Inactivation of hydrogenase-3 leads to enhancement of 1,3-propanediol and 2,3-butanediol production by Klebsiella pneumoniae.

Klebsiella pneumoniae can use glucose or glycerol as carbon sources to produce 1,3-propanediol or 2,3-butanediol, respectively. In the metabolism of Klebsiella pneumoniae, hydrogenase-3 is responsible for H2 production from formic acid, but it is not directly related to the synthesis pathways for 1,3-propanediol and 2,3-butanediol. In the first part of this research, hycEFG, which encodes subunits of the enzyme hydrogenase-3, was knocked out, so K. pneumoniae ΔhycEFG lost the ability to produce H2 during cultivation using glycerol as a carbon source. As a consequence, the concentration of 1,3-propanediol increased and the substrate (glycerol) conversion ratio reached 0.587 mol/mol. Then, K. pneumoniae ΔldhAΔhycEFG was constructed to erase lactic acid synthesis which led to the further increase of 1,3-propanediol concentration. A substrate (glycerol) conversion ratio of 0.628 mol/mol in batch conditions was achieved, which was higher compared to the wild type strain (0.545 mol/mol). Furthermore, since adhE encodes an alcohol dehydrogenase that catalyzes ethanol production from acetaldehyde, K. pneumoniae ΔldhAΔadhEΔhycEFG was constructed to prevent ethanol production. Contrary to expectations, this did not lead to a further increase, but to a decrease in 1,3-propanediol production. In the second part of this research, glucose was used as the carbon source to produce 2,3-butanediol. Knocking out hycEFG had distinct positive effect on 2,3-butanediol production. Especially in K. pneumoniae ΔldhAΔadhEΔhycEFG, a substrate (glucose) conversion ratio of 0.730 mol/mol was reached, which is higher compared to wild type strain (0.504 mol/mol). This work suggests that the inactivation of hydrogenase-3 may have a global effect on the metabolic regulation of K. pneumoniae, leading to the improvement of the production of two industrially important bulk chemicals, 1,3-propanediol and 2,3-butanediol.

Bioconversion of Grape Pomace with Rhizopus oryzae under Solid-State Conditions: Changes in the Chemical Composition and Profile of Phenolic Compounds.

Grape pomace is a sustainable source of bioactive phenolic compounds used in various industries. The recovery of phenolic compounds could be improved by biological pretreatment of grape pomace, as they are released from the lignocellulose structure by the activity of the enzymes produced. The influence of grape pomace pretreatment with Rhizopus oryzae under solid-state conditions (SSF) on the phenolic profile and chemical composition changes was studied. SSF was performed in laboratory jars and in a tray bioreactor for 15 days. Biological pretreatment of grape pomace resulted in an increase in the content of 11 individual phenolic compounds (from 1.1 to 2.5-fold). During SSF, changes in the chemical composition of the grape pomace were observed, including a decrease in ash, protein, and sugar content, and an increase in fat, cellulose, and lignin content. A positive correlation (r > 0.9) was observed between lignolytic enzymes and the hydrolytic enzyme's xylanase and stilbene content. Finally, after 15 days of SSF, a weight loss of GP of 17.6% was observed. The results indicate that SSF under experimental conditions is a sustainable bioprocess for the recovery of phenolic compounds and contributes to the zero-waste concept by reducing waste.

Antioxidant and antiproliferative potentials of phenolic-rich extracts from biotransformed grape pomace in colorectal Cancer.

BACKGROUND: Colorectal carcinoma is one of the most commonly diagnosed malignancies worldwide. Consumption of dietary supplements and nutraceuticals such as phenolic compounds may help combat colorectal carcinoma. The effect of two phenolic-rich extracts prepared from biotransformed grape pomace on the antioxidant properties and antiproliferative activity against two colorectal cancer cell lines (Caco-2 and SW620) were investigated. METHODS: A 15-day solid-state fermentation with the white-rot fungi Phanerochaete chrysosporium and Trametes gibbosa was used to biotransform grape pomace. Solid-liquid extraction was then performed to extract bioactive compounds. The extract was analyzed for the determination of phenolic compounds by ultra-high performance liquid chromatography and in vitro assays of biological activities (antioxidant activity, antiproliferative activity, cell cycle analysis). RESULTS: The 4 days of solid-state fermentation proved to be the optimal period to obtain the maximum yield of phenolic compounds. The tested extracts showed significant antioxidant and antiproliferative activities. Grape pomace treated with P. chrysosporium and T. gibbosa reduced cancer cell growth by more than 60% at concentrations (solid/liquid ratio) of 1.75 mg/mL and of 2.5 mg/mL, respectively. The cell cycle perturbations induced by the grape pomace extracts resulted in a significant increase in the number of cells in the S (9.8%) and G2/M (6.8%) phases of SW620 exposed to T. gibbosa after 48 hours, while P. chrysosporium increased the percentage of cells in the G1 phase by 7.7%. The effect of grape pomace extracts on Caco-2 was less pronounced. CONCLUSIONS: The obtained results suggest the presence of bioactive compounds in biotransformed grape pomace as a residue from winemaking, which could be used to prevent colon cancer.

Improvement of the Nutraceutical Profile of Brewer's Spent Grain after Treatment with Trametes versicolor.

Brewer's spent grain (BSG) is an important secondary raw material that provides a readily available natural source of nutraceuticals. It finds its largest application as animal feed and part of the human diet, while the future perspective predicts an application in the production of value-added products. In order to investigate a sustainable BSG treatment method, two BSG samples (BSG1 and BSG2) were evaluated as substrates for the production of hydrolytic (xylanase, ß-glucosidase and cellulase) and lignolytic enzymes (laccase, manganese peroxidase and lignin peroxidase) by solid-state fermentation (SSF) with Trametes versicolor while improving BSG nutritional value. The biological treatment was successful for the production of all hydrolytic enzymes and laccase and manganese peroxidase, while it was unsuccessful for the production of lignin peroxidase. Because the two BSGs were chemically different, the Trametes versicolor enzymes were synthesized at different fermentation times and had different activities. Consequently, the chemical composition of the two BSG samples at the end of fermentation was also different. The biological treatment had a positive effect on the increase in protein content, ash content, polyphenolic compounds, and sugars in BSG1. In BSG2, there was a decrease in the content of reducing sugars. Cellulose, hemicellulose, and lignin were degraded in BSG1, whereas only cellulose was degraded in BSG2, and the content of hemicellulose and lignin increased. The fat content decreased in both samples. The safety-related correctness analysis showed that the biologically treated sample did not contain any harmful components and was therefore safe for use in nutritionally enriched animal feed.

The Potential of Brewer's Spent Grain in the Circular Bioeconomy: State of the Art and Future Perspectives.

Brewer's spent grain (BSG) accounts for approximately 85% of the total mass of solid by-products in the brewing industry and represents an important secondary raw material of future biorefineries. Currently, the main application of BSG is limited to the feed and food industry. There is a strong need to develop sustainable pretreatment and fractionation processes to obtain BSG hydrolysates that enable efficient biotransformation into biofuels, biomaterials, or biochemicals. This paper aims to provide a comprehensive insight into the availability of BSG, chemical properties, and current and potential applications juxtaposed with the existing and emerging markets of the pyramid of bio-based products in the context of sustainable and circular bioeconomy. An economic evaluation of BSG for the production of highly valuable products is presented in the context of sustainable and circular bioeconomy targeting the market of Central and Eastern European countries (BIOEAST region).

A Comparative Study of the Influence of Various Fungal-Based Pretreatments of Grape Pomace on Phenolic Compounds Recovery.

Wineries produce considerable amounts of grape pomace, which is a readily available natural source of bioactive phenolic compounds. In this study, grape pomace was used as a substrate for the cultivation of eleven filamentous fungi (Trametes versicolor TV6, Trametes versicolor TV8, Trametes versicolor AG613, Trametes gibbosa, Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Pleurotus eryngii, Ganoderma lucidum, Ganoderma resinaceum, Humicola grisea, and Rhizopus oryzae) under solid-state conditions (SSF) for 15 days with the aim of improving the recovery of the individual phenolic compounds. Twenty-one phenolic compounds were quantified and the recovery of seventeen of them (gallic acid, ellagic acid, p-hydroxybenzoic acid, syringic acid, vanillic acid, 3,4-dihydroxybenzoic acid, ferulic acid, o-coumaric acid, p-coumaric acid, epicatechin gallate, galocatechin gallate, quercetin, kaempferol, procyanidin B1, procyanidin B2, resveratrol, and ε-viniferin) were positively affected by SSF. Ellagic acid is the most recovered compound, whose content increased 8.8-fold after 15 days of biological treatment with Ceriporiopsis subvermispora compared to the untreated initial sample. Among the microorganisms tested, the fungi Pleurotus eryngii and Rhizopus oryzae proved to be the most effective in increasing the recovery of most phenolic compounds (1.1-4.5-fold). In addition, the nutrient composition (proteins, ash, fats) of grape pomace was positively affected by the biological treatments.

The roles of diol dehydratase from pdu operon on glycerol catabolism in Klebsiella pneumoniae.

The dha operon of Klebsiella pneumoniae is responsible for glycerol catabolism and 1,3-propanediol formation. Subunits of glycerol dehydratase and the large subunit of glycerol dehydratase reactivating factor are encoded by dhaBCE and dhaF, respectively. Proteins of pdu operon form a microcompartment (bacteria organelle) and responsible for 1,2-propanediol catabolism. In this operon, pduCDE and pduG encode subunits of diol dehydratase and its reactivating factor. Diol dehydratase is an isofunctional enzyme of glycerol dehydratase, but its role in glycerol catabolism was not entirely clear. In this study, dhaBCE, pduCDE, dhaF, and pduG in K. pneumoniae were knocked out individually or combinedly. These strains were cultured with glycerol as a substrate, and dehydratase activities in the cytoplasm and microcompartment were detected. Results showed that glycerol dehydratase and diol dehydratase were simultaneously responsible for glycerol catabolism in K. pneumoniae. Besides being packaged in microcompartment, large amounts of diol dehydratase was also presented in the cytoplasm. However, the Pdu microcompartment reduced the accumulation of 3-hydroxypropionaldehyde in the fermentation broth. PduG can cross reactivate glycerol dehydratase instead of DhaF. However, DhaF is not involved in reactivation of diol dehydratase. In conclusion, diol dehydratase and Pdu microcompartment play important roles in glycerol catabolism in K. pneumoniae.

1,2-Propanediol production from glycerol via an endogenous pathway of Klebsiella pneumoniae.

Klebsiella pneumoniae is an important microorganism and is used as a cell factory for many chemicals production. When glycerol was used as the carbon source, 1,3-propanediol was the main catabolite of this bacterium. K. pneumoniae ΔtpiA lost the activity of triosephosphate isomerase and prevented glycerol catabolism through the glycolysis pathway. But this strain still utilized glycerol, and 1,2-propanediol became the main catabolite. Key enzymes of 1,2-propanediol synthesis from glycerol were investigated in detail. dhaD and gldA encoded glycerol dehydrogenases were both responsible for the conversion of glycerol to dihydroxyacetone, but overexpression of the two enzymes resulted in a decrease of 1,2-propanediol production. There are two dihydroxyacetone kinases (I and II), but the dihydroxyacetone kinase I had no contribution to dihydroxyacetone phosphate formation. Dihydroxyacetone phosphate was converted to methylglyoxal, and methylglyoxal was then reduced to lactaldehyde or hydroxyacetone and further reduced to form 1,2-propanediol. Individual overexpression of mgsA, yqhD, and fucO resulted in increased production of 1,2-propanediol, but only the combined expression of mgsA and yqhD showed a positive effect on 1,2-propanediol production. The process parameters for 1,2-propanediol production by Kp ΔtpiA-mgsA-yqhD were optimized, with pH 7.0 and agitation rate of 350 rpm found to be optimal. In the fed-batch fermentation, 9.3 g/L of 1,2-propanediol was produced after 144 h of cultivation, and the substrate conversion ratio was 0.2 g/g. This study provides an efficient way of 1,2-propanediol production from glycerol via an endogenous pathway of K. pneumoniae.Key points• 1,2-Propanediol was synthesis from glycerol by a tpiA knocked out K. pneumoniae• Overexpression of mgsA, yqhD, or fucO promote 1,2-propanediol production• 9.3 g/L of 1,2-propanediol was produced in fed-batch fermentation.

A Comprehensive Review on Valorization of Agro-Food Industrial Residues by Solid-State Fermentation.

Agro-food industrial residues (AFIRs) are generated in large quantities all over the world. The vast majority of these wastes are lignocellulosic wastes that are a source of value-added products. Technologies such as solid-state fermentation (SSF) for bioconversion of lignocellulosic waste, based on the production of a wide range of bioproducts, offer both economic and environmental benefits. The versatility of application and interest in applying the principles of the circular bioeconomy make SSF one of the valorization strategies for AFIRs that can have a significant impact on the environment of the wider community. Important criteria for SSF are the selection of the appropriate and compatible substrate and microorganism, as well as the selection of the optimal process parameters for the growth of the microorganism and the production of the desired metabolites. This review provides an overview of the management of AFIRs by SSF: the current application, classification, and chemical composition of AFIRs; the catalytic function and potential application of enzymes produced by various microorganisms during SSF cultivation on AFIRs; the production of phenolic compounds by SSF; and a brief insight into the role of SSF treatment of AFIRs for feed improvement and biofuel production.

Trametes versicolor in lignocellulose-based bioeconomy: State of the art, challenges and opportunities.

Although Trametes versicolor is one of the most investigated white-rot fungi, the industrial application of this fungus and its metabolites is still far from reaching its full potential. This review aims to highlight the opportunities and challenges for the industrial use of T. versicolor according to the principles of circular bioeconomy. The use of this fungus can contribute significantly to the success of efforts to valorize lignocellulosic waste biomass and industrial lignocellulosic byproducts. Various techniques of T. versicolor cultivation for enzyme production, food and feed production, wastewater treatment, and biofuel production are listed and critically evaluated, highlighting bottlenecks and future perspectives. Applications of T. versicolor crude laccase extracts in wastewater treatment, removal of lignin from lignocellulose, and in various biotransformations are analyzed separately.

Transesterification of Sunflower Oil over Waste Chicken Eggshell-Based Catalyst in a Microreactor: An Optimization Study.

The statistical experimental design (DoE) and optimization (Response Surface Methodology combined with Box-Behnken design) of sunflower oil transesterification catalyzed by waste chicken eggshell-based catalyst were conducted in a custom-made microreactor at 60 °C. The catalyst was synthesized by the hydration-dehydration method and subsequent calcination at 600 °C. Comprehensive characterization of the obtained catalyst was conducted using: X-ray powder diffractometry (XRD), X-ray fluorescence (XRF), Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), N2 physisorption, and Hg-porosimetry. Structural, morphological, and textural results showed that the obtained catalyst exhibited high porosity and regular dispersity of plate-like CaO as an active species. The obtained optimal residence time, catalyst concentration, and methanol/oil volume ratio for the continuous reaction in microreactor were 10 min, 0.1 g g-1, and 3:1, respectively. The analysis of variance (ANOVA) showed that the obtained reduced quadratic model was adequate for experimental results fitting. The reaction in the microreactor was significantly intensified compared to a conventional batch reactor, as seen through the fatty acid methyl esters (FAMEs) content after 10 min, which was 51.2% and 18.6%, respectively.

Natural deep eutectic solvent as a unique solvent for valorisation of orange peel waste by the integrated biorefinery approach.

This research investigates the use of seven natural deep eutectic solvents (NADESs) for valorisation of orange peel waste, with the final goal to propose a unique NADES for integrated biorefinery. Initial screening of NADESs revealed the excellent ability of cholinium-based NADES with ethylene glycol as hydrogen bond donor (ChEg50) to serve as a medium for orange peel-catalysed kinetic resolution (hydrolysis) of (R,S)-1-phenylethyl acetate with high enantioselectivity (ee = 83.2%, X  = 35%), as well as it's stabilizing effect on the hydrolytic enzymes (hydrolytic enzymes within ChEg50 peel extract were stabile during 20 days at 4 °C). The ChEg50 also showed a satisfactory capacity to extract D-limonene (0.5 mg gFW-1), and excellent capacity to extract polyphenols (45.7 mg gFW-1), and proteins (7.7 mg gFW-1) from the peel. Based on the obtained results, the integrated biorefinery of orange peel waste using ChEg50 in a multistep process was performed. Firstly, enantioselective kinetic resolution was performed (step I; ee = 83.2%, X  = 35%), followed by isolation of the product 1-phenylethanol (step II; h = 82.2%) and extraction of polyphenols (step III; h = 86.8%) from impoverished medium. Finally, the residual orange peel was analysed for sugar and lignin content, and results revealed the potential of waste peel for the anaerobic co-digestion process. The main bottlenecks and futures perspective of NADES-assisted integrated biorefinery of orange peel waste were outlined through SWOT analysis.

Pulsed electric field: An emerging pretreatment technology in a biogas production.

This review focuses on current status of pulsed electric field (PEF) technology and its implementation in biogas production. First, basic principles of PEF and a schematic overview of typical PEF processing system were provided. Thereafter, lab- and pilot-scale PEF pretreatments of sludge with subsequent anaerobic digestion (AD) were provided. Furthermore, PEF technology, as an emerging technology for the lignocellulose (LC) pretreatment in biogas production which is still predominantly used at lab-scale, was outlined. Eventually, conclusion together with future perspectives and challenges were outlined.

Role of the Encapsulation in Bioavailability of Phenolic Compounds.

Plant-derived phenolic compounds have multiple positive health effects for humans attributed to their antioxidative, anti-inflammatory, and antitumor properties, etc. These effects strongly depend on their bioavailability in the organism. Bioaccessibility, and consequently bioavailability of phenolic compounds significantly depend on the structure and form in which they are introduced into the organism, e.g., through a complex food matrix or as purified isolates. Furthermore, phenolic compounds interact with other macromolecules (proteins, lipids, dietary fibers, polysaccharides) in food or during digestion, which significantly influences their bioaccessibility in the organism, but due to the complexity of the mechanisms through which phenolic compounds act in the organism this area has still not been examined sufficiently. Simulated gastrointestinal digestion is one of the commonly used in vitro test for the assessment of phenolic compounds bioaccessibility. Encapsulation is a method that can positively affect bioaccessibility and bioavailability as it ensures the coating of the active component and its targeted delivery to a specific part of the digestive tract and controlled release. This comprehensive review aims to present the role of encapsulation in bioavailability of phenolic compounds as well as recent advances in coating materials used in encapsulation processes. The review is based on 258 recent literature references.

Enhancement of the anti-inflammatory properties of grape pomace treated by Trametes versicolor.

The application of solid-state fermentation for the production of value-added products from the agro- and food-industry residues has been recently investigated greatly. The white-rot basidiomycete Trametes versicolor is a widely used fungi for the degradation lignocellulosic material in solid-state conditions. Grape pomace constitutes the major by-product of Vitis vinifera L. and is a source of compounds with recognized health benefits. In this study, a process for treating grape pomace with Trametes versicolor for 15 days under solid-state conditions was developed, and the phenolic profile and anti-inflammatory potential of the grape pomace extracts before and after treatment was studied. The anti-inflammatory potential of the grape pomace extracts was studied via tests based on the inhibition of 5-lipoxygenase and hyaluronidase, two key enzymes in inflammatory processes. A total of 24 phenolic compounds were identified and quantified by HPLC methods. With the exception of anthocyanins, an increase in phenolic acids, flavan-3-ols and the flavonol rutin was observed after a treatment period of 1-4 days with T. versicolor. Moreover, the increase in the phenolic content was accompanied by an enhancement in the anti-inflammatory activity of the grape pomace extracts, which was confirmed by the strong correlation between them. This is the first study providing evidence of the benefits of the application of fungal-based solid-state fermentation as an environmentally friendly process for the enhancement of the phenolic composition and anti-inflammatory potential of grape pomace, increasing the possibility of profiting from the great waste produced by the grape-processing industry.

Cost analysis of oil cake-to-biodiesel production in packed-bed micro-flow reactors with immobilized lipases.

Biodiesel production depends to a great extent on the use of cheap raw materials, since biodiesel itself is a mass product, not a high-value product. New processing methods, such as micro-flow continuous processing combined with enzymatic catalysis, open doors to the latter. As reported here, the window of opportunity in enzyme-catalyzed biodiesel production is the conversion of waste cooking oil. The main technological challenge for this is to obtain efficient immobilization of the lipase catalyst on beads. The beads can be filled into tubular reactors where designed packed-bed provide porous channels, forming micro-flow. It turns out, that in this way, the immobilization costs become the decisive economic factor. This paper reports a solution to that issue. The use of oil cake enables economic viability, which is not given by any of the commercial polymeric substrates used so far for enzyme immobilization. The costs of immobilization are mirrored in the earnings and cash flow of the new biotechnological process.

Electroporation of harvest residues for enhanced biogas production in anaerobic co-digestion with dairy cow manure.

The aim of this research was to develop a method for pretreatment of lignocellulose (LC) substrates (harvest residues (HR)) via electroporation (EP) for the purpose of improving the biogas production process. In addition, pretreated LC substrates were analyzed by scanning electron microscopy (SEM) and the energy balance of the total process was calculated. After the conducted pretreatment and anaerobic co-digestion with dairy cow manure (DCM), the statistical data analysis showed statistically significant differences in biogas and/or methane yield for all three LC substrates and their fractions. It was concluded that, after the pretreatment of LC substrates via EP, it is possible to improve the anaerobic co-digestion process and to achieve positive energy balance of the total process.

Corn silage fungal-based solid-state pretreatment for enhanced biogas production in anaerobic co-digestion with cow manure.

The objective of this research was to use white-rot fungus Trametes versicolor for corn silage pretreatment and to investigate the effect of pretreatment on biogas productivity. Semi-continuous pilot-scale experiment, comprised of two experimental phases, was carried out. In the first phase, operational conditions of the full-scale biogas plant were reproduced at pilot-scale. In that phase, the reactor was daily fed with the mixture of cow manure, digestate from industrial postfermentor, corn grits and ensiled corn silage, and the average methane generation rate was 0.167 m3CH4 kgVS-1. In the second phase, corn grits and ensiled corn silage were replaced with corn silage pretreated with T. versicolor, and the average methane generation rate increased up to 0.236 m3CH4 kgVS-1. The results of this study suggest that application of fungal-based solid-state pretreated corn silage has positive effect on pH stability and increase the biogas productivity.

Proximate analysis of cold-press oil cakes after biological treatment with Trametes versicolor and Humicola grisea.

In order to increase the current knowledge on cold-press oil cakes composition, the present study aims to determine the chemical composition of oil cakes from hull-less pumpkin (Cucurbita pepo L.), flax (Linum usitatissimum L.), and hemp (Canabis sativa L.) before and after the biological treatment with Trametes versicolor and Humicola grisea using fungal-based solid-state technology. After 10 days of treatment, the content of ash, total nitrogen, total proteins, and total organic carbon increased in all the three oil cakes, while the content of ether extracts decreased. After treatment, the concentration of soluble carbohydrates decreased in pumpkin and hemp seed oil cakes, whereas it increased in flaxseed oil cake. During treatment with T. versicolor, the content of fructose significantly increased in hull-less pumpkin seed oil cake. Fiber content decreased in pumpkin and flaxseed oil cakes after treatment with both of the fungi, whereas it increased in flaxseed oil cake.