Joyce
Cavalcante
ab and
Gyorgy
Szekely
*abc
aAdvanced Membranes and Porous Materials Center, Physical Science and Engineering Division (PSE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia. E-mail: gyorgy.szekely@kaust.edu.sa; www.SzekelyGroup.com; Tel: +966128082769
bMaterials Science and Engineering Program, Physical Science and Engineering Division (PSE), King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
cChemical Engineering Program, Physical Science and Engineering Division (PSE), King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
First published on 26th October 2023
The increasing demand for effective oil–water separation materials has encouraged the exploration of sustainable and ecofriendly solutions. In this study, we investigate the surface engineering of a superamphiphilic, self-growing fibrous membrane derived from the Pleurotus ostreatus mycelium. A nanoporous membrane contactor facilitated the harvest of a fibrous Janus membrane by blocking the contact between the mycelium and the growth medium yet allowing nutrient transport to the mycelium. The analysis of the hydrophilicity of the membrane surface and at the mycelium–membrane interface revealed improved wettability and surface fine-tuning. The hydrophilic side was observed at the membrane–mycelium interface, whereas the hydrophobic side displayed a dense layer of closely packed fibrous hyphae. A gradient in the hypha density was revealed through Z-stack fluorescence confocal microscopy and two-dimensional segmentation analysis. The selectivity in oil–water separation was fine-tuned, which provided a sustainable and ecofriendly approach for addressing environmental challenges. The findings of this study emphasize the significance of harnessing natural compounds and self-growing fibrous mycelium as an innovative approach to surface engineering for advanced separation technologies. For the first time, we have successfully demonstrated a new application of membrane contactors for developing superamphiphilic mycelium materials.
Janus membranes are materials used for separation that have distinct characteristics on their two opposite sides. These materials have contrasting physical, chemical, or functional attributes on their surfaces, such as amphiphilicity.7 Amphiphilicity refers to the dual affinity of a molecule or material toward both polar and nonpolar liquids. Superamphiphilic materials exhibit an extremely high contact angle with water (equal to or greater than 140°) on one side while having an extremely low contact angle with water (equal to or smaller than 10°) on the other side.8,9 An important alternative to oil–water separation systems is the fine-tuning of a membrane surface toward superhydrophobicity and/or superhydrophilicity.10
Hydrophobic biomaterials have gained attention for their potential use in oil separation applications11,12 due to their capacity to repel water while selectively adsorbing and retaining oils. Superhydrophobic biomaterials have an inherent affinity toward hydrophobic substances such as oils and hydrocarbons. They can selectively sorb and retain oils while repelling water, making them effective in oil spill remediation. The Sustainable Development Goals of the United Nations—Goal #6 on Clean Water and Sanitation, Goal #11 on Sustainable Cities and Communities, and Goal #13 on Climate Action—are all supported by adopting sustainable alternatives for oil cleanup materials. Fungi mycelium (Fig. 1), which is a network of thread-like structures called hyphae, has begun to attract interest as a biomaterial in recent years. Fungi mycelium can be grown into various shapes and forms, making it highly versatile for different applications. It can be molded into packaging materials,13 building insulation,14 and furniture.15 Moreover, it can even be used as a substitute for leather.16 The characteristics of fungi mycelium-based materials can be altered by adjusting their growth conditions, substrate composition, and environmental factors. As a result, its properties can be modified and tailored to fit particular requirements and applications. Mycelium materials align with the principles of the circular economy by utilizing waste streams as inputs, leaving a small environmental footprint and being compostable at the end of their life cycle.
Naturally existing biological systems offer promising solutions for developing smart surfaces with superamphiphilic properties (Fig. 1). One such instance is filamentous fungi, which generate hydrophobin, a specialized surface amphiphilic protein.17 Hydrophobins play a crucial role in enabling fungi to escape their aqueous environment and facilitate the dispersal of spores through self-assembly mechanisms.18 As structural elements of fungal growth, hydrophobins also play a part in how fungi interact with their surroundings. They have, for instance, been found to be crucial for aerial growth and for the attachment of fungi to solid supports.19 This class of proteins presents a characteristic sequence of eight cysteine residues with conserved spacings in their primary sequence. Because of this residue spacing pattern, hydrophobins are surface-active proteins with a noncentrosymmetric arrangement of hydrophilic and hydrophobic patches.20 Harnessing the properties of hydrophobins enables us to explore new avenues for creating intelligent surfaces with enhanced water-repellent characteristics.
At present, fungal mycelium-based products have attracted considerable research interest, leading to their commercialization. These remarkable materials have emerged as novel and sustainable alternatives that can replace petroleum-based products in various applications, ranging from leather making,21 packaging,22 to building construction.23 In this study, mycelium was used for the first time in nanotechnology to create a superamphiphilic self-growing Janus membrane. The membrane was inoculated on the surface of a membrane contactor, and its physicochemical characteristics were examined (Table 1). Traditionally, hydrophilic membranes have been predominantly utilized for their water permeability24 and separation capabilities.25 Our study, however, reveals a novel strategy that uses the hydrophilic nature of the membrane contactor to produce a unique biomembrane with exceptional superamphiphilic properties. Oil sorption tests were performed, and surface modifications in terms of topography and water–oil affinity were assessed.
Membrane | Definition |
---|---|
Mycelium | Pleurotus ostreatus mycelium |
PBI | Nanoporous 16-wt% PBI membrane contactor |
PBIM | Mycelium on top of PBI (composite) |
PP | Macroporous nonwoven PP membrane contactor |
PPM | Mycelium on top of PP (composite) |
This study presents a significant advancement in membrane technology by introducing the first fully biobased and self-growing Janus membrane, achieved without the use of additives or blending. We have developed an innovative interface design that not only facilitates mycelium growth but also enables easy harvesting of the resulting membrane. Furthermore, this research marks the pioneering use of hydrophobins for the development of Janus membranes. Leveraging a membrane contactor, we have successfully grown a superamphiphilic mycelium material, opening up new possibilities for membrane engineering. Importantly, this work showcases the first-ever application of mycelium in the realm of oil–water separation, offering a sustainable and efficient solution, and the resulting self-growing membrane exhibits rapid sorption characteristics. These achievements collectively highlight the groundbreaking nature of our research and its potential to revolutionize the field of membrane technology.
Thermogravimetric analysis (TGA) results revealed that the first zone of decomposition of the Mycelium was in the range of 157–320 °C (Fig. 2b), where the decomposition of proteins, lipids, chitin, and amino acids occurred.29 Above 320 °C, a single decomposition step occurred, which indicated the pyrolysis of cellulose and hemicellulose.30 A single decomposition step at ∼330 °C was observed in case of the PP membrane contactor, attributed to the thermal degradation of the covalent bonds within the polypropylene chains. The PBI membrane contactor exhibited a double degradation step related, attributed to the presence of the polymer backbone. The double degradation step observed in case of PBIM and PPM demonstrated the presence of various decomposition compounds originating from both the contactor and the mycelium. As observed in other works, the TGA curves did not plateau, indicating that the decomposition process was incomplete at the investigated temperature range.27,31
Differential scanning calorimetry (DSC) analysis of the PBI membrane contactor (Fig. 2c) revealed a single endothermic peak associated with the polymer's glass transition temperature (Tg), which was observed at 85 °C. Similarly, Mycelium exhibited a single endothermic peak at 70 °C, which is its Tg. DSC analysis of PBIM revealed the Tg endothermic peak of the composite material at an intermediate temperature of 77 °C between the Tg peaks of PBI and Mycelium. PP, which serves as a casting support for the PBI membrane contactor, showed a melting temperature at 129 °C, where the second endothermic peak was observed. Additionally, the exothermic peak during cooling at 118 °C could be associated with the temperature at which PP crystallizes.28PPM exhibited distinctive transition peaks, which were attributed to the constituents of both PP and mycelium. An exothermic peak at 118 °C and an endothermic peak at 129 °C were identified in case of PP, along with an endothermic peak at 70 °C corresponding to Mycelium, which reflected the copresence of these materials.
The growth rate of mycelium was investigated from the day of inoculation, and the results are presented in Fig. 2d. Three stages of analysis were conducted: the first stage spanned the first 3 days, the second stage encompassed the following 7 days, and the third stage spanned the first 21 days. Fig. S1† contains additional images of mycelium development. PDA plays a crucial role in the growth and cultivation of fungi mycelium. It provides a nutrient-rich environment that supports the growth of a variety of fungal species. At all stages of the experiment, direct contact with the growth medium (PDA) led to the fastest growth. However, when the microporous PP membrane contactor was introduced, there was a slight reduction in growth, which was primarily attributed to a decrease in nutrient transport from the PDA to the mycelium. The use of the nanoporous PBI membrane contactor resulted in the further reduction in mycelium growth rate. The mycelium cycle of growth on the PBI membrane contactor on the day of inoculation and after the third stage of growth is shown in Fig. 2e and f.
The incorporation of a membrane contactor between the growth media and the mycelium facilitates the harness of mycelium hyphae via the detachment of hyphae from the soft gel-like growth medium (Fig. 2g and h). We refer to the α surface as the top side of the mycelium and the β surface as the bottom side of the mycelium, which is at the mycelium–membrane contactor interface. The top surface of the membrane contactor after the removal of the mycelium is designated as a γ2 surface. The membrane contactor facilitates the transfer of nutrients from the growth medium and prevents hyphae from penetrating it. This is the first report on such an application of membrane contactors, and we encourage the community to learn more about how it can be utilized to control growth, nutrient feeding, and hyphae diameter.
In the cross-section micrographs (Fig. 3a, d, g, and j), there was no evidence of mycelium penetrating through the membrane contactor, and only hyphae were found on its surface (Fig. 3h–k). Interestingly, the surface property of the PBI membrane contactor was altered when the Mycelium was removed. The surface of the PBI became more hydrophilic, as demonstrated by the change in its wettability, and its water contact angle (WCA) decreased from 82° to 47° due to the growth of mycelium on its surface (Fig. 3b and e). Moreover, the Mycelium membrane exhibited remarkable duality in its surface characteristics: it displayed superhydrophobic behavior on its α surface with a WCA of 140°, while forming a contrasting superhydrophilic β surface (WCA = 9°) at the interface.
Differences in surface texture were observed and quantified through atomic force microscopy (AFM) topography measurements (Fig. 3c, f, i and l). The roughness value (Ra) of the membrane contactor's γ1 surface was 0.038 μm. Upon mycelium growth, the γ2 surface presented a roughness value of Ra = 0.051 μm. The surface roughness values of both sides of the mycelium membrane were measured: the hydrophilic α surface displayed a roughness of Ra = 1.508 μm, while the hydrophobic β side displayed a roughness of Ra = 0.962 μm. The roughness values on the more hydrophilic surface were consistently higher. This phenomenon is in accordance with previous findings, demonstrating that rough surfaces enhance hydrophilicity in biofilms.32 By applying the Wenzel33 and Cassie–Baxter models,34 we were able to conduct a quantitative analysis and obtain additional insights into these phenomena. These models provide a deeper understanding on the surface roughness and wetting characteristics of our unique mycelium–PBI. The reasons for the observed surface behavior can be attributed to two factors. First, the wetting of water onto the hydrophilic surface is encouraged by the rough surface, which may increase the effective contact area of the solid–liquid interface. Second, the rough surface offers additional adsorption sites.35
The fact that Mycelium is extremely amphiphilic provides evidence that our membrane surface modification method, which was inspired by nature, improved wettability because of the unique and rough fibrous microstructure of the hyphae. The enhanced water-repellent capability of the Mycelium–membrane surface has great potential for various applications, including self-cleaning surfaces,36 antifouling coatings,37 and water-resistant textiles.38 Our study opens a path for utilizing the intricate design principles found in nature, which include patterns, structures, and processes that have developed over millions of years to improve effectiveness, sustainability, and adaptability in the natural world. By taking inspiration from nature, we can develop useful and adaptable materials that mimic the ingenuity of nature, which leads to more efficient and sustainable solutions for various applications.
To gain a deeper understanding of the potential factors that contribute to the increase in hydrophobicity, we investigated the hydrophobin proteins in PBIM through optical fluorescence microscopy (Fig. 4). This analysis involved Z-stack two-dimensional (2D) (Fig. 4a and d) and three-dimensional (3D) (Fig. 4b and c) reconstructions of the top surface (α) and bottom surface (β) of Mycelium. In a Z-stack optical fluorescence measurement, the optical path length varies according to sample density.39 Greater 3D thickness and volume were produced as a result of the identification of more focal planes due to the sparse hyphal arrangement. In contrast, a more compact hyphae arrangement produced fewer focal planes and a shorter optical path length, which produced a thinner three-dimensional Z-stack.
We found a distinct correlation between the hydrophobicity of the self-grown mycelium and the presentation of a dense and thin layer (thickness = 35 μm and volume = 5.17 × 108 μm3) of tightly packed fibrous hyphae on its α surface. The hydrophobic α side of the Mycelium contrasted with its hydrophilic β side because the Z-stack revealed a sparse arrangement of hyphae on the interphase with the nanoporous membrane contactor (which led to a 3D thickness = 143 μm and volume = 8.66 × 108 μm3). The microscopy results provided valuable visual evidence of the structural differences between the hydrophobic and hydrophilic sides of the Mycelium membrane, offering key insights into the mechanisms behind the observed increase in hydrophobicity.
Images of PBIM captured via Z-stack optical microscopy and labeled with Congo red fluorescence dye revealed a distinct accumulation of hydrophobin proteins on the exterior of the hyphae fibers (as shown in Fig. 4f). An optical image is also provided to show the macroscopic perspective of the mycelium fibers (Fig. 4e). In previous studies on fungi, the same staining agent was used to verify hydrophobin activity.40,41 Utilizing 2D segmentation via Fiji (ImageJ), a gradient in the density of hyphae between the α and β surfaces of the mycelium fibers was identified. The optical analysis (Fig. S5 and Table S2†) revealed hyphae fiber coverage of 48.2% on the α surface of the Mycelium membrane as opposed to 39.9% on the β surface. The presence of surface-active hydrophobin proteins throughout the self-grown hyphae arrangement can be directly attributed to the observed gradient in fiber density. These proteins were essential in creating an amphiphilic surface, paving the way for novel insights into the surface engineering of self-growing mycelium.
A significant WCA modification, in relation to the membrane contactor, provided additional proof of the amphiphilic nature of hydrophobins. The γ1 surface of the pristine membrane contactor presented a WCA value of 82°, whereas the γ2 surface (PBI surface after mycelium growth) presented a WCA value of 47°. The pronounced modification of the PBI surface using a natural compound indicates that the surface can be fine-tuned through the hydrophobin content of mycelium. The wettability enhancement of the PBI surface was outstanding, as demonstrated by a 42% decrease in the WCA (Fig. 3). The decrease in WCA upon mycelium growth was a clear indication that the PBI surface had become hydrophilic. These findings provide additional evidence for the amphiphilic nature of the hydrophobin protein, which includes both hydrophilic and hydrophobic patches. The Mycelium membrane exhibited superhydrophobic behavior (WCA = 140°) on its α surface, whereas it formed a superhydrophilic β surface (WCA = 9°) at the interface with the PBI membrane contactor (Fig. 3b and c). During the mycelium growth, the hydrophilic patches of the hydrophobin protein were able to attach to the surface of the membrane contactor, which oriented the hydrophobic patches of the protein in the opposite direction. The precise alignment of hydrophobins was instrumental in the creation of a superamphiphilic Janus membrane from mycelium. This alignment mechanism resulted in the formation of a membrane with distinct hydrophobic and hydrophilic surfaces. Our findings support previous observations on hydrophobins and their application as stabilizing agents for foams and emulsions.42 We showed how the unique and versatile properties of mycelium can be used as effective surface engineering tools, offering exciting opportunities for designing innovative materials with tailored wetting behavior.
Our developed composite materials are not well-suited for high-pressure applications, which led us to emphasize its potential for oil sorption and conduct relevant sorption tests. In a qualitative assessment, we found that the material exhibits excellent foldability; however, it lacks the necessary stability for tension pulling. These findings underscore its unique attributes, making it a promising candidate for specific applications where its mechanical limitations are less critical, such as in oil sorption and related environmental remediation tasks.
Amphiphilic materials have demonstrated considerable potential for efficient oil–water separation.43–45 These materials possess both hydrophobic (water-repelling) and hydrophilic (water-attracting) surfaces, allowing them to selectively adsorb or repel oil and separate it from water. Janus structures enhance the efficiency of the oil-water separation processes since superamphiphilic materials create a stable interface between the oil and water phases. This selective affinity enables the material to adsorb oil onto its hydrophobic surfaces while repelling water away from these surfaces. For all oil types, including olive oil, soybean oil, diesel, gasoline, and crude oil, the sorption kinetics of our materials showed rapid oil-uptake behavior (Fig. 4g), with no discernible variation in oil sorption after the first minute of treatment. In particular, within the first minute of water treatment, the Mycelium membrane reached its maximum sorption capacity. According to Fig. 4h, the presence of Mycelium caused a 152% increase in the sorption of crude oil and a 67% decrease in water-uptake behavior in case of PBIM when compared with the pristine PBI. Oil–water selectivity (S) values, which measure the material's preference for adsorbing oil molecules over water molecules, provide a more precise quantification of this behavior. PBI demonstrated an S of 2.6, while PBIM achieved S of 20.1. Remarkably, Mycelium displayed a remarkable S of 622.5, demonstrating a considerably higher affinity for oil adsorption over water adsorption. Janus membranes previously reported in the literature displayed a considerably lower selectivity coefficient of 4.05.46 The optical photos depict the conditions before, during, and after the treatment process, showing the sequential stages of treating crude oil spillage on water (Fig. 4i–k). Our findings present new opportunities for the use of mycelium-based materials in oil–water separation. We created the first Janus membrane for oil–water separation owing to the amphiphilic properties of hydrophobins. This membrane has a superhydrophobic surface on one side and a superhydrophilic surface on the other.
As PP is widely used in oil spill remediation,47,48 we investigated its surface modification and potential oil-uptake enhancement using mycelium. Compared with the original PP, PPM showed a significant change in oil and water sorption (Fig. S7†). When compared with the results obtained using the microporous PP membrane contactor, mycelium was found to be responsible for an unprecedented 445% increase in crude oil sorption and a 99.6% decrease in water sorption. We took advantage of the unique wetting behavior of mycelium, which allows for the selective repulsion of water while attracting and retaining oil. The coated PPM demonstrated a remarkable selectivity of 388.6, compared to PP's selectivity of 0.2. The reason for the remarkably higher selectivity of the composite PPM compared to PP lies in the presence of a superamphiphilic mycelium Janus membrane in PPM. The superhydrophobic component of the Janus coating attracts and adsorbs oil molecules effectively due to their similar nature. The presence of the Janus mycelium membrane creates a stable interface between the oil and water phases. This interface minimizes the possibility of oil droplets coalescing and re-entering the water phase, thus maintaining effective separation. The high selectivity that was achieved can be used to create cutting-edge separation materials for various oil–water mixtures, such as those used for wastewater treatment, oil spill cleanup, or industrial processes that require oil–water separation. In addition to offering a more environmentally friendly and sustainable approach, the improved performance of this material in oil–water separation also demonstrates the potential of using natural substances such as Pleurotus ostreatus fungi mycelium to address urgent environmental challenges. Thus, this material holds promise for contributing to the development of more efficient and environmentally friendly oil–water separation technologies.
The morphology of the fabricated membranes was examined using a scanning electron microscope (SEM) (Magellan SEM, FEI). Samples were placed in the Electron Microscopy Vacuum Cryo Manipulation System (EM VCM, Leica Microsystems) and plunge frozen in liquid nitrogen prior to analysis. They were then transferred via the Leica VCT500 shuttle under cryogenic temperature into the Leica ACE900, where they were automatically fractured, etched at −100 °C for 2 min, and then coated with 4 nm Pt. Fluorescence microscopy images were acquired using the Zeiss LSM 710 Confocal Microscope. Before analysis, materials were stained with a 1 mM Congo Red (SunYoung Chemical Ltd.) aqueous solution. WCA measurements were performed in triplicate on the membranes using the Young Laplace method and a KRÜSS Scientific Drop Shape Analyzer DSA100E. Tapping-mode AFM was used to describe the surface topographies of the membranes (Dimension Icon SPM, Bruker, RTESPA-300 probe).
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Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3ta05220f |
This journal is © The Royal Society of Chemistry 2023 |