For more than 10 years, batch dilute acid pretreatment techniques have been extensively evaluated at NREL on biomass feedstocks, including several species of hardwoods, herbaceous crops, and agricultural residues (39-42). The pretreatment objectives have focused largely on a prehydrolysis approach, in which the hemicellulose component of biomass is hydrolyzed via dilute acid catalysis, leaving the cellulose fraction in an insoluble form for subsequent enzymatic hydrolysis by cellulase enzymes. A detailed process economic analysis of a base-case bioethanol production process, utilizing continuous cocurrent dilute acid prehydrolysis technology, has been conducted at NREL (43). Pretreatment conditions and performance parameters used in this evaluation were based on bench-scale batch, dilute acid pretreatment experimental data for hardwood poplar species. Since that time, pilot-scale batch (44) and cocurrent pretreatment reactors have been installed and operated to collect larger-scale reactor data for the pretreatment performance of selected feedstocks. For the most part, pretreatment performance data collected in these larger reactors have been comparable to results obtained in bench-scale batch dilute acid pretreatments (44).

Numerous of sensitivity analyses were performed as a part of this base case process engineering and economic analysis. The most significant parameter in improving the overall economics of bioethanol production was maximizing the yield of ethanol from a unit of biomass. Clearly, improving the yields of each individual unit operation will contribute to increasing the overall yield of biomass. The pretreatment step is very significant in this respect, as an improved pretreatment process would result in higher yields of soluble sugars from the hemicellulose fraction and a more digestible cellulose fraction, resulting in greater conversion of cellulose via enzymatic hydrolysis by cellulase. Because bench — and pilot-scale batch and cocurrent dilute acid pretreatment methods show definite limitations in the yields of soluble sugars from hemicellulose (about 80%-85% of theoretical) and in the yields of ethanol from cellulose in pretreated biomass solids from a simultaneous saccharification and fermentation (SSF) process (about 70%-75% of theoretical), new pretreatment approaches aimed at achieving higher yields are being developed and evaluated.

During the past few years, dilute acid prehydrolysis R&D at NREL has focused on the developing processes that exploit the biphasic kinetics of xylan hydrolysis from the hemicellulose fraction of biomass. In most species being considered for bioethanol conversion, xylan is the overwhelming majority of the hemicellulose component. The kinetics of xylan hydrolysis have been widely reported to be biphasic, with an "easy-to — remove" fraction that can be removed under relatively mild conditions and a "hard-to — remove" fraction that requires more severe conditions (45-47). The hydrolysis reaction mechanism for fast — and slow-reacting xylan fractions assumes conversion first to soluble xylose oligomers, followed by conversion to monomeric xylose and finally, to xylose degradation products. Three prehydrolysis reactor configurations (batch, cocurrent, and countercurrent) have been evaluated in a model of xylan hydrolysis kinetics to determine the best configuration for maximizing xylan hydrolysis yields, while minimizing degradation product formation (48). This work showed that a countercurrent configuration is the best design for achieving high recovery of xylose equivalents, especially at high conversion levels of xylan, which is critical to the economic viability of any bioethanol conversion process. This conclusion is somewhat intuitive, as in a countercurrent configuration, the residence time of the xylose from the "easy-to-remove" xylan fraction is short compared to a batch or cocurrent configuration, resulting in less degradation product formation.

Two-Stage Countercurrent Pretreatment. A large number of pretreatment data have been collected at NREL using hardwood yellow poplar sawdust in a system of percolation reactors operated in such a way as to simulate a two-stage countercurrent dilute acid process. The selection of the reaction conditions and the details of the operation of what is now referred to as the reverse-flow two-stage system have been described in detail (49). A factorial experimental design and subsequent reaction condition optimization was conducted in this study. The conditions selected as a result of this optimization were a 10-min residence time of the solids in each stage, an acid concentration of 0.07% (w/w) sulfuric acid, a first-stage solids temperature of 174°C, and a second-stage solids temperature of 204°C. A recovery of 97.0% of xylose equivalents in the liquor stream from the xylan content of the feedstock was achieved, with about 60% in the form of soluble oligomeric xylose and 40% as monomeric xylose. Less than 3% of the xylan degraded to furfural. About 10% of the glucan was solubilized, but with much lower levels of oligomeric glucose relative to monomeric glucose. About 35% of the lignin was solubilized. The structure of the soluble lignin compounds was not determined. The degree of lignin solubilization is significantly higher than with batch or cocurrent dilute acid pretreatments. It is believed that the reverse-flow mode of operation, in which the liquor is separated from the residual solids while temperatures are still high, prevents re-precipitation and/or re-condensation of solubilized lignin back on the pretreated solids particles.

As the reverse-flow procedure includes a hot water washing of the residual solids, with the wash liquor combined with the actual preydrolysis liquor, the resulting residual solids are free of soluble compounds, including sugars, oligomers, and soluble inhibitory compounds from lignin breakdown or sugar degradation. Thus, the residual solids can proceed to the SSF conversion process without any further washing or other detoxification procedure. Standard SSF conversion studies were performed using the conditions described by Torget and co-workers (49), including a cellulase loading of 25 FPU/g cellulose. For the reverse-flow two-stage optimization run described above, an ethanol yield from cellulose of 91% of theoretical was achieved in 55 h. Both the yield and the rate are substantially higher than those reported for similar feedstocks subjected to batch dilute acid pretreatments. This represents a substantial potential improvement to the overall process economics.

Because of the near quantitative yield of xylose equivalents, the partial solubilization of glucan to glucose equivalents, and the complete displacement of soluble compounds from the residual solids during the washing step, a significant fraction of the initial carbohydrates found in the feedstock end up in the prehydrolyzate liquor. Based on the initial composition of yellow poplar sawdust and the pretreatment performance achieved in the optimization run, more than 40% of the total available carbohydrates are found in this liquor. In the past, the evaluation of pretreatment performance has been based on the enzymatic digestibility and/or SSF production of ethanol from washed, pretreated solids only. It has largely been assumed that the soluble carbohydrates in the liquid fraction of the pretreated slurry or in a separate prehydrolyzate liquor, could be converted to ethanol at assumed yields and rates and would not require any type of post-treatment prior to either a separate or cofermentation.

Hydrolyzate Detoxification. The base-case process economic evaluation described above did not include any prehydrolyzate detoxification step. It has become recognized that some type of detoxification of at least the liquid fraction will likely be required. This is even more likely in flow-through, dilute acid pretreatments compared to batch or cocurrent pretreatments because of the higher lignin solubilization. Certain compounds that result from the solubilization of lignin, including certain organic acids, higher alcohols, and phenolic-based compounds, are known to be inhibitory to fermentative microorganisms. Also, feedstocks that have highly acetylated hemicellulose in their structure are likely to release inhibitory levels of acetic acid upon hemicellulose hydrolysis. Significant efforts are under way to develop cost-effective detoxification processes that can selectively remove or convert such compounds into non-toxic forms. At the same time, modified prehydrolysis conditions that result in similar pretreatment performance levels, but that produce fewer toxic compounds, are being evaluated.

Challenges to Sugar Yield. As stated above, the analysis of the carbohydrates in the optimized prehydrolyzate liquor shows that about 60% of the xylose equivalents in the liquor are in the form of oligomeric xylose. Previous process economic analyses have assumed that solubilized carbohydrates from pretreatment would be fermentable, i. e. monomeric sugars. Options for converting oligomeric xylose to monomeric xylose have been reported (48). Two options were investigated in work performed in the laboratory of Y. Y. Lee at Auburn University (48). The first was a mild-temperature hold of the prehydrolysis liquor at the pH (~2.2) of the liquor as it leaves the pretreatment reactor. A range of hold times and temperatures was examined. The best conditions were a temperature of 130°C and a hold time of 11 h, where the monomeric xylose content increases to 97% of total xylose equivalents and the oligomeric xylose content decreases to virtually zero. The furfural content increased only marginally, indicating little increased sugar degradation under these relatively mild conditions.

The other option evaluated for xylose oligomer hydrolysis was to determine whether sufficient xylanase activity exists in cellulase preparations to allow for enzymatically catalyzed hydrolysis of xylan oligomers to monomers under typical SSF conditions. In tests where Genencor Cytolase CL was added to prehydrolyzate liquor in amounts equivalent to 10 and 25 FPU/g cellulose as if 8% (w/v) cellulose was present, significant conversion to xylose monomer was achieved (Elander, R., unpublished results, 1996). Starting from an initial monomer level of 41% of the total xylose equivalents, the monomeric xylose level increased to 87% of total xylose equivalents with a 10 FPU/g assumed cellulose loading at 61 h, and 95% of total xylose equivalents with a 25 FPU/g assumed cellulose loading at 61 h. The 61-h time is similar to the 55-h SSF time required to achieve a 91% ethanol yield in SSF, as reported above for the reverse-flow, two-stage pretreated solids. The unknown of these experiments is whether these levels of conversion would be maintained when process concentrations of cellulose and lignin are present in the system.

In addition, the work of Elander and co-workers (48) included an analysis of potential process improvements that could result in higher concentrations of carbohydrates feeding into the fermentation steps, which would presumably result in a higher ethanol concentration exiting the fermentation step(s) and lower ethanol recovery costs. Using the volume of liquor generated from pretreatment, which was 2.2 reactor void volumes per biomass-packed reactor (49), and an assumed washed pretreated solids concentration of 40%, process analysis indicated that in a simultaneous saccharification and cofermentation (SSCF) process, in which the washed solids are combined with detoxified liquor and yields of ethanol from cellulose (49) and from xylose are known (43), an ethanol concentration exiting SSF of 3.05% (w/v) could be expected. This is somewhat lower that the base case value of -4.5% (w/v) assumed by Hinman and co-workers (43), and indicates that the volumes of liquor generated could increase ethanol recovery costs. Also, the high liquor volumes result in increased fermentation tankage requirements and would require higher steam demands in pretreatment, which would be exacerbated by the higher temperature requirements that have been used in reverse-flow prehydrolysis. This situation will certainly have an impact on the energy balance of the process and will affect overall process economics.

Reactor Design. The ultimate ability to reduce prehydrolyzate liquor volumes, but still maintain prehydrolysis sugar yields and pretreated solids enzymatic digestibility, becomes a reactor design issue, not only at the bench-scale level but, more importantly, in an engineering-scale system that is representative of a commercial-scale countercurrent prehydrolysis reactor system. The ability to achieve the contacting and movement of liquid and solids effectively in a large-scale device is a major challenge and will likely determine the ultimate commercial success of this technology. Efforts are currently under way to work with equipment suppliers to design, develop, and test engineering — scale countercurrent dilute acid prehydrolysis reactor systems. Although biomass particle sizes, reaction conditions, and the ultimate goals of the reaction are somewhat different, an extensive body of knowledge in reactor design issues related to biomass solubilization exists within the pulp and paper industry. Collaborations with pulping equipment manufacturers are being investigated as a means to more rapidly develop commercial — scale countercurrent prehydrolysis reactor systems.

New Directions at NREL. To date, the only feedstock extensively investigated using the dilute acid countercurrent process is yellow poplar sawdust. This particular feedstock is representative of a near-term waste biomass source from sawmill operations, but also is similar to potential hardwood energy crop species; thus, it provides useful information for near — and long-term applications. Ultimately, feedstock options, including other waste biomass materials, such as agricultural residues and industrial lignocellulosic wastes, and energy crop feedstocks, such as herbaceous grasses and fast-growing hardwoods, will be investigated.

In addition to dilute acid, the feasibility of a pressurized hot water countercurrent prehydrolysis process that requires no added acid catalyst is being investigated on appropriate feedstocks. Eliminating the acid catalyst requirement has a number of potentially beneficial impacts on the process, including reduced chemicals costs, potentially less expensive metals for pretreatment reactor construction because of less corrosive conditions, and reduced formation of insoluble compounds, such as gypsum, upon neutralization. Such a process is also more environmentally benign than those that utilize harsh acids, bases, or solvents. This work is being conducted using a flow-through percolation system at NREL. In addition, the investigation of a neutral pH-controlled process via a NREL-sponsored subcontract at Purdue University is currently being pursued. Other workers (50-52) have recently reported promising findings with pressurized hot water prehydrolysis approaches, but the economic impacts of some reported process conditions, such as very small biomass particle size and relatively large volumes of pressurized hot water, have not been thoroughly evaluated.

A number of alternate pretreatment methods have also recently been investigated through a series of NREL-sponsored subcontracts. These include the use of various acid catalysts in a dilute acid prehydrolysis mode, such as nitric acid, phosphoric acid, and phosphoric acid/organosolv; alkaline-based methods, such as lime pretreatment and ammonia recycle percolation; and physicochemical methods, such as hydrogen peroxide extrusion and ammonia freeze explosion. Many of these methods have been widely discussed in the literature, but it is difficult to compare the relative effectiveness of these various approaches because of variation in feedstocks tested, analytical methods, and pretreatment performance measurement and reporting protocols. A common-basis evaluation of these approaches that will determine the relative effectiveness and process economic implications of these various pretreatment approaches is currently being conducted. This can serve as a basis for making rational choices in pretreatment technologies for given feedstock and process applications. Promising pretreatment technologies will likely be further developed from additional bench-scale work and initial integration with subsequent detoxification and fermentation steps, potentially leading to engineering scale equipment design and testing in the future. Another key goal of this evaluation is to determine which pretreatments are best suited to various feedstocks.

Complete Hydrolysis Studies. Very recently, the general concept of dilute acid countercurrent prehydrolysis developed at NREL has been extended to investigate the possibility of a full hydrolysis of both hemicellulose and cellulose to soluble sugars. This concept has been motivated by the fact that thus far, costs of commercial cellulase preparations from industrial enzyme suppliers have been prohibitive for use in bioethanol processes (43у 53). Although ongoing process development and research plans at NREL show promise in substantially reducing cellulase production costs, the ultimate costs of cellulase production are still unclear. Thus, a full hydrolysis process that substantially reduces (or even eliminates) cellulase requirements is an option worthy of investigation.

The use of dilute sulfuric acid to totally hydrolyze the carbohydrates in lignocellulosic feedstocks for ethanol production was widely abandoned by 1990 due to the relatively low yields of glucose (50%-60%) as compared to potentially high yields (80%-95%) from enzymatic hydrolysis of cellulose (54-56). Even though kinetic modeling exercises conducted at NREL (57) suggested that yields as high as 88% could be obtained using a countercurrent reactor configuration, laboratory studies could only demonstrate total sugar yields (C5 and C6 sugars) of about 60% (56). However, several observations were made to explain the low yields. The material of construction of the experimental reactor (Carpenter Cb20-3), when subjected to 1% acid at elevated temperatures, leached chromium ions at a concentration that leads to degradation of glucose formed in the reactor. Additionally, as the biomass was hydrolyzed, the packed bed collapsed, which led to non-ideal fluid dynamics that caused increased degradation of the glucose.

The renewed interest in total hydrolysis dilute acid processes has been motivated by modeling exercises, which indicate that with a hydrolysis liquor residence time about one-seventh of the solid residence time, yields of glucose could approach those obtained with an enzymatic process (57). Such a process could result in high volumes of hydrolyzate liquor relative to countercurrent dilute acid prehydrolysis. The impact of producing liquor volumes even greater than those for countercurrent dilute acid prehydrolysis is to diminish the overall process efficiency. Although overall cost savings may be realized through reduced cellulase requirement, process improvements to reduce the quantity of the resulting liquor volumes is required. Again, innovative pretreatment reactor design aimed at construction feasibility and reducing required liquor volumes will play a key role in determining the viability of a dilute acid total hydrolysis process.