Self-Grown Bacterial Cellulose Capsules Made through Emulsion Templating

Microcapsules made of synthetic polymers are used for the release of cargo in agriculture, food, and cosmetics but are often difficult to be degraded in the environment. To diminish the environmental impact of microcapsules, they use the biofilm-forming ability of bacteria to grow cellulose-based biodegradable microcapsules. The present work focuses on the design and optimization of self-grown bacterial cellulose capsules. In contrast to their conventionally attributed pathogenic role, bacteria and their self-secreted biofilms represent a multifunctional class of biomaterials. The bacterial strain used in this work, Gluconacetobacter xylinus, is able to survive and proliferate in various environmental conditions by forming biofilms as part of its lifecycle. Cellulose is one of the main components present in these self-secreted protective layers and is known for its outstanding mechanical properties. Provided enough nutrients and oxygen, these bacteria and the produced cellulose are able to self-assemble at the interface of any given three-dimensional template and could be used as a novel stabilization concept for water-in-oil emulsions. Using a microfluidic setup for controlled emulsification, they demonstrate that bacterial cellulose capsules can be produced with tunable size and monodispersity. Furthermore, they show that successful droplet stabilization and bacterial cellulose formation are functions of the bacteria concentration, droplet size, and surfactant type. The obtained results represent the first milestone in the production of self-assembled biodegradable cellulose capsules to be used in a vast range of applications such as flavor, fragrance, agrochemicals, nutrients, and drug encapsulation.

Self-grown microcapsules produced by G. xylinus. (a) The bacteria are enclosed in the water phase, surrounded by an oxygen-permeable oil phase. Over time and with nutrients such as sugar, the bacteria produce the cellulose within the emulsion template, resulting in bacterial cellulose capsules. (b) After the removal of the oil phase, the capsules/spheres can be resuspended in water as nutrients or drug carriers.

Microcapsules are used to protect and release active ingredients in cosmetics and agriculture.Most of these encapsulation systems are made of non-degradable polymers, which guarantee control over the mechanical and release properties.However, these non-degradable systems accumulate over time in soil and fresh water,potentially threatening the environment. Furthermore, their fabrication often involves harsh chemical reactions and several intensive processing steps. The ideal sustainable microcapsule system should thus be made by readily accessible polymers, be easy to produce, and be made by materials of biological origin. A common solution to making biodegradable microcapsules is the use of biopolymers in the form of proteins and polysaccharides such as alginate and chitosan that form strong enough capsules to withstand harsh conditions while being effortlessly degraded in the environment. Synthetic capsules can be assembled using a multitude of techniques, such as emulsification followed by polymerization,layer-by-layer assembly, conservation, or internal phase separation. Most biopolymer assembly techniques are currently limited to layer-by-layer assembly and coacervation.

Customized flow-focusing device for transient double emulsions. The microscopy images show bacterial droplets produced using flow rates of 200 μL/h, 800 μL/h, and 20 mL/h for the inner, middle, and continuous phases, respectively. The droplets have a diameter of approximately 150 μm with decane as the continuous phase, the culture medium as the inner phase, and a 100% concentrated bacterial solution as the middle phase.

A more autonomous approach exploits the natural design ability provided by living systems such as bacteria that synthesize and assemble biopolymers in biofilms during growth. Gluconacetobacter xylinus, also known as Acetobacter xylinum, is one of the strongest biofilm formers and bacteria cellulose producers. In this work, the aerobic bacterial strain G. xylinus is used due to its ability to produce large quantities of bacterial cellulose. Glucose chains are produced inside the cell body and subsequently extruded out through pores present in the bacteria’s cell wall. The final result is an intricate network of nanofibers composed of repeated units of β-1,4-lined d-glucose.

(a,b) Setup of the interfacial shear rheometer measurements using a custom-made biconical geometry with thicker edges. (c) Time sweep (t = 0–60 h) at a constant frequency and amplitude of mannitol-based media with bacteria inoculated at 0.2, 2, and 5 v/v %. (d) Time sweep (t = 0–20 h) indicating the cross-over points of the viscoelastic moduli [close-up of (c), frequency = 1 rad/s and strain amplitude = 1%]. (e,f) Bulk amplitude sweep and frequency sweep measured on a bacterial pellicle using a plate–plate geometry. The amplitude sweep is measured at 1 rad/s and the frequency sweep at a strain amplitude of 0.1%.

SEM images of a single bacterial cellulose capsule at three magnifications, showing the cellulose fiber network.

Bacterial cellulose has gained increasing interest over the last few years as a biomaterial thanks to its remarkable material properties such as tensile strength, water holding capacity, and biocompatibility. The biofilm formation at the air–water interface has been used to directly grow macroscopic structures such as 3D-printed face masks,foams, implant envelopes, or food packaging.

Because of this structure and properties, applications in the field of biomedicine have also been identified, such as cellulose-based bioscaffolds for cellular adhesion and proliferation or cellulose patches used for skin tissue repair for severe skin burns.This biofilm formation can also be exploited by confining cellulose-producing bacteria to an emulsion droplet to form capsules for biomedical applications by gelling the inner phase or by using a gelled oil phase to stabilize oil droplets.

(a) Normalized fluorescence intensity and droplet/capsule diameter as a function of time. (b–d) Confocal microscopy images during the growth of cellulose in an emulsion droplet, showing the increasing cellulose fibers accumulating at the droplet interface (the image width is 200 μm, with 2% initial bacterial concentration, and an initial diameter of 700 μm, decreased to 270 μm during the experiment).

However, methods of incorporating living microorganisms in well-defined geometries and maintaining their functionality and ability to produce their biofilms are limited. The challenge lies in ensuring the necessary environmental conditions, yet providing a defined template to allow the assembly into functional geometries. In this work, they exploit the possibility of using bacterial cellulose to engineer a stable emulsion droplet. Oxygen is provided by diffusion through an oxygen-permeable oil phase (decane), while nutrients are dissolved in the water phase. Capsule growth was studied by confocal and electron microscopy, whereas bacterial cellulose growth kinetics was monitored via interfacial rheology. To upscale the production of monodisperse capsules, microfluidic techniques were used to create high throughputs of uniform water-in-oil emulsion droplets as templates for the formation of cellulose capsules.

Optical microscopy images of cellulose capsules under (a,b) bright-field, (c,d) polarized, and (e,f) fluorescent light for capsules obtained by flow-focusing (left column) and step emulsification (right column) devices. The averaged final capsule diameter is about 80 μm for flow focusing and 140 μm for step emulsification. Capsules generated in flow-focusing and step emulsification techniques used 5 and 25 5 v/v % bacterial suspensions, respectively. As the oil phase, decane was used with a 5% Span 85 surfactant to increase the stability. All images were taken after 5 days of growth.

SEM images showing the capsules formed as a result of step emulsification.

  1. Self-Grown Bacterial Cellulose Capsules Made through Emulsion Templating Martina Pepicelli, Marco R. Binelli, André R. Studart, Patrick A. Rühs, and Peter Fischer ACS Biomaterials Science & Engineering Article ASAP DOI: 10.1021/acsbiomaterials.1c00399