Research Article: A technique for lyopreservation of Clostridium ljungdahlii in a biocomposite matrix for CO absorption

Date Published: July 5, 2017

Publisher: Public Library of Science

Author(s): Mark J. Schulte, Jason Solocinski, Mian Wang, Michelle Kovacs, Ryan Kilgore, Quinn Osgood, Lukas Underwood, Michael C. Flickinger, Nilay Chakraborty, Xiaoming He.


A system capable of biocatalytic conversion of distributed sources of single carbon gases such as carbon monoxide into hydrocarbons can be highly beneficial for developing commercially viable biotechnology applications in alternative energy. Several anaerobic bacterial strains can be used for such conversion. The anaerobic carbon monoxide-fixing bacteria Clostridium ljungdahlii OTA1 is a model CO assimilating microorganism that currently requires cryogenic temperature for storage of the viable strains. If these organisms can be stabilized and concentrated in thin films in advanced porous materials, it will enable development of high gas fraction, biocomposite absorbers with elevated carbon monoxide (CO) mass transfer rate, that require minimal power input and liquid, and demonstrate elevated substrate consumption rate compared to conventional suspended cell bioreactors. We report development of a technique for dry-stabilization of C. ljungdahlii OTA1 on a paper biocomposite. Bacterial samples coated onto paper were desiccated in the presence of trehalose using convective drying and stored at 4°C. Optimal dryness was ~1g H2O per gram of dry weight (gDW). CO uptake directly following biocomposite rehydration steadily increases over time indicating immediate cellular metabolic recovery. A high-resolution Raman microspectroscopic hyperspectral imaging technique was employed to spatially quantify the residual moisture content. We have demonstrated for the first time that convectively dried and stored C. ljungdahlii strains were stabilized in a desiccated state for over 38 days without a loss in CO absorbing reactivity. The Raman hyperspectral imaging technique described here is a non-invasive characterization tool to support development of dry-stabilization techniques for microorganisms on inexpensive porous support materials. The present study successfully extends and implements the principles of dry-stabilization for preservation of strictly anaerobic bacteria as an alternative to lyophilization or spray drying that could enable centralized biocomposite biocatalyst fabrication and decentralized bioprocessing of CO to liquid fuels or chemicals.

Partial Text

Energy efficient stabilization of anaerobic bacteria capable of fixing single carbon gases, such as carbon monoxide (CO), into hydrocarbons can be highly beneficial for developing commercially viable biotechnology applications [1, 2]. While dry-stabilization techniques have been successfully developed for preservation of microbial biocatalysts such as lactic acid bacteria [3] and yeast [4], no such technique has been developed for stabilization of anaerobic microorganisms that can assimilate gaseous carbon compounds other than carbon dioxide. Stabilization of anaerobic bacteria that are capable of assimilating single carbon gases, such as C. ljungdahlii, and other clostridia, could prove to be highly beneficial in developing bioprocess-oriented large scale gas treatment technologies [5–7].

The efficacy of the C. ljungdahlii OTA1 cells stabilized in the paper biocomposites was evaluated following rehydration by measuring the CO uptake using GC techniques [5]. In an effort to develop an optimized desiccation scheme for these otherwise desiccation- sensitive microorganisms, first the optimum drying time for stabilization of C. ljungdahlii in paper biocomposites was investigated (Fig 2A). The optimum drying time was determined by desiccating the paper biocomposites containing C. ljungdahlii OTA1 cells for different time durations. Following desiccation, the samples were rehydrated and 18 hr post-rehydration CO uptake rate was used as an indicator of cellular viability as shown in Fig 2A. It was found that exposure to convective drying conditions for 25 min produced the highest level of post-rehydration CO uptake. Exposure to convective drying conditions both shorter and longer than 25 min yielded significantly diminished CO uptake rates, possibly indicating cellular injury due to rate dependent cumulative osmotic stress.

We have demonstrated, for the first time, dry storage of C. ljungdahlii by convective drying using argon for over 38 days without any loss in CO absorbing reactivity. The extent of desiccation of the cell samples have a direct relationship with the cellular viability. The injury may be due to over-desiccation effects which leads to permanent loss of viability. The optimal length of convective drying was determined to be approximately 25 minutes with further drying toward the glassy regime only decreasing reactivity (Fig 1). Significantly diminished CO uptake rates following rehydration for convective drying conditions shorter and longer than 25 min possibly indicates significant cellular injury. However, there is a strong possibility that the nature of the injury for the longer duration of drying is different from shorter duration of drying. For shorter duration of drying, the injury may come from the accumulation of osmotic stress [27], whereas at a longer duration of drying, the injury may be due to over-desiccation effects. The pH of the media for drying and rehydration was found to not have a significant effect if it was within the normal growth range. Reducing the storage temperature to 4°C increased reactivity after rehydration indicating that some intercellular biochemical reactions likely remain active following desiccation to ~1 gH2O/gDW and that these reactions lead to cell death.




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