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Abstract Background There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency. Results We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.). Conclusion Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality.

The ultrastructural characteristics of different cellulase and cellulase-xylanase multiprotein complexes that form the cellulase-xylanase system of Clostridium papyrosolvens C7 were studied by transmission electron microscopy. Findings show that the cellulase-xylanase system is composed of at least seven diverse high-molecular-weight multiprotein complexes. The interactions between C. papyrosolvens C7 and cellulose are not the same as those for Clostridium thermocellum.

KEY WORDS: cellulase, cellulose fermentation, microbial interactions, cellulolytic microoganisms, carbon cycle CONTENTS INTRODUCTION THE SUBSTRATE CELLULOLYTIC ENZYME SYSTEMS OF ANAEROBES CELLULOSE DEGRADATION IN SOILS. SEDIMENTS, AND AQUATIC ENVIRONMENTS The Environments [...]

The hydrolysis of crystalline cellulase and xylan in Clostridium papyrosolvens is caused by an enzyme system comprising at least seven multiprotein complexes with varied structural and enzymatic profiles. Three of the complexes interact synergistically to digest crystalline cellulase, while two others predominantly act upon xylan. Glycoproteins with high molecular weight and apparently lacking any enzymatic function are the proteins common to all seven complexes.

A dual-fluorescent-dye protocol to visualize and quantify Clostridium phytofermentans ISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-type Sorghum bicolor, or a reduced-lignin S. bicolor double mutant (bmr-6 bmr-12 double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Lignocellulosic biomass is a source of low cost polysaccharides that some microbes can deconstruct and convert into liquid transportation fuel. Feedstocks vary in their ease of use depending on their source and handing. Estimating conversion amenability is useful to determine the effects of biomass pretreatment and genetic potential for the purposes of energy crop breeding and genetics. Here we describe a small-scale high-throughput assay that measures ethanol production from a culture of plant biomass and the ethanologen Clostridium phytofermentans.