Acrox UH3 Driver
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Acrox UH3 Driver
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DNA amplification was checked Acrox UH3 an agarose gel and imaged with a c Azure Biosystems imager. Phylogenetic reconstruction was performed using MEGA7 v 7.
ITS1 and LSU sequences were analyzed with the maximum likelihood method using a Tamura Nei nucleotide substitution model with bootstrap replications to estimate the confidence in node Acrox UH3. Growth curve analyses for characterizing the substrate range of Piromyces sp.
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UH Fungal growth was tracked according to the method introduced by Thedorou et al. Briefly, Hungate tubes containing anaerobic medium C and untreated substrate were autoclaved prior to assessing growth Additional file 1: Table S1 [ 16 ]. Every substrate was tested at Acrox UH3 in triplicate for growth. Additionally, duplicate uninoculated tubes were used as negative controls for each substrate.
Specific growth rates were determined by performing a linear regression of a semi-log plot of accumulated pressure in psig versus time in hours. The data points in the exponential phase that were linearly increasing and had an R2 of approximately 0. We prepared fresh media as Acrox UH3 above. Tubes were inoculated in a random order to prevent systematic bias in inoculum quality. The growth of UH on wild type and genetically modified lines of poplar was tested to evaluate the effect of lignin composition on fungal Acrox UH3 Additional file 1: Tables S2, S3 [ 1719 ].
For all analyses, individual growth rates and total accumulated pressures were calculated.
Acrox UH3 data normalization to glucose Fig. The error for these measurements was propagated accordingly. For data normalization to wild-type poplar Fig. Isolation of the carbohydrate active enzymes CAZymes We used a pull-down purification protocol similar to the one by Solomon et al.
This procedure exploits the cellulose-binding domains of CAZymes to isolate lignocellulose degrading enzymes [ 13 ]. These proteins were also tested for activity Acrox UH3 zymography with 0. The SDS was removed from the gel with slight modification to the procedure of Tseng et al. The gels were rinsed Acrox UH3 ddH2O and placed in 0.
The zymograms were then washed with 0. Zymograms were then stained in 0. We fixed the zymograms with 0. Sugar reducing Acrox UH3 for xylanase activity UH xylanase activity was measured after harvesting the cellulose-binding proteins as discussed above.
However, we used 0. All samples were measured in triplicate and normalized by total protein.
Analyzing the composition of lignocellulosic material after fungal growth To test the effect of syringyl lignin composition on sugar consumption by Piromyces sp. Three different poplar constructs were Acrox UH3 The sugar composition of the spent biomass was determined according to the standard methods Additional file 1: Table S4 Acrox UH3 29 — 31 ]. The differences in glucan and xylan composition between the raw and spent biomass were calculated and one-way ANOVA analyses were performed to evaluate the differences in composition.
Results Isolation of a biomass-degrading anaerobic gut fungus from a donkey To identify more robust and efficient CAZymes and microbial systems that may be used for bioenergy applications, we isolated a previously uncharacterized microbe from the fecal samples of a donkey. Light microscopy revealed the presence of non-planktonic microorganisms that grew Acrox UH3 into the plant substrates, reminiscent of a mature fungal sporangium Fig.
Additionally, this isolate exhibits endogenous zoosporangial development, where the zoosporangium retains its nuclei. The slow growth, zoospore presence, and the well-differentiated stages of a life cycle Fig. DAPI staining of the nucleic acid in the developing fungal Acrox UH3 revealed that this isolate was monocentric has nuclei only within the zoosporangium Fig.
Taxonomic classification of our novel fungal isolate was confirmed via phylogenetic analysis [ 2432 ]. Figure S1 [ 33 ].