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.

Accounting for all sugars produced during integrated production of ethanol from lignocellulosic biomass.

Accurate mass balance and conversion data from integrated operation is needed to fully elucidate the economics of biofuel production processes. This study explored integrated conversion of corn stover to ethanol and highlights techniques for accurate yield calculations. Acid pretreated corn stover (PCS) produced in a pilot-scale reactor was enzymatically hydrolyzed and the resulting sugars were fermented to ethanol by the glucose-xylose fermenting bacteria, Zymomonas mobilis 8b. The calculations presented here account for high solids operation and oligomeric sugars produced during pretreatment, enzymatic hydrolysis, and fermentation, which, if not accounted for, leads to overestimating ethanol yields. The calculations are illustrated for enzymatic hydrolysis and fermentation of PCS at 17.5% and 20.0% total solids achieving 80.1% and 77.9% conversion of cellulose and xylan to ethanol and ethanol titers of 63g/L and 69g/L, respectively. These procedures will be employed in the future and the resulting information used for techno-economic analysis.

Performance and techno-economic assessment of several solid-liquid separation technologies for processing dilute-acid pretreated corn stover.

Solid-liquid separation of pretreated lignocellulosic biomass slurries is a critical unit operation employed in several different processes for production of fuels and chemicals. An effective separation process achieves good recovery of solute (sugars) and efficient dewatering of the biomass slurry. Dilute acid pretreated corn stover slurries were subjected to pressure and vacuum filtration and basket centrifugation to evaluate the technical and economic merits of these technologies. Experimental performance results were used to perform detailed process simulations and economic analysis using a 2000 tonne/day biorefinery model to determine differences between the various filtration methods and their process settings. The filtration processes were able to successfully separate pretreated slurries into liquor and solid fractions with estimated sugar recoveries of at least 95% using a cake washing process. A continuous vacuum belt filter produced the most favorable process economics.

Characterization of pilot-scale dilute acid pretreatment performance using deacetylated corn stover.

BACKGROUND: Dilute acid pretreatment is a promising process technology for the deconstruction of low-lignin lignocellulosic biomass, capable of producing high yields of hemicellulosic sugars and enhancing enzymatic yields of glucose as part of a biomass-to-biofuels process. However, while it has been extensively studied, most work has historically been conducted at relatively high acid concentrations of 1 - 4% (weight/weight). Reducing the effective acid loading in pretreatment has the potential to reduce chemical costs both for pretreatment and subsequent neutralization. Additionally, if acid loadings are sufficiently low, capital requirements associated with reactor construction may be significantly reduced due to the relaxation of requirements for exotic alloys. Despite these benefits, past efforts have had difficulty obtaining high process yields at low acid loadings without supplementation of additional unit operations, such as mechanical refining. RESULTS: Recently, we optimized the dilute acid pretreatment of deacetylated corn stover at low acid loadings in a 1-ton per day horizontal pretreatment reactor. This effort included more than 25 pilot-scale pretreatment experiments executed at reactor temperatures ranging from 150 - 170°C, residence times of 10 - 20 minutes and hydrolyzer sulfuric acid concentrations between 0.15 - 0.30% (weight/weight). In addition to characterizing the process yields achieved across the reaction space, the optimization identified a pretreatment reaction condition that achieved total xylose yields from pretreatment of 73.5% ± 1.5% with greater than 97% xylan component balance closure across a series of five runs at the same condition. Feedstock reactivity at this reaction condition after bench-scale high solids enzymatic hydrolysis was 77%, prior to the inclusion of any additional conversion that may occur during subsequent fermentation. CONCLUSIONS: This study effectively characterized a range of pretreatment reaction conditions using deacetylated corn stover at low acid loadings and identified an optimum reaction condition was selected and used in a series of integrated pilot scale cellulosic ethanol production campaigns. Additionally, several issues exist to be considered in future pretreatment experiments in continuous reactor systems, including the formation of char within the reactor, as well as practical issues with feeding herbaceous feedstock into pressurized systems.

Degradation of carbohydrates during dilute sulfuric acid pretreatment can interfere with lignin measurements in solid residues.

The lignin content measured after dilute sulfuric acid pretreatment of corn stover indicates more lignin than could be accounted for on the basis of the untreated corn stover lignin content. This phenomenon was investigated using a combination of (13)C cross-polarization/magic-angle spinning (CP/MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy and lignin removal using acid chlorite bleaching. Only minimal contamination with carbohydrates and proteins was observed in the pretreated corn stover. Incorporating degradation products from sugars was also investigated using (13)C-labeled sugars. The results indicate that sugar degradation products are present in the pretreatment residue and may be intimately associated with the lignin. Studies comparing whole corn stover (CS) to extractives-free corn stover [CS(Ext)] clearly demonstrated that extractives are a key contributor to the high-lignin mass balance closure (MBC). Sugars and other low molecular weight compounds present in plant extractives polymerize and form solids during pretreatment, resulting in apparent Klason lignin measurements that are biased high.

Conversion of residual organics in corn stover-derived biorefinery stream to bioenergy via a microbial fuel cell.

A biorefinery process typically uses about 4-10 times more water than the amount of biofuel generated. The wastewater produced in a biorefinery process contains residual sugars, 5-furfural, phenolics, and other pretreatment and fermentation byproducts. Treatment of the wastewater can reduce the need for fresh water and potentially add to the environmental benefits of the process. Use of microbial fuel cells (MFCs) for conversion of the complete range of phenolic compounds and furan aldehyde derivatives present in a postfermentation biorefinery stream is reported here. The consortium was capable of removing the molecules simultaneously with sugars, which were present at 2 orders of magnitude higher concentrations. Organic loading in a fed-batch MFC affected Coulombic efficiency, which decreased from 40% at 0.66 g/L loading to 1.8% at 66.4 g/L loading. Power density increased with loading reaching 1180 mW/m(2) at 5.3 g/L (8% dilution), but decreased thereafter. Excessive loading leads to poor electrogenic performance; therefore, operation of an MFC at an intermediate loading using dilution and recirculation of the process stream can enable effective treatment with bioenergy recovery.

Membrane extraction for detoxification of biomass hydrolysates.

Membrane extraction was used for the removal of sulfuric acid, acetic acid, 5-hydroxymethyl furfural and furfural from corn stover hydrolyzed with dilute sulfuric acid. Microporous polypropylene hollow fiber membranes were used. The organic extractant consisted of 15% Alamine 336 in: octanol, a 50:50 mixture of oleyl alcohol:octanol or oleyl alcohol. Rapid removal of sulfuric acid, 5-hydroxymethyl and furfural was observed. The rate of acetic acid removal decreased as the pH of the hydrolysate increased. Regeneration of the organic extractant was achieved by back extraction into an aqueous phase containing NaOH and ethanol. A cleaning protocol consisting of flushing the hydrolysate compartment with NaOH and the organic phase compartment with pure organic phase enabled regeneration and reuse of the module. Ethanol yields from hydrolysates detoxified by membrane extraction using 15% Alamine 336 in oleyl alcohol were about 10% higher than those from hydrolysates detoxified using ammonium hydroxide treatment.

Fermentation of reactive-membrane-extracted and ammonium-hydroxide-conditioned dilute-acid-pretreated corn stover.

Acid-pretreated biomass contains various compounds (acetic acid, etc.) that are inhibitory to fermentative microorganisms. Removing or deactivating these compounds using detoxification methods such as overliming or ammonium hydroxide conditioning (AHC) improves sugar-to-ethanol yields. In this study, we treated the liquor fraction of dilute-acid-pretreated corn stover using AHC and a new reactive membrane extraction technique, both separately and in combination, and then the sugars in the treated liquors were fermented to ethanol with the glucose-xylose-fermenting bacterium, Zymomonas mobilis 8b. We performed reactive extraction with mixtures of octanol/Alamine 336 or oleyl alcohol/Alamine 336. The best ethanol yields and rates were achieved for oleyl alcohol-extracted hydrolysates followed by AHC hydrolysates, while octanol-extracted hydrolysates were unfermentable because highly toxic octanol was found in the hydrolysate. Adding olive oil significantly improved yields for octanol-extracted hydrolysate. Additional work is underway to determine if this technology is a cost-effective alternative to traditional hydrolysate conditioning processes.

Conditioning of dilute-acid pretreated corn stover hydrolysate liquors by treatment with lime or ammonium hydroxide to improve conversion of sugars to ethanol.

Dilute-acid pretreatment of lignocellulosic biomass enhances the ability of enzymes to hydrolyze cellulose to glucose, but produces many toxic compounds that inhibit fermentation of sugars to ethanol. The objective of this study was to compare the effectiveness of treating hydrolysate liquor with Ca(OH)2 and NH4OH for improving ethanol yields. Corn stover was pretreated in a pilot-scale reactor and then the liquor fraction (hydrolysate) was extracted and treated with various amounts of Ca(OH)2 or NH4OH at several temperatures. Glucose and xylose in the treated liquor were fermented to ethanol using a glucose-xylose fermenting bacteria, Zymomonas mobilis 8b. Sugar losses up to 10% occurred during treatment with Ca(OH)2, but these losses were two to fourfold lower with NH4OH treatment. Ethanol yields for NH4OH-treated hydrolysate were 33% greater than those achieved in Ca(OH)2-treated hydrolysate and pH adjustment to either 6.0 or 8.5 with NH4OH prior to fermentation produced equivalent ethanol yields.

Mass spectral analyses of corn stover prehydrolysates to assess conditioning processes.

Flow injection electrospray (FIE) and LC-tandem mass spectrometry techniques were used to characterize corn stover acid hydrolysates before and after overliming and ammonia conditioning steps. Analyses were performed on samples without fractionation (dilution only) in an effort provide an inventory of ionizable substances. Statistical evaluation of the results indicates that the ammonia-treated and crude hydrolysates were more similar to one another than any other pairing, with conditioning leading to a decrease in malate levels. LC-tandem mass spectrometry studies were also developed to characterize the oligosaccharides present in each hydrolysate utilizing a hydrophilic interaction chromatographic separation method. Neutral and acidic pentose-based oligosaccharides (xylodextrins) with degrees of polymerization between 2 and 5 were quantified with 4-O-methyl glucuronic acid-containing dimer and trimers predominating. Conditioning had little effect on the quantified oligosaccharide pool.

Economic impact of total solids loading on enzymatic hydrolysis of dilute acid pretreated corn stover.

In process integration studies of the biomass-to-ethanol conversion process, it is necessary to understand how cellulose conversion yields vary as a function of solids and enzyme loading and other key operating variables. The impact of solids loading on enzymatic cellulose hydrolysis of dilute acid pretreated corn stover slurry was determined using an experimental response surface design methodology. From the experimental work, an empirical correlation was obtained that expresses monomeric glucose yield from enzymatic cellulose hydrolysis as a function of solids loading, enzyme loading, and temperature. This correlation was used in a technoeconomic model to study the impact of solids loading on ethanol production economics. The empirical correlation was used to provide a more realistic assessment of process cost by accounting for changes in cellulose conversion yields at different solids and enzyme loadings as well as enzyme cost. As long as enzymatic cellulose conversion drops off at higher total solids loading (due to end-product inhibition or other factors), there is an optimum value for the total solids loading that minimizes the ethanol production cost. The optimum total solids loading shifts to higher values as enzyme cost decreases.

Impact of corn stover composition on hemicellulose conversion during dilute acid pretreatment and enzymatic cellulose digestibility of the pretreated solids.

This study assessed the impact of corn stover compositional variability on xylose conversion yields during dilute acid pretreatment and on enzymatic cellulose digestibility of the resulting pretreated solids. Seven compositionally-different stovers obtained from various locations throughout the United States were pretreated at three different conditions in triplicate in a pilot-scale continuous reactor. At the same pretreatment severity, a 2-fold increase in monomeric xylose yield and a 1.5-fold increase in enzymatic cellulose digestibility from their lowest values were found. Similar results were observed at the other pretreatment conditions. It was found that xylose conversion yields decreased with increasing acid neutralization capacity or soil content of the corn stover. Xylose yields also increased with increasing xylan content. No other significant correlations between corn stover's component concentrations and conversion yields were found.

An economic comparison of different fermentation configurations to convert corn stover to ethanol using Z. mobilis and Saccharomyces.

Numerous routes are being explored to lower the cost of cellulosic ethanol production and enable large-scale production. One critical area is the development of robust cofermentative organisms to convert the multiple, mixed sugars found in biomass feedstocks to ethanol at high yields and titers without the need for processing to remove inhibitors. Until such microorganisms are commercialized, the challenge is to design processes that exploit the current microorganisms' strengths. This study explored various process configurations tailored to take advantage of the specific capabilities of three microorganisms, Z. mobilis 8b, S. cerevisiae, and S. pastorianus. A technoeconomic study, based on bench-scale experimental data generated by integrated process testing, was completed to understand the resulting costs of the different process configurations. The configurations included whole slurry fermentation with a coculture, and separate cellulose simultaneous saccharification and fermentation (SSF) and xylose fermentations with none, some or all of the water to the SSF replaced with the fermented liquor from the xylose fermentation. The difference between the highest and lowest ethanol cost for the different experimental process configurations studied was $0.27 per gallon ethanol. Separate fermentation of solid and liquor streams with recycle of fermented liquor to dilute the solids gave the lowest ethanol cost, primarily because this option achieved the highest concentrations of ethanol after fermentation. Further studies, using methods similar to ones employed here, can help understand and improve the performance and hence the economics of integrated processes involving enzymes and fermentative microorganisms.

Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol.

Both the current corn starch to ethanol industry and the emerging lignocellulosic biofuels industry view recycling of spent fermentation broth or stillage as a method to reduce fresh water use. The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute-acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose-xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively poorer as the sugar concentration increased and fraction of the stillage recycled increased. At an equivalent corn stover slurry concentration of 20% with 25% recycled stillage the ethanol yield was only 5%. For this microorganism with dilute-acid pretreated corn stover, recycling a large fraction of the stillage had a significant negative impact on fermentation performance. Although this finding is of concern for biochemical-based lignocellulose conversion processes, other microorganism/pretreatment technology combinations will likely perform differently.

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.

Modeling sucrose hydrolysis in dilute sulfuric acid solutions at pretreatment conditions for lignocellulosic biomass.

Agricultural and herbaceous feedstocks may contain appreciable levels of sucrose. The goal of this study was to evaluate the survivability of sucrose and its hydrolysis products, fructose and glucose, during dilute sulfuric acid processing at conditions typically used to pretreat lignocellulose biomass. Solutions containing 25g/l sucrose with 0.1-2.0% (w/w) sulfuric acid concentrations were treated at temperatures of 160-200 degrees C for 3-12min. Sucrose was observed to completely hydrolyze at all treatment conditions. However, appreciable concentrations of fructose and glucose were detected and glucose was found to be significantly more stable than fructose. Different mathematical approaches were used to fit the kinetic parameters for acid-catalyzed thermal degradation of these sugars. Since both sugars may survive dilute acid pretreatment, they could provide an additional carbon source for production of ethanol and other bio-based products.

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%).