Perylenequinones (PQs) from bambusicolous Shiraia fungi serve as excellent photosensitizers for photodynamic therapy. However, the lower yield of PQ production in mycelium cultures is an important bottleneck for their clinical application. Light has long been recognized as a pivotal regulatory signal for fungal secondary metabolite biosynthesis. In this study, we explored the role of nitric oxide (NO) in the growth and PQ biosynthesis in mycelium cultures of Shiraia sp. S9 exposed to red light. The continuous irradiation with red light (627 nm, 200 lx) suppressed fungal conidiation, promoted hyphal branching, and elicited a notable increase in PQ accumulation. Red light exposure induced NO generation, peaking to 81.7 μmol/g FW on day 8 of the culture, with the involvement of nitric oxide synthase (NOS)- or nitrate reductase (NR)-dependent pathways. The application of a NO donor sodium nitroprusside (SNP) restored conidiation of Shiraia sp. S9 under red light and stimulated PQ production, which was mitigated upon the introduction of NO scavenger carboxy-PTIO or soluble guanylate cyclase inhibitor NS-2028. These results showed that red light-induced NO, as a signaling molecule, was involved in the regulation of growth and PQ production in Shiraia sp. S9 through the NO-cGMP-PKG signaling pathway. While mycelial H₂O₂ content exhibited no significant alternations, a transient increase of intracellular Ca²⁺ and extracellular ATP (eATP) content was detected upon exposure to red light. The generation of NO was found to be interdependent on cytosolic Ca²⁺ and eATP concentration. These signal molecules cooperated synergistically to enhance membrane permeability and elevate the transcript levels of PQ biosynthetic genes in Shiraia sp. S9. Notably, the combined treatment of red light with 5 μM SNP yielded a synergistic effect, resulting in a substantially higher level of hypocrellin A (HA, 254 mg/L), about 3.0-fold over the dark control. Our findings provide valuable insights into the regulation of NO on fungal secondary metabolite biosynthesis and present a promising strategy involving the combined elicitation with SNP for enhanced production of photoactive PQs and other valuable secondary metabolites in fungi.

Development of nano-enabled fertilizers from green waste is one of the effective options to enhance global agricultural productions and minimize environmental pollution. In this study, novel, eco-friendly and cost-effective nano- enabled fertilizers (NEF) were synthesized using the planetary ball milling procedure. The NEF (nDPF1and nDPF2) were prepared by impregnation of nanostructured date palm pits (nDPP) with (KH₂PO₄ + MgO) at 1:1 and 3:1 (w/w) ratios respectively. The nDPP, nDPF1 and nDPF2 were extensively characterized. The produced nano-fertilizers enhanced soil water retention capacity with nDPF2 being the most effective. The water retention capacity of nDPF2 treated soil was 5.6 times higher than that of soil treated with conventional fertilizers. In addition, the nDPF2 exhibited superior sustained lower release rates of P, K and Mg nutrients for longer release periods in comparison with the conventional fertilizers. For instance, P cumulative release percentages from conventional fertilizers, nDPF1 and nDPF2 in soil reached 22.41%, 10.82 and 8.9% respectively within 384 h. Findings from FTIR and XPS analyses suggested that hydrogen bonding and ligand exchange were the main interaction mechanisms of PO₄-K-Mg ions with nDPP surface. The released kinetics data of the NEF revealed that power function was the best suitable model to describe the kinetics of P, K and Mg release data from NEF in water and soil. Pot study ascertained that the nano-enabled fertilizers (nDPF1 and nDPF2) significantly promoted biomass production and nutrient uptake of maize plants as compared to commercial fertilizer treated plants. The present work demonstrated the potential of NEF to increase nutrients uptake efficiency, mitigate moisture retention problem in arid soils and reduce nutrients loss through leaching and safeguard the environment.

Lignocellulose pretreated using pyrolysis can yield clean energy (such as bioethanol) via microbial fermentation, which can significantly contribute to waste recycling, environmental protection, and energy security. However, the acids, aldehydes, and phenols present in bio-oil with inhibitory effects on microorganisms compromise the downstream utilization and conversion of lignocellulosic pyrolysates. In this study, we constructed a microbial electrolysis cell system for bio-oil detoxification and efficient ethanol production using evolved Escherichia coli to overcome the bioethanol production and utilization challenges highlighted in previous studies. In electrically treated bio-oil media, the E. coli-H strain exhibited significantly higher levoglucosan consumption and ethanol production capacities compared with the control. In undetoxified bio-oil media containing 1.0% (w/v) levoglucosan, E. coli-H produced 0.54 g ethanol/g levoglucosan, reaching 94% of the theoretical yield. Our findings will contribute to developing a practical method for bioethanol production from lignocellulosic substrates, and provide a scientific basis and technical demonstration for its industrialized application.

Cytidine triphosphate (CTP), as a substance involved in the metabolism of phospholipids, proteins and nucleic acids, has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline. In this study, we established an in vitro six-enzyme cascade system to generate CTP. To avoid thermodynamic bottlenecks, we employed a circuitous and two-stage reaction strategy. Using cytidine as the key substrate, the final product CTP is obtained via the deamination and uridine phosphorylation pathways, relying on the irreversible reaction of cytidine triphosphate synthase to catalyze the amination of uridine triphosphate. Several extremophilic microbial-derived deaminases were screened and characterized, and a suitable cytidine deaminase was selected to match the first-stage reaction conditions. In addition, directed evolution modification of the rate-limiting enzyme CTP synthetase in the pathway yielded a variant that successfully relieved the product feedback inhibition, along with a 1.7-fold increase in activity over the wild type. After optimizing the reaction conditions, we finally carried out the catalytic reaction at an initial cytidine concentration of 20 mM, and the yield of CTP exceeded 82% within 10.0 h.

The Thermomyces lanuginosus lipase (TLLs) was successfully immobilized within a novel hydrogel matrix through a two-step crosslinking method. TLLs were initially crosslinked through the Schiff base reaction by oxidized carboxymethyl cellulose (OCMC). The water-soluble OCMC@TLLs complex was subsequently crosslinked by carboxymethyl chitosan (CMCSH) in a microfluidic apparatus to form the CMCHS/OCMC@TLLs microspheres. The CD (Circular Dichroism, CD) and FT-IR (Fourier Transform infrared spectroscopy, FT-IR) spectra demonstrated that the crosslinking of TLLs with OCMC resulted in a less significant impact on their structure compared to that with glutaraldehyde. CMCHS/OCMC@TLLs showed decreased catalytic performance due to the mass transfer resistance, while its thermal stability was greatly improved. The CMCHS/OCMC@TLLs were used to catalyze the lauroylation of arbutin in tetrahydrofuran. After 12 h of reaction under optimal conditions, the yield of 6′-O-lauryl arbutin reached an impressive 92.12%. The prepared 6′-O-lauryl arbutin has high lipophilicity and exhibits similar tyrosinase inhibitory activity and higher antioxidant activity compared to its parent compound.

Generally wastewater such agricultural runoff is considered a nuisance; however, it could be harnessed as a potential source of nutrients like nitrates and phosphates in integrated biorefinery context. In the current study, microalgae Chlorella sp. S5 was used for bioremediation of agricultural runoff and the leftover algal biomass was used as a potential source for production of biofuels in an integrated biorefinery context. The microalgae Chlorella sp. S5 was cultivated on Blue Green (BG 11) medium and a comprehensive optimization of different parameters including phosphates, nitrates, and pH was carried out to acquire maximum algal biomass enriched with high lipids content. Dry biomass was quantified using the solvent extraction technique, while the identification of nitrates and phosphates in agricultural runoff was carried out using commercial kits. The algal extracted lipids (oils) were employed in enzymatic trans-esterification for biodiesel production using whole-cell biomass of Bacillus subtilis Q4 MZ841642. The resultant fatty acid methyl esters (FAMEs) were analyzed using Fourier transform infrared (FTIR) spectroscopy and gas chromatography coupled with mass spectrometry (GC–MS). Subsequently, both the intact algal biomass and its lipid-depleted algal biomass were used for biogas production within a batch anaerobic digestion setup. Interestingly, Chlorella sp. S5 demonstrated a substantial reduction of 95% in nitrate and 91% in phosphate from agricultural runoff. The biodiesel derived from algal biomass exhibited a noteworthy total FAME content of 98.2%, meeting the quality standards set by American Society for Testing and Materials (ASTM) and European union (EU) standards. Furthermore, the biomethane yields obtained from whole biomass and lipid-depleted biomass were 330.34 NmL/g VS_added and 364.34 NmL/g VS_added, respectively. In conclusion, the findings underscore the potent utility of Chlorella sp. S5 as a multi-faceted resource, proficiently employed in a sequential cascade for treating agricultural runoff, producing biodiesel, and generating biogas within the integrated biorefinery concept.

The bioconversion of 4-hydroxy-2-keto acid derivatives via aldol condensation of formaldehyde and pyruvate has received substantial attention as potential source of chemicals for production of amino acids, hydroxy carboxylic acids, and chiral aldehydes. We developed an environmentally friendly biocatalyst consisting of a novel thermostable class II pyruvate aldolase from Deinococcus radiodurans with maltose-binding protein (MBP-DrADL), which has specific activity of 46.3 µmol min^–1 mg^–1. Surprisingly, MBP-DrADL maintained over 60% of enzyme activity for 4 days at 50 to 65 °C, we used MBP-DrADL as the best candidate enzyme to produce 2-keto-4-hydroxybutyrate (2-KHB) from formaldehyde and pyruvate via aldol condensation. The optimum reaction conditions for 2-KHB production were 50 °C, pH 8.0, 5 mM Mg²⁺, 100 mM formaldehyde, and 200 mM pyruvate. Under these optimized conditions, MBP-DrADL produced 76.5 mM (8.94 g L^–1) 2-KHB over 60 min with a volumetric productivity of 8.94 g L^–1 h^–1 and a specific productivity of 357.6 mg mg-enzyme^–1 h^–1. Furthermore, 2-KHB production was improved by continuous addition of substrates, which produced approximately 124.8 mM (14.6 g L^–1) of 2-KHB over 60 min with a volumetric productivity and specific productivity of 14.6 g L^–1 h^–1 and 583.4 mg mg-enzyme^–1 h^–1, respectively. MBP-DrADL showed the highest specific productivity for 2-KHB production yet reported. Our study provides a highly efficient biocatalyst for the synthesis of 2-KHB and lays the foundation for large-scale production and application of high-value compounds from formaldehyde.

This study delves into the aroma characteristics and microbial composition of filler tobacco leaves (FTLs) sourced from six distinct cigar-growing regions within Yunnan, China, following standardized fermentation. An integrated approach using gas chromatography-mass spectrometry (GC–MS), electronic nose (E-nose), and microbiome analysis was employed for comprehensive profiling. Results derived from Linear Discriminant Analysis (LDA) using E-nose data confirmed the presence of notable variability in flavor substance profiles among the FTLs from six regions. Additionally, GC–MS was used to discern disparities in volatile organic compound (VOC) distribution across FTLs from these regions, identifying 92, 81, 79, 58, 69, and 92 VOCs within each respective sample set. Significantly, 24 VOCs emerged as pivotal determinants contributing to the heterogeneity of flavor profiles among FTLs from diverse origins, as indicated by Variable Importance for the Projection (VIP) analysis. Furthermore, distinctions in free amino acid content and chemical constituents were observed across FTLs. Of noteworthy significance, solanone, isophorone, durene, (-)-alpha-terpineol, and 2,3'-bipyridine exhibited the strongest correlations with microbiome data, with fungal microorganisms exerting a more pronounced influence on metabolites, as elucidated through two-way orthogonal partial least-squares (O2PLS) modeling. These findings provide a theoretical and technical basis for accurately evaluating the synchronization of FTLs in aromas and fermentation processes, and they will enhance the quality of fermented FTLs and foster the growth of the domestic cigar tobacco industry ultimately.

The evaluation of plant-based feedstocks is an important aspect of biorefining. Nicotiana glauca is a solanaceous, non-food crop that produces large amounts of biomass and is well adapted to grow in suboptimal conditions. In the present article, compatible sequential solvent extractions were applied to N. glauca leaves to enable the generation of enriched extracts containing higher metabolite content comparing to direct leaf extracts. Typically, between 60 to 100 metabolite components were identified within the fractions. The occurrence of plant fatty acids, fatty acid alcohols, alkanes, sterols and terpenoids was detected by gas liquid chromatography–mass spectrometry (GC–MS) and metabolite identification was confirmed by comparison of physico-chemical properties displayed by available authentic standards. Collectively, co-products such waxes, oils, fermentable sugars, and terpenoids were all identified and quantified. The enriched fractions of N. glauca revealed a high level of readily extractable hydrocarbons, oils and high value co-products. In addition, the saccharification yield and cell wall composition analyses in the stems revealed the potential of the residue material as a promising lignocellulosic substrate for the production of fermentable sugars. In conclusion a multifractional cascade for valuable compounds/commodities has been development, that uses N. glauca biomass. These data have enabled the evaluation of N. glauca material as a potential feedstock for biorefining.

Traditional autoclaving, slow degradation rate and preservation of biomass treated by fungi are the main factors restricting biological treatment. In our previous studies, strains with high efficiency and selective lignin degradation ability were obtained. To further solve the limiting factors of biological treatment, this paper proposed a composite treatment technology, which could replace autoclaves for fungal treatment and improve the preservation and utilization of fungal-pretreated straw. The autoclaved and expanded buckwheat straw were, respectively, degraded by Irpex lacteus for 14 days (CIL, EIL), followed by ensiling of raw materials (CK) and biodegraded straw of CIL and EIL samples with Lactobacillus plantarum for different days, respectively (CP, CIP, EIP). An expansion led to lactic acid bacteria, mold, and yeast of the samples below the detection line, and aerobic bacteria was significantly reduced, indicating a positive sterilization effect. Expansion before I. lacteus significantly enhanced lignin selective degradation by about 6%, and the absolute content of natural detergent solute was about 5% higher than that of the CIL. Moreover, EIL decreased pH by producing higher organic acids. The combination treatment created favorable conditions for ensiling. During ensiling, EIP silage produced high lactic acid about 26.83 g/kg DM and the highest acetic acid about 22.35 g/kg DM, and the pH value could be stable at 4.50. Expansion before I. lacteus optimized the microbial community for ensiling, resulting in EIP silage co-dominated by Lactobacillus, Pediococcus and Weissella, whereas only Lactobacillus was always dominant in CP and CIP silage. Clavispora gradually replaced Irpex in EIP silage, which potentially promoted lactic acid bacteria growth and acetic acid production. In vitro gas production (IVGP) in EIL was increased by 30% relative to CK and was higher than 24% in CIL. The role of expansion was more significant after ensiling, the IVGP in EIP was increased by 22% relative to CP, while that in CIP silage was only increased by 9%. Silage of fungal-treated samples reduced methane emissions by 28% to 31%. The study demonstrated that expansion provides advantages for fungal colonization and delignification, and further improves the microbial community and fermentation quality for silage, enhancing the nutrition and utilization value. This has practical application value for scaling up biological treatment and preserving the fungal-treated lignocellulose.

Tobacco polysaccharides were extracted by hot water extraction, and purified and separated using DEAE-52 cellulose chromatography columns, and three purified polysaccharide fractions, YCT-1, YCT-2, and YCT-3, were finally obtained. The physicochemical properties of the three fractions were analyzed by ultraviolet spectroscopy, high-performance liquid chromatography and high-performance gel chromatography. The in vitro antioxidant activity of tobacco polysaccharides was compared among different fractions by using DPPH radical, hydroxyl radical scavenging assay and potassium ferricyanide method. The in vitro hypoglycemic activity was compared using α-amylase and α-glucosidase activity inhibition assay. And the in vitro hypolipidemic activity were investigated by using pancreatic lipase activity inhibition assay and HepG-2 intracellular lipid accumulation assay. All the results showed that the constituent monosaccharides of the three tobacco polysaccharide fractions were similar, but the molar percentages of each monosaccharide were different. The average molecular weights of the three components were 27,727 Da, 27,587 Da, and 66,517 Da, respectively, and the scavenging activities on DPPH radicals and hydroxyl radicals were at a high level with good quantitative-effect relationships. The reducing power were much lower than that of the positive control VC, and the three polysaccharide fractions had a weak inhibitory ability on α-amylase activity, but showed excellent inhibitory ability on α-glucosidase and pancreatic lipase activity. In addition, the results of cellular experiments showed that all three fractions were able to inhibit lipid over-accumulation in HepG-2 cells by increasing the mRNA expression levels of PPAR-α, CPT-1A, and CYP7A1 genes, and the tobacco polysaccharide YCT-3 showed the best effect. The mechanism by which YCT-3 ameliorated the over-accumulation of intracellular lipids in HepG-2 cells was found to be related to its influence on the expression of miR-155-3p and miR-17-3p in the exosomes of HepG-2 cells.

Cell immobilization plays an important role in biocatalysis for high-value products. It is necessary to maintain the viability of immobilized cells for bioconversion using viable cells as biocatalysts. In this study, a novel polyester nonwoven chemostat was designed for cell immobilization to investigate biofilm formation and the dynamic balance between adsorption and desorption of cells on polyester nonwoven. The polyester nonwoven was suitable for cell immobilization, and the cell numbers on the polyester nonwoven can reach 6.5 ± 0.38 log CFU/mL. After adding the polyester nonwoven to the chemostat, the fluctuation phenomenon of free bacterial cells occurred. The reason for this phenomenon was the balance between adsorption and desorption of bacterial cells on the polyester nonwoven. Bacterial cells could adhere to the surface of polyester nonwoven via secreting extracellular polymeric substances (EPS) to form biofilms. As the maturation of biofilms, some dead cells inside the biofilms can cause the detachment of biofilms. This process of continuous adsorption and desorption of cells can ensure that the polyester nonwoven chemostat has lasting biological activity.

This comprehensive review systematically examines the multifarious aspects of Nelumbo nucifera, elucidating its ecological, nutritional, medicinal, and biomimetic significance. Renowned both culturally and scientifically, Nelumbo nucifera manifests remarkable adaptability, characterized by its extensive distribution across varied climatic regions, underpinned by its robust rhizome system and prolific reproductive strategies. Ecologically, this species plays a crucial role in aquatic ecosystems, primarily through biofiltration, thereby enhancing habitat biodiversity. The rhizomes and seeds of Nelumbo nucifera are nutritionally significant, being rich sources of dietary fiber, essential vitamins, and minerals, and have found extensive culinary applications. From a medicinal perspective, diverse constituents of Nelumbo nucifera exhibit therapeutic potential, including anti-inflammatory, antioxidant, and anti-cancer properties. Recent advancements in preservation technology and culinary innovation have further underscored its role in the food industry, highlighting its nutritional versatility. In biomimetics, the unique "lotus effect" is leveraged for the development of self-cleaning materials. Additionally, the transformation of Nelumbo nucifera into biochar is being explored for its potential in sustainable environmental practices. This review emphasizes the critical need for targeted conservation strategies to protect Nelumbo nucifera against the threats posed by climate change and habitat loss, advocating for its sustainable utilization as a species of significant value.

Biliverdin, a bile pigment hydrolyzed from heme by heme oxygenase (HO), serves multiple functions in the human body, including antioxidant, anti-inflammatory, and immune response inhibitory activities. Biliverdin has great potential as a clinical drug; however, no economic and efficient production method is available currently. Therefore, the production of biliverdin by the biotransformation of exogenous heme using recombinant HO-expressing yeast cells was studied in this research. First, the heme oxygenase-1 gene (HO1) encoding the inducible plastidic isozyme from Arabidopsis thaliana, with the plastid transport peptide sequence removed, was recombined into Pichia pastoris GS115 cells. This resulted in the construction of a recombinant P. pastoris GS115-HO1 strain that expressed active HO1 in the cytoplasm. After that, the concentration of the inducer methanol, the induction culture time, the pH of the medium, and the concentration of sorbitol supplied in the medium were optimized, resulting in a significant improvement in the yield of HO1. Subsequently, the whole cells of GS115-HO1 were employed as catalysts to convert heme chloride (hemin) into biliverdin. The results showed that the yield of biliverdin was 132 mg/L when hemin was added to the culture of GS115-HO1 and incubated for 4 h at 30 °C. The findings of this study have laid a good foundation for future applications of this method for the economical production of biliverdin.

Chitooligosaccharides (COS) find numerous applications due to their exceptional properties. Enzymatic hydrolysis of chitosan by chitosanase is considered an advantageous route for COS production. Heterologous expression of chitosanase holds significant promise, yet studies using commonly employed Escherichia coli and Pichia pastoris strains encounter challenges in subsequent handling and industrial scalability. In this investigation, we opted for using the safe yeast strain Saccharomyces cerevisiae (GRAS), obviating the need for methanol induction, resulting in successful expression. Ultimately, utilizing the GTR-CRISPR editing system, shake flask enzyme activity reached 2 U/ml. The optimal chitosanase activity was achieved at 55℃ and pH 5, with favorable stability between 30 and 50 °C. Following a 2-h catalytic reaction, the product primarily consisted of chitobiose to chitotetraose, predominantly at the chitotriose position, with a slight increase in chitobiose content observed during the later stages of enzymatic hydrolysis. The results affirm the feasibility of heterologous chitosanase expression through Saccharomyces cerevisiae, underscoring its significant industrial potential.

Osteoarthritis (OA) of the knee is a common degenerative articular disorder and is one of the main causes of pain and functional disability. Cartilage damage is frequently linked to elevated osteoarthritis incidence. Supercritical carbon dioxide (scCO₂) decellularized cartilage graft produced from the porcine cartilage is an ideal candidate for cartilage tissue engineering. In the present study, we derived collagen type II (Col II) solution from the scCO₂ decellularized porcine cartilage graft (dPCG) and compared its efficacy with hyaluronic acid (HA) in the surgical medial meniscectomy (MNX) induced post-traumatic osteoarthritis (PTOA) model. Dose-dependent attenuation of the OA (12.3 ± 0.8) progression was observed in the intra‐articular administration of Col II solution (7.3 ± 1.2) which significantly decreased the MNX-induced OA symptoms similar to HA. The pain of the OA group (37.4 ± 2.7) was attenuated dose-dependently by Col II solution (45.9 ± 4.1) similar to HA (43.1 ± 3.5) as evaluated by a capacitance meter. Micro‐CT depicted a dose-dependent attenuation of articular cartilage damage by the Col II solution similar to HA treatment. A significant (p < 0.001) dose-dependent elevation in the bone volume was also observed in Col II solution-treated OA animals. The protective competence of Col II solution on articular cartilage damage is due to its significant (p < 0.001) increase in the expression of type II collagen, aggrecan and SOX‐9 similar to HA. To conclude, intra‐articular administration of type II collagen solution and HA reestablished the injured cartilage and decreased osteoarthritis progression in the experimental PTOA model.

Hydrolysis at changing hydraulic retention time, recirculation, bedding straw content in the feed, bioaugmentation and the impact of those changes on gradient formation in the liquid phase in plug-flow reactors (PFRs) was examined. The pH-value, conductivity and oxidation–reduction potential (ORP) were monitored at three spots along the PFRs to study potential correlations to process performance during a total process time of 123 weeks. The on-line monitoring showed good correlations to acidogenesis: namely, the pH and ORP to the acidification, to butyric (and lactic) acid concentration and to the acid yield. The ORP (measured at the inlet) showed the most stable correlation to acidogenesis under dynamic operation, while the conductivity (at the outlet) correlated to the acid concentration in dependence on the feedstock. Multiple measurement spots as used in this study allow to gain more information about acidogenic fermentation than a single spot, simplifying process control and automation attempts with recalcitrant feedstock.

Butyric acid is a volatile saturated monocarboxylic acid, which is widely used in the chemical, food, pharmaceutical, energy, and animal feed industries. This study focuses on producing butyric acid from pre-treated rape straw using simultaneous enzymatic hydrolysis semi-solid fermentation (SEHSF). Clostridium beijerinckii BRM001 screened from pit mud of Chinese nongxiangxing baijiu was used. The genome of C. beijerinckii BRM001 was sequenced and annotated. Using rape straw as the sole carbon source, fermentation optimization was carried out based on the genomic analysis of BRM001. The optimized butyric acid yield was as high as 13.86 ± 0.77 g/L, which was 2.1 times higher than that of the initial screening. Furthermore, under optimal conditions, non-sterile SEHSF was carried out, and the yield of butyric acid was 13.42 ± 0.83 g/L in a 2.5-L fermentor. This study provides a new approach for butyric acid production which eliminates the need for detoxification of straw hydrolysate and makes full use of the value of fermentation waste residue without secondary pollution, making the whole process greener and more economical, which has a certain industrial potential.

Monoclonal antibodies (mAbs) require a high level of purity for regulatory approval and safe administration. High-molecular weight (HMW) species are a common impurity associated with mAb therapies. Hydrophobic interaction chromatography (HIC) resins are often used to remove these HMW impurities. Determination of a suitable HIC resin can be a time and resource-intensive process. In this study, we modeled the chromatographic behavior of seven mAbs across 13 HIC resins using measurements of surface hydrophobicity, surface charge, and thermal stability for mAbs, and hydrophobicity and zeta-potential for HIC resins with high fit quality (adjusted R ² > 0.80). We identified zeta-potential as a novel key modeling parameter. When using these models to select a HIC resin for HMW clearance of a test mAb, we were able to achieve 60% HMW clearance and 89% recovery. These models can be used to expedite the downstream process development for mAbs in an industry setting.

The use of enzymes to catalyze Henry reaction has advantages of mild reaction conditions and low contamination, but low enzyme activity of promiscuous catalysis limits its application. Here, rational design was first performed to identify the key amino acid residues in Henry reaction catalyzed by Lactococcal multidrug resistance Regulator (LmrR). Further, non-canonical amino acids were introduced into LmrR, successfully obtaining variants that enhanced the catalytic activity of LmrR. The best variant, V15CNF, showed a 184% increase in enzyme activity compared to the wild type, and was 1.92 times more effective than the optimal natural amino acid variant, V15F. Additionally, this variant had a broad substrate spectrum, capable of catalyzing reactions between various aromatic aldehydes and nitromethane, with product yielded ranging from 55 to 99%. This study improved enzymatic catalytic activity by enhancing affinity between the enzyme and substrates, while breaking limited types of natural amino acid residues by introducing non-canonical amino acids into the enzyme, providing strategies for molecular modifications.

In this work, the properties of biochar produced from green macroalga Ulva intestinalis by pyrolysis were studied at temperatures of 300, 500, and 700 °C. This biochar was characterized in terms of multielemental composition, BET surface area, total pore volume, and biosorption properties toward phosphate ions. Biochar produced at 700 °C–25 m²/g had the highest surface area. The kinetics and isotherms of sorption processes of phosphate ions as sorbate by these sorbents were investigated. Modified biochar was able to remove 84.3% of phosphate ions from wastewater, whereas non-modified biochar—only 40.6%. Hence, biochar enriched with phosphate ions can serve as a valuable soil amendment. Pot experiments performed on winter wheat (Triticum aestivum) with a 3% addition of dry Ulva intestinalis, pristine biochar, and Mg-modified biochar enriched with phosphate ions showed that these amendments stimulated plant growth (length and fresh weight of plants) as well as enlarging the chlorophyll content in leaves. Our results indicate that the production of biochar (pristine and Mg-impregnated) is a sustainable option to valorize the biomass of seaweeds, and to recycle phosphorus from wastewater.

The endophytic fungus Aspergillus sp. SPH2 was isolated from the stems of the endemic plant Bethencourtia palmensis and its extracts were found to have strong fungicidal effects against Botrytis cinerea and ixodicidal effects against Hyalomma lusitanicum at different fermentation times. In this study, the fungus was grown using three different culture media and two methodologies, Microparticulate Enhancement Cultivation (MPEC) and Semi-Solid-State Fermentation (Semi-SSF), to increase the production of secondary metabolites during submerged fermentation. The addition of an inert support to the culture medium (Semi-SSF) resulted in a significant increase in the extract production. However, when talcum powder was added to different culture media, unexpected results were observed, with a decrease in the production of the biocompounds of interest. Metabolomic analyses showed that the production of aspergillic, neoaspergillic, and neohydroxyaspergillic acids peaked in the first few days of fermentation, with notable differences observed among the methodologies and culture media. Mellein production was particularly affected by the addition of an inert support to the culture medium. These results highlight the importance of surface properties and morphology of spores and mycelia during fermentation by this fungal species.

Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides, which are advantageous for plant immunity, animal nutrition and health. However, thermophilic endo-chitinases are scarce and the transformation from exo- to endo-activity of chitinases is still a challenging problem. In this study, to enhance the endo-activity of the thermophilic chitinase Chi304, we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses. Four effective single-point mutants were identified among 28 designed mutations. The ratio of (GlcNAc)₃ to (GlcNAc)₂ quantity (DP_3/2) in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89, 1.65, 1.24, and 1.38 times that of Chi304, respectively. When combining to double-point mutants, the DP_3/2 proportions produced by F79A/W140R, F79A/M264L, F79A/W272R, and M264L/W272R were 2.06, 1.67, 1.82, and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity. When applied to produce chitooligosaccharides (DP ≥ 3), F79A/W140R accumulated the most (GlcNAc)₄, while M264L/W272R was the best to produce (GlcNAc)₃, which was 2.28 times that of Chi304. The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate. Overall, this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides.

Almond pruning biomass is an important agricultural residue that has been scarcely studied for the co-production of sugars and solid biofuels. In this work, the production of monosaccharides from almond prunings was optimised by a two-step process scheme: pretreatment with dilute sulphuric acid (0.025 M, at 185.9–214.1 ℃ for 0.8–9.2 min) followed by enzyme saccharification of the pretreated cellulose. The application of a response surface methodology enabled the mathematical modelling of the process, establishing pretreatment conditions to maximise both the amount of sugar in the acid prehydrolysate (23.4 kg/100 kg raw material, at 195.7 ℃ for 3.5 min) and the enzymatic digestibility of the pretreated cellulose (45.4%, at 210.0 ℃ for 8.0 min). The highest overall sugar yield (36.8 kg/100 kg raw material, equivalent to 64.3% of all sugars in the feedstock) was obtained with a pretreatment carried out at 197.0 ℃ for 4.0 min. Under these conditions, moreover, the final solids showed better properties for thermochemical utilisation (22.0 MJ/kg heating value, 0.87% ash content, and 72.1 mg/g moisture adsorption capacity) compared to those of the original prunings.

GABA (Gamma-aminobutyric acid), a crucial neurotransmitter in the central nervous system, has gained significant attention in recent years due to its extensive benefits for human health. The review focused on recent advances in the biosynthesis and production of GABA. To begin with, the investigation evaluates GABA-producing strains and metabolic pathways, focusing on microbial sources such as Lactic Acid Bacteria, Escherichia coli, and Corynebacterium glutamicum. The metabolic pathways of GABA are elaborated upon, including the GABA shunt and critical enzymes involved in its synthesis. Next, strategies to enhance microbial GABA production are discussed, including optimization of fermentation factors, different fermentation methods such as co-culture strategy and two-step fermentation, and modification of the GABA metabolic pathway. The review also explores methods for determining glutamate (Glu) and GABA levels, emphasizing the importance of accurate quantification. Furthermore, a comprehensive market analysis and prospects are provided, highlighting current trends, potential applications, and challenges in the GABA industry. Overall, this review serves as a valuable resource for researchers and industrialists working on GABA advancements, focusing on its efficient synthesis processes and various applications, and providing novel ideas and approaches to improve GABA yield and quality.

Unspecific peroxygenases (UPOs) are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide (H₂O₂) as the oxygen donor. However, their poor heterologous expression has hindered their practical application. Here, a novel UPO from Marasmius fiardii PR910 (MfiUPO) was identified and heterologously expressed in Pichia pastoris. By employing a two-copy expression cassette, the protein titer reached 1.18 g L⁻¹ in a 5 L bioreactor, marking the highest record. The glycoprotein rMfiUPO exhibited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation, epoxidation and alcohol oxidation. Moreover, the peroxidative activity was enhanced by 150% after exposure to 50% (v/v) acetone for 40 h. A semi-preparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2% isolated yield with 95% purity. With its high expression level, rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution, thereby meeting the necessary criteria for practical application.

Escherichia coli MLB (MG1655 ΔpflB ΔldhA), which can hardly grow on glucose with little succinate accumulation under anaerobic conditions. Two-stage fermentation is a fermentation in which the first stage is used for cell growth and the second stage is used for product production. The ability of glucose consumption and succinate production of MLB under anaerobic conditions can be improved significantly by using acetate as the solo carbon source under aerobic condition during the two-stage fermentation. Then, the adaptive laboratory evolution (ALE) of growing on acetate was applied here. We assumed that the activities of succinate production related enzymes might be further improved in this study. E. coli MLB46-05 evolved from MLB and it had an improved growth phenotype on acetate. Interestingly, in MLB46-05, the yield and tolerance of succinic acid in the anaerobic condition of two-stage fermentation were improved significantly. According to transcriptome analysis, upregulation of the glyoxylate cycle and the activity of stress regulatory factors are the possible reasons for the elevated yield. And the increased tolerance to acetate made it more tolerant to high concentrations of glucose and succinate. Finally, strain MLB46-05 produced 111 g/L of succinic acid with a product yield of 0.74 g/g glucose.

Cell separation using microfluidics has become an effective method to isolate biological contaminants from bodily fluids and cell cultures, such as isolating bacteria contaminants from microalgae cultures and isolating bacteria contaminants from white blood cells. In this study, bacterial cells were used as a model contaminant in microalgae culture in a passive microfluidics device, which relies on hydrodynamic forces to demonstrate the separation of microalgae from bacteria contaminants in U and W-shaped cross-section spiral microchannel fabricated by defocusing CO₂ laser ablation. At a flow rate of 0.7 ml/min in the presence of glycine as bacteria chemoattractant, the spiral microfluidics devices with U and W-shaped cross-sections were able to isolate microalgae (Desmodesmus sp.) from bacteria (E. coli) with a high separation efficiency of 92% and 96% respectively. At the same flow rate, in the absence of glycine, the separation efficiency of microalgae for U- and W-shaped cross-sections was 91% and 96%, respectively. It was found that the spiral microchannel device with a W-shaped cross-section with a barrier in the center of the channel showed significantly higher separation efficiency. Spiral microchannel chips with U- or W-shaped cross-sections were easy to fabricate and exhibited high throughput. With these advantages, these devices could be widely applicable to other cell separation applications, such as separating circulating tumor cells from blood.

Single-cell oils (SCO) produced by oleaginous yeast hold promise as a sustainable alternative for producing nutritionally and pharmaceutically valuable lipids. However, the accumulation of oils varies substantially between yeast spp. Consequently, identifying well-suited producers with a high innate capacity for lipids biosynthesis is paramount. Equally important is optimizing culturing and processing conditions to realize the total lipids production potential of selected strains. The marine Rhodotorula mucilaginosa and Lodderomyces elongisporus yeast were investigated to explore their potential for polyunsaturated fatty acids (PUFAs) production on high glucose media (HGM) using two-stage culture mode. Both strains accumulated > 20% (w/w) of their dry cell weight as lipids when grown on HGM using a two-stage culture system. Both yeast isolates exhibited a maximal lipid/biomass coefficient (Y_L/X) of 0.58–0.66 mg/mg at 7 °C and 0.49–0.53 mg/mg at 26 °C when grown on 8% glucose and produced monounsaturated and PUFAs similar to that of Menhaden and Salmon marine oils. For the first time, significant amounts of Eicosapentaenoic acid (19%) and Eicosadienoic acid (19.6%) were produced by L. elongisporus and R. mucilaginosa, respectively. Thus, the SCO derived from these wild strains possesses significant potential as a substitute source for the industrial-scale production of long-chain PUFAs, making them a promising contender in the market.

As an alternative to antibiotics in response to antimicrobial-resistant infections, bacteriophages (phages) are garnering renewed interest in recent years. However, the massive preparation of phage is restricted using traditional pathogens as host cells, which incurs additional costs and contamination. In this study, an opportunistic pathogen, Klebsiella pneumoniae used to convert glycerol to 1,3-propanediol (1,3-PDO), was reused to prepare phage after fermentation. The phage infection showed that the fed-batch fermentation broth containing 71.6 g/L 1,3-PDO can be directly used for preparation of phage with a titer of 1 × 10⁸ pfu/mL. Then, the two-step salting-out extraction was adopted to remove most impurities, e.g. acetic acid (93.5%), ethanol (91.5%) and cells (99.4%) at the first step, and obtain 1,3-PDO (56.6%) in the top phase as well as phage (97.4%) in the middle phase at the second step. This integrated process provides a cheap and environment-friendly manner for coproduction of 1,3-PDO and phage.

In this work, a beneficial approach for efficient depolymerization of lignin and controllable product distribution is provided. Lignin, an abundant aromatic biopolymer, has the potential to produce various biofuels and chemical adsorption agents and is expected to benefit the future circular economy. Microwave-ultrasonic (MW/US) assisted efficient depolymerization of lignin affords some aromatic materials used in manufacturing the starting material to be investigated. Some nano organometallic surfactants (NOMS) based on Ni²⁺, Cu²⁺, Co²⁺, Fe³⁺, and Mn²⁺ besides 2-hydroxynaphth-sulphanilamide are synthesized to enhance oil recovery (EOR). In this work, the assessment of the NOMS’s efficiency was improving the heavy oil recovery via the study of the dynamic interfacial tension (IFT), contact angle, and chemical flooding scenarios. The NOMS-Ni²⁺ exhibited the maximum reduction of viscosity and yield values. Dropping the viscosity to 819.9, 659.89, and 499.9 Pa s from blank crude oil viscosity of 9978.8, 8005.6, and 5008.6 Pa s respectively at temperatures of 40, 60, and 80 °C was investigated. The reduction of τ_B values was obtained also by OMS-Ni²⁺. The minimum IFT was recorded against the Ni²⁺ derivatives (0.1 × 10^–1 mN m⁻¹). The complete wettability alteration was achieved with the NOMS-Ni²⁺ surfactant (ɵ $\cong 6.01).$ The flooding test has been steered in 3 sets using the sand-packed model as a porous media at surfactant concentrations (1, 1.5, 2 and 2.5%) at 50 °C and 499 psi as injection pressure. The best value (ORs) formed for NOMS-Ni²⁺ were 62, 81, 85.2, and 89% respectively as compared with other NOMS-M²⁺ at the same concentrations. The mechanism of alternating wettability was described in the text. The rheology of the used heavy crude oil was investigated under temperatures of 40, 60, and 80 °C.

Integrating hydrothermal treatment processes and anaerobic digestion (AD) is promising for maximizing resource recovery from biomass and organic waste. The process water generated during hydrothermal treatment contains high concentrations of organic matter, which can be converted into biogas using AD. However, process water also contains various compounds that inhibit the AD process. Fingerprinting these inhibitors and identifying suitable mitigation strategies and detoxification methods is necessary to optimize the integration of these two technologies. By examining the existing literature, we were able to: (1) compare the methane yields and organics removal efficiency during AD of various hydrothermal treatment process water; (2) catalog the main AD inhibitors found in hydrothermal treatment process water; (3) identify recalcitrant components limiting AD performance; and (4) evaluate approaches to detoxify specific inhibitors and degrade recalcitrant components. Common inhibitors in process water are organic acids (at high concentrations), total ammonia nitrogen (TAN), oxygenated organics, and N-heterocyclic compounds. Feedstock composition is the primary determinant of organic acid and TAN formation (carbohydrates-rich and protein-rich feedstocks, respectively). In contrast, processing conditions (e.g., temperature, pressure, reaction duration) influence the formation extent of oxygenated organics and N-heterocyclic compounds. Struvite precipitation and zeolite adsorption are the most widely used approaches to eliminate TAN inhibition. In contrast, powdered and granular activated carbon and ozonation are the preferred methods to remove toxic substances before AD treatment. Currently, ozonation is the most effective approach to reduce the toxicity and recalcitrance of N and O-heterocyclic compounds during AD. Microaeration methods, which disrupt the AD microbiome less than ozone, might be more practical for nitrifying TAN and degrading recalcitrant compounds, but further research in this area is necessary.

Formolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde or C3 dihydroxyacetone (DHA). FLS lays the foundation for several artificial carbon fixation and valorization pathways, such as the artificial starch anabolic pathway. However, the application of FLS is limited by its low catalytic activity and product promiscuity.

FLS, designed and engineered based on benzoylformate decarboxylase from Pseudomonas putida, was selected as a candidate for modification. To evaluate its catalytic activity, 25 residues located within an 8 Å distance from the active center were screened using single-point saturation mutagenesis. A screening approach based on the color reaction of the DHA product was applied to identify the desired FLS variants. After screening approximately 5,000 variants (approximately 200 transformants per site), several amino acid sites that were not identified by directed evolution were found to improve DHA formation. The serine-to-phenylalanine substitution at position 236 improved the activity towards DHA formation by 7.6-fold. Molecular dynamics simulations suggested that the mutation increased local hydrophobicity at the active site, predisposing the cofactor-C2 intermediate to nucleophilic attack by the third formaldehyde molecule for subsequent DHA generation.

This study provides improved FLS variants and valuable information into the influence of residues adjacent to the active center affecting catalytic efficiency, which can guide the rational engineering or directed evolution of FLS to optimize its performance in artificial carbon fixation and valorization.

Side streams from the milling industry offer excellent nutritional properties for animal feed; yet their use is constrained by the elevated phosphorus (P) content, mainly in the form of phytate. Biotechnological P recovery fosters sustainable P management, transforming these streams into P-depleted animal feed through enzymatic hydrolysis. The enzymatic P mobilization not only enables P recovery from milling by-products but also supports the valorization of these streams into P-depleted animal feeds. Our study presents the scalability and applicability of the process and characterizes the resulting P-depleted rye bran as animal feed component. Batch mode investigations were conducted to mobilize P from 100 g to 37.1 kg of rye bran using bioreactors up to 400 L. P reductions of 89% to 92% (reducing from 12.7 g_P/kg to 1.41–1.28 g_P/kg) were achieved. In addition, High Performance Ion Chromatography (HPIC) analysis showed complete depletion of phytate. The successful recovery of the enzymatically mobilized P from the process wastewater by precipitation as struvite and calcium hydrogen phosphate is presented as well, achieving up to 99% removal efficiency. Our study demonstrates a versatile process that is easily adaptable, allowing for a seamless implementation on a larger scale.