In the twenty-first century, water contamination with pharmaceutical residues is becoming a global phenomenon and a threat. Antibiotic residues and antibiotic resistance genes (ARGs) are recognized as new emerging water pollutants because they can negatively affect aquatic ecosystems and human health, thereby posing a complex environmental problem. These nano-adsorbents of the next generation can remove these pollutants at low concentrations. This study focuses on the chemical synthesis of copper oxide nanoparticles (CuONPs) and nano-zero-valent iron (nZVI) used as nano-adsorbents for levofloxacin removal from water samples and antibiotic-resistant genes. The CuONPs and nZVI are initially characterized by transmission electron microscopy, scanning electron microscopy, and X-ray diffraction. The levofloxacin adsorption isotherm on the CuONPS and nZVI shows the best fit with the Langmuir isotherm model, exhibiting correlation coefficients (R ²) of 0.993 and 0.999, respectively. The adsorption activities of CuONPS and nZVI were fitted to a pseudo-second-order kinetic model with correlation coefficients (R ²) of 0.983 and 0.994, respectively. The maximum levofloxacin removal capacity was observed at (89%), (84%), (89%), (88%) and (71.6) at pH 7 and adsorbent dose(0.06 mg/L), initial LEV concentration (1 mg/L), temperature 25 °C, and contact time 120 min for CuONPs. Removal efficiency was (91%), (90.6%), (91%), (89%), and (80%), at pH 7, adsorbent dose(0.06), initial LEV concentration (1 mg/L), temperature 35 °C, and contact time 120 min. The levofloxacin adsorption is an exothermic process for nZVI and CuONPs, according to thermodynamic analysis. A thermodynamic analysis indicated that each adsorption process is spontaneous. Several genera, including clinically pathogenic bacteria (e.g., Acinetobacter_baumannii, Helicobacter_pylori, Escherichia_coli, Pseudomonas_aeruginosa, Clostridium_beijerinckii, Escherichia/Shigella_coli, Helicobacter_cetorum, Lactobacillus_gasseri, Bacillus_cereus, Deinococcus_radiodurans, Rhodobacter_sphaeroides, Propionibacterium_acnes, and Bacteroides_vulgatus) were relatively abundant in hospital wastewater. Furthermore, 37 antibiotic resistance genes (ARGs) were quantified in hospital wastewater. The results demonstrated that 95.01% of nZVI and 91.4% of CuONPs are effective adsorbents for removing antibiotic-resistant bacteria from hospital effluent. The synthesized nZVI and CuONPs have excellent reusability and can be considered cost effective and eco-friendly adsorbents.

Physcion is an anthraquinone compound observed dominantly in medicinal herbs. This anthraquinone possesses a variety of pharmaceutically important activities and has been developed to be a widely used antifungal biopesticide. Herein, we report on the effective preparation of 3R-torosachrysone (4), a tetrahydroanthracene precursor of physcion, in Aspergillus oryzae NSAR1 by heterologous expression of related genes mined from the phlegmacins-producing ascomycete Talaromyces sp. F08Z-0631. Conditions for converting 4 into physcion were studied and optimized, leading to the development of a concise approach for extracting high-purity physcion from the alkali-treated fermentation broth of the 4-producing A. oryzae strain.

Microbial electrosynthesis (MES) is a promising technology for CO₂ fixation and electrical energy storage. Currently, the low current density of MES limits its practical application. The H₂-mediated and non-biofilm-driven MES could work under higher current density, but it is difficult to achieve high coulombic efficiency (CE) due to low H₂ solubility and poor mass transfer. Here, we proposed to enhance the hydrogen mass transfer by adding silica nanoparticles to the reactor. At pH 7, 35 ℃ and 39 A·m^− 2 current density, with the addition of 0.3wt% silica nanoparticles, the volumetric mass transfer coefficient (k_La) of H₂ in the reactor increased by 32.4% (from 0.37 h^− 1 to 0.49 h^− 1), thereby increasing the acetate production rate and CE of the reactor by 69.8% and 69.2%, respectively. The titer of acetate in the reactor with silica nanoparticles (18.5 g·L^− 1) was 56.9% higher than that of the reactor without silica nanoparticles (11.8 g·L^− 1). Moreover, the average acetate production rate of the reactor with silica nanoparticles was up to 2.14 g·L^− 1·d^− 1 in the stable increment phase, which was much higher than the other reported reactors. These results demonstrated that the addition of silica nanoparticles is an effective approach to enhancing the performance of H₂-mediated MES reactors.

Apolipoprotein A-I_Milano (Apo A-I_Milano) is a natural mutant of Apolipoprotein. It is currently the only protein that can clear arterial wall thrombus deposits and promptly alleviate acute myocardial ischemia. Apo A-I_Milano is considered as the most promising therapeutic protein for treating atherosclerotic diseases without obvious toxic or side effects. However, the current biopharmaceutical platforms are not efficient for developing Apo A-I_Milano. The objectives of this research were to express Apo A-I_Milano using the genetic transformation ability of N. tabacum. The method is to clone the coding sequence of Apo A-I_Milano into the plant binary expression vector pCHF3 with a Flag/His6/GFP tag. The constructed plasmid was transformed into N. tabacum by a modified agrobacterium-mediated method, and transformants were selected under antibiotic stress. PCR, RT-qPCR, western blot and co-localization analysis was used to further verify the resistant N. tabacum. The stable expression and transient expression of N. tabacum were established, and the pure product of Apo A-I_Milano was obtained through protein A/G agarose. The results showed that Apo A-I_Milano was expressed in N. tabacum with a yield of 0.05 mg/g leaf weight and the purity was 90.58% ± 1.65. The obtained Apo A-I_Milano protein was subjected to amino acid sequencing. Compared with the theoretical sequence of Apo A-I_Milano, the amino acid coverage was 86%, it is also found that Cysteine replaces Arginine at position 173, which indicates that Apo A-I_Milano, a mutant of Apo A-I, is accurately expressed in N. tabacum. The purified Apo A-I_Milano protein had a lipid binding activity. The established genetic modification N. tabacum will provide a cost-effective system for the production of Apo A-I_Milano. Regarding the rapid propagation of N. tabacum, this system provides the possibility of large-scale production and accelerated clinical translation of Apo A-I_Milano.

In order to meet the contemporary concept of sustainable development, the reuse of biological waste has also been emphasized. Lots of papers nowadays study the extraction of primary residues. The disposal of secondary residues is often neglected. The chemical composition and biological activity of secondary residues of Turkish Gall (SRTG) were researched in this paper. We selected five methods to extract the SRTG, and the extraction conditions were water, hydrochloric acid buffer (pH = 2), artificial gastric juice (pH = 2), phosphate buffer (pH = 6.8), and artificial intestinal solution (pH = 6.8). The changes of phenolic components were determined by spectrophotometry and high-performance liquid chromatography. The acid-base environment did not affect total polyphenols contents and gallic acid ethyl ester contents in SRTG. But it affected the gallic acid contents in SRTG. The contents of gallic acid in the hydrochloric acid buffer extraction groups were 1.63 times that of the water extraction group. The SRTG were extracted by hydrochloric acid buffer also had better inhibition on Escherichia coli and Staphylococcus aureus. In addition, SRTG showed positive effects on 1,1-Diphenyl-2-picrylhydrazyl Free, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), ·OH radicals, and Ferric ion reducing antioxidant power. Some active components of SRTG can be effectively released through the digestion of simulated gastric juices in vitro. The change of active ingredients affects the antibacterial and antioxidant capacity. The results provide data support for the conversion of secondary residues into products, such as feed additives. The SRTG has certain contributes to the value of the circular economy.

Cellulose extraction from gloss art paper (GAP) waste is a recycling strategy for the abundance of gloss art paper waste. Here, a study was conducted on the impact of ultrasonic homogenization for cellulose extraction from GAP waste to improve the particle size, crystallinity, and thermal stability.

At treatment temperature of 75.8 °C, ultrasonic power level of 70.3% and 1.4 h duration, cellulose with properties of 516.4 nm particle size, 71.5% crystallinity, and thermal stability of 355.2 °C were extracted. Surface modification of cellulose GAP waste with H₃PO₄ hydrolysis and 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) oxidation was done followed by starch reinforcement. Surface hydrophobicity and mechanical strength were increased for H₃PO₄ hydrolysis and TEMPO oxidation starch–cellulose. No reduction of thermal properties observed during the treatment, while increment of crystallinity index up to 47.65–59.6% was shown. Neat starch film was more transparent, followed by starch–TEMPO film and starch–H₃PO₄ film, due to better homogeneity.

The cellulose GAP reinforced starch film shows potential in developing packaging materials and simultaneously provide an alternative solution of GAP waste recycling.

Cellulose is the most abundant renewable bioresources on earth, and the biodegradation and utilization of cellulose would contribute to the sustainable development of global environment. Sporocytophaga species are common aerobic cellulose-degrading bacteria in soil, which can adhere to the surface of cellulose matrix and motile by gliding. In this study, a differential transcriptome analysis of Sporocytophaga sp. CX11 was performed and a total of 4,217 differentially expressed genes (DEGs) were identified. Gene Ontology enrichment results showed that there are three GO categories related to cellulose degradation function among the annotated DEGs. A total of 177 DEGs were identified as genes encoding carbohydrate-active enzymes (CAZymes), among which 54 significantly upregulated CAZymes were mainly cellulases, hemicellulases, pectinases, etc. 39 DEGs were screened to associate with gliding function. In order to explore unannotated genes potentially related to cellulose metabolism, cluster analysis was performed using the Short-Time Series Expression Miner algorithm (STEM). 281 unannotated genes were predicted to be associated with the initial-middle stage of cellulose degradation and 289 unannotated genes might function in the middle-last stage of cellulose degradation. Sporocytophaga sp. CX11 could produce extracellular endo-xylanase, endo-glucanase, FPase and β-glucosidase, respectively, according to different carbon source conditions. Altogether, this study provides valuable insights into the transcriptome information of Sporocytophaga sp. CX11, which would be useful to explore its application in biodegradation and utilization of cellulose resources.

Synthetic biology has rapidly advanced from the setup of native genetic devices to the design of artificial elements able to provide organisms with highly controllable functions. In particular, genetic switches are crucial for deploying new layers of regulation into the engineered organisms. While the assembly and mutagenesis of native elements have been extensively studied, limited progress has been made in rational design of genetic switches due to a lack of understanding of the molecular mechanism by which a specific transcription factor interacts with its target gene. Here, a reliable workflow is presented for designing two categories of genetic elements, one is the switch element-MtlR box and the other is the transcriptional regulatory element- catabolite control protein A (CcpA) box. The MtlR box was designed for ON/OFF-state selection and is controlled by mannitol. The rational design of MtlR box-based molecular structures can flexibly tuned the selection of both ON and OFF states with different output switchability in response to varied kind effectors. Different types of CcpA boxes made the switches with more markedly inducer sensitivities. Ultimately, the OFF-state value was reduced by 90.69%, and the maximum change range in the presence of two boxes was 15.31-fold. This study presents a specific design of the switch, in a plug-and-play manner, which has great potential for controlling the flow of the metabolic pathway in synthetic biology.

Vibrio species (Vibrio sp.) is a class of Gram-negative aquatic bacteria that causes vibriosis in aquaculture, which have resulted in big economic losses. Utilization of antibiotics against vibriosis has brought concerns on antibiotic resistance, and it is essential to explore potential antibiotic alternatives. In this study, seven compounds (compounds 1–7) were isolated from the Arctic endophytic fungus Penicillium sp. Z2230, among which compounds 3, 4, and 5 showed anti-Vibrio activity. The structures of the seven compounds were comprehensively elucidated, and the antibacterial mechanism of compounds 3, 4, and 5 was explored by molecular docking. The results suggested that the anti-Vibrio activity could come from inhibition of the bacterial peptide deformylase (PDF). This study discovered three Penicillium-derived compounds to be potential lead molecules for developing novel anti-Vibrio agents, and identified PDF as a promising antibacterial target. It also expanded the bioactive diversity of polar endophytic fungi by showing an example in which the secondary metabolites of a polar microbe were a good source of natural medicine.

The effect of fermentation by Saccharomyces cerevisiae on biological properties of cinnamon (Cinnamomum cassia) was investigated. The study demonstrated that the extract of S. cerevisiae-fermented cinnamon (S.C.FC) has antioxidants higher than non-fermented one. The optimum results for antioxidant yield were noted with 10⁷ CFU S. cerevisiae/10 g cinnamon and 70 mL of dH₂O at pH 6 and incubated for 3 d at 35 °C. Under optimum conditions, ABTS, DPPH, and H₂O₂ radical-scavenging activity increased by 43.8, 61.5, and 71.9%, respectively. Additionally, the total phenols and flavonoids in S.C.FC were increased by 81.3 and 415% compared by non-fermented one. The fermented cinnamon had antimicrobial activity against L. monocytogenes, S. aureus, E. coli, S. typhi, and C. albicans. Also, the anti-inflammatory properties were increased from 89 to 92% after fermentation. The lyophilized extract of S.C.FC showed positive effect against Huh7 cancer cells which decreased by 31% at the concentration of 700 µg/mL. According to HPLC analysis, p-hydroxybenzoic acid, gentisic acid, catechin, chlorogenic acid, caffeic acid, and syringic acid were increased by 116, 33.2, 59.6, 50.6, 1.6, and 16.9%, respectively. Our findings suggest the applicability of cinnamon fermentation using S. cerevisiae as a useful tool for processing functional foods to increase their antioxidant and anti-inflammatory content.

Pulse meal should be a valuable product in the animal feed industry based on its strong nutritional and protein profiles. However, it contains anti-nutritional compounds including phenolics (large and small molecular weight), which must be addressed to increase uptake by the industry. Microbial fermentation is currently used as a strategy to decrease larger molecular weight poly-phenolics, but results in the undesirable accumulation of small mono-phenolics. Here, we investigate cell-free biocatalytic reduction of phenolic content in faba bean (Vicia faba L.) meal. A representative phenolic ring-breaking catechol dioxygenase, Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O) was used in this proof-of concept based on its known stability and broad substrate specificity. Initially, large-scale fermentative recombinant production and purification of BLC23O was carried out, with functionality validated by in vitro kinetic analysis. When applied to faba bean meal, BLC23O yielded greatest reductions in phenolic content in a coarse air classified fraction (high carbohydrate), compared to either a fine fraction (high protein) or the original unfractionated meal. However, the upstream hydrolytic release of phenolics from higher molecular weight species (e.g. tannins, or complexes with proteins and carbohydrates) likely remains a rate limiting step, in the absence of other enzymes or microbial fermentation. Consistent with this, when applied to a selection of commercially available purified phenolic compounds, known to occur in faba bean, BLC23O was found to have high activity against monophenolic acids and little if any detectable activity against larger molecular weight compounds. Overall, this study highlights the potential viability of the biocatalytic processing of pulse meals, for optimization of their nutritional and economical value in the animal feed industry.

Fish swim bladders used to be considered as byproducts or waste in fishery; however, they are potential materials for biological medicine with abundant collagen. In this work, an efficient noncytotoxic decellularization process using sodium dodecyl sulfate (SDS) ternary system assisted with supercritical carbon dioxide (scCO₂) as the green extraction fluid and ethanol (ET) as the cosolvent has been developed to harvest acellular fish swim bladders (AFSBs). The experimental results show that the tissue treated by SDS assisted with scCO₂ and ethanol at 37 °C and 25 MPa can be decellularized thoroughly and maintains intact fibers and uniform pore distribution, which resulting in a tensile strength of 5.61 MPa and satisfactory biocompatibility. Meanwhile, the residual SDS content in scCO₂/SDS/ET ternary system is 0.0122% which is significantly lower than it in scCO₂/SDS system due to the enhanced mass transfer rate of SDS in tissues by scCO₂ with ethanol. The synergy between SDS and ethanol can enhance the diffusion coefficient and the solubility of SDS in scCO₂, which reduced the contact time between SDS and tissues. Meaningfully, the results obtained in this work can not only provide a novel strategy to produce acellular matrix with superior properties, but also offer a further understanding of the decellularization through scCO₂ extraction processing with the synergy of suitable detergent/cosolvent.

Tetrahydroisoquinoline alkaloids (THIQAs) are ubiquitous compounds with important pharmaceutical and biological activity. Their key N-heterocyclic structural motifs are synthesised via Pictet–Spengler (P–S) reaction by norcoclaurine synthases (NCS) in plants. The synthesis of 1-aryl-tetrahydroisoquinoline alkaloids has attracted increasing attention due to their antitumor and antivirus activities. Herein, the L68T/M97V mutant of NCS from Thalictrum flavum with improved activity was developed by semi-rational design. This mutant not only showed higher catalytic performance (> 96% conversion) toward benzaldehyde and dopamine over the wild-type enzyme, but also catalysed the P–S reaction of the bulky substrate 4-biphenylaldehyde and dopamine with high conversion (> 99%) for the effective synthesis of 1-aryl-THIQA. In terms of stereoselectivity, all products synthesised by the L68T/M97V mutant showed high optical purity (92–99% enantiomeric excess).

Lipids produced by oleaginous yeasts are considered as sustainable sources for the production of biofuels and oleochemicals. The red yeast Rhodosporidium toruloides can accumulate lipids to over 70% of its dry cell mass. To facilitate lipid extraction, a recombinant β-1,3-glucomannanase, MAN5C, has been applied to partially breakdown R. toruloides cell wall. In this study, R. toruloides NP11 was engineered for secretory expression of MAN5C to simplify the lipid extraction process. Specifically, a cassette contained a codon-optimized gene MAN5C was integrated into the genome of R. toruloides by Agrobacterium-mediated transformation. The engineered strain NP11-MAN5C was found with proper expression and secretion of active MAN5C, yet no notable compromise in terms of cell growth and lipid production. When NP11-MAN5C cell cultures were extracted with ethyl acetate without any pretreatment, 20% of total lipids were recovered, 4.3-fold higher than that of the parental strain NP11. When the cells were heat-treated followed by extraction with ethyl acetate in the presence of the culture broth supernatants, up to 93% of total lipids were recovered, confirming beneficial effects of MAN5C produced in situ. This study provides a new strategy to engineer oleaginous yeasts for more viable lipid extraction and down-stream processes.

A novel extracellular polymeric substance (EPS) with flocculating activity produced by Pseudomonas fluorescein isolated from soil was studied in this paper. Firstly, atmospheric and room temperature plasma (ARTP) was applied to get a mutant of P. fluorescein with higher EPS production. A mutant T4-2 exhibited a 106.48% increase in flocculating activity compared to the original strain. The maximum EPS yield from T4-2 was enhanced up to 6.42 g/L, nearly 10 times higher than the original strain on a 3.6-L bioreactor with optimized fermentation conditions. Moreover, the flocculating activity of the mutant reached 3023.4 U/mL, 10.96-fold higher than that of T4. Further identification showed that EPS from mutant T4-2 was mainly composed of polysaccharide (76.67%) and protein (15.8%) with a molecular weight of 1.17 × 10⁵ Da. The EPS showed excellent adsorption capacities of 80.13 mg/g for chromium (VI), which was much higher than many reported adsorbents such as chitosan and cellulose. The adsorption results were described by Langmuir isotherm and pseudo-second-order kinetic model. The thermodynamic parameters (ΔG ⁰, ΔH ⁰ and ΔS ⁰) revealed that the adsorption process was spontaneous and exothermic. Adsorption mechanisms were speculated to be electrostatic interaction, reduction, and chelation.

Mesenchymal stem cells (MSCs) are highly important in biomedicine and hold great potential in clinical treatment for various diseases. In recent years, the capabilities of MSCs have been under extensive investigation for practical application. Regarding therapy, the efficacy usually depends on the amount of MSCs. Nevertheless, the yield of MSCs is still limited due to the traditional cultural methods. Herein, we proposed a three-dimensional (3D) scaffold prepared using poly lactic-co-glycolic acid (PLGA) nanofiber with polylysine (PLL) grafting, to promote the growth and proliferation of MSCs derived from the human umbilical cord (hUC-MSCs). We found that the inoculated hUC-MSCs adhered efficiently to the PLGA scaffold with good affinity, fast growth rate, and good multipotency. The harvested cells were ideally distributed on the scaffold and we were able to gain a larger yield than the traditional culturing methods under the same condition. Thus, our cell seeding with a 3D scaffold could serve as a promising strategy for cell proliferation in the large-scale production of MSCs. Moreover, the simplicity and low preparation cost allow this 3D scaffold to extend its potential application beyond cell culture.

Indigo is an economically important dye, especially for the textile industry and the dyeing of denim fabrics for jeans and garments. Around 80,000 tonnes of indigo are chemically produced each year with the use of non-renewable petrochemicals and the use and generation of toxic compounds. As many microorganisms and their enzymes are able to synthesise indigo after the expression of specific oxygenases and hydroxylases, microbial fermentation could offer a more sustainable and environmentally friendly manufacturing platform. Although multiple small-scale studies have been performed, several existing research gaps still hinder the effective translation of these biochemical approaches. No article has evaluated the feasibility and relevance of the current understanding and development of indigo biocatalysis for real-life industrial applications. There is no record of either established or practically tested large-scale bioprocess for the biosynthesis of indigo. To address this, upstream and downstream processing considerations were carried out for indigo biosynthesis. 5 classes of potential biocatalysts were identified, and 2 possible bioprocess flowsheets were designed that facilitate generating either a pre-reduced dye solution or a dry powder product. Furthermore, considering the publicly available data on the development of relevant technology and common bioprocess facilities, possible platform and process values were estimated, including titre, DSP yield, potential plant capacities, fermenter size and batch schedule. This allowed us to project the realistic annual output of a potential indigo biosynthesis platform as 540 tonnes. This was interpreted as an industrially relevant quantity, sufficient to provide an annual dye supply to a single industrial-size denim dyeing plant. The conducted sensitivity analysis showed that this anticipated output is most sensitive to changes in the reaction titer, which can bring a 27.8% increase or a 94.4% drop. Thus, although such a biological platform would require careful consideration, fine-tuning and optimization before real-life implementation, the recombinant indigo biosynthesis was found as already attractive for business exploitation for both, luxury segment customers and mass-producers of denim garments.

Lignin has enormous potential as a renewable feedstock for depolymerizing to numerous high-value chemicals. However, lignin depolymerization is challenging owing to its recalcitrant, heterogenous, and limited water-soluble nature. From the standpoint of environmental friendliness and sustainability, enzymatic depolymerization of lignin is of great significance. Notably, laccases play an essential role in the enzymatic depolymerization of lignin and are considered the ultimate green catalysts. Deep eutectic solvent (DES), an efficient media in biocatalysis, are increasingly recognized as the newest and utmost green solvent that highly dissolves lignin. This review centers on a lignin depolymerization strategy by harnessing the good lignin fractionating capability of DES and the high substrate and product selectivity of laccase. Recent progress and insights into the laccase–DES interactions, protein engineering strategies for improving DES compatibility with laccase, and controlling the product selectivity of lignin degradation by laccase or in DES systems are extensively provided. Lastly, the challenges and prospects of the alliance between DES and laccase for lignin depolymerization are discussed. The collaboration of laccase and DES provides a great opportunity to develop an enzymatic route for lignin depolymerization.

Chloroethenes are widely used as solvent in the metal industry and the dry cleaning industry, but their spillage into soil and groundwater due to improper handling has negatively impacted human health. Bioremediation using microorganisms is one of the technologies to clean up soil and groundwater contaminated with chloroethenes. In this study, we examined the bioremediation of chloroethene-contaminated soil using wine pomace extract (WPE). WPE is a liquid containing seven major carboxylic acids and other substances extracted from grape pomace produced in winemaking. WPE clearly promoted the anaerobic bioremediation of chloroethenes. In the tetrachloroethene (PCE) degradation test that used fractions derived from WPE, the water-eluted fraction containing l-lactic acid, l-tartaric acid, and others promoted the dechlorination of PCE, whereas the methanol-eluted fraction containing mainly syringic acid did not. In another PCE degradation test that used l-lactic acid, l-tartaric acid, and syringic acid test solutions, l-lactic acid and l-tartaric acid enhanced the dechlorination of PCE, but syringic acid did not. The results suggest that l-lactic acid and l-tartaric acid in WPE function as hydrogen donors in the anaerobic microbial degradation of chloroethene. This technology realizes environmental remediation through the effective use of food by-products.

Hydrothermal carbonization (HTC) reacts with biomass in water at a high temperature and pressure to produce hydrochar with a higher heating value (HHV) and lower ash content than dry torrefaction. The high potassium content in biomass can promote thermochemical conversion; however, it lowers the melting temperature of the ash, causing slugging and fouling. Therefore, this study, investigated the effect of potassium on the HTC of sorghum bagasse by comparing the removal of potassium by washing with the addition of K₂CO₃. Consequently, the ash content was the highest in the potassium-added hydrochar and was 3.81% at a reaction time of 2 h. Elemental analysis showed that the lower the potassium content, the higher the carbon content, and the hydrochar with potassium removed by water washing at a reaction time of 3 h had the highest carbon content at 68.3%. Fourier transform infrared spectrometer showed dehydration and decarboxylation reactions due to HTC, but no significant differences were observed between the potassium concentrations. The mass yield decreased with increasing potassium content, and was 27.2% for the potassium-added hydrochar after 3 h. This trend was more pronounced with increasing reaction temperature. On the other hand, HHV was not affected by the potassium content. Therefore, the energy yield was similar to the weight yield. Thermal gravimetry and derivative thermal gravimetry (TG-DTG) analysis showed that higher potassium tended to accelerate the decomposition of lignin and decrease the oxidation temperature.

Using enzymes to hydrolyze and recycle poly(ethylene terephthalate) (PET) is an attractive eco-friendly approach to manage the ever-increasing PET wastes, while one major challenge to realize the commercial application of enzyme-based PET degradation is to establish large-scale production methods to produce PET hydrolytic enzyme. To achieve this goal, we exploited the industrial strain Pichia pastoris to express a PET hydrolytic enzyme from Caldimonas taiwanensis termed CtPL-DM. In contrast to the protein expressed in Escherichia coli, CtPL-DM expressed in P. pastoris is inactive in PET degradation. Structural analysis indicates that a putative N-glycosylation site N181 could restrain the conformational change of a substrate-binding Trp and hamper the enzyme action. We thus constructed N181A to remove the N-glycosylation and found that the PET hydrolytic activity of this variant was restored. The performance of N181A was further enhanced via molecular engineering. These results are of valuable in terms of PET hydrolytic enzyme production in industrial strains in the future.

Alkaline protease is widely used in the food, detergent, and pharmaceutical industries because of its comparatively great hydrolysis ability and alkali tolerance. To improve the ability of the recombinant Bacillus licheniformis to produce alkaline protease, single-factor experiments and response surface methodology (RSM) were utilized to determine and develop optimal culture conditions. The results showed that three factors (corn starch content, soybean meal content, and initial medium pH) had significant effects on alkaline protease production (P < 0.05), as determined through the Plackett‒Burman design. The maximum enzyme activity was observed with an optimal medium composition by central composite design (CCD): corn starch, 92.3 g/L; soybean meal, 35.8 g/L; and initial medium pH, 9.58. Under these optimum conditions, the alkaline protease activity of strain BL10::aprE was 15,435.1 U/mL, 82% higher than that in the initial fermentation medium. To further investigate the application of the optimum fermentation medium, the overexpressed strain BL10::aprE/pHYaprE was cultured using the optimized medium to achieve an enzyme activity of 39,233.6 U/mL. The present study achieved the highest enzyme activity of alkaline protease by B. licheniformis at the shake-flask fermentation level, which has important application value for large-scale production.

Salvianic acid A (SAA), used for treating cardiovascular and cerebrovascular diseases, possesses several pharmacological properties. However, the current methods for the enzymatic synthesis of SAA show low efficiency. Here, we constructed a three-enzyme cascade pathway in Escherichia coli BL21 (DE3) to produce SAA from l-dihydroxyphenylalanine (L-DOPA). The phenylpyruvate reductase (LaPPR) from Lactobacillus sp. CGMCC 9967 is a rate-limiting enzyme in this process. Therefore, we employed a mechanism-guided protein engineering strategy to shorten the transfer distances of protons and hydrides, generating an optimal LaPPR mutant, LaPPR^Mu2 (H89M/H143D/P256C), with a 2.8-fold increase in specific activity and 9.3-time increase in k_cat/K_m value compared to that of the wild type. Introduction of the mutant LaPPR^Mu2 into the cascade pathway and the optimization of enzyme levels and transformation conditions allowed the obtainment of the highest SAA titer (82.6 g L⁻¹) ever reported in vivo, good conversion rate (91.3%), excellent ee value (99%) and the highest productivity (6.9 g L⁻¹ h⁻¹) from 90 g L⁻¹ L-DOPA in 12 h. This successful strategy provides a potential new method for the industrial production of SAA.

The first-cured tobacco contains macromolecular substances with negative impacts on tobacco products quality, and must be aged and fermented to mitigate their effects on the tobacco products quality. However, the natural fermentation takes a longer cycle with large coverage area and low economic efficiency. Microbial fermentation is a method to improve tobacco quality. The change of chemical composition of tobacco during the fermentation is often correlated with shapes of tobacco. This study aimed to investigate the effects of tobacco microorganisms on the quality of different shapes of tobacco. Specifically, Bacillus subtilis B1 and Cytobacillus oceanisediminis C4 with high protease, amylase, and cellulase were isolated from the first-cured tobacco, followed by using them for solid-state fermentation of tobacco powder (TP) and tobacco leaves (TL). Results showed that strains B1 and C4 could significantly improve the sensory quality of TP, enabling it to outperform TL in overall texture and skeleton of tobacco products during cigarette smoking. Compared with the control, microbial fermentation could increase reducing sugar; regulate protein, starch, and cellulose, reduce nicotine, improve total aroma substances, and enable the surface of fermented TP and TL to be more loose, wrinkled, and porous. Microbial community analysis indicated that strains B1 and C4 could change the native structure of microbial community in TP and TL. LEfSe analysis revealed that the potential key biomarkers in TP and TL were Bacilli, Pseudonocardia, Pantoea, and Jeotgalicoccus, which may have cooperative effects with other microbial taxa in improving tobacco quality. This study provides a theoretical basis for improving tobacco fermentation process for better cigarettes quality.

The 3-Hydroxypropionic acid (3-HP) pathway is one of the six known natural carbon fixation pathways, in which the carbon species used is bicarbonate. It has been considered to be the most suitable pathway for aerobic CO₂ fixation among the six natural carbon fixation pathways. Mesaconate is a high value-added derivative in the 3-HP pathway and can be used as a co-monomer to produce fire-retardant materials and hydrogels. In this study, we use mesaconate as a reporting compound to evaluate the construction and optimization of the sub-part of the 3-HP pathway in Saccharomyces cerevisiae. Combined with fine-tuning of the malonyl-CoA reductase (MCR-C and MCR-N) expression level and optimization of 3-Hydroxypropionyl-CoA synthase, the 3-HP sub-pathway was optimized using glucose or ethanol as the substrate, with the productions of mesaconate reaching 90.78 and 61.2 mg/L, respectively.

Pyrolysis, a thermal decomposition without oxygen, is a promising technology for transportable liquids from whole fractions of lignocellulosic biomass. However, due to the hydrophilic products of pyrolysis, the liquid oils have undesirable physicochemical characteristics, thus requiring an additional upgrading process. Biological upgrading methods could address the drawbacks of pyrolysis by utilizing various hydrophilic compounds as carbon sources under mild conditions with low carbon footprints. Versatile chemicals, such as lipids, ethanol, and organic acids, could be produced through microbial assimilation of anhydrous sugars, organic acids, aldehydes, and phenolics in the hydrophilic fractions. The presence of various toxic compounds and the complex composition of the aqueous phase are the main challenges. In this review, the potential of bioconversion routes for upgrading the aqueous phase of pyrolysis oil is investigated with critical challenges and perspectives.

Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino acid embedding, glycoprotein production, and more. To realize the construction of an efficient eukaryotic CFPS platform with the advantages of low cost and short time, a CFPS system based on the yeast Pichia pastoris was built in this study. The internal ribosomal entry site (IRES) can independently initiate translation and thus promote protein synthesis. The Kozak sequences can facilitate translation initiation. Therefore, the screening of IRES and its combination with Kozak was performed, in which cricket paralysis virus (CRPV) exhibited as the best translation initiation element from 14 different IRESs. Furthermore, the system components and reaction environment were explored. The protein yield was nearly doubled by the addition of RNase inhibitor. The cell extract amount, energy regeneration system (phosphocreatine and phosphocreatine kinase), and metal ions (K⁺ and Mg²⁺) were optimized to achieve the best protein synthesis yield. This P. pastoris CFPS system can extend the eukaryotic CFPS platform, providing an enabling technology for fast prototyping design and functional protein synthesis.

It is of great significance to utilize CO₂ as feedstock to synthesize biobased products, particularly single cell protein (SCP) as the alternative food and feed. Bioelectrochemical system (BES) driven by clean electric energy has been regarded as a promising way for Cupriavidus necator to produce SCP from CO₂ directly. At present, the key problem of culturing C. necator in BES is that reactive oxygen species (ROS) generated in cathode chamber are harmful to bacterial growth. Therefore, it is necessary to find a solution to mitigate the negative effect of ROS. In this study, we constructed a number of C. necator strains displayed with superoxide dismutase (SOD), which allowed the decomposition of superoxide anion radical. The effects of promoters and signal peptides on the cell surface displayed SOD were analyzed. The proteins displayed on the surface were further verified by the fluorescence experiment. Finally, the growth of C. necator CMS incorporating a pBAD-SOD-E-tag-IgAβ plasmid could achieve 4.9 ± 1.0 of OD₆₀₀ by 7 days, equivalent to 1.7 ± 0.3 g/L dry cell weight (DCW), and the production rate was 0.24 ± 0.04 g/L/d DCW, around 2.7-fold increase than the original C. necator CMS (1.8 ± 0.3 of OD₆₀₀). This study can provide an effective and novel strategy of cultivating strains for the production of CO₂-derived SCP or other chemicals in BES.

β-Elemene, an active ingredient found in medicinal plants like turmeric and zedoary, is a sesquiterpene compound with antitumor activity against various cancers. However, its current mode of production through plant extraction suffers from low efficiency and limited natural resources. Recently, there has been an increased interest in establishing microbial cell factories to produce germacrene A, which can be converted to β-elemene by a one-step reaction in vitro. In this study, we constructed an engineered Pichia pastoris cell factory for producing germacrene A. We rerouted the fluxes towards germacrene A biosynthesis through the optimization of the linker sequences between germacrene A synthase (GAS) and farnesyl pyrophosphate synthase (ERG20), overexpression of important pathway genes (i.e., IDI1, tHMG1, and ACS), and multi-copy integration of related expression cassettes. In combination with medium optimization and bioprocess engineering, the final titer of germacrene A in a 1 L fermenter reached 1.9 g/L through fed-batch fermentation. This represents the first report on the production of germacrene A in P. pastoris and demonstrates its advantage in producing terpenoids and other value-added natural products.

Terpenoids are pervasive in nature and display an immense structural diversity. As the largest category of plant secondary metabolites, terpenoids have important socioeconomic value in the fields of pharmaceuticals, spices, and food manufacturing. The biosynthesis of terpenoid skeletons has made great progress, but the subsequent modifications of the terpenoid framework are poorly understood, especially for the functionalization of inert carbon skeleton usually catalyzed by hydroxylases. Hydroxylase is a class of enzymes that plays an important role in the modification of terpenoid backbone. This review article outlines the research progress in the identification, molecular modification, and functional expression of this class of enzymes in the past decade, which are profitable for the discovery, engineering, and application of more hydroxylases involved in the plant secondary metabolism.

Solar radiation varies quantitatively and qualitatively while penetrating through the seawater column and thus is one of the most important environmental factors shaping the vertical distribution pattern of phytoplankton. The haploid and diploid life-cycle phases of coccolithophores might have different vertical distribution preferences. Therefore, the two phases respond differently to high solar photosynthetically active radiation (PAR, 400–700 nm) and ultraviolet radiation (UVR, 280–400 nm). To test this, the haploid and diploid Emiliania huxleyi were exposed to oversaturating irradiance. In the presence of PAR alone, the effective quantum yield was reduced by 10% more due to the higher damage rate of photosystem II in haploid cells than in diploid cells. The addition of UVR resulted in further inhibition of the quantum yield for both haploid and diploid cells in the first 25 min, partly because of the increased damage of photosystem II. Intriguingly, this UVR-induced inhibition of the haploid cells completely recovered half an hour later. This recovery was confirmed by the comparable maximum quantum yields, maximum relative electron transport rates and yields of the haploid cells treated with PAR and PAR + UVR. Our data indicated that photosynthesis of the haploid phase was more sensitive to high visible light than the diploid phase but resistant to UVR-induced inhibition, reflecting the ecological niches to which this species adapts.

There is increasing attention to the production of cellulose nanocrystals (CNCs) from lignocellulosic biomass by enzymatic hydrolysis with cellulase. In this study, the feasibility of the application of a cellulase system from engineered strain Penicillium oxalicum cEES in the production of CNCs was assessed. Using commercial eucalyptus dissolving pulp (EDP) as substrate, the CNCs were successfully obtained by enzymatic hydrolysis with the cellulase cEES, and the total yields of CNCs reached 15.7% through three-step enzymatic hydrolysis of total 72 h (24 h for each step). The prepared CNCs were characterized and found that their crystallinity and thermal stability were higher than that of EDP. In the later stage of enzymatic hydrolysis, the process efficiency of enzymatic preparation of CNCs greatly decreased because of the high crystallinity of cellulosic substrate, and a simple homogenization treatment can promote the enzymatic hydrolysis, as well as produce fusiform CNCs with more uniform size and more fermentable sugar that could be further converted into fuels and bulk chemicals through fermentation. This study provides a feasible enzymatic preparation process for CNCs with engineered cellulase and commercial cellulosic materials.

In Germany alone, more than 5·10⁶ tons of municipal green waste is produced each year. So far, this material is not used in an economically worthwhile way. In this work, grass clippings and tree pruning as examples of municipal green waste were utilized as feedstock for the microbial production of platform chemicals. A pretreatment procedure depending on the moisture and lignin content of the biomass was developed. The suitability of grass press juice and enzymatic hydrolysate of lignocellulosic biomass pretreated with an organosolv process as fermentation medium or medium supplement for the cultivation of Saccharomyces cerevisiae, Lactobacillus delbrueckii subsp. lactis, Ustilago maydis, and Clostridium acetobutylicum was demonstrated. Product concentrations of 9.4 g_ethanol L⁻¹, 16.9 g_lactic acid L⁻¹, 20.0 g_{itaconic acid} L⁻¹, and 15.5 g_solvents L⁻¹ were achieved in the different processes. Yields were in the same range as or higher than those of reference processes grown in established standard media. By reducing the waste arising in cities and using municipal green waste as feedstock to produce platform chemicals, this work contributes to the UN sustainability goals and supports the transition toward a circular bioeconomy.

In this study, the sequential extraction of the three types of biochemicals from microalgae is employed, which is a more realistic and practical solution for large-scale extraction of bioproducts. The drying, grinding, organic solvent treatment, and ultra-sonication were combined to disrupt cells and sequentially extract bioproducts from three microalgae strains, Chlorella sorokiniana IG-W-96, Chlorella sp. PG-96, and Chlorella vulgaris IG-R-96. As the drying is the most energy-intensive step in cell disruption and sequential extraction, the effect of this step on sequential extraction deeply explored. The results show that total ash-plus contents of biochemicals in freeze-dried samples (95.4 ± 2.8%, 89.3 ± 3.9%, and 77.5 ± 4.2 respectively) are higher than those in oven-dried samples (91.0 ± 2.8%, 89.5 ± 3.0%, 71.4 ± 4.8%, respectively) showing the superiority of freeze drying over oven drying merely for Chlorella vulgaris IG-R-96 (p-value = 0.003) and non-significant variation for Chlorella sorokiniana IG-W-96 (p-value = 0.085) and Chlorella sp. PG-96 (p-value = 0.466). Variation among biochemical contents of strains is due to the difference in cell wall strength confirmed by TEM imaging. The freeze-dried samples achieved higher lipid yields than oven-dried samples. The total carbohydrate yields followed the same pattern. The extraction yields of total protein were higher in freeze-dried samples than in oven-dried. Total mass balance revealed that drying-based sequential extraction of value-added bioproducts could better demonstrate the economic potential of sustainable and renewable algal feedstock than independent assays for each biochemical.

A significant distinction between cigar production and tobacco lies in the necessary aging process, where intricate microbial growth, metabolic activities, enzymatic catalysis, and chemical reactions interact. Despite its crucial role in determining the final quality of cigars, our comprehension of the underlying chemical and biological mechanisms within this process remains insufficient. Biomass and alkaloids are the primary constituents that influence the flavor of cigars. Consequently, investigating the entire aging process could begin by exploring the involvement of microbes and enzymes in their biodegradation. In this study, handmade cigars were aged under different conditions. Metagenomic sequencing was employed to identify the microbes and enzymes responsible for the degradation of biomass and alkaloids derived from tobacco leaves. The results revealed that various environmental factors, including temperature, humidity, duration time, and turning frequency, yielded varying contents of total sugar and alkaloids in the cigars. Significant correlations were observed between microbial communities and starch, reducing sugars, total sugars, and alkaloids. Key species involved in the breakdown of biomass constituents, such as starch (Bacillus pumilus, Pseudomonas sp. 286, and Aspergillus cristatus), reducing sugars and total sugars (Aspergillus cristatus and Nitrolancea hollandica), were identified. Furthermore, Corynespora cassiicola and Pseudomonas fulva were found to potentially contribute to the degradation of alkaloid compounds, specifically nornicotine and neonicotinoid. Our work contributes to a deeper understanding of the microbial roles in the aging of cigars. Moreover, the selection of specific microbial strains or starter cultures can be employed to control and manipulate the aging process, thereby further refining the flavor development in cigar products.

Bacterioruberin and its rare glycosylated derivatives are produced by Arthrobacter agilis as an adaptation strategy to low temperature conditions. The high antioxidant properties of bacterioruberin held great promise for different future applications like the pharmaceutical and food industries. Microbial production of bacterioruberin via a cost-effective medium will help increase its commercial availability and industrial use. The presented study aims to optimize the production of the rare C₅₀ carotenoid bacterioruberin and its derivatives from the psychotrophic bacteria Arthrobacter agilis NP20 strain on a whey-based medium as a cost effective and readily available nutritious substrate. The aim of the study is extended to assess the efficiency of whey treatment in terms of estimating total nitrogen content in treated and untreated whey samples. The significance of medium ingredients on process outcome was first tested individually; then the most promising factors were further optimized using Box Behnken design (BBD). The produced carotenoids were characterized using UV–visible spectroscopy, FTIR spectroscopy, HPLC–DAD chromatography and HPLC-APCI-MS spectrometry. The maximum pigment yield (5.13 mg/L) was achieved after a 72-h incubation period on a core medium composed of 96% sweet whey supplemented with 0.46% MgSO₄ & 0.5% yeast extract and inoculated with 6% (v/v) of a 24 h pre-culture (10⁹ CFU/mL). The cost of the formulated medium was 1.58 $/L compared with 30.1 $/L of Bacto marine broth medium. The extracted carotenoids were identified as bacterioruberin, bis-anhydrobacteriouberin, mono anhydrobacterioruberin, and glycosylated bacterioruberin. The presented work illustrates the possibility of producing bacterioruberin carotenoid from Arthrobacter agilis through a cost-effective and eco-friendly approach using cheese whey-based medium.

This study examined potential of the extracts obtained from the byproducts generated at commercial pecan nut-shelling operations in cancer treatment. The subcritical water extracts obtained from two varieties, Native and Pawnee, were analyzed for their phenolic contents and compositions. Effects of the extracts on viability and IC50 of the human cell lines representing a broad range of cancer types, cervical, lung, skin, breast, colon and prostate cancers, were investigated. Although the effect of the temperature on the phenolic contents and compositions of the extracts was not statistically significant, the influence of the variety was extensive. The pecan shell extracts were not cytotoxic to the healthy cell line Vero in the concentration range examined. Some of the pecan shell extracts had greater efficay than Doxorubicin, a drug used in cancer chemotherapy, in reducing cancer cell viability. This study is novel and practical implications of the data generated in this study are noteworthy, because this is the first report on the beneficial effects of subcritical water extracts obtained from pecan shelling industry byproducts on a broad range of cancer cell lines. It is likely that the experimental data presented in this study will support and encourage future research on the biological pathways involved in the interactions of the cancer cells and the extracts. The findings of this study will facilitate research on downstream processing and purification of the crude extracts exhibiting high cancer cell cytotoxcity, potentially improving the final product efficacy and lead to commercial applications.

Ursodeoxycholic acid (UDCA) is not only safer than chenodeoxycholic acid in the treatment of hepatobiliary diseases, but also has a wide range of applications in Acute Kidney Injury and Parkinson’s Disease. The purpose of this experiment is to improve the conversion rate of 7-ketocholic acid (7K-LCA) and the yield of ursodeoxycholic acid in aprotic solvents during electrochemical reduction process. Three aprotic solvents were investigated as electrolytes. 1,3-Dimethyl-2-imidazolidinone (DMI) has a stable five-membered ring structure, and 7K-LCA has undergone two nucleophilic reactions and “Walden” inversion, the 7K-LCK was stereoselectively reduced to UDCA. Hexamethylphosphoramide (HMPA) and 1,3-methyl-3,4,5,6-Tetrahydro-2(1H)-pyrimidinone (DMPU) can be attacked by chloride ions to produce by-products. Molecular orbital theory-based simulations were conducted to study the reducibility of three aprotic solvents [hexamethylphosphoramide (HMPA), 1,3-methyl-3,4,5,6-Tetrahydro-2(1H)-pyrimidinone (DMPU), and 1,3-Dimethyl-2-imidazolidinone (DMI)] in combination with experiments. Choose the best solvent based on the simulation results, the electrolysis reaction can be carried out by applying current and voltage when lithium chloride is used as electrolytes. Calculations using Materials Studio showed that Cu, Pb, Hg–Cu, and Ni exhibited the highest binding energies to the substrate in this system. Using Cu as the electrode when the solvent is a 1:1 mix of DMI and HMPA, the conversion rate of 7-ketocholic acid (could reach 98%, the yield of ursodeoxycholic acid was up to 80%. Under the same conditions, linear voltammetry was performed on the electrochemical workstation to study the electrolysis behavior, and the obtained results were consistent with the experiment.

Biochar modified by metal ions—particularly Mg—is typically used for the effective recovery of phosphorous. In this study, MgO-modified biochars were synthesized via the direct co-pyrolysis of MgO and raw materials such as rice straw, corn straw, Camellia oleifera shells, and branches from garden waste, which were labeled as MRS, MCS, MOT, and MGW, respectively. The resulting phosphate (PO) adsorption capacities and potential adsorption mechanisms were analyzed. The PO adsorption capacities of the biochars were significantly improved after the modification with MgO: MRS (24.71 ± 0.32 mg/g) > MGW (23.55 ± 0.46 mg/g) > MOT (15.23 ± 0.19 mg/g) > MCS (14.12 ± 0.21 mg/g). PO adsorption on the modified biochars was controlled by physical adsorption, precipitation, and surface inner-sphere complexation processes, although no electrostatic attraction was observed. Furthermore, PO adsorbed on modified biochars could be released under acidic, alkaline, and neutral conditions. The desorption efficiency of MRS was modest, indicating its suitability as a slow-release fertilizer.

The concept of biorefinery has been advancing globally and organosolv pretreatment strategy has seen an upsurge in research due to its efficiency in removing the recalcitrant lignin and dissolution of cellulose. The high-performance organosolv system uses green solvents and its reusability contributes concurrently to the biorefinery sector and sustainability. The major advantage of the current system involves the continuous removal of lignin to enhance cellulose accessibility, thereby easing the later biorefinery steps, which were immensely restricted due to the recalcitrant lignin. The current system process can be further explored and enhanced via the amalgamation of new technologies, which is still a work in progress. Thus, the current review summarizes organosolv pretreatment and the range of solvents used, along with a detailed mechanistic approach that results in efficient pretreatment of LCB. The latest developments for designing high-performance pretreatment systems, their pitfalls, and advanced assessments such as Life Cycle Assessment along with Techno-Economic Assessment have also been deliberated to allow an insight into its diverse potential applicability towards a sustainable future.