The valorization of hempseed oil pomace (HP) using two techniques, solid-state fermentation (SSF) and hydrothermal carbonization (HTC), to produce value-added products was investigated. HP was first pretreated with Thermomyces lanuginosus under SSF for 10 days to produce two enzymes (lipase and xylanase) and fermented HP (a nutritionally enriched, mycelium-based product). The structural and chemical properties of the biomass were characterized, and lipase and xylanase activities were monitored daily throughout the fermentation process. Samples collected after 4, 7, and 10 days of fermentation were further subjected to HTC to produce hydrochar (HC) and a liquid fraction (LF), and to study the effect of SSF pretreatment on the properties of HTC products. Structural analysis of fermented HP using NMR, SEM–EDX and FTIR indicated substantial structural modification and partial degradation of the lignocellulosic matrix following SSF pretreatment. High activities of lipase (up to 0.29 U/mL) and xylanase (up to 79.34 U/mL) were obtained during fermentation, while fermented HP exhibited improved nutritional properties, particularly being enriched with free amino acids, among which glutamic and aspartic acids were the most abundant, each exceeding 5000 µg/g. HC from fermented HP exhibited great energetic and fuel properties (calorific values between 28.9 and 29.7 MJ/kg), comparable to HC from non-fermented HP. SSF treatment affected energy yield, volatile matter, and carbon and ash contents, resulting in increased fixed carbon content and an improved fuel ratio. LF obtained from HTC of fermented HP showed reduced toxicity and increased organic acids, total nitrogen and potassium content. Both HC and LF showed potential for fertilizer-oriented applications, although LF would require appropriate dilution or post-treatment before practical use. To the best of the authors’ knowledge, the application of T. lanuginosus for the integrated production of lipase and xylanase, together with the subsequent generation of hydrochar and biofertilizer from hempseed oil pomace or other oilseed pomaces, has not previously been reported.

The influence of Thermomyces lanuginosus on the chemical structure of biopolymers and lipids from hemp pomace during solid-state fermentation (SSF) was studied using Nuclear Magnetic Resonance (NMR) spectroscopy. The samples of hemp pomace before, during and after SSF were used for the isolation of biopolymers (lignin, cellulose, hemicellulose) and lipids, which were subsequently analyzed using solution and solid-state NMR spectroscopy. It was observed that SSF significantly alters the composition and quality of individual biopolymers. An increase in the carbonyl group content in lignin was noted. In lipid samples, a significant reduction in mono-, di-, and triglycerides occurred accompanied by an increase in glycerol and unsaturated fatty acids. SSF also notably impacted phosphorus-containing compounds in the hemp pomace. These results demonstrate that SSF enables targeted modification of key chemical components in hemp pomace. By altering the structure of lignin, breaking down complex lipids, and affecting phosphorus-containing compounds, SSF improves the chemical quality and functionality of the biomass. This highlights its potential as an effective method for upgrading agricultural residues into more valuable and versatile materials supporting sustainable biorefinery applications.

1,3-Propanediol (1,3-PDO) is a bulk chemical that can be produced by Klebsiella pneumoniae using glycerol as a substrate. In the 1,3-PDO synthesis pathway, part of the glycerol is oxidised to maintain intracellular NADH balance. Consequently, the theoretical maximum yield of 1,3-PDO from glycerol was lower than 1 mol/mol. In this study, engineered K. pneumoniae strains were constructed to direct all glycerol toward 1,3-PDO synthesis, with NADH being supplied through the catabolism of glucose. However, glycerol utilisation was inhibited in the presence of glucose. To alleviate this carbon catabolite repression (CCR), ptsG and crr were individually knocked out. The dha pathway is responsible for 1,3-PDO synthesis. Key genes in the oxidation branch of this pathway, including dhaK, dhaL, dhaD, and gldA, were knocked out to block this pathway. However, the expression of the dha operon was impaired in these strains, resulting in low 1,3-PDO production. In contrast, knocking out dhaM, which encodes a subunit of dihydroxyacetone kinase II, effectively blocked the glycerol oxidation pathway while maintaining the activity of the dha operon. Additionally, glpK was knocked out to block the sn-glycerol-3-phosphate formation from glycerol. Glucose to glycerol with the ratio of 0.5:1 mol/mol was the optimal value for 1,3-PDO production by K. pneumoniae ∆dhaM∆ptsG∆glpK, leading to a balance of NADH generation and consumption. Microaerobic conditions were favourable for 1,3-PDO production than anaerobic or aerobic conditions. In fed-batch fermentations, this strain produced 58.6 g/L of 1,3-PDO after 70 h, achieving a yield of 0.93 mol/mol glycerol, 2 mol/mol glucose, 0.63 mol/mol substrate. A highly efficient 1,3-PDO production technology that using glycerol and glucose as co-substrates was established.

Klebsiella pneumoniae is a commonly known 2,3-butanediol producer. 2,3-Butanediol synthesis and branched-chain amino acid (BCAA) synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. Those two pathways do not interfere with each other in the wild-type strain. Knocking out budA (encoding α-acetolactate decarboxylase) blocks the 2,3-butanediol synthesis pathway. Meanwhile, metabolites of the BCAA synthesis pathway (valine, 2-ketoisovalerate, 2,3-dihydroxyisovalerate and 2-hydroxyisovalerate) are accumulated. However, the mechanism underlying the metabolite changes resulting from the inactivation of budA remains unclear. In this study, both ex vivo and in vitro experiments were conducted to elucidate this mechanism. Kinetic parameters of BudA and acetohydroxy acid isomeroreductase (IlvC) were determined. BudA has a higher affinity toward α-acetolactate and has a higher catalytic constant (Km = 3.66 mM, kcat = 7.8 s-1) compared to IlvC (Km = 17.98 mM, kcat = 0.68 s-1). ex vivo experiments showed that IlvC activities were not influenced by knocking out budA and vice versa. IlvC activities were improved in the cells in which ilvC was overexpressed, but this did not lead to the accumulation of metabolites of the BCAA synthesis pathway. The activities of IlvC in the cell were not affected by the accumulation of 2,3-dihydroxyisovalerate, 2-ketoisovalerate, or valine in the broth. These results indicated that the competitiveness of BudA and IlvC in the cell determines the metabolites distribution between those two pathways. The inactivation of BudA and intact IlvC led to the exceeded α-acetolactate flow into the BCAA synthesis pathway, which caused the accumulation of metabolites of the BCAA synthesis pathway.

Grape pomace (GP), the solid residues remained after production of the continental and coastal Croatian red wines, has been studied by solid-state NMR and ATR-FTIR methods. After collection, drying and milling, GP samples have been analysed by IR spectroscopy and characteristic vibrational bands have been assigned. The observed differences in some functional group vibrations have been detected for continental and coastal GPs as well as for different vintages. 13C cross-polarization magic angle spinning (CP MAS) NMR experiments provided further information on chemical composition of GPs and percentages of different compounds present in the samples such as polysaccharides and phenolic compounds. 31P MAS spectra gave valuable quantitative information on the phosphorus content. The GPs from the continental varieties contain much larger portion of phosphorus compared to coastal ones, which is important for their potential use as an attractive raw material and value-added compounds for industrial applications.

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 0.587mol/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 0.628mol/mol in batch conditions was achieved, which was higher compared to the wild type strain (0.545mol/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 0.730mol/mol was reached, which is higher compared to wild type strain (0.504mol/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.

Lignocellulosic biomass (LB) is promising feedstock for the production of various bio-based products. However, due to its heterogenous character, complex chemical structure and recalcitrance, it is necessary to know its structural composition in order to optimize pretreatment process and further (bio)conversion into bio-based products. Nuclear Magnetic Resonance (NMR) spectroscopy is a fast and reliable method that can provide advanced data on the molecular architecture and composition of lignocellulosic biomass. In this brief overview, characteristic examples of the use of high-resolution NMR spectroscopy for the investigation of various types of LB and their structural units are given and the main drawbacks and future perspectives are outlined.

Lignocellulosic biorefineries (LBRs) are platforms for the production of a variety of bio-based products such as biofuels, biomaterials, biochemicals, food, and feed using lignocellulosic biomass (LB) as feedstock. LBRs are still rare worldwide. Their commercialization depends on challenges associated with the entire feedstock supply chain, efficiency, sustainability, and scale-up of pretreatment methods, as well as isolation and purification of value-added products. Each step within LBRs requires the development of new technologies or the improvement of existing ones, considering all three sustainability dimensions, environmental, social, and economic. Machine learning (ML) methods are widely used in various industrial fields, including biotechnology. The merging of biotechnology and ML has driven scientific progress and opened new opportunities for the development of LBRs as well. In this review, ML methods and their efficiency, used in biotechnology (metabolic engineering, bioprocess development, and environmental engineering), are presented, followed by their application in various phases of LB valorization.

Grape pomace (GP) is considered a natural source of bioactive compounds. To improve the extractability of bioactive compounds, in this work, GP was biologically treated for 15 days with the white-rot fungus Trametes versicolor in laboratory jars and a tray bioreactor under solid-state fermentation (SSF) conditions. During SSF, the activity of lignolytic (laccase and manganese peroxidase) and hydrolytic (xylanase, cellulase, β-glucosidase, and invertase) enzymes was measured, with the activities of laccase (2.66 U/gdb in jars and 0.96 U/gdb in the bioreactor) and xylanase (346.04 U/gdb in jars and 200.65 U/gdb in the bioreactor) being the highest. The effect of the complex enzyme system was reflected in the changes in the chemical composition of GP with increasing ash, crude protein, and free fat content: 28%, 10%, and 17% in the laboratory jars, and 29%, 11%, and 7% in the bioreactor, respectively. In addition, the biological treatment improved the extractability of 13 individual phenolic compounds. Therefore, the applied SSF technique represents an effective strategy to improve the profile of phenolic compounds and the nutritional composition of GP, promoting their valorization and opening the door for potential applications in the food industry and other sectors.

2-Hydroxyisovalerate is a valuable chemical that can be used in the production of biodegradable polyesters. In nature, it was only produced at a very low level by Lactococcus lactis. 2-Ketoisovalerate is an intermediate metabolite of the branched-chain amino acid biosynthesis pathway, and Klebsiella pneumoniae ΔbudAΔldhA (Kp ΔbudAΔldhA) was a 2-ketoisovalerate producing strain. In this research, 2-hydroxyisovalerate was identified as a metabolite of Kp ΔbudAΔldhA, and its synthesis pathway was revealed. It was found that 2-ketoisovalerate and 2-hydroxyisovalerate were produced by Kp ΔbudA and Kp ΔbudAΔldhA, but not by Kp ΔbudAΔldhAΔilvD in which the 2-ketoisovalerate synthesis was blocked. budA, ldhA, and ilvD encode α-acetolactate decarboxylase, lactate dehydrogenase, and dihydroxy acid dehydratase, respectively. Thus, it was deduced that 2-hydroxyisovalerate was synthesized from 2-ketoisovalerate. Isoenzymes of ketopantoate reductase PanE, PanE2, and IlvC were suspected of being responsible for this reaction. Kinetic parameters of these enzymes were detected, and they all hold the 2-ketoisovalerate reductase activities. PanE and PanE2 use both NADH and NADPH as co-factors. While IlvC only uses NADH as a co-factor. Over-expression of panE, panE2, or ilvC in Kp ΔbudAΔldhA all enhanced the production of 2-hydroxyisovalerate. Accordingly, 2-hydroxyisovalerate levels were reduced by knocking out panE or panE2. In fed-batch fermentation, 14.41 g/L of 2-hydroxyisovalerate was produced by Kp ΔbudAΔldhA-panE, with a substrate conversion ratio of 0.13 g/g glucose.

Caragana korshinskii kom. (CKK) waste, a common forestry byproduct in northwest of China, presents challenges in its transformation into alternative ruminant feed due to its initial nutritional limitations and unappealing palatability. Conventional strategies, such as ensiling and fungal-based solid-state fermentation (SSF) cannot effectively address this issue in practice. Herein, a two-stage bioaugmentation (TBA) process was devised, leveraging the benefits of ensiling and SSF. During the anaerobic ensiling phase, CKK waste was inoculated with Lactiplantibacillus plantarum LP1, effectively suppressing potential animal pathogens such as Aspergillus and Nocardiopsis while enriching the material with potential probiotics like Pediococcus and Lactiplantibacillus, reaching an abundance of 95.7%. In the subsequent aerobic SSF stage, the ensiled CKK underwent inoculation with the white-rot fungus Irpex lacteus F17, which became enriched to 87.9%. Comprehensive multi-omics analysis identified Irpex as the key taxon, possessing an extensive redox enzyme system that led to the improvement in nutrient composition, reduction of astringent phenolic substances, and mitigation of mycotoxins. As a result, the crude protein content of the CKK increased by 39.2%, while lignin, total phenolic substances, and tannic acid content decreased by 24.4%, 52.2%, and 51.4%, respectively. The mycotoxin levels, including aflatoxin B1, zearalenone, and vomitoxin, were rendered negligible, confirming the safety. Overall, this study demonstrates the TBA strategy can successfully transform challenging and unpalatable CKK waste into a nutrient-enriched and safe mycelium-based bioproduct, thereby enabling the valorization of a previously underutilized forestry resource as a promising alternative feed.

In this study, the hydrothermal co-carbonization (co-HTC) of residues from the vegetable oil industry (pumpkin oil cake - PC, hemp oil cake - HC) and sewage sludge (SS) was investigated for the first time. The co-HTC was performed at 250 degrees C and a treatment time of 5 h. The effects of the mass ratio of the feedstocks (1:1, 1:3 and 3:1) on the properties of the HTC products were investigated using various analytical methods (NMR, XRD, 3D-EEM, FTIR, etc.). The co-HTC of SS with oil cakes resulted in improved fuel properties of the hydrochar and an increase in C content from 36.9 to 53.7 wt%, and an increase in the higher heating value (HHV) from 14.8 to 23.6 MJ/kg. The combination with HC gave hydrochars with a higher HHV and higher C content than the combination with PC. The hydrochar yield varied in the range of 39.4-55.3 wt%. NMR analysis revealed a higher proportion of aliphatic ( 60 %) than aromatic compounds ( 35 %) in the hydrochars, as well as a high content of orthophosphate and unsaturated fatty acids. The liquid fractions were rich in nutrients and organic compounds, but toxic to aquatic organisms. The hydrochars and liquid fractions performed well in the germination test with plant species.

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.

Lignocellulose biomass, as a renewable and biodegradable carbon source, provides a wide range of valuable bioproducts. Their utilization requires an efficient conversion process to break down the complex and variable chemical structure of lignocellulose. In this work, a solid-state fermentation-based pretreatment method for the release of fermentable sugars from corn silage was investigated. The optimal process conditions for water-soluble sugar extraction were initially explored by response surface methodology with the aim to achieve the maximum sugar concentration in the extracts. The optimal extraction conditions were determined: t = 30 min; the liquid-solid ratio L/S = 25 mL g–1; n = 170 rpm; and T = 30 °C. The changes in the content of water-soluble sugars (glucose, fructose, sucrose, maltose, maltotriose) were analyzed during seven days of fungal treatment.

Natural deep eutectic solvents (NADES) have been recognised as a promising alternative to conventional aqueous media in biodiesel production because of their dual function, as lipase extractants and transesterification en-hancers. To use lipase in NADES, lyophilized lipase must be dissolved in NADES or lipase must be extracted from liquid lipase formulations in NADES. The extraction processes, although efficient, is time consuming because it is highly limited by mass transfer. To enhance the extraction process and shift it from batch to continuous, the aqueous two-phase extraction (ATPS) based on NADES was tested as a possible tool for continuous extraction of lipase from raw liquid formulation to NADES in a microextractor. The fast screening of NADES was performed in a batch extractor and betaine-urea NADES-based was selected for further research. The process was then transferred to a microextractor where the influence of residence time, ultrasound, temperature and channel diameter were studied. Room temperature (T = 25 degrees C) and 1000 mu m channel diameter were optimal for continuous microextraction. The extraction process was significantly intensified in a microextractor. The lipase extraction efficiency obtained in a batch reactor was 94.70% for 30 min, while an extraction efficiency of 98.50% was achieved for a residence time of 30 s in a microextractor. Finally, the extracted lipase was used for biodiesel synthesis in a batch reactor, obtaining a biodiesel yield of 96.11%. The reusability of the extracted lipase was also investigated, and the enzyme was successfully reused in three cycles, after which the biodiesel yield decreased.

Spent brewer's yeast (SBY) is a byproduct of the brewing industry traditionally used as a feed additive, although it could have much broader applications. In this paper, a comprehensive review of valorization of SBY for the production of high-value products, new materials, and biofuels, as well as environmental application, is presented. An economic perspective is given by mirroring marketing of conventional SBY with innovative high-value products. Cascading utilization of fine chemicals, biofuels, and nutrients such as proteins, carbohydrates, and lipids released by various SBY treatments has been proposed as a means to maximize the sustainable and circular economy.

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.

Lignocellulosic biorefineries play an important role in the development of a circular and sustainable bioeconomy worldwide. Although a wide variety of different value-added products and bioenergy can be derived from lignocellulosic materials, the key Research Topic is to find the methods that are economically sustainable. In order to gain new knowledge in the field of lignocellulosic biorefineries, especially in terms of sustainable production of bioactive compounds, we have been working on the Research Topic “Sustainable Production of Bioactive Compounds within Lignocellulosic Biorefineries”. However, the Research Topic not only includes bioactive compounds, but also findings on the area of biomaterials. It includes five articles (one review paper, one mini-review and three original research articles) written by scientists from Brazil, China, Croatia, Czech Republic, Germany, Greece, and Slovenia. We hereby express our sincere gratitude for the efforts and contributions of all those involved in this Research Topic. Two papers present possibilities to convert brewer’s spent grain and sugarcane bagasse into a variety of bioactive compounds. The first paper (Zeko-Pivač et al.) is a review paper which provides a comprehensive overview of the temporary use of brewer’s grains and future goals for its better exploitation in the production of value-added products, targeting the Central and Eastern European markets. It includes market size and growth rate of selected value-added products, namely lactic acid, ferulic acid; 2,3-butanediol, gibberellic acid, xylitol, citric acid, PHB, natural red pigment, ascorbic acid and cordycepin. The second paper is an original research paper by authors from Chile, which presents information on the production of a wide range of bioproducts (such as p-hydroxycinnamates and lignin or lignin-carbohydrate complexes) from sugarcane bagasse enzymatically treated with endoxylanase. In regards to biomaterials, two papers deal with the production of the new generation of biomaterials. A mini-review paper by scientists from China (Lai et al.) looks at enzymatically catalyzed reactions to produce polymer hydrogels, which are expected to be the next-generation of biomaterials for tissue engineering and regenerative medicine. The possibility of producing elastin-like proteins as renewable biobased high-performance polymers with intriguing mechanical properties was explored in another research paper from scientists from Germany (Haas et al.) OPEN ACCESS