Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates

Abstract

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. Microbial decomposition of plant biomass is central to nutrient cycling in numerous varied environments, and this process plays a key role in the cycling of carbon on the planet. In terrestrial environments, carbon storage occurs primarily in forests where soils are estimated to store 1,200 gigatons of carbon, approximately two thirds more carbon than found in the atmosphere (1). A major thrust of terrestrial microbial ecology is centered on understanding the composition and function of microbial communities in order to assess their influence on carbon cycling (2). In anoxic environments rich in decaying plant material, diverse communities of interacting microbes are responsible for the degradation of cellulose, complex polysaccharides, and other abundantly produced plant cell wall components. Given that most of these substrates are insoluble, microbial decomposition of plant biomass occurs extracellularly, and breakdown products may become available to other community members, forming a basis for multifarious interactions that occur in these environments (3).Anoxic decomposition of plant biomass is tied to health and nutrition in animals by way of gastrointestinal tract microbial communities. Most animals lack the enzymatic capacity required to digest cellulose and m...

Custom fabrication of biomass containment devices using 3-D printing enables bacterial growth analyses with complex insoluble substrates

“…Colony forming unit (CFU) counting is also a time-delayed measurement, even more so than protein-based measures, and also requires the removal of sample. It should be noted that, despite the drawbacks described above, these methods are used successfully with microbes that adhere to insoluble polysaccharides, particularly anaerobes (Alvelo-Maurosa et al, 2016, Yang et al, 2010.…”