Bacteria play a role in myriad industrial processes from fermentation to cleaning
up environmental pollution. But floating freely in solution, the microbial cells
constantly multiply, generating biomass that must be removed periodically, causing
downtime. Additionally, the microorganisms cannot be localized to a specific
region of interest.
This scanning electron micrograph shows rod-shaped Pseudomonas fluorescens bacterium completely encased within the polymer fibers of an open-weave, porous hydrogel formed by electrospinning. In these bio-hybrid materials, the bacteria remain immobilized but viable for applications in biotechnology. The white scale bar in the lower right corner measures 1 micrometer."
Now, scientists at the U.S.
Department of Energy's (DOE) Brookhaven National Laboratory and Stony
Brook University have devised a way to encapsulate bacteria in a synthetic polymer
hydrogel. These new, stable, bio-hybrid materials maintain the microbes'
ability to exchange nutrients and metabolic products with their environment,
and could find widespread applications, for example, as biosensors, catalysts,
drug-delivery systems, or in wastewater treatment. The method and results are
described in a paper published online by the Proceedings of the National Academy
of Sciences the week of August 3, 2009.
"In many ways, our research is trying to mimic the biofilms many microorganisms
form in nature," said Brookhaven Lab materials scientist Dev Chidambaram,
corresponding author on the study. "These complex and dynamic communities
form when microbes encapsulate themselves in an extracellular polymer matrix,
which offers them considerable protection from environmental challenges such
as changes in acidity or salinity, and even antimicrobial agents.
"Our goal is to develop synthetic biofilms, in the form of bioactive
materials that could be produced reliably on an industrial scale, and used or
reused continuously for a range of applications. This study, which reports the
generation of a very thin polymeric fibrous material in which microbes maintain
their ability to function, represents a significant step toward achieving that
goal."
Previous attempts to encapsulate viable bacteria in insoluble materials suffered
from several shortcomings, according to the researchers. Foremost, the encapsulating
materials were usually orders of magnitude larger than thin films. Because nutrients
or reactants had to diffuse far into these materials to reach the microbes,
activity - and microbe viability - suffered as a consequence.
To overcome these problems, the Brookhaven-Stony Brook team used a technique
called electrospinning, which uses electrostatic force to produce polymer filaments.
In this process, a polymer solution containing the microorganism of interest
is spun to create fibers.
One challenge was developing a polymer-solvent system that would not be toxic
to the bacteria. Another was achieving a structure with enough porosity to facilitate
the transfer of materials such as nutrients and waste products between the microbes
and their environment. Additionally, the final material must be made insoluble
so it would remain intact in the watery environments envisioned for many potential
applications.
The scientists met these challenges through a series of experiments to develop
a method for producing their fibers. They achieved their objective - an
insoluble, fibrous polymeric material in which industrially relevant bacteria
were successfully encapsulated and remained viable - using a nontoxic,
non-biodegradable, water-soluble polymer known as FDMA as the encapsulating
agent, and by cross-linking the fibers in a glycerol solution after encapsulation
to prevent the material from dissolving in aqueous environments.
Scanning electron microscope and fluorescent microscopy images reveal the rod-shaped
bacteria completely encased within the tiny polymer fibers. The fibers form
a mesh-like random weave with an open pore structure ideal for use as electrodes,
membranes, or filters. Additional tests showed that a high percentage of the
bacteria remained viable for up to several months, and their metabolic activity
was not affected by immobilization. Yet the encapsulated bacterial cells do
not replicate. Therefore no removal of accumulated biomass would be necessary.
The bacteria chosen for this study - from the genera Pseudomonas, Zymomonas,
and Escherichia - already have industrial applications, such as fermenting
glucose to produce ethanol (a key reaction of biofuel production from plant
matter). Insoluble materials containing such bacteria could also be used to
develop sophisticated, reusable biosensors, stable drug-delivery systems, and
permeable reactive barriers for cleaning up contaminated groundwater.
In addition to Chidambaram, collaborators on the research described in the
PNAS paper are: Ying Liu (graduate student) and Miriam Rafailovich of both the
Advanced Energy Research & Technology Center and Stony Brook University, and
Ram Malal and Daniel Cohn of The Hebrew University of Jerusalem. A patent application
has been filed for this method of producing biohybrid hydrogels as well as various
applications. Those interested in licensing the technology should contact Dorene
Price, 631 344-4153, price@bnl.gov.
This research was funded by the Laboratory Directed Research and Development
program at Brookhaven National Laboratory and by the Goldhaber distinguished
fellowship program at the Lab. This work is the result of a collaboration between
the Brookhaven National Laboratory, Advanced Energy Research and Technology
Center (AERTC), Stony Brook University and The Hebrew University of Jerusalem.