Full text loading...
Chapter 44 : Surface Microbiology of Cellulolytic Bacteria
Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase
In this chapter, biofilm structure and cell behavior in fully hydrated, and undisturbed biofilms of Clostridium thermocellum are considered as a means to study biofilm formation by anaerobic thermophiles. Recent evidence demonstrates the strong correlation between first-order hydrolysis rates of cellulose with the concentration of sessile bacteria rather than with the concentration of total or planktonic biomass after inoculation with enriched leachate and rumen fluid. A study aimed to correlate the imaging of cellulolytic biofilms with process performance data in real time, and the results suggested that the rumen culture formed thicker and more stable biofilms than the inoculum from digester leachate, which were consistent with the higher rates of cellulose solubilization in the rumen reactors. Cellulolytic organisms occupy a special niche where (the primarily) insoluble cellulose serves both as the carbon source and as biofilm solid support, theoretically providing optimal conditions for biomass accumulation with preferential access to the primary substrate. C. thermocellum is one of the potential candidates for strain development through recombinant and classical strategies for application in consolidated bioprocessing aimed at large-scale conversion of renewable biomass into biofuels and other products. The numerous examples of biofilm formation in the literature, and the observed association between C. thermocellum cells and substrate, make this organism a suitable model to delineate cell-cellulosic substratum associations and to optimize the tools to study this phenomenon in other cellulolytic cultures and consortia with potential for application in biomass conversion.
Key Concept Ranking
- Confocal Laser Scanning Microscopy
Schematic flow cell design showing top view (A) and lateral view (B) and typical C. thermocellum continuous-flow culture growth within flow cells showing the initial growth (dark areas) pattern around the inoculation site (C) with subsequent spreading to uncolonized regions and the depletion of its only cellulosic carbon source, which serves as the attachment substratum (D).
Schematic diagram of a continuous-flow system used under normal atmosphere for growth of anaerobic, thermophilic C. thermocellum cultures. A sampling port allows the aseptic collection of liquid effluent samples, and a “CO2 exchange reactor” purged with N2 gas and coupled to an infrared analyzer allows real-time measurements of dissolved CO2 in the effluent. GFM, gas mass flowmeter.
Confocal laser scanning micrographs of C. thermocellum biofilms growing on solid cellulosic substratum. Attachment to abiotic surfaces was recorded when the solid cellulosic support had been considerably depleted. Cells were stained with Syto9, and cellulose was stained with wheat germ agglutinin-TRITC.
Confocal laser scanning micrograph of C. ther-mocellum cells colonizing a cellulose substrate (fiber) showing spores (arrows) with distinct end-on attachment on the non-sporulating side. Cells were stained with Syto9, and cellulose was stained with wheat germ agglutinin-TRITC. The circles indicate dividing cells with parallel orientation to the attachment interface. Objective, 63 × 1.2 water immersion.
Confocal laser scanning micrograph of C. thermocellum biofilm developing on a cellulose fiber, seen from the top-down view of a three-dimensional projection (top); sectioning through the projection along the x and y axes in the z plane (bottom) revealed the distance between cells and substrate, which was recorded to be lower than the 0.44-μm z-scaling limit of the scanning microscope. Cells were stained with Syto9, and cellulose was stained with wheat germ agglutinin-TRITC.
List of stain stocks and working volumes used on continuous-flow cultures of C. thermocellum in confocal laser scanning microscopy a