Biologically-inspired hybrid systems will pave the way for the next generation of smart and responsive materials. Bacteria, found in virtually every environment on Earth, offer a wide array of biosynthetic pathways that make them invaluable in diverse fields such as food science, health, [1,2] biocatalysis, environmental remediation, agriculture, and energy [3], [4], [5]. The tendency of bacteria to come together in vast multicellular colonies known as biofilms makes them particularly suited for material engineering and fabrication. The most notable hallmark of these bacterial consortia is a range of collective behaviors exhibited by the bacteria. These include member-to-member communication, co-metabolism, and labor sharing, and mutual protection. This biofilm lifestyle has been shown to protect the microorganisms from predators, toxic substances, and environmental effects such as temperature fluctuations, acidity etc․ [6,7]. Owing to all of the above, biologically-inspired hybrid systems endowed with active properties of living organisms on the one hand and robustness of solid materials on the other, are expected to find applications in a variety of fields. Biofilm-based materials hold exciting promise in such areas as biosensors, bio-electrochemical systems, solid-state bioreactors and biotechnology in general. In healthcare, these materials are drawing interest for use in medical devices, drug and gene delivery systems, and applications that promotes colonization by commensal microorganisms or preventing infection by use of antagonistic bacteria. Such materials may also find application in food and agriculture as smart packaging and living fertilizers [6,7]. Despite these advantages, the mechanical fragility of bacterial biofilms-based materials presents a significant challenge. The extracellular matrix that holds together the biofilm-forming cells is primarily composed of biopolymers such as bacterial alginate and cellulose. It provides only limited mechanical support, precluding the use of biofilm-based materials as self-standing materials and devices. For this reason, current biofilm-based devices require support materials for biofilms to grow on, which raises concerns about compatibility and environmental impact, particularly in medicinal and agricultural applications. Thus, additional methods for the fabrication of self-supporting and mechanically stable biofilm materials are constantly searched for [6,7]. Recent studies in the field of living materials have demonstrated the integration of nanomaterials as an effective strategy to reinforce biofilm structures and modulate their functional properties [8]. These advances highlight the emerging niche of bio-hybrid systems where 2D nanomaterials contribute to both mechanical robustness and biological activity, reinforcing the importance and timeliness of our study.

Various strategies have been developed for assembling bacterial cells into stable, functional films. One common approach involves electrostatic interactions between negatively charged bacterial cell walls and positively charged polymers, such as in Layer-by-Layer (LbL) assembly [6,7]. This technique allows the formation of ordered multilayered structures with tuneable thickness and composition. However, despite its scientific appeal, LbL assembly remains limited by its low throughput, labour-intensive protocols, and challenges in upscaling, making it impractical for widespread application in biofilm engineering. In parallel, biological processes such as biomineralization have garnered interest as natural routes for biofilm reinforcement. Biogenic mineral layers, such as calcium carbonate (CaCO₃), can enhance the mechanical integrity of biofilms and influence their metabolic microenvironments [9], [10], [11]. These processes have inspired new directions in the development of bio-hybrid materials. Nonetheless, the spontaneous and environment-dependent nature of biomineralization poses challenges for reproducibility and control, which currently limits its utility in rational material design [12]. Currently, these limitations highlight the need for alternative fabrication strategies that are both accessible and scalable, while still enabling the integration of structural reinforcement into bacterial biofilms.

Two-dimensional (2D) nanomaterials have attracted significant attention due to their unique physical and chemical properties. Among the popular 2D materials, graphene remains one of the most extensively studied due to its good mechanical strength, electrical conductivity, thermal stability, and large surface area. These properties have made graphene a leading candidate for applications in electronics, sensors, energy storage, and composites [13]. However, despite its remarkable features, graphene and its derivatives face significant challenges including biocompatibility issues, difficulties in large-scale production, high cost, and poor dispersibility in aqueous environments, which limit their applicability in biological and environmental contexts [14]. Layered Double Hydroxides (LDHs) represent another important class of 2D materials with distinctive advantages for bio-related applications. LDHs offer excellent film-forming capabilities due to their high aspect ratio and surface area [15], [16], [17], [18].

LDH are a prominent and intensively studied group of 2D materials consisting of positively charged brucite-like layers held together by counter anions inside the interlayer space [19]. The chemical composition of LDH is described by the formula [MII1−xMIIIx (OH)2]x+(An−)x/n·mH2O, where MII and MIII are divalent and trivalent metal cations, respectively, and An− is an n-valent anion. Due to their intercalation and anion-exchange properties, these materials find many applications in such diverse areas as catalysis, [20], [21], [22], [23] energy devices, [24] and adsorbents. Most relevantly for this study, LDH-derived 2D nanosheets have been applied to the fabrication of smart and functional films and coatings [25], [26], [27], [28]. Importantly, LDH are recognized as GRAS (Generally Recognized as Safe) by both FDA and EU regulatory authorities, making them suitable for applications in food, medicine, and agriculture [29], [30], [31].

In this study, we used Bacillus Pumilus as the model microorganism. Bacillus Pumilus is a Gram-positive, spore-forming bacterium with a well-documented range of applications in fields such as biocatalysis, [32,33] plant growth promotion, [34,35] and animal probiotics [36]. There is substantial evidence supporting the potential applicability of materials based on this microorganism in areas like green chemistry, biotechnology, medicine, food and sustainable agriculture [37], [38], [39], [40].