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Fig. 4 | Biotechnology for Biofuels

Fig. 4

From: Process relevant screening of cellulolytic organisms for consolidated bioprocessing

Fig. 4

Method scheme: comparison of OTR-based cellulase activity measurement with freeze assay. a The RAMOS technique allows the quantification of the released sugars based on the oxygen consumption rate (OTR) during growth on cellulose. The fungus produces cellulases to degrade cellulose into soluble sugars. The sugar and oxygen uptake are assumed to be stoichiometrically coupled. As the conversion of cellulose is the process-limiting step, no monomeric sugars accumulate. Based on the total amount of oxygen consumed, the amount of sugars released can be estimated. b The freeze assay allows the quantification of the initial carbon release rate (CRRFreeze) under target process conditions. A sample is mixed with a fixed amount of substrate and 1 M itaconic acid buffer at pH 3.7, and then frozen to inactivate the fungus. Then the sample is thawed and incubated for 2 h at fermentation temperature to mimic the in situ conditions. Finally, the supernatant is analyzed by HPLC for soluble sugars to quantify the initial CRRFreeze. c The in situ CRR is dependent on the actual cellulose concentration (Cellt), the enzyme concentration, e and cellulose digestibility d. These factors change dynamically over the cultivation time: new enzyme is produced, the cellulose is consumed, and the digestibility decreases due to various effects. To compare different fungi under equal conditions, the freeze assay is performed. Because the hydrolysis time is short in comparison to the cultivation time and a defined amount of fresh cellulose is added, the influencing factors cellulose concentration, enzyme concentration, and digestibility can be considered constant. Thus, the freeze assay quantifies the initial hydrolysis rate (CRRFreeze) under target process conditions at high cellulose loading. To compare the results of both methods, the CRRFreeze can be transformed into in situ CRR by incorporating the difference in initial cellulose concentration in the fermentation and the freeze assay by a logarithmic correlation of the specific carbon release rate with initial enzyme/cellulose ratio (see Fig. 5b). The decrease in digestibility is included using a fractal kinetic model (see Fig. 5c)

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