| Global energy demand, climate change, and environmental pollution caused by fossil fuel combustion have heightened the need for renewable energy sources. Lignocellulosic biomass, the most abundant form of biopolymers on earth, is readily renewable and a promis-ing source of raw materials for biofuels synthesis. C. thermocellum has great potential for ef-fective bioconversion of low-value cellulosic feedstocks because of its capability in hydroly-sis of different cellulosic materials. T. aotearoense is hemicellulolytic in contrast to the C. thermocellum which are cellulytic. Thus, these bacteria could be promising candidates for CBP biofuels production. In order to monitor such a co-culture system composed of two dif-ferent strains, new protocol was tested. In this study, a quantitative real-time PCR assay was developed for the direct growth detection of C. thermocellum and T. aotearoense at the sin-gle-cell level in insoluble lignocellulosic biomasses.The assay targeted the cip A and xyl A gene and q PCR primer sets were developed for C. thermocellum and T. aotearoense, respectively. These markers are present in single copies in the genome, whereas there is no copy in the other strain’s genome of the co-culture system. This was able to distinguish C. thermocellum and T. aotearoense from each other. The recom-binant plasmid which contained cip A and xyl A gene was used as standards to generate a stan-dard curve. The sensitivity, reproducibility and specificity of the assay were evaluated, and practical samples in insoluble lignocellulosic biomasses were detected using the assay.The assay has good reproducibility and specificity which quantitative detection limit was 10 cell equivalents(CE) per reaction. OD600-based counting and q PCR quantification of C. thermocellum and T. aotearoense respectively cultured in soluble medium were compared and an excellent consistency was revealed, indicating the appropriateness of the developed q PCR method. Analysis based on yellow affinity substrate and fermentation products may incor-rectly estimate its population.Paper sludge rich in Ca CO3 has buffering effect on the stability and hydrogen production of the anaerobic process that helps in improving hydrogen fermentation performance. The op-timum parameters for bio-hydrogen production from paper sludge were 7% inoculum size, 5g/L yeast extract concentration, 15g/L urea concentration and 2% substrate concentration. The maximum hydrogen yield was obtained at 110.61 mmol/L.Although the growth patterns of C. thermocellum and T. aotearoense during the fermen-tation showed differences, it was apparent that the two strains were able to coexist throughout the experimental period with lignocellulosic biomasses as the substrate. C. thermocellum was detected as the dominant strain for co-culture of C. thermocellum-T. aotearoense in lignocel-lulosic biomass media. The fermentation products concentration trend well represents the cell concentration trend of the dominant strain. The q PCR data showed a higher growth rate of T. aotearoense in the co-culture compared to its mono-culture, which suggest the presence of synergism between these two species, while C. thermocellum metabolic activity significantly allows T. aotearoense to grow better under harsh co-culture conditions, which do not allow T. aotearoense to grow well in lignocellulosic biomasses independently.The developed q PCR assay can serve as a specific, sensitive and reproducible method for the detection of C. thermocellum and T. aotearoense in lignocellulosic biomass at the sin-gle-cell level as well as for routine monitoring of the dynamics of artificial co-culture system. |
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