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.

FIG 1

Diagrammatic overview of dual-staining protocol.

FIG 2

Differential interference contrast (DIC) and epifluorescence micrographs of C. phytofermentans cultured for 3 days with filter paper, wild-type S. bicolor (WT sorgum), or reduced-lignin S. bicolor double mutant (bmr-6 bmr-12 sorghum). Cultures were prepared for microscopy using the dual-staining procedure. For each sample, one field is shown imaged by DIC microscopy (A), and fluorescence microscopy at 385 to 400 nm showing cellulose fibers stained with calcofluor white (B) and at 480 nm showing cells stained with Syto 9 (C). The epifluorescence images in rows B and C are merged in the row D images. Stained culture preparations were diluted with sterile basal culture medium prior to slide preparation: the filter paper culture was diluted 1:3, and sorghum cultures were diluted 1:1. Bar, 10 µm.

FIG 3

Two-dimensional rendering of a 3D image of C. phytofermentans cultured for 5 days on medium with filter paper as the growth substrate. The culture was prepared for microscopy using the dual-staining procedure. Multiple focal plane images at a depth of 1 μm were obtained and used to generate 3D images.

FIG 4

Cell growth (A) and ethanol production (B and C) by C. phytofermentans, and cell numbers (D), cell attachment to substrate (E) and ethanol concentration (F) after 3 days of growth with filter paper, wild-type S. bicolor, or reduced-lignin S. bicolor bmr-6 bmr-12 double mutant as the growth substrate. The concentration of ethanol in cultures is expressed as millimolar concentration (B and F), and the amount of ethanol produced per cell is expressed as nanomoles per cell (C). Error bars represent standard deviations of the means (n = 3). Statistically significant differences are represented with bars and asterisks; one asterisk for P ≤ 0.05, two asterisks for P ≤ 0.01, and three asterisks for P ≤ 0.001.

FIG 5

Correlation of ethanol production with cell number during growth. Ethanol production is given as a function of the cell number in cultures with filter paper (A), wild-type S. bicolor (B), and reduced-lignin S. bicolor bmr-6 bmr-12 double mutant (C) as the growth substrate. Error bars represent standard deviations of the means.