Dry,
ambient storage of C. ljungdahlii paper-based biocomposites: steps
toward continuous, modular, high intensity bioprocessing of syngas into liquids

font-family:"Times New Roman",serif'>                                                                                   

 Mark
J. Schulte and Michael C. Flickinger

Chemical
and Biomolecular Engineering

line-height:115%;font-family:"Times New Roman",serif'>Despite the product
flexibility and specificity offered by cells as biocatalysts, bioprocessing has
not made much penetration into the conversion of gases to commodity chemicals.
This is due to the inherently slower rates of transport of poorly soluble gases
into water and the slow growth rate of many microbes that assimilated C₁
gases (CO, CO₂, CH₄). This is especially true considering
the long time to scale-up culture volume from freezer stock to production scale
bioreactors to achieve high cell density for rapid gas processing. We are
creating a novel continuous bioprocessing system using coating technology, inexpensive
composite flexible fibrous materials and ambient drying. By combining these
methods, we are engineering biocatalysts for falling film gas absorbers which
enable concentrated microorganisms to be stored dry for extended periods at
ambient conditions and rehydrated immediately prior to use. This is
accomplished by adding carbohydrate excipients prior to coating/drying, controlling
drying rate, controlling storage headspace composition and humidity, and
rehydration conditions. font-family:"Times New Roman",serif'>Our model system, C. ljungdahlii OTA1,
takes up CO/H₂ and forms C-C bonds secreting ethanol and acetate. Ambient
drying is achieved using late exponential phase cell pellets resuspended in a
drying formulation then extrusion coated onto filter paper. The coated paper biocomposites
are dried (25 inches Hg vacuum, 20 min) and stored in an anaerobic chamber in a
controlled humidity box for 80 hours at 25°C. The dried biocomposites are placed
in the gas phase of horizontal tube gas absorbers, the paper pores hydrated by
capillary action from a limited liquid phase, and assayed for consumption of CO
over time. The extent of drying/rehydration and its effect on the cellular CO
absorption is investigated by Raman micro-spectroscopy.   Both trehalose and
non-fat dry milk provide desiccation protection to the C. ljungdahlii. Quick
drying under vacuum is essential for dry stabilization without loss of
reactivity.  No reactivity is observed when rehydrating C. ljungdahlii
slowly.   Significant reactivity was seen after storage at 11% relative
humidity compared to 76%. Higher reactivity was observed when cells were
rehydrated with growth-limiting media at 25°C compared to media warmed to 37°C.
Paper gas absorbing biocomposites lost <1 log of reactivity after optimized drying
and rehydration.  The goal of this work is stabilizing highly concentrated
cells (>10¹² cells/m²) within a high intensity gas
absorbing reactor module that can be stored dry. This will enable centralized
manufacture of the modules for distribution to the gas source without a cold
chain for bioprocessing of gaseous carbon emissions (CO₂, CH₄,
CO) into commodity chemicals. A laboratory scale model paper biocomposite batch
falling film reactor has demonstrated comparable gas-to-liquid mass transfer
(~40h^-1) to some CSTR configurations at 1 to 2 logs less power
(<10W/m³).  This technology may lead to a paradigm shift in gas bioprocessing
using high intensity dry stabilized biocatalysts and novel gas-processing
reactor designs.