Bioconversion of waste cellulose by using an attrition bioreactor

A new type of reactor, an attrition bioreactor, was tested to achieve a higher rate and extent of enzymatic saccharification of cellulose than is possible with conventional methods. The reactor consisted of a jacketted stainless-steel vessel with shaft, stirrer, and milling media, which combined the effect of the mechanical action of wet milling with cellulose hydrolysis. The substrates tested were newsprint and white-pine heartwood. The performance of the reactor was excellent. The extent and rate of enzymatic hydrolysis could be markedly improved over other methods. The power consumption of the attrition bioreactor was also measured. The cellulase enzyme deactivation during attrition milling was not significant.     

Investigating the role of mechanics in lignocellulosic biomass degradation during hydrolysis

Enzymes and mechanics play major roles in lignocellulosic biomass deconstruction in biorefineries by catalyzing chemical cleavage or inducing physical breakdown of biomass, respectively. At industrially relevant substrate concentrations mechanical agitation is also a driving force for mass transfer as well as agglomeration of elongated biomass particles. Contrary to the physically induced particle attrition, which typically facilitates feedstock handling, particle agglomeration tends to hinder mass transfer and in the worst case induces processing difficulties like pipe blockage. Understanding the complex interplay between mechanical agitation and enzymatic degradation during hydrolysis is therefore critical and was the aim of this study. Particle size analyses revealed that neither mechanical agitation alone nor enzymatic treatment without mechanical agitation had any noteworthy effect on flax fiber attrition. Similarly, successive treatment, where mechanical agitation was either preceded or proceeded by enzymatic hydrolysis, did not induce any substantial segmentation of flax fibers. Simultaneous enzymatic and mechanical treatment on the other hand was found to promote fast fiber shortening. Higher hydrolysis yields, however, were obtained from nonagitated samples after prolonged enzymatic treatment, indicating that mechanical agitation in the long run reduces activity of the cellulolytic enzymes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2754, 2019.     

Liquefaction of lignocellulose at high-solids concentrations

To improve process economics of the lignocellulose to ethanol process a reactor system for enzymatic liquefaction and saccharification at high-solids concentrations was developed. The technology is based on free fall mixing employing a horizontally placed drum with a horizontal rotating shaft mounted with paddlers for mixing. Enzymatic liquefaction and saccharification of pretreated wheat straw was tested with up to 40% (w/w) initial DM. In less than 10 h, the structure of the material was changed from intact straw particles (length 1-5 cm) into a paste/liquid that could be pumped. Tests revealed no significant effect of mixing speed in the range 3.3-11.5 rpm on the glucose conversion after 24 h and ethanol yield after subsequent fermentation for 48 h. Low-power inputs for mixing are therefore possible. Liquefaction and saccharification for 96 h using an enzyme loading of 7 FPU/g.DM and 40% DM resulted in a glucose concentration of 86 g/kg. Experiments conducted at 2%-40% (w/w) initial DM revealed that cellulose and hemicellulose conversion decreased almost linearly with increasing DM. Performing the experiments as simultaneous saccharification and fermentation also revealed a decrease in ethanol yield at increasing initial DM. Saccharomyces cerevisiae was capable of fermenting hydrolysates up to 40% DM. The highest ethanol concentration, 48 g/kg, was obtained using 35% (w/w) DM. Liquefaction of biomass with this reactor system unlocks the possibility of 10% (w/w) ethanol in the fermentation broth in future lignocellulose to ethanol plants.     

The role of endoglucanase and endoxylanase in liquefaction of hydrothermally pretreated wheat straw

The role of endocellulases and endoxylanase during liquefaction and saccharification of hydrothermally pretreated wheat straw was studied. The use of a flow‐loop setup with in‐line magnetic resonance imaging enabled frequent measurements of viscosity at 55°C during saccharification at 6% total solids content. Viscosity data were complemented with off‐line measurements of fiber lengths and release of soluble sugars. A clear correlation between fiber attrition and a decrease in viscosity was found. Fiber lengths and viscosity dropped quickly within the first hour and then stagnated, while sugar yields increased substantially thereafter, illustrating that liquefaction and saccharification are separate mechanisms. Both endoglucanase and endoxylanase were shown to have a significant effect on viscosity during liquefaction while the addition of endoxylanase also increased sugar yield. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:923–931, 2014     

Enzymatic liquefaction and saccharification of pretreated corn stover at high-solids concentrations in a horizontal rotating bioreactor

A self-designed horizontal rotating bioreactor (HRR) was applied for enzymatic hydrolysis of pretreated corn stover to improve the process economics of ethanol production. The mixing principle was based on gravity and free fall employed with tank-rotating. The liquefaction performances using the HRR and the vertical stirred-tank reactor (VSTR) with a helical impeller were compared and analyzed by measuring rheological properties of the slurry. During the enzymatic hydrolysis, viscosity decreased dramatically in the initial phase for both bioreactors and more pronouncedly for the HRR. Rheological parameters fitted to the power law showed that shear thinning properties of the slurry weakened during the reaction. The glucose concentration was used to define the efficiency of the saccharification reaction. The HRR also proved to be more efficient for glucose release with both the constant and fed-batch substrate addition modes. Liquefaction and saccharification at 25 % w/w dry matter (DM) and enzyme loading of 7 FPU/g DM resulted in the optimal glucose concentration of 86 g/kg. Results revealed a decrease in cellulose conversion at increasing initial DM, which was slighter in the HRR compared with that in the VSTR.     

Enhancement of enzymatic hydrolysis by simultaneous attrition of cellulosic substrates

It has been shown that simultaneous attrition of cellulose in an attritor containing stainlesssteel beads results in a substantial enhancement of the enzymatic hydrolysis. The attrition exerts two opposing effects, continuous delamination and comminution of the substrate with formation of new reactive sites and a gradual denaturation and inactivation of the enzyme. Consequently, the hydrolysis proceeds very rapidly at first and levels off at about 70% saccharification of the substrate. Accumulation of hydrolysis products is also responsible for inhibition of the enzyme. The attrition method is effective for the saccharification of cottonwood in which the cellulosic microfibrils are embedded in a matrix of lignin and hemicelluloses. A comparison between the saccharification of wood, lignocellulose, holocellulose, and cellulose with simultaneous attrition showed that the lignin component provided more hindrance toward the saccharification process than hemicelluloses, which are themselves subject to enzymatic hydrolysis.     

Enhanced enzymatic hydrolysis of mild alkali pre-treated rice straw at high-solid loadings using in-house cellulases in a bench scale system

In the present study, scale-up systems for cellulase production and enzymatic hydrolysis of pre-treated rice straw at high-solid loadings were designed, fabricated and tested in the laboratory. Cellulase production was carried out using tray fermentation at 45 °C by Aspergillus terreus in a temperature-controlled humidity chamber. Enzymatic hydrolysis studies were performed in a horizontal rotary drum reactor at 50 °C with 25 % (w/v) solid loading and 9 FPU g^?1 substrate enzyme load using in-house as well commercial cellulases. Highly concentrated fermentable sugars up to 20 % were obtained at 40 h with an increased saccharification efficiency of 76 % compared to laboratory findings (69.2 %). These findings demonstrate that we developed a simple and less energy intensive bench scale system for efficient high-solid saccharification. External supplementation of commercial β-glucosidase and hemicellulase ensured better hydrolysis and further increased the saccharification efficiency by 14.5 and 20 %, respectively. An attempt was also made to recover cellulolytic enzymes using ultrafiltration module and nearly 79–84 % of the cellulases and more than 90 % of the sugars were recovered from the saccharification mixture.     

The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understood. Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry, particularly when it comes to up-scaling of processes based on insoluble feed stocks.     

Process development of starch hydrolysis using mixing characteristics of Taylor vortices

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor–Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.     

Enzyme production by the mixed fungal culture with nano‐shear pretreated biomass and lignocellulose hydrolysis

Cellulase, xylanase, and β‐glucosidase production was studied on novel nano‐shear pretreated corn stover by the mixed fungi culture. The high shear force from a modified Tayor‐Couette nano‐shear mixing reactor efficiently disintegrated corn stover, resulting in a homogeneous watery mash with particles in much reduced size. Scanning electron microscope study showed visible mini‐pores on the fiber cell wall surface, which could improve the accessibility of the pretreated corn stover to microorganisms. Mixed fungal culture of Trichoderma reesei RUT‐C30 and Aspergillus niger produced enzymes with higher cellulolytic and xylanolytic activities on corn stover pretreated with nano‐shear mixing reactor, in comparison with other pretreatment methods, including acid and ammonia fiber explosion (AFEX) pretreatment. The hydrolytic potential of the whole fermentation broth from the mixed fungi was studied, and the possibility of applying the whole cell saccharification concept was also investigated to further reduce the cost of lignocellulose hydrolysis. Biotechnol. Bioeng. 2013; 110: 2123–2130. © 2013 Wiley Periodicals, Inc.     

Pretreatment and saccharification of rice hulls for the production of fermentable sugars

Hydrolytic conditions of rice hulls by acid and alkaline treatments before enzymatic saccharification were optimized in this study. Based on the results of single-factor experiments and an orthogonal array experiment, reaction time was found to be the most important factor for the acidic hydrolysis of rice hulls. Maximum yield of sugars from 1 g of rice hulls by acidic treatment under optimized conditions was 213.6 mg. The yield of lignin removal from acidic pretreated rice hulls by alkaline treatment increased with increase in reaction temperature and time. The amount of sugars obtained from 1 g of pretreated rice hulls by enzymatic saccharification was 307.7 mg, and the conversion rate of sugars from crude fibers in pretreated rice hulls was about 72%. Instrumental analyses with FTIR and SEM indicated that lignin in rice hulls was partially removed by alkaline treatment, and the structure of rice hulls became deformed and more fibers were exposed to cellulases after acidic treatment.     

Effects of the surfactant tween 80 on enzymatic hydrolysis of newspaper

The effects of Tween 80 on the enzymatic hydrolysis of newspaper were tested. By monitoring sugar production it was found that the surfactant (0.1%) increased the rate and extent of cellulose saccharification. Consequently, the rate of enzyme usage in the hydrolysis reactor was improved by 33%. In addition, in the presence of surfactant the recovery of enzymes was higher. Analysis of the enzyme solution showed that with Tween 80 present larger fractions of enzyme remained in solution throughout hydrolysis. Thus, it appears that the surfactant hindered the immobilization of the enzymes on the substrates by reducing the strength of adsorption.     

The pattern of cell wall deterioration in lignocellulose fibers throughout enzymatic cellulose hydrolysis

Cell wall deterioration throughout enzymatic hydrolysis of cellulosic biomass is greatly affected by the chemical composition and the ultrastructure of the fiber cell wall. The resulting pattern of cell wall deterioration will reveal information on cellulose activity throughout enzymatic hydrolysis. This study investigates the progression and morphological changes in lignocellulose fibers throughout enzymatic hydrolysis, using (transmission electron microscopy) TEM and field emission scanning electron microscopy (FE‐SEM). Softwood thermo‐mechanical pulp (STMP) and softwood bleached kraft pulp (SBKP), lignocellulose substrates containing almost all the original fiber composition, and with lignin and some hemicellulose removed, respectively, was compared for morphology changes throughout hydrolysis. The difference of conversion between STMP and SBKP after 48 h of enzymatic hydrolysis is 11 and 88%, respectively. TEM images revealed an even fiber cell wall cross section density, with uneven middle lamella coverage in STMP fibers. SKBP fibers exhibited some spaces between cell wall and lamella layers due to the removal of lignin and some hemicellulose. After 1 h hydrolysis in SBKP fibers, there were more changes in the fiber cross‐sectional area than after 10 h hydrolysis in STMP fibers. Cell wall degradation was uneven, and originated in accessible cellulose throughout the fiber cell wall. FE‐SEM images illustrated more morphology changes in SBKP fibers than STMP fibers. Enzymatic action of STMP fiber resulted in a smoother fiber surface, along with fiber peeling and the formation of ribbon‐disjunction layers. SBKP fibers exhibited structural changes such as fiber erosion, fiber cutting, and fiber splitting throughout enzymatic hydrolysis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Enzymatic hydrolysis of cellulose in a membrane bioreactor: assessment of operating conditions

The optimization of operating conditions for cellulose hydrolysis was systemically undertaken using an ultra-scaled down membrane bioreactor based on the parameter scanning ultrafiltration apparatus. The bioconversion of cellulose saccharification was carried out with freely suspended cellulase from Aspergillus niger as the biocatalyst. The polyethersulfone ultrafiltration membranes with a molecular weight cutoff of 10 kDa were used to construct the enzymatic membrane bioreactor, with the membrane showing a complete retaining of cellulase and cellobiase. The influence of solution pH, temperature, salt (NaCl) concentration, presence of cellobiase, cellulose-to-enzyme ratio and stirring speed on reducing sugar production was examined. The results showed that the addition of an appropriate amount of NaCl or cellobiase had a positive effect on reducing sugar formation. Under the identified optimal conditions, cellulose hydrolysis in the enzymatic membrane bioreactor was tested for a long period of time up to 75 h, and both enzymes and operation conditions demonstrated good stability. Also, the activation energy (E _a) of the enzymatic hydrolysis, with a value of 34.11 ± 1.03 kJ mol⁻¹, was estimated in this study. The operational and physicochemical conditions identified can help guide the design and operation of enzymatic membrane bioreactors at the industrial scale for cellulose hydrolysis.     

Particle concentration and yield stress of biomass slurries during enzymatic hydrolysis at high‐solids loadings

Effective and efficient breakdown of lignocellulosic biomass remains a primary barrier for its use as a feedstock for renewable transportation fuels. A more detailed understanding of the material properties of biomass slurries during conversion is needed to design cost‐effective conversion processes. A series of enzymatic saccharification experiments were performed with dilute acid pretreated corn stover at initial insoluble solids loadings of 20% by mass, during which the concentration of particulate solids and the rheological property yield stress (τ_y) of the slurries were measured. The saccharified stover liquefies to the point of being pourable (τ_y ≤ 10 Pa) at a total biomass conversion of about 40%, after roughly 2 days of saccharification for a moderate loading of enzyme. Mass balance and semi‐empirical relationships are developed to connect the progress of enzymatic hydrolysis with particle concentration and yield stress. The experimental data show good agreement with the proposed relationships. The predictive models developed here are based on established physical principles and should be applicable to the saccharification of other biomass systems. The concepts presented, especially the ability to predict yield stress from extent of conversion, will be helpful in the design and optimization of enzymatic hydrolysis processes that operate at high‐solids loadings. Biotechnol. Bioeng. 2009; 104: 290–300 © 2009 Wiley Periodicals, Inc.     

Kinetic analysis of bioconversion of cellulose in attrition bioreactor

The attrition bioreactor (ABR) combines wet ball milling and enzymatic hydrolysis in one process step. It was found that the ABR did not accelerate enzyme deacti-vation. Interfacial forces, not shear forces, caused the most deactivation. Elimination of the air-liquid interface by covering the reactor substantially increased enzyme stability. A simple exponential kinetic model was tested to predict the cellulose conversion in an ABR. Kinetic parameters were estimated from batch runs performed at various enzyme and substrate concentrations.     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

Enzymatic saccharification of solid residue of olive mill in a batch reactor

This paper describes the enzymatic hydrolysis of solid residue of olive mill (OMRS) in a batch reactor with the Trichoderma reesei enzyme. Before enzymatic saccharification, crude lignocellulosic material is submitted to alkaline pre-treatment with NaOH. Optimum conditions of the pre-treatment (temperature of T=100 degrees C and OMRS-NaOH concentration ratio of about R=20) were determined. The optimum enzymatic conditions determined were as follows: pH of about 5, temperature of T=50 degrees C and enzyme to mass substrate mass ratio E/S=0.1g enzyme (g OMRS)(-1). The maximum saccharification yield obtained at optimum experimental conditions was about 50%. The experimental results agree with Lineweaver Burk's formula for low substrate concentrations. At substrate concentrations greater than 40gdm(-3), inhibitory effects were encountered. The kinetic constants obtained for the batch reactor were K(m)=0.1gdm(-3)min(-1) and V(m)=800gdm(-3).     

Fiber fractionation to understand the effect of mechanical refining on fiber structure and resulting enzymatic digestibility of biomass

Mechanical refining results in fiber deconstruction and modifications that enhance enzyme accessibility to carbohydrates. Further understanding of the morphological changes occurring to biomass during mechanical refining and the impacts of these changes on enzymatic digestibility is necessary to maximize yields and reduce energy consumption. Although the degree of fiber length reduction relative to fibrillation/delamination can be impacted by manipulating refining variables, mechanical refining of any type (PFI, disk, and valley beater) typically results in both phenomena. Separating the two is not straightforward. In this study, fiber fractionation based on particle size performed after mechanical refining of high-lignin pulp was utilized to successfully elucidate the relative impact of fibrillation/delamination and fiber cutting phenomena during mechanical refining. Compositional analysis showed that fines contain significantly more lignin than larger size fractions. Enzymatic hydrolysis results indicated that within fractions of uniform fiber length, fibrillation/delamination due to mechanical refining increased enzymatic conversion by 20–30 percentage points. Changes in fiber length had little effect on digestibility for fibers longer than ~0.5 mm. However, the digestibility of the fines fractions was high for all levels of refining even with the high-lignin content.