Ammonia Recycled Percolation Research Articles

Reflowed Aqua Ammonia Explosion Pretreatment for Corn Stover

Abstract: Based on ammonia recycled percolation (ARP) and combined with the advantage of ammonia fiber explosion (AFEX), a set of reflowed aqua ammonia explosion pretreatment (RAAEP) equipment was developed. According to the previous orthogonal experiment experimental results, the optimal pretreatment conditions were at 90°C, 9 min retention time, water to dry corn stover loading of 0.8:1(w/w), and aqua ammonia flow rate is 1.5L/min. The cellulose content of the pretreated corn stover was 50.46 % and the glucan and xylan enzymatic digestibility was 86.45 % and 95.77% respectively. The reflowed aqua ammonia not only caused fiber swelling and an efficient delignification (up to 62.5%) of biomass, but also altered the cellulose crystalline structure. The SEM picture and FTIR spectra confirmed swelling and significant delignification effects of the RAAEP process. The X-ray crystallography data indicated that the rigid structure of corn stover was disrupted with more crystallization fiber being exposed after the pretreatment process, but the crystallinity increased due to the removal of amorphous materials (lignin and hemicelluloses).

Comparison of inhibition effects of various isolated lignins on enzymatic hydrolysis of cellulose

Various isolated soluble and insoluble lignins isolated from ammonia recycle percolation (ARP), dilute sulfuric acid, and hot-water pretreatments and two commercially available lignins (alkali and organo-solv lignin) were tested to evaluate their inhibitory effects on the enzymatic hydrolysis of cellulose. The purpose of this study was to compare the effects by adding various isolated lignins in the enzyme hydrolysis. The results showed that enzyme reaction was generally inhibited by addition of lignins. The order of lignins from most to least inhibitory effects were alkali, ARP-soluble, water-soluble, acid-soluble, ARP-insoluble, and organo-solv lignins. With 0.344 g ARP-soluble or alkali lignin/g-glucan lignin loadings, 72-h digestibilities were decreased by 16% and 20%, respectively. It was also observed that the digestibilities were decreased, as the lignin loadings increased. The hindering effect by ARP-insoluble or organosolv lignin was not significant.

Sequential hydrolysis of hemicellulose and lignin in lignocellulosic biomass by two-stage percolation process using dilute sulfuric acid and ammonium hydroxide

To obtain the total fractionation and pretreatment of the corn stover, two-stage percolation process was investigated. This process consists of two steps: use of 0.07 wt% sulfuric acid for hemicellulose recovery in first stage and ARP (ammonia recycled percolation) in the following stage for lignin recovery. Among tested conditions, the best conditions of two-stage process were as follows: 1st stage; 170 °C, 2.5 ml/min, 30 minutes using 0.07 wt% sulfuric acid and 2nd stage; 170 °C, 5.0 ml/min, 60minutes using 15 wt% ammonium hydroxide. At above two-stage treatment conditions, the hemicellulose in corn stover was easily hydrolyzed (95%) and recovered with high yields (86%) and the extent of the lignin removal was 81%. After two-stage process, the treated biomass contained nearly pure glucan (85%). Two-stage treatment brought about enzymatic digestibility of 90% and 89% with 60 and 15 FPU/g glucan cellulase enzyme loadings, respectively.

Summary of findings from the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI): corn stover pretreatment

The Biomass Refining Consortium for Applied Fundamentals and Innovation, with members from Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, has developed comparative data on the conversion of corn stover to sugars by several leading pretreatment technologies. These technologies include ammonia fiber expansion pretreatment, ammonia recycle percolation pretreatment, dilute sulfuric acid pretreatment, flowthrough pretreatment (hot water or dilute acid), lime pretreatment, controlled pH hot water pretreatment, and sulfur dioxide steam explosion pretreatment. Over the course of two separate USDA- and DOE-funded projects, these pretreatment technologies were applied to two different corn stover batches, followed by enzymatic hydrolysis of the remaining solids from each pretreatment technology using identical enzyme preparations, enzyme loadings, and enzymatic hydrolysis assays. Identical analytical methods and a consistent material balance methodology were employed to develop comparative sugar yield data for each pretreatment and subsequent enzymatic hydrolysis. Although there were differences in the profiles of sugar release, with the more acidic pretreatments releasing more xylose directly in the pretreatment step than the alkaline pretreatments, the overall glucose and xylose yields (monomers + oligomers) from combined pretreatment and enzymatic hydrolysis process steps were very similar for all of these leading pretreatment technologies. Some of the water-only and alkaline pretreatment technologies resulted in significant amounts of residual xylose oligomers still remaining after enzymatic hydrolysis that may require specialized enzyme preparations to fully convert xylose oligomers to monomers.

Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies

Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO(2)) and pure Avicel glucan was measured at 4 degrees C, as were adsorption and desorption of cellulase and adsorption of beta-glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (sigma(solids) = 195 mg/g solid) followed by dilute acid (sigma(solids) = 170.0 mg/g solid) and lime pretreated solids (sigma(solids) = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (sigma(solids) = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (sigma(cellulose) = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (sigma(lignin) = 57 mg/g lignin) followed by dilute acid lignin (sigma(lignin) = 74 mg/g lignin). AFEX lignin also had the lowest beta-glucosidase capacity (sigma(lignin) = 66.6 mg/g lignin), while lignin from SO(2) (sigma(lignin) = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO(2) followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute acid pretreated solids inhibited Avicel hydrolysis less than ARP and flowthrough lignin, whereas acid lignin from unpretreated poplar inhibited enzymes the most. Irreversible binding of cellulase to lignin varied with pretreatment type and desorption method.

Does change in accessibility with conversion depend on both the substrate and pretreatment technology?

The accessibility of cellulase and xylanase enzymes to glucan and xylan, respectively, and its change with conversion were measured for pure Avicel glucan and poplar solids that had been pretreated by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), dilute acid, and lime. Avicel and pretreated solids were digested to various degrees by cellulase together with β-glucosidase enzymes and then cleaned of residual protein via a biological method using Protease. Glucan accessibility was determined by purified CBHI (Cel7A) adsorption at 4 °C, and 4 and 24 h hydrolysis yields were determined for solids loading containing equal amounts of glucan (1.0% w/v) and lignin (1.0% w/v), in two separate sets of experiments. Consistent with our previous study and in contrast to some in the literature, little change in glucan accessibility was observed with conversion for Avicel, but glucan and xylan accessibility for real biomass varied with the type of pretreatment. For example, AFEX pretreated solids showed a negligible change in glucan accessibility for conversion up to 90%, although xylan accessibility seemed to decline first and then remained constant. On the other hand, a substantial decline in glucan and xylan accessibility with conversion was observed for lime pretreated poplar solids, as shown by initial hydrolysis rates. Yet, an increase in CBHI adsorption with conversion for lime pretreated poplar solids suggested the opposite trend, possibly due to increased lignin exposure and/or reduced effectiveness of adsorbed enzyme.

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO 2) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with β-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and β-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and β-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase–xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.

Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies

In order to investigate changes in substrate chemical and physical features after pretreatment, several characterizations were performed on untreated (UT) corn stover and poplar and their solids resulting pretreatments by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough, lime, and SO 2 technologies. In addition to measuring the chemical compositions including acetyl content, physical attributes determined were biomass crystallinity, cellulose degree of polymerization, cellulase adsorption capacity of pretreated solids and enzymatically extracted lignin, copper number, FT-IR responses, scanning electron microscopy (SEM) visualizations, and surface atomic composition by electron spectroscopy of chemical analysis (ESCA). Lime pretreatment removed the most acetyl groups from both corn stover and poplar, while AFEX removed the least. Low pH pretreatments depolymerized cellulose and enhanced biomass crystallinity much more than higher pH approaches. Lime pretreated corn stover solids and flowthrough pretreated poplar solids had the highest cellulase adsorption capacity, while dilute acid pretreated corn stover solids and controlled pH pretreated poplar solids had the least. Furthermore, enzymatically extracted AFEX lignin preparations for both corn stover and poplar had the lowest cellulase adsorption capacity. ESCA results showed that SO 2 pretreated solids had the highest surface O/C ratio for poplar, but for corn stover, the highest value was observed for dilute acid pretreatment with a Parr reactor. Although dependent on pretreatment and substrate, FT-IR data showed that along with changes in cross linking and chemical changes, pretreatments may also decrystallize cellulose and change the ratio of crystalline cellulose polymorphs (Iα/Iβ).

Session 6: Advances in Enzyme Science and Technology

As ethanol from lignocellulosic materials progresses to commercial fruition, finer details of the hydrolysis process are being investigated. In this session, papers were presented that dealt with several aspects of biomass conversion including substrate effects, additives to the saccharification reaction, and high performance enzymes. The influence of each one of these aspects on hydrolysis performance was analyzed by different authors. Rajeev Kumar from Dartmouth College described how the choice of pretreatment technology influences cellulose accessibility. He investigated how cellulose accessibility in poplar solids changed depending on the pretreatment method. The pretreatment methods used were ammonia fiber expansion (AFEX), ammonia recycle percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide. The main conclusion was that cellulase enzyme site accessibility is influenced by the type of pretreatment as well as by the type and composition of the substrate. Maobing Tu from the Pulp and Paper Research Institute of Canada investigated the influence of added surfactant (Tween 80) on cellulose conversion of steam-exploded lodgepole pine and ethanol-pretreated lodgepole pine. He showed that the presence of Tween 80 resulted in a significant increase in the cellulose-to-glucose yield for the steamexploded substrate, but had no effect for the ethanol-treated substrate. In addition, the presence of surfactant increased the amount of free enzyme for both substrates implying that surfactant might be minimizing non-productive binding of cellulases to the surface. Lastly, the presence of surfactant did not affect final ethanol yields in SSF reactions with yeast. Feng Xu from Novozymes observed that cellulose oxidation had a profound impact on the rate and extent of cellulose hydrolysis by single enzymes (cellobiohydrolases) and complete cellulase mixtures (Trichoderma reesei-secreted proteins). Thermochemical pretreatments may negatively affect enzymatic hydrolysis by producing inhibitory Appl Biochem Biotechnol (2009) 154:302–303 DOI 10.1007/s12010-009-8613-0

Pretreatment of hybrid poplar by aqueous ammonia

Enzymatic hydrolysis of hybrid poplar treated by ammonia recycle percolation (ARP) was studied applying cellulase enzyme supplemented with additional xylanase or pectinase. The effect of xylanase addition was much more significant than pectinase addition. Conversion of ARP-treated hybrid poplar to ethanol was carried out by simultaneous saccharification and fermentation (SSF) and SS and cofermentation (SSCF). The maximum ethanol yield observed from the SSCF experiment was 78% of theoretical maximum based on the total carbohydrate (glucan + xylan). The same feedstock was also treated by soaking in aqueous ammonia (SAA), a batch pretreatment process with lower severity than ARP. The test results indicated that relatively high severity is required to attain acceptable level of digestibility of hybrid poplar. In order to lower the severity of the pretreatment, addition of H(2)O(2) was attempted in the SAA. Addition of H(2)O(2) significantly enhanced delignification of hybrid poplar due to its oxidative degradation of lignin. Several different H(2)O(2) feeding schemes and different temperature profiles were attempted in operation of the SAA to investigate the effects of H(2)O(2) on degradation of lignin and carbohydrates in hybrid poplar. More than 60% of lignin in hybrid poplar was removed with stepwise-increase of temperature (60-120 degrees C after 4h of reaction). Increase of carbohydrate degradation was also observed under this condition.

Effects of cellulase and xylanase enzymes on the deconstruction of solids from pretreatment of poplar by leading technologies

Comparative data is presented on glucose and xylose release for enzymatic hydrolysis of solids produced by pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough (FT), lime, and sulfur dioxide (SO(2)) technologies. Sugar solubilization was measured for times of up to 72 h using cellulase supplemented with beta-glucosidase at an activity ratio of 1:2, respectively, at combined protein mass loadings of 5.8-116 mg/g of glucan in poplar wood prior to pretreatment. In addition, the enzyme cocktail was augmented with up to 11.0 g of xylanase protein per gram of cellulase protein at combined cellulase and beta-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose to evaluate cellulase-xylanase interactions. All pretreated poplar solids required high protein loadings to realize good sugar yields via enzymatic hydrolysis, and performance tended to be better for low pH pretreatments by dilute sulfuric acid and sulfur dioxide, possibly due to higher xylose removal. Glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments, xylanase leverage on glucan removal decreased at high cellulase loadings. Washing the solids improved digestion for all pretreatments and was particularly beneficial for controlled pH pretreatment. Furthermore, incubation of pretreated solids with BSA, Tween 20, or PEG6000 prior to adding enzymes enhanced yields, but the effectiveness of these additives varied with the type of pretreatment.

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO(2)) pretreatments were measured at 4 degrees C. Langmuir adsorption parameters were then estimated by non-linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4 degrees C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO(2) pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness.

Effect of additives on the digestibility of corn stover solids following pretreatment by leading technologies

Bovine serum albumin (BSA), Tween-20, and polyethylene glycol (PEG6000) were added to washed corn stover solids produced by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), dilute sulfuric acid (DA), lime, controlled pH, and sulfur dioxide (SO(2)) pretreatments and to untreated corn stover (UT) and pure Avicel glucan prior to adding cellulase supplemented with beta-glucosidase at an activity ratio of 1:2/g and a moderate enzyme loading of 16.1 mg/g glucan in the raw corn stover. The additives were applied individually at 150, 300, and 600 mg/g glucan in the pretreated solids and in combinations of equal amounts of each that totaled 600 mg/g. The greatest increase in total sugar release was by Tween-20 with SO(2) pretreated solids followed by PEG6000 with ARP solids and Tween-20 with lime solids. The effectiveness of the additives was observed to depend on the type of sugars left in the solids, suggesting that it may be more beneficial to use the mixture of these additives to realize a high total sugar yield. In addition, little enhancement in sugar release was possible beyond a loading of 150 mg additives/g glucan for most pretreatments, and combinations did not improve sugar release much over use of additives alone for all except SO(2). Additives were also found to significantly increase concentrations of cellobiose and cellooligomers after 72 h of Avicel hydrolysis.

Substrate Dependency and Effect of Xylanase Supplementation on Enzymatic Hydrolysis of Ammonia-Treated Biomass

Pretreatment based on aqueous ammonia was investigated under two different modes of operation: soaking in aqueous ammonia and ammonia recycle percolation. These processes were applied to three different feedstocks with varied composition: corn stover, high lignin (HL), and low lignin (LL) hybrid poplars. One of the important features of ammonia-based pretreatment is that most of the hemicellulose is retained after treatment, which simplifies the overall bioconversion process and enhances the conversion efficiency. The pretreatment processes were optimized for these feedstocks, taking carbohydrate retention as well as sugar yield in consideration. The data indicate that hybrid poplar is more difficult to treat than corn stover, thus, requires more severe conditions. On the other hand, hybrid poplar has a beneficial property that it retains most of the hemicellulose after pretreatment. To enhance the digestibility of ammonia-treated poplars, xylanase was supplemented during enzymatic hydrolysis. Because of high retention of hemicellulose in treated hybrid poplar, xylanase supplementation significantly improved xylan as well as glucan digestibility. Of the three feedstocks, best results and highest improvement by xylanase addition was observed with LL hybrid poplar, showing 90% of overall sugar yield.

Pretreatment of Corn Stover by Low-Liquid Ammonia Recycle Percolation Process

A pretreatment method using aqueous ammonia was investigated with the intent of minimizing the liquid throughput. This process uses a flowthrough packed column reactor (or percolation reactor). In comparison to the ammonia recycle percolation (ARP) process developed previously in our laboratory, this process significantly reduces the liquid throughput to one reactor void volume in packed bed (2.0-4.7 mL of liquid/g of corn stover) and, thus, is termed low-liquid ARP (LLARP). In addition to attaining short residence time and reduced energy input, this process achieves 59-70% of lignin removal and 48-57% of xylan retention. With optimum operation of the LLARP to corn stover, enzymatic digestibilities of 95, 90, and 86% were achieved with 60, 15, and 7.5 filter paper units/g of glucan, respectively. In the simultaneous saccharification and fermentation test of the LLARP samples using Saccharomyces cerevisiae (NREL-D5A), an ethanol yield of 84% of the theoretical maximum was achieved with 6% (w/v) glucan loading. In the simultaneous saccharification and cofermentation (SSCF) test using recombinant Escherichia coli (KO11), both the glucan and xylan in the solid were effectively utilized, giving an overall ethanol yield of 109% of the theoretical maximum based on glucan, a clear indication that the xylan content was converted into ethanol. The xylooligomers existing in the LLARP effluent were not effectively hydrolyzed by cellulase enzyme, achieving only 60% of digestibility. SSCF of the treated corn stover was severely hampered when the substrate was supplemented with the LLARP effluent, giving only 56% the overall yield of ethanol. The effluent appears to significantly inhibit cellulase and microbial activities.

Process and economic analysis of pretreatment technologies

Five pretreatment processes (dilute acid, hot water, ammonia fiber explosion (AFEX), ammonia recycle percolation (ARP), and lime) for the liberation of sugars from corn stover are compared on a consistent basis. Each pretreatment process model was embedded in a full bioethanol facility model so that systematic effects for variations in pretreatment were accounted in the overall process. Economic drivers influenced by pretreatment are yield of both five and six carbon sugars, solids concentration, enzyme loading and hemicellulase activity. All of the designs considered were projected to be capital intensive. Low cost pretreatment reactors in some pretreatment processes are often counterbalanced by higher costs associated with pretreatment catalyst recovery or higher costs for ethanol product recovery. The result is little differentiation between the projected economic performances of the pretreatment options. Additional process performance data, especially involving the identification of optimal enzyme blends for each pretreatment approach and conditioning requirements of hydrolyzates at process-relevant sugar concentrations resulting from each pretreatment may lead to greater differentiation in projected process economics.

Pretreatment and fractionation of corn stover by ammonia recycle percolation process

Corn stover was pretreated with aqueous ammonia in a flow-through column reactor, a process termed as Ammonia Recycle Percolation (ARP). The aqueous ammonia causes swelling and efficient delignification of biomass at high temperatures. The ARP process solubilizes about half of xylan, but retains more than 92% of the cellulose content. Enzymatic digestibility of ARP-treated corn stover is 93% with 10 FPU/g-glucan enzyme loading. The SEM pictures and FTIR spectra confirm swelling and delignification effects of the ARP process. The X-ray crystallography data indicate that the basic crystalline structure of the cellulosic component of corn stover is not altered by the ARP treatment. Low-liquid ARP can reduce the liquid throughput and residence time to 3.3 mL/g-biomass and 10–12 min, without adversely affecting the overall effectiveness. The low-water ARP achieved 73.4% delignification and 88.5% digestibility with 15 FPU/g-glucan. The ethanol yield from the SSF of low-liquid ARP-treated corn stover using Saccharomyces cerevisiae reached 84% of the theoretical maximum. Successive operation of a hot-water treatment and the ARP was applied as a method of biomass fractionation. The two-stage process separated xylan in the first stage (84%) and lignin in the second stage (75%), resulting treated solid that contains 79% glucan.

Pretreatment of corn stover by aqueous ammonia

Corn stover was pretreated with aqueous ammonia in a flow-through column reactor, a process termed ammonia recycled percolation (ARP). This method was highly effective in delignifying of the biomass, reducing the lignin content by 70–85%. Most lignin removal occurred within the first 20 min of the process. Lignin removal by ARP was further confirmed by FTIR analysis and lignin staining. The ARP process solubilized 40–60% of the hemicellulose but left the cellulose intact. The solubilized carbohydrate existed in oligomeric form. Carbohydrate decomposition during the pretreatment was insignificant. Corn stover treated for 90 min exhibited enzymatic digestibility of 99% with 60 FPU/g of glucan enzyme loading, and 92.5% with 10 FPU/g of glucan. The digestibility of ARP treated corn stover was substantially higher than that of α-cellulose. The enzymatic digestibility was related with the removal of lignin and hemicellulose, perhaps due to increased surface area and porosity. The SEM pictures indicated that the biomass structure was deformed and its fibers exposed by the pretreatment. The crystallinity index increased with pretreatment reflecting removal of the amorphous portion of biomass. The crystalline structure of the cellulose in the biomass, however, was not changed by the ARP treatment.

Ammonia recycled percolation as a complementary pretreatment to the dilute-acid process

A two-stage dilute-acid percolation (DA) was investigated as a pretreatment method for switchgrass. With use of extremely low acid (0.078 wt% sulfuric acid) under moderate temperature (145-170 degrees C), hemicellulose in switchgrass was completely solubilized showing no sugar decomposition. The treated switchgrass contained about 70% glucan and 30% lignin. The high lignin content in the treated feedstock raises a concern that it may cause a high enzyme consumption because of irreversible adsorption of cellulase enzymes to lignin. This problem may be amplified in the SSF operation since it is usually run in fed-batch mode and the residual lignin is accumulated. The DA pretreatment was, therefore, combined with the ammonia recycled percolation (ARP) process that has been proven to be effective in delignification. The combined pretreatment essentially fractionated the switchgrass into three major components. The treated feedstock contained about 90% glucan and 10% lignin. The digestibility of these samples was consistently higher that that of DA treated samples. Further study on the interaction of cellulase with xylan and that with lignin has shown that the enzymatic hydrolysis of cellulose is inhibited by lignin as well as xylan. The external xylan was found to be a noncompetitive inhibitor to cellulose hydrolysis. The cellulase used in this study was proven to have the xylanase activity.

Ammonia-recycled percolation process for pretreatment of biomass feedstock

An ammonia-based biomass pretreatment method named ammonia recycled percolation (ARP) process was investigated. The process characteristics are: 1. Aqueous ammonia is used as a pretreatment reagent; 2. A packed bed flowthrough-type reactor (percolation reactor) is used in recirculation mode; 3. ARP-Ammonia is continuously recycled.