Genome-wide transcriptional response to silver stress in extremely halophilic archaeon Haloferax alexandrinus DSM 27206 T.

BACKGROUND: The extremely halophilic archaeon Haloferax (Hfx.) alexandrinus DSM 27206 T was previously documented for the ability to biosynthesize silver nanoparticles while mechanisms underlying its silver tolerance were overlooked. In the current study, we aimed to assess the transcriptional response of this haloarchaeon to varying concentrations of silver, seeking a comprehensive understanding of the molecular determinants underpinning its heavy metal tolerance. RESULTS: The growth curves confirmed the capacity of Hfx. alexandrinus to surmount silver stress, while the SEM-EDS analysis illustrated the presence of silver nanoparticles in cultures exposed to 0.5 mM silver nitrate. The RNA-Seq based transcriptomic analysis of Hfx. alexandrinus cells exposed to 0.1, 0.25, and 0.5 mM silver nitrate revealed the differential expression of multiple sets of genes potentially employed in heavy-metal stress response, genes mostly related to metal transporters, basic metabolism, oxidative stress response and cellular motility. The RT-qPCR analysis of selected transcripts was conducted to verify and validate the generated RNA-Seq data. CONCLUSIONS: Our results indicated that copA, encoding the copper ATPase, is essential for the survival of Hfx. alexandrinus cells in silver-containing saline media. The silver-exposed cultures underwent several metabolic adjustments that enabled the activation of enzymes involved in the oxidative stress response and impairment of the cellular movement capacity. To our knowledge, this study represents the first comprehensive analysis of gene expression in halophillic archaea facing increased levels of heavy metals.

Insights into the Synergistic Antibacterial Activity of Silver Nitrate with Potassium Tellurite against Pseudomonas aeruginosa.

The constant, ever-increasing antibiotic resistance crisis leads to the announcement of "urgent, novel antibiotics needed" by the World Health Organization. Our previous works showed a promising synergistic antibacterial activity of silver nitrate with potassium tellurite out of thousands of other metal/metalloid-based antibacterial combinations. The silver-tellurite combined treatment not only is more effective than common antibiotics but also prevents bacterial recovery, decreases the risk of future resistance chance, and decreases the effective concentrations. We demonstrate that the silver-tellurite combination is effective against clinical isolates. Further, this study was conducted to address knowledge gaps in the available data on the antibacterial mechanism of both silver and tellurite, as well as to give insight into how the mixture provides synergism as a combination. Here, we defined the differentially expressed gene profile of Pseudomonas aeruginosa under silver, tellurite, and silver-tellurite combination stress using an RNA sequencing approach to examine the global transcriptional changes in the challenged cultures grown in simulated wound fluid. The study was complemented with metabolomics and biochemistry assays. Both metal ions mainly affected four cellular processes, including sulfur homeostasis, reactive oxygen species response, energy pathways, and the bacterial cell membrane (for silver). Using a Caenorhabditis elegans animal model we showed silver-tellurite has reduced toxicity over individual metal/metalloid salts and provides increased antioxidant properties to the host. This work demonstrates that the addition of tellurite would improve the efficacy of silver in biomedical applications. IMPORTANCE Metals and/or metalloids could represent antimicrobial alternatives for industrial and clinical applications (e.g., surface coatings, livestock, and topical infection control) because of their great properties, such as good stability and long half-life. Silver is the most common antimicrobial metal, but resistance prevalence is high, and it can be toxic to the host above a certain concentration. We found that a silver-tellurite composition has antibacterial synergistic effect and that the combination is beneficial to the host. So, the efficacy and application of silver could increase by adding tellurite in the recommended concentration(s). We used different methods to evaluate the mechanism for how this combination can be so incredibly synergistic, leading to efficacy against antibiotic- and silver-resistant isolates. Our two main findings are that (i) both silver and tellurite mostly target the same pathways and (ii) the coapplication of silver with tellurite tends not to target new pathways but targets the same pathways with an amplified change.

Microplastic Fibers Increase Sublethal Effects of AgNP and AgNO3 in Daphnia magna by Changing Cellular Energy Allocation.

The effects of combined exposure to microplastics and contaminants are still not completely understood. To fill this gap, we assessed the effects of polyethylene terephthalate microplastic fibers (100 mg/L; 360 µm average length) on the toxicity of silver nanoparticles (AgNPs; 32 nm) and silver nitrate (AgNO3 ; 0.1-10 µg Ag/L) to Daphnia magna. Acute immobilization (median effect concentration [EC50]) and cellular energy allocation (CEA; ratio between available energy and energy consumption) were determined in neonates (<24 h old) and juveniles (7 d old), respectively. The 48-h EC50 for AgNP and AgNO3 (2.6 and 0.67 µg Ag/L, respectively) was not affected by the presence of microplastic fibers (2.2 and 0.85 µg Ag/L, respectively). No decrease in the available energy was observed: lipid, carbohydrate, and protein contents were unaffected. However, a significant increase in energy consumption was observed in animals exposed to AgNO3 (250% compared with control) and to the combination of microplastic fibers with AgNP (170%) and AgNO3 (260%). The exposure to microplastic fibers alone or in combination with both Ag forms decreased the CEA (values were 55-75% of control values). Our results show that after short-term exposure (48 h), microplastic fibers increased Ag toxicity at a subcellular level (i.e., CEA), but not at the individual level (i.e., immobilization). These results highlight the importance of combining different levels of biological organization to fully assess the ecotoxicological effects of plastics in association with environmental contaminants. Environ Toxicol Chem 2022;41:896-904. © 2021 SETAC.

Synthesis of silver nanoparticles using white-rot fungus Anamorphous Bjerkandera sp. R1: influence of silver nitrate concentration and fungus growth time.

Currently, silver nanoparticles (AgNPs) constitute an interesting field of study in medicine, catalysis, optics, among others. For this reason, it has been necessary to develop new methodologies that allow a more efficient production of AgNPs with better antimicrobial and biological properties. In this research growth time effects Anamorphous Bjerkandera sp. R1 and the silver nitrate (AgNO3) concentration over AgNPs synthesis were studied. Through the protocol used in this work, it was found that the action of the capping proteins on the surface of the mycelium played a determining role in the reduction of the Ag+ ion to Ag0 nanoparticles producing a particle size that oscillated between 10 and 100 nm. The progress of the reaction was monitored using visible UV-Vis spectroscopy and the synthesized AgNPs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier transform infrared radiation (FTIR) spectroscopy. The best synthetic properties were found at 1 mM of AgNO3 concentration, growth time of 8 days, and reaction time of 144 h. Nanometals obtention from microorganisms could be considered as a new method of synthesis, due to reducing abilities of metal ions through its enzymatic system and represents low-cost synthesis that reduces the generation of harmful toxic wastes.

Dietary bioaccumulation potential of silver nanomaterials compared to silver nitrate in wistar rats using an ex vivo gut sac technique.

Chronic dietary bioaccumulation tests with rodents are required for new substances, including engineered nanomaterials (ENMs), in order to provide information on the potential hazards to human health. However, screening tools are needed to manage the diversity of ENMs and alternative methods are desirable with respect to animal welfare. Here, an ex vivo gut sac method was used to estimate the dietary bioaccumulation potential of silver nanomaterials. The entire gastrointestinal tract (except the caecum) was removed and filled with a gut saline containing 1 mg L-1 of Ag as either AgNO3, silver nanoparticles (Ag NPs) or silver sulphide nanoparticles (Ag2S NPs), and compared to controls with no added Ag. The gut sacs were incubated for 4 h, rinsed to remove excess media, and the total Ag determined in the mucosa and muscularis. There was no detected Ag in the control treatments. Within the Ag treatments, 1.4-22% of the exposure dose was associated with the tissues and serosal saline. Within the mucosa of the AgNO3 treatment, the highest Ag concentration was associated with the intestinal regions (3639-7087 ng g-1) compared to the stomach (639 ± 128 ng g-1). This pattern was also observed in the Ag NP and Ag2S NP treatments, but there was no significant differences between any Ag treatments for the mucosa. However, differences between treatments were observed in the muscularis concentration. For example, both the Ag NP (907 ± 284 ng g -1) and Ag2S NP (1482 ± 668 ng g-1) treatments were significantly lower compared to the AgNO3 treatment (2514 ± 267 ng g-1). The duodenum demonstrated serosal accumulation in both the AgNO3 (~10 ng mL-1) and Ag NP (~3 ng mL-1) treatments. The duodenum showed some of the highest Ag accumulation with 41, 61 and 57% of the total Ag in the mucosa compared to the muscularis for the AgNO3, Ag NP and Ag2S NP treatments, respectively. In conclusion, the ex vivo gut sac method demonstrates the uptake of Ag in all Ag treatments, with the duodenum the site of highest accumulation. Based on the serosal saline accumulation, the ranked order of accumulation is AgNO3 > Ag NPs > Ag2S NPs.

Antioxidant Enzyme Activity and Lipid Peroxidation in Aporrectodea caliginosa Earthworms Exposed to Silver Nanoparticles and Silver Nitrate in Spiked Soil.

Silver nanoparticles (AgNPs) from industrial use, discharged via the land application of sewage sludge, are interacting with soil biota, including earthworms. In affected organisms, excessive production of reactive oxygen species can result in lipid peroxidation, shifting the balance between oxidants and antioxidants to cause oxidative stress. We determined selected lower-tier biomarkers such as antioxidant responses and lipid peroxidation in Aporrectodea caliginosa earthworms exposed to soils spiked with AgNPs or silver nitrate (AgNO3 ). Aporrectodea caliginosa were exposed to AgNPs at 0 (control), 0.3, 3, 30, and 300 mg/kg or Ag+ (as AgNO3 ) at 0, 0.03, 0.3, 3, and 10 mg/kg in soil for 4 wk. At 1, 2, 3, and 4 wk, the activity of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, as well as lipid peroxidation (malondialdehyde content), increased as a function of concentration, with a much larger response for Ag+ than AgNPs. Given the likelihood of ever-increasing AgNP concentrations in soil, where AgNPs can transform to ionic Ag (Ag+ ), our findings of antioxidant response to oxidative stress in a common indicator organism even at an environmentally realistic exposure concentration of 0.03 mg/kg demonstrate that AgNPs may affect soil fertility and, thus, agricultural production. Evaluating selected lower-tier biomarkers offers a meaningful assessment of AgNPs and Ag+ effects on terrestrial earthworms. Environ Toxicol Chem 2020;39:1257-1266. © 2020 SETAC.

Establishment and characterization of silver-resistant Enterococcus faecalis.

Enterococcus faecalis is a Gram-positive facultative anaerobe involved in many fatal or refractory infections of humans. Silver, often used as silver ions (Ag+) or nanoparticles (AgNPs), is a strong and broad-spectrum antibacterial agent, but E. faecalis shows resistance against it. Despite this, the knowledge about the resistance of E. faecalis against silver is still lacking. In this study, the silver-resistant E. faecalis strains (AgR and ANR E. faecalis) were established through a serial selection method. Their biological and silver-resistant features as well as the Gene Ontology (GO) in comparison with the original E. faecalis were evaluated. The results showed that the silver-resistant E. faecalis could proliferate as original bacteria and had strong resistance against both Ag+ and AgNPs. The minimum bactericidal concentrations (MBCs) of AgNO3 on original, AgR, and ANR E. faecalis were 400 mg/L, 600 mg/L, and 500 mg/L, and the MBCs of AgNPs on these strains were 80 mg/L, 110 mg/L, and 130 mg/L, respectively. GO analysis revealed significant difference (P < 0.05) in gene expressions of biological process (BP), cellular component (CC), and molecular function (MF) among original, AgR, and ANR E. faecalis. These findings provided a significant basis for further understanding and managing the silver-resistance of E. faecalis in infection-control environments. The mechanism behind Ag+/AgNPs resistance of E. faecalis needs to be further investigated.

New decolorization method produces more information from tissue sections stained with hematoxylin and eosin stain and masson-trichrome stain.

Histological analysis is a fundamental and principal method used in biological research and even for disease diagnosis. The result shows the status of cells and tissues in organs and enables us to infer the condition of the whole body. The tissue staining method known as hematoxylin and eosin staining (HE) is one of the most general methods of investigating the status of cells and tissues. Hematoxylin stains the nucleus violet and eosin stains cytosol pink. HE staining shows the unique morphologies of tissues and cells. However, after being stained with HE, tissues are very difficult to use in another histological analysis because hematoxylin is hard to remove from the sections due to its stain stability. Therefore, serial sections of the tissue are used to obtain more information through another staining, including immunohistochemistry. The adjacent tissue section is not the same as the HE-stained section, however, so the results from the adjacent sections can cause confusion or ambiguity. The present study showed that our decolorization solution can decolor the hematoxylin or iron hematoxylin stain from stained structures, including the nucleus, and the decolored section could be stained again in another staining, including immunohistochemistry. This decolorization method is very valuable, in that it can determine the accurate distribution of substances and features in cells and tissues, and thus it can improve the robustness of the resulting data.

Impact of in vitro digestion on gastrointestinal fate and uptake of silver nanoparticles with different surface modifications.

Nanomaterials, especially silver nanoparticles (AgNPs), are used in a broad range of products owing to their antimicrobial potential. Oral ingestion is considered as a main exposure route to AgNPs. This study aimed to investigate the impact of the biochemical conditions within the human digestive tract on the intestinal fate of AgNPs across an intestinal in vitro model of differentiated Caco-2/HT29-MTX cells. The co-culture model was exposed to different concentrations (250-2500 µg/L) of pristine and in vitro digested (IVD) AgNPs and silver nitrate for 24 h. ICP-MS and spICP-MS measurements were performed for quantification of total Ag and AgNPs. The AgNPs size distribution, dissolution, and particle concentration (mass- and number-based) were characterized in the cell fraction and in the apical and basolateral compartments of the monolayer cultures. A significant fraction of the AgNPs dissolved (86-92% and 48-70%) during the digestion. Cellular exposure to increasing concentrations of pristine or IVD AgNPs resulted in a concentration dependent increase of total Ag and AgNPs content in the cellular fractions. The cellular concentrations were significantly lower following exposure to IVD AgNPs compared to the pristine AgNPs. Transport of silver as either total Ag or AgNPs was limited (<0.1%) following exposure to pristine and IVD AgNPs. We conclude that the surface chemistry of AgNPs and their digestion influence their dissolution properties, uptake/association with the Caco-2/HT29-MTX monolayer. This highlights the need to take in vitro digestion into account when studying nanoparticle toxicokinetics and toxicodynamics in cellular in vitro model systems.

Cyanobacterial extract-mediated synthesis of silver nanoparticles and their application in ammonia sensing

Green route for silver nanoparticle synthesis has gained increasing attention. Cyanobacteria are one of the promising organisms to produce a number of secondary metabolites that are capable of reducing silver ions to small-sized silver nanoparticles. In the present study, we employed an aqueous extract of the cyanobacterium Haloleptolyngbya alcalis KR2005/106 isolated from a soda lake for biosynthesis of silver nanoparticles (AgNPs). The extract acted as a reducing agent for AgNPs synthesis and resulted formation of nanoparticles <50 nm in size. In this study, synthesis of AgNPs obtained only in the sample exposed to photosynthetically active radiation (PAR) while the synthesis of AgNPs was not observed in the samples kept in dark. The biogenic fabrication of AgNPs was carried out by optimizing several governing parameters such as concentration of the silver nitrate solution, pH, temperature, and amount of biomass. Results obtained through different analytical techniques revealed that cyanobacterial taxon H. alcalis isolated from saline-alkaline habitat is a potential candidate for biosynthesis of optimum-sized spherical AgNPs. Surface plasmon resonance (SPR) property of AgNPs was exploited for aqueous ammonia sensing and revealed that AgNPs synthesized using aqueous extract of cyanobacterium H. alcalis could be employed for colorimetric detection of dissolved ammonia for monitoring quality of water

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Cyanobacterial extract-mediated synthesis of silver nanoparticles and their application in ammonia sensing.

Green route for silver nanoparticle synthesis has gained increasing attention. Cyanobacteria are one of the promising organisms to produce a number of secondary metabolites that are capable of reducing silver ions to small-sized silver nanoparticles. In the present study, we employed an aqueous extract of the cyanobacterium Haloleptolyngbya alcalis KR2005/106 isolated from a soda lake for biosynthesis of silver nanoparticles (AgNPs). The extract acted as a reducing agent for AgNPs synthesis and resulted formation of nanoparticles < 50 nm in size. In this study, synthesis of AgNPs obtained only in the sample exposed to photosynthetically active radiation (PAR) while the synthesis of AgNPs was not observed in the samples kept in dark. The biogenic fabrication of AgNPs was carried out by optimizing several governing parameters such as concentration of the silver nitrate solution, pH, temperature, and amount of biomass. Results obtained through different analytical techniques revealed that cyanobacterial taxon H. alcalis isolated from saline-alkaline habitat is a potential candidate for biosynthesis of optimum-sized spherical AgNPs. Surface plasmon resonance (SPR) property of AgNPs was exploited for aqueous ammonia sensing and revealed that AgNPs synthesized using aqueous extract of cyanobacterium H. alcalis could be employed for colorimetric detection of dissolved ammonia for monitoring quality of water.

Protocol identification and preservation of myxozoan parasites for microscopy with silver nitrate (Klein's dry) staining technique.

The present study, discusses a first report of staining techniques with silver nitrate (AgNO3 ) stain for the preservation and identification of myxozoans globally. The silver nitrate stain was used to prepare permanent slide preparation of myxozoans with some adaptations made in our laboratory. Fresh air dried smear were stained with silver nitrate stain ensuing dark brown color polar capsules and light brown color of spore-wall. The stain is everlasting for years differing to other stains like Geimsa, iron-heamotoxylin Zeihl-Neelsen Blue, and trichome stains. RESEARCH HIGHLIGHTS: Stains usually fade with time and fail to disclose the morphological characters of the specimen. Present staining method helps to detect less infection in the tissue locating the myxospores. The Klein dry method (1958) is useful and suitable for long term preservation of the myxozoan slides and morphological description.

Route of exposure has a major impact on uptake of silver nanoparticles in Atlantic salmon (Salmo salar).

The potential impact of silver nanoparticles (Ag NPs) on aquatic organisms is to a large extent determined by their bioavailability through different routes of exposure. In the present study juvenile Atlantic salmon (Salmo salar) were exposed to different sources of radiolabeled Ag (radiolabeled 110m Ag NPs and 110m AgNO3 ). After 48 h of waterborne exposure to 3 µg/L citrate stabilized 110m Ag NPs or 110m AgNO3 , or a dietary exposure to 0.6 mg Ag/kg fish (given as citrate stabilized or uncoated 110m Ag NPs, or 110m AgNO3 ), Ag had been taken up in fish regardless of route of exposure or source of Ag (Ag NPs or AgNO3 ). Waterborne exposure led to high Ag concentrations on the gills, and dietary exposure led to high concentrations in the gastrointestinal tract. Silver distribution to the target organs was similar for both dietary and waterborne exposure, with the liver as the main target organ. The accumulation level of Ag was 2 to 3 times higher for AgNO3 than for Ag NPs when exposure was through water, whereas no significant differences were seen after dietary exposure. The transfer (Bq/g liver/g food or water) from exposure through water was 4 orders of magnitude higher than from feed using the smallest, citrate-stabilized Ag NPs (4 nm). The smallest NPs had a 5 times higher bioavailability in food compared with the larger and uncoated Ag NPs (20 nm). Despite the relatively low transfer of Ag from diet to fish, the short lifetime of Ag NPs in water and their transfer to sediment, feed, or sediment-dwelling food sources such as larvae and worms could make diet a significant long-term exposure route. Environ Toxicol Chem 2018;37:2895-2903. © 2018 SETAC.

Antiproliferative effect of p-Coumaric acid targets UPR activation by downregulating Grp78 in colon cancer.

p-CA is a naturally occurring phenolic acid present in most plants and in all commonly consumed vegetables and fruits. Here we demonstrated the anti-cancer effect of the food borne phytochemical p-CA both in vitro and in vivo models of colon cancer using growth rate and tumor incidence as endpoints. Glucose regulated protein (GRP78) induction and UPR activation plays a key role in oncogenic progression, therefore increased dependence of cancer cells on these UPR signaling pathways for survival can be exploited for anti-cancer research. Hence we investigated the effect of p-CA on Grp78 a molecular chaperone often upregulated in colon cancer and its impact on unfolded protein response (UPR). Administration of the procarcinogen 1,2- dimethylhydrazine (DMH) causes Grp78 upregulation and tumor adaptation via UPR activation. The adaptive activity of UPR activates antiapoptotic NF-κB that results in upregulation of the markers of inflammation and angiogenesis. Supplementation of p-CA downregulated Grp78 and activated UPR mediated apoptosis both in in vitro and in vivo models of colon cancer. Further we observed that p-CA significantly reduced inflammation by decreasing the expression of cytokines COX-2, IL-6, TNF-α and PGE2 as analyzed by q-PCR and also reduced the expression of p-p65 and p-IκBα as analyzed by western blot. Further mechanistic insights revealed that p-CA inhibits Grp78 upregulation in cancer cells through activation of PERK-eIF2α-ATF-4-CHOP pathway that culminates in apoptosis inducing effect of p-CA.

Comparing ex vivo and in vitro translocation of silver nanoparticles and ions through human nasal epithelium.

Silver nanomaterials are widely used in clinically approved devices, consumer goods, and over-the-counter nutraceutical products. Despite the increase in silver nanomaterial research, few investigations have specifically distinguished the biological effects resulting from silver nanoparticles (AgNPs) versus silver ions released from AgNPs. This is in part, due to the complex analytical methods required to characterize silver ion release from AgNPs in biological media. This study sought to analyze silver ion release from AgNPs in biological media, compare silver transport from soluble AgNO3 and AgNPs through ex vivo full thickness sinus human tissue explants and human nasal epithelium and determine fractional AgNP internalization by human nasal epithelial cells. Rapid silver ion release is observed from AgNPs in human nasal epithelial cell medium over 3 h (9.6% of total silver mass). Significantly lower translocation of AgNPs is observed through human nasal epithelial cell monolayers and ex vivo human sinus tissue explants compared to silver ion (AgNO3). AgNP internalization is directly observed in AgNP-exposed human nasal epithelial cell monolayers by live cell scanning transmission electron microscopy (STEM), providing one potential mechanism for AgNP transcytosis. However, in vitro AgNP dissolution experiments suggest that silver in human nasal epithelium is primarily silver ion. Ionic AgNO3 produces significantly higher silver translocation, supporting previous results claiming silver ion as primarily responsible for biological effects of AgNPs.

Nanoparticles synthesis by Agaricus soil basidiomycetes.

We examined the effect of various concentrations of HAuCl4, AgNO3, Na2SeO3, Na2SiO3, and GeO2 on mycelial growth of the soil basidiomycetes Agaricus bisporus and A. arvensis in submerged and solid media. Fungal mycelial extracts and cell-free culture filtrates were able to reduce ions of Au, Ag, Se, Si, and Ge compounds, forming Au0, Ag0, Se0, Si0/SiO2 and Ge0/GeO2 nanoparticles. The physical characteristics of the mycogenic nanoparticles differed depending on the species of Agaricus and the type of extract. Au nanospheres obtained with cell-free culture filtrates were of 2-5 nm diameter in A. bisporus and of 2-10 nm in A. arvensis. Nanoparticles produced by extracts of mycelia were several times larger and highly heterogenous. Ag nanoparticles produced by cell-free culture filtrates were spherical or irregular-shaped and agglomerated, whereas with extracts of mycelia, small homogenous nanospheres of 1-10 nm were formed. Se nanospheres obtained with cell-free culture filtrates were of 100-250 nm diameter in A. bisporus and of 150-550 nm diameter in A. arvensis. The particles synthesized with extracts of mycelia were of 40-140 nm in A. bisporus and of 100-250 nm in A. arvensis. Incubation of Na2SiO3 with cell-free culture filtrates resulted in porous Si nanoparticles of 30-65 nm in A. bisporus and of 50-200 nm in A. arvensis. Ge nanoparticles synthesized by both Agaricus species were mostly spheres of 50-250 nm diameter.

Biosynthesis of silver nanoparticles using novel Bacillus sp. SBT8.

Biosynthesis of silver nanoparticles (AgNPs) using microorganisms is an important application of nanobiotechnology and green chemistry because of interest by pharmaceutical and food manufacturers. In this study, biosynthesis of AgNPs by a novel Bacillus strain isolated from a soil sample from Sakarya district in Turkey was investigated. Biosynthesis was performed using cell-free supernatant of the bacterium following 24 h growth. Effects of varying AgNO3 concentration (1-10 mM), pH (5-10), and temperature (30-40°C) on the synthesis of AgNPs were determined. Formation of AgNPs was monitored by UV-VIS spectroscopy. Field emission scanning electron microscopy was used to compare morphologies among the various culture conditions. The peaks created by surface plasmon resonance (SPR) of metals were obtained only at 4 and 6 mM AgNO3 concentrations and the maximum concentration for the biosynthesis was observed at 6 mM. The highest yield was achieved at pH 10 and larger nanoparticles were obtained at this pH. The optimum temperatures for biosynthesis were 33 and 37°C. Fourier transform infrared spectroscopy analysis and transmission electron microcopy images confirmed that the proteins served as capping. Energy-dispersive spectroscopy analysis validated the formation of AgNPs. AgNPs exhibited antibacterial activity toward Gram-positive and Gram-negative pathogens.

Enhanced production of total flavones from Inonotus baumii by multiple strategies.

As one kind of important secondary metabolites produced by Inonotus baumii, flavones can be applied in food, medicine, and other industries due to their biological activities such as antioxidant, anticancer, and antibacterial activity. To enhance total flavone production in submerged fermentation of I. baumii, three different strategies, optimization of fermentation parameters by statistical designs including Plackett-Burman design and response surface methodology, addition of precursors and elicitors, and two-phase culture, were used. The production of total flavones (PTF) reached 1532.83 mg/L when the optimized medium was used. All precursors and elicitors can increase the PTF. The maximum PTF (2184.06 mg/L, up to 1.57-fold) was obtained with the addition of both AgNO3 and glutathione in fermentation media. Interestingly, when 0.5% (w/v) DM130 macroporous resin as adsorbent was added to fermentation broth on day 4 of culture, the highest production reached 2407.79 mg/L with this two-phase culture strategy. These methods can be further applied to large-scale industrial production and broaden the application of flavones.

Extracellular biosynthesis of silver nanoparticles using the cell-free filtrate of nematophagous fungus Duddingtonia flagrans.

The biosynthesis of metallic nanoparticles (NPs) using biological systems such as fungi has evolved to become an important area of nanobiotechnology. Herein, we report for the first time the extracellular synthesis of highly stable silver NPs (AgNPs) using the nematophagous fungus Duddingtonia flagrans (AC001). The fungal cell-free filtrate was analyzed by the Bradford method and 3,5-dinitrosalicylic acid assay and used to synthesize the AgNPs in the presence of a 1 mM AgNO3 solution. They have been characterized by UV-Vis spectroscopy, X-ray diffraction, transmission electron microscopy, dynamic light scattering, Zeta potential measurements, Fourier-transform infrared, and Raman spectroscopes. UV-Vis spectroscopy confirmed bioreduction, while X-ray diffractometry established the crystalline nature of the AgNPs. Dynamic light scattering and transmission electron microscopy images showed approximately 11, 38 nm monodisperse and quasispherical AgNPs. Zeta potential analysis was able to show a considerable stability of AgNPs. The N-H stretches in Fourier-transform infrared spectroscopy indicate the presence of protein molecules. The Raman bands suggest that chitinase was involved in the growth and stabilization of AgNPs, through the coating of the particles. Our results show that the NPs we synthesized have good stability, high yield, and monodispersion.

The effect of soil properties on the toxicity and bioaccumulation of Ag nanoparticles and Ag ions in Enchytraeus crypticus.

Standard natural Lufa soils (2.2, 2.3 and 5M) with different organic carbon contents (0.67-1.61%) and pHCaCl2 (5.5-7.3) were spiked with ionic Ag (AgNO3) and polyvinyl pyrrolidone (AgNP-PVP) and citrate (AgNP-Cit) coated Ag nanoparticles (NPs). Enchytraeus crypticus were exposed for 21 days to assess effects on survival and reproduction. Soil, pore water and animals were analyzed for Ag. AgNP-Cit had a strong increasing effect on soil pH, leading to high enchytraeid mortality at concentrations higher than 60-100mg Ag/kg dry soil which made it impossible to determine the influence of soil properties on its toxicity. LC50s were lower for AgNO3 than for AgNP-PVP (92-112 and 335-425mg Ag/kg dry soil, respectively) and were not affected by soil properties. AgNO3 and AgNP-PVP had comparable reproductive toxicity with EC50s of 26.9-75.2 and 28.2-92.3mg Ag/kg dry soil, respectively; toxicity linearly increased with decreasing organic carbon content of the soils but did not show a clear effect of soil pH. Ag uptake in the enchytraeids was higher at higher organic carbon content, but could not explain differences in toxicity between soils. This study indicates that the bioavailability of both ionic and nanoparticulate Ag is mainly affected by soil organic carbon, with little effect of soil pH.