A variety of microbial communities are thought to have unique lignocellulolytic capabilities making them of great interest for the development of second and third generation biofuels. Among the natural degraders of lignocellulose are microbial communities that are found e. g. in the gut of wood-degrading insects such as termites, in ruminants, in compost or soil. Electron microscopy with its ability to conduct an analysis at a spatial resolution of about a nanometer allows the investigation of the interplay between the different bacterial cells that constitute a community, its association with the plant material as well as their macromolecular inventory and degradation strategies.

SEM can only be conducted on surfaces, e. g. plant part from the digestive system of a cellulose degrading animal containing the microbial community, or microbes filtered out of a microbial culture suspension. Since SEM imaging typically requires sample fixation, dehydration and critical point drying, followed by sputter coating, the fine details tend to get lost. The microscopy on those samples in a high-resolution SEM (typically, 2 — 10 kV accelerating voltage are used) can visualize the shape of microorganisms, thus sometimes enabling an identification of certain species, and show both interaction between microbes and between microbes and the surface (e. g. plant material). It can thus be a valuable first insight into the composition and functioning of a microbial community, facilitated by the fact that the technique is rather quick and does not require extensive preparations. However, the technique does not enable a more detailed analysis of the microbial community and is limited to the surface of the samples, thus possibly representing only a small fraction of the sample that might not be typical for the sample in general.

If a more in-depth analysis of the microbial community is the goal, one has to use TEM instead of additional SEM. The analysis of a microbial community with TEM can visualize a lot of interesting traits in this community, including frequent cell-cell interactions, either directly or via a variety of microstructures such as pili and flagella. Even more interesting in this case are the interactions between microbial cells and the plant biomass, which is present in the sample. In studies of such communities (Knierim et al., in preparation) we have found different strategies of cell wall attachment and biomas digestion. An unresolved challenge is the direct identification of species in TEM or SEM. There have been recent advances in this field, combining TEM with Catalized Reporter Deposition Fluorescence In-Situ Hybridization (CARD-FISH), which can identify either groups of bacteria or even certain species (Knierim et al., submitted). However, this technique is extremely tedious and requires some compromises of the TEM imaging quality in order to enable the identification via CARD-FISH.

The described electron microscopy techniques are not only applicable to the understanding of microbial communities but can also be utilized for the analysis of bacteria that have been engineered for certain capabilities. Such capabilities that are developed by synthetic biologists can be the increased production of fuels, the production of different fuels (especially those with longer carbon chains which are more valuable than ethanol) or the assessment of toxicity of the produced biofuels. When combined with tag-based labeling TEM also allows a rational monitoring of protein expression levels, cell-to cell variation and subcellular localization. Thus the application of SEM and TEM to those samples can facilitate the re-engineering of microorganisms that are needed for the production of second and third generation biofuels.

A natural limitation of TEM usually is its coverage of very small sample volumes covered by thin sections. To overcome this problem, FIB/SEM can be employed, which provides a three dimensional view of large volumes of a microbial community at a resolution comparable to electron tomography. For FIB/SEM a similar sample preparation protocol as described above for TEM can be used, but one has to ensure a high internal contrast inside the plastic blocks. We have had very good experience with tannic acid for this purpose. During

FIB/SEM imaging one must pay attention to the area of interest careful, as this technique is very time intensive and hence currently can only be applied to a limited number of volumes. Typically, volumes of 10 x 10 x 5 gm can be covered at an estimated resolution around 10-15 nm. The datasets that are produced this way easily get very large (in the several Gigabyte range) and require careful 3D reconstruction and analysis using software packages with those capabilities such as UCSF Chimera (http://www. cgl. ucsf. edu/chimera) or Amira (http://www. amira. com). We are currently drafting a manuscript on the FIB/SEM 3D analysis of the termite hindgut microbial community, where we classify and quantify constituent microbial community member according to size, shape and internal density characteristics, and map out their distribution with respect to the biomass (Knierim et al., in preparation).

An understanding of the functioning of microbial communities that are capable of lignocellulose degradation will ultimately lead to the development of better techniques for lignocellulose degradation in an industrial setting such as a biorefinery for second or third generation biofuels. We are still in the early steps of this understanding due to the high complexity and variability of these microbial communities, but if we can reduce the complexity by identifying a small set of microbes with valuable capabilities, we may be able to speed up this process. As the principles that are present in these microbial communities have been developed by evolution over billions of years, we can assume that they are very energy efficient, thus providing a maximal energy output while taking up a small amount of energy themselves — an important challenge for the design of industrial processes for the production of biofuels

6. Outlook

Improving feedstocks properties as well as optimizing each step of the deconstruction process and the fuels synthesis production step will decrease the production cost and is therefore key for replacing fossil fuels with biofuels. To accomplish such needed technological advances, one needs to resort to a variety of different biophysical techniques, typically carried out by specialists, that can quantify the effect of experimental intervention and lead to a detailed understanding of the physical, chemical and biological processes of lignocellulosic biofuel production.

7. Acknowledgements

This work was part of the DOE Joint BioEnergy Institute (http://www. jbei. org) supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy. This work was in part supported in part by the Energy Biosciences Institute grant 007-G18.