Enzymatic cellulose hydrolysis in an attrition bioreactor combined with an aqueous two-phase system

Cellulose was hydrolyzed in the attrition bioreactor (ABR) with enzyme recycling by employing an aqueous two-phase system (composed of dextran and polyethylene glycol) and an ultrafiltration unit. The ABR combines wet ball milling and enzymatic hydrolysis in one process step. The cellulase enzymes were more stable in the two-phase system than in the normal buffer solution. With the initial substrate concentration (Solka Floe BW200) of 40 g/L and intermittent addition of cellulose, sugar was semicontinuously produced at dilution rates of 0.06 h(-1) and productivities of 2.1 g/L h, which is approximately a 10-fold increase of the previously reported values performed in a regular stirred reactor with an aqueous two-phase system. The conversion of the substrate was 86%.     

Disruption of Saccharomyces cerevisiae using enzymatic lysis combined with high-pressure homogenization

The disruption of commercially-available pressed Bakers' yeast (Saccharomyces cerevisiae) was studied using a relatively new high-pressure homogenizer (the Microfluidizer). Initial experiments using only mechanical disruption generally gave low disruption yields (i.e., less than 40% disruption in 5 passes). Consequently combinations of two disruption methods, namely enzymatic lysis and subsequent homogenization, were tested to identify achievable levels of disruption. The enzyme preparation employed was Zymolyase, which has been shown to effectively lyse the walls of viable yeast. Yeast cell suspensions ranging in concentration from 0.6 to 15 gDW/L were disrupted with and without enzymatic pre-treatment. Final total disruption obtained using the combined protocol approached 100% with 4 passes at a pressure of 95 MPa, as compared to only 32% disruption with 4 passes at 95 MPa using only homogenization. A model is presented to predict the fraction disrupted while employing this novel enzymatic pretreatment.Nomenclature a exponent of pressure (-) - b exponent of number of passes (-) - K disruption constant (MPa^-a) - N number of passes (-) - P pressure (MPa) - R total fraction of cells disrupted (-) - Ro fraction of cells disrupted after enzymatic pre-treatment (-) - X cell concentration (dry weight) (gDW/L) abbreviation DW dry weight     

Enzymatic hydrolysis and recrystallization behavior of initially amorphous cellulose

Cellulose samples from cotton and wood pulps with varying low degrees of crystallinity (mechanically decrystallized) were studied. The influence of initial cellulose crystallinity on sugar yield after enzymatic hydrolysis was determined by two different methods. As expected, samples with low crystallinity were much more accessible to enzymatic attack and glucose yields were higher than were samples of high initial crystallinity. Hydrolysis of cellulose seems more dependent on cellulose crystallinity than on the source of cellulose. It is known that decrystallized or amorphous cellulose can recrystallize under proper conditions, e.g., during acid hydrolysis. The data reported here also reveal some recrystallization during enzymatic hydrolysis which probably occurs simulataneously with a selective enzymatic attack on the amorphous regions of cellulose. In all cases, the amorphous celluloses recrystallized in the original lattice form, that of native cellulose.     

Enzymatic hydrolysis of cellulose and various pretreated wood fractions

Three strains of Trichoderma-T. reesei C30, T. reesei QM9414, and Trichoderma species E-58-were used to study the enzymatic hydrolysis of pretreated wood substrates. ach of the culture filtrates was incubated with a variety of commercially prepared cellulose substrates and pretreated wood substrates. Solka floc was the most easily degraded commercial cellulose. The enzyme accessibility of steam-exploded samples which had been alkali extracted and then stored wet decreased with the duration of the steam treatment. Air drying reduced the extent of hydrolysis of all the samples but had a greater effect on the samples which had previously shown the greatest hydrolysis. Mild pulping using 2% chlorite increased the enzymatic hydrolysis of all the samples. Steam explosion was shown to be an excellent pretreatment. The results indicate that the distribution of the lignin as well as the surface area of the cellulosic substrate are important features in enzymatic hydrolysis.     

Enzymatic hydrolysis of cellulose coupled with electricity generation in a microbial fuel cell

Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from a variety of biodegradable substrates, including cellulose. Particulate materials have not been extensively examined for power generation in MFCs, but in general power densities are lower than those produced with soluble substrates under similar conditions likely as a result of slow hydrolysis rates of the particles. Cellulases are used to achieve rapid conversion of cellulose to sugar for ethanol production, but these enzymes have not been previously tested for their effectiveness in MFCs. It was not known if cellulases would remain active in an MFC in the presence of exoelectrogenic bacteria or if enzymes might hinder power production by adversely affecting the bacteria. Electricity generation from cellulose was therefore examined in two-chamber MFCs in the presence and absence of cellulases. The maximum power density with enzymes and cellulose was 100 +/- 7 mW/m(2) (0.6 +/- 0.04 W/m(3)), compared to only 12 +/- 0.6 mW/m(2) (0.06 +/- 0.003 W/m(3)) in the absence of the enzymes. This power density was comparable to that achieved in the same system using glucose (102 +/- 7 mW/m(2), 0.56 +/- 0.038 W/m(3)) suggesting that the enzyme successfully hydrolyzed cellulose and did not otherwise inhibit electricity production by the bacteria. The addition of the enzyme doubled the Coulombic efficiency (CE) to CE = 51% and increased COD removal to 73%, likely as a result of rapid hydrolysis of cellulose in the reactor and biodegradation of the enzyme. These results demonstrate that cellulases do not adversely affect exoelectrogenic bacteria that produce power in an MFC, and that the use of these enzymes can increase power densities and reactor performance.     

Increased disruption resistance of Candida utilis grown from survivors of enzymatic lysis combined with high-pressure homogenization

The resistance of Candida utilis (ATCC 9226) to disruption as a result of enzymatic pretreatment combined with high-pressure homogenization was found to increase when the yeast was grown from an inoculum which had previously been subjected to enzymatic pretreatment combined with high-pressure homogenization. The inoculum thus consisted of a mixture of undisrupted, viable cells and non-viable cells. The enzyme preparation employed was Zymolyase, which depolymerizes various components of the cell walls of viable yeast. A Microfluidizer was used for the high-pressure homogenization step. In order to obtain the 'disruption-resistant' cell fraction for use as an inoculum, 'normal' C. utilis was enzymatically pretreated, and subsequently homogenized (herein referred to as Microfluidization) using either three or 10 passes through the Microfluidizer at an operating pressure of 95 MPa. Yeast grown from the survivors of the enzyme/3-pass treatment were found to be somewhat more resistant to disruption by either enzymatic pretreatment alone or to enzymatic pretreatment followed by Microfluidization. Cells grown from enzyme/ 10-pass treated inocula exhibited the highest resistance to disruption. The 'disruption-resistant' fraction exhibited this characteristic through three serial re-cultivations. Possible mechanisms for the increased 'disruption-resistance' of this isolated population of C. utilis are presented.     

Enzymatic hydrolysis and fermentation of corn for fuel alcohol

The integration of enzyme saccharification with fermentation reduces the total time required to produce acceptable levels of ethanol. The use of a more concentrated mash (84.8 L total mash/bu corn) results in a 26.6% increase in ethanol productivity and a 21.4% increase in beer ethanol concentration compared to standard corn mash (96.6 L total mash/bu corn). Thus, the energy requirement and cost of distillation can be reduced. The addition of waste cola syrup at 30 g invert sugar/L total mash gave a 19% increase in ethanol concentration in the final beer and required only a small increase in the period of fermentation. Surplus laundry starch can replace 30-50% of the weight of corn normally used in fermentation without influencing ethanol production or the time required for fermentation. Both of these waste materials reduce the unit cost of ethanol and demonstrate the value of such substances in ethanol systems.     

Chimeric lactase capable of spontaneous and strong immobilization on cellulose and development of a continuous-flow system for lactose hydrolysis at high temperatures

Recombinant plasmids containing fusion proteins composed of two different modules were constructed and expressed in Escherichia coli. The modules encoded the lactase LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus and the cellulase CelD, a cellulose-binding module (CBM) from Anaerocellum thermophilum. The CelD CBM provides a spontaneous and strong sorption of the fusion proteins onto a cellulose carrier. The enzymatic activities of both the free LacA protein and LacA-CelD CBM fusion proteins immobilized onto the cellulose carrier were assessed. The LacA activity of the fusion protein was dependent upon its position with respect to the CBM. The highest level of lactase activity and stability was observed when the lactase domain was localized at its N terminus. A continuous-flow column reactor of lactase immobilized on a cellulose carrier was constructed, and its activity was assessed. The lactose hydrolysis rate for a 150 mM (5%) solution at a flow rate of 1 reactor volume per min was 75%, which is a value optimal for further whey transformation into glucose/galactose syrup.     

Kinetics of cellulose regeneration from cellulose--NaOH--water gels and comparison with cellulose--N-methylmorpholine-N-oxide--water solutions

The regeneration kinetics of cellulose from cellulose--NaOH--water gels immersed in a nonsolvent bath is studied in detail. Cellulose concentration, bath type, and temperature were varied, and diffusion coefficients were determined. The results were compared with data measured and taken from the literature on the regeneration kinetics of cellulose from cellulose--N-methylmorpholine-N-oxide (NMMO) monohydrate solutions. Different theories developed for the transport behavior of solutes in hydrogels or in porous media were tested on the systems studied. While the diffusion of NaOH from cellulose--NaOH--water gels into water has to be described with "porous media" approaches, the interpretation of NMMO diffusion is complicated because of the change of NMMO's state during regeneration (from solid crystalline to liquid) and the high concentration of NMMO in the sample. The activation energies were calculated from diffusion coefficient dependence on temperature for both systems and compared with the ones obtained from the rheological measurements. The activation energy of cellulose--NaOH--water systems does not depend on cellulose concentration or the way of measurement. This result shows that whatever the system is, pure NaOH--water solution, cellulose--NaOH--water solution, or cellulose--NaOH--water gel, it is NaOH hydrate with or without cellulose in solution, which is moving in the system. The swelling of cellulose in different nonsolvent liquids such as water or different alcohols during regeneration was investigated and interpreted using the Hildebrand parameter.     

Preparation of chitin/cellulose composite gels and films with ionic liquids

In this study, we performed preparation and characterizations of the chitin/cellulose composite gels and films using the two ionic liquids, 1-allyl-3-methylimidazolium bromide and 1-butyl-3-methylimidazolium chloride. First, chitin and cellulose were dissolved in each appropriate ionic liquid. Then, the two liquids were mixed in the desired ratios at 100 °C to give the homogeneous mixtures. The gels were obtained by standing the mixtures for 4 days. On the other hand, the films were obtained by casting the mixtures on glass plates, followed by soaking in water and drying. The obtained gels and films were characterized by XRD and TGA measurements. The mechanical properties of the gels and films were evaluated under compressive and tensile modes, respectively.     

Engineering and characterization of carbohydrate-binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nk_on) and desorption (k_off) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.     

Enzymatic hydrolysis of rice straw and corn stalks for monosugars production

Rice straw and corn stalks were used as fermentation substrate for the evaluation of cellulases activity secreted by different organisms. The substrates were pretreated with alkaline hydrogen peroxide (AHP) for 6 h at 30 and 60 °C. From the fermentation studies, rice straw and corn stalks substrates showed the highest cellulases activity after 96 h at 60 °C of pre-treatment.     

Pretreatment of wheat straw using combined wet oxidation and alkaline hydrolysis resulting in convertible cellulose and hemicellulose

The wet oxidation process of wheat straw has been studied as a pretreatment method to attain our main goal: To break down cellulose to glucose enzymatic, and secondly, to dissolve hemicellulose (e.g., for fermentation) without producing microbial inhibitors. Wet oxidation combined with base addition readily oxidizes lignin from wheat straw facilitating the polysaccharides for enzymatic hydrolysis. By using a specially constructed autoclave system, the wet oxidation process was optimized with respect to both reaction time and temperature. The best conditions (20 g/L straw, 170 degrees C, 5 to 10 min) gave about 85% w/w yield of converting cellulose to glucose. The process water, containing dissolved hemicellulose and carboxylic acids, has proven to be a direct nutrient source for the fungus Aspergillus niger producing exo-beta-xylosidase. Furfural and hydroxymethyl-furfural, known inhibitors of microbial growth when other pretreatment systems have been applied, were not observed following the wet oxidation treatment. (c) 1996 John Wiley & Sons, Inc.     

Mathematical model for enzymatic hydrolysis and fermentation of cellulose by Trichoderma

This paper describes a mathematical model for the enzymatic hydrolysis and fermentation of cellulose by Trichoderma reesei. The principal features of the model are the assumption of two forms of cellulose (crystalline and amorphous), two sugars (cellobiose and glucose), and two enzymes (cellulase and β-glucosidase). An inducer–repressor–messenger RNA mechanism is used to predict enzyme formation, and pH effects are included. The model consists of 12 ordinary differential equations for 12 state variables and contains 38 parameters. The parameters were estimated from four sets of experimental data by optimization. The results appear satisfactory, and the computer programs permit simulation of a variety of system changes.     

Characterization of acid catalytic domains for cellulose hydrolysis and glucose degradation

Cellulolytic enzymes consist of a catalytic domain, a linking peptide, and a binding domain. The paper describes research on carboxylic acids that have potential as catalytic domains for constructing organic macromolecules for use in cellulose hydrolysis that mimic the action of enzymes. The tested domains consist of the series of mono-, di-, and tricarboxylic acids with a range of pK(a)'s. This paper systematically characterizes the acids with respect to hydrolysis of cellobiose, cellulose in biomass, and degradation of glucose and compares these kinetics data to dilute sulfuric acid. Results show that acid catalyzed hydrolysis is proportional to H+ concentration. The tested carboxylic acids did not catalyze the degradation of glucose while sulfuric acid catalyzed the degradation of glucose above that of water alone. Consequently, overall yields of glucose obtained from cellobiose and cellulose are higher for the best carboxylic acid tested, maleic acid, when compared to sulfuric acid at equivalent solution pH.     

Comparison of steam and ammonia pretreatment for enzymatic hydrolysis of cellulose

Summary Aspenwood, wheat straw, wheat chaff and alfalfa stems were treated under pressure with either steam or ammonia. The material was then water or methanol/water extracted. The extent of enzymatic hydrolysis of the cellulose portion of the treated substrates was compared using two different cellulases, a commercial preparation, Celluclast, and those from the fungus Trichoderma harzianum. Both steam and ammonia treatment enhanced the accessibility of the cellulose as measured by hydrolysis. Methanol extraction of steamed material generally reduced the access of the enzyme to the cellulose, whereas methanol extraction of ammonia-treated material increased accessibility. The optimum combinations of pretreatment and extraction method depended on the substrate and on the enzyme system; no treatment suitable for all situations could be selected.     

Characterization and immobilization of liposome-bound cellulase for hydrolysis of insoluble cellulose

The liposome-bound cellulase was prepared by covalently coupling cellulase with the enzyme-free liposomes bearing aldehyde groups so that cellulase was located solely on the outer membrane of liposomes. The modified cellulase possessed the higher activity efficiency and lipid-based specific activity than the cellulase-containing liposomes reported previously. The enzyme-free liposomes bearing aldehyde groups were covalently immobilized with the chitosan gel beads and the free cellulase was coupled with the treated gel beads to prepare the immobilized liposome-bound cellulase. The activity efficiency of the immobilized liposome-bound cellulase was much higher than that of the conventionally immobilized cellulase. The results on reusability of the immobilized liposome-bound cellulase in the hydrolysis of either soluble or insoluble cellulose showed that the immobilized liposome-bound cellulase had the higher remaining cellulase activity and reusability than the conventionally immobilized cellulase for the hydrolysis of either type of cellulose. The liposomal membrane was suggested to be efficient in maintaining the cellulase activity during the hydrolysis.     

Enzymatic hydrolysis of sugarcane bagasse and straw mixtures pretreated with diluted acid

Abstract

In Brazil, sugarcane biomass is generated in large amounts. Sugarcane bagasse and straw are considered as an important feedstock for renewable energy and biorefinery. This paper aims to study the generation of monosaccharides (C5 and C6) from sugarcane biomass via processing bagasse or straw and mixtures of both materials (bagasse:straw 3:1, 1:1 and 1:3). Samples were pretreated with sulfuric acid which resulted in approximately 90% of hemicellulose solubilization, corresponding to around 58 g L^{? 1} of xylose. Pretreated straw showed greater susceptibility to enzymatic hydrolysis in comparison to bagasse, as shown by glucose yields of 76% and 65%, respectively, whereas the mixtures showed intermediate yields. Thus, one strategy to balance sugarcane biomass availability and possibly increasing 2G ethanol production would be to use bagasse–straw mixtures in appropriate ratios according to market fluctuations. Untreated and pretreated samples were analyzed using X-ray diffraction, but there was no relationship to enzymatic hydrolysis.     

Application of high-voltage electrical discharges and high-pressure homogenization for recovery of intracellular compounds from microalgae Parachlorella kessleri

Treatments with high-voltage electrical discharges (HVED) and high-pressure homogenization (HPH) were studied and compared for the release of ionic components, carbohydrates, proteins, and pigments from microalgae Parachlorella kessleri (P. kessleri). Suspensions (1% w/w) of microalgae were treated by HVED (40 kV/cm, 1–8 ms) or by HPH (400–1200 bar, 1–10 passes). Particle-size distribution (PSD) and microscopic analyses were used to detect the disruption and damage of cells. HVED were very effective for the extraction of ionic cell components and carbohydrates (421 mg/L after 8 ms of the treatment). However, HVED were ineffective for pigments and protein extraction. The concentration of proteins extracted by HVED was just 750 mg/L and did not exceed 15% of the total quantity of proteins. HPH permitted an effective release overall of intracellular compounds from P. kessleri microalgae including a large quantity of proteins, whose release (at 1200 bar) was 4.9 times higher than that obtained by HVED. Consequently, HVED can be used at the first step of the overall extraction process for the selective recovery of low-molecular-weight components. HPH can be then used at the second step for the recovery of remaining cell compounds.