Aspergillus oryzae is a widely used host for heterologous expression of fungal natural products. However, the vectors previously developed are not convenient for use and screening positive transformants by PCR and fermentation is time- and effort-consuming. Hence, three plug-and-play vectors were developed here for multi-gene expression and liquid chromatography mass spectrometry detection was introduced to screen positive transformants. Using rug BGC for verification, we demonstrated that the vectors we developed perform well and liquid chromatography mass spectrometry detection is feasible to screen positive transformants. For deleterious gene expression, PxyrA rather than PamyB was employed. Utilizing the toolkit described here to express natural products, dozen days can be saved.
The global trend toward carbon neutrality and sustainability calls for collaborative efforts in both the basic and applied research sectors to utilize mushroom mycelia as environmentally friendly and sustainable materials. Fungi, along with animals and plants, are one of the major eukaryotic life forms. They have long been utilized in traditional biotechnology sectors, such as food fermentation, antibiotic production, and industrial enzyme production. Some fungi have also been consumed as major food crops, such as the fruiting bodies of various mushrooms. Recently, new trends have emerged, shifting from traditional applications towards the innovative use of mushroom mycelium as eco-friendly bioresources. This approach has gained attention in the development of alternative meats, mycofabrication of biocomposites, and production of mycelial leather and fabrics. These applications aim to replace animal husbandry and recycle agricultural waste for use in construction and electrical materials. This paper reviews current research trends on industrial applications of mushroom mycelia, covering strain improvements and molecular breeding as well as mycelial products and the production processes. Key findings, practical considerations, and valorization are also discussed.
Amino acids are important bio-based products with a multi-billion-dollar market. The development of efficient high-throughput screening technologies utilizing biosensors is essential for the rapid identification of high-performance amino acid producers. However, there remains a pressing need for biosensors that specifically target certain critical amino acids, such as L-threonine and L-proline. In this study, a novel transcriptional regulator-based biosensor for L-threonine and L-proline was successfully developed, inspired by our new finding that SerE can export L-proline in addition to the previously known L-threonine and L-serine. Through directed evolution of SerR (the corresponding transcriptional regulator of SerE), the mutant SerRF104I which can recognize both L-threonine and L-proline as effectors and effectively distinguish strains with varying production levels was identified. Subsequently, the SerRF104I-based biosensor was employed for high-throughput screening of the superior enzyme mutants of L-homoserine dehydrogenase and γ-glutamyl kinase, which are critical enzymes in the biosynthesis of L-threonine and L-proline, respectively. A total of 25 and 13 novel mutants that increased the titers of L-threonine and L-proline by over 10% were successfully identified. Notably, six of the newly identified mutants exhibited similarities to the most effective mutants reported to date, indicating the promising application potential of the SerRF104I-based biosensor. This study illustrates an effective strategy for the development of transcriptional regulator-based biosensors for amino acids and other chemical compounds.
Kaempferol and quercetin possess various biological activities, making them valuable in food and medicine. However, their production via traditional methods is often inefficient. This study aims to address this gap by engineering the yeast Yarrowia lipolytica to achieve high yields of these flavonoids. We designed a kaempferol biosynthetic pathway by integrating multiple-copy fusion enzyme expression modules, F3H-(GGGGS)2-FLS, into the genome with an optimized linker (GGGGS)2 to enhance kaempferol production from naringenin. To synthesize quercetin de novo, we introduced the FMOCPR gene into the kaempferol-synthesizing strain using the optimized pFBAin promoter. Notably, increasing glucose concentration effectively boosted the production of both flavonoids. Our results demonstrated kaempferol and quercetin titers reaching 194.30 ± 7.69 and 278.92 ± 11.58 mg/L, respectively, in shake-flask cultures. These findings suggest that Y. lipolytica is a promising platform for the efficient production of flavonoid-derived products.
This research investigated the acidogenic fermentation (AF) of sugar cane molasses in an up-flow anaerobic sludge blanket (UASB) reactor for the production of carboxylates. The first step was to assess the optimum process temperature (25, 35 or 55 ºC) using two different granular inocula, one from a brewery company (BGS) and other from a paper plant company (PGS). These experiments determined that the most suitable temperature for carboxylates production was 25 ºC, obtaining higher bioconversions (27.3 ± 0.3% using PGS and 39.2 ± 0.2% using BGS), despite the low pH value recorded (4.0-4.2). Then, both inocula were tested in UASB reactors. As a consequence of the operational conditions (25 ºC, pH = 5.5-6, organic loading rate (OLR) = 3 gCOD·L-1·d-1 and hydraulic retention time (HRT) = 10 d), the microbial communities changed from those typical for biogas production to those specialised in the production of volatile fatty acids (VFAs). Indeed, the highest bioconversion efficiency (70.1%) was obtained with BGS, where uncultured Eubacteriaceae family microorganisms (56.0%) prevailed, enhancing the production of butyric acid (59.5 ± 2.4%w/w). Consequently, this inoculum was used to further identify the OLR threshold that should not be exceeded to attain optimal carboxylates production. OLR of 6 gCOD·L-1·d-1 resulted in a decrease in bioconversion efficiency (59.5%). The VFAs pool was dominated by butyric acid (63.0 ± 1.4%w/w at an OLR of 4.5 gCOD·L-1·d-1 and 52.8 ± 2.2%w/w at 6 gCOD·L-1·d-1). The microbial community became even more specialised, increasing the presence of Firmicutes and Actinobacteriota phyla, proving that the imposed conditions favoured the production of VFAs when operating semicontinuously fed UASB reactors.
The current study attempted to evaluate the influence of three extraction processes such as Soxhlet, ultrasound-assisted extraction (UAE,) and cold maceration on one hand and solvent polarities (ethanol 70%, ethyl acetate, and water) on the other hand, vs. phytoconstituents and biofunctional properties of Mentha longifolia L. Noteworthy, all extracts were examined in terms of their chemical components, phenolic and flavonoid content, antioxidant and antimicrobial potentials. Notably, high-performance liquid chromatography coupled with array detector analysis (HPLC–DAD) showed the existence of many phenolic compounds. Hydro-ethanol extract (ETOH 70% (v/v)) prepared with maceration and Soxhlet process showed the ultimate rate of phenolic compounds coupled with the most powerful antioxidant and antimicrobial capacity. Notably, ETOH extract obtained with UAE showed important nutritional properties, particularly soluble carbohydrate (50.1 ± 0.70 mg/g dry weight), and soluble protein (26.5 ± 0.5 mg/g dry weight), while aqueous extract obtained by use of UAE extraction maximized pigment content. The outcome of this work showed that Soxhlet and maceration extraction processes with many polar solvents are more appropriate for M. longifolia extraction. M. longifolia possesses promising phytochemicals, which can be used in different sectors like food, pharmaceutical, and cosmetic formulations.
This study evaluated the ability of six purple non-sulfur bacteria (PNSB) to convert olive oil by-products into poly-β-hydroxybutyrate (PHB). Strains were first independently cultivated in synthetic media with different carbon sources (acetic, lactic and malic acid) to assess their physiology and PHB production. Subsequently, their growth and PHB production using ingested pâté olive cake (IPOC) as a substrate were investigated. Transmission electron microscopy (TEM) observations were conducted on strains cultivated on IPOC to investigate their cell morphologies and inclusion bodies presence and size. Rhodopseudomonas palustris strains accumulated up to 6.8% w PHB/w cells with acetate and 0.86% w PHB/w cells with a daily productivity of 0.54 mg PHB L⁻1 culture d⁻1 on IPOC. In contrast, Cereibacter johrii and Cereibacter sphaeroides reached 58.64% w PHB/w cells and 65.45% w PHB/w cells with acetate, respectively, while C. sphaeroides achieved 21.48% w PHB/w cells and a daily productivity of 10.85 mg PHB L⁻1 culture d⁻1 when cultivated on IPOC. All strains exhibited growth and PHB accumulation in both synthetic media and IPOC substrate. Specifically, R. palustris strains 42OL, AV33 and CGA009 displayed growth capability in all substrates, while C. johrii strains 9Cis and PISA 7, and C. sphaeroides F17 showed promising PHB synthesis capabilities. TEM observations revealed that R. palustris strains, with smaller cell and inclusion body sizes, exhibited lower PHB accumulations, while C. johrii and C. sphaeroides strains, characterized by larger cells and inclusion bodies, demonstrated higher PHB production, recognizing them as promising candidates for PHB production using olive oil by-products. Further investigations under laboratory-scale conditions will be necessary to optimize operating parameters and develop integrated strategies for simultaneous PHB synthesis and the co-production of value-added products, thereby enhancing the economic feasibility of the process within a biorefinery framework.
The valorization of vinasses into short-chain fatty acids (SCFAs) via anaerobic fermentation (AF) is an emerging approach that remains under research. Given the diverse microbial metabolisms simultaneously occurring in AF, the control of operational parameters is essential to avoid process destabilization. To unravel their effect, the novelty of this investigation relied on the evaluation of the robustness of AF process against operational perturbation deliberately set (i.e. hydraulic retention time (HRT) and temperature increase). Regardless the applied perturbation, similar yields (0.5–0.6 g COD-SCFAs/g VSin) were attained. However, the selected perturbations exerted an effect on microbiome development. Whereas the temperature increase mediated a 49.70% microbiome dissimilarity, only a 21.91% dissimilarity was caused by the HRT increase. Microbial analysis revealed Clostridiales, Prevotella and Megasphaera as key bacteria in vinasses degradation. The similar bioconversion obtained despite the different microbiomes developed after each perturbation suggested a functional redundancy highlighting the AF robustness. These findings evidenced AF as a feasible biotechnology to further valorize vinasse into SCFAs, demonstrating the process stability against common perturbations that might be encountered at industrial scale.
The cyanobacterium, Spirulina sp. is a photosynthetic blue-green alga with essential nutrients, vitamins nucleic acids, proteins, carbohydrates, fatty acids and pigments carotenes; and phycocyanins are the significant components having immunomodulatory, anti-inflammatory properties, which are used in food and cosmetics industries. Spirulina sp. can play an important role in human and animal nutrition for potential health benefits due to their phycochemical and pharmaceutical significance. This study highlights antibacterial, antifungal, antiviral, antioxidant, nephroprotective, cardioprotective, anticancer, neuroprotective, anti-aging, anti-inflammatory, and immunomodulatory properties. It highlights anti-anemic, antidiabetic, probiotic, anti-malarial, anti-obesity and weight loss, anti-genotoxicity, anti-thrombic, radioprotective, and detoxifying effects of Spirulina sp. Pharmaceutical studies indicate it may improve heart health and add to the treatment of diabetes, obesity and weight loss. It can play a major role in protecting the environment by recycling wastewater and providing food for humans and animals. Spirulina sp. can supply ingredients for aquaculture and agricultural feeds, pigments, antioxidants, and essential omega-3 oils, among other human health and wellness products. The amino acid of Spirulina is among the greatest qualititavely of any plant, even higher than that of soybean. Furthermore, cyanobacterium Spirulina sp. could be a future antimicrobial drug agent.
Natural products of plant origin are being explored as safe alternatives for illness management. Their extraction processes play a crucial role in determining their phytochemical and pharmacological properties. In this context, Euphorbia trigona was extracted using Supercritical Fluid Extraction with CO2 (SFE-CO2) at two operating temperatures: 20 °C and 40 °C. Phytochemical characterization was performed via HPLC, along with anti-yeast evaluation using the well diffusion method, anticancer assessment using the MTT assay, wound healing analysis via the scratch assay, and anti-obesity evaluation through the lipase assay of the E. trigona extract. The results indicated that SFE-CO2 at 40 °C extracted a greater quantity (0.198 g) of E. trigona than SFE-CO2 at 20 °C (0.156 g). Several compounds, such as rosmarinic acid, gallic acid, daidzein, ellagic acid, naringenin, and ferulic acid, were identified at high concentrations of 10,034.29, 1,800.33, 750.22, 748.11, 462.15, and 207.05 µg/mL, respectively, in the E. trigona extract obtained using SFE-CO2 at 40 °C, compared to the extract obtained using SFE-CO2 at 20 °C. High inhibition zones of 24 ± 1.5, 24 ± 0.5, and 23 ± 0.33 mm were recorded against C. albicans, C. tropicalis, and G. candidum, respectively, using the extract from SFE-CO2 at 40 °C, compared to the inhibition zones of 24 ± 1.5, 24 ± 0.5, and 23 ± 0.33 mm obtained from the extract using SFE-CO2 at 20 °C. Moreover, the extract from SFE-CO2 at 40 °C exhibited lower MIC and MFC values against the tested yeasts compared to the efficacy of the extract from SFE-CO2 at 20 °C. The ultrastructure of the examined yeasts was severely affected by the extract from SFE-CO2 at 40 °C. A lower IC50 (98.87 ± 1.26 µg/mL) was recorded for the extract from SFE-CO2 at 40 °C compared to the IC50 (333.87 ± 1.8 µg/mL) of the extract from SFE-CO2 at 20 °C against cancer cells (A431). The wound closure level was 84.08% using the extract from SFE-CO2 at 40 °C, while it was 71.27% using the extract from SFE-CO2 at 20 °C. Lipase was inhibited by the extract obtained via SFE-CO2 at 40 °C and 20 °C, with IC50 values of 15.77 and 28.14 µg/mL, respectively. Molecular docking indicated that rosmarinic acid is a suitable inhibitor for the tested yeasts.
This study investigates the in silico anti-arboviral potential of zoochemicals derived from the methanolic extract of Charybdis natator shell, alongside their larvicidal efficacy against Aedes aegypti 4th instar larvae. Through GC–MS analysis, 27 zoochemicals were identified, demonstrating promising in silico activity against molecular antiviral targets: DENV2 protease (PDB: 6MO1) for anti-dengue, RNA polymerase (PDB: 5U04) for anti-Zika, and nsP2 protease (PDB: 3TRK) for anti-chikungunya. A strong positive correlation (r = 0.726–0.889) in binding affinities (kcal/mol) suggests a consistent inhibitory mechanism across these targets. Furthermore, PASS analysis indicates higher probabilities of activity (Pa) for insecticidal properties compared to antiviral efficacy, highlighting their dual potential as larvicidal agents and antiviral candidates. The methanolic extract of Charybdis natator shell exhibited potent larvicidal activity against Aedes aegypti (LC₅₀ = 81.001 µg/mL) in a dose-dependent manner (R2 = 0.968). In silico analysis further elucidated its inhibitory action on key growth regulators of A. aegypti, underscoring its potential to disrupt larval development. These findings highlight the dual utility of C. natator shell extract in vector management and in mitigating the transmission of arboviral diseases such as Dengue, Zika, and Chikungunya. The extract's promise as an eco-friendly, cost-effective source for developing novel insecticidal and antiviral agents merits further exploration.
Andrographolide (AG), one of the main active components of Andrographis paniculata (Burm.f.) Wall. ex Nees, has been proved to possess the pharmacological function of anti-inflammation in multiple disease including asthma. But the potential mechanism is still not clear. In this study, network pharmacology, molecular docking and experimental validation were utilized to explore the molecular mechanism of AG in the treatment of asthma. AG-related targets and asthma-related targets were screened by Swiss Target Prediction, DrugBank, STITCH, OMIM, Genecards and TTD databases. A protein–protein interaction (PPI) network was obtained through the STRING Database. The plug-in of “Network Analyzer” in Cytoscape 3.7.1 software was used to conduct the topological analysis. GO enrichment and KEGG pathway analysis were achieved by Metascape database and Bioinformatics platform. The target-pathway network was acquired by Cytoscape 3.7.1 software. The binding affinity between AG and the target genes was evaluated by Molecular docking with AutoDockTools 1.5.6. Flow cytometry was also used to verify the mechanism behind the treatment of asthma by AG, which was predicted in network pharmacology. In total, 38 targets were identified as potential targets of AG against asthma. The top 10 targets revealed by PPI are: IL-6, IL-1B, NFKB1, MMP9, CDK2, CREBBP, MAP2K1, JAK1, AR, PRKCA. GO and KEGG analysis showed that AG treatment of asthma mainly involved protein phosphorylation, peptidyl-serine phosphorylation, peptidyl-amino acid modification and other biological processes. The main signaling pathways are Th17 cell differentiation, JAK-STAT signaling pathway and PI3K-Akt signaling pathway. Molecular docking showed that AG has higher affinity with MMP9, PRKCA, JAK2, LTGAL and LRRK2. Flow cytometry showed that Th17 cell differentiation may be the potential target of AG in the treatment of asthma. This study successfully revealed the underlying target genes and mechanism involved in the treatment of asthma for AG, providing a reference and guidance for future mechanism research.
Lignocellulose is a promising renewable resource for producing platform chemicals, such as acetone, butanol, and ethanol, via ABE fermentation by solventogenic clostridia. This study investigates the effects of common lignocellulose derived inhibitory compounds: ferulic acid, coumaric acid, and furfural on Clostridium beijerinckii. Dual-staining with propidium iodide and CFDA, combined with flow cytometry, was employed to assess physiological variability. The results showed that phenolic acid-induced stress helped maintain a higher proportion of viable cells during the production phase, enhancing solvent yields and reducing sporulation. At 0.4 g/L, ferulic and coumaric acids did not reduce cell viability; however, coumaric acid exposure led to an acid-crash profile. Conversely, a more robust inoculum exposed to both phenolic acids simultaneously exhibited effects similar to ferulic acid alone, including slower viability decline, reduced growth and sporulation, and improved solvent production. Furfural exposure at 1.5 g/L resulted in immediate viability loss in 20% of the population, though the overall decline accompanied by the highest sporulation rate occurred later than in the control. Additionally, furfural transformation was slower, suppressing butyrate production and reducing solvent production by 13%. This study suggests that delaying cell death mechanism may explain the stimulatory effects of inhibitors, advancing lignocellulose use in the future.
β-Glucanases are a series of glycoside hydrolases (GHs) that are of special interest for various medical and biotechnological applications. Numerous β-glucanases were produced by different types of microorganisms. Particularly, bacterial β-glucanases have the privilege of being stable, easily produced, and suitable for large-scale production. This study aimed for finding potent β-glucanase producing bacterial strains and optimizing its production. Soil samples from Egyptian governorates were screened for such strains, and 96 isolates were collected. The β-glucanase activity was qualitatively assessed and quantitatively measured using 3,5-dinitrosalicylic acid (DNS) method. The highest β-glucanase producing strain (0.74 U/ml) was identified as Streptomyces albogriseolus S13-1. The optimum incubation period and temperature, determined one-variable at a time, were estimated as 4 d and 45 ͦ C, respectively. Similarly, yeast β-glucan and beef extract were selected as the best carbon and nitrogen sources, with enzymatic activities of 0.74 and 1.12 U/ml, respectively. Other fermentation conditions were optimized through response surface methodology (RSM); D-optimal design (DOD) with a total of 28 runs. The maximum experimental β-glucanase activity (1.3 U/ml) was obtained with pH 6.5, inoculum volume of 0.5% v/v, agitation speed of 100 rpm, carbon concentration of 1% w/v, and nitrogen concentration of 0.11% w/v. This was 1.76-fold higher compared to unoptimized conditions. Using the same experimental matrix, an artificial neural network (ANN) was built to predict β-glucanase production by the isolated strain. Predicted β-glucanase levels by RSM and ANN were 1.79 and 1.32 U/ml, respectively. Both models slightly over-estimated production levels, but ANN showed higher predictivity and better performance metrics. The enzyme was partially purified through acetone precipitation, characterized, and immobilized on chitosan-coated iron oxide microparticles. The optimal pH and temperature for enzyme activity were 5 and 50 °C, respectively. The immobilized enzyme showed superior characters such as higher stability at temperatures 50, 60, and 70 °C compared to the free enzyme, and satisfactory reusability, losing only 30% of activity after 6 cycles of reuse.
The biosynthesis of bimetallic nanoparticles using plant extracts has garnered significant attention due to their eco-friendly and cost-effective nature. This study aimed to biosynthesize magnesium oxide-zinc oxide nanocomposite (MgO-ZnO nanocomposite) using Pluchea indica leaf extract for the first time, with a focus on characterizing its physicochemical properties and evaluating its biological activities. The biosynthesized MgO-ZnO nanocomposite was fully characterized, revealing an absorbance peak at 300 nm using UV–vis spectroscopy. Transmission electron microscopy (TEM) confirmed particle stability within the size range of 5–35 nm. Cytotoxicity analysis on the Wi 38 normal cell line demonstrated an IC50 value of 179.13 µg/mL, indicating good biosafety. The nanocomposite exhibited potent anticancer activity, with IC50 values of 73.61 µg/mL and 31.25 µg/mL against Hep-G2 and MCF-7 cancer cell lines, respectively. Antibacterial assays revealed activity against Klebsiella pneumoniae, Escherichia coli, Bacillus cereus, Staphylococcus aureus, and Candida albicans, with minimum inhibitory concentrations (MICs) ranging from 31.25 to 250 µg/mL. Furthermore, the nanocomposite displayed antioxidant activity with an IC50 value of 175 µg/mL, as determined by the DPPH assay. In conclusion, the successful synthesis of the MgO-ZnO nanocomposite using P. indica leaf extract demonstrates its potential as a safe and effective agent for concentration-dependent antioxidants, antibacterial, and anticancer applications. This study highlights the versatility of plant-mediated biosynthesis in developing functional nanomaterials for biomedical use.
The search for safe, natural antimicrobial agents has received significant attention for controlling postharvest diseases in fruits. This study evaluated the antifungal activity of pomegranate peel extracts (PPE) against 9 pathogenic fungi by different methods. Additionally, the key antifungal polyphenol compounds were identified. Results revealed that the n-hexane partitioned fraction of ethanolic extract exhibited the highest inhibition efficacy. A total 36 polyphenols, including 10 newly discovered compounds in pomegranate peel were recognized. Among these, 9 specific polyphenols were found to be relatively abundant in the n-hexane fraction, highlighting their potential as primary antifungal agents. Notably, the newly identified polyphenol compound nobiletin demonstrated the strongest inhibitory effects against Colletotrichum gloeosporioides, Rhizopus stolonifer and Aspergillus niger with inhibition diameters of 12.2, 12.0, and 12.5 mm, respectively. Salidroside exhibited significant antifungal activity against Monilinia fructicola and A. niger (inhibition diameters > 12.0 mm). Furthermore, cinnamic acid that was enriched in hexane fraction, showed the highest inhibitory zones against Alternaria alternata, C. gloeosporioides and Botryosphaeria dothidea (11.8 ~ 12.7 mm). Caffeic acid and protocatechuic acid both displayed the lowest minimum inhibitory concentration (MIC) on Penicillium expansum. The study demonstrated the potential of PPE, particularly the n-hexane fraction, as a broad-spectrum natural antifungal agent for postharvest disease management.
To explore the chemical and biological diversities of diterpenoids from the fungus Talaromyces adpressus, a previously unknown biosynthetic gene cluster (BGC, tdn) for sordarin (a well-known fungal antibiotics) was discovered by leveraging the genome mining method. Heterologous expressions of key genes of tdn in Aspergillus oryzae, led to the determination of one new diterpenoid, cycloaraneosene-9-ol-8-one (4), and three known diterpenoids, cycloaraneosene (1), cycloaraneosene-9-ol (2), cycloaraneosene-8,9-diol (3). The structures of 1–4 was elucidated well via detailed analysis of 1D and 2D NMR, GCMS, HRESIMS, IR data, and comparison with reported data. Structurally, compounds 1–4 were belonging to fusicoccane diterpenoids with a classical tricyclic 5/8/5 ring system, which are participated in the biosynthesis of sordarin. Compound 4 maybe a key precursor for a Baeyer–Villiger like reaction with C8–C9 bond cleavage in the biosynthetic pathway of sordarin. Moreover, all isolates were evaluated for their bioactivities, compounds 3, and 4 exhibited inhibitory activities against the human cancer cell lines with IC50 values ranging from 7.8 to 32.4 µM. 3 and 4 promote cell apoptosis of HCT-116 and HepG2 cells, and suppress cell migration of HepG2 cells. As well, 3 and 4 also decrease gene expression of cell proliferation related molecules BCL-2 and cyclin D1, while increase expression of cell apoptosis related gene BAX. Targets predication and molecular docking indicate that compound 4 exhibits stronger affinity for DBL, suggesting its excellent binding potential. This finding will be enriched the structures and bioactivities of diterpenoids with a tricyclic 5/8/5 ring system, most importantly, will provide new strategies for the synthetic biological research of sordarins.
Biomass recalcitrance makes pretreatment process a key step for efficient bioconversion process. In this study, differential effects of promising acid (AP) and alkaline pretreatments (ALP) on enzymatic hydrolysis of diverse herbaceous and woody wastes were systematically investigated. Four biomass samples were separately pretreated and sugar recovery was then recorded in the subsequent hydrolysis. Results showed that both dilute AP and ALP exhibited efficacy in the removal of hemicellulose. Specifically, soybean straw AP demonstrated the highest recovery of soluble sugars at the pretreatment stage [270 mg/g raw stalk (RS)], against 71–212 mg/g RS achieved in AP and ALP of other wastes. Compared with herbaceous soybean straw, both AP and ALP of more recalcitrant woody biomass (e.g., bamboo and poplar) showed much lower enzymatic sugar yields. Among tested samples, ALP soybean straw produced stronger structure modification, morphological changes and higher delignification, which increased its availability to cellulases. As a result, the sugar yield of ALP soybean straw using 1.5% NaOH reached 787 mg/g, which is much higher than those of other tested AP & ALP biomass wastes. The present study revealed differential responses of diverse biomass wastes to AP & ALP, hence providing valuable information for the development of effective bioconversion process of these promising biomass. Looking ahead, these classic AP and ALP will be further investigated together with other potential and emerging pretreatments (e.g., green solvent pretreatments) to provide a foundation for high value utilization of biomass.
Exploring novel amylolytic enzymes and understanding their biochemical properties are essential for advancing starch-based industries. This study focused on identifying the effective extracellular enzymes produced by Priestia koreensis HL12, a novel starch-converting bacterium, through a combined proteomics and genomics approach. Genome annotation revealed 82 genes belonging to various CAZyme families. Among the ten media tested for amylolytic enzyme production, raw cassava pulp was identified as the most effective carbon source, yielding a remarkable starch-degrading activity of 452.6 ± 2.944 U/mg protein against cassava starch. The extracellular enzymes demonstrated significant potential for high-solid enzymatic hydrolysis of raw cassava pulp (5% w/v), achieving a conversion rate of 72% (719.1 mg/g biomass) at 45 °C, pH 5.0 for 24 h of hydrolysis. Proteomics analysis provided insights into the specific enzymes responsible for efficient starch breakdown and modification. These findings position P. koreensis HL12 as a highly effective bacterium for cellulase/xylanase-free amylase production. This work not only highlights the unique enzymatic profile of P. koreensis HL12 but also emphasizes its significant role in optimizing starch bioconversion processes, ultimately contributing to the development of more sustainable biorefineries.
Trimethylene carbonate (TMC) is an innovative modifier for polylactic acid and a promising biodegradable polymer monomer with broad application potential. However, industrial production of TMC faces challenges such as high catalyst costs, safety issues, and environmental impacts. Enzymatic catalysis offers a potential alternative, but its low product yields have hindered progress. In this study, we introduce a novel synthesis route for TMC using bio-based 1,3-propanediol (1,3-PDO) and dimethyl carbonate (DMC) as substrates. This process involves lipase-catalyzed formation of the intermediate 3-hydroxypropyl methyl carbonate (P1), which is then cyclized to produce TMC. Notably, the by-product, C,C’-1,3-propanediyl C,C’-dimethyl ester (P2), reacts with 1,3-PDO to regenerate P1, further enhancing the overall TMC yield. The mechanism exploration reveals that 1,3-PDO acts as both a reactant and an acid catalyst, initiating a nucleophilic substitution reaction on P2 to produce P1. Under optimized conditions, we achieved a total TMC yield of 88%, the highest reported to date.This study provides a novel green synthesis route for TMC that holds great promise for industrial application, given its safer conditions and competitive yields.
This study investigated the potential use of pyrolysis liquid from bark as an anti-fungal substance against food decaying fungi. Four different fractions of pyrolysis liquid were collected during variable temperature phases of the pyrolysis process: F1 (25–260 °C), F2 (260–512 °C), F3 (512–800 °C), and F4 (800–25 °C). The thermal degradation of bark material was assessed using TGA analysis. The concentration, pH, total phenolic content, and functional groups of the liquid samples were determined. Additionally, the molecular composition was examined using UHPLC and QToF mass spectrometry methods. Fungal species were isolated from bell pepper and animal fat and identified through microscopic observation and DNA sequencing. The anti-fungal activity of the liquid fractions was evaluated using the disk diffusion test. The obtained degradation thermograms had a typical shape characteristic of lignocellulosic materials, revealing different thermal degradation phases of the bark. These phases served as a basis for the collection of the pyrolysis liquid in fractions, which were expected to differ in properties and molecular composition. In the fractions collected above 260 ºC (F2, F3, F4), the pyrolysis liquid presented an acidic character, resulting from the complex thermochemical reactions that occur during the slow pyrolysis of bark. F2 had the highest concentration of total phenolic compounds (6.46 mg GAE/g extract) while F1 and F4 contained only negligible amounts. The FTIR spectra of F2 displayed additional peaks compared to other samples which provided information on the occurrence of various compounds. The reversed phase UHPLC-UV analysis revealed that furfural, 5-hydroxymethyl furfural, and 5-methyl furfural were the most abundant compounds, and F2 had the highest concentration of summed furans (570 µg/mL) among all samples. The morphological assessment and DNA sequence analysis of the fungal strains revealed that Penicillium crustosum and Cladosporium sp were isolated from fat and bell pepper, respectively. The antifungal activity of the liquid fractions was limited due to their low concentration (ranging from 0.24% to 0.01% (v/v)), with only minor inhibition observed for F2, indicated by a small inhibition zone of approximately 1 mm around the 10 mm filter paper. However, concentrating the fraction F2 up to 1% (v/v) demonstrated a stronger inhibitory zone against Cladosporium pseudocladosporioides and Penicillium sp., indicating its antifungal potential at higher concentrations. Overall, the pyrolysis liquid demonstrated promising antifungal activity, particularly after concentration, with F2 exhibiting the highest bioactivity and strongest inhibition effect. These findings highlight its potential for controlling food-decaying fungi while emphasizing the need for further purification, toxicity assessments, and application studies to ensure its feasibility for agro-industrial applications.
Qu-aroma is a critical quality indicator for medium–high temperature Daqu (MT-Daqu). Due to the differences in regional and environmental conditions, these differences are likely to cause variations in the Qu-aroma of MT-Daqu. Despite this, comprehensive research on the variations in Qu-aroma across different regions has been lacking. This study aims to address this gap to analyze the differences in Qu-aroma characteristics of MT-Daqu from seven distinct production areas. Sensory analysis identified seven broad aroma categories and 25 specific aromas characterizing the Qu-aroma of MT-Daqu samples. Chen aroma and roasted aroma were notably more prevalent in MT-Daqu samples from Sichuan compared to those from other regions (P < 0.05). In total, 123 volatile compounds were detected in MT-Daqu, with 42 classified as aroma-active compounds. Among these, 21 aroma-active compounds were identified as markers distinguishing Sichuan MT-Daqu from those produced in non-Sichuan regions, showing significant differences in their concentrations (P < 0.05). Additionally, six major aroma-active compounds were found to significantly contribute to the aroma differences between Sichuan and non-Sichuan MT-Daqu. This study delineates the distinct Qu-aroma characteristics of MT-Daqu from various SAB production areas. The insights gained from this research offer valuable guidance for optimizing flavor profiles in MT-Daqu production.
The aging is a crucial stage in tobacco processing, which contributes to the reduction of impurities and irritation, and the stabilization of the internal chemical composition of the leaves. However, it usually takes a long time (2–3 years) for the nature aging process of tobacco (20 °C–30 °C, relative humidity of 65–75%), which seriously affects the processing efficiency of tobacco. Microorganisms play an important role in the change of chemical composition and characteristic aromatic substances of tobacco. Acinetobacter, Sphingomonas Aspergillus, Bacilli, and Pseudonocardia is the main microorganism in the aging process of tobacco, which increasing the aromatic substances (such as alcohols, ketones, and esters) by the action of the enzymes and metabolites, and degrade the harmful components (such as alkaloid, nicotine and nitrosamines in tobacco). This review systematically summarizes recent advancements in understanding the primary microbial composition and the changes in chemical composition during tobacco aging. This knowledge is helpful for screening functional strains, and control the process of tobacco aging by the inoculation of these strains.
Polysaccharides derived from Pleurotus eryngii possess various bioactive properties, including antioxidant, antidiabetic, anti-inflammatory, and immunomodulatory effects. In this study, polysaccharides were extracted from P. eryngii fruiting bodies and exposed to gamma irradiation at doses of 50 and 100 kGy, with a dose rate of 5 kGy/h. The surface morphology of the polysaccharide irradiated at 100 kGy exhibited numerous pores and a smaller flake structure compared to those irradiated at 50 kGy and the non-irradiated sample. 1H and 13C NMR spectra of all samples indicated that both irradiated and non-irradiated polysaccharides exhibited α- and β-configurations, with signals corresponding to C1–C5 clearly observed. HPLC analysis of the polysaccharides revealed that glucose (75.23%), galactose (4.96%), glucuronic acid (1.38%), ribose (0.94%), rhamnose (2.35%), and mannose (3.87%) are the main components. All polysaccharides demonstrated antioxidant activity, which increased with concentration. Both non-irradiated and irradiated polysaccharides exhibited antidiabetic effects, significantly reducing blood glucose levels, and restoring insulin level with superiority of irradiated polysaccharides. Additionally, they significantly elevated body weight, slightly reduced MDA levels, and markedly enhanced catalase activity in treated rats compared to diabetic controls. The antidiabetic effects of the polysaccharides were further confirmed by histopathological examination of the pancreas and liver sections from polysaccharide-treated diabetic rats. This suggests that irradiation, by reducing the molecular weight of polysaccharides, enhances their bioavailability and efficacy in modulating glucose metabolism.
The development of newfangled bioprocess strategies for the capture of C1-gases (CO and CO2) and their bioconversion into valuable products is currently one of the main focuses of research in order to achieve a more resilient world. This work analyses the viability of the co-culture C. carboxidivorans and C. beijerinkii to produce bioproducts (bioalcohols and organic acids) in mixotrophic conditions. In this way, the bioconversion of C1 gases (CO and CO2), in the presence of Fe0, using mixotrophic co-culture fermentation by C. carboxidivorans and C. beijerinkii, was evaluated, analyzing the influence of the ratio between both microorganisms, the pH, and the presence of Fe0. As a result, up to 7 g/L of butanol were achieved at pH 7, 12.5 g/L Fe0, and using a 1:1 ratio of C. carboxidivorans: C. beijerinkii, also improving the production of ethanol, acetic acid, and butyric acid as compared to individual culture fermentations. Finally, the operation in a bioreactor, comparing discontinuous and continuous gas feeding operation modes, was also studied, with better C1-gases utilization and overall fermentation efficiency (7 vs 4.6 g/L butanol) in continuous gas operation mode.
Astaxanthin, a red carotenoid with potent antioxidant properties, holds significant value in the feed, cosmetics, and nutraceutical industries. While traditionally sourced from microalgae, Corynebacterium glutamicum, a well-established industrial microorganism, has been engineered to serve as an efficient host for astaxanthin production. As astaxanthin integrates into the cellular membrane, effective extraction methods are essential to access this valuable compound. In this study, a sustainable batch extraction process using supercritical carbon dioxide (scCO₂) as a green solvent was developed. The effects of cosolvent concentration (0–9% (w/w)), temperature (50–75 °C), and pressure (450–650 bar) were investigated with regard to the extraction yield. An optimized extraction was achieved with 9% (w/w) ethanol as a cosolvent, at 68 °C and 550 bar, allowing the extraction of 67.5 ± 3.7% of the cellular astaxanthin within 0.5 h. Prolonging the extraction time further increased the recovery to 93.3%, which is comparable to processes that have been established for the extraction of astaxanthin from microalgae and yeast. This approach provides a scalable and environmentally friendly solution for industrial astaxanthin recovery.
Laccases with high catalytic efficiency and high thermostability can drive a broader application scope. However, the structural distribution of key amino acids capable of significantly influencing the performance of laccases has not been explored in depth. Thirty laccase 13B22 mutants with changes in amino acids at distances of 5 Å (first shell), 5–8 Å (second shell), and 8–12 Å (third shell) from the active center were validated experimentally with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate. Twelve of these mutants (first shell, 1; second shell, 4; third shell, 7) showed higher catalytic efficiency than the wild-type enzyme. Mutants D511E and I88L-D511E showed 5.36- and 10.58-fold increases in kcat/Km, respectively, with increases in optimal temperature of 15 °C and optimal pH from 7.0 to 8.0. Furthermore, both mutants exhibited greater thermostability compared to the wild-type, with increases of 3.33 °C and 5.06 °C in Tm and decreases of 0.39 J and 0.59 J in total structure energy, respectively. The D511E mutation resides in the third shell, while I88L is in the second shell, and their performance enhancements were attributed to alterations in the rigidity or flexibility of specific protein structural domains. Both mutants showed enhanced degradation efficiency for benzo[a]pyrene and zearalenone. These findings highlight the importance of the residues located far from the active center in the function of laccase (second shell and third shell), suggesting broader implications for enzyme optimization and biotechnological applications.
In the present study, a novel technology called intermittent solvent-free microwave extraction (ISFME), which integrates intermittent microwave treatment with solvent-free extraction, was proposed. Taking Nanfeng citrus peel as the research object, the extraction efficiency and composition of its essential oil were evaluated and compared with conventional hydrodistillation (HD) and solvent-free microwave extraction (SFME). Results from single-factor experiments and response surface method optimization indicate that the optimal parameters for SFME are a power of 660 W, a duration of 20 min, and a water content of 60%, yielding an essential oil output of 3.47%. In contrast, the optimal parameters for ISFME, determined through single-factor experiments, are a moisture content of 50%, a power setting of 380 W, a reaction time of 6 min, a 15 min interval, and 4 reaction cycles, resulting in an essential oil yield of 3.51%. Notably, the extraction efficiencies of ISFME and SFME are significantly superior to that of HD, which requires 240 min and yields only 3.28% essential oil. Furthermore, ISFME demonstrates comparable performance to SFME in terms of reaction time, extraction rate, and oil quality, while operating at a substantially lower power requirement of 380 W compared to the 660 W needed for SFME. Additionally, the carbon dioxide emissions and energy consumption associated with ISFME are markedly lower than those of traditional methods, amounting to only one-third of the energy consumption of SFME. These findings underscore that ISFME not only enhances the efficiency and quality of essential oil extraction but also minimizes environmental impact, thereby highlighting its significant advantages in terms of sustainability and ecological responsibility.
Pinus radiata is the dominant tree species in exotic plantation forestry of New Zealand producing timber for construction and pulp and paper. Additionally, the processing yields large amounts of bark as a byproduct that is either left at the harvest site or used for landscaping. P. radiata bark is rich in biochemical extractives containing polyphenols and waxes on sequential extraction with hydrophilic and lipophilic solvents, respectively. Previous studies have exclusively focussed on the effect of parameters such as solvent type, bark to solvent ratio, and extraction time on the yield of extractives. However, two parameters were always maintained constant: solvent order (lipophilic to hydrophobic) and particle size. This work investigated the the combined impact of these two parameters on total yield and product quality by using two solvents- water and hexane. Total extractives were highest when water was used first (11.74% and 9.45%) compared to hexane (10.53% and 6.53%). The individual yields of hexane extractives were in the range of 2.25–2.9% while those of water were 4.30–9.24%. Chemical analyses of the extracts and residues showed no qualitative differences, indicating the order in which bark is extracted does not alter the extract composition. Moreover, the results have successfully established that extracting bark with water first followed by hexane will increase the total yield of extractives and increasing particle size decreases the total yield of the sequential extraction.
Valorization of food byproducts, especially fruits and vegetables, has recently attracted considerable attention, mostly due to their high wastage rates. Exploitation of these byproducts, including the non-edible parts of crucifer vegetables, may provide value-added opportunities in the food, functional food, and nutraceutical industries as well as in non-food applications such as therapeutics, biofuels, and paper pulp production. This review focuses on the state-of-the-art valorization practices of crucifer vegetable agro-food wastes including those of broccoli, cauliflower, cabbage, kale, Brussels sprouts, collards, watercress and radish constituting the main cultivated crucifer vegetables worldwide and suggests potential novel uses through upcycling. A detailed comparative phytochemical composition of crucifer vegetable waste products as potential sources of raw materials in promising applications including the production of food enhancers, and antioxidants is presented. Different extraction techniques combining downstream and white biotechnology processes for the optimum utilization of such agro-food waste are discussed. The valorization of cruciferous vegetables by-products is shown to be economical, sustainable and a viable approach to unlock novel applications across diverse industries. To fully maximize the potential of these underutilized resources and promote an ecological bioeconomy, more research and development into extraction methods and upcycling techniques is needed
Global population growth underlies the need to explore alternative materials to address pressing challenges in food security, medicine, energy, and environmental pollution. Spirulina is a nutrient dense cyanobacteria that offers promising solutions to the aforementioned challenges, mainly due to its rich composition of proteins, vitamins, minerals, and bioactive compounds such as β-carotene and phycocyanin. These compounds confer various health benefits, including antioxidant, anticancer, anti-diabetic, antimicrobial, and anti-inflammatory properties, which make Spirulina a valuable dietary and therapeutic supplement. Essential fatty acids and its rapid growth rate also makes Spirulina a potential source of biodiesel for energy related applications. Additionally, Spirulina's high porosity and variable functional groups endow it with remarkable biosorption properties for soil and wastewater remediation applications. The chemical structure and unique properties of Spirulina have been utilized to produce biotemplates for nanomaterials as well as the fabrication of functional composites for various applications. Thus, in this review, we have highlighted the broad potentials of Spirulina in diverse applications, emphasizing its eco-friendliness, economic viability, challenges, and the prospects of its biomass for sustainable, nutraceutical, therapeutic, energy related, and environmental applications.
This study aims to synthesize calcium oxide nanoparticles by employing green synthetic methods and explore their potential as nano-catalyst based upon the utilization of waste into a value-based product. Waste orange peel extract has been utilized as a reducing medium. The reaction was optimized by varying the reactants’ molar ratio to obtain calcium carbonate microparticles that were calcined to obtain calcium oxide nanoparticles with a particle size ranging from 70 to 100 nm. Various spectrochemical techniques analyzed the composition and morphology of the nano-catalyst. The nano-catalyst was further exploited in the one-pot transesterification of waste cooking oil. The biodiesel was analyzed for the presence of methyl ester groups by FTIR and GCMS analysis. The impact of varying reaction constraints, including temperature, contact time, nano-catalyst concentration, and methanol-oil molar ratio, were critically analyzed to optimize biodiesel yield. The study provided an economical and environmentally benign technique to successfully synthesize calcium oxide nano-catalyst to obtain biodiesel with 93.4% yield and effective waste minimization.
Atherosclerosis is a chronic vascular disease characterized by failure to resolve inflammation and forming plaque within the arterial wall. Atherosclerosis and its related cardiovascular diseases are the major causes of death worldwide. Our previous preliminary study showed that anthocyanin-rich extract (ACNE) from Xinjiang wild cherry plum (Prunus divaricata Ledeb) fruit peels exhibited anti-atherosclerotic effect. However, the potential mechanism of this health-beneficial effect remains unclear. Here, network pharmacology combined with molecular docking was used to tentatively address this issue. The ACNE mainly contains cyanidin, cyanidin 3-glucoside (Cy3Glu), Cyanidin 3-(6’’-acetylglucoside) (Cy3AcGlu), cyanidin 3-galactoside (Cy3Gal), cyanidin 3-xyloside (Cy3Xyl), and cyanidin 3-rutinoside (Cy3Rut). Seven key targets, EGFR, VEGFA, HSP90AA1, SRC, HIF1A, CXCR4 and IGF1R were identified from core protein-protein interaction (PPI) network. Anthocyanins interacting on key targets were initially demonstrated by molecular docking, particularly Cy3Rut and Cy3Xyl having highest affinity with most key targets. Biological function analysis suggested that key targets were involved in several biological processes, including positive regulation of cell migration, positive regulation of phosphorylation, inflammatory response, response to hypoxia, etc. The significantly enriched pathways, such as HIF-1 signaling pathway, calcium signaling pathway, macrophage stimulating protein MSP signaling network map, were closely related to atherosclerosis. Altogether, based on the comprehensive analysis and discussion, we revealed that TLR4/EGFR and IGF1R-CXCL12/CXCR4 pathways were at least partially implicated in the anti-atherosclerotic effects of anthocyanins through affecting inflammation, endothelial homeostasis, and foam cell formation. This study served as a theoretical basis for further validating the underlying anti-atherosclerotic mechanism of anthocyanins via in vitro and in vivo experiments.
Storage of economic fruits is a fatal economic and nutritional factor for most countries. Edible coating played a restricted role in this manner, forcing good usability with many limitations. In this work, Apple (Malus domestica var. Anna) was coated using bioactive nanopackaging films formulated to overcome the economic limitations and drawbacks of conventional coating. The formulated bioactive nanopackaging was based on nanochitosan prepared from shrimp shells and orange peel waste, which is used to produce nanocellulose (white part) and extract active ingredients (orange part). The formulated bioactive nanopackaging based on nanochitosan and nanocellulose (2:1) and orange peel waste extract with ratios of 1, 3, and 5% (w/w) based on nanochitosan dry weight and called T3, T4, and T5, respectively. Characteristics of bioactive nanopackaging films and their precursor materials were characterized physicochemically and topographically as well. The waste orange peel waste extract was characterized phytochemically. According to the orange peel extract, the formulated bioactive nanopackaging films observed antioxidant and antimicrobial activity. The results revealed that all treatments outperformed the control, especially treatments T4 (1% Nano Chitosan + 1% Nano Cellulose + 3% Orange Peel Waste extract) and T5 (1% Nano Chitosan + 1% Nano Cellulose + 5% Orange Peel Waste extract), in terms of fruit decay percentage (11.72 ± 11.4 C and 12.33 ± 10.83 C, respectively), weight loss (3.81 ± 2.29B and 3.77 ± 2.22B, respectively), TSS/acidity (17.07 ± 1.14 A and 16.77 ± 1.18 A, respectively), fruit firmness (12.83 ± 1.19B and 13.48 ± 0.91 A, respectively), total sugars (7.98 ± 0.21 A and 8.21 ± 0.29 A, respectively) and total anthocyanin (0.21 ± 0.03B and 0.25 ± 0.05 A, respectively).
In recent years, there has been growing attention towards developing renewable energy and materials derived from abundant biomass resources. 5-Hydroxymethylfurfural (HMF) is recognized as a promising bio-based platform compound for synthesizing value-added chemicals and materials due to its versatile reactivity. HMF can be directly synthesized from carbohydrates and various raw biomass through the acid-hydrolysis reaction. Heterogeneous catalysts have gained prominence in biomass conversion owing to their environmental friendliness, facile separation from reaction mixtures, high catalytic efficiency, and reduced corrosivity toward equipment. This review systematically examines the reaction pathways and mechanisms involved in HMF synthesis from fructose, glucose, cellulose, and raw biomass using heterogeneous catalysts. Then we give an introduction to the preparation of furandicarboxylic acid (FDCA) from HMF with different catalytic methods. FDCA is an important degradable bio-material monomer for polyethylene furanoate to replace petroleum-based polyethylene terephthalate. This review ends with a prospect on the challenges and opportunities of HMF synthesis in the near future.
A new green composite material with antioxidant properties and specific-targeting capabilities was developed using Montmorillonite Clay in modified forms for rosemary essential oil gaseous adsorption without harmful chemicals or excessive heat. The modified clays were tested for their adsorption capabilities, with the sodic clay showing the highest affinity for the essential oil. The study also found that the adsorbed essential oil on sodic clay had the highest antioxidant activity compared to the organic and pillared clays. Adsorption isotherms revealed values of 40, 30, and 18 mg g−1 for sodic, organic, and pillared clay respectively. To assess the experimental data three models were used: First Order, Second Order, and Intra-particular Diffusion. According to the models’ data, the Second Order model fits the experimental results. These results suggest combining modified clays with essential oils can create materials suitable for controlled release systems in food and pharmaceutical applications.
Highlights
| • | The capacity of gaseous adsorption of REO on sodic, organic, and pillared clays depends essentially on the interaction between the clay’s external surface and the essential oil-active molecules. |
| • | Adsorbed essential oil on clays’ surfaces without any direct contact shows an antioxidant activity against DPPH that can be explored in further research concerning food packaging, drug delivery…etc. |
| • | The desorbed amount of REO is practically less than half of the adsorbed quantity which involves both physisorption and chemisorption interactions. |
5-Aminopentanol (5-AP) is a valuable amino alcohol with potential applications in polymer synthesis and bioplastics. Conventional production methods rely on petroleum-based feedstocks and metal catalysts, which raise environmental and sustainability concerns. In this study, a de novo biosynthetic pathway for 5-AP production from l-lysine was developed in Escherichia coli. The engineered pathway consisted of lysine decarboxylase 2 (LdcC), putrescine aminotransferase (PatA), and tested aldehyde reductase (YahK, YihU, YqhD). Among the tested reductases, aldehyde reductase exhibited the highest catalytic efficiency, producing 44.5 ± 2.6 mM of 5-AP (0.44 ± 0.03 mol5 − AP/mol l−lysine). The replacement of the expression system with a T7-based dual-plasmid platform, pET24ma::ldcC, and pCDFDuet-1::yqhD::patA co-transformed into E. coli, increased the production to 60.7 ± 5.8 mM, accompanied by reduced cadaverine accumulation. Further enhancement was achieved by increasing the gene dosage of PatA, leading to 68.5 ± 4.2 mM 5-AP and reduced by 40% in cadaverine levels. Cadaverine is a precursor in the production of 5-AP, and its accumulation is an important factor in the limitation of conversion to 5-AP. Intracellular cofactor regeneration is expected to cause an indirect supply of α-KG, a cofactor, to enhance conversion to 5-AP. To support intracellular cofactor regeneration, glucose supplementation and increased aeration were applied, resulting in a final titer of 78.5 ± 1.2 mM 5-AP and improved precursor utilization. This study is the first report of selective microbial 5-AP production and highlights the importance of PatA expression in pathway optimization. The newly established l-lysine (C6) valorization process which converts l-lysine to high-value materials such as 1,5-PDO, glutarate, and 5-AP offers a promising route for the sustainable biosynthesis of amino alcohols, laying the groundwork for future improvements through enzyme engineering and metabolic design.
The accumulation of plastic waste presents a significant worldwide environmental challenge. This study aimed to isolate polyethylene-degrading bacteria from marine samples containing plastic waste. Four culturable bacterial isolates: Micrococcus luteus, Bacillus cereus, Enterococcus faecalis, and Actinomyces sp. were assessed for their biofilm formation, biosurfactant, and protease production. Gamma irradiation was used to induce structural changes and promote bacterial colonization and biofilm formation on low-density polyethylene microplastics (LDPE MPs). Optimal biofilm formation was achieved in minimal media supplemented with 30% tryptic soy broth, 10% biosurfactant, and 300 µM calcium chloride. The factorial design experiment demonstrated that adding media supplementation significantly improved bacterial colonization and biofilm formation when compared to gamma irradiation. This was supported with Scanning Electron Microscopy (SEM), Energy Dispersive X-ray (EDX) mapping, and Fourier Transform Infrared Spectroscopy (FTIR). The optimized LDPE MP degradation was achieved through a multi-step protocol: (1) samples are pre-treated to 40 kGy gamma irradiation, which resulted in 5.7% Gravimetric weight loss and structural and morphological changes, (2) incubation in biofilm inducing media overnight, and (3) further incubation in minimal media for 30 days. This approach resulted in a total weight loss of 22.5%. In conclusion, synergistic pre-treatment is recommended to promote biofilm and improve biodegradation of LDPE MPs by marine bacteria.
The sugarcane bagasse was analyzed for Particle Size Distribution (PSD) with a mean geometric diameter of 0.722 mm. Various standard techniques assessed its physical and chemical properties, including density measurements, higher heating value (HHV), thermogravimetric analysis (TGA/DTA), and compositional, proximate, ultimate, and CHNS/O analysis. The raw bagasse showed higher volatile matter, fixed carbon, ash content, and HHV of 16 MJ/kg, with lower moisture content (8.71%). Thermal analysis indicated a peak degradation temperature for organic matter at 310–330 °C, and bagasse exhibited a higher combustion index than fossil fuels and other biomasses. Logarithmic models were obtained to determine the real, particle, and apparent densities of bagasse with the mean particle size within the 0.075–9.5 mm range, showing adequate results for particles with a mean diameter greater than 0.15 mm. For smaller particles, the reported errors were 12.6%, 8.23%, and 28%, respectively. These findings highlight sugarcane bagasse's significant potential for thermochemical conversion systems and its importance in selecting and designing fluidized bed technologies like pneumatic conveying, drying, combustion, and gasification equipment.
High-molecular weight heparosan (HMW-heparosan) is a member of the glycosaminoglycan family. It possesses various chemical and physical properties suitable for a range of high-quality tissue engineering biomaterials, gels, scaffolds, and drug delivery systems. In this study, the HMW-heparosan biosynthesis pathway was engineered in Corynebacterium glutamicum through the introduction of heparosan synthase PmHS2 from Pasteurella multocida combined with overexpression of the key genes ugdA and galU, resulting in the generation of a stable HMW-heparosan-producing strain. Subsequently, to address metabolic flux competition, endogenous glycosyltransferases were systematically deleted to minimize UDP-glucose consumption, leading to a significant increase in HMW-heparosan accumulation. Additionally, cell growth was optimized by overexpressing transcriptional regulators whcD and PnkB, which was found to improve cell growth while creating an improved intracellular environment for biosynthesis. Notably, the critical enzyme heparosan synthase PmHS2 was relocated to the cell membrane by cell membrane display motifs porB, with its stability and catalytic efficiency being significantly enhanced so that the titer of HMW-heparosan reached 1.40 g/L in shake-flasks. Ultimately, the engineered strain was demonstrated to achieve HMW-heparosan production at 7.02 g/L with an average molecular weight (Mw) of 801 kDa in 5 L fed-batch bioreactor. These results demonstrate combinatorial optimization of cell factories, especially cell morphology and membrane localization of key enzymes, is efficacious and likely applicable for the production of other biopolymers.
Catha edulis (Khat) waste (KW) is one of the challenging waste managements in Ethiopian urban areas. While biochar from other biomass sources has been studied, the effect of pyrolysis conditions on Catha edulis waste-based biochar yield and quality remains unexplored. Therefore, this study aims to optimize the biochar production process from Catha edulis waste for high yield and desirable characteristics. The KW and biochar were characterized using FTIR, BET, proximate analysis and other key parameters. The results indicated that KW possesses favorable properties for thermochemical conversion, with low ash content (4.35% wt. dry basis) and significant organic constituents (46.89% cellulose, 28.53% lignin, 19.62% hemicellulose, 4.96% extractives). The effect of pyrolysis process variables embracing reaction temperature, reaction time, and particle size on biochar yield and quality was optimized using response surface methodology (RSM) coupled with central composite design (CCD). The biochar was desirably characterized by a pH of 8.96, fixed carbon of 60.08%, ash content of 10.55%, and a yield of 45.12% at the optimum production processes of 390 °C, 44 min, and 0.7 mm particle size. Moreover, the study found that pyrolysis temperature was the most influential factor across all responses (yield and quality). Consequently, the biochar (yield and quality) was significantly (p < 0.05) influenced by pyrolysis temperature. In conclusion, the study inferred that KW holds substantial potential for biochar production with remarkable soil amendment characteristics.
Atmospheric and Room Temperature Plasma (ARTP) mutagenesis has emerged as a novel and powerful physical mutation technology for microbial strain improvement recently. ARTP operates at atmospheric pressure and room temperature using a helium plasma jet, inducing widespread genomic mutations through reactive species and DNA damage. Compared to traditional mutagenesis methods, ARTP is safer, more efficient, and capable of producing high mutation rates without genetic modification, making it a valuable and sophisticated tool in biomanufacturing. This review outlines the principles and diverse applications of ARTP technology for enhancing enzyme activity, metabolite yields, and stress tolerance across various organisms. It also provides a comprehensive discussion of underlying biological mechanisms, workflow, optimization parameters, and potential cellular instability associated with ARTP-induced mutagenesis. Finally, current breakthroughs and future perspectives of ARTP mutagenesis are addressed, emphasizing its role in advancing next-generation microbial platforms for industrial biotechnology and environmental sustainability.
As the world’s top producers of oil palm (Elaeis guineensis), Indonesia and Malaysia are urged to propose a value-added valorization of its lignocellulosic biomass, oil palm empty fruit bunches (OPEFB). Meanwhile, the nations’ signature ‘batik’ textile industries are in dire need of optimum remediation treatments of their wastewater high in harmful dyes and chemicals. Organic–inorganic hybrid systems of mixed matrix membranes (MMMs) for heavy metals removal were prepared using OPEFB-based cellulose acetate (CA) and zinc oxide (ZnO; 0.5, 0.75, 1%, w/v) in N-methyl pyrrolidinone (NMP; 89, 90, 91%, v/v). The high crystallinity (62.42%) and fibrils’ web-like structure of OPEFB-CA were confirmed. Microscopic observation of OPEFB CA-NMP-ZnO membranes evidenced the porous yet smooth surface due to the use of plasticizing NMP, as well as uniform dispersion of ZnO particles. MMM 2 (0.75%ZnO; 90%NMP) was the best-performing membrane mechanically with excellent tensile strength (1.78 MPa), Young’s modulus (0.13 GPa), and elongation-at-break (2.59%), while thermal stability (Td,5%, 291 °C) improvement was also highlighted. Pores characteristics on size, volume, and surface area were discussed, too. Remediation performance was excellent even at high (20%) effluent concentration reaching 28% and 65% removal of Cu and Pb, respectively, by MMM 1 (0.5%ZnO; 89%NMP). These findings confirmed the promising prospect of the developed membranes as a wastewater remediation treatment, including in textile industries.
The display of enzymes on bacterial surfaces is an interesting approach for immobilising industrially important biocatalysts. In recent years, non-recombinant surface display using food-grade bacteria, such as lactic acid bacteria (LAB), have gained interest because of their safety, simplicity, and cost-effectiveness. β-Xylosidase is one of the many biocatalytic enzymes targeted for immobilisation due to its key role in the complete saccharification of lignocellulosic biomass, including xylan hemicellulose. Recently, the xylose-tolerant β-xylosidase, LfXyl43, was identified in Limosilactobacillus fermentum. LfXyl43 is capable of producing xylose from the degradation of xylo-oligosaccharides (XOS) and beechwood xylan. This study aimed to immobilise this new biocatalyst on the surface of LAB-derived bacteria-like particles (BLP) and investigate its applicability and reusability in the degradation of xylan hemicellulose. Additionally, the influence of the anchor position and the presence of linker peptides on the display and activity of the β-xylosidase was investigated. Four expression vectors were constructed to express different anchor-xylosidase fusion proteins. Upon expression and purification, all anchor-xylosidase fusion proteins were active towards the artificial substrate p-nitrophenyl-β-D-xylopyranoside. In addition, all anchor-xylosidase fusion proteins were successfully displayed on the surface of BLP. However, only the β-xylosidases with linker peptide showed hydrolytic activity after immobilisation on BLP. BLP displaying β-xylosidases demonstrated high activity against XOS and beechwood xylan, thereby producing high amounts of xylose. Moreover, the immobilised enzyme demonstrated reusability across several bioconversion cycles. Overall, this study highlights the potential industrial application of surface-displayed β-xylosidase for the effective degradation of lignocellulosic biomass.
Plant probiotics are bacteria that play a significant role in enhancing plant growth and health. To understand the interactions between plant probiotics and host plants, a comprehensive approach of antagonistic activity and analytical methods such as high-performance liquid chromatography (HPLC), gas chromatography‒mass spectrometry (GC‒MS), and Fourier transform infrared (FT‒IR) spectroscopy, were employed. The previously isolated bacterial strains, namely, Corynebacterium accolens strain CNTC Th1/57, Bacillus rugosus strain SPB7, Lactobacillus pasteurii DSM 23907 and Cytobacillus firmus strain NBRC 15306, were exposed to antagonistic testing against Botrytis cinerea and Fusarium oxysporum. Considering the results of the antagonistic activity both in vitro and statistically, the bacterial strains Bacillus rugosus strain SPB7 and Lactobacillus pasteurii DSM 23907 presented greater zones of inhibition. Hence these bacteria were moved to obtain comprehensive insights into the chemical composition. HPLC and GC‒MS resulted in the identification of phenols and organic acids. These results were further confirmed by FT-IR, which revealed a peak at 3500 cm−1 for Bacillus rugosus strain SPB7, where O–H, aromatic C-H and aromatic C = C stretching vibrations were also observed at 3069 and 1549 cm−1. The peak at 1736 cm−1 corresponds to the carboxyl group (-COOH) as the functional group with respect to Lactobacillus pasteurii DSM 23907. Further confirmation was performed by observing the other absorption bands at 3451 cm−1 and 2958 cm−1, indicating the presence of hydroxyl group (O–H) and alkyl group (C-H) stretching vibrations, thus confirming their potential for the production of phenols and organic acids, respectively, by bacteria. This findings would make a way to explore plant diseases, tolerance against pathogens, and also study ecological role of these bacteria in plant communities.
Galactomannan oligosaccharides (GMOS), composed of 2–10 mannose units linked with β-1, 4 glycosidic bond as the main chain and galactose linked with α-1, 6 glycosidic bond as the side chain, are crucial for probiotic food synthesis due to their ability to promote the growth and activity of beneficial intestinal microbiota, enhance the host immune system, and improve nutrient digestion. GMOS is usually obtained by hydrolyzing plants such as locust bean gum and guar gum with mannanase. β-mannanase ManA from Alkaliphilic Bacillus sp. N16-5 can hydrolyze β-1, 4 glycosidic bond of galactomannan. In this study, an immobilization system was employed utilizing polyhydroxyalkanoate (PHA) biopolymers, which naturally have an affinity mainly mediated by hydrophobic interaction for PhaP protein. Fusion protein combining ManA with PhaP from Aeromonas hydrophila, was subsequently immobilized on PHA support to form a multi-enzyme complex, facilitating the hydrolysis of locust bean gum to generate GMOS. This immobilized enzyme enhances enzyme stability and reusability, can be reused up to 32 times while maintaining ~ 80% of its activity, offering substantial cost savings through in-situ enzyme and product separation. Additionally, the different PHA forms were developed to hydrolyze locust bean gum to produce GMOS, such as nano PHA particles, PHA electrospun materials, while these preliminary investigations show promise, further research is needed to optimize their performance and practical application.
Rising environmental concerns and fossil fuel depletion necessitate the search for sustainable, alternative energy sources. Biodiesel is emerging as a viable alternative owing to its biodegradability, low toxicity, and reduced greenhouse gas emissions. This review highlights the environmental and economic advantages of biodiesel, with a special emphasis on microbial lipids from wastewater as a promising third-generation feedstock. The novelty of this review is the comprehensive evaluation of wastewater and sewage sludge as low-cost, nutrient-rich substrates for microbial lipid production. In this review, the biodiesel production by direct lipid extraction and microbial conversion routes is discussed. Biodiesel production from different types of industrial wastewaters, such as municipal, dairy, pulp and paper, brewery, and textile wastewater using oleaginous microorganisms is discussed as well. This review revealed that lipid content in different microbes ranged from 27 to 90% when different wastewaters were used as substrates. Further, this review compares the fuel properties of biodiesel derived from plant oils, animal fats, and microbial lipids, their fatty acid profiles, and estimated production costs, and it varies from 0.1 to 0.83 USD/L. In addition, this review also focuses on the biodiesel production from wastewater using microalgae, fungi, and bacteria. Biodiesel with a high cetane number of 64.47 was produced by Cryptococcus curvatus. Finally, key challenges in producing biodiesel from wastewater and future prospects are discussed, emphasizing the potential of wastewater as a substrate for sustainable biodiesel production.