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.

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.

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.

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.

Modelling the ex situ bioremediation of diesel-contaminated soil in a slurry bioreactor using a hydrocarbon-degrading inoculant.

Bioremediation is a soil clean-up technique which exploits the metabolic capacity of microorganisms to degrade soil contaminants. A model was developed to simulate the ex situ bioremediation of a diesel-contaminated soil in a bio-slurry reactor inoculated with a diesel-degrading bacterial strain. Mass transfer processes involving desorption of diesel from soil to water and volatilization of diesel from water, and biodegradation by the bacterial inoculant were included in the model by using Weibull sigmoid kinetics and logistic/Monod kinetics respectively. Model parameters were estimated in batch-based abiotic and biodegradation experiments. Sensitivity analysis revealed the importance of maintaining a high bacterial density in the system for maximum bioremediation efficiency. The model was validated using a pilot bioreactor monitored for 528 h, which removed almost 90% of the diesel present in the system. The results revealed the capacity of the model to predict the bioremediation efficiency under different scenarios by adapting the input parameters to each system.

Polymerization of coniferyl alcohol by Mn3+ -mediated (enzymatic) oxidation: Effects of H2 O2 concentration, aqueous organic solvents, and pH.

The objective of this study was to evaluate the ability of one versatile peroxidase and the biocatalytically generated complex Mn(III)-malonate to polymerize coniferyl alcohol (CA) to obtain dehydrogenation polymers (DHPs) and to characterize how closely the structures of the formed DHPs resemble native lignin. Hydrogen peroxide was used as oxidant and Mn2+ as mediator. Based on the yields of the polymerized product, it was concluded that the enzymatic reaction should be performed in aqueous solution without organic solvents at 4.5 ≤ pH ≤ 6.0 and with 0.75 ≤ H2 O2 :CA ratio ≤ 1. The results obtained from the Mn3+ -malonate-mediated polymerization showed that the yield was almost 100%. Reaction conditions had, however, effect on the structures of the formed DHPs, as detected by size exclusion chromatography and pyrolysis-GC/MS. It can be concluded that from the structural point of view, the optimal pH for DHP formation using the presently studied system was 3 or 4.5. Low H2 O2 /CA ratio was beneficial to avoid oxidative side reactions. However, the high frequency of ß-ß linkages in all cases points to dimer formation between monomeric CA rather than endwise polymerization. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:81-90, 2018.

Application of flow cytometry for monitoring the production of poly(3-hydroxybutyrate) by Halomonas boliviensis.

In this study, a flow cytometry (FC) protocol was implemented to measure poly(3-hydroxybutyrate) (PHB) content in a halophilic bacterium, to have a faster and easier control of the process. The halophilic bacterium Halomonas boliviensis was stained with BODIPY 493/503 and analyzed using FC. Bacterial polymer accumulation induced by two different nutrient limitations during the operation of a 2 L bioreactor was studied using traditional gas chromatography (GC) analysis and FC. The application of this rapid and straightforward method is useful to obtain complex and precise information about PHB accumulation that could be used for the monitoring, control and optimization of the production of PHB. A clear correlation between the PHB concentration determined by GC and the fluorescence signal obtained from stained bacteria by using FC was observed. Additionally, the heterogeneity of bacterial population as a function of PHB content was measured. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:276-284, 2017.

Effect of nitrogen and/or oxygen concentration on poly(3-hydroxybutyrate) accumulation by Halomonas boliviensis.

The behaviour of Halomonas boliviensis during growth in fed-batch culture under different kind of nutrient restrictions was examined. The metabolic switch between growth and accumulation phase is determined by the limitation in one or more essential nutrient for bacterial growth. The aim of this study was to test the effect of applying limitations of a essential nutrient, such as nitrogen, and the influence of different O2 concentrations on poly(3-hydroxybutyrate) (PHB) production during the accumulation phase. Single limitations of nitrogen and oxygen provoke PHB accumulations of 45 and 37 % (g g(-1)), respectively, while N limitation with low O2 supply causes the highest PHB accumulation of 73 %. The characterization of the PHB production with the strain H. boliviensis would allow a better optimization of the process and enrich the knowledge about the PHB production from strains different than Cupriavidus necator.

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.