Enzymatic Hydrolysis in the Green Production of Bacterial Cellulose Nanocrystals

Abstract

In this study, we extensively describe experimental models, with correlating experimental conditions, which were used to investigate the enzymatic hydrolysis of bacterial cellulose (BC) to obtain nanocrystals. Cellulase from Trichoderma reesei was used in five enzyme/BC ratios over a period of h. The turbidity data was modeled using both logistic regression and empirical regression to determine the fractal kinetics, resulting in unique kinetic patterns for the mixtures that were richest in BC and in enzymes. The evolution of the yield was inversely related to the turbidity, as confirmed through a semi-empirical approach that was adopted to model the experimental data. The yield values after 74 h of hydrolysis were higher for the substrate-rich mixtures (~20%) than for the enzyme-rich mixtures (~5%), as corroborated by cellobiose and glucose quantification. Transmission electron microscopy and atomic force microscopy analyses revealed a shift from a fibril network to a needlelike morphology (i.e., aggregated nanocrystals or individual nanocrystals ~6 nm width and 200-800 nm in length) as the enzyme/BC ratios went from lower to higher. These results were explained in terms of the heterogeneous substrate model and the erosion model. This work initiated a promising, environmental-friendly method that could serve as an alternative to the commonly used chemical hydrolysis routes.

Enzymatic Hydrolysis of Bacterial Cellulose for the Production of Nanocrystals for the Food Packaging Industry

“…The production of BC followed the same procedure used by Rovera et al (2016) [12]. Briefly, BC was produced by static fermentation (seven days at 30 • C in a Hestrin and Schramm culture medium) using Komagataeibacter sucrofermentans DSM 15973 strain (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig Germany).…”

“…The evolution of the enzymatic hydrolysis was monitored at 3 h intervals in turbidity experiments, over a temporal window of 145 h, according to the procedure reported in our previous work [12]. Spectrophotometric measurements were performed using a Lambda 25 spectrophotometer (PerkinElmer, Waltham, MA, USA) at wavelengths between 380 and 800 nm in transmittance mode.…”

“…Turbidity experiments via spectrophotometric measurements represent a viable approach to monitor the evolution of the enzyme-mediated hydrolysis of cellulose, the basic principle being that a decrease in turbidity (i.e., an increased transmittance) is expected for extended hydrolysis times, because of the reduced size of the BC fibrils [12]. Figure 1a shows the hydrolysis kinetics, expressed as transmittance evolution over time, for both the endo-1,4-β-glucanase-and the cellulase-containing systems.…”

“…In our previous work, we demonstrated that cellulases facilitate the obtaining of bacterial cellulose nanocrystals (BCNCs), with the best outcome (in terms of morphology and size distribution) obtained using a high amount of enzymes [12]. However, the overall yield of the process was very low (≈5%), which was ascribed to the high intensity of EGs' and CBHs' synergistic action, up to the obtainment of cellobiose and glucose.…”

A review of current and future food applications of natural hydrocolloids

“…Cellulose nanocrystals (CNCs), interchangeably known as nanocrystalline cellulose, cellulose whiskers, cellulose nanowhiskers and cellulose microcrystals, are mostly obtained by acid hydrolysis of native cellulose, although alternative methods (e.g. enzymatic hydrolysis) have been proposed recently (Rovera et al, 2018b). These processes allow removing the amorphous domains, thus yielding a totally crystalline substance in the form of rods (Fig.…”