The impacts of pretreatment on the fermentability of pretreated lignocellulosic biomass: a comparative evaluation between ammonia fiber expansion and dilute acid pretreatment
© Lau et al. 2009
Received: 15 August 2009
Accepted: 4 December 2009
Published: 4 December 2009
Pretreatment chemistry is of central importance due to its impacts on cellulosic biomass processing and biofuels conversion. Ammonia fiber expansion (AFEX) and dilute acid are two promising pretreatments using alkaline and acidic pH that have distinctive differences in pretreatment chemistries.
Comparative evaluation on these two pretreatments reveal that (i) AFEX-pretreated corn stover is significantly more fermentable with respect to cell growth and sugar consumption, (ii) both pretreatments can achieve more than 80% of total sugar yield in the enzymatic hydrolysis of washed pretreated solids, and (iii) while AFEX completely preserves plant carbohydrates, dilute acid pretreatment at 5% solids loading degrades 13% of xylose to byproducts.
The selection of pretreatment will determine the biomass-processing configuration, requirements for hydrolysate conditioning (if any) and fermentation strategy. Through dilute acid pretreatment, the need for hemicellulase in biomass processing is negligible. AFEX-centered cellulosic technology can alleviate fermentation costs through reducing inoculum size and practically eliminating nutrient costs during bioconversion. However, AFEX requires supplemental xylanases as well as cellulase activity. As for long-term sustainability, AFEX has greater potential to diversify products from a cellulosic biorefinery due to lower levels of inhibitor generation and lignin loss.
Cellulosic ethanol, in comparison with first generation biofuels, is substantially more advantageous with regard to feedstock abundance and greenhouse gas reduction [1, 2]. However, unlike the corn ethanol industry, lignocellulosic biomass processing requires higher severity pretreatments due to the inherent recalcitrance of plant material . The selection of pretreatment method has a far-reaching impact on the overall process, including feedstock handling, biological conversions, and downstream processing . The ability to generate steam and electricity from residual lignin is also crucial to maximize the economic profitability and environmental benefits of this industry .
Among potential pretreatment processes, dilute acid pretreatment and ammonia fiber expansion (AFEX) are regarded as promising candidates for large-scale cellulosic biofuel production. Dilute acid pretreatment has been extensively investigated and developed both in the laboratory and at pilot scale [5, 6] to pretreat lignocellulosic biomass for fuel production. This pretreatment is a dry-to-slurry process which effectively hydrolyzes hemicellulose to soluble sugars in the liquor stream . In contrast, AFEX is a dry-to-dry process at alkaline pH using anhydrous ammonia as the reaction catalyst. Although the macrostructure of the pretreated materials is preserved, AFEX reduces the degree of polymerization of cellulose and hemicellulose to increase enzyme accessibility for hydrolysis . High sugar recoveries for corn stover (CS) can be achieved by both pretreatments, as shown by a previous comparative study .
However, a comprehensive comparison of pretreatments with regard to their impacts on important processing units is required. Lignocellulosic biomass is a complex material consisting primarily of cellulose, hemicellulose, lignin, and protein . An ideal pretreatment should produce reactive biomass while minimizing the generation of inhibitory compounds that complicate bioconversions and downstream processes [4, 11]. Furthermore, lignin and biomass nutrients must be preserved for coproduct generation.
In this report, we examine the impacts of these two pretreatments from an overall process perspective. Specifically, we evaluate the interactions of dilute acid pretreatment and AFEX with enzyme requirements, hydrolysate fermentability and lignin preservation. The microbial platform used for the pretreatment comparison involves Saccharomyces cerevisiae 424A(LNH-ST) and Escherichia coli KO11. Comprehensive mass balances were also constructed around each pretreatment.
CS was supplied by the National Renewable Energy Laboratory (NREL, Golden, CO, USA). It was milled and passed through a 4 mm screen. The moisture content was approximately 7% (total weight basis). The milled CS was kept at 4°C for long-term storage. This CS contains 34.1% cellulose, 20.4% xylan, 3.3% arabinan and 2.3% protein on a dry weight basis.
Dilute acid pretreated CS from pilot scale continuous (Sund) reactor at NREL
This dilute acid pretreatment was carried out as described previously . Pretreatment was conducted at 190°C for a residence time of 45 to 75 s. The solids and sulfuric acid loading of the pretreatment were reported as 30% (w/w) and 0.048 g/g dry CS, respectively. The whole slurry from the reactor was used in this study.
The AFEX pretreatment was performed in a 2.0 L pressure vessel (Parr Instruments, Moline, IL, USA). The reactor was equipped with thermocouples and a pressure sensor. AFEX on CS was conducted at 62.5% solids loading. The reactor was preheated to 100 to 110°C and prewetted CS (150 g dry CS + 90 g distilled water) was loaded into the vessel. The lid was bolted shut. Anhydrous ammonia (150 g) was preheated in a 500 mL stainless steel cylinder (Parker Instrumentation, Jacksonville, AL, USA) until the pressure reached 4.48 MPa (650 psi). Heated ammonia was then transferred into the reactor to initiate the reaction. The initial and final temperatures of the pretreatment were 130 ± 5°C and 110 ± 5°C, respectively. The reactor pressure was quickly released after 15 min through an exhaust value. AFEX-pretreated CS was then air dried in a fume hood overnight.
Bench scale dilute acid pretreatment
The dilute acid pretreatment was performed with a 1.0 L Parr reactor made of Hastelloy C (Parr Instruments, Moline, IL, USA) equipped with a thermocouple (Extech Instruments, Waltham, MA, USA) and a helical impeller (8.89 cm (3.5 inches)) on a two-piece shaft. The impeller was driven by a variable speed DC motor assembly (Parr Instruments). CS was presoaked in 1.0% w/v dilute sulfuric acid solution at 5.0% and 7.5% solids (w/w) overnight. The total weight of the pretreatment mixture was 800 g. The presoaked slurry was transferred into the reactor, which was then sealed and fitted to the impeller driver motor. The impeller speed was set at 150 rpm. The reactor was heated rapidly (within 2 min) to an internal temperature of 140 ± 2°C and maintained at 140 ± 2°C in a fluidized heating bath for 40 min. At the end of the reaction time, the reactor was cooled to below 50°C in a water bath. The combined severity factor of the pretreatment is 35.5. The diluted acid pretreated CS slurry was filtered through Whatman no. 1 filter paper. Details on the apparatus, experimental procedure and combined severity calculation are as described previously .
Fermentation on water extract of soluble compounds from pretreated CS
Water extract/pretreatment liquor preparation
Four water extract/pretreatment liquors of pretreated CS were prepared for fermentation studies; they were prepared by (i) washing AFEX-CS pretreated CS, (ii) concentrating pretreatment liquor from dilute acid-CS pretreated CS, which was conducted at 5.0% solids loading in the bench scale reactor, (iii) concentrating/using pretreatment liquor from dilute acid-CS pretreated CS, which was conducted at 7.5% solids loading in the bench scale reactor and (iv) diluting pretreatment liquor from dilute acid-CS pretreated CS, which was conducted at 30.0% solids loading in a continuous pilot reactor (Sund). Solids-free water extracts were used for fermentation. The procedure to prepare the water extracts from different pretreatment was as follows.
Where [Xyl], f and i denote for concentration of xylose, final and initial condition, respectively.
For CS from the Sund reactor, distilled water was added so that the mixture contained 5 mL of liquid to 0.51 g of dry water-insoluble pretreated CS. The diluted slurry was mixed by rigorous shaking and centrifuged at 6 000 g. The supernatant was at 20% solids-loading equivalent. No mass balance around Sund pretreatment was made available, therefore it was assumed that the percentage of input CS remaining as water-insoluble solids after the pretreatment in the Sund reactor was the same as that of bench scale dilute acid pretreatment (that is, 51%).
Seed culture preparation
Seed cultures of E. coli KO11 and S. cerevisiae 424A(LNH-ST) were prepared in 100 mL of complex media YEP_GX (5 g/L bacto yeast extract + 10 g/L bacto peptone + 30 g/L glucose + 20 g/L xylose) by inoculating frozen (-80°C) culture stock at an initial cell density of 0.1 unit optical density (OD) 600 nm using a UV/Vis spectrophotometer (Beckman Coulter, DU720, Brea, CA, USA). The culture temperatures and periods for KO11 and 424A(LNH-ST) were 37°C, 18 h and 30°C, 18 h, respectively. The cultures were conducted under microaerophilic conditions and mixed at 150 rpm agitation. The grown cells were used to initiate fermentations
Fermentations of E. coli KO11 and S. cerevisiae 424A(LNH-ST) in wash streams of 7.5% and 15.0% solids-loading equivalent of the three types of pretreated CS were conducted in 24-well cell culture microplates (BD Falcon #353047, San Jose, CA, USA). The media were supplemented with wash stream, yeast nitrogen base (YNB) with ammonium sulfate (MP Biomedicals, lot no.s 4027512-119914, Solon, OH, USA), glucose and xylose in appropriate buffer (50 mM) at final concentrations of 16.7 g/L, 9 g/L and 35 g/L, respectively. Distilled water was added to dilute the wash streams to 7.5% and 15.0% solid loading equivalent. Chloramphenicol (50 mg/L) was added to reduce the risk of contamination.
Each well contained 2.0 mL media and a glass bead was added (6 mm in diameter) to aid stirring. Seed cultures were prepared as described above and the microplate cell culture was initiated at OD 600 nm of 0.5. The microplate was sealedand fixed on the microplate clamp system (Applikon Inc, Springfield, IL, USA) in an incubator shaker (150 rpm). An opening (about 1 mm diameter) was made on the seal to vent CO2 produced. The initial pH for E. coli KO11 was at 7.0 and at 5.5 for S. cerevisiae 424A(LNH-ST). The incubation temperature was the same as seed culture conditions. The fermentations were conducted for a designated period (E. coli KO11, S. cerevisiae 424A(LNH-ST): 24 h). Cell density was measured using a spectrophotometer at an OD of 600 nm. Sugars and fermentation products were analyzed using a high-performance liquid chromatography (HPLC) system with a Biorad Aminex HPX-87 H column as described previously . Error bars shown in the results are standard deviations of triplicates.
Shake flask fermentation
Fermentations of KO11 and 424A(LNH-ST) were further conducted in 250 mL shake flasks with a 70 mL working volume capped with a rubber stopper, pierced with a needle to vent CO2 formed during fermentation. Wash stream from AFEX and dilute acid pretreatment were supplemented with 1 g/L yeast extract and 2 g/L peptone with 3-(N-morpholino)propanesulfonic acid (MOPS)/phosphate buffer. Sugar levels were adjusted to about 10 g/L glucose and 50 g/L xylose. Final solids-loading equivalents were 7.5%. Inoculum was added to achieve an initial cell density of 0.1 OD 600 nm. Fermentations of E. coli KO11 and S. cerevisiae 424A(LNH-ST) were conducted at 37°C and 30°C, respectively, at 150 rpm agitation. KO11 fermentation was pH adjusted every 24 h using 6 M KOH to pH 7.0. 424A(LNH-ST) fermentation was not pH adjusted because the pH was stable at between 5.2 to 5.5 throughout the fermentation. Fermentation samples were taken at designated points throughout the 120 h culture.
Enzymatic hydrolysis of water-insoluble solids of the pretreated CS
To prepare water-insoluble materials, pretreated CS from both pretreatments was washed with distilled water at a ratio 1 dry g (input CS to pretreatment) to 50 mL of water. For bench scale dilute acid pretreated CS, the designated amount of distilled water was poured into a filter system with Whatman filter paper (no. 4) under vacuum. The solids remaining on the filter paper were dried under vacuum at 60°C. For AFEX-pretreated CS, the washing was achieved in two stages: (1) incubation at 250 rpm, 50°C for 24 h at 5% solids-loading equivalent and (2) two cycles of centrifugation at 6 000 g. After each cycle of centrifugation, the supernatant was decanted through the filter system. The total weight of water-insoluble solids was measured and the carbohydrate content of the solids was analyzed using NREL protocol LAP-002.
The water-insoluble materials were enzymatically hydrolyzed using either (i) cellulase mixtures or (ii) cellulase + hemicellulase mixtures at pH 4.8, 50°C for 144 h. The cellulase mixture consisted of Spezyme CP (86.7 mL/kg CS; 15 FPU/g cellulose) and Novozyme 188 (87.5 mL/kg CS; 64 pNPGU/g cellulose). The hemicellulase mixture was Multifect Xylanase (12.7 mL/kg CS) and Multifect Pectinase (12.7 mL/kg CS). The spectra of activities for the commercial enzymes were as reported . The Spezyme and Multifect enzymes were obtained from Genencor Inc. (Palo Alto, CA, USA) and Novozyme 188 was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Enzymatic hydrolysis was conducted at 5.1% glucan loading. Glucose and xylose in both monomeric and oligomeric forms were measured. Error bars shown are standard deviations of triplicates.
Mass balance construction
Carbohydrate mass balance around pretreatment
Klason lignin mass balance around pretreatment
The dry matter mass of the input and output materials around pretreatment and enzymatic hydrolysis were recorded. The total percentage of Klason lignin in the dry matters before and after pretreatment was analyzed using NREL protocol LAP-002. The final acid concentration, temperature and residence time for the assay was 4% sulfuric acid, 121°C and 60 min, respectively. The Klason lignin content was calculated by multiplying the total dry matter by the percentage of Klason lignin.
Residual solids analysis and heat value estimation
This is done by assuming 90% of the total residual solids is lignin and the rest of 10% has negligible heat value. The heat value of lignin used (25.4 kJ/g) was as reported .
Sugar and lignin preservation during AFEX and dilute acid pretreatment
With regard to the Klason lignin content (at assay condition: 4% sulfuric acid, 121°C, 60 min), AFEX pretreatment did not remove Klason lignin from the solids. For dilute acid pretreatment, 12% of the Klason lignin was removed.
Cell growth and fermentation in soluble extract of the pretreated CS
Enzymatic hydrolysis of washed pretreated solids
Energy content of non-carbohydrate residual solids
Dilute acid pretreatment reduces maximum possible product yield by 10%
The viability of a commercial process is highly dependent on overall process yield. Hence, efforts to increase ethanol yield per unit mass of biomass (CS) at a given product titer deserve the highest priority. In this regard, AFEX preserves all carbohydrates while effectively increasing the susceptibility of the pretreated CS to hydrolytic enzymes. Unlike AFEX, acid-catalyzed pretreatment hydrolyzed hemicellulose almost completely. Monomeric pentoses are further degraded to byproducts such as furfural under acid treatment conditions. In our investigations, about 13% of xylan was lost through chemical degradation. However, a greater degree of degradation (20% to 30%) was reported at a higher solids loading of dilute acid pretreatment . This reduces the maximum product yield by 10%. In any mature chemical process for commodities, raw material is the dominant factor in the processing costs . Therefore, selection of a pretreatment that highly preserves plant carbohydrates is critical for long-term success in this industry.
Pretreatment dictates the fermentability of pretreated biomass
Apart from the preservation of carbohydrate, an ideal pretreatment reduces the generation of inhibitory degradation compounds. AFEX-pretreated CS is highly fermentable using both bacteria and yeast. In certain cases, the soluble fraction of AFEX-pretreated CS has been shown to be beneficial to microbial growth . In contrast, CS hydrolysate from dilute acid pretreatment is substantially more inhibitory. The nitrogenous (amides and amines) reaction products formed during ammonia-lignocellulose reactions are generally non-inhibitory toward microbial growth. These degradation products would otherwise be organic (aliphatic and phenolic) acids in acid-catalyzed reactions . Fermentation at higher initial cell density, nutrient supplementation and/or detoxification are likely needed to alleviate or overcome their inhibitory effects of acid pretreatment [18, 19].
Pretreatment determines feasible biomass-processing configurations
Due to the nature of pretreatment, particularly with respect to the degree of hemicellulose solubilization, inhibitor generation and nutrient preservation, different biomass-processing strategies that maximize the advantages of each pretreatment should be exploited. Dilute acid pretreatment is reported to be well suited for softwood materials  and effectively hydrolyze hemicellulose, eliminating the need for hemicellulases during enzymatic hydrolysis. Nevertheless, the hemicellulase stream is inhibitory toward enzymes and microorganisms. Therefore, separation of solids and the hemicellulose stream as previously proposed  is essential to minimize the adverse effects of the inhibitors from the bioconversion of the remaining solids. However, important technical issues need to be solved in a cost-effective fashion, including (i) separation of solids and liquid with low fresh water use and (ii) effective fermentation of the hemicellulose stream at high sugar concentration without significant conditioning.
AFEX-centered biomass processing can be performed in a straightforward manner where the pretreated biomass (cellulose and hemicellulose) can be converted to ethanol after enzymatic hydrolysis and fermentation without washing or stream separation . In this report, washing was done in AFEX-pretreated CS to establish a basis for comparison. Due to high fermentability of AFEX-pretreated biomass, washing, nutrient supplementation and high initial cell density are not required during the fermentation stage . In comparison to dilute acid pretreatment, a relatively large portion of oligomeric xylose is present in AFEX hydrolysate. Exploitation of hemicellulase-secreting strains such as Thermoanaerobacterium saccharolyticum to biologically process AFEX-pretreated materials could address this issue without added cost of hemicellulase .
AFEX enhances coproduct generation and diversity
Reduction in greenhouse gas emissions by cellulosic ethanol E85 relative to petroleum gasoline is projected to be 68% to 102%, and this is largely due to the heating value of residual solids (primarily lignin) to generate steam or electricity as a coproduct [2, 23]. Our results indicate that AFEX-centered cellulosic technology is expected to have about 17% more available energy from the insoluble lignin residue compared to dilute acid. This also implies that the selection of pretreatment directly affects the magnitude of environmental benefits brought about by a cellulosic ethanol plant beyond the direct impact of the pretreatment process. Nevertheless, a definitive conclusion on the impact of different pretreatments on various environmental benefits can only be made after careful life cycle analysis based on these experimental data.
Lignin removal is a function of severity in terms of acid concentration, temperature and residence time , and part of the solubilized lignin can be recovered . However, the recovery process will inevitably increase the processing cost relative to a production process where lignin is preserved in the solid residue.
AFEX, a dry-to-dry pretreatment process, completely preserves Klason lignin and carbohydrate. In comparison, 13% of the xylan was degraded to byproduct and 12% of the Klason lignin was not preserved in the dilute acid pretreated CS. Categorically, streams resulting from AFEX-CS displayed significantly better fermentability than those from dilute acid. While dilute acid pretreatment eliminates the need for hemicellulolytic enzymes for hydrolysis, AFEX-centered cellulosic technology simplifies production steps, reduces the requirement for nutrient supplementation, increases the diversity of coproducts and potentially enhances the environmental benefits beyond the direct impact of the pretreatment processes. This is largely due to the nature of the pretreatment chemistries, which reduces inhibitory degradation compound generation and preserves lignin in solid residues while being effective in overcoming biomass recalcitrance that increases the susceptibility of biomass constituents (carbon or nitrogen sources) for digestion.
This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494). We thank Professor Charles Wyman, Dr Bin Yang and Ms Qing Qing for providing facility and training for dilute acid pretreatment. The authors acknowledge Purdue University for granting access to 424A(LNH-ST) and the National Renewable Energy Laboratory (NREL) for dilute acid pretreated (Sund) CS. Thanks to the members of the Biomass Conversion Research Laboratory (BCRL) at Michigan State University for general assistance in the research work particularly Mr Derek Marshall and Mr Charles Donald Jr for preparing AFEX-pretreated CS. We are grateful to Genencor Inc. for supplying enzymes used in this research. We also thank Mr Bryan D Bals for important critical comments on the manuscript.
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