles information on integrated multi-biorefinery processes from agro-industrial wastes to shed light on the path towards sustainable development and circular bioeconomy.

Microwave-assisted autohydrolysis of avocado seed for the recovery of antioxidant phenolics and glucose.

This study describes the valorization of avocado seed (AS) within a green biorefinery concept using microwave-assisted autohydrolysis. After the treatment at temperatures of 150-230 °C for 5 min, the resulting solid and liquor were characterized. The temperature of 220 °C led to the simultaneous optimal values of antioxidant phenolics/flavonoids (42.15 mg GAE/g AS, 31.89 RE/g AS, respectively) and glucose + glucooligosaccharides (38.82 g/L) in the liquor. Extraction with ethyl acetate allowed the recovery of the bioactive compounds while maintaining the polysaccharides in the liquor. The extract was rich in vanillin (99.02 mg/g AS) and contained several phenolic acids and flavonoids. The solid phase and the phenolic-free liquor were subjected to enzymatic hydrolysis to produce glucose, reaching values of 9.93 and 105 g glucose/L, respectively. This work demonstrates that microwave-assisted autohydrolysis is a promising technology to obtain fermentable sugars and antioxidant phenolic compounds from avocado seeds following a biorefinery scheme.

Valorization of bioethanol by-products to produce unspecific peroxygenase with Agrocybe aegerita: Technological and proteomic perspectives.

Unspecific peroxygenase (UPO) presents a wide range of biotechnological applications. This study targets the use of by-products from bioethanol synthesis to produce UPO by Agrocybe aegerita. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase in SmF using vinasse as nutrients source. Optimized UPO production of 331 U/L was achieved in 50% (v:v) vinasse with an inoculum grown for 14 days. These conditions were scaled-up to a 4 L reactor, achieving a UPO activity of 265 U/L. Fungal proteome expression was analyzed before and after UPO activity appeared by shotgun mass spectrometry proteomics. Laccase, dye-decolorizing peroxidases (DyP), lectins and proteins involved in reactive oxygen species (ROS) production and control were detected (in addition to UPO). Interestingly, the metabolism of complex sugars and nitrogen sources had a different activity at the beginning and end of the submerged fermentation.

Avoiding acid crash: From apple pomace hydrolysate to butanol through acetone-butanol-ethanol fermentation in a zero-waste approach.

Apple pomace (AP) is a lignocellulosic residue from the juice and cider industries that can be valorized in a multi-product biorefinery to generate multiple value-added compounds, including biofuels such as butanol. Butanol is produced biologically by acetone-butanol-ethanol (ABE) fermentation using bacteria of the genus Clostridium from sugar-based feedstocks. In this study, AP hydrolysate was used as a substrate for producing butanol by ABE fermentation. Various environmental factors influence the amount of butanol produced, but only under certain conditions the so-called 'acid crash', an undesirable phenomenon characterized by a total arrest of cell growth and solvent production, can be avoided. Operational parameters that may influence the prevention of acid crash occurrence, such as pH, CaCO3 concentration and culture temperature, were optimized in C. beijerinckii CECT 508 cultures applying a Box-Behnken experimental design. The mathematical model of the fermentation found the optimal conditions of pH 7, 6.8 g/L of CaCO3 and 30 °C, and this was validated in an independent experiment carried out at the optimal conditions, reaching 10.75 g/L of butanol. Also, the comparison of butanol production between the supernatant of the AP hydrolysate (10.57 g/L) and the full hydrolysate with solids (11.69 g/L) indicated that it is possible to eliminate the centrifugation step after hydrolysis, which may allow to reduce process costs and the full utilization of apple pomace, aiming a zero-waste approach.

Potential and prospects for utilization of avocado by-products in integrated biorefineries.

The industrial processing of avocado to extract oil, and produce guacamole or sauces generates enormous quantities of peels and seeds (around 2 million tons worldwide in 2019) without commercially valuable applications. However, various studies have suggested the presence of a wide range of interesting compounds in the composition of these by-products. This review depicts a thorough outline of the capacity of avocado residues to be converted into a portfolio of commodities that can be employed in sectors such as the food, cosmetics, pharmaceuticals, environment, and energy industries. Therefore, a novel biorefinery strategy to valorize avocado-processing residues to obtain a polyphenolic extract, pectooligosaccharides, and succinic acid was presented. Additionally, the prospects and challenges facing a biorefinery based on the valorization of avocado residues are presented, particularly its techno-economic feasibility on an industrial scale, aiming for a resource-efficient circular bio-economy.

3D Printing: An Emerging Technology for Biocatalyst Immobilization.

Employment of enzymes as biocatalysts offers immense benefits across diverse sectors in the context of green chemistry, biodegradability, and sustainability. When compared to free enzymes in solution, enzyme immobilization proposes an effective means of improving functional efficiency and operational stability. The advance of printable and functional materials utilized in additive manufacturing, coupled with the capability to produce bespoke geometries, has sparked great interest toward the 3-dimensional (3D) printing of immobilized enzymes. Printable biocatalysts represent a new generation of enzyme immobilization in a more customizable and adaptable manner, unleashing their potential functionalities for countless applications in industrial biotechnology. This review provides an overview of enzyme immobilization techniques and 3D printing technologies, followed by illustrations of the latest 3D printed enzyme-immobilized industrial and clinical applications. The unique advantages of harnessing 3D printing as an enzyme immobilization technique will be presented, alongside a discussion on its potential limitations. Finally, the future perspectives of integrating 3D printing with enzyme immobilization will be considered, highlighting the endless possibilities that are achievable in both research and industry.

Bundling the removal of emerging contaminants with the production of ligninolytic enzymes from residual streams.

Enzymes offer interesting features as biological catalysts for industry: high specificity, activity under mild conditions, accessibility, and environmental friendliness. Being able to produce enzymes in large quantities and having them available in a stable and reusable form reduces the production costs of any enzyme-based process. Agricultural residues have recently demonstrated their potential as substrates to produce ligninolytic enzymes by different white rot fungi. In this study, the biotechnological production of a manganese peroxidase (MnP) by Irpex lacteus was conducted through solid-state fermentation (SSF) with wheat straw as substrate and submerged fermentation (SmF) employing wheat straw extract (WSE). The obtained enzyme cocktail also showed manganese-independent activity (MiP), related to the presence of a short MnP and a dye-decolorizing peroxidase (DyP) which was confirmed by shotgun proteomic analyses. In view of the enhanced production of ligninolytic enzymes in SmF, different parameters such as WSE concentration and nitrogen source were evaluated. The highest enzyme titers were obtained with a medium formulated with glucose and peptone (339 U/L MnP and 15 U/L MiP). The scale-up to a 30 L reactor achieved similar activities, demonstrating the feasibility of enzyme production from the residual substrate at different production scales. Degradation of five emerging pollutants was performed to demonstrate the high oxidative capacity of the enzyme. Complete removal of hormones and bisphenol A was achieved in less than 1 h, whereas almost 30% degradation of carbamazepine was achieved in 24 h, which is a significant improvement compared to previous enzymatic treatments of this compound. KEY POINTS: • Wheat straw extract is suitable for the growth of I. lacteus. • The enzyme cocktail obtained allows the degradation of emerging contaminants. • Mn-dependent and Mn-independent activities increases the catalytic potential.

A customizable 3D printed device for enzymatic removal of drugs in water.

The infiltration of drugs into water is a key global issue, with pharmaceuticals being detected in all nearly aqueous systems at often alarming concentrations. Pharmaceutical contamination of environmental water supplies has been shown to negatively impact ecological equilibrium and pose a risk to human health. In this study, we design and develop a novel system for the removal of drugs from water, termed as Printzyme. The device, fabricated with stereolithography (SLA) 3D printing, immobilises laccase sourced from Trametes Versicolor within a poly(ethylene glycol) diacrylate hydrogel. We show that SLA printing is a sustainable method for enzyme entrapment under mild conditions, and measure the stability of the system when exposed to extremes of pH and temperature in comparison to free laccase. When tested for its drug removal capacity, the 3D printed device substantially degraded two dissolved drugs on the European water pollution watch list. When configured in the shape of a torus, the device effectively removed 95% of diclofenac and ethinylestradiol from aqueous solution within 24 and 2 h, respectively, more efficiently than free enzyme. Being customizable and reusable, these 3D printed devices could help to efficiently tackle the world's water pollution crisis, in a flexible, easily scalable, and cost-efficient manner.

Hydrothermal treatment of avocado peel waste for the simultaneous recovery of oligosaccharides and antioxidant phenolics.

Avocado industrial processing generates huge quantities of residues that are currently wasted without any valuable commercial application. This work deals with autohydrolysis of Avocado peel (AP) for the concomitant recovery of oligosaccharides and polyphenolics. Temperature of 150 °C allowed the highest recovery of oligosaccharides (14.3 g oligosaccharides/100 g AP) and high recovery of antioxidant phenolics (3.48 g gallic acid equivalents/100 g AP and 10.80 g Trolox equivalents/100 g AP measured with ABTS●+ assay). The liquor obtained at this temperature was characterized by TGA and FTIR to study its thermal stability and functional groups. UHPLC-TOF MS analysis of an ethyl acetate extract of AP liquor enabled the tentative identification of 43 compounds, belonging to various metabolite families, including flavonoids, phenolic acids, organic acids, lignans and fatty acids. These findings demonstrated that autohydrolysis of AP is a suitable technology to obtain bioactive agents with potential uses in food and cosmetic industries.

Chemical and thermal stabilization of CotA laccase via a novel one-step expression and immobilization in muNS-Mi nanospheres.

A methodology that programs eukaryotic or bacterial cells to encapsulate proteins of any kind inside micro/nanospheres formed by muNS-Mi viral protein was developed in our laboratory. In the present study such "in cellulo" encapsulation technology is utilized for immobilizing a protein with an enzymatic activity of industrial interest, CotA laccase. The encapsulation facilitates its purification, resulting in a cost-effective, one-step way of producing immobilized enzymes for industrial use. In addition to the ability to be recycled without activity loss, the encapsulated protein showed an increased pH working range and high resistance to chemical inactivation. Also, its activity was almost unaffected after 30 min incubation at 90 °C and 15 min at the almost-boiling temperature of 95 °C. Furthermore, the encapsulated laccase was able to efficiently decolorate the recalcitrant dye RB19 at room temperature.

Energy requirements and economics of acetone-butanol-ethanol (ABE) extractive fermentation: a solvent-based comparative assessment.

The reindustrialization of acetone-butanol-ethanol (ABE) fermentation is hampered by its significant production cost, linked to high product inhibition and low product yield. ABE fermentation can be significantly enhanced by integrating in situ liquid-liquid extraction. In this study, hybrid simulations using Excel® and ASPEN Plus® were performed based on solvent-dependent experimental data (product titer, yield and productivity) to consider the physiological response of the microorganism in specific extractive ABE fermentations, and to quantify the energy requirements and the economic improvement of the overall process. Four scenarios, based on two different solvents (2-butyl-1-octanol, 2B1O, and a vegetable oil, VO) applied in batch or fed-batch operation, were compared with the batch conventional process. Total energy demand decreased in all extractive configurations and the greatest energy savings (61%) were reached with the VO-based fed-batch operation. However, the highest profit increase was achieved with 2B1O in fed-batch mode, reducing the minimum butanol selling price by 29% over the base case, along with 34% savings in raw materials and 80% wastewater reduction. The techno-economical solvent-based comparative evaluation is a useful tool to identify key challenges to be tackled when revisiting ABE extractive fermentation.

Valorization of horse chestnut burs to produce simultaneously valuable compounds under a green integrated biorefinery approach.

A biorefinery scheme for the valorization of horse chestnut biowastes (a municipal solid waste) into added value bioactive compounds is proposed in this work. The bur fraction of horse chestnut was evaluated as a novel and cheap renewable feedstock to obtain valuable compounds suitable for their use in industrial applications. The integrated valorization scheme comprised an initial hydroethanolic extraction of antioxidant compounds (optimized through surface response methodology), the alkaline delignification of the exhausted solid to obtain a lignin-enriched fraction, and the enzymatic digestibility of the remaining cellulose fraction to produce fermentable sugars. In addition, the structural characterization of the extract by FT-IR and TGA was performed, and the analysis by UPLC-DAD-ESI-MS allowed the tentative identification of eleven antioxidant phenolic compounds. The application of this multiproduct valorization approach led to the production of 13 kg antioxidant extracted compounds, 33.2 kg lignin and 14.5 kg glucose per each 100 kg of horse chestnut burs, which demonstrates the great potential of this residue as a biorefinery substrate.

Green sustainable process to revalorize purple corn cobs within a biorefinery frame: Co-production of bioactive extracts.

Purple corn (Zea mays L.) is used for the preparation of traditional drinks and desserts, generating great quantities of residues. The scarce information about purple corn cob (PCC) is encouraging an interest in exploring its potential as a valuable source of bioactive compounds with benefits for human health. In this study, a green method based on hydrothermal processing was used for the simultaneous extraction of oligosaccharides and phenolic compounds from PCC. For this purpose, the effects of three factors (time, temperature and pH) on the oligosaccharide content (OSC), total phenolic content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC), as well as on the antioxidant activity measured with three different methods (DPPH, ABTS and FRAP) were evaluated. The bioactive extract obtained under optimal conditions presented a high content of bioactive compounds exhibiting a notable antioxidant capacity and moderate inhibitory activities towards xanthine oxidase. This extract was also structurally characterized by FTIR, HPAEC-DAD, MALDI-TOF-MS and TGA, and the HPLC-ESI-MS analysis led to the tentative identification of 15 antioxidant phenolic compounds. Thus, this research demonstrated that this residue from the food industry has a high potential for obtaining several bioactive compounds that can be utilized as multi-functional ingredients in the food and pharmaceutical industries.

Valorisation of olive agro-industrial by-products as a source of bioactive compounds.

A large amount of olive-derived biomass is generated yearly in Spain, which could be used as a potential source of bioactive compounds. The present work evaluates the recovery of natural antioxidants from olive tree pruning (OTP) and olive mill leaves (OML). For this purpose, the effect of different solvents on the total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity was evaluated. The solvent was found to have a significant effect (p < 0.05) on the TPC, TFC, and the DPPH, ABTS, and FRAP activity, affording similar results for the extracts from the two by-products. The extracts obtained using 50% ethanol showed high TPC (23.85 and 27.54 mg GAE/gdw for OTP and OML, respectively) and TFC (52.82 and 52.39 mg RE/gdw for OTP and OML, respectively). Also, the OTP and OML extracts exhibited notable antioxidant activity as measured by the ABTS method (45.96 and 42.71 mg TE/gdw, respectively). Using pyrolysis-gas chromatography/mass spectrometry, 30 bioactive compounds were detected in both extracts. Additionally, UPLC-DAD-ESI-MS allowed the identification of 15 compounds in the samples. Furthermore, the antioxidant extracts were found to inhibit the growth of several food pathogenic bacteria. This research demonstrates that these by-products from olive grove farming are a good source of antioxidant compounds with antibacterial properties, which have potential applications in the food and pharmaceutical industries.

Hydrothermal treatment of chestnut shells (Castanea sativa) to produce oligosaccharides and antioxidant compounds.

Hydrothermal treatment is an environmentally friendly technology that allows the solubilisation of hemicellulosic oligosaccharides with potential for their use as prebiotics. The purpose of this study was to solubilize oligosaccharides and antioxidant compounds from chestnut shells by a hydrothermal processing. The highest content of oligosaccharides (18.3 g/L), with a relatively low level of monosaccharides (2.4 g/L) and degradation products (0.5 g/L) was obtained at 180 °C (severity of 3.08). In addition, the liquors presented a high content of phenolic and flavonoid compounds with good antioxidant properties. The GC-MS revealed that the most abundant phenolic compound was pyrogallol (13.2%). The molecular weight distribution of the solubilization products showed that a 26.5% presented an apparent Mw of 6077 g/mol and a 73.5% presented an apparent Mw of 586 g/mol with a high polydispersity index. MALDI-TOF, FTIR, and TGA analyses revealed structural information of these compounds and their thermal stability.

Bifidobacterial growth stimulation by oligosaccharides generated from olive tree pruning biomass.

This work aims to evaluate the prebiotic potential of oligosaccharides (OS) obtained from autohydrolysis of olive tree pruning biomass (OTPB). Two selected fractions (F1 and F2) were characterized and used in in vitro fermentations by two Bifidobacterium spp. (B. adolescentis and B. longum) and one fecal inoculum. The fraction F1 presented a lower average degree of polymerization (DP) mainly with OS ranging from 3 to 6 DP, whereas the fraction F2 corresponded to a pool of unsubstituted and acetylated oligomers with DP between 4 and 19. In the fermentation by Bifidobacterium, F1 supported a higher biomass formation, OS consumption and organic acids production than F2. With the fecal inoculum, the accumulation of organic acids, as the sum of acetate, propionate and butyrate, was similar for F1 and F2 (107 and 101mM, respectively). The bifidobacteria counts also increased during the incubation time for both OS fractions.

Continuous removal of endocrine disruptors by versatile peroxidase using a two-stage system.

The oxidant Mn(3+) -malonate, generated by the ligninolytic enzyme versatile peroxidase in a two-stage system, was used for the continuous removal of endocrine disrupting compounds (EDCs) from synthetic and real wastewaters. One plasticizer (bisphenol-A), one bactericide (triclosan) and three estrogenic compounds (estrone, 17ß-estradiol, and 17α-ethinylestradiol) were removed from wastewater at degradation rates in the range of 28-58 µg/L·min, with low enzyme inactivation. First, the optimization of three main parameters affecting the generation of Mn(3+) -malonate (hydraulic retention time as well as Na-malonate and H2 O2 feeding rates) was conducted following a response surface methodology (RSM). Under optimal conditions, the degradation of the EDCs was proven at high (1.3-8.8 mg/L) and environmental (1.2-6.1 µg/L) concentrations. Finally, when the two-stage system was compared with a conventional enzymatic membrane reactor (EMR) using the same enzyme, a 14-fold increase of the removal efficiency was observed. At the same time, operational problems found during EDCs removal in the EMR system (e.g., clogging of the membrane and enzyme inactivation) were avoided by physically separating the stages of complex formation and pollutant oxidation, allowing the system to be operated for a longer period (∼8 h). This study demonstrates the feasibility of the two-stage enzymatic system for removing EDCs both at high and environmental concentrations.

Removal of estrogenic compounds from filtered secondary wastewater effluent in a continuous enzymatic membrane reactor. Identification of biotransformation products.

In the present study, a novel and efficient technology based on the use of an oxidative enzyme was developed to perform the continuous removal of estrogenic compounds from polluted wastewaters. A 2 L enzymatic membrane reactor (EMR) was successfully operated for 100 h with minimal requirements of laccase for the transformation of estrone (E1), 17ß-estradiol (E2), and 17α-ethinylestradiol (EE2)from both buffer solution and real wastewater (filtered secondary effluent). When the experiments were performed at high and low concentrations of the target compounds, 4 mg/L and 100 µg/L, not only high removal yields (80-100%) but also outstanding reduction of estrogenicity (about 84-95%) were attained. When the EMR was applied for the treatment of municipal wastewaters with real environmental concentrations of the different compounds (0.29-1.52 ng/L), excellent results were also achieved indicating the high efficiency and potential of the enzymatic reactor system. A second goal of this study relied on the identification of the transformation products to elucidate the catalytic mechanism of estrogens' transformation by laccase. The formation of dimers and trimers of E1, E2, and EE2, as well as the decomposition of E2 into E1 by laccase-catalyzed treatment, has been demonstrated by liquid chromatography atmospheric pressure chemical ionization (LC-APCI) analysis and confirmed by determination of accurate masses through liquid chromatography electrospray time-of-flight mass spectrometry (LC-ESI-TOF). Dimeric products of E2 and EE2 were found even when operating at environmental concentrations. Moreover, the reaction pathways of laccase-catalyzed transformation of E2 were proposed.

Biocatalytic generation of Mn(III)-chelate as a chemical oxidant of different environmental contaminants.

The objective of this study was to investigate the enzymatic generation of the Mn(3+) -malonate complex and its application to the process of oxidizing several organic compounds. The experimental set-up consisted of an enzymatic reactor coupled to an ultrafiltration membrane, providing continuous generation of Mn(3+) -malonate from a reaction medium containing versatile peroxidase (an enzyme produced by Bjerkandera adusta strain BOS55), H(2) O(2) , MnSO(4,) and malonate. The effluent of the enzymatic reactor was introduced into a batch-stirred reactor to oxidize three different classes of compounds: an azo dye (Orange II), three natural and synthetic estrogens, and a polycyclic aromatic hydrocarbon (anthracene). The enzymatic reactor provided the Mn(3+) complex under steady-state conditions, and this oxidative species was able to transform the three classes of xenobiotics considerably (90-99%) with negligible loss of activity.

Ex vivo expansion of human mesenchymal stem cells on microcarriers.

Due to the very low titers of human mesenchymal stem cells (MSC) in their niches, namely the bone marrow, an effective approach to isolate and expand those cells ex vivo is required to meet the needs of the increasing MSC clinical applications (e.g., therapy-resistant severe acute graft-versus-host disease). Herein we describe a microcarrier-based stirred culture system protocol for the efficient ex vivo expansion of human bone marrow-derived MSC. This protocol is potentially adaptable to different culture conditions, namely focusing the use of serum-free medium formulations, other sources of MSC, or different types of microcarriers.