AFEX Pretreatment Research Articles

Ammonia Fiber Expansion (Afex) Pretreatment Combined with White Rot Fungi Treatment to Improve Enzymatic Hydrolysis of Lignocellulose

Ammonia Fiber Expansion (Afex) Pretreatment Combined with White Rot Fungi Treatment to Improve Enzymatic Hydrolysis of Lignocellulose

Combined Effects of Ammonia Fiber Expansion (Afex) Pretreatment Severity and Enzymatic Hydrolysis Time on Microporous Structure of Corn Stover

Combined Effects of Ammonia Fiber Expansion (Afex) Pretreatment Severity and Enzymatic Hydrolysis Time on Microporous Structure of Corn Stover

A high solids field-to-fuel research pipeline to identify interactions between feedstocks and biofuel production

BackgroundEnvironmental factors, such as weather extremes, have the potential to cause adverse effects on plant biomass quality and quantity. Beyond adversely affecting feedstock yield and composition, which have been extensively studied, environmental factors can have detrimental effects on saccharification and fermentation processes in biofuel production. Only a few studies have evaluated the effect of these factors on biomass deconstruction into biofuel and resulting fuel yields. This field-to-fuel evaluation of various feedstocks requires rigorous coordination of pretreatment, enzymatic hydrolysis, and fermentation experiments. A large number of biomass samples, often in limited quantity, are needed to thoroughly understand the effect of environmental conditions on biofuel production. This requires greater processing and analytical throughput of industrially relevant, high solids loading hydrolysates for fermentation, and led to the need for a laboratory-scale high solids experimentation platform.ResultsA field-to-fuel platform was developed to provide sufficient volumes of high solids loading enzymatic hydrolysate for fermentation. AFEX pretreatment was conducted in custom pretreatment reactors, followed by high solids enzymatic hydrolysis. To accommodate enzymatic hydrolysis of multiple samples, roller bottles were used to overcome the bottlenecks of mixing and reduced sugar yields at high solids loading, while allowing greater sample throughput than possible in bioreactors. The roller bottle method provided 42–47% greater liquefaction compared to the batch shake flask method for the same solids loading. In fermentation experiments, hydrolysates from roller bottles were fermented more rapidly, with greater xylose consumption, but lower final ethanol yields and CO2 production than hydrolysates generated with shake flasks. The entire platform was tested and was able to replicate patterns of fermentation inhibition previously observed for experiments conducted in larger-scale reactors and bioreactors, showing divergent fermentation patterns for drought and normal year switchgrass hydrolysates.ConclusionA pipeline of small-scale AFEX pretreatment and roller bottle enzymatic hydrolysis was able to provide adequate quantities of hydrolysate for respirometer fermentation experiments and was able to overcome hydrolysis bottlenecks at high solids loading by obtaining greater liquefaction compared to batch shake flask hydrolysis. Thus, the roller bottle method can be effectively utilized to compare divergent feedstocks and diverse process conditions.

Pre-senescence Harvest of Switchgrass Inhibits Xylose Utilization by Engineered Yeast

Proper timing of switchgrass harvest for bioenergy is important to maximize yield and optimize end use conversion. Proposed windows range from peak biomass to the following spring after overwintering in the field. There are various pros and cons associated with harvest timing: earlier harvests maximize yield but can remove nutrients from the field that may require replacement, while later harvests have reduced biomass yields due to weathering but maximize nutrient resorption in belowground tissues. Switchgrass composition changes during the harvest period, with losses of potential fermentation nutrients (amino acids and minerals), and sources of pretreatment-derived inhibitors (soluble sugars), which could affect downstream conversion by microorganisms. For this work we investigated whether switchgrass harvest could be timed to maximize beneficial impacts on fermentation. Switchgrass samples were harvested from five replicate field plots in Wisconsin, roughly every 2-3 weeks from peak biomass (Aug. 20) until after the killing frost (Nov. 7). Cell wall composition showed little consistent variation with harvest date while bulk biomass analysis showed a relative increase in cell wall content (lignin and structural sugars) and loss of extractives (minerals, protein, soluble sugars, and others). Following high or low severity AFEX pretreatment and high solids enzymatic hydrolysis (6% glucan loading), two field replicates were fermented using Saccharomyces cerevisiae 424A, a strain engineered to utilize xylose in addition to glucose. For both pretreatment severities, S. cerevisiae 424A grown in hydrolysates from the three earlier harvests utilized only a small fraction of available xylose, while almost complete utilization occurred within 96 hr for the last three harvest dates. Detailed analysis of the hydrolysate low molecular weight aromatics did not indicate any compounds potentially responsible for the inhibition, with most of the observed variation in their concentration due to pretreatment severity. Amino acid composition also did not appear to be limiting. Current indications point to a plant-generated compound that degrades during senescence, which future work will attempt to identify. Ultimately this work demonstrates that, although an attractive option to maximize yield, harvesting switchgrass before it begins senescing could have a negative effect on downstream conversion processes.

Techno-economic comparison of centralized versus decentralized biorefineries for two alkaline pretreatment processes

In this work, corn stover subjected to ammonia fiber expansion (AFEX™)1AFEX™, is a trademark of MBI International.1 pretreatment or alkaline pre-extraction followed by hydrogen peroxide post-treatment (AHP pretreatment) were compared for their enzymatic hydrolysis yields over a range of solids loadings, enzymes loadings, and enzyme combinations. Process techno-economic models were compared for cellulosic ethanol production for a biorefinery that handles 2000tons per day of corn stover employing a centralized biorefinery approach with AHP or a de-centralized AFEX pretreatment followed by biomass densification feeding a centralized biorefinery. A techno-economic analysis (TEA) of these scenarios shows that the AFEX process resulted in the highest capital investment but also has the lowest minimum ethanol selling price (MESP) at $2.09/gal, primarily due to good energy integration and an efficient ammonia recovery system. The economics of AHP could be made more competitive if oxidant loadings were reduced and the alkali and sugar losses were also decreased.

Spatially Explicit Life Cycle Analysis of Cellulosic Ethanol Production Scenarios in Southwestern Michigan

Modeling the life cycle of fuel pathways for cellulosic ethanol (CE) can help identify logistical barriers and anticipated impacts for the emerging commercial CE industry. Such models contain high amounts of variability, primarily due to the varying nature of agricultural production but also because of limitations in the availability of data at the local scale, resulting in the typical practice of using average values. In this study, 12 spatially explicit, cradle-to-refinery gate CE pathways were developed that vary by feedstock (corn stover, switchgrass, and Miscanthus), nitrogen application rate (higher, lower), pretreatment method (ammonia fiber expansion [AFEX], dilute acid), and co-product treatment method (mass allocation, sub-division), in which feedstock production was modeled at the watershed scale over a nine-county area in Southwestern Michigan. When comparing feedstocks, the model showed that corn stover yielded higher global warming potential (GWP), acidification potential (AP), and eutrophication potential (EP) than the perennial feedstocks of switchgrass and Miscanthus, on an average per area basis. Full life cycle results per MJ of produced ethanol demonstrated more mixed results, with corn stover-derived CE scenarios that use sub-division as a co-product treatment method yielding similarly favorable outcomes as switchgrass- and Miscanthus-derived CE scenarios. Variability was found to be greater between feedstocks than watersheds. Additionally, scenarios using dilute acid pretreatment had more favorable results than those using AFEX pretreatment.

Evaluation of agave bagasse recalcitrance using AFEX™, autohydrolysis, and ionic liquid pretreatments

A comparative analysis of the response of agave bagasse (AGB) to pretreatment by ammonia fiber expansion (AFEX™), autohydrolysis (AH) and ionic liquid (IL) was performed using 2D nuclear magnetic resonance (NMR) spectroscopy, wet chemistry, enzymatic saccharification and mass balances. It has been found that AFEX pretreatment preserved all carbohydrates in the biomass, whereas AH removed 62.4% of xylan and IL extracted 25% of lignin into wash streams. Syringyl and guaiacyl lignin ratio of untreated AGB was 4.3, whereas for the pretreated biomass the ratios were 4.2, 5.0 and 4.7 for AFEX, AH and IL, respectively. Using NMR spectra, the intensity of β-aryl ether units in aliphatic, anomeric, and aromatic regions decreased in all three pretreated samples when compared to untreated biomass. Yields of glucose plus xylose in the major hydrolysate stream were 42.5, 39.7 and 26.9kg per 100kg of untreated AGB for AFEX, IL and AH, respectively.

Saccharification of newspaper waste after ammonia fiber expansion or extractive ammonia.

The lignocellulosic fractions of municipal solid waste (MSW) can be used as renewable resources due to the widespread availability, predictable and low pricing and suitability for most conversion technologies. In particular, after the typical paper recycling loop, the newspaper waste (NW) could be further valorized as feedstock in biorefinering industry since it still contains up to 70 % polysaccharides. In this study, two different physicochemical methods—ammonia fiber expansion (AFEX) and extractive ammonia (EA) were tested for the pretraetment of NW. Furthermore, based on the previously demonstrated ability of the recombinant enzymes endocellulase rCelStrep, α-l-arabinofuranosidase rPoAbf and its evolved variant rPoAbf F435Y/Y446F to improve the saccharification of different lignocellulosic pretreated biomasses (such as corn stover and Arundo donax), in this study these enzymes were tested for the hydrolysis of pretreated NW, with the aim of valorizing the lignocellulosic fractions of the MSW. In particular, a mixture of purified enzymes containing cellulases, xylanases and accessory hemicellulases, was chosen as reference mix and rCelStrep and rPoAbf or its variant were replaced to EGI and Larb. The results showed that these enzymatic mixes are not suitable for the hydrolysis of NW after AFEX or EA pretreatment. On the other hand, when the enzymes rCelStrep, rPoAbf and rPoAbf F435Y/Y446F were tested for their effect in hydrolysis of pretreated NW by addition to a commercial enzyme mixture, it was shown that the total polysaccharides conversion yield reached 37.32 % for AFEX pretreated NW by adding rPoAbf to the mix whilst the maximum sugars conversion yield for EA pretreated NW was achieved 40.80 % by adding rCelStrep. The maximum glucan conversion yield obtained (45.61 % for EA pretreated NW by adding rCelStrep to the commercial mix) is higher than or comparable to those reported in recent manuscripts adopting hydrolysis conditions similar to those used in this study.

Scaling up and benchmarking of ethanol production from pelletized pilot scale AFEX treated corn stover using Zymomonas mobilis 8b

ABSTRACTThis report outlines the recent scale-up of AFEX pretreatment from the laboratory to pilot scale. Sugar yields were improved by 19 and 15% for glucose and xylose, respectively. Further improvement was achieved when scaling up the hydrolysis and fermentation of AFEX-treated corn stover to 2500 L working volume. Benchmarking was performed using CTec 3 and HTec 3 enzymes along with Zymomonas mobilis 8b. Subsequently, the seed train was modified to use a hydrolysate-based medium as a replacement for pure sugars and nutrients. Fermentation performance was comparable following this change. Economic analysis showed a 19% reduction in MESP for the new process over the previous benchmark process.

Chemical Composition of Native and Ammonia Fiber Expansion Pretreated Rice Straw-Unextracted versus Extractives-free Material

Characterization of chemical composition for lignocellulosic biomasss (LCB) is essential in the conversion process of LCB to bioenergy and biochemicals. Accurate quantification of chemical composition allows for better determination of the LCB conversion and product yield in LCB processing. However, the presence of the extractable material is stated to strongly influence the compositional analysis of the LCB. This study was conducted to analyze and compare the chemical composition of (A) native-whole unextracted rice straw of (1) untreated rice straw (URS) and (2) AFEX pretreated rice straw (AC1RS and AC2RS) as well as (B) the extractives-free rice straw of (1) URS and (2) AFEX pretreated rice straw (AC1RS and AC2RS). The effect of the AFEX pretreatment on the composition of rice straw was determined using the extractives-free material of URS, AC1RS and AC2RS. The actual reported composition of native-whole rice straw of URS and AFEX pretreated rice straw were made based on the corrected values of the extractives-free rice straw of URS and AFEX pretreated rice straw. The rice straw was analyzed for structural constituents such as glucan, xylan, arabinan and Klason lignin as well as non-structural constituents. The results demonstrated that the extractives in native-whole unextracted rice straw significantly interfere with the analysis of Klason lignin. The lignin content of the rice straw was overestimated if the extractives were not removed prior to the compositional analysis. The extractives in rice straw statistically did not affect the carbohydrate analyses. However, some soluble sugars were removed from the rice straw during the extraction process.

Bioethanol production from hemicellulose rich Populus nigra involving recombinant hemicellulases from Clostridium thermocellum

Bioethanol was produced from poplar leafy biomass rich in hemicelluloses content involving recombinant Clostridium thermocellum hemicellulases and pentose sugar utilizing Candida shehatae. FT-IR analysis revealed effective AFEX pretreatment of poplar leaves. Repetitive batch strategy yielded ∼1.5-fold rise in cell biomass and specific activity of both, acetylxylanesterase (Axe) and GH43 hemicellulase. TLC and HPAEC exhibited xylose and arabinose release from hydrolyzed biomass. SSF trial with 1% (wv−1) pretreated poplar and mixed enzymes showed ∼1.5-fold higher ethanol titre as compared with SHF. The shake flask SSF with 5% (wv−1) pretreated poplar furnished 4.56 and 5.43gL−1 ethanol with Axe and mixed enzymes, respectively. Whereas, bioreactor scale-up exhibited ∼1.25-fold increase in ethanol titres (5.68, 6.75gL−1) as compared with shake flask with an yield of 0.295 (gg−1) and 0.351 (gg−1), respectively with Axe and mixed enzymes.

Effects of compositional changes of AFEX-treated and H-AFEX-treated corn stover on enzymatic digestibility

Corn stover is one of the main agricultural residues being considered as a cellulosic ethanol feedstock. This work evaluated the effectiveness of AFEX™(1) pretreatment for converting corn stover to fermentable sugars, both with and without pre-soaking in hydrogen peroxide. The compositional changes and enzymatic digestibility of AFEX-treated and H-AFEX-treated biomass were investigated. Results showed that most of the polysaccharides remained intact following each of these two methods. Compared with AFEX pretreatment, the H-AFEX process enhanced delignification and enzymatic hydrolysis yields of both glucose and xylose. The maximum glucan and xylan digestibility of H-AFEX process were 87.78% and 90.64%, respectively, and were obtained using 0.7 (w/w) water loading, 1.0 (w/w) ammonia loading, 0.5 (w/w) 30wt.% hydrogen peroxide loading, and 130°C for 10min. The results of the present work show that H-AFEX is a feasible pretreatment to improve the enzymatic saccharification of corn stover for bioethanol production.

Enzymatic hydrolysis of pelletized AFEX™‐treated corn stover at high solid loadings

Ammonia fiber expansion (AFEX™) pretreatment can be performed at small depots, and the pretreated biomass can then be pelletized and shipped to a centralized refinery. To determine the feasibility of this approach, pelletized AFEX-treated corn stover was hydrolyzed at high (18-36%) solid loadings. Water absorption and retention by the pellets was low compared to unpelletized stover, which allowed enzymatic hydrolysis slurries to remain well mixed without the need for fed-batch addition. Glucose yields of 68% and xylose yields of 65% were obtained with 20 mg enzyme/g glucan and 18% solid loading after 72 h, compared to 61% and 59% for unpelletized corn stover. Pelletization also slightly increased the initial rate of hydrolysis compared to unpelletized biomass. The ease of mixing and high yields obtained suggests that pelletization after AFEX pretreatment could have additional advantages beyond improved logistical handling of biomass.

Liquid AFEX Pretreatment and Enzymatic Hydrolysis of Switchgrass from Different Harvest and Storage Conditions

Abstract. Switchgrass harvest and storage conditions increasingly affected a representative biochemical process and theoretical ethanol yield. The approach used a novel liquid ammonia fiber explosion (LAFEX) pretreatment technique implemented through engineered introduction of ammonia in liquid phase to biomass with controlled pressures, temperatures, and a mechanical liquid pump. The LAFEX pretreatment involved pressurizing an AFEX reactor with nitrogen prior to adding ammonia, and then nitrogen pressure was increased as the reactor temperature was increased to 100°C after the addition of ammonia. LAFEX pretreatment improved Alamo switchgrass xylan digestibility up to 63.3% compared to AFEX pretreatment that had published levels of xylan digestibility up to 38.3%, all using Accellerase enzyme for enzymatic hydrolysis. This improvement occurred at less water loading (0.8 vs. 2.0 g H 2 O per g dry switchgrass), less ammonia loading (1.0 vs. 1.6 and 2.0 g NH 3 per g dry switchgrass), lower temperature (100°C vs. 160°C and 150°C), and increased pretreatment time (120 vs. 30 and 25 min) for LAFEX vs. AFEX pretreatments, respectively. Additional potential for LAFEX pretreatment was noted based on published studies documenting enzymes that outperform the enzyme selected for this study. A wide range of switchgrass field harvest conditions, including one-cut versus two-cut strategies and harvest times ranging from June to January, indicated relatively small differences in fresh switchgrass cellulose and hemicellullose levels (64% to 68%) determined with wet chemistry, and associated trends in lignin increases (21.9% to 25.7%) and ash decreases (5.3% to 2.2%) across samples obtained at harvest time, not including storage effects. Baseline theoretical ethanol yield for freshly harvested samples ranged from 462 to 485 L per dry Mg. Field storage conditions included covered and uncovered bales and piles of field-chopped switchgrass and ensiled field-chopped switchgrass. Field storage conditions affected cellulose and hemicellulose levels, as determined by wet chemistry, for theoretical ethanol yield (243 to 477 L per dry Mg) to a greater degree than variation due to field harvest conditions. Implementation of enzymatic hydrolysis for 72 h with Accellerase 1000 introduced to LAFEX-pretreated samples represented a biorefinery process for theoretical ethanol yield based on fresh harvest conditions (232 to 270 L per dry Mg) and revealed an exaggerated effect of storage conditions on theoretical ethanol yield (79 to 283 L per dry Mg). Essentially, switchgrass that got wet and stayed wet, compared to switchgrass that stayed dry or was allowed a drying cycle if exposed to precipitation during storage, reduced available cellulose and hemicelluloses (to ~50%) and further reduced the available sugars from enzymatic hydrolysis (to ~30%). It should be noted that no amount of improved pretreatment or enzymatic hydrolysis can overcome or recover the degraded sugars that occurred at the bale bottom under wet conditions. Overall results demonstrated the supply logistics challenge to deliver biomass feedstock with predictable, uniform conditions year-round to allow consistent biochemical conversion at commercial scale. Harvesting and storage conditions could also affect biochemical processes associated with ethanol fermentation that were not investigated.

Hydrogen peroxide presoaking of bamboo prior to AFEX pretreatment and impact on enzymatic conversion to fermentable sugars

Bamboo is a fast growing plant found worldwide that has high potential as an energy crop. This project evaluated the effectiveness of AFEX pretreatment for converting moso bamboo (Phyllostachys heterocycla var. pubescens) to fermentable sugars, both with and without pre-soaking in hydrogen peroxide. Pretreatment conditions including temperature, water loading, residence time, ammonia loading, and hydrogen peroxide loadings were varied to maximize hydrolysis yields. The optimal conditions for AFEX were 150°C, 0.8 or 2.0 (w/w) water loading, 10-30 min residence time, and 2.0-5.0 (w/w) ammonia loading. The optimal conditions for H-AFEX were same AFEX conditions with 0.7-1.9 (w/w) 30% (wt) hydrogen peroxide solutions loading. Using 15 FPU/g glucan cellulase and under optimal conditions, AFEX pretreatment achieved a theoretical sugars yield of 64.8-72.7% and addition of hydrogen peroxide presoaking increased the yield to 83.4-92.1%. It is about 5-fold and 7-fold increase in sugars yield for AFEX-treated and H-AFEX-treated bamboo respectively.

Low Temperature and Long Residence Time AFEX Pretreatment of Corn Stover

Low temperature and long residence time pretreatments have been proposed as an alternative to conventional pretreatments within a centralized biorefinery, allowing for a decentralized pretreatment without high energy costs. Ammonia fiber expansion (AFEX™) pretreatment may be uniquely suitable for decentralized pretreatment, and this study considers the possibility of decreasing the temperature in AFEX pretreatment of corn stover. AFEX pretreatment at 40°C and 8 h produced comparable sugar and ethanol yields as conventional AFEX pretreatment at high temperatures and short residence time during subsequent hydrolysis and fermentation. Increasing the ammonia loading at these temperatures tends to increase digestibility, although the moisture content of the reaction has little effect. This study suggests a greater flexibility in AFEX pretreatment conditions than previously thought, allowing for an alternative approach for decentralized facilities if the economic conditions are appropriate.

Enzymatic digestibility and pretreatment degradation products of AFEX‐treated hardwoods (Populus nigra)

There is a growing need to find alternatives to crude oil as the primary feed stock for the chemicals and fuel industry and ethanol has been demonstrated to be a viable alternative. Among the various feed stocks for producing ethanol, poplar (Populus nigra x Populus maximowiczii) is considered to have great potential as a biorefinery feedstock in the United States, due to their widespread availability and good productivity in several parts of the country. We have optimized AFEX pretreatment conditions (180 degrees C, 2:1 ammonia to biomass loading, 233% moisture, 30 minutes residence time) and by using various combinations of enzymes (commercical celluloses and xylanases) to achieve high glucan and xylan conversion (93 and 65%, respectively). We have also identified and quantified several important degradation products formed during AFEX using liquid chromatography followed by mass spectrometry (LC-MS/MS). As a part of degradation product analysis, we have also quantified oligosaccharides in the AFEX water wash extracts by acid hydrolysis. It is interesting to note that corn stover (C4 grass) can be pretreated effectively using mild AFEX pretreatment conditions, while on the other hand hardwood poplar requires much harsher AFEX conditions to obtain equivalent sugar yields upon enzymatic hydrolysis. Comparing corn stover and poplar, we conclude that pretreatment severity and enzymatic hydrolysis efficiency are dictated to a large extent by lignin carbohydrate complexes and arabinoxylan cross-linkages for AFEX.