Regenerated Silk Fibroin Solution Research Articles

In Situ ATR-FTIR Studies on the β-Sheet Formation of Native and Regenerated Bombyx mori Silk Material in Solution and Its Potential for Drug Releasing Coatings.

Dynamic attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy at both solutions and coatings of a semicrystalline silk material derived from Bombyx mori was applied to monitor the β-sheet conformation, which is known to correlate with silk protein crystallinity. The secondary structure-sensitive Amide I band was analyzed. Two silk protein samples were studied: native-based silk buffer fibroin (NSF) was extracted from silk glands and regenerated silk fibroin (RSF) was extracted from degummed cocoons. Solutions of both NSF and RSF at 2 mg/mL featured low initial β-sheet contents of 5-12%, which further increased to 47-53% after 24 h. RSF and NSF solutions at 23 mg/mL also featured low initial β-sheet contents of 9-10%, which yet only slightly increased to 16-17% after 24 h. Coatings deposited from RSF solutions showed high surface integrity (Q > 99%) after rinsing in mineralized water, enabling interfacial drug delivery applications. RSF coatings were post-treated with either formic acid (FA) or pure methanol (MeOH) vapor to showcase inducibility of crystalline domains in RSF coatings. Such coatings were loaded with the model antibiotic drugs tetracycline (TCL) and streptomycin (STRP), and the sustained release of TCL was followed in contact with (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer. RSF/TCL coatings post-treated with formic acid (FA) vapor followed by methanol (MeOH) vapor showed a significantly lower (52%) initial burst of rather hydrophobic TCL compared to untreated RSF/TCL coatings (72%), while no such significant release difference was observed for hydrophilic STRP. This was rationalized by a specific interaction between nonpolar TCL and hydrophobic crystalline RSF domains.

The production of soluble regenerated silk fibroin powder with high molecular weight and silk protein-based materials

The aqueous solution of regenerated silk fibroin (RSF) is considered as the raw material to produce various silk protein-based materials, including hydrogel, film, rod and fiber, etc. with significant mechanical properties. However, the aqueous solution of RSF is usually unstable within a few days or even hours in terms of its essential properties, because the conformation of the RSF chain would spontaneously transfer from random coil to β-sheet in water. In this work, we developed a way to harvest the RSF powder through an optimized spray drying method via rapid drying at a relatively low temperature. It was demonstrated that no severe degradation and conformational transition of the RSF chain occurred during powder preparation, and the RSF powder exhibited remarkable solubility in water and long stability at room temperature. Importantly, there are no obvious differences in the mechanical properties of the silk protein-based materials made from the aqueous solution from the spray-dried RSF powder (Sp-RSF) and from fresh RSF solution. Indeed, such amorphous Sp-RSF powder, in which the protein chain was dominated by random coil conformation, not only promised as the raw material for large-scale silk protein-based products in various applications but also provided the basis for fabricating bulk silk protein materials via the untraditional processing of silk fibroin, such as molding with the help of heat and moisture.

Silk‐Based Functional Inks with Color‐Tunable Light Upconversion for Printing on Arbitrary Surfaces

AbstractA biocompatible and versatile silk‐based functional ink with hydrophilic upconversion nanoparticles (UCNPs) that can be printed or painted on arbitrary substrates, including biological surfaces is demonstrated. Hydrophilic UCNPs, ready to be dispersed in aqueous silk fibroin solution, are synthesized in large quantities via facile hydrothermal synthesis using citric acid. Based on a systematic study of dopant control to achieve robust red emission from citrate‐capped UCNPs, three primary colors are obtained, which serve as the basis for the expansion of the color palette. The synthesized UCNPs are homogeneously dispersed in regenerated silk fibroin solutions derived from Bombyx mori cocoons to produce water‐based functional inks. Cyan, magenta, and yellow (CMY) inks are created by combining red, green, and blue (RGB) inks. Demonstrator devices, where a variety of colorful patterns and codes are revealed only under near‐infrared irradiation, are realized through stencil printing or brush painting on flexible polymer films, fruit, leaves, silkworm cocoons, and pig skin. The patterns and codes are highly heat resistant (≈230 °C) and can be easily washed off with water after use. The printable/paintable inks and functions proposed here will provide new opportunities for applications where sustainability, biocompatibility, and intentional luminescence are simultaneously required.

Partially insoluble regenerated silk fibroin film induced by UV irradiation for electronic skins

The regenerated silk fibroin (RSF) film has been regarded as an ideal substrate for biocompatible, flexible, and biodegradable electronic skin (e-skin) devices. However, it is still a great challenge to balance the flexibility and solubility of the RSF film by adjusting its secondary structure. Herein, a film prepared with the hydrolyzed RSF was exposed to the 254 nm ultraviolet (UV) light to prepare a crosslinked and partially water-insoluble substrate for a strain-sensing e-skin. The hydrolyzed low-molecular-weight RSF was produced by heating the LiBr-silk fibroin solution at 85 °C for a certain duration. The film cast with the hydrolyzed RSF solution could be thoroughly dissolved in water rapidly. The UV irradiation could induce the crosslinking of the low-molecular-weight RSF to form insoluble substances, thus producing a partially insoluble RSF film. After silver nanowires painting, an e-skin strain sensor was successfully constructed based on the UV-irradiated film. The sensor shows a fast response time (2.01 s), high sensitivity (GF = 1.03 within 0%–40% strain range), and good stability. The device could be tightly attached to human skin with a drop of water. The finger, wrist, elbow, and knee bending could be sensitively detected in real-time. The head nodding and mouth opening could also be sensed by sticking the e-skin at the neck and cheek, respectively. This work may provide a facile way to prepare a stretchable and stickable RSF film, which could serve as an ideal substrate of low-cost, biodegradable, direct-to-skin sensors for wearable applications.

Continuous Wet Spinning of Regenerated Silk Fibers from Spinning Dopes Containing 4% Fibroin Protein.

The wet spinning of fibers from regenerated silk fibroin has long been a research goal. Due to the degradation of the molecular structure of the fibroin protein during the preparation of the regenerated silk fibroin solution, fibroin concentrations with at least 10% protein content are required to achieve sufficient viscosity for wet spinning. In this study, a spinning dope formulation of regenerated silk fibroin is presented that shows a rheological behavior similar to that of native silk fibroin isolated from the glands of B. mori silkworm larvae. In addition, we present a wet-spinning process that enables, for the first time, the continuous wet spinning of regenerated silk fibroin with only 4% fibroin protein content into an endless fiber. Furthermore, the tensile strength of these wet-spun regenerated silk fibroin fibers per percentage of fibroin is higher than that of all continuous spinning approaches applied to regenerated and native silk fibroin published so far.

Long-Lasting Thixotropic Natural Polymeric Hydrogel Based on Silk Nanofibrils.

Hydrogels are considered to be ideal biomedical materials as their physical properties are similar to the physiological tissue environment. In particular, thixotropic hydrogels have received increasing attention from researchers because of their injectability. Herein, a simple and rapid method was developed for the preparation of a regenerated silk fibroin (RSF) hydrogel with long-lasting and excellent thixotropy. The thixotropic RSF hydrogel was readily formed by ultrasonic treatment of the pretreated RSF solution for 2 min followed by incubation at 40 °C for 10 min. The storage modulus of the RSF hydrogels recovered to more than 90% of the original value within 20 s after withstanding 1000% shear strain. By avoiding complicated chemical or physical treatments and by addition of crosslinking agents and/or other chemical components, the obtained RSF hydrogels maintained excellent biocompatibility. Hence, the cells implanted inside the hydrogel can grow and proliferate normally. By virtue of ultrasonic treatment during the preparation, functional nanoparticles can be uniformly dispersed in the RSF solution to prepare RSF-based hybrid hydrogels with various functions. As an application example, hydroxyapatite (HAP) with osteoinductivity was mixed with RSF solution to prepare the RSF/HAP hybrid hydrogel. The RSF/HAP hybrid hydrogel maintained biocompatibility and thixotropy of the original RSF hydrogel and promoted osteoblastic differentiation of cells owing to the addition of HAP. Therefore, the RSF hydrogel prepared in this work has a strong application prospect in the biomedical field including, but not limited to, bone repair.

Local injection of adipose-derived mesenchymal stem cells in silk fibroin solution on the regeneration of lower esophageal sphincter in an animal model of GERD.

Presently, various tissue engineering methods using adult stem cells and biomaterials are being confirmed to regenerate vessels, cardiac muscle, bladder, and intestines. However, there are few studies about the repair of the lower esophageal sphincter (LES) may help alleviate the symptoms of gastroesophageal reflux disease (GERD). This study aims to determine whether Adipose-Derived Stem Cells (ADSCs) combined with regenerated silk fibroin (RSF) solution could regenerate the LES. In vitro, the ADSCs were isolated, identified, and then cultured with an established smooth muscular induction system. In vivo, in the experimental groups, CM-Dil labeled ADSCs or induced ADSCs mixed with RSF solution were injected into the LES of rats after the development of the animal model of GERD respectively. The results showed that ADSCs could be induced into smooth muscular-like cells with the expression of h-caldesmon, calponin, α-smooth muscle actin, and a smooth muscle-myosin heavy chain in vitro. In vivo, the thickness of LES in the experiment rats was much thicker than those in the controlled groups. This result indicated that ADSCs mixed with RSF solution might contribute to the regeneration of the LES, thus reducing the occurrence of GERD.

Controllable Production of Natural Silk Nanofibrils for Reinforcing Silk-Based Orthopedic Screws

As a natural high-performance material with a unique hierarchical structure, silk is endowed with superior mechanical properties. However, the current approaches towards producing regenerated silk fibroin (SF) for the preparation of biomedical devices fail to fully exploit the mechanical potential of native silk materials. In this study, using a top-down approach, we exfoliated natural silk fibers into silk nanofibrils (SNFs), through the disintegration of interfibrillar binding forces. The as-prepared SNFs were employed to reinforce the regenerated SF solution to fabricate orthopedic screws with outstanding mechanical properties (compression modulus > 1.1 GPa in a hydrated state). Remarkably, these screws exhibited tunable biodegradation and high cytocompatibility. After 28 days of degradation in protease XIV solution, the weight loss of the screw was ~20% of the original weight. The screws offered a favorable microenvironment to human bone marrow mesenchymal stem cell growth and spread as determined by live/dead staining, F-action staining, and Alamar blue staining. The synergy between native structural components (SNFs) and regenerated SF solutions to form bionanocomposites provides a promising design strategy for the fabrication of biomedical devices with improved performance.

Design of Volumetric Nanolayers via Rapid Proteolysis of Silk Fibroin for Tissue Engineering.

Various methods have been studied to make a regenerated silk fibroin solution. However, most of them take too much time and effort to liquefy. Here, we report that a regenerated silk fibroin solution could be prepared within seconds through acid proteolysis for the first time. The solubilized fibroin could be applied to advanced tissue engineering. Our method shortened the production time to one day (more than 10 times) compared to the general fibroin solution preparation method. It was confirmed that the initial protein affinity nearly doubled from 0.028 to 0.076 μg·mm-2 in FF(ac) compared to FF(aq). A fibroin nanofiber layer having a volumetric hierarchical structure was prepared by electrospinning an acid-proteolyzed fibroin solution, followed by gas foaming. In vitro results of cell adhesion and proliferation capacity of the gas-foamed scaffold were not significantly different compared to the two-dimensional (2D) fibroin nanofiber membrane, overcoming the limitations of volumetric nanofiber scaffolds. We are confident that our research will greatly contribute to the development of regenerative engineering using other proteins.

A facile and green approach toward precise fabrication of silk fibroin-based microfluidic devices using water as etchant

Regenerated silk fibroin (RSF) is considered as a superior substrate material for biocompatible and biodegradable microfluidic devices. However, the manufacture of RSF-based microfluidic chips in a simple, low-cost, and environmentally friendly manner remains a difficulty. We devised a tape mask-assisted approach for preparing RSF-based microfluidic chips for cell adhesion and growth utilizing water as an eco-friendly etchant. The RSF solution was pre-hydrolyzed to assure the RSF film's water-solubility. The UV–visible spectra reveal that the RSF film, as made, is easily dissolved in water. The film could be turned into a water-insoluble state after being treated with ethanol for 40 min. Concave and convex micro-patterns may be formed on the RSF film using tape mask-assisted water etching and ethanol-induced insolubilization. Controlling the etching period allowed the tape-mask designs to be carefully transferred to the RSF film. Unlike LiBr etching, water treatment creates channels that are covered by a smooth RSF hydrogel layer, which may aid cell adhesion and development. The creation of the hydrogel layer might be explained by the insoluble RSF chains exposed after water etching and the ethanol-induced conformation change. Human umbilical vein endothelial cells were seeded in a blood vessel-like RSF-based channel, demonstrating the quick attachment and growth tendency. After 7 days of growth, a micro-vessel-like structure was visible in the channel, suggesting the device's high biocompatibility and vast potential. This work introduces a low-cost, facile and eco-friendly approach to fabricating RSF-based microfluidic devices for cell- or tissue-based assays.

Recent Research Progress of Ionic Liquid Dissolving Silks for Biomedicine and Tissue Engineering Applications.

Ionic liquids (ILs) show a bright application prospect in the field of biomedicine and energy materials due to their unique recyclable, modifiability, structure of cation and anion adjustability, as well as excellent physical and chemical properties. Dissolving silk fibroin (SF), from different species silkworm cocoons, with ILs is considered an effective new way to obtain biomaterials with highly enhanced/tailored properties, which can significantly overcome the shortcomings of traditional preparation methods, such as the cumbersome, time-consuming and the organic toxicity caused by manufacture. In this paper, the basic structure and properties of SF and the preparation methods of traditional regenerated SF solution are first introduced. Then, the dissolving mechanism and main influencing factors of ILs for SF are expounded, and the fabrication methods, material structure and properties of SF blending with natural biological protein, inorganic matter, synthetic polymer, carbon nanotube and graphene oxide in the ILs solution system are introduced. Additionally, our work summarizes the biomedicine and tissue engineering applications of silk-based materials dissolved through various ILs. Finally, according to the deficiency of ILs for dissolving SF at a high melting point and expensive cost, their further study and future development trend are prospected.

Polydopamine-Induced Multilevel Engineering of Regenerated Silk Fibroin Fiber for Photothermal Conversion.

Solid photothermal materials with favorable biocompatibility and modifiable mechanical properties demonstrate obvious superiority and growing demand. In this work, polydopamine (PDA) induced functionalization of regenerated silk fibroin (RSF) fibers has satisfactory photothermal conversion ability and flexibility. Based on multilevel engineering, RSF solution containing PDA nanoparticles is wet spun to PDA-incorporating RSF (PDA@RSF) fibers, and then the fibers are coated with PDA via oxidative self-polymerization of dopamine to form PDA@RSF-PDA (PRP) fibers. During the wet spinning process, PDA is to adjust the mechanical properties of RSF by affecting its hierarchical structure. Meanwhile, coated PDA gives the PRP fibers extensive absorption of near-infrared light and sunlight, which is further fabricated into PRP fibrous membranes. The temperature of PRP fibrous membranes can be adjusted and increases to about 50°C within 360 s under 808nm laser irradiation with a power density of 0.6Wcm-2 , and PRP fibrous membranes exhibit effective photothermal cytotoxicity both in vitro and in vivo. Under the simulated sunlight, the temperature of PRP fiber increases to more than 200°C from room temperature and the material can generate 4.5V voltage when assembled with a differential thermal battery, which means that the material also has the potential for flexible wearable electronic devices.

A Fast and Reliable Process to Fabricate Regenerated Silk Fibroin Solution from Degummed Silk in 4 Hours.

Silk fibroin has a high potential for use in several approaches for technological and biomedical applications. However, industrial production has been difficult to date due to the lengthy manufacturing process. Thus, this work investigates a novel procedure for the isolation of non-degraded regenerated silk fibroin that significantly reduces the processing time from 52 h for the standard methods to only 4 h. The replacement of the standard degumming protocol by repeated short-term microwave treatments enabled the generation of non-degraded degummed silk fibroin. Subsequently, a ZnCl2 solution was used to completely solubilize the degummed fibroin at only 45 °C with an incubation time of only 1 h. Desalting was performed by gel filtration. Based on these modifications, it was possible to generate a cytocompatible aqueous silk fibroin solution from degummed silk within only 4 h, thus shortening the total process time by 48 h without degrading the quality of the isolated silk fibroin solution.

Randomly Induced Phase Transformation in Silk Protein-Based Microlaser Arrays for Anticounterfeiting.

Anticounterfeiting labels based on physical unclonable functions (PUFs) exhibit high security with unreplicable code outputs, making them an ideal platform to realize unbreakable anticounterfeiting. Although various schemes are proposed for PUF labels, the utilization of natural randomness suffers from unpredictable signal extraction sites, which poses a challenge to efficient and convenient authentication for practical anticounterfeiting applications. Here, a covert optical PUF-based cryptographic protocol from silk protein-based microlaser (SML) arrays that possess hidden randomness of lasers for unclonable lasing signals as well as a defined location for efficient identification is proposed. The initial SMLs are patterned by casting laser dye-doped regenerated silk fibroin solution, resulting in a uniform microlaser array with regulated positions. With the SML array as substrate, random methanol microdroplets are stochastically sprayed on the SML array, which eventually induces uneven lasing signal changes of the patterned microlasers. The treated SML array possesses the deterministic readout sites of laser signals and unrepeatable signal distribution characteristics, which can guarantee efficient authentication and high security when serving as an anticounterfeiting label.

A Comparative Study on the Dissolution of Argema mimosae Silk Fibroin and Fabrication of Films and Nanofibers.

Limited studies have been done on silk fibroins of wild silkworm species owing to their relative insolubility in many solvents. In this study, the solubility of Argema mimosae wild silk fibroin in different salts (LiBr, LiCl, Ca(NO3)2, and CaCl2) dissolved in formic acid under varying temperatures was investigated. The dissolution conditions under which the solubility was optimum were optimized using a central composite design approach. The optimum range for solvation of the fibroin were visualized using contour plots. The influence of temperature and salt concentration were found to significantly influence the solvation of the fibroin. Following the successful dissolution of the fibroin, the regenerated silk fibroin solutions were cast to obtain water insoluble films which were used in investigating optimum electrospinning conditions. Average nanofiber diameters in the 110–141 nm range were obtained under optimum electrospinning conditions. The silk forms were characterized using the FTIR, TGA, XRD, and SEM to understand their properties. The investigations revealed that formic acid—salt solvents were effective in the solvation of the wild silk fibroin. Some of the dissolution conditions induced mild effects on the silk fibroin while others were harsh. Furthermore, processing to nanofibers resulted in the degradation of the β-sheets producing nanofibers rich in α-helices. However, post-treatment using methanol and water vapor were effective in restoring β-sheet crystallinity.

Preparation of silk fibroin-based microspheres under high-gravity field

The possibility of preparing regenerated silk fibroin (RSF)-based microspheres by a high-gravity anti-solvent precipitation method was explored. The high-gravity environment was generated by a rotating packed bed (RPB) reactor. Aqueous RSF solutions and ethanol were used as solvent and anti-solvent, respectively. The effects of liquid flow rates and anti-solvent/solvent ratios, as well as centrifugation and freezing time, on the formation, size distribution and morphology of RSF microspheres were investigated. The results showed that RSF microspheres, with diameters of 0.1–1.6 μm, can be prepared under high-gravity field via self-assembly of RSF molecular chains induced by ethanol and quenched by freezing and lyophilization. The mechanism of RSF microsphere formation was also discussed. In addition, rhodamine B was used as a model drug for evaluation of drug delivery capability of the RSF microspheres. The merits of this study lie in a simple low-cost apparatus with ease for scale-up and thereby potential high volume continuous production capacity, which is of practical advantages and industrial significance in preparation of protein microspheres for commercial mass production.

Challenges and Advances in Aerosol Jet Printing of Regenerated Silk Fibroin Solutions

AbstractSilk fibroin materials are manufactured using printing and coating techniques at resolutions 1–2 µm. However, current processes are unstable, of low printability and versatility, and of limited feature size, and often require use of additives to process, which can impact material functionality and performance. Although there exist well established material synthesis and formulation approaches for making processable solutions from silkworm cocoons, these approaches do not translate to the emerging fabrication processes, such as aerosol jet printing (AJP). Here, a new approach is introduced to formulate silk‐worm solutions for AJP and subsequently analyze the processing limits, due to defects such as overspray, pooling, and cloudiness. It is found that the degumming step is critical and can lead to defects such as gelling and pooling. Furthermore, it is found that there exists a narrow processing window (sheath rate as a function of ink rate) for AJP formulations without defects. As with other materials (such as metal inks), overspray is an issue during the fabrication process; however, it is minimized within the processing window. This work stands to open a pathway for manufacturing new and emerging biodegradable materials suitable for pharmaceuticals, food packaging, and electronics, among others.

Colorless Silk/Copper Sulfide Hybrid Fiber and Fabric with Spontaneous Heating Property under Sunlight.

With the increasing demand for comfort, thinness, and warmth of fabrics, various functional fibers have emerged. However, natural silkworm silk, as one of the most widely used natural fibers in textile, faces the issue that it cannot be modified during the spinning process like synthetic fibers. Herein, copper sulfide nanoparticles (CuS NPs) with a near-infrared (NIR) absorption property were first prepared by using regenerated silk fibroin (RSF) as the biological template. Then, trace CuS NPs prepared in RSF solution (no more than 100 ppm) were added into the RSF spinning dope to prepare colorless RSF/CuS hybrid fibers via wet-spinning process. The tensile test of the RSF/CuS hybrid fibers showed that the toughness was improved with the addition of CuS NPs, which completely met the requirements of textile development. The temperature of RSF/CuS hybrid fiber bundles could increase 18.5 °C within 3 min under 1064 nm laser irradiation with power density of 1.0 W/cm2. Finally, these RSF/CuS hybrid fiber bundles were woven into silk fabric or embroidered on a cotton fabric. Under the simulated sunlight, the temperature of RSF/CuS fabric could increase to more than 40 °C from room temperature. Also, as per the infrared images, the pattern of embroidery displayed a significant difference in temperature increase as compared to cotton matrix. Based on these results, an almost colorless RSF/CuS hybrid fiber that can be mass produced by wet spinning may have great potential in the fabrication of dyeable, light, and comfortable silk functional fabric with spontaneous heating characteristics under sunlight.

Microstructure Transitions and Dry-Wet Spinnability of Silk Fibroin Protein from Waste Silk Quilt.

With excellent biocompatibility and biodegradability, silk fibroin has been developed into many protein materials. For producing regenerated silk fibroin (RSF) fibers, the conformation transition of silk fibroin needs to be thoroughly studied during the spinning process. Since the many silk fabrics that are discarded comprise an increasing waste of resources and increase the pressure on the environment, in this paper, waste silk fiber was recycled in an attempt to prepare regenerated fibroin fiber by dry-wet spinning. Ethanol was the coagulation bath. The rheological properties of all the RSF solutions were investigated to acquire rheology curves and non-Newtonian indexes for spinnability analysis. Four stages of the spinning process were carried out to obtain RSF samples and study their conformation transitions, crystallization, and thermal properties by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction, and differential scanning calorimetry. Quantitative analysis of the FTIR results was performed to obtain specific data regarding the contents of the secondary structures. The results showed that higher concentration spinning solutions had better spinnability. As the spinning process progressed, random coils were gradually converted into β-sheets and crystallization increased. Among the different influencing factors, the ethanol coagulation bath played a leading role in the conformation transitions of silk fibroin.

Comparisons of the restoring and reinforcement effects of carboxymethyl chitosan-silk fibroin (Bombyx Mori/Antheraea Yamamai/Tussah) on aged historic silk

This work presents the results of the reinforcement effects of regenerated silk fibroin solutions (SF) of Bombyx-Mori, Antheraea-Yamamai and Tussah on aged historic silk. Furthermore, Carboxymethyl-chitosan (CMC) was utilized as reinforcement and antibacterial filler to further improving the mechanical properties and antibacterial effects. To clarify the rationale behind this process, comprehensive characterization was applied, and a speculative explanation was provided. The results showed that Bombyx-mori and Tussah have better restoring effects than Antheraea-yamamai. CMC has good compatibility to the SF, and the addition of CMC has significantly contributed to the improvement the mechanical properties and thermal stability of the restored silk, which is due to the formation of chemical bonding, strong hydrogen bonding and the construction of polymer network structure. The enhancement of crystallinity and reduction of β-turns structure indicate that the micro-defects in the crystallization zone of the aged silk has been restored, and the ordered arrangement in the long-range ordered structure has been improved within a certain range. It was found that the CMC acted as antifungal agents when introduced on the aged historic silk, reducing the growth of Aspergillus niger, Aspergillus flavus and Paecilomyces variotii to a certain extent, which were commonly found in storage areas of libraries.