Current Opinion in Pulmonary Medicine

To prevent the unique difficulty of hygromycin-based gene editing on industrial A. niger strain and increase the working efficiency, the local pyrG marker was removed by well-designed dual sgRNAs and repair template through Cas9-ribonucleoprotein (RNP) strategy in this study. The positive rate of the desired pyrG auxotroph construction was 100%, while no transformant was observed using the traditional methods. The complementation strain showed similar fermentation character as the starting strain. Moreover, an efficient and seamless knock out plasmid-based strategy was established, achieving positive rate at 90% and 50% for challenging Δku70 and Δku80 respectively. Further, combined hygromycin markers and miniaturization cultivation were conducted to select the poor growth strain. Finally, skillfully designed sgRNA and amdS counter-selection repair template were used to obtain ERG3Tyr185 mutation. A highly-efficient precise strategy was established for A. niger through a diagnostic PCR method, with nearly 100% positive rate. Highly- precise desired point mutation was achieved by the developed gene toolbox.

The removal of nutrients from wastewater to reduce the toxicity of these compounds to the environment requires more space in wastewater treatment plants to establish anaerobic, anoxic and aerobic treatment stages. To address this limitation, researchers have developed practical, intensive hybrid treatment systems that enhance nutrient removal performance while requiring less space. However, the implementation of hybrid systems within a reactor introduces the interaction between the attached and suspended growth that can influence the microbial community structure and the performance of the system, so it is crucial to understand the composition of the microbial communities involved in hybrid growth to optimize control strategies in these systems. This study investigated the microbial community structure of the integrated moving bed membrane bioreactor (IMBMBR) system and its impact on nutrient removal in municipal wastewater. The findings demonstrated that the effluent quality was improved with the IMBMBR. The efficiency of removing COD, BOD5, $${\text{NH}}_{4}^{+}\text{-N}$$ and $${\text{PO}}_{4}^{3-}\text{-P}$$ in the IMBMBR were 91 ± 4.0%, 95 ± 4.0%, 99 ± 0.2% and 24 ± 3.0%, respectively. The IMBMBR had better nitrite oxidation and complete nitrification by increasing the diversity and abundance of effective bacteria. The abundance of Proteobacteria, Bacteroidetes and Nitrospira was enhanced in IMBMBR. Coexistence of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in IMBMBR led to increased nutrient removal. The study results suggest that IMBMBR can be an effective process for nutrient removal, achieving quality standards that comply with legal requirements for wastewater in municipal and industries with limited space for establishing treatment facilities. Additionally, this process can be quickly implemented as an upgrade to existing wastewater treatment plants, avoiding the need to develop an entirely new system.

Present study deals with the green fabrication of copper oxide nanoparticles (CuO NPs) employing cell-free aqueous extract of Cladophora glomerata (L.) Kuetz, freshwater algal species. The UV–visible, FTIR, XRD, FESEM, HRTEM, EDX, BET, XPS and Raman spectroscopic techniques were used to confirm and characterize the biosynthesized CuO NPs. The UV–Vis analysis revealed a sharp peak at 264 nm with a band gap of 3.7 eV, which was attributable to the fabrication of CuO NPs. FESEM and HRTEM detect the spherical-shaped morphology with size between 40 and 50 nm. The biochemical profiling of cell free extract of the C. glomerata by Gas chromatography–mass spectrometry (GC–MS) revealed the presence of various bioactive biomolecules that may acts as a precursor for the fabrication of CuO NPs. The antibacterial study of fabricated CuO NPs revealed significant growth inhibitory potential against selected bacterial strains Klebsiella pneumoniae and Bacillus cereus with an IC50 value of 10 μg/ml. The synthesized CuO NPs also displayed strong DPPH radical scavenging (IC50 value 11.25 mg/L) and total antioxidant (IC50 value 11 mg/L) properties. Further, the anticancer activity of fabricated CuO NPs was studied employing a human hepatocellular carcinoma (HepG2) cell line by MTT assay, which marks their ability to diminish the 50% cell with IC50 value of 168.6 µg/ml. Overall, the findings confirmed that CuO NPs fabricated employing cell-free extract of C. glomerata have the potential to be used as active agent in various biomedical applications after further detailed clinical investigations.

Abstract This study assesses the antimicrobial, antibiofilm, and antiurease properties of selenium (Se), zinc (Zn), and zinc selenide (ZnSe) nanoparticles (NPs) against clinically pathogenic strains of Streptococcus salivarius and Proteus mirabilis. The Se, Zn, and ZnSe NPs, synthesized by Pseudomonas aeruginosa OG1, were characterized using transmission electron microscopy (TEM) revealing average sizes of approximately 30 ± 10 nm, 30 ± 15 nm, and 40 ± 10 nm, respectively. Atomic force microscopy (AFM) was used to examine the morphological and topological characteristics of the NPs. The structural and crystal characteristics were investigated using X-ray diffraction (XRD). Among the evaluated NPs, Zn NPs at a concentration of 200 mg/mL exhibited the most substantial growth inhibition zone against S. salivarius. Conversely, the highest antibiofilm activity was observed against P. mirabilis, notably with 200 µg/mL Zn NPs. In the context of antiurease activity, both 100 μg Zn and ZnSe NPs caused complete urease inhibition (100%) in P. mirabilis within the initial 5 h, with notable inhibition rates of 94% and 80%, respectively, observed against S. salivarius. Significantly, in the current landscape of NP research primarily focused on antimicrobial and antibiofilm properties, our study stands out due to its pioneering exploration of antiurease activities with these NPs. This distinctive emphasis on antiurease effects contributes original and unique value to our study, offering novel insights into the broader spectrum of NP applications, and paving the way for potential therapeutic advancements.

The enrichment of galactooligosaccharides (GOS), synthesized by whole cells of Kluyveromyces marxianus 3551 in a 5.0-L bioreactor, was investigated in this study. The synthesized sugar mixture containing 17.89% (w/w of total carbohydrates) of GOS with 15.57% (w/w of total carbohydrates) of lactose, and 66.54% (w/w of total carbohydrates) monosaccharides as impurities, was subjected to nanofiltration for enrichment of GOS. Three distinct spiral wound membranes, namely, NFPS-01(polysulfone), NFCA-02 (cellulose acetate), and NFPES-03 (polyethersulfone) were tested out of which the NFPES-03 performed the best for fractionation of the GOS mixture. The polyethersulphone membrane (cut-off 400–1000 Da) was evaluated at 30 ℃ and 50 ℃, at different transmembrane pressures or TMP (15, 20, 25 bar) and a combination of high temperature (50 ℃) and low pressure (15 bar) gave the greatest difference in the trisaccharide and disaccharide/monosaccharide rejection percentages, resulting in enrichment of GOS. An analysis of the sugar concentrations in the retentate samples by high-performance liquid chromatography indicated the percentage recovery of GOS in the integrated process to be 88.8%. Measurement of the growth profile of several microbes on the nano-filtered GOS demonstrated its effectiveness as a prebiotic source.

Abstract Enzymatic degradation of polyethylene terephthalate (PET) represents a sustainable approach to reducing plastic waste and protecting fossil resources. The cost efficiency of enzymatic PET degradation processes could be substantially improved by reusing the enzymes. However, conventional immobilisation strategies, such as binding to porous carriers, are challenging as the immobilised enzyme can only interact with the macromolecular solid PET substrate to a limited extent, thus reducing the degradation efficiency. To mitigate this challenge, this work compared different immobilisation strategies of the PET-degrading cutinase ICCGDAQI. Immobilisation approaches included enzyme fixation via linkers to carriers, the synthesis of cross-linked enzyme aggregates with different porosities, and immobilisation on stimulus-responsive polymers. The highest degradation efficiencies were obtained with the pH-responsive material Kollicoat®, where 80% of the initial enzyme activity could be recovered after immobilisation. Degradation of textile PET fibres by the cutinase-Kollicoat® immobilisate was investigated in batch reactions on a 1 L-scale. In three consecutive reaction cycles, the product yield of the released terephthalic acid exceeded 97% in less than 14 h. Even in the fifth cycle, 78% of the maximum yield was achieved in the same reaction time. An advantage of this process is the efficient pH-dependent recovery of the immobilisate after the reaction, which integrates seamlessly into the terephthalic acid recovery by lowering the pH after hydrolysis. This integration therefore not only simplifies the downstream processing, but also provides a cost-effective and resource-efficient solution for both enzyme reuse and product separation after PET degradation, making it a promising approach for industrial application.

Membrane bioreactors (MBRs) have been widely used in the field of wastewater treatment because of their small footprint and high treatment efficiency. In this research, 10 rural wastewater treatment sites in China that employ the MBR process were systematically studied. Specifically, treatment of actual domestic wastewater using MBRs was examined by high-throughput 16S rRNA gene sequencing to explore the microbial community composition and perform function prediction. The data of water quality parameters revealed high removal rates of chemical oxygen demand and NH4+–N in all the sites. Proteobacteria were absolutely dominant in all the sites. Thauera, Nitrospira, Ferribacterium, and Dechloromonas were the main functional genera responsible for nitrogen and phosphorus removal at the tested sites. Nitrospira includes conventional nitrite-oxidizing bacteria and complete ammonia-oxidizing bacteria. Among them, 26 genes related to nitrogen metabolism were retrieved according to gene prediction, which verified the good NH4+–N removal efficiency at the tested sites. This study focuses on the analysis of microbial community structure and functional characteristics of MBR-based treatment systems for rural wastewater treatment, thereby providing a microbial basis for improving rural wastewater treatment processes.

p-Coumaric acid (p-CA), an invaluable phytochemical, has novel bioactivities, including antiproliferative, anxiolytic, and neuroprotective effects, and is the main precursor of various flavonoids, such as caffeic acid, naringenin, and resveratrol. Herein, we report the engineering of Escherichia coli for de novo production of p-CA via the PAL-C4H pathway. As the base strain, we used the E. coli H-02 strain, which was previously engineered for sufficient supplementation of L-phenylalanine, the main precursor of p-CA. For the bioconversion of L-Phe to p-CA, we constructed and optimized an expression system for phenylalanine ammonia lyase (SmPAL), codon-optimized cinnamate 4-hydroxylase (AtC4H), and its redox partner, cytochrome P450 reductase (AtCPR1). We confirmed that the engineered cell showed higher production of p-CA at 30 °C and the addition of 0.5 mM 5-aminolevulinic acid could increase the production titer further. Subsequently, the main pathways of acetic acid (poxB and pta-ackA) were eliminated to reduce its accumulation and restore cell growth. Next, to increase the available pool of cofactor (NADPH), the co-expression system of the zwf gene in the pentose phosphate pathway (PPP) was integrated into genome and the expression level was optimized with synthetic promoters. Finally, by optimizing fed-batch culture in a 5 L-scale bioreactor, the engineered strain achieved 1.5 g/L p-CA with a productivity of 31.8 mg/L/h.

AbstractCellobiose lipids (CBLs) are a class of glycolipid biosurfactants produced by various fungal strains. These compounds have gained significant interest due to their surface-active and antifungal properties, which are comparable to traditional synthetic surfactants and antimicrobials. Despite their potential applicability in various cosmetic, pharmaceutical, and agricultural formulations, significantly less research has been focused on their production and purification in comparison to other glycolipid biosurfactants, such as mannosylerythritol lipids (MELs) and sophorolipids. Hence, this work proposes the development of a bioprocess that involves the microbial production and high-level chromatographic purification of CBLs from a submerged culture of Ustilago maydis DSM 4500. After a highly purified CBL product was obtained, the factors affecting the production of this glycolipid were investigated. It was demonstrated that U. maydis DSM 4500 produces a specific structural variant of CBLs at a concentration of 1.36 g/L on an optimized the growth medium. Also, it was established that when the C/N ratio was decreased, the CBL titer increased by 2.3-fold. Furthermore, supplementing the culture with ZnSO4 at a concentration of 0.04 mg/L further increased CBL concentration to 4.95 g/L, representing the highest CBL titer achieved in a single-stage bioprocess to date. This study developed a methodology for utilizing U. maydis as a high-level CBL producer, which could challenge other familiar CBL producers, such as Sporisorium scitamineum and Cryptococcus humicola.

Ascomycin (FK520) is a 23-membered macrolide antibiotic primarily produced by the Streptomyces hygroscopicus var. ascomyceticus. Structurally similar to tacrolimus and rapamycin, it serves as an effective immunosuppressant widely used in the treatment of rejection reactions after organ transplantation and certain autoimmune diseases. Currently, FK520 is mainly produced through microbial fermentation, but its yield remains low. Since the gene fkbR2 is a regulatory gene within the FK520 biosynthesis gene cluster that has not been studied, this paper focuses on the overexpression of the gene fkbR2 in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 (WT). By constructing a strain with overexpressed fkbR2 gene, we initially obtained a high-yield strain R2-17 through shake flask fermentation, with a 28% increase in yield compared to WT. In the process of further improving the stability of the high-yield strain, this paper defines two indices: high-yield index and stability index. After two consecutive rounds of natural breeding, strain R2-17 achieved a high-yield index of 100% and a stability index of 80%. Finally, the high-yield strain R2-17–3-10 was successfully screened, and the yield was increased by 34% compared with the strain WT, reaching 686.47 mg/L. A comparative analysis between the high-yield strain R2-17–3-10 and the original strain WT revealed differences in fermentation process parameters such as FK520 synthesis rate, pH, bacterial growth, glycerol consumption rate, ammonia nitrogen level, and ammonium ion concentration. In addition, the transcription levels of genes involved in the synthesis of precursors 4,5-dihydroxycyclohex-1-enecarboxylic acid (fkbO), ethylmalonyl-CoA (fkbE, fkbU, fkbS), and pipecolic acid (fkbL), as well as pathway-specific regulatory genes (fkbN, fkbR1), were significantly increased at different time points in the high-yield strain R2-17–3-10. EMSAs analysis showed that the FkbR2 protein could not bind to the promoter region of above genes. This suggests that the gene fkbR2 may enhance the supply of FK520 synthetic precursors by indirectly regulating the transcription levels of these genes, thereby promoting an increase in FK520 production. These results demonstrate that modifying genes within the biosynthetic gene clusters of natural products can be successfully applied to increase the yields of industrially and clinically important compounds. However, it is found that fkbR2 gene is a regulatory gene that has not been fully studied, and it is worth further studying its regulatory mechanism.

The ubiquitous presence of pharmaceuticals and personal care products (PPCPs) in the environment has become a significant concern due to their persistence, bioaccumulation potential in biota, and diverse implications for human health and wildlife. This review provides an overview of the current state-of-the-art in environmental bioremediation techniques for reducing pharmaceutical residues, with a special emphasis on microbial physiological aspects. Numerous microorganisms, including algae, bacteria or fungi, can biodegrade various pharmaceutical compounds such as antibiotics, analgesics and beta-blockers. Some microorganisms are capable of transferring electrons within the cell, and this feature can be harnessed using Bio Electrochemical Systems (BES) to potentiate the degradation of pharmaceuticals present in wastewater. Moreover, researchers are evaluating the genetic modification of microbial strains to improve their degradation capacity and expand list of target compounds. This includes also discuss how environment changes, such as fluctuations in temperature or pH, may affect bioremediation efficiency. Furthermore, the presence of pharmaceuticals in the environment is emphasised as a major public health issue because it increases the chance for antibiotic-resistant bacteria emerging. This review combines existing information and outlines needed research areas for improving bioremediation technologies in the future.

Abstract The purpose of this review is to gain attention about intro the advanced and green technology that has dual action for both clean wastewater and produce energy. Water scarcity and the continuous energy crisis have arisen as major worldwide concerns, requiring the creation of ecologically friendly and sustainable energy alternatives. The rapid exhaustion of fossil resources needs the development of alternative energy sources that reduce carbon emissions while maintaining ecological balance. Microbial fuel cells (MFCs) provide a viable option by producing power from the oxidation of organic and biodegradable chemicals using microorganisms as natural catalysts. This technology has sparked widespread attention due to its combined potential to cleanse wastewater and recover energy. The review presents a complete examination of current advances in MFCs technology, with a focus on the crucial role of anode materials in improving their performance. Moreover, different anode materials and their nanoscale modifications are being studied to boost MFC efficiency. This current review also focused on the effects of surface modifications and different anode compositions on power generation and system stability. It also investigates the electrochemical principles behind these enhancements, providing insights into the economic potential of MFCs. MFCs provide a long-term solution to energy and environmental issues by addressing both wastewater treatment and energy production.

Membrane fouling is a common and complex challenge with cell culture perfusion process in biopharmaceutical manufacturing that can have detrimental effects on the process performance. In this study, we evaluated a method to calculate the hollow fiber membrane resistance at different time points for water and supernatant. In addition, the number of subvisible particles of < 200 nm. diameter suspended in the supernatant were quantified using a nano-flow cytometry method. A computational fluid dynamics (CFD) model was developed to evaluate the impact of feed flow rate and particle count on the transmembrane pressure (TMP). Then a steady-state discrete phase model was applied to incorporate particles into the model and simulate the particles deposition over the membrane wall. The results showed an increase in the number of particles and the membrane resistance along the time course of the perfusion process. The CFD model illustrated that more particle deposition was observed at lower feed stream flow rates. The fraction of deposited particle was reduced by > 50% when the feed flow rate was increased from 35 ml/min to 300 ml/min. Our findings suggest that the total number of subvisible particles has a significant impact on TMP and membrane resistance and, thus, could play a major role in the mechanism of membrane fouling. CFD modeling can be a useful tool to predict the behavior of a process in a specific membrane. CFD simulations could also be used to optimize process parameters to improve membrane cleanability, reduce particle deposition, and reduce the risk of membrane fouling.

In recent years, zinc oxide nanoparticles (ZnO NPs) have gained much attention in biomedical applications because of their distinctive physicochemical features such as low toxicity and biocompatible properties. Traditional methods to produce ZnO NPs sometimes include harmful substances and considerable energy consumption, causing environmental issues and potential health risks. Nowadays, the concern of ZnO production has moved toward environmentally friendly and sustainable synthesis methods, using natural extracts or plant-based precursors. This review discusses the green synthesis of ZnO NPs utilizing various plant extracts for wound healing applications. Moreover, ZnO NPs have antibacterial characteristics, which can prevent infection, a substantial obstacle in wound healing. Their ability to maintain inflammation, proliferation, oxidative stress, and promote angiogenesis proves their critical role in wound closure. In addition, ZnO NPs can also be easily and ideally incorporated with wound dressings and scaffolds such as hydrogel, chitosan, cellulose, alginate, and other materials, due to their exceptional mechanical properties. The latest publication of green synthesis of ZnO NPs and their applications for wound healing has been discussed. Therefore, this review provides a current update of knowledge on the sustainable and biocompatible ZnO NPs for specific applications, i.e., wound healing applications. In addition, the green synthesis of ZnO NPs using plant extracts also provides a particular approach in terms of material preparation, which is different from previous review articles.