Multiphase Under‐Liquid Biofabrication With Living Soft Matter: A Route to Customize Functional Architectures With Microbial Nanocellulose

The growth of aerobic microbes at air–water interfaces typically leads to biofilm formation. Herein, a fermentative alternative that relies on oil–water interfaces to support bacterial activity and aerotaxis is introduced. The process uses under‐liquid biofabrication by structuring bacterial nanocellulose (BNC) to achieve tailorable architectures. Cellulose productivity in static conditions is first evaluated using sets of oil homologues, classified in order of polarity. The oils are shown for their ability to sustain bacterial growth and BNC production according to air transfer and solubilization, both of which impact the physiochemical properties of the produced biofilms. The latter are investigated in terms of their morphological (fibril size and network density), structural (crystallinity) and physical–mechanical (surface area and strength) features. The introduced under‐liquid biofabrication is demonstrated for the generation of BNC‐based macroscale architectures and compartmentalized soft matter. This can be accomplished following three different routes, namely, 3D under‐liquid networking (multi‐layer hydrogels/composites), emulsion templating (capsules, emulgels, porous materials), and anisotropic layering (Janus membranes). Overall, the proposed platform combines living matter and multi‐phase systems as a robust option for material development with relevance in biomedicine, soft robotics, and bioremediation, among others.


Introduction
Bacterial nanocellulose (BNC) is synthesized by gram-negative microorganisms [1] to form an extracellular biofilm following extrusion of fibrils or ribbons that fortify the system against carbon/nutrients available in the aqueous culture medium. [6]So far, the potential of BNC in advancing material biofabrication has been achieved by using solid molds, [7] aerotaxis under controlled air permeability, [8] or bubble templating. [9]These methods are respectively challenged by the difficulties associated with a strong attachment to solid surfaces, non-uniform oxygen access (for example, under the gradient of hydrostatic pressure), and lack of shape control.
Noting that soft matter systems can modulate bacterial activity, it is reasonable to consider biofabrication following the example of Nature, where living structures form as complex organizations. [10]A synthetic counterpart includes viscoelastic bacteria-laden BNC hydrogels to generate skinned complex materials via 3D printing. [11]9a,12] The first case, foam-guided BNC growth, requires a high-viscosity medium to maintain long-term stability.The second case, the generation of liquid marbles, requires a large number of solid microparticles.Finally, the available protocols for 3D biofabrication with BNC demand high humidity conditions to prevent water drainage during prolonged cultivation time.
Overall, the prevalent biofabrication of 3D structures based on BNC requires manipulation of air-water interfaces in lowcurvature geometries (e.g., surface of the culture medium in a container) and with high bulk viscosity and reduced surface tension.Herein, we relax these constraints by producing BNC architectures at oil-water interfaces where oxygen transport through the organic phase, allowing for "under-liquid biofabrication."We evaluate the universality of this approach by testing several types of the organic phase (hydrocarbons, silicone, and perfluorocarbon oils) according to the yield and properties of the formed BNC biofilms.The versatility of the produced BNC structures is further demonstrated in multilayered hydrogels, spherical capsules, tubular emulgels, and Janus membranes.Overall, this work fills the research gaps related to bacterial activity at oilwater interfaces and introduces under-liquid biofabrication as a convenient approach to 3D shaping, most relevant to advanced materials.

Under-Liquid Biofabrication
The growth of BNC requires i) a supply of oxygen and ii) a source of essential nutrients. [6]In our under-liquid biofabrication, oxygen is transported through the hydrophobic oil phase to the culture medium containing the bacteria along with the essential nutrients (Figure 1a).Consequently, the BNC is produced by the microorganism at the oil-water interface.Compared to traditional open-air cultivation, the under-liquid biofabrication (Figure 1b) incorporates a physical barrier (the oil phase) that prevents water evaporation (as noted in a week-long cultivation process, Figure S1a, Supporting Information).8b,13] Consequently, the resultant BNC structures exhibit a high fidelity with respect to the precursor geometry (e.g., capsules from droplets in water-in-oil emulsions or porous networks from oil-in-water emulsions), allowing the form fidelity required for biomaterials.In addition, the presence of oil reduces or prevents cross-contamination and molding.Indeed, BNC pellicles grown in an open chamber easily become moldy, in contrast with mold-free structures cultivated under oil after 7 days (see the case of silicone oil in Figure S1b, Supporting Information).
The under-liquid biofabrication facilitates biosynthesis of 3D nanocellulosic structures, owing to the fact that the oil-water interfacial tension (IFT) is notably lower than that of the air-water interface.This makes the oil-water system suitable for producing non-planar configurations, relaxing the need for high bulk viscoelasticity.Besides, liquid interfaces (e.g., emulsions) are usually more robust compared to the air-water counterparts (e.g., foams) due to the lower density difference between the phases, which reduces gravity drainage (and phase separation).This enhanced stability is pivotal in preserving interfacial features and transferring them to the BNC structure.Overall, the introduced under-liquid biofabrication is amenable to direct formation of 3D BNC structures (Figure 1b).
In the context of under-liquid biofabrication, it is reasonable to propose that multi-phase systems are effective in creating environments that incorporate living bacteria.Indeed, the traditional multiphase systems (emulsions, foams, and dispersions) are brought to a new level by their implementation (in our case, emulsions and oil-water interfaces) for soft templating, for which we show as a convenient route to guide microbial activities (leading to BNC production in by-design structures).Therefore, we consider that the versatility of the under-liquid biofabrication is a key innovation, generating numerous opportunities for future studies (e.g., functionalization, encapsulation, and micro-/macro-morphology control).Our emulsion biofabrication is inspired by traditional emulsion polymerization.BNC capsules and emulgels are successfully synthesized by using this approach, as discussed next.
Moreover, biofabrication in a colloidal multiphase system is anticipated to facilitate the production of closed and homogeneous cellulose structures in a colloidal system.For example, our previous efforts in the area of 3D biofabrication [9b] showed that the areal density of BNC was reduced, going away from the surface of the culture medium in a container used to template BNC structures at the solid-water interface (in the vicinity of the superhydrophobic surface of the container).This was due to the increased hydrostatic pressure (and reduced oxygen access).A new opportunity was advanced in the present work where oxygen was supplied through an oil phase (oil-water interface), which made the BNC production outcome indifferent to pressure or gravitational effects.Therefore, homogeneous distribution of BNC is possible across the entire structure templated by the oil-water interface.

Effect of Oil Type on BNC Growth
We evaluated the bacterial activity at oil-water interfaces (in the absence of direct air contact) using oils of high oxygen solubility, including perfluorocarbons (PFC). [14]Specifically, we considered perfluorodecaline (F-decalin), which dissolved as much as 403 mL of O 2 per liter [15] and had been used as artificial blood and underwater breathing. [16]Silicone oils were also tested, given  S1, Supporting Information.Note: a hydrofluorocarbon oil, HFE7500, showed a 10-day BNC yield (1.33 ± 0.15 mg) similar to that of F-decalin but it is not shown because no oxygen solubility data are available.
that it dissolved oxygen and had been shown to act as vector in bioreactors. [17]Finally, long-chain hydrocarbon oils were investigated, given that they dissolved oxygen at various degrees. [18]wo experimental configurations were considered next, depending on the density of the oil (Figure 1c).The sole source of oxygen was the one dissolved in the given oil phase, thereby driving the aerobic bacteria to migrate and concentrate at the oil-water interface.Given a sufficient cultivation time, robust biofilms developed at the oil-water interface (Figure 1c), demonstrating that dissolved oxygen supports the interfacial growth of BNC.
The effect of oil type on under-liquid biofabrication was assessed by measuring the dry weight of produced BNC as a function of the incubation time (Figure 1d), with the expectation that the oils could be ranked following their Henry's constant.Interestingly, BNC growth at the water-F-decalin interface showed a more comparable growth profile to that of open-air incubation, compared to other oils.14b] Both silicone oil and dodecane showed reduced BNC growth, especially during the first week, primarily attributed to the limited availability of oxygen at the interface.Nevertheless, the 10-day dry weight of BNC reached significant levels.
We mapped the microbial growth of cellulose using the oxygen solubility in homologue hydrocarbon and silicone series, as well as PFC oil during a 10-day cultivation (Figure 2).PFC oil, which has the highest oxygen solubility, produced the highest BNC yield.It is worth mentioning that many PFC oils can be chemically and biologically inert. [19]Together with the inherent biocompatibility of BNC, the as-biofabricated materials under PFC oils emerge as suitable options for biomedical applications.14a] Viable alternatives to PFC oils are hydrofluorocarbon oils, which are more cost-effective and commonly used.Previous literature indicates the production of a biofilm (Bacillus subtilis) using a hydrocarbon lubricant (HFE7500) microenvironment. [20]or BNC production at the HFE7500-water interface, our experiments showed a 10-day dry weight of 1.33 ± 0.15 mg, similar to that measured for F-decalin (1.37 ± 0.14 mg).Overall, the oils discussed so far are appealing candidates for under-liquid biofabrication.
The silicone oil series featured good oxygen solubility, which is sufficient to sustain bacterial activity and BNC production.Oils of low viscosity (1-50 mPa s) showed no significant difference as far as BNC yield.The 10-day dry weight of BNC pellicles grown under silicone oils (1.18-1.20 mg) was ≈89% of the BNC yield at the F-decalin-water interface.A reduced cellulose productivity had been reported for highly viscous silicone oils (10 5 mPa s), which was likely related to inhibitory effects of oxygen diffusion and bacteria movement. [21]It is also noteworthy that oils of high viscosity might cause difficulties in their removal.Given their relatively low cost and biological inertness, [19] silicone oils offer good prospects for under-liquid biofabrication.In addition, they are non-volatile, which allows precise biofabrication, as discussed in Section 2.1.
As far as the hydrocarbon oil series, we noted that the carbon chain length had a significant impact on the 10-day BNC dry weight.Decane and dodecane enabled a high microbial activity, leading to the highest BNC yield.This aligns with the fact that decane and dodecane are commonly used as oxygen carriers for cell culturing applications. [18]Heavier oils (larger chains, for example, 14-carbon tetradecane, 16-carbon hexadecane) showed a reduced yield.On the other hand, hexane and cyclohexane did not support under-liquid biofabrication, most likely due to biotoxicity to the microbial community. [22]o prove the critical role of oxygen in BNC cultivation, we cultivated bacteria at the oil-water interface in air-tight conditions (using a sealed tube, with no oxygen transport from the environment).The Komagataeibacter medellinensis strain used in this study was an obligatory aerobe, which was known to be more active in proliferation and biosynthesis with a better supply of oxygen. [23]The yield of microbial nanocellulose was significantly inhibited within the air-tight system due to lack of oxygen access (Figure S2, Supporting Information).It is also worth noting that the bacterial activity (e.g., proliferation and cellulose production) was evaluated by monitoring the dry weight of BNC biofilms; however, techniques that offer non-invasive, non-destructive, and real-time monitoring of biofilm formation are highly desirable.

BNC Films
We selected F-decalin, silicone oil (1 mPa s), and dodecane as reference oils to investigate the characteristics of BNC films cultivated at the oil-water interface.This selection was based on the 10-day BNC yield, as well as considering the economic feasibility and availability.The results were compared with BNC samples that were cultivated with direct air contact (i.e., no oil).The surface morphology of the biofilms was revealed by scanning electron microscopy (SEM, Figure 3a).The BNC grown in air showed the thickest fibrils and the highest density on both sides of the pellicle (air and water sides).On the other hand, following under-liquid biofabrication, the BNC fibrils ex-hibited a reduced thickness and density, attributed to the less available oxygen supply at the interface.Among all, BNC produced under F-decalin displayed a morphology that most closely resembled the one produced in open air.For silicone and dodecane oil conditions, freeze-dried BNC pellicles showed more voids and larger pores, and the cellulose nanofibers were significantly thinner (see image analysis of nanofiber diameter, Figure 3b).
The exceptional oxygen solubility in F-decalin led to the thickest fibrils.On the other hand, silicone and dodecane oils enabled fibrils with diameters that were half of that grown in air.The changes in fibril morphology affected the surface area and porosity of the pellicles, as revealed by BET measurements, Figure S3, Supporting Information.Most freeze-dried BNC pellicles exhibited similar BET surface area (air, silicone oil, and dodecane), despite the differences in fibril size.Interestingly, BNC produced in the presence of F-decalin led to the highest surface area, even compared to air-grown BNC that showed a similar yield.This is a result of the smaller fibril diameter.
The properties of nanofibers produced by under-liquid biofabrication resulted in the corresponding mechanical performance of the BNC biofilms, as evaluated by the tensile strength in the wet state (Figure 3c).5c,24] The tensile curve of the biofilm formed in F-decalin was almost identical to that of the air-grown counterpart, except for the reduced strain at break.This confirms that the PFC oil-water interface more closely resembled the open-air condition for BNC cultivation.
In general, the biofilms cultivated under oils exhibited lower breaking strain than the open-air reference due to the size of the cellulose fibrils.Interestingly, the biofilms produced by under-liquid biofabrication presented a higher Young's modulus: 2.79 MPa (silicone oil) and 2.57 MPa (F-decalin) compared to that from open air (1.78 MPa).We propose that the reduced oxygen accessibility at the oil-water interface influenced biosynthesis, endowing increased cellulose crystallinity (see X-Ray diffraction data in Figure S4, Supporting Information) and resistance to elastic deformation.However, the biological details related to biofilm rigidity in the under-liquid biofabrication process remained unclear.
The main data, namely, yield, fibril diameter, crystallinity, pore sizes, Young's modulus, and oil cost, are given in radar maps, Figure 3e, and the detailed values are listed in Table S2, Supporting Information.The costs were provided considering the possibility of actual implementation and scaleup.In general, BNC biofilms cultivated with direct air contact showed the highest yield and fibril diameter.The biofilms produced with F-decalin were comparable with the air-grown counterpart, owing to the highest oxygen solubility.Yet, better crystallinity index and Young's modulus were shown, which are key advantages of under-liquid biofabrication.For the silicone oil, the values of yield and fibril diameter were significantly lower than the air-grown BNC.However, they possessed the highest crystallinity and Young's modulus.Finally, BNC cultured in dodecane oils showed the weakest overall performance across all parameters.

3D Biofabrication Roadmap
Modern developments in advanced multi-phase systems have facilitated the design of living soft matter systems.As such, with the gained understanding of under-liquid biofabrication, various nanocellulosic architectures were demonstrated.More specifically, we answer the research question of how to manipulate the shape and position of oil-water interfaces.For this purpose, we discuss three routes to achieve advanced nanocellulosic materials by using under-liquid biofabrication, namely, 1) 3D under-liquid networking, 2) emulsion templating, and 3) anisotropic layering.

3D Under-Liquid Networking (Multi-Layer Hydrogels/Composites)
Cultivating BNC simultaneously at multiple interfaces (e.g., oilwater and air-water interfaces) opens up the possibility for a single-step biofabrication of multilayered hydrogels.Using oils denser than water (e.g., F-decalin), it is possible to generate two interfaces with access to the oxygen needed for BNC production.Figure 4a illustrates a simple bioreactor wherein BNC biofilms are produced on both the top and bottom sides.Intriguingly, the natural movement of bacteria between these two interfaces induces the formation of interconnected and entangled cellulose nanofibers in the bulk phase, connecting the top and bottom of the biofilm.Consequently, the resultant material is a sandwichlike hydrogel (Figure 4b).The thickness of both BNC layers was determined by the cultivation time; while, the mid-layer thickness was controlled by the volume of the culture medium (Figure S5, Supporting Information).It is noteworthy that three-layer hydrogels were only structurally and mechanically robust if the mid-layer was in the appropriate range of thickness.A narrow size (e.g., <2 mm) caused the collapse of the top and bottom biofilms.A large gap (e.g., >5 cm) led to diminished mechanical strength in the mid-layer due to reduced fibril density.
Such a multilayer biofabrication platform allows facile particle loading in the middle layer by simply dispersing nanoparticles in the culture medium.In this study, we demonstrated the compartmentalization of magnetic nanoparticles (MNPs, 0.5 wt% Fe 3 O 4 nanoparticles).A three-layer hydrogel was successfully obtained with the middle layer embedding the MNPs (Figure 4c).Such systems were shown to be ultra-stable; no leaching of nanoparticles was observed from the hydrogel after a 1-month immersion in water.The strategy is not limited to MNPs but also suitable to other water-dispersible nanoparticles as long as they are not toxic to bacteria.The maximum loading, determined by the dispersion limit of the nanoparticle in the culture medium, reaches a remarkable value, compared to most conventional methods.
The multi-layer hydrogels can be transformed into thin composites following air drying (Figure 4d): a sandwich architecture is preserved with encapsulated nanoparticles remaining in the middle layer, flanked by protective biofilms (and with morphologies similar to those of air-dried BNC; see SEM in Figure S6, Supporting Information).The high MNP loading in the middle layer of the composite is illustrated qualitatively in Figure 4e.Such a  d) SEM images of a broken BNC capsule to show the outer (①) and inner (②) surface morphologies.Shown is also the cross-section view (③).e) BNC capsules used as carriers of metal organic framework (MOF) particles.f) BNC cultivation in structured liquids in f-i) spiral and f-ii) zigzag shapes loaded with magnetic nanoparticles (MNP).f-iii) The produced magnetic-responsive BNC structure (in the form of a filament) is easily removed from the liquid medium and shown to support its own weight.
membrane holds a significant interest in applications such as hemodialysis. [25]Moreover, BNC surfaces can serve as biocompatible layers; while, nanoparticles in the middle layer can be incorporated for given functions, for example, filtration and adsorption.Interestingly, a cross-section SEM image shows that most MNPs are firmly enveloped and localized within BNC nanofibers (Figure 4f), which explains the water stability of the multi-layer hydrogel.We propose that bacteria move around the nanoparticles; while, extruding cellulose nanofibers, producing a tight particle-fibril structure.Interestingly, such a particle-cellulose architecture is similar to supraparticle assemblies reported previously; for instance, those demonstrated for a superior performance in CO 2 capture applications. [26]Finally, the dynamic interactions between living bacteria and nanoparticles, as well as the biological behavior, affect the properties of the system, which are topics that deserve future attention.

Emulsion Templating (Capsules, Filaments, Emulgels, and Porous Materials)
Akin to emulsion polymerization to produce porous polymer networks, [27] a pertinent feature of this work is the possibility of synthesizing 3D nanocellulose structures via emulsionbased under-liquid biofabrication.This platform offers a sustainable option for material development by using either water-in-oil (W/O) or oil-in-water (O/W) templating. [28]We first demonstrate the emulsion biofabrication of capsules using W/O emulsions, Figure 5a.The continuous oil phase supplies oxygen to bacterialaden water droplets that evolve into BNC capsules, whose size is regulated by the initial size of the droplet (Figure 5b).For each BNC capsule, the fibrils reside near the oil-water interface (see confocal fluorescence images, Figure 5c), leaving the core of the capsules available for cargo loading.The surface morphologies of the BNC capsule are elucidated by SEM imaging, which displays exterior, interior, and cross-sectional areas composed of given BNC nanofiber structures (Figure 5d).Similar to the 3D under-liquid networking strategy, BNC capsules can be loaded with a wide range of nanoparticles without disrupting the homogeneity of the biofilm.As a proof-of-concept, we enveloped metal-organic framework (MOF) particles within BNC capsules (Figure 5e).It is anticipated that a wide range of functional materials can be carried within the BNC capsules for delivery and controlled-release applications.While BNC capsules have been reported by using W/O emulsions, [21,29] our contribution to the oxygen regulation-dependent bacterial activity adds to the customizability of the material.
More complex BNC architectures could be achieved via advanced emulsion morphologies, for example, templating in W/O structured liquids. [30]Structured liquids utilize the rapid assembly of a rigid interfacial film to enable the extrusion of the tubular water phase in an oil bath.Combined with 3D printing techniques, various liquid patterns could be generated with desired shapes (Figure 5f-i-ii).Herein, we inoculated the living bacteria and magnetic nanoparticles (MNPs) within the aqueous phase, leading to the biofabrication of magnetic-responsive BNC filaments (Figure 5f-iii).With the growth of BNC, the mechanical integrity of the structured liquids was strengthened, allowing detachment from the oil phase and sustaining load and external magnetic forces (Videos S1 and S2, Supporting Information).These magnetic-responsive BNC filaments offer potential in soft robotics for biomedical applications.Most importantly, we highlight the synergy between structured liquids and under-liquid biofabrication.The former provides a customizable soft template for cellulose-producing activities; while, living matter enhances both the mechanical strength and the biocompatibility.
We next present the biofabrication of bulky BNC O/W emulgels (Figure 6a).Therein, oil droplets are dispersed in the culture medium, continuously releasing oxygen to the surrounding environment and supporting bacteria activity near the interface.To maximize the oxygen supply, the oil is saturated with oxygen before emulsification.During cultivation, bacteria move freely in the aqueous phase; while, simultaneously extruding cellulose nanofibers, continuously bridging the oil droplets.Eventually, BNC fibrils strengthen the emulsion templates, forming a viscoelastic emulgel, Figure 6b.Note that the O/W emulsion precursor (in the absence of living bacteria) shows a liquid-like behavior, given that no BNC is formed.Cross-sectional SEM images of the freeze-dried samples reveal interconnected BNC fibrils within the continuous aqueous phase, formed from the dissolved oxygen supplied in the dispersed oil droplets (Figure 6d).Surface view of the emulgel still maintains the morphology of a typical BNC film (Figure S7, Supporting Information).Depending on the container, freeze-dried BNC porous material can easily be molded into corresponding shapes (Figure 6c).These BNC emulgels structures can be developed for food and cosmetic applications.Similar BNC structures are achieved from a foam template (air-in-water system). [12]However, foams are more difficult to stabilize in the long term due to the density difference between air and water and the high air-water surface tension.Our biofabrication route based on BNC emulgels is simple, does not require viscosifiers to reinforce the emulsion precursor, and allows direct encapsulation of MNPs (Figure S8, Supporting Information).
The porosity of BNC emulgels can be adjusted via the oil-towater (O:W) volume ratio.Figure 6d exhibits the SEM images of the BNC emulgels originating from emulsions of different oil content (O:W ratio).The porosity of BNC emulgels increases from 31.9% to 60.0% when the O:W ratio changes from 1:9 (Figure 6d-i) to 5:5 (Figure 6d-iii).As anticipated, oil droplets act as oxygen resource and porogen during the BNC emulgel formation.Therefore, a higher oil content leads to structures of higher porosity.
BNC thickness decreases significantly by increasing oil content (see the weakest BNC scaffold in Figure 6d-iii for the highest O:W ratio), which negatively impacts material integrity in the emulgel and porous materials.With increasing oil content, the total interfacial area is increased; while, the number of inoculated bacteria is reduced (due to the fixed value of bacteria concentration used in the aqueous phase), leading to the dilution of bacteria per interfacial area.Although, in principle, a higher volume fraction of oil means increased oxygen availability at the oil-in-water interface, it is likely that the excess amount of oil hampers the BNC yields in an undesired manner.It is also likely that bacterial activity is influenced by the spatial confinement between oil droplets.
The O/W emulsion precursor of 3:7 O:W ratio is found optimal for emulgel formation (Figure 6d-ii), giving a robust BNC scaffold and a relatively high porosity (50.9%).The micro-morphology of such BNC emulgel is shown in Figure 6e, which reveals a robust BNC film at the oil-water interface (Figure 6e-ii) and a strongly interconnected nanocellulose fibril network in between (Figure 6e-iii).The effect of O:W ratio in BNC emulgel biofabrication and its relationship with bacteria locomotion within a confined environment are subjects that deserve further exploration.

Anisotropic Layering ( Janus Membranes)
We investigated the under-liquid biofabrication one-pot route toward anisotropic Janus membranes (Figure 7).In this approach, a superhydrophobic electrospun membrane (polyvinylidene fluoride, PVDF) was first prepared [31] and placed in contact with the living culture medium, submersed in oil for 10 days.A biofilm was generated on the inner sections of the membrane.Given the hydrophilicity of cellulose, the water contact angle of the membrane on the BNC-grown side was decreased to as low as ≈11.7°.Meanwhile, the other side of the membrane (contacting the oil) kept its hydrophobic character (water contact angle of ≈125°).SEM images show that the BNC side was fully covered by cellulose fibrils; while, the hydrophobic side corresponded to the electrospun web.Thus, an anisotropic modification was successfully achieved via biofabrication, leading to Janus membranes.These structures can be useful in numerous applications, such as oil-water separation, energy harvesting, and fog collection. [32]verall, we demonstrate living systems in the field of soft matter, creating 3D architectures using multi-phase systems where the oil phase not only supplies the needed oxygen but acts as a supporting medium that is not consumed; and therefore, can be reused over the long term.

Conclusion
We introduce a new under-liquid approach for precise biofabrication and 3D shaping of soft matter.Bacterial activity takes place at the oil-water interface where the oil phase dissolves, transports, and supplies oxygen, resulting in interfacially-grown nanocellulose biofilms.Three types of oils (perfluorocarbon, silicone, and hydrocarbon oils) are evaluated in under-liquid biofabrication.Perfluorocarbon oil provides an interfacial environment that leads to BNC structures that more strongly match those formed on the surface of water.Under-liquid biofabrication with silicone produces high-strength BNC biofilms; those produced with hydrocarbons present structures whose properties track with their chain length.Finally, three under-liquid bio-fabrication platforms are demonstrated, namely, multi-layer hydrogels/composites, BNC capsules, and emulgels (emulsion biofabrication), noting that microbial interactions with porous surfaces enable anisotropic membranes.In summary, under-liquid biofabrication offers vast opportunities to achieve new advanced nanocellulose architectures.

Figure 1 .
Figure 1.a) Schematic illustration of BNC growth at oil-water interfaces via access to dissolved oxygen.b) Some advantages of the introduced underliquid biofabrication includes: b-i) a physical barrier that prevents evaporation and delays cross-contamination, b-ii) 3D shaping ability, and b-iii) compatibility with traditional soft matter techniques.c) Experimental setups and visual observations of BNC growth at oil-water interfaces.Top: light oils (e.g., silicone oil).Bottom: heavy oils (e.g., F-decalin).d) BNC growth profiles for different types of oils after a 10-day cultivation period.Data were fitted with a Hill growth function.

Figure 2 .
Figure 2. Map of the effect of oil type on the 10-day dry weight of BNC films produced by under-liquid biofabrication: homologue hydrocarbon and silicone series (according to carbon chain and viscosity, respectively) as well as a PFC oil, F-decalin.Physiochemical parameters of oils are provided in TableS1, Supporting Information.Note: a hydrofluorocarbon oil, HFE7500, showed a 10-day BNC yield (1.33 ± 0.15 mg) similar to that of F-decalin but it is not shown because no oxygen solubility data are available.

Figure 3 .
Figure 3.Comparison of BNC biofilms cultivated in open-air and by under-liquid biofabrication (using the given oils).Significant differences are shown in a) BNC morphology (SEM images), b) diameter of the fibrils, c) tensile strength profile of the biofilms, and d) respective Young's modulus.e) Comparison of the BNC biofilm characteristics for three under-liquid biofabrication conditions.The properties of the air-cultivated BNC are indicated with a black solid line.

Figure 4 .
Figure 4. Multi-interface biofabrication of sandwiched hydrogel and thin composites.a) An illustration of the multi-interface biofabrication, utilizing BNC layers at the air-water and oil-water interface.b) Purified layered BNC hydrogels.Yellow dash lines are used as a guide to the eye to show the connection between two BNC biofilms.c) Compartmentalization of magnetic nanoparticles (MNPs) within the multi-layer hydrogel.d) MNPs-encapsulated thin composite after air drying the hydrogel precursor.e) SEM images of the cross-section of the dried composite.f) A high magnification SEM cross-section image showing MNP loading embedded in cellulose nanofibers.

Figure 5 .
Figure 5. Biofabrication by emulsion templating (water-in-oil, W/O, systems).a) W/O emulsions produce BNC capsules that are b) customized by the initial droplet size.c) Fluorescence image of a BNC capsule and the corresponding fluorescence intensity along a center line (horizontal dashed line).d)SEM images of a broken BNC capsule to show the outer (①) and inner (②) surface morphologies.Shown is also the cross-section view (③).e) BNC capsules used as carriers of metal organic framework (MOF) particles.f) BNC cultivation in structured liquids in f-i) spiral and f-ii) zigzag shapes loaded with magnetic nanoparticles (MNP).f-iii) The produced magnetic-responsive BNC structure (in the form of a filament) is easily removed from the liquid medium and shown to support its own weight.

Figure 6 .
Figure 6.Emulsion biofabrication in oil-in-water (O/W) systems.a) Oil-in-water (O/W) emulsions are used to produce BNC emulgels and porous materials: b) tubular BNC construct based on emulgels and c) freeze-dried BNC porous materials.d) SEM images of the freeze-dried BNC porous materials templated by O/W emulsions at different oil-to-water (O:W) ratio.e-i)Detailed structures of porous materials, showing e-ii) BNC microstructures on the wall and e-iii) interconnected fibrils in between.

Figure 7 .
Figure 7. Under-liquid biofabrication for anisotropic membrane modification.a) Conceptual illustration of the direct biofabrication of a Janus membrane, utilizing an electrospun superhydrophobic film.Performance and behaviors of the b) hydrophilic BNC-grown side and c) hydrophobic side are shown by the followings: c-i) Water contact angle, c-ii) a photo image of a water drop sitting on either side of the membrane, and c-iii,iv) the SEM images of the membrane surfaces on both sides.