Growth Medium Research Articles

Polyvinyl alcohol assisted citrate based reduction of gold(III) ions: Theoretical design and experimental study on green synthesis of spherical and biocompatible gold nanoparticles

In this study, we demonstrate the synthesis of small gold nanoparticles (typically 8-10 nm) through a green synthesis approach. This method involves utilizing chloroauric acid as the gold precursor, trisodium citrate as a mild reducing and co-capping agent, and polyvinyl alcohol (PVA), as a co-capping and shape-directing agent. This novel synthesis method stands alone as a protocol that involves just mixing the reagents at room temperature and allowing the reaction to proceed at ambient temperature without any disturbance. In the absence of PVA, spherical particles are not formed and the reaction mixture slowly turns black followed by precipitation.The synthesis process was meticulously tracked using time-resolved monitoring of the growth of the localized surface plasmon resonance (LSPR) by UV-vis spectroscopy and transmission electron microscopy. The as-prepared colloidal gold solution was bright red, attributed to the small size (≤ 10 nm) and spherical geometry of nanoparticles. This colloidal solution could be stored indefinitely at room temperature without precipitation or a change in its absorption profile. The nanoparticles remained stable in adverse conditions such as 100 mM NaCl solution, 1×PBS and cell culture medium. Additionally, they could be easily loaded with drugs like doxorubicin by adsorption. The particles were thoroughly characterized using a range of techniques, including UV-vis spectroscopy, attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), high-resolution transmission electron microscopy (HRTEM), hyperspectral-enhanced dark field microscopy (HEDFM), dynamic light scattering (DLS), and X-ray photoelectron spectroscopy (XPS). Cell viability tests conducted on 2D cell lines and 3D spheroids revealed negligible toxicity even at high concentrations. Furthermore, we demonstrate that an advanced version of conventional diffusion-limited aggregation (DLA) schemes, which allows individual clusters to move freely within a domain to form larger aggregates, can effectively replicate the intricate interactions between chloroauric acid, trisodium citrate, and PVA, the agents involved in the synthesis of gold nanoparticles.

Flavonoids modulate regenerative-related cellular events in LPS-challenged dental pulp cells

ObjectiveTo investigate the effects of quercetin (QU), hesperetin (HT), and taxifolin (TX) on human dental pulp cells (hDPCs) chronically exposed to lipopolysaccharide (LPS). MethodsFirst, the cytotoxicity (alamarBlue) and bioactivity (biomineralization, Alizarin Red) of QU, HT, and TX concentrations were evaluated on healthy hDPCs. Then, the effects of non-cytotoxic and bioactive concentrations were investigated on hDPCs after previous stimulation with E. coli LPS (10 µg/mL) for 7 days. Cell culture media with and without LPS were used as positive and negative controls, respectively. Cell viability (alamarBlue), NF-κB activation (immunofluorescence), reactive oxygen species production (ROS, H2DCFDA probe), cell migration (Transwell), inflammatory gene expression (RT-qPCR), and odontogenic differentiation (RT-qPCR and alizarin red) were evaluated (n=8). Data were analyzed using confidence intervals and ANOVA (α=5%). ResultsThe concentrations of 20 µM QU, 20 µM HT, and 200 µM TX reduced cell viability by more than 30%. The 5 µM QU, 10 µM HT, and 100 µM TX concentrations were cytocompatible and stimulated biomineralization by healthy hDPCs. These concentrations were tested under the LPS challenge, and cell viability and odontogenic differentiation were significantly increased, while ROS production and inflammatory response were significantly decreased. In addition, the flavonoids significantly stimulated cell migration, reduced NF-κB activation, and increased biomineralization by LPS-challenged hDPCs compared to cells exposed to LPS alone and without any other treatment. ConclusionFlavonoids can modulate the metabolism of hDPCs chronically exposed to LPS in vitro, stimulating cellular events compatible with stem cell-based regenerative processes. Clinical SignificanceFlavonoids may be explored as adjuvant therapeutic agents during pulp capping to counteract chronic inflammatory conditions and stimulate regeneration of the dentin-pulp complex in caries-affected teeth, thereby preserving tooth vitality.

Effects of anti-inflammatory drugs on distinct endotypes of chronic rhinosinusitis without nasal polyps: comparison using an ex-vivo model.

The objectives of this study were to characterize the endotypes of different chronic rhinosinusitis without nasal polyps (CRSsNP) samples, to investigate the effects of certain anti-inflammatory drugs on these endotypes, and to investigate the effect of the same drugs on recently identified CRSsNP marker proteins. Initially, ethmoid tissues (ETs) from CRSsNP patients (n=12) were dissected into sections and incubated with the addition of mometasone, verapamil, cenicriviroc, and dupilumab. Cell culture media were collected after 24 hours, and the contents of the secreted proteins interferon-gamma (IFN-γ), interleukin (IL)-5, IL-17A, macrophage inflammatory protein-1 beta (MIP-1β), resistin and platelet (P)-selectin were measured using enzyme-linked immunosorbent assay (ELISA). The endotypes were characterized using the unstimulated samples. The fold changes of protein secretion caused by the analyzed substances were calculated. For each protein, the samples of the distinct endotypes were compared with the remaining samples. Both single and mixed endotypes were identified within the CRSsNP samples, whereas none of the typical endotype-defining cytokines were elevated in a significant portion of the samples. All of the incubated medicaments greatly reduced the tissue secretions of IFN-γ and IL-5 in type 1 CRS while causing a lower secretion of IL-17A in all endotypes compared to the remaining samples. Among the analyzed CRSsNP marker proteins, the distinct endotypes revealed different reactions to the drugs. Dupilumab induced more effects among the examined cytokines than the marker proteins but did not stand out from the other substances overall. Medications used to treat CRS may have different effects on distinct CRS endotypes.

Effects of Particle Size and Shape on the Texture Property of a Solid-Liquid Dispersion System With Gel Particles.

Food texture is one of the most important factors for assessing the quality and acceptability of food. However, the study of food texture has been delayed compared with other factors, such as flavor and taste, due to the difficulty of quantitative analysis related to real physiological senses. Furthermore, the numerical and systematic evaluation of the texture property of dispersion systems, in which solid particles are dispersed in a liquid medium, is very difficult, despite most foods being in a solid-liquid dispersion state during mastication in the human mouth. In this study, the texture property of a solid-liquid dispersion system with spherical and cubic gel particles of agar and konjac was examined to evaluate the physical behavior of food during mastication using the back extrusion method. The yield stress of the system strongly depended on the size and shape of the particles, the mixing ratio of particles of different sizes and shapes, and the concentration of components in the particles. The proposed index, reflecting the size, shape, and number of particles and the yield stress of a single particle, expressed well the measured yield stress of the entire dispersion system. However, the adhesiveness and recoverability showed relatively little dependence on particle size. The findings obtained in this study will contribute to elucidating the texture property of various foods and to the development of new and novel food products and cuisines, thereby benefiting food science and industry.

Potential of a novel marine microalgae Dunaliella sp. MASCC-0014 as feed supplements for sustainable aquaculture

Microalgae, as live feed organisms in aquatic ecosystems, play a crucial role in enhancing water quality by providing oxygen and nutrients. The accumulation of heavy metals in microalgae, however, can pose significant threats to environmental sustainability and human health. In this study, we focused on the marine microalgae Dunaliella sp. MASCC-0014, which exhibited resilience to a wide range of salinities from 0.5 to 4.5 M NaCl, with optimal growth at 0.5 M NaCl. Phylogenetic analysis revealed that MASCC-0014 is closely related to Dunaliella salina CCAP 19/25. Biosorption assay indicated that MASCC-0014 had limited capacity to absorb cadmium, cobalt, and nickel from the liquid medium, while Dunaliella salina CCAP 19/18, Dunaliella tertiolecta FACHB-821 and Dunaliella bardawil FACHB-847 showed varied degrees of cadmium biosorption. When exposed to cadmium stress, MASCC-0014 exhibited suppressed growth and increased activities of catalase (CAT) and peroxidase (POD), suggesting cadmium still triggered cellular responses in MASCC-0014. Furthermore, MASCC-0014 displayed reduced chlorophyll content per cell but increased biomass in warmer conditions, indicating its preference of tropical water over temperate water. This unique microalga strain, which is rich in protein but with limited heavy metal biosorption capabilities, shows promise as a sustainable feed source in aquaculture. The findings in this study offer valuable insights into mitigating the risks of heavy metal enrichment in the food chain through algae-based feeds.

In Vitro Rooting of <em>Gyrinops walla</em> in Activated Charcoal-Containing Semi-Solid Culture and Filter Paper-Bridged Liquid Culture Systems

Plant tissue culture is the best option in producing planting material for cultivation programmes of Gyrinops walla. Perfection of in vitro rooting protocol for this species is the most decisive step in micropropagation. In the present study the effect of physical state of substrate, different concentrations of sucrose, IBA and NAA in media containing activated charcoal on in vitro rooting was investigated. In experiment-1, 0.2% (w/v) activated charcoal was added in to the ½ MS medium fortified with 30 or 40 g/L sucrose and varying concentrations of IBA or NAA (0, 0.1, 0.5, 1.0 and 2.0 mg/L). In experiment-2, filter paper-bridged liquid medium was used with 40 g/L sugar and varying concentrations of IBA or NAA (0, 0.1, 0.5, 1.0 and 2.0 mg/L). The treatments were arranged in completely randomized design with three replicates. Percentage of rooted microshoots and number of roots per shoot were recorded. The ½ MS semi-solid medium with 0.2% activated charcoal, 40 g/L sucrose and 0.1 mg/L NAA resulted in the significantly highest rooting percentage (50%)while the significantly highest number of roots (6.0) were noted in ½ MS charcoal added (0.2%) basal medium with 40 g/L sucrose and 0.5 mg/L NAA. Shoots supported with filter paper bridge on ½ MS liquid medium, supplemented with 0.5 mg/L IBA, had the highest rooting percentage (66.7%) and the highest mean number of roots per shoot (3.0) (P<0.05). Results confirmed that in vitro rooting of G. walla could be perfected by optimizing the culture medium and its constituents.

Sensitive and real-time monitoring of microbial growth using a dielectric sensor with a 65-GHz LC-oscillator array and polytetrafluoroethylene membrane

In this study, we report a sensitive real-time microbial growth monitoring technique using a complementary metal-oxide semiconductor (CMOS) dielectric sensor with a polytetrafluoroethylene (PTFE) membrane. The sensor comprised an LC oscillator array operating at 65-GHz, whose resonant frequency was altered according to the dielectric properties of the region approximately 15 μm from the surface. We previously reported the rapid detection of viable Escherichia coli suspended in a liquid medium using the dielectric sensor; however, sensing growing cells was challenging owing to their tendency to float outside the effective sensing area in the suspended medium. To address this, we propose a new method to enhance the sensitivity of the device using a PTFE membrane that retains cells inside the effective area during measurement. Experiments using Escherichia coli suggested that the use of the membrane more than doubled sensitivity, reducing inspection times for practical applications. Furthermore, experiments with Lactococcus lactis, Staphylococcus epidermidis, and Saccharomyces cerevisiae demonstrated that this method can be used to monitor the growth of various microbes. In addition, variations in the output values of each oscillator facilitated the determination of microbial characteristics, such as cell size and growth distribution. This microbial growth monitoring technique is expected to find applications across a wide range of fields, such as food inspection, environmental hygiene monitoring, antibiotic susceptibility testing, new drug discovery, and the exploration of beneficial microbes.

Engineering living root with mechanical stimulation derived from reciprocating compression in a double network hydrogel as elastic soil

The root system actively reacts to mechanical stimuli in its environment, transmitting mechanical signals to optimize the utilization of environmental resources. While the mechanical impedance created by the growth medium serves as the primary source of stimulation for the roots, extensive research has focused on the roots' response to static mechanical stimulation. However, the impact of dynamic mechanical stimulation on root phenotype remains underexplored. In this study, we utilized a low acyl gellan gum/polyacrylamide (GG/PAM) double network elastic hydrogel as the growth medium for rapeseed. We constructed a mechanical device to investigate the effects of reciprocating extrusion stimulation on the growth of the rapeseed root system. After three weeks of mechanical stimulation, the root system exhibited a significant increase in lateral roots. This branching enhanced the roots' anchoring and penetration into the hydrogel, thereby improving the root system's adaptability to its environment. Our findings offer valuable data and insights into the effects of reciprocating mechanical stimulation on root growth, providing a new way for engineering root phenotype.

Growth and physiology responses of Samanea saman inoculated with arbuscular mycorrhizal fungi in silica post-mining soil media using biodegradable pots

Arbuscular mycorrhizal fungi (AMF) and biodegradable pots are environmentally friendly and enhance plant growth in adverse soil conditions. These studies explored AMF interactions and biodegradable pots in physiology, growth, and the uptake of nutrients in Samanea saman seedlings. The present research interactive effects of biodegradable pots with different compositions and raw material sizes with and without AMF inoculation on S. saman grown in silica post-mining soil media. Results indicated AMF inoculations significantly improved leaf chlorophyll content, photosynthetic rate, heights, diameters, biomass, as well as nutrient absorption of S. saman as compared with non-inoculated plants in biodegradable pots. AMF and biodegradable pots composed of 15% used newspaper, 70% compost, 5% cocopeat, and 10% rock phosphate showed the best results and increased the leaf chlorophyll content, photosynthesis rate, height, diameter, and total biomass of the plants by 161.1%, 208.7%, 263.6%, 118.1%, and 269.9%, respectively, compared to biodegradable pots composed only of used newspaper. Additionally, uptake of the nutrients nitrogen, phosphate, potassium, and magnesium increased by 365%, 800%, 369%, and 250%, respectively. By the fourteenth week after transplanting, the C/N ratio of the organic pot decreased significantly. Thus, AMF and biodegradable pots containing compost and rock phosphate interact positively and enhance the growth of S. saman under adverse soil conditions. These findings suggest that biodegradable pots containing compost and rock phosphate show potential as more environmentally friendly replacements for plastic bags (polybags) as growth medium containers for seedling production.

Experimental setup for comprehensive study of non-stationary heat transfer in complex liquid media.

This paper presents a setup for studying non-stationary heat exchange in liquid media on a scale of small times and sizes at high heat flux densities created using a high-speed precision power controller. A platinum wire with a diameter of 20μm and a length from 0.5 to 1cm is used as a probe. The heating time ranged from 1 to 300ms, and the heat flux densities from 1 to 20 MW/m2 were achieved in the experiments. Strictly specified conditions for heat release in the probe are confirmed by maintaining a constant power in a series of pulses with an accuracy of 0.05%. Acquiring a primary electrical signal of thermograms is synchronized with high-speed video recording, allowing an accurate relationship between the applied thermal load and the mechanics of processes in the studied liquid. The setup capabilities were shown by comparing the boiling patterns of a simple substance, such as ethanol, and a solution with a lower critical solution temperature, a 30 vol.% solution of polypropylene glycol-425 in water, which have similar thermograms. As a result, a qualitative difference between the heating and boiling patterns observed using high-speed video and the presented setup fitting its purpose has been shown.

Investigation of Antimicrobial Compounds Produced by Endolichenic Fungi in Different Culture Media.

Continuous use of synthetic fungicides has led to explosive emergence of fungicide-resistant microbes. Therefore, there are urgent needs for environmentally friendly antimicrobial agents with novel modes of action. This study investigated endolichenic fungi (ELF) as a source of antimicrobial compounds against various plant pathogens. We utilized an One Strain MAny Compounds (OSMAC) approach to enhance the chemical diversity of fourteen ELF. This involved cultivation of ELF in four growth media and subsequently assessing antimicrobial activities of culture extracts. Nearly half of the culture extracts exhibited antimicrobial activity against a Gram-positive bacterium, but showed minimal activity against Gram-negative bacteria tested. Notably, culture extracts from two ELF, Chaetomium globosum and Nodulisporium sp., demonstrated significant inhibitory effects against plant pathogenic fungi. LC-MS/MS-based metabolome profiling confirmed the presence of known bioactive compounds like cyclic dipeptides and chaetoglobosins. These findings highlight the effectiveness of combining OSMAC and metabolomics for identifying antimicrobial agents for agricultural use.

Biological response guided by hybrid additive manufacturing for Ti6Al4V titanium alloy

This research evaluates the cellular response from Ti6Al4V scaffolds fabricated by hybrid additive manufacturing (AM) with the goal of improving bone tissue growth and biocompatibility of titanium implants. Since cellular adhesion and viability are subject to microstructure, hybrid AM was sought to dictate biological response by creating a dispersed and gradient microstructure within implants. The objective was to evaluate the effect of gradient microstructures within Ti6Al4V scaffolds on cellular metabolic activity. Gradient microstructure was incorporated within scaffolds by hybrid AM coupling directed energy deposition (DED) with interlayer milling. The cellular response from the scaffolds was evaluated using cell viability assays in two stages: the Ti6Al4V powder used for DED was first evaluated, followed by the fabricated scaffolds. Cell viability on hybrid AM scaffolds was compared with as–printed, i.e., DED-fabricated and annealed Ti6Al4V scaffolds. The cell viability assays demonstrated that Ti6Al4V powder was non-toxic as cells cultured with powder exhibited metabolic activity similar to cells in growth media without powder. The cell viability study on scaffolds indicated, on average, cell adhesion on hybrid AM scaffolds outperformed as–printed and annealed scaffolds. This study provides preliminary data demonstrating the use of hybrid AM to dictate cell activity. A larger sample set would be required in future work to understand the effects of grain refinement through coldworking on biological response.

3D bioprinting meniscus tissue onboard the International Space Station

We bioprinted meniscus tissue on the International Space Station (ISS) using the onboard BioFabrication Facility (BFF). The three dimensional (3D) printing bioink, cells, culture media and fixative were delivered to the ISS on NG-18 and SpX-27 vehicles and stored prior to the printing operation. The meniscus tissue was fabricated from ink composed of collagens type I and II, chondroitin sulfate and mesenchymal stem cells. Following printing, the meniscus tissue was cultured for 2 weeks in growth media, then stored at 4 °C and returned to earth for analysis. The print showed good overall shape fidelity, and dimensions were comparable to control meniscus tissue printed on Earth. Young's modulus of the ISS printed meniscus was approximately 4-fold lower than the control. Histologic evaluation showed good cell distribution within the print. Though logistical challenges were encountered during payload delivery to the ISS and operational challenges limited the cell culture portion of this study, this investigation demonstrated the feasibility for 3D printed musculoskeletal tissue in microgravity. The completed meniscus tissue print is the largest tissue engineered model 3D printed on the ISS to date, the first to be 3D bioprinted using an ink similar in composition to native tissue, and the first to be fabricated on the ISS in an anatomically relevant shape. These experiments help advance the field of tissue engineering in low or microgravity where 3D bioprinting may have a role in future long term space flight or extraterrestrial habitation.

Enhancing Resistance to Corrosion and Fouling Using Epoxy Coatings with Superhydrophobic Cells

AbstractAs maritime transport becomes increasingly important, metal corrosion results in significant economic losses. Superhydrophobic materials have significant potential for corrosion prevention. However, poor compatibility between superhydrophobic materials and resin reduces the long‐term corrosion resistance. Interfacial strengthening cells (IS‐Cells) are designed to maintain superhydrophobicity and retain active hydroxyl groups participating in resin‐curing. IS‐Cells also contribute to achieving the maze effect. A novel anti‐corrosion IS coating is fabricated by compatible IS‐Cells and epoxy. IS coatings feature smooth surfaces and vertical compactness, protecting against liquid and gaseous corrosive medium. Strong diffusion resistance and extended diffusion distance ensure long‐term corrosion resistance of IS coatings. Approximately 85 µm of IS coatings remain corrosion‐free after 112 days of immersion in the NaCl solution. The low‐frequency impedance, self‐corrosion potential, and self‐corrosion current density are 1.68 × 109 Ω cm2, −0.0708 V, and 5.38 × 10−11 A cm−2, respectively. IS coatings can also withstand 2400 h of neutral salt spray testing without corrosion in the artificial scratches. Due to the electrostatic repulsion between the hydrophobic C–F chains onto IS‐Cells and protein molecules, IS coatings maintain anti‐fouling properties after dry‐wet immersion tests in the natural marine environment for 90 days. IS coatings show great potential for marine anti‐corrosion applications.

Shattering the Water Window: Comprehensive Mapping of Faradaic Reactions on Bioelectronics Electrodes.

It is generally accepted that for safe use of neural interface electrodes, irreversible faradaic reactions should be avoided in favor of capacitive charge injection. However, in some cases, faradaic reactions can be desirable for controlling specific (electro)physiological outcomes or for biosensing purposes. This study aims to systematically map the basic faradaic reactions occurring at bioelectronic electrode interfaces. We analyze archetypical platinum-iridium (PtIr), the most commonly used electrode material in biomedical implants. By providing a detailed guide to these reactions and the factors that influence them, we offer a valuable resource for researchers seeking to suppress or exploit faradaic reactions in various electrode materials. We employed a combination of electrochemical techniques and direct quantification methods, including amperometric, potentiometric, and spectrophotometric assays, to measure O2, H2, pH, H2O2, Cl2/OCl-, and soluble platinum and iridium ions. We compared phosphate-buffered saline (PBS) with an unbuffered electrolyte and complex cell culture media containing proteins. Our results reveal that the "water window"─the potential range without significant water electrolysis─varies depending on the electrolyte used. In the culture medium that is rich with redox-active species, a window of potentials where no faradaic process occurs essentially does not exist. Under cathodic polarizations, significant pH increases (alkalization) were observed, while anodic water splitting competes with other processes in media, preventing prevalent acidification. We quantified the oxygen reduction reaction and accumulation of H2O2 as a byproduct. PtIr efficiently deoxygenates the electrolyte under low cathodic polarizations, generating local hypoxia. Under anodic polarizations, chloride oxidation competes with oxygen evolution, producing relatively high and cytotoxic concentrations of hypochlorite (OCl-) under certain conditions. These oxidative processes occur alongside PtIr dissolution through the formation of soluble salts. Our findings indicate that the conventional understanding of the water window is an oversimplification. Important faradaic reactions, such as oxygen reduction and chloride oxidation, occur within or near the edges of the water window. Furthermore, the definition of the water window significantly depends on the electrolyte composition, with PBS yielding different results compared with culture media.

Endoplasmic reticulum stress alters myelin associated protein expression and extracellular vesicle composition in human oligodendrocytes.

Myelination of the central nervous system is mediated by specialized glial cells called oligodendrocytes (OLs). Multiple sclerosis (MS) is characterized by loss of myelination and subsequent clinical symptoms that can severely impact the quality of life and mobility of those affected by the disease. The major protein components of myelin sheaths are synthesized in the endoplasmic reticulum (ER), and ER stress has been observed in patients with MS. Extracellular vesicles (EVs) have been shown to carry bioactive cargo and have the potential to be utilized as noninvasive biomarkers for various diseases. In the current study, we sought to determine how ER stress in OLs affected the production of key myelination proteins and EV release and composition. To achieve this, tunicamycin was used to induce ER stress in a human oligodendroglioma cell line and changes in myelination protein expression and markers of autophagy were assessed. EVs were also separated from the conditioned cell culture media through size exclusion chromatography and characterized. Significant reductions in the expression of myelination proteins and alterations to autophagosome formation were observed in cells undergoing ER stress. EVs released from these cells were slightly smaller relative to controls, and had strong expression of LC3B. We also observed significant upregulation of miR-29a-3p in ER stress EVs when compared to controls. Taken together, these data suggest that ER stress negatively impacts production of key myelination proteins and induces cells to release EVs that may function to preemptively activate autophagic pathways in neighboring cells.

Fungal degradation of phenylacetate focusing on CRISPR/Cas9-assisted characterization of two oxidative enzyme genes of Akanthomyces muscarius AM1091

The degradation of phenylacetate (PA) was investigated as a model to explore aromatic compound breakdown in the fungal system. Fungal strains capable of utilizing PA as their sole carbon source were isolated using a minimal solid medium supplemented with 0.5 % PA. Subsequent cultivation in minimum liquid medium revealed that selected fungal strains, including Trametes versicolor TV0876 and TV3295, Paecilomyces hepiali PH4477, and Akanthomyces muscarius AM1091, efficiently removed PA within 24 h. HPLC analysis of culture supernatants from various fungal strains revealed a time-dependent accumulation of 2-hydroxyphenylacetate (2-HPA) and 4-hydroxyphenylacetate (4-HPA), two key major metabolic products primarily found in ascomycetes and basidiomycetes, respectively. This suggests that the first hydroxylation of PA is catalyzed by two distinct hydroxylases, one for each fungal group. Furthermore, fungal species that make 4-HPA also produce phenylethanol (PE), indicating a distinct catabolic mechanism to remove PA by direct reduction of PA to PE. A. muscarius AM1091, identified as the most efficient PA degrader in this study, was studied further to determine the biochemical pathway of PA degradation. RNA-Seq and RT-PCR analyses of AM1091 revealed two oxidative enzyme genes, CYP1 and DIO4, upregulated in the presence of PA. Targeted disruption utilizing preassembled Cas9-gRNA ribonucleoprotein complexes and homologous DNAs harboring the URA3 gene as an auxotrophic marker resulted in the cyp1 and dio4 mutant strains. The cyp1 mutant was incapable of converting PA to 2-HPA, indicating its involvement in the C2 hydroxylation, whereas the dio4 mutant was unable to degrade 2,5-dihydroxyphenylacetate (2,5-DHPA), resulting in the accumulation of 2,5-DHPA. Our findings indicate that A. muscarius AM1091 degrades PA through the activities of CYP1 and DIO4 for the C2 hydroxylation and subsequent ring-opening reactions, respectively.

Biodegradation of imidacloprid and diuron by Simplicillium sp. QHSH-33

Imidacloprid (IMI) and diuron (DIU) are widely used pesticides in agricultural production. However, their excessive use and high residues have caused harm to the ecological environment and human health. Microbial remediation as an efficient and low-toxic method has become a research hotspot for controlling environmental pollutants. A fungus QHSH-33, identified as Simplicillium sp., has the ability to degrade neonicotinoids IMI and phenylurea DIU. When QHSH-33 and pesticide were co-cultured in liquid medium for 7 days, the degradation rates of IMI and DIU by QHSH-33 in simulated field soil microenvironment were 50.19 % and 70.57 %, respectively. Through HPLC-MS analysis, it was found that the degradation of IMI mainly involved nitro reduction, hydroxylation and other reactions. Three degradation pathways and eight degradation products were identified, among which two metabolites were obtained by microbial transformation of IMI for the first time. The degradation of DIU mainly involved demethylation and dehalogenation reactions, and two degradation pathways and four degradation products were identified, one of which was a new degradation product of DIU. Toxicity assessment demonstrated that most of the degradation products might be considerably less harmful than IMI and DIU. Whole genome sequencing of QHSH-33 revealed a genome size of 33.2 Mbp with 11,707 genes. The genome of QHSH-33 was annotated by KEGG to reveal 128 genes related to exogenous degradation and metabolism. After local blast with reported IMI and DIU degrading enzymes, seven IMI-degrading related genes and seven DIU-degrading related genes were identified in the QHSH-33 genome. The results of this study will help to expand our knowledge on the microbial decomposition metabolism of IMI and DIU, and provide new insights into the degradation mechanism of IMI and DIU in soil and pure culture system, laying a foundation for QHSH-33 strain applied to the removal, biotransformation or detoxification of IMI and DIU.

A novel, cost-effective approach for the production of hydrogenase enzymes and molecular hydrogen from recycled whey-based by-products

Cupriavidus necator produces O2-tolerant [NiFe]-hydrogenases (Hyds), and is a valuable biocatalyst for biological fuel cells, while Escherichia coli produces H2 during mixed-acid fermentation. A mixture of curd and cheese whey (CW) was used to explore two-phase growth with C. necator H16 and E. coli. Enhanced biomass, as well as Hyd activity and electrical potential (∼0.65 V) were shown for growth of C. necator in the CW medium with added glycerol. The residual growth medium made available after the cultivation of C. necator and removal of cells was then used for cultivation of E. coli. Maximum fermentative growth of E. coli was attained after 72 h and with a H2 yield of ∼6 mmol/L/g dry whey after 48 h. This study demonstrates the economically viable production of biomass, hydrogenase enzymes and H₂ using cheap, industrially produced whey within the 3R concept.

Chemical profiling of fungal metabolites via the OSMAC approach: Novel identification of Brianthein W from an endophytic fungus, Hypomontagnella monticulosa Zg15SU

In the previous report, we reported that Hypomontagnella monticulosa originating from the rhizome of Zingiber griffithii was known to produce a marine-derived natural product. An OSMAC-based approach was designed by modifying the nutritional composition of the growth medium to investigate any possible new metabolites produced by the strain. The culture filtrate and biomass were conditioned through the use of three basal media, namely, Czapek-dox, potato dextrose, and Wickersham broth medium. GC-MS and multivariate analysis was performed to distinguish the chemicals and determine their composition in the tested extracts. Antimicrobial activity was tested against selected human pathogenic microbes using the disk-diffusion method. The MeOH extract of both culture filtrate and biomass from different fermentation media revealed that the majority of identified compounds (n=40) were found in Wickerham medium (n=23), which is later termed as MeOH-Wi. The chemical composition of MeOH-Wi was fatty acids (21.74%), followed by terpenoids (17.38%), cyclosiloxane (13.04%), aldehydes (13.04%), alkenes (8.7%), hydrocarbons (8.7%), esters (8.7%), alkaloids (4.35%), and an unclassified compound, the 9,9-Dimethyl-3,7-diazabicyclo[3.3.1]nonane. The numerous chemical compounds in MeOH-Wi corresponded with its antimicrobial activities against Micrococcus luteus NBRC 13867 (±11 mm), Candida maltosa NBRC 1977 (±9 mm), and Escherichia coli JM 109 (±5 mm) which were higher than the other tested extracts. Further purification using HPLC RP-C18 using NP-SiO2/n-Hex–EtOAc (10:2) to yield compound (1). Compound (1) was determined as an analog of briarian W—a diterpene mostly found in marine sponge, Briareum spp.—as a pure compound in the MeOH extract of H. monticulosa based on the 1D and 2D NMR analysis. The anticancer activity (IC50) of compound (1) was 0.077, 0.080, and 0.102 µg/mL against the growth of HCT-116, NBT-T2, and Panc-1, respectively. As far as we are aware, this is the first report on finding a briarian diterpene that originates from an endophytic fungus, especially by H. monticulosa.