Quantification of Cell and Cellulase Mass Concentrations during Anaerobic Cellulose Fermentation: Development of an Enzyme-Linked Immunosorbent Assay-Based Method with Application to Clostridium thermocellum Batch Cultures

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Abstract
A methodology was developed to determine the mass concentrations of cellulase and cells applicable to studies of microbial cellulose utilization in systems for which a substantial fraction of cellulase is cell-associated. Antibodies raised against a 14-amino acid synthetic peptide with sequence taken from the cohesin domain of the scaffoldin protein of Clostridium thermocellum ATCC 27405 were used to develop an indirect ELISA protocol. Six cellulase calibration standards were prepared using affinity digestion (Morag, E.; Bayer, E. A.; Lamed, R. Enzyme Microb. Technol. 1992, 14, 289−292.). These included supernatant and pellet samples from an Avicel-grown culture with fractional cellulose conversion (χ) = 0.98, as well as supernatant, pellet, cell-associated, and cellulose-associated samples from an Avicel-grown culture with χ = 0.8. All six standards displayed a very similar absorbance versus concentration relationship when subjected to ELISA, essentially identical SDS−PAGE banding patterns, and similar cellulase specific activity in relation to both other purified cellulase preparations and crude samples. Coefficients of variation for cellulase concentration measurements were 5.2% for supernatant samples and 5.9% for pellet samples. The ELISA method was applied to batch cultures of C. thermocellum grown on Avicel. Cell concentration was calculated from the pellet protein concentration and the cell protein fraction of a cellobiose-grown control. Two alternative methods appeared to overpredict the cell concentration and were not capable of quantifying cells as distinct from cellulase. Cellulase protein production by Avicel-grown batch cultures represented ∼20% of cell mass exclusive of cellulase. It is concluded that the reported protocols establish a reasonable methodological basis for quantitative determination of the mass concentration of cellulase protein produced by C. thermocellum and for calculation of cell mass concentration as distinct from cellulase concentration.