Sustainable structural materials with excellent impact-resistance properties are urgently needed but challenging to produce, especially in a scalable fashion and with control over 3D shape. Here, we show that bacterial cellulose (BC) and bacterially precipitated calcium carbonate self-assemble into a layered structure reminiscent of tough biomineralized materials in nature (nacre, bone, dentin). The fabrication method consists of biomineralizing BC to form an organic/inorganic mixed slurry, in which calcium carbonate crystal size is controlled with bacterial poly(γ-glutamic acid) and magnesium ions. This slurry self-assembles into a layered material that combines high toughness and high impact and fire resistance. The rapid fabrication is readily scalable, without involving toxic chemicals. Notably, the biomineralized BC can be repeatedly recycled and molded into any desired 3D shape and size using a simple kitchen blender and sieve. This fully biodegradable composite is well suited for use as a component in daily life, including furniture, helmets, and protective garments.

@en

Graphene oxide (GO) has recently been highlighted as a promising multipurpose two-dimensional material. However, free-standing graphene oxide films suffer from poor strength and flexibility, which limits scaling-up of production and lifetime structural robustness in applications. Inspired by the relationship between the organic and inorganic components of the hierarchical structure of nacre found in mollusk shells, we have fabricated self-assembled, layered graphene-based composite films. The organic phase of our composite is produced via environmentally friendly and economical methods based on bacterial production of γ-poly(glutamic acid) (PGA). Composite films made of GO, PGA, and divalent cations (Ca2+) were prepared through a slow solvent evaporation method at ambient temperature, resulting in a nacre-like layered structure. These biobased nanocomposite films showed impressive mechanical properties, which resulted from a synergistic combination of hydrogen bonding with the bacterially produced PGA and ionic bonding with calcium ions (Ca2+). The GO/PGA/Ca2+ composite films possessed a high strength of 150 ± 51.9 MPa and a high Young's modulus of 21.4 ± 8.7 GPa, which represents an increase of 120% and over 70% with respect to pure GO films. We provide rational design strategies for the production of graphene-based films with improved mechanical performance, which can be applied in filtration purification of wastewater in the paper, food, beverage, pigment, and pharmaceuticals industries, as well as for manufacturing of functional membranes and surface coatings.

@en

The impressive mechanical properties of natural composites, such as nacre, arise from their multiscale hierarchical structures, which span from nano- to macroscale and lead to effective energy dissipation. While some synthetic bioinspired materials have achieved the toughness of natural nacre, current production methods are complex and typically involve toxic chemicals, extreme temperatures, and/or high pressures. Here, the exclusive use of bacteria to produce nacre-inspired layered calcium carbonate-polyglutamate composite materials that reach and exceed the toughness of natural nacre, while additionally exhibiting high extensibility and maintaining high stiffness, is introduced. The extensive diversity of bacterial metabolic abilities and the possibility of genetic engineering allows for the creation of a library of bacterially produced, cost-effective, and eco-friendly composite materials.

@en

In this study, a method for the bacterial production of a nacre-mimicking composite material was developed. Nacre (mother-of-pearl) is an organic-inorganic composite found in the inner lining of many mollusk shells and in pearls. It has a brick-and-mortar structure consisting of 95% aragonite (calcium carbonate) platelets and 5% organic matrix. Serving as a protective structure against e.g. predators, nacre has developed into an extremely strong and tough material, despite largely consisting of ceramic calciumcarbonate. Numerous mechanisms have been proposed to explain the outstanding mechanical properties of nacre, such as crack deflection and local strain hardening. Many groups are pursuing the aim of developing new materials which mimic nacre’s structure and mechanical properties. Nacre is produced by mollusks at ambient temperatures with easily obtainable materials and with low expenditure of energy. In contrast, human methods usually require extensive energy input, high temperatures and/or pressures, and environmentally damaging chemicals. @en

Nature is based on complex self-assembling systems that span from the nanoscale to the macroscale. We have already begun to design biomimetic systems with properties that have not evolved in nature, based on designed molecular interactions and regulation of biological systems. Synthetic biology is based on the principle of modularity, repurposing diverse building modules to design new types of molecular and cellular assemblies. While we are currently able to use techniques from synthetic biology to design self-assembling molecules and re-engineer functional cells, we still need to use guided assembly to construct biological assemblies at the macroscale. We review the recent strategies for designing biological systems ranging from molecular assemblies based on self-assembly of (poly)peptides to the guided assembly of patterned bacteria, spanning 7 orders of magnitude.

@en

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.

@en

Biofilms can grow on virtually any surface available, with impacts ranging from endangering the lives of patients to degrading unwanted water contaminants. Biofilm research is challenging due to the high degree of biofilm heterogeneity. A method for the production of standardized, reproducible, and patterned biofilm-inspired materials could be a boon for biofilm research and allow for completely new engineering applications. Here, we present such a method, combining 3D printing with genetic engineering. We prototyped a low-cost 3D printer that prints bioink, a suspension of bacteria in a solution of alginate that solidifies on a calcium-containing substrate. We 3D-printed Escherichia coli in different shapes and in discrete layers, after which the cells survived in the printing matrix for at least 1 week. When printed bacteria were induced to form curli fibers, the major proteinaceous extracellular component of E. coli biofilms, they remained adherent to the printing substrate and stably spatially patterned even after treatment with a matrix-dissolving agent, indicating that a biofilm-mimicking structure had formed. This work is the first demonstration of patterned, biofilm-inspired living materials that are produced by genetic control over curli formation in combination with spatial control by 3D printing. These materials could be used as living, functional materials in applications such as water filtration, metal ion sequestration, or civil engineering, and potentially as standardizable models for certain curli-containing biofilms.

@en

Sustainable and personally tailored materials production is an emerging challenge to society. Living organisms can produce and pattern an extraordinarily wide range of different molecules in a sustainable way. These natural systems offer an abundant source of inspiration for the development of new environmentally friendly materials production techniques. In this paper, we describe the first steps toward the 3-dimensional printing of bacterial cultures for materials production and patterning. This methodology combines the capability of bacteria to form new materials with the reproducibility and tailored approach of 3D printing systems. For this purpose, a commercial 3D printer was modified for bacterial systems, and new alginate-based bioink chemistry was developed. Printing temperature, printhead speed, and bioink extrusion rate were all adapted and customized to maximize bacterial health and spatial resolution of printed structures. Our combination of 3D printing technology with biological systems enables a sustainable approach for the production of numerous new materials.

@en

Nacre (mother of pearl) is an attractive model for the development of new materials. Its sheet structure of alternating layers of calcium carbonate and an organic matrix confers it highly desirable properties such as high toughness and strength. In this study, we produce a nacre-inspired composite material using only bacterially-produced components. Calcium carbonate is crystallized via the action of ureolytic bacteria. After each crystallization event, we apply bacterially produced γ-polyglutamate (PGA) to the sample, which promotes layering compared to the PGA-free control. We show that the combination of these two compounds yields a layered material reminiscent of nacre, showing a way towards the biotechnological production of new, nacre-inspired materials.@en