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

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 Avicelgrown culture with fractional cellulose conversion (X) = 0.98, as well as supernatant, pellet, cell-associated, and cellulose-associated samples from an Avicel-grown culture with X = 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 approximately 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.

Cellulose utilization by Clostridium thermocellum : Bioenergetics and hydrolysis product assimilation

“…All chemicals were reagent grade and were obtained from Sigma unless indicated otherwise. C. thermocellum ATCC 27405 has been maintained in our lab since 1983, as described (21)(22)(23). Chemically defined, MTC medium was prepared by combining six sterile solutions under a nitrogen atmosphere, as described (21)(22)(23).…”

“…C. thermocellum ATCC 27405 has been maintained in our lab since 1983, as described (21)(22)(23). Chemically defined, MTC medium was prepared by combining six sterile solutions under a nitrogen atmosphere, as described (21)(22)(23). Cellodextrins were prepared by mixed acid hydrolysis and separated chromatographically, as reported (24).…”

“…The mass concentrations of cellulase and cells growing on Avicel and cellodextrins were calculated based on an indirect ELISA by using antibody raised against a sequence from the C. thermocellum scaffoldin protein, as described (21,23). Noncellulase extracellular protein is defined as supernatant protein concentration minus the supernatant cellulase concentration as determined by ELISA.…”

“…Noncellulase extracellular protein is defined as supernatant protein concentration minus the supernatant cellulase concentration as determined by ELISA. The dry weight, cellulose conversion, and fermentation product concentration of culture samples were measured as described (21,23).…”

“…Radiolabeled cellulose (50 Ci; 1 Ci ϭ 37 GBq), purchased from American Radiolabeled Chemicals (St. Louis), was extracted by using 90% ethanol followed by boiling, dilution using 40 mg of Avicel PH105, and then treated with 83% phosphoric acid to obtain a radiolabeled amorphous cellulose suspension (21). Radiolabeled cellobiose was prepared by using cellobiose phosphorylase in conjunction with radiolabeled glucose (Sigma) and glucose-1-phosphate, as described (25,26).…”