Effects of dilute-acid pretreatment conditions on filtration performance of corn stover hydrolyzate.

The reaction conditions used during dilute-acid pretreatment of lignocellulosic biomass control the carbohydrate digestion yield and also hydrolyzate properties. Depending on the conversion route of interest, solid-liquid separation (SLS) may be required to split the hemicellulose-rich liquor from the cellulose-rich insoluble solids, and slurry properties are important for SLS. Corn stover was pretreated at different reaction conditions and the slurries were assessed for conversion yield and filtration performance. Increasing pretreatment temperature reduced the solids mean particle size and resulted in slower slurry filtration rates when vacuum filtered or pressure filtered. Corn stover pretreated at 165°C for 10min and with 1% H2SO4 exhibited the highest xylose yield and best filtration performance with a no-wash filtration rate of 80kg/hm2 and cake permeability of 15x10-15.

Assessing pretreatment reactor scaling through empirical analysis.

BACKGROUND: Pretreatment is a critical step in the biochemical conversion of lignocellulosic biomass to fuels and chemicals. Due to the complexity of the physicochemical transformations involved, predictively scaling up technology from bench- to pilot-scale is difficult. This study examines how pretreatment effectiveness under nominally similar reaction conditions is influenced by pretreatment reactor design and scale using four different pretreatment reaction systems ranging from a 3 g batch reactor to a 10 dry-ton/days continuous reactor. The reactor systems examined were an automated solvent extractor (ASE), steam explosion reactor (SER), ZipperClave®Reactor (ZCR), and large continuous horizontal screw reactor (LHR). To our knowledge, this is the first such study performed on pretreatment reactors across a range of reaction conditions and at different reactor scales. RESULTS: The comparative pretreatment performance results obtained for each reactor system were used to develop response surface models for total xylose yield after pretreatment and total sugar yield after pretreatment followed by enzymatic hydrolysis. Near- and very-near-optimal regions were defined as the set of conditions that the model identified as producing yields within one and two standard deviations of the optimum yield. Optimal conditions identified in the smallest scale system (the ASE) were within the near-optimal region of the largest scale reactor system evaluated. The maximum total sugar yields for the ASE and LHR were [Formula: see text], while [Formula: see text] was the optimum observed in the ZipperClave. CONCLUSIONS: The optimum condition identified using the automated and less costly to operate ASE system was within the very-near-optimal space for the total xylose yield of both the ZCR and the LHR, and was within the near-optimal space for total sugar yield for the LHR. This indicates that the ASE is a good tool for cost effectively finding near-optimal conditions for operating pilot-scale systems. Additionally, using a severity factor approach to optimization was found to be inadequate compared to a multivariate optimization method. Finally, the ASE and the LHR were able to enable significantly higher total sugar yields after enzymatic hydrolysis relative to the ZCR, despite having similar optimal conditions and total xylose yields. This underscores the importance of mechanical disruption during pretreatment to improvement of enzymatic digestibility.

Assessing the protein concentration in commercial enzyme preparations.

Although a poor indicator of how a cellulase preparation will perform on biomass, the filter paper unit (FPU) still finds wide use in the literature as an apparent measure of performance efficacy. In actuality, the assessment of commercial enzyme preparation performance in terms of biomass conversion or solubilization of insoluble polysaccharides is largely dependent on the substrate composition, which cannot be easily standardized. Commercial cellulase preparations are evaluated based upon their performance or specific activity. The ability to compare commercial enzyme preparation efficacy across a wide variety of different preparations requires defining the amount of enzyme protein required in milligrams per gram of cellulose to achieve a targeted level of cellulose hydrolysis in a specified timeframe. Since biomass substrates are highly variable, reproducible and accurate protein determination is as important as performance testing to be able to rank order the effectiveness of diverse preparations. This chapter describes a protocol that overcomes many of the difficulties encountered with determining the protein concentration in commercial cellulase preparations.

Comparative performance of precommercial cellulases hydrolyzing pretreated corn stover.

BACKGROUND: Cellulases and related hydrolytic enzymes represent a key cost factor for biochemical conversion of cellulosic biomass feedstocks to sugars for biofuels and chemicals production. The US Department of Energy (DOE) is cost sharing projects to decrease the cost of enzymes for biomass saccharification. The performance of benchmark cellulase preparations produced by Danisco, DSM, Novozymes and Verenium to convert pretreated corn stover (PCS) cellulose to glucose was evaluated under common experimental conditions and is reported here in a non-attributed manner. RESULTS: Two hydrolysis modes were examined, enzymatic hydrolysis (EH) of PCS whole slurry or washed PCS solids at pH 5 and 50°C, and simultaneous saccharification and fermentation (SSF) of washed PCS solids at pH 5 and 38°C. Enzymes were dosed on a total protein mass basis, with protein quantified using both the bicinchoninic acid (BCA) assay and the Bradford assay. Substantial differences were observed in absolute cellulose to glucose conversion performance levels under the conditions tested. Higher cellulose conversion yields were obtained using washed solids compared to whole slurry, and estimated enzyme protein dosages required to achieve a particular cellulose conversion to glucose yield were extremely dependent on the protein assay used. All four enzyme systems achieved glucose yields of 90% of theoretical or higher in SSF mode. Glucose yields were reduced in EH mode, with all enzymes achieving glucose yields of at least 85% of theoretical on washed PCS solids and 75% in PCS whole slurry. One of the enzyme systems ('enzyme B') exhibited the best overall performance. However in attaining high conversion yields at lower total enzyme protein loadings, the relative and rank ordered performance of the enzyme systems varied significantly depending upon which hydrolysis mode and protein assay were used as the basis for comparison. CONCLUSIONS: This study provides extensive information about the performance of four precommercial cellulase preparations. Though test conditions were not necessarily optimal for some of the enzymes, all were able to effectively saccharify PCS cellulose. Large differences in the estimated enzyme dosage requirements depending on the assay used to measure protein concentration highlight the need for better consensus methods to quantify enzyme protein.

Calculating sugar yields in high solids hydrolysis of biomass.

Calculation of true sugar yields in high solids enzymatic hydrolysis of biomass is challenging due to the varying liquid density and liquid volume resulting from solid solubilization. Ignoring these changes in yield calculations can lead to significant errors. In this paper, a mathematical method was developed for the estimation of liquid volume change and thereafter the sugar yield. The information needed in the calculations include the compositions of the substrate, initial solids loading, initial liquid density, and sugar concentrations before and after hydrolysis. All of these variables are measurable with conventional laboratory procedures. This method was validated experimentally for enzymatic hydrolysis of dilute sulfuric acid pretreated corn stover at solid loadings up to 23% (w/w). The maximum relative error of predicted glucose yield from the true value was less than 4%. Compared to other methods reported in the literature, this method is relatively easy to use and provides good accuracy.

Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility.

Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF-pretreated corn stover but reached only approximately 60% for the DA-pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were measured for the first time. The COSLIF-pretreated corn stover had a CAC of 11.57 m(2)/g, nearly twice that of the DA-pretreated biomass (5.89 m(2)/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA- and COSLIF-pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility.

Model-based fed-batch for high-solids enzymatic cellulose hydrolysis.

While many kinetic models have been developed for the enzymatic hydrolysis of cellulose, few have been extensively applied for process design, optimization, or control. High-solids operation of the enzymatic hydrolysis of lignocellulose is motivated by both its operation decreasing capital costs and increasing product concentration and hence separation costs. This work utilizes both insights obtained from experimental work and kinetic modeling to develop an optimization strategy for cellulose saccharification at insoluble solids levels greater than 15% (w/w), where mixing in stirred tank reactors (STRs) becomes problematic. A previously developed model for batch enzymatic hydrolysis of cellulose was modified to consider the effects of feeding in the context of fed-batch operation. By solving the set of model differential equations, a feeding profile was developed to maintain the insoluble solids concentration at a constant or manageable level throughout the course of the reaction. Using this approach, a stream of relatively concentrated solids (and cellulase enzymes) can be used to increase the final sugar concentration within the reactor without requiring the high initial levels of insoluble solids that would be required if the operation were performed in batch mode. Experimental application in bench-scale STRs using a feed stream of dilute acid-pretreated corn stover solids and cellulase enzymes resulted in similar cellulose conversion profiles to those achieved in batch shake-flask reactors where temperature control issues are mitigated. Final cellulose conversions reached approximately 80% of theoretical for fed-batch STRs fed to reach a cumulative solids level of 25% (w/w) initial insoluble solids.

Rheology of corn stover slurries at high solids concentrations--effects of saccharification and particle size.

The rheological characteristics of untreated and dilute acid pretreated corn stover (CS) slurries at high solids concentrations were studied under continuous shear using plate-plate type measurements. Slurry rheological behavior was examined as a function of insoluble solids concentration (10-40%), extent of pretreatment (0-75% removal of xylan) and particle size (-20 and -80 mesh). Results show that CS slurries exhibit shear-thinning behavior describable using a Casson model. Further, results demonstrate that the apparent viscosity and yield stress increase with increasing solids concentration (which corresponds to a decrease in free water). Dilute acid pretreatment leads to lower viscosity and yield stresses at equivalent solids concentrations, as does smaller particle size. Taken together, these findings are consistent with the hypothesis that the availability of free water in the slurry plays a significant role in determining its rheological behavior. In particular, as the free water content of the slurry decreases, e.g., with increasing solids concentration, the greater interaction among particles likely increases the apparent viscosity and yield stress properties of the slurry. The results also suggest that the availability of free water, and thereby slurry rheological properties, depend on the chemical composition of the corn stover as well as its physical characteristics such as particle size and porosity. Hydrophilic polymers within the cell wall, such as xylan or pectin, or larger pores within bigger particles, facilitate sequestration of water in the solid phase resulting in decreased availability of free water. Thus, dilute acid pretreated slurries, which contain smaller size particles having significantly lower xylan content than slurries of untreated milled stover, exhibit much lower viscosities and yield stresses than untreated slurries containing large particles at similar solid concentrations.

Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose.

The rates and extents of enzymatic cellulose hydrolysis of dilute acid pretreated corn stover (PCS) decline with increasing slurry concentration. However, mass transfer limitations are not apparent until insoluble solids concentrations approach 20% w/w, indicating that inhibition of enzyme hydrolysis at lower solids concentrations is primarily due to soluble components. Consequently, the inhibitory effects of pH-adjusted pretreatment liquor on the enzymatic hydrolysis of PCS were investigated. A response surface methodology (RSM) was applied to empirically model how hydrolysis performance varied as a function of enzyme loading (12-40 mg protein/g cellulose) and insoluble solids concentration (5-13%) in full-slurry hydrolyzates. Factorial design and analysis of variance (ANOVA) were also used to assess the contribution of the major classes of soluble components (acetic acid, phenolics, furans, sugars) to total inhibition. High sugar concentrations (130 g/L total initial background sugars) were shown to be the primary cause of performance inhibition, with acetic acid (15 g/L) only slightly inhibiting enzymatic hydrolysis and phenolic compounds (9 g/L total including vanillin, syringaldehyde, and 4-hydroxycinnamic acid) and furans (8 g/L total of furfural and hydroxymethylfurfural, HMF) with only a minor effect on reaction kinetics. It was also demonstrated that this enzyme inhibition in high-solids PCS slurries can be approximated using a synthetic hydrolyzate composed of pure sugars supplemented with a mixture of acetic acid, furans, and phenolic compounds, which indicates that generally all of the reaction rate-determining soluble compounds for this system can be approximated synthetically.

How biotech can transform biofuels.

For cellulosic ethanol to become a reality, biotechnological solutions should focus on optimizing the conversion of biomass to sugars.

Methodological analysis for determination of enzymatic digestibility of cellulosic materials.

Accurate measurement of enzymatic cellulose digestibility (X) is important in evaluating the efficiency of lignocellulose pretreatment technologies, assessing the performance of reconstituted cellulase mixtures, and conducting economic analysis for biorefinery processes. We analyzed the effect of sugars contained in enzymes solutions, usually added as a preservative, and random measurement errors on the accuracy of X calculated by various methods. The analysis suggests that exogenous sugars at levels measured in several commercial enzyme preparations significantly bias the results and that this error should be minimized by accounting for these sugars in the calculation of X. Additionally, a method of calculating X equating the ratio of the soluble glucose equivalent in the liquid phase after hydrolysis to the sum of the soluble glucose equivalent in the liquid phase and the insoluble glucose equivalent in the residual solid after hydrolysis was found to be the most accurate, particularly at high conversion levels (>ca. 50%).

Catalyst transport in corn stover internodes: elucidating transport mechanisms using Direct Blue-I.

The transport of catalysts (chemicals and enzymes) within plant biomass is believed to be a major bottleneck during thermochemical pretreatment and enzymatic conversion of lignocellulose. Subjecting biomass to size reduction and mechanical homogenization can reduce catalyst transport limitations; however, such processing adds complexity and cost to the overall process. Using high-resolution light microscopy, we have monitored the transport of an aqueous solution of Direct Blue-I (DB-I) dye through intact corn internodes under a variety of impregnation conditions. DB-I is a hydrophilic anionic dye with affinity for cellulose. This model system has enabled us to visualize likely barriers and mechanisms of catalyst transport in corn stems. Microscopic images were compared with calculated degrees of saturation (i.e., volume fraction of internode void space occupied by dye solution) to correlate impregnation strategies with dye distribution and transport mechanisms. Results show the waxy rind exterior and air trapped within individual cells to be the major barriers to dye transport, whereas the vascular bundles, apoplastic continuum (i.e., the intercellular void space at cell junctions), and fissures formed during the drying process provided the most utilized pathways for transport. Although representing only 20-30% of the internode volume, complete saturation of the apoplast and vascular bundles by fluid allowed dye contact with a majority of the cells in the internode interior.

Measurement and analysis of intracellular ATP levels in metabolically engineered Zymomonas mobilis fermenting glucose and xylose mixtures.

Intracellular adenosine-5'-triphosphate (ATP) levels were measured in a metabolically engineered Zymomonas mobilis over the course of batch fermentations of glucose and xylose mixtures. Fermentations were conducted over a range of pH (5-6) in the presence of varying initial amounts of acetic acid (0-8 g/L) using a 10% (w/v) total sugar concentration (glucose only, xylose only, or 5% glucose/5% xylose mixture). Over the design space investigated, ethanol process yields varied between 56.6% and 92.3% +/- 1.3% of theoretical, depending upon the test conditions. The large variation in process yields reflects the strong effect pH plays in modulating the inhibitory effect of acetic acid on fermentation performance. A corresponding effect was observed on maximum cellular specific growth rates, with the rates varying between a low of 0.15 h(-1) observed at pH 5 in the presence of 8 g/L acetic acid to a high of 0.32 +/- 0.02 h(-1) obtained at pH 5 or 6 when no acetic acid was initially present. While substantial differences were observed in intracellular specific ATP concentration profiles depending upon fermentation conditions, maximum intracellular ATP accumulation levels varied within a relatively narrow range (1.5-3.8 mg ATP/g dry cell mass). Xylose fermentations produced and accumulated ATP at much slower rates than mixed sugar fermentations (5% glucose, 5% xylose), and the ATP production and accumulation rates in the mixed sugar fermentations were slightly slower than in glucose fermentations. Results demonstrate that higher levels of acetic acid delay the onset and influence the extent of intracellular ATP accumulation. ATP production and accumulation rates were most sensitive to acetic acid at lower values of pH.

Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis.

Zymomonas mobilis engineered to express four heterologous enzymes required for xylose utilization ferments xylose along with glucose. A network of pentose phosphate (PP) pathway enzymatic reactions interacting with the native glycolytic Entner Doudoroff (ED) pathway has been hypothesized. We have investigated this putative reaction network by developing a kinetic model incorporating all of the enzymatic reactions of the PP and ED pathways, including those catalyzed by the heterologous enzymes. Starting with the experimental literature on in vitro characterization of each enzymatic reaction, we have developed a kinetic model to enable dynamic simulation of intracellular metabolite concentrations along the network of interacting PP and ED metabolic pathways. This kinetic model is useful for performing in silico simulations to predict how varying the different enzyme concentrations will affect intracellular metabolite concentrations and ethanol production rate during continuous fermentation of glucose and xylose mixtures. Among the five enzymes whose concentrations were varied as inputs to the model, ethanol production in the continuous fermentor was optimized when xylose isomerase (XI) was present at the highest level, followed by transaldolase (TAL). Predictions of the model that the interconnecting enzyme phosphoglucose isomerase (PGI) does not need to be overexpressed were recently confirmed through experimental investigations. Through such systematic analysis, we can develop efficient strategies for maximizing the fermentation of both glucose and xylose, while minimizing the expression of heterologous enzymes.

Development and validation of a kinetic model for enzymatic saccharification of lignocellulosic biomass.

A multireaction kinetic model was developed for closed-system enzymatic hydrolysis of lignocellulosic biomass such as corn stover. Three hydrolysis reactions were modeled, two heterogeneous reactions for cellulose breakdown to cellobiose and glucose and one homogeneous reaction for hydrolyzing cellobiose to glucose. Cellulase adsorption onto pretreated lignocellulose was modeled via a Langmuir-type isotherm. The sugar products of cellulose hydrolysis, cellobiose and glucose, as well as xylose, the dominant sugar prevalent in most hemicellulose hydrolyzates, were assumed to competitively inhibit the enzymatic hydrolysis reactions. Model parameters were estimated from experimental data generated using dilute acid pretreated corn stover as the substrate. The model performed well in predicting cellulose hydrolysis trends at experimental conditions both inside and outside the design space used for parameter estimation and can be used for in silico process optimization.

Dilute-sulfuric acid pretreatment of corn stover in pilot-scale reactor: investigation of yields, kinetics, and enzymatic digestibilities of solids.

Corn stover is a domestic feedstock that has potential to produce significant quantities of fuel ethanol and other bioenergy and biobased products. However, comprehensive yield and carbon mass balance information and validated kinetic models for dilute-sulfuric acid (H2SO4) pretreatment of corn stover have not been available. This has hindered the estimation of process economics and also limited the ability to perform technoeconomic modeling to guide research. To better characterize pretreatment and assess its kinetics, we pretreated corn stover in a continuous 1 t/d reactor. Corn stover was pretreated at 20% (w/w) solids concentration over a range of conditions encompassing residence times of 3-12 min, temperatures of 165- 195 degrees C, and H2SO4 concentrations of 0.5-1.4% (w/w). Xylan conversion yield and carbon mass balance data were collected at each run condition. Performance results were used to estimate kinetic model parameters assuming biphasic hemicellulose hydrolysis and a hydrolysis mechanism incorporating formation of intermediate xylo-oligomers. In addition, some of the pretreated solids were tested in a simultaneous saccharification and fermentation (SSF) process to measure the reactivity of their cellulose component to enzymatic digestion by cellulase enzymes. Monomeric xylose yields of 69-71% and total xylose yields (monomers and oligomers) of 70-77% were achieved with performance level depending on pretreatment severity. Cellulose conversion yields in SSF of 80-87% were obtained for some of the most digestible pretreated solids.

Availability of corn stover as a sustainable feedstock for bioethanol production.

The amount of corn stover that can be sustainably collected is estimated to be 80-100 million dry tonnes/yr (t/yr), a majority of which would be available to ethanol plants in the near term as only a small portion is currently used for other applications. Potential long-term demand for corn stover by non-fermentative applications in the United States is estimated to be about 20 million dry t/yr, assuming that corn stover-based products replace 50% of both hardwood pulp and wood-based particleboard, and that 50% of all furfural production is from corncobs. Hence, 60-80 million dry t/yr of corn stover should be available to fermentative routes. To achieve an ethanol production potential of 11 billion L (3 billion gal) per year (a target level for a non-niche feedstock), about 40% of the harvestable corn stover is needed. This amount should be available as long as the diversion of corn stover to non-ethanol fermentative products remains limited.

Carbon mass balance evaluation of cellulase production on soluble and insoluble substrates.

A methodology is described and applied for performing carbon mass balances across cellulase enzyme production processes using both soluble sugar and insoluble cellulose substrates. The fungus Trichoderma reesei was grown on either glucose, lactose, or cellulose in aerobic batch mode, and the evolution of the main carbonaceous components (cell mass, cellulose, soluble protein, adsorbed protein, sugars, and carbon dioxide) was followed. A variety of analytical techniques were utilized to measure these components, including (i) gravimetric analysis, (ii) near-infrared spectroscopy, (iii) bicinchoninic acid based soluble protein measurement, (iv) gas mass spectrometry and flow rate, (v) CHNS/O elemental analyses, and (vi) high-performance liquid chromatography. The combined set of measurements allowed carbon mass balances across the cellulase production process to be assessed to determine the consistency of the underlying kinetic data. Results demonstrate the capability to determine the levels and distribution of all major carbonaceous components during the cellulase production process on both soluble and insoluble substrates. Average carbon mass balance closures were near 100% during early stages (<72 h) of the cultivations using glucose, lactose, or cellulose as the substrates, but carbon mass closures trended high later in the cultivation. Analysis of carbon allocation results suggests that an error in the gas mass flow rate measurement was the primary cause for carbon mass balance closures to exceed 110% late in the process.