Incorporating microorganisms into polymer layers provides bioinspired functional living materials

Abstract

Artificial two-dimensional biological habitats were prepared from porous polymer layers and inoculated with the fungus Penicillium roqueforti to provide a living material. Such composites of classical industrial ingredients and living microorganisms can provide a novel form of functional or smart materials with capability for evolutionary adaptation. This allows realization of most complex responses to environmental stimuli. As a conceptual design, we prepared a material surface with self-cleaning capability when subjected to standardized food spill. Fungal growth and reproduction were observed in between two specifically adapted polymer layers. Gas exchange for breathing and transport of nutrient through a nano-porous top layer allowed selective intake of food whilst limiting the microorganism to dwell exclusively in between a confined, well-enclosed area of the material. We demonstrated a design of such living materials and showed both active (eating) and waiting (dormant, hibernation) states with additional recovery for reinitiation of a new active state by observing the metabolic activity over two full nutrition cycles of the living material (active, hibernation, reactivation). This novel class of living materials can be expected to provide nonclassical solutions in consumer goods such as packaging, indoor surfaces, and in biotechnology.

Fig. 1.

Design of a living surface. The simplest form of a living surface is composed of three layers. The base layer (light blue) may be an inert support foil (e.g. a PVC film), a linker—e.g. an adhesive—or a surface that shall be coated directly with a living surface. The second layer (white, with black CFU) is the living layer hosting microorganisms. The top layer (black, wireframe) is responsible for the confinement of the fungi, for nutrient, gas, and product transportation, and mechanical, chemical, and biological stability. (A) In the waiting state the number of potent/living fungi slowly degrades, but may survive for a long time e.g. in the form of spores. (B) When a nutrient [e.g. a food spill (yellow)] is on the surface, the organisms within the living layer start growing and consuming the food. (C) Active phase: Here the microorganisms grow until all food is consumed. The membrane is responsible for the transport of food, gas, and secondary metabolites. (D) Secondary metabolites (e.g. penicillin) interacting with the surrounding allow complex functions of such materials.

Fig. 2.

Layered structure of a living material on (A) macroscopic and (B) microscopic level. The base layer consists of a sprayed 100 μm thick polyacrylic layer. The living layer mainly consists of agar and Penicillium roqueforti as active organism. The top layer is a thin porous polycarbonate membrane. (C) Scanning electron microscopy image of the porous top membrane. (D) Microscopic top view of a partially grown living layer showing the two-dimensional hyphae network.

Fig. 3.

Behavior of a living layer exposed to differently concentrated potato glucose broths. Initially 2,000 CFU were present in a 7 cm² sized living surface (left). Exposure to 2 mL of indicated media (diluted PD from 0.00–47.5 g glucose/L) led to the here shown CFU values after 8 d. On the secondary y-axis the amount of consumed glucose is shown. Insets: Photographs of the extracted 8 d old living layers. The more food was provided, the more CFU were found. This correlates to the increasing optical density shown. A constant ratio glucose consumed to CFU formed indicates that the growth is limited by the available food and not by the habitat itself.

Fig. 4.

Cyclic behavior of a living surface (7 cm²). At time zero, the living layer had 1,700 CFU/cm² and was fed with 2 mL diluted PD (4.41 g glucose/L). For every time point two samples were analyzed. When the glucose level reached 0.0 g/L, one week of regeneration time was granted. At day 21 the surfaces were fed again with 2 mL PD (4.34 g/L). As in the first cycle, the glucose content goes to zero, the CFU count goes up. The fast increase of CFU after that the renewed addition of glucose is attributed to spore formation due to starvation. The glucose consumption rate and therefore the fungal activity were even higher in the second cycle. At the end of the second cycle, the CFU count increased disproportionately strong due to sporulation.

Fig. 6.

Demonstration of robustness through pretreatment of a ready-to-use living layer with disinfection agents, soap, or a 10 d starvation under humid conditions which had no influence on the glucose consumption capacity (i.e. a measure for fungal proliferation). Control experiments: If no food was provided, the CFU count slowly decreased. With no fungi in the living layer, no glucose was consumed. Complete dehydration over 10 d killed the function (no glucose consumption) of the living surface although potent fungi were detected within the living layer. *Below detection limit (CFU < 10; glucose < 0.1 mg).

Fig. 5.

(A) A ready-to-use living surface is activated locally by the application of a PD drop (model for a food spill). Exclusively under the drop the living layer is activated. (B) Photograph and microscopic magnifications of a 300 cm² sized living surface (top layer removed for images) after 5 d feeding with PD (a food spill area, 6 cm in diameter, was placed in the bottom left corner). The fungi grew exclusively where food (PD) was present. No growth was observed if only sodium chloride solution was applied.