Effect of Reaction Temperature on Properties of Torrefied Kenaf.

Torrefaction is a thermal treatment method used to achieve solid-phase biofuel. Raw biomass generally have low heating value and high moisture content; thus, these characteristics should be enhanced before using it as a fuel. In this study, herbaceous biomass kenaf was torrefied at 220, 260, 300, and 340 °C under nitrogen atmosphere for 30 min to investigate the effect of temperature on its properties. The properties of torrefied kenaf were classified into two groups: physical properties such as mass and energy yields, moisture content, and proximate analysis and chemical properties such as functional groups and chemical compositions of sugars and lignin. The mass and energy yield of torrefied kenaf decreased as the reaction temperature increased. In addition, an increase in carbon content and a rapid decrease in oxygen content were observed in torrefied kenaf, which indicated the degradation of compounds such as hemicellulose and cellulose. Elemental analysis, proximate analysis, thermal analysis, Fourier transform infrared spectroscopy, and chemical composition analysis were performed to further investigate the characteristics of torrefied kenaf.

Recycling of bottom ash derived from combustion of cattle manure and its adsorption behaviors for Cd(II), Cu(II), Pb(II), and Ni(II).

Bottom ash generated by the combustion of cattle manure (BA-CCM) was investigated as an adsorbent for the removal of heavy metals such as Cd(II), Cu(II), Pb(II), and Ni(II) from aqueous solutions. When cattle manure was used as fuel, the thermal efficiency of the boiler was 88.7%, and the CO and CO2 concentrations in the exhaust gas were 2.3 ppm and 12.1%, respectively. The percentage of remaining solids was 31 wt.% after combustion at 900 °C. X-ray fluorescence analyses showed that the elemental composition of the BA-CCM was mainly CaO (43.3%), SiO2 (15.8%), CO2 (13.0%), and P2O5 (10.3%). The kinetic adsorption of Cd(II), Cu(II), Pb(II), and Ni(II) by BA-CCM reached equilibrium after 12 h, and the pseudo-second-order model fitted the experimental data well. The maximum amount of Cd(II), Cu(II), Pb(II), and Ni(II) adsorbed by the bottom ash was 5.4, 72.6, 88.2, and 24.6 mg/g, respectively. The equilibrium adsorption of metals onto BA-CCM was well-described by the Freundlich model. Thermodynamic analysis showed that the adsorption onto the bottom ash was endothermic and that the Gibbs free energy decreased as the temperature increased. The presence of cations such as Na+, Ca2+, and Al3+ was found to reduce the amount of metals adsorbed onto the BA-CCM, and Cd(II) adsorption was found to be more dependent on ionic strength than adsorption of Cu(II), Pb(II), and Ni(II). This study demonstrates the feasibility of producing heat energy by burning cattle manure and removing heavy metals from aqueous solutions using the generated bottom ash as an adsorbent.

Fractionation and characterization of lignins from Miscanthus via organosolv and soda pulping for biorefinery applications.

The structural complexity of lignins necessitates characterization and isolation methodologies for assessing their appropriateness for thermo-chemical systems and material applications. Lignins prepared via two pulping methods (organosolv and soda) were comprehensively investigated by analyzing the properties, including lignin purity, yield, and thermal and chemical properties. The extracted organosolv lignin has high purity (93.13-98.12%), however, the purity of soda lignin was relatively low (87.58-89.61%). Organosolv lignin produced the highest heating value of 26.79-26.95 MJ kg-1, with a fixed carbon content of 39.47-41.06 wt%, high purity, and low ash content, making it suitable for biofuel applications. The content of total phenolic OH groups was higher for the organosolv lignins; however, for the phenolic OH groups, the 4-vinylphenol content was significantly higher in the soda lignins, and increased with increasing NaOH concentration. Overall, the thermal and chemical properties related to the lignin structure changed with the fractionation method and solvent concentration, which in turn influences the design of lignin valorization strategies for prospective depolymerization and material applications.

Delignification of Pinecone and Extraction of Formic Acid in the Hydrolysate Produced by Alkaline Fractionation.

The objectives of our research are to investigate the concept of delignification from pinecone through alkaline fractionation and then extraction of formic acid from the hydrolysate through esterification using ethanol. The pinecone is considered a promising material because of its relatively higher lignin content (35.80%) than other lignocellulosic biomass. The recovery yield of acid insoluble lignin (AIL) reached its maximum value of 79.20% at 8% NaOH, and the concentration of formic acid in the hydrolysate had its highest value under the same conditions. Moreover, the glucan content in fractionated solid remained high. The hydrolysate was subjected to esterification with ethanol under various reaction conditions for formic acid extraction, with solvent mixing ratio range: 1:1-1:4 v/v, reaction temperature range: 30-45 °C, and reaction time range: 60-100 min. Subsequently, the ethanol mixture (ethanol and ethyl formate) was recovered through distillation. The formic acid was extracted with more than 85% at mixing ratio of 1:2 and 45 °C for all reaction times. Furthermore, salt compounds composed mainly of Na and S were recovered because of its properties not soluble in ethanol solution.

Acetic Acid Removal from Pre-Pulping Wood Extract with Recovery and Recycling of Extraction Solvents.

Pre-pulping extraction is a means of deriving a hemicellulose-rich process stream from the front end of a kraft pulp mill. When the extraction is carried out using green liquor, pulp quality and quantity can be retained while still releasing hemicelluloses and acetic acid (HAc) for recovery as bioprocessing feedstock or chemical products. The HAc that is present in the wood extraction is inhibitory to microorganisms and can hinder fermentation. HAc is also a commodity chemical that may provide sufficient value to justify recovery and purification. In this study, a liquid-liquid extraction (LLE) method is applied to extract HAc from a green liquor pre-pulping hardwood extract (GLE). The HAc removal is carried out after acid hydrolysis and prior to fermentation. Two organic solutions: trioctylphosphine oxide (TOPO) diluted in undecane and trioctylamine (TOA) diluted in octanol were tested for their abilities to extract HAc from GLE and to be recycled back through the process. GLE was contacted with the organic solvents, phase separated by centrifugation, and the organic phase was then distilled to recover the acetic acid. The solvent was then recycled back for a subsequent extraction of fresh GLE. It was found that TOA was a superior extractant, but failed to easily release its HAc through distillation. It also quickly became contaminated with other compounds in the wood extracts and lost its extraction efficiency after only a few recycles. The TOPO solvent did release its HAc through distillation but also lost extraction capacity with recycling. Back extraction of the TOPO solvent with sodium hydroxide solution restored the performance of the TOPO solvent.

High-solid enzymatic hydrolysis and fermentation of solka floc into ethanol.

To lower the cost of ethanol distillation of fermentation broths, a high initial glucose concentration is desired. However, an increase in the substrate concentration typically reduces the ethanol yield because of insufficient mass and heat transfer. In addition, different operating temperatures are required to optimize the enzymatic hydrolysis (50 degrees C) and fermentation (30 degrees C). Thus, to overcome these incompatible temperatures, saccharification followed by fermentation (SFF) was employed with relatively high solid concentrations (10% to 20%) using a portion loading method. In this study, glucose and ethanol were produced from Solka Floc, which was first digested by enzymes at 50 degrees for 48 h, followed by fermentation. In this process, commercial enzymes were used in combination with a recombinant strain of Zymomonas mobilis (39679:pZB4L). The effects of the substrate concentration (10% to 20%, w/v) and reactor configuration were also investigated. In the first step, the enzyme reaction was achieved using 20 FPU/g cellulose at 50 degrees C for 96 h. The fermentation was then performed at 30 degrees C for 96 h. The enzymatic digestibility was 50.7%, 38.4%, and 29.4% after 96 h with a baffled Rushton impeller and initial solid concentration of 10%, 15%, and 20% (w/v), respectively, which was significantly higher than that obtained with a baffled marine impeller. The highest ethanol yield of 83.6%, 73.4%, and 21.8%, based on the theoretical amount of glucose, was obtained with a substrate concentration of 10%, 15%, and 20%, respectively, which also corresponded to 80.5%, 68.6%, and 19.1%, based on the theoretical amount of the cell biomass and soluble glucose present after 48 h of SFF.

Optimization of the process for biodiesel production using a mixture of immobilized Rhizopus oryzae and Candida rugosa lipases.

In this study, the enzymatic process for biodiesel production was optimized using a mixture of immobilized Rhizopus oryzae and Candida rugosa lipases. The optimal temperature and agitation speed for biodiesel production were 45oC and 300 rpm, respectively. The optimal ratio of R. oryzae and C. rugosa lipases in the mixture was 3:1 (w:w). When 3mmol of methanol was the initial reaction medium and 3mmol of methanol was added every 1.5 h during biodiesel production, biodiesel conversion was over 98% at 4 h. In addition, when the immobilized lipase mixture was reused, biodiesel conversion exceeded 80% after 5 reuses.

Dilute sulfuric acid fractionation of Korean food waste for ethanol and lactic acid production by yeast.

Fermentation of food waste biomass can be used to produce biochemicals such as lactic acid and ethanol in a cost-effective manner. Korean food waste (KFW) dewatered by a screw press contains 23.1% glucan on a dry basis and is a potential raw material for the production of ethanol and lactic acid through fermentation. This study was conducted to optimize the dilute acid fractionation conditions for KFW fermentation with respect to the H2SO4 concentration (0-0.8% w/v), temperature (130-190 °C), and residence time (1-128 min) using response surface methodology. Dilute sulfuric acid fractionation was carried out using a 30-mL stainless steel reactor under conditions, and then the dilute acid fractionation was scaled-up in 1-L and 7-L stainless steel reactors under the optimal conditions. The hydrolysate was concentrated, liquid-liquid extracted and neutralized for lactic acid and ethanol production. The highest concentration of glucose obtained from the KFW was 26.4 g/L using fractionation with 0.37% w/v H2SO4 at 156 °C for 123.6 min. Using recombinant Saccharomyces cerevisiae containing a codon-optimized lactate dehydrogenase, the yield of lactic acid and ethanol was 77% of the theoretical yield for 17.4 g/L of fermentable sugar at pH 5.5. Additionally, the yield of ethanol produced by Issatchenkia orientalis was 89% of the theoretical yield for 25 g/L of fermentable sugar at pH 3.

Comparison of hemicellulose extracts from two pulping woodchips with green liquor followed by scale-up pre-hemicellulose extraction.

This study investigates the effects of different pre-pulping extraction conditions on two types of raw material, hardwood oak chips and softwood pine chips. The chips were air-dried, and 2-g samples were extracted in a Dionex ASE-200 with alkaline solutions of a partially recovered form of kraft pulping liquor called green liquor (GL). The GL was applied at different alkali charges of 0-5 % on dry wood weight. The extractions were performed at various H-factors from 500 to 1100 with temperatures ranging from 170 to 190 °C. As the alkali charge decreased, the amount of monosaccharides in the extracts increased while the remaining solids decreased. The highest xylose + mannose + galactose (xmg) concentrations (12.73 and 11.12 g/L for extracts derived from hardwood and softwood, respectively) were detected with a hot water extraction (GL 0 %) at 180 °C and an H-factor of 900. However, low amounts of remaining solids were obtained under these conditions (65.77% and 74.42%, for hard- and soft woods, respectively). We also performed an extraction that was scaled-up in a 1-L Parr reactor with 80 g of woodchips under pre-optimized condition through ASE-200. In this condition, the xmg and acetic acid concentrations of the hardwood extracts were 9.74 and 9.94 g/L, respectively, whereas their concentrations in the softwood extracts were 3.59 and 3.76 g/L, respectively.

Acetic acid-assisted hydrothermal fractionation of empty fruit bunches for high hemicellulosic sugar recovery with low byproducts.

Xylose, mannose, and galactose (xmg) recovery from empty fruit bunches using acetic acid-assisted hydrothermal (AAH) fractionation method was investigated. Acetic acid has been demonstrated to be effective in xmg recovery in comparison with the liquid hot-water (LHW) fractionation. The maximum xmg recovery yield (50.7 %) from the empty fruit bunch (EFB) was obtained using AAH fractionation at optimum conditions (6.9 wt.% acetic acid at 170 °C and for 18 min); whereas, only 16.2 % of xmg recovery was obtained from the LHW fractionation at the same reaction conditions (170 °C and 18 min). Releasing out the glucose from EFB was kept at low level (<1.0 %) through all tested conditions and consequently negligible 5-HMF and formic acid were analyzed in the hydrolyzate. The production of furfural was also resulted with extremely low level (1.0 g/L).

Enhancement of ß-Glucosidase Activity from a Brown Rot Fungus Fomitopsis pinicola KCTC 6208 by Medium Optimization.

ß-Glucosidase, which hydrolyzes cellobiose into two glucoses, plays an important role in the process of saccharification of the lignocellulosic biomass. In this study, we optimized the activity of ß-glucosidase of brown-rot fungus Fomitopsis pinicola KCTC 6208 using the response surface methodology (RSM) with various concentrations of glucose, yeast extract and ascorbic acid, which are the most significant nutrients for activity of ß-glucosidase. The highest activity of ß-glucosidase was achieved 3.02% of glucose, 4.35% of yeast extract, and 7.41% ascorbic acid where ascorbic acid was most effective. The maximum activity of ß-glucosidase predicted by the RSM was 15.34 U/mg, which was similar to the experimental value 14.90 U/mg at the 16th day of incubation. This optimized activity of ß-glucosidase was 23.6 times higher than the preliminary activity value, 0.63 U/mg, and was also much higher than previous values reported in other fungi strains. Therefore, a simplified medium supplemented with a cheap vitamin source, such as ascorbic acid, could be a cost effective mean of increasing ß-glucosidase activity.

Effect of sulfuric and phosphoric acid pretreatments on enzymatic hydrolysis of corn stover.

The pretreatment of corn stover with H2SO4 and H3PO4 was investigated. Pretreatments were carried out from 30 to 120 min in a batch reactor at 121 degrees C, with acid concentrations ranging from 0 to 2% (w/v) at a solid concentration of 5% (w/v). Pretreated corn stover was washed with distilled water until the filtrate was adjusted to pH 7.0, followed by surfactant swelling of the cellulosic fraction in a 0-10% (w/v) solution of Tween-80 at room temperature for 12 h. The dilute acid treatment proved to be a very effective method in terms of hemicellulose recovery and cellulose digestibility. Hemicellulose recovery was 62-90%, and enzymatic digestibility of the cellulose that remained in the solid was >80% with 2% (w/v) acid. In all cases studied, the performance of H2SO4 pretreatment (hemicellulose recovery and cellulose digestibility) was significantly better than obtained with H3PO4. Enzymatic hydrolysis was more effective using surfactant than without it, producing 10-20% more sugar. Furthermore, digestibility was investigated as a function of hemicellulose removal. It was found that digestibility was more directly related to hemicellulose removal than to delignification.

The influence of screw configuration on the pretreatment performance of a continuous twin screw-driven reactor (CTSR).

A combination of a continuous twin screw-driven reactor (CTSR) and a dilute acid pretreatment was used for the pretreatment of biomass with a high cellulose content and high monomeric xylose hydrolyzate. With the newly modified CTSR screw configuration (Config. 3), the influences of the screw rotational speed (30-60 rpm), of the pretreatment conditions such as acid concentration (1-5%) and reaction temperature (160-175 °C) at the operating condition of biomass feeding rate (1.0 g/min) and acid feeding rate (13.4 mL/min) on the pretreatment performance were investigated. The cellulose content in the pretreated rape straw was 67.1% at the following optimal conditions: barrel temperature of 165 °C, acid concentration of 3.0% (w/v), and screw rotational speed of 30 rpm. According to the three screw configurations, the glucose yields from enzymatic hydrolysis were 70.1%, 72.9%, and 78.7% for screw Configs. 1, 2, and 3, respectively.

Chemicals effect on the enzymatic digestibility of rape straw over the thermo-mechanical pretreatment using a continuous twin screw-driven reactor (CTSR).

Rape straw pretreated by a continuous twin screw-driven reactor (CTSR) with hot water presented a distinctive particle-size distribution profile as a function of the operating temperature. The relative amount of finer particle size dramatically increased as the ratio of solid to liquid was increased. Size reduction through physical CTSR process effectively promoted the enzymatic hydrolysis of pretreated rape straw. Meanwhile, the crystallinity of the physically pretreated straw was not a greater factor affecting the enzyme digestibility. The glucose conversion from the enzymatic hydrolysis of the straw pretreated by CTSR with hot water was maximized at 52%. Using the chemicals as catalyst have affected considerably for increasing the digestibility at same condition with hot water pretreatment. The enzymatic digestibilities of the straw pretreated by CTSR with sodium hydroxide and sulfuric acid were 60% and 77%, respectively.

Effect of pretreatment severity on accumulation of major degradation products from dilute acid pretreated corn stover and subsequent inhibition of enzymatic hydrolysis of cellulose.

The concept of reaction severity, which combines residence time and temperature, is often used in the pulp and paper and biorefining industries. The influence of corn stover pretreatment severity on yield of sugar and major degradation products and subsequent effects on enzymatic cellulose hydrolysis was investigated. The pretreatment residence time and temperature, combined into the severity factor (Log R(o)), were varied with constant acid concentration. With increasing severity, increasing concentrations of furfural and 5-hydroxymethylfurfural (5-HMF) coincided with decreasing yields of oligosaccharides. With further increase in severity factor, the concentrations of furans decreased, while the formation of formic acid and lactic acid increased. For example, from severity 3.87 to 4.32, xylose decreased from 6.39 to 5.26 mg/mL, while furfural increased from 1.04 to 1.33 mg/mL; as the severity was further increased to 4.42, furfural diminished to 1.23 mg/mL as formate rose from 0.62 to 1.83 mg/mL. The effects of dilute acid hydrolyzate, acetic acid, and lignin, in particular, on enzymatic hydrolysis were investigated with a rapid microassay method. The microplate method gave considerable time and cost savings compared to the traditional assay protocol, and it is applicable to a broad range of lignocellulosic substrates.

Mass balance on green liquor pre-pulping extraction of northeast mixed hardwood.

A forest biorefinery configuration employing a hemicellulose pre-pulping extraction is being investigated that will retain pulp yields, reduce the organic and inorganic load for liquor recovery, and create a hemicellulose feed stream for the generation of biofuels and biomaterials. Current efforts are focused on developing extract production and conditioning processes that will result in fermentable sugars suitable for conversion to fuel alcohols or organic acid chemical products. As efforts move the process closer to commercial demonstration, it is apparent that a high level of confidence is needed in the analysis of the partitioning of fresh wood into its extracted wood and liquid extract fractions. Of particular interest is the partitioning of the carbohydrates, as these constitute the feedstock for bioconversion to fuels and chemicals. The extraction method employed utilizes green liquor derived from the kraft pulping process for pretreatment of the woodchips. To enable analysis, green liquor extraction was followed by 4% sulfuric acid hydrolysis to complete hydrolysis of the oligomers that were still present. High performance anion-exchange chromatography (HPAEC-PAD) and high performance liquid chromatography (HPLC) methods were used to analyze the carbohydrates in northern hardwood and its extract fractions. The Bio-Rad Aminex HPX-87H column did not separate mannose, xylose, and galactose, but the area of the collective peak corresponds well to the sum of these components as measured by HPAEC. In addition to sugars, standard methods were employed for quantification of the individual components (e.g., lignin, ash, nitrogen, carbon, extractives, uronic and acetic acid). The analytical mass balance closure was 102.2% and 103.6% for raw wood, 99.3% and 102.3% for extracted wood, and 94.7% and 95.6% for hemicellulose extract from the HPAEC and HPLC, respectively. The extraction mass balance was 96.9% and 98.2% for HPAEC and HPLC, respectively. The data generated by this analysis are important to further design work in commercializing the pulp and biorefinery processes.

Evaluation of enzyme mixtures in releasing fermentable sugars from pre-pulping extracts of mixed northeast hardwoods.

One near-term option to developing a forest product biorefinery is to derive pre-pulping extract from incoming wood chips before the main pulping step. The release of monomer sugars from a xylan-rich extract, creating a fermentable substrate is a prerequisite for utilization of pre-pulping extract for production of ethanol or other value-added products. This study examined the individual and mixture efficiencies of two hemicellulolytic microbial enzymes and two xylanase preparations in catalyzing degradation of green liquor (GL) and hot water (HW) pre-pulping extracts. The effects of four commercial enzyme preparations were determined by assessing yields of xylose + galactose + mannose (xmg) obtained under different reaction conditions. Of the individual enzyme preparations tested, a sample NS 50012 was superior to the other enzyme preparations in releasing xmg under conditions optimized for separate hydrolysis and fermentation and for simultaneous saccharification and fermentation. In comparison to pre-pulping extracts treated with HW, extract treated with GL was found to inhibit the action of all tested enzymes. This inhibition may be related to higher salt and lignin phenol in the GL extract. On both types of extracts, the mixture constituted by NS 50012 and NS 50030 provided the highest yield of hemicellulose conversion at 55 degrees C and pH 5.5. The generated digestibility thus signified that the synergistic effectiveness in xylan + galactan + mannan (XMG) hydrolysis between NS 50012 (from Aspergillus aculeatus) and NS 50030 (from Aspergillus oryzae) is the result of an interaction mechanism involving different XMG-degrading enzyme activities in the two enzyme preparations.

Optimizing dilute-acid pretreatment of rapeseed straw for extraction of hemicellulose.

Biological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars prior to fermentation. Hydrolysis can be performed enzymatically or with mineral acids. In this study, dilute sulfuric acid was used as a catalyst for the pretreatment of rapeseed straw. The purpose of this study is to optimize the pretreatment process in a 15-mL bomb tube reactor and investigate the effects of the acid concentration, temperature, and reaction time. These parameters influence hemicellulose removal and production of sugars (xylose, glucose, and arabinose) in the hydrolyzate as well as the formation of by-products (furfural, 5-hydroxymethylfurfural, and acetic acid). Statistical analysis was based on a model composition corresponding to a 3(3) orthogonal factorial design and employed the response surface methodology to optimize the pretreatment conditions, aiming to attain maximum xylan, mannan, and galactan (XMG) extraction from hemicellulose of rapeseed straw. The obtained optimum conditions were: H2SO4 concentration of 1.76% and temperature of 152.6 degrees C with a reaction time of 21 min. Under these optimal conditions, 85.5% of the total sugar was recovered after acid hydrolysis (78.9% XMG and 6.6% glucan). The hydrolyzate contained 1.60 g/L glucose, 0.61 g/L arabinose, 10.49 g/L xylose, mannose, and galactose, 0.39 g/L cellobiose, 0.94 g/L fructose, 0.02 g/L 1,6-anhydro-glucose, 1.17 g/L formic acid, 2.94 g/L acetic acid, 0.04 g/L levulinic acid, 0.04 g/L 5-hydroxymethylfurfural, and 0.98 g/L furfural.

Acid hydrolysis of hemicellulose in green liquor pre-pulping extract of mixed northern hardwoods.

Forest biomass is a promising resource for future biofuels and bioproducts. Pre-pulping extraction of hemicellulose by alkaline (Green Liquor) pretreatment produces a neutral-pH extract containing hemicellulose-derived oligomers. A near-term option for use of this extract is to hydrolyze the oligomers to fermentable monomer sugars. Chips of mixed northern hardwoods were cooked in a rocking digester at 160 degrees C for 110 min in Green Liquor at a concentration of 3% Na2O equivalent salts on dry wood. The mass of wood extracted into the Green Liquor extract was approximately 11.4% of the debarked wood mass, which resulted in a dilute solution of oligomeric hemicelluloses sugars. The concentration of the extract was increased through partial evaporation prior to hydrolysis. Dilute sulfuric acid hydrolysis was applied at conditions ranging from 100 to 160 degrees C, 2% to 6% (w/v) H2SO4, and 2- to 258-min residence time. The maximum fermentable sugar concentration achieved from evaporated extract was 10.7 g/L, representing 90.7% of the maximum possible yield. Application of the biomass pretreatment severity function to the hydrolysis results proved to offer a relatively poor prediction of temperature and reaction time interaction. The combined severity function, which incorporates reaction time, temperature, and acid concentration, did prove to provide a useful means of trading off the combined effects of these three variables on total sugar yields.

A comparison of simple rheological parameters and simulation data for Zymomonas mobilis fermentation broths with high substrate loading in a 3-L bioreactor.

Traditionally, as much as 80% or more of an ethanol fermentation broth is water that must be removed. This mixture is not only costly to separate but also produces a large aqueous stream that must then be disposed of or recycled. Integrative approaches to water reduction include increasing the biomass concentration during fermentation. In this paper, experimental results are presented for the rheological behavior of high-solids enzymatic cellulose hydrolysis and ethanol fermentation for biomass conversion using Solka Floc as the model feedstock. The experimental determination of the viscosity, shear stress, and shear rate relationships of the 10 to 20% slurry concentrations with constant enzyme concentrations are performed with a variable speed rotational viscometer (2.0 to 200 rpm) at 40 degrees C. The viscosities of enzymatic suspension observed were in range of 0.0418 to 0.0144, 0.233 to 0.0348, and 0.292 to 0.0447 Pa s for shear rates up to 100 reciprocal seconds at 10, 15, and 20% initial solids (w/v), respectively. Computational fluid dynamics analysis of bioreactor mixing demonstrates the change in bioreactor mixing with increasing biomass concentration. The portion-loading method is shown to be effective for processing high-solids slurries.