Integrated analysis of hydrothermal flowthroughpretreatment

ABSTRACT: BACKGROUND: The impact of hydrothermal flowthrough (FT) pretreatment severity on pretreatment and solubilization performance metrics was evaluated for three milled feedstocks (corn stover, bagasse, and poplar) and two conversion systems (simultaneous saccharification and fermentation using yeast and fungal cellulase, and fermentation by Clostridium thermocellum). RESULTS: Compared to batch pretreatment, FT pretreatment consistently resulted in higher xylan recovery, higher removal of non-carbohydrate components and higher glucan solubilization by simultaneous saccharification and fermentation (SSF). Xylan recovery was above 90% for FT pretreatment below 4.1 severity but decreased at higher severities, particularly for bagasse. Removal of non-carbohydrate components during FT pretreatment increased from 65% at low severity to 80% at high severity for corn stover, and from 40% to 70% for bagasse and poplar. Solids obtained by FT pretreatment were amenable to high conversion for all of the feedstocks and conversion systems examined. The optimal time and temperature for FT pretreatment on poplar were found to be 16 minutes and 210 oC. At these conditions, SSF glucan conversion was about 85%, 94% of the xylan was removed, and 62% of the non carbohydrate mass was solubilized. Solubilization of FT-pretreated poplar was compared for C. thermocellum fermentation (10% inoculum), and for yeast-fungal cellulase SSF (5% inoculum, cellulase loading of 5 and 10 FPU/g glucan supplemented with beta-glucosidase at 15 and 30 U/g glucan). Under the conditions tested, which featured low solids concentration, C. thermocellum fermentation achieved faster rates and more complete conversion of FT-pretreated poplar than did SSF. Compared to SSF, solubilization by C. thermocellum was 30% higher after 4 days, and was over twice as fast on ball-milled FT-pretreated poplar. CONCLUSIONS: Xylan removal trends were similar between feedstocks whereas glucan conversion trends were significantly different, suggesting that factors in addition to xylan removal impact amenability of glucan to enzymatic attack. Corn stover exhibited higher hydrolysis yields than bagasse or poplar, which could be due to higher removal of non-carbohydrate components. Xylan in bagasse is more easily degraded than xylan in corn stover and poplar. Conversion of FT-pretreated substrates at low concentration was faster and more complete for C.thermocellum than for SSF.     

Influence of xylan on the enzymatic hydrolysis of steam‐pretreated corn stover and hybrid poplar

The focus of this study was to alter the xylan content of corn stover and poplar using SO₂‐catalyzed steam pretreatment to determine the effect on subsequent hydrolysis by commercial cellulase preparations supplemented with or without xylanases. Steam pretreated solids with xylan contents ranging from ～1 to 19% (w/w) were produced. Higher xylan contents and improved hemicellulose recoveries were obtained with solids pretreated at lower severities or without SO₂‐addition prior to pretreatment. The pretreated solids with low xylan content (<4% (w/w)) were characterized by fast and complete cellulose to glucose conversion when utilizing cellulases. Commercial cellulases required xylanase supplementation for effective hydrolysis of pretreated substrates containing higher amounts of xylan. It was apparent that the xylan content influenced both the enzyme requirements for hydrolysis and the recovery of sugars during the pretreatment process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Short‐term lime pretreatment of poplar wood

Short‐term lime pretreatment uses lime and high‐pressure oxygen to significantly increase the digestibility of poplar wood. When the treated poplar wood was enzymatically hydrolyzed, glucan and xylan were converted to glucose and xylose, respectively. To calculate product yields from raw biomass, these sugars were expressed as equivalent glucan and xylan. To recommend pretreatment conditions, the single criterion was the maximum overall glucan and xylan yields using a cellulase loading of 15 FPU/g glucan in raw biomass. On this basis, the recommended conditions for short‐term lime pretreatment of poplar wood follow: (1) 2 h, 140°C, 21.7 bar absolute and (2) 2 h, 160°C, and 14.8 bar absolute. In these two cases, the reactivity was nearly identical, thus the selected condition depends on the economic trade off between pressure and temperature. Considering glucose and xylose and their oligomers produced during 72 h of enzymatic hydrolysis, the overall yields attained under these recommended conditions follow: (1) 95.5 g glucan/100 g of glucan in raw biomass and 73.1 g xylan/100 g xylan in raw biomass and (2) 94.2 g glucan/100 g glucan in raw biomass and 73.2 g xylan/100 g xylan in raw biomass. The yields improved by increasing the enzyme loading. An optimal enzyme cocktail was identified as 67% cellulase, 12% β‐glucosidase, and 24% xylanase (mass of protein basis) with cellulase activity of 15 FPU/g glucan in raw biomass and total enzyme loading of 51 mg protein/g glucan in raw biomass. Ball milling the lime‐treated poplar wood allowed for 100% conversion of glucan in 120 h with a cellulase loading of only 10 FPU/g glucan in raw biomass. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Enzymatic digestion of liquid hot water pretreated hybrid poplar

Liquid hot (LHW) water pretreatment (LHW) of lignocellulosic material enhances enzymatic conversion of cellulose to glucose by solubilizing hemicellulose fraction of the biomass, while leaving the cellulose more reactive and accessible to cellulase enzymes. Within the range of pretreatment conditions tested in this study, the optimized LHW pretreatment conditions for a 15% (wt/vol) slurry of hybrid poplar were found to be 200^oC, 10 min, which resulted in the highest fermentable sugar yield with minimal formation of sugar decomposition products during the pretreatment. The LHW pretreatment solubilized 62% of hemicellulose as soluble oligomers. Hot‐washing of the pretreated poplar slurry increased the efficiency of hydrolysis by doubling the yield of glucose for a given enzyme dose. The 15% (wt/vol) slurry of hybrid poplar, pretreated at the optimal conditions and hot‐washed, resulted in 54% glucose yield by 15 FPU cellulase per gram glucan after 120 h. The hydrolysate contained 56 g/L glucose and 12 g/L xylose. The effect of cellulase loading on the enzymatic digestibility of the pretreated poplar is also reported. Total monomeric sugar yield (glucose and xylose) reached 67% after 72 h of hydrolysis when 40 FPU cellulase per gram glucan were used. An overall mass balance of the poplar‐to‐ethanol process was established based on the experimentally determined composition and hydrolysis efficiencies of the liquid hot water pretreated poplar. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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 H₂O₂ was attempted in the SAA. Addition of H₂O₂ significantly enhanced delignification of hybrid poplar due to its oxidative degradation of lignin. Several different H₂O₂ feeding schemes and different temperature profiles were attempted in operation of the SAA to investigate the effects of H₂O₂ 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°C after 4h of reaction). Increase of carbohydrate degradation was also observed under this condition. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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 alpha-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.     

Fractionation of corn stover by hot-water and aqueous ammonia treatment

The efficiency of biomass utilization can be significantly improved by fractionation of biomass. A two-stage percolation process was investigated for pretreatment and fractionation of corn stover. The two-stage process is composed of hot water treatment followed by treatment with aqueous ammonia, both applied in a flow-through (percolation) reactor. The first stage processing is intended for hemicellulose removal whereas the second stage is intended for delignification. The pretreated material was nearly pure cellulose and both reagents are cheap and environmentally friendly. The conditions that achieve satisfactory level of biomass fractionation and acceptable enzymatic hydrolysis were identified in terms of reaction temperature, flow rate (retention time) and reaction time for each stage. With proper operation of two-stage treatment, fractionation of biomass was achieved to the extent that the xylan fraction is hydrolyzed with 92-95% conversion, and recovered with 83-86% yields; and the lignin removal is 75-81%. The remaining solid after two-stage treatment contained 78-85% cellulose. The two-stage treatments enhanced the enzymatic digestibility to 90-96% with 60 FPU/g of glucan, and 87-89% with 15 FPU/g of glucan. In two-stage treatment, the composition and digestibility data indicate that the lignin content in the biomass is one of the major factors controlling the enzymatic digestibility.     

玉米秸秆不同部位的组成及其对预处理的影响

不同玉米秸秆部位的成分组成及分布对预处理和酶解影响显著。研究表明:韧皮部与髓芯的成分相近,但叶子的差异较大,其木聚糖和总糖的质量分数最高,分别为29.48%和66.15%,而木质素的质量分数最低,因而叶子更容易预处理。玉米秸秆在稀酸预处理过程中可回收96.9%葡聚糖和50.0%～70.0%木聚糖,其中50.0%～60.0%木聚糖水解成木糖溶出;不同部位的木聚糖损失率与初始的木聚糖含量正相关;经稀酸预处理后,叶子中葡聚糖的质量分数最高,达72.40%,叶子和髓芯易于被纤维素酶水解生成葡萄糖,而韧皮部困难。不同部位的酶解得率与自身的葡聚糖含量正相关,与酸不溶木质素含量负相关,同时受原料的物理结构、葡聚糖和木质素大分子的化学组成等影响。     

Ethanol and furfural production from corn stover using a hybrid fractionation process with zinc chloride and simultaneous saccharification and fermentation (SSF)

A two-stage hybrid fractionation process was investigated to produce cellulosic ethanol and furfural from corn stover. In the first stage, zinc chloride (ZnCl₂) was used to selectively solubilize hemicellulose. During the second stage, the remaining treated solids were converted into ethanol using commercial cellulase and Saccharomyces cerevisiae or recombinant Escherichia coli, KO11. This hybrid fractionation process recovered 93.8% of glucan, 89.7% of xylan, 71.1% of arabinan, and 74.9% of lignin under optimal reaction conditions (1st stage: 5% acidified ZnCl₂, 7.5 ml/min, 150 °C (10 min) and 170 °C (10 min); 2nd stage: simultaneous saccharification and fermentation (SSF) using S. cerevisiae). The furfural yield from the hemicellulose hydrolysates was 58%. The SSF of the treated solids resulted in 69–98% of the theoretical maximum ethanol yields based on the glucan content in the treated solids. After fermentation, the solid residues contained primarily lignin. Based on the total lignin in untreated corn stover, the lignin recovery yield was 74.9%.     

Novel Single-step Pretreatment of Steam Explosion and Choline Chloride to De-lignify Corn Stover for Enhancing Enzymatic Edibility

It is important to develop efficient and economically feasible pretreatment methods for lignocellulosic biomass, to increase annual biomass production. A number of pretreatment methods were introduced to promote subsequent enzymatic hydrolysis of biomass for green energy processes. Pretreatment with steam explosion removes the only xylan at high severity but increases lignin content. In this study, corn stover soaked in choline chloride solution before the steam explosion is economically feasible as it reduced cost. Enzymatic hydrolysis of de-lignified corn stover is enhanced by combinatorial pretreatments of steam explosion and choline chloride. Corn stover pretreated with choline chloride at the ratio of 1:2.2 (w/w), 1.0 MPa, 184 °C, for 15 min efficiently expelled 84.7% lignin and 78.9% xylan. The residual solid comprised of 74.59% glucan and 7.51% xylan was changed to 84.2% glucose and 78.3% xylose with enzyme stacking of 10FPU/g. This single-step pretreatment had ∼ 4.5 and 6.4 times higher glucose yield than SE-pretreated and untreated corn stover, respectively. Furthermore, SEM, XRD and FTIR indicated the porosity, crystalline changes, methoxy bond-cleavage respectively due to the lignin and hemicellulose expulsion. Thus, the released acetic acid during this process introduced this novel strategy, which significantly builds the viability of biomass in short pretreatment time.     

Lime pretreatment and enzymatic hydrolysis of corn stover

Corn stover was pretreated with an excess of calcium hydroxide (0.5 g Ca(OH)2/g raw biomass) in non-oxidative and oxidative conditions at 25, 35, 45, and 55 degrees C. The optimal condition is 55 degrees C for 4 weeks with aeration. Glucan (91.3%) and xylan (51.8%) were converted to glucose and xylose respectively, when the treated corn stover was enzymatically hydrolyzed with 15 FPU/g cellulose. Only 0.073 g Ca(OH)2 was consumed per g of raw corn stover. Of the initial lignin, 87.5% was maximally removed. Almost all acetyl groups were removed. After 4 weeks at 55 degrees C with aeration, some cellulose and hemicellulose were solubilized as monomers and oligomers in the pretreatment liquor. When considering the dissolved fragments of glucan and xylan in the pretreatment liquor, the overall yields of glucose and xylose were 93.2% and 79.5% at 15 FPU/g cellulose. The pretreatment liquor has no inhibitory effect on ethanol fermentation.     

Neural network prediction of biomass digestibility based on structural features

Plots of biomass digestibility are linear with the natural logarithm of enzyme loading; the slope and intercept characterize biomass reactivity. The feed-forward back-propagation neural networks were performed to predict biomass digestibility by simulating the 1-, 6-, and 72-h slopes and intercepts of glucan, xylan, and total sugar hydrolyses of 147 poplar wood model samples with a variety of lignin contents, acetyl contents, and crystallinity indices. Regression analysis of the neural network models indicates that they performed satisfactorily. Increasing the dimensionality of the neural network input matrix allowed investigation of the influence glucan and xylan enzymatic hydrolyses have on each other. Glucan hydrolysis affected the last stage of xylan digestion, and xylan hydrolysis had no influence on glucan digestibility. This study has demonstrated that neural networks have good potential for predicting biomass digestibility over a wide range of enzyme loadings, thus providing the potential to design cost-effective pretreatment and saccharification processes.     

Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25 mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO₂, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188.     

Effect of structural features on enzyme digestibility of corn stover

Corn stover was pretreated with excess calcium hydroxide (0.5 g Ca(OH)2/g raw biomass) in non-oxidative and oxidative conditions at 25, 35, 45, and 55 degrees C. The enzymatic digestibility of lime-treated corn stover was affected by the change of structural features (acetylation, lignification, and crystallization) resulting from the treatment. Extensive delignification required oxidative treatment and additional consumption of lime (up to 0.17 g Ca(OH)2/g biomass). Deacetylation reached a plateau within 1 week and there were no significant differences between non-oxidative and oxidative conditions at 55 degrees C; both conditions removed approximately 90% of the acetyl groups in 1 week at all temperatures studied. Delignification highly depended on temperature and the presence of oxygen. Lignin and hemicellulose were selectively removed (or solubilized), but cellulose was not affected by lime pretreatment in mild temperatures (25-55 degrees C), even though corn stover was contacted with alkali for a long time, 16 weeks. The degree of crystallinity slightly increased from 43% to 60% with delignification because amorphous components (lignin, hemicellulose) were removed. However, the increased crystallinity did not negatively affect the 3-d sugar yield of enzymatic hydrolysis. Oxidative lime pretreatment lowered the acetyl and lignin contents to obtain high digestibility, regardless of crystallinity. The non-linear models for 3-d hydrolysis yields of glucan (Y(g)), xylan (Y(x)), and holocellulose (Y(gx)) were empirically established as a function of the residual lignin (L) for the corn stover pretreated with lime and air.     

Liquid hot water and alkaline pretreatment of soybean straw for improving cellulose digestibility

Soybean straw was pretreated with either liquid hot water (LHW) (170-210 °C for 3-10 min) or alkaline soaking (4-40 g NaOH/100 g dry straw) at room temperature to evaluate the effects on cellulose digestibility. Nearly 100% cellulose was recovered in pretreated solids for both pretreatment methods. For LHW pretreatment, xylan dissolution from the raw material increased with pretreatment temperature and time. Cellulose digestibility was correlated with xylan dissolution. A maximal glucose yield of 70.76%, corresponding to 80% xylan removal, was obtained with soybean straw pretreated at 210 °C for 10 min. NaOH soaking at ambient conditions removed xylan up to 46.37% and the subsequent glucose yield of pretreated solids reached up to 64.55%. Our results indicated LHW pretreatment was more effective than NaOH soaking for improving cellulose digestibility of soybean straw.     

Optimization of pH controlled liquid hot water pretreatment of corn stover

Controlled pH, liquid hot water pretreatment of corn stover has been optimized for enzyme digestibility with respect to processing temperature and time. This processing technology does not require the addition of chemicals such as sulfuric acid, lime, or ammonia that add cost to the process because these chemicals must be neutralized or recovered in addition to the significant expense of the chemicals themselves. Second, an optimized controlled pH, liquid hot water pretreatment process maximizes the solubilization of the hemicellulose fraction as liquid soluble oligosaccharides while minimizing the formation of monomeric sugars. The optimized conditions for controlled pH, liquid hot water pretreatment of a 16% slurry of corn stover in water was found to be 190 degrees C for 15 min. At the optimal conditions, 90% of the cellulose was hydrolyzed to glucose by 15FPU of cellulase per gram of glucan. When the resulting pretreated slurry, in undiluted form, was hydrolyzed by 11FPU of cellulase per gram of glucan, a hydrolyzate containing 32.5 g/L glucose and 18 g/L xylose was formed. Both the xylose and the glucose in this undiluted hydrolyzate were shown to be fermented by recombinant yeast 424A(LNH-ST) to ethanol at 88% of theoretical yield.     

Low-liquid pretreatment of corn stover with aqueous ammonia

A low-liquid pretreatment method of corn stover using aqueous ammonia was studied to reduce the severity and liquid throughput associated with the pretreatment step for ethanol production. Corn stover was treated at 0.5-50.0 wt.% of ammonia loading, 1:0.2-5.0 (w/w) of solid-to-liquid ratio, 30 °C for 4-12 weeks. The effects of these conditions on the composition and enzyme digestibility of pretreated corn stover were investigated. Pretreatment of corn stover at 30 °C for four weeks using 50 wt.% of ammonia loading and 1:5 solid-to-liquid ratio resulted in 55% delignification and 86.5% glucan digestibility with 15 FPU cellulase + 30 CBU β-glucosidase/g-glucan.Simultaneous saccharification and fermentation of corn stover treated at 30 °C for four weeks using 50 wt.% ammonia loading and 1:2 solid-to-liquid ratio gave an ethanol yield of 73% of the theoretical maximum based on total carbohydrates (glucan + xylan) present in the untreated material.     

Effect of biological pretreatment with Trametes hirsuta yj9 on enzymatic hydrolysis of corn stover

In this study, a newly isolated Trametes hirsuta yj9 was used to pretreat corn stover in order to enhance enzymatic digestibility. T. hirsuta yj9 preferentially degraded lignin to be as high as 71.49% after 42-day pretreatment. Laccase and xylanase was the major ligninolytic and hydrolytic enzyme, respectively and filter paper activity (FPA) increased gradually with prolonged pretreatment time. Sugar yields increased significantly after pretreatment with T. hirsuta yj9, reaching an enzymatic digestibility of 73.99% after 42 days of pretreatment. Scanning electron microscopy (SEM) showed significant structural changes in pretreated corn stover, the surface of pretreated corn stover became increasingly coarse, the gaps between cellulose fibers were visible, and many pores were developed. Correlation analysis showed that sugar yields were inversely proportional to the lignin contents, less related to cellulose and hemicellulose contents.     

Bioconversion of acid-hydrolyzed poplar hemicellulose to acetic acid byClostridium thermoaceticum

Summary Clostridium thermoaceticum was used to ferment carbohydrate released from pretreated oat splet xylan and hemicellulose isolated from hybrid poplar. Hydrolysis with dilute sulfuric acid (2.5% (v/v) for oat spelt xylan and 4.0% (v/v) for poplar hemicellulose) at 100°C for 60 min was found to release the highest concentration of fermentable substrate.C. thermoaceticum, when grown in non-pH controlled batch culture at 55°C under a headspace of 100% CO₂, typically produced 14gl^–1 acetic acid during a 48 h fermentation in medium containing 2% xylose. In fed-batch fermentations this organism was able to produce 42gl^–1 acetic acid after 116h when the concentration of xylose was maintained at approximately 2% and the pH was controlled at 7.0.     

Mild pretreatment of yellow poplar biomass using sequential dilute acid and enzymatically-generated peracetic acid to enhance cellulase accessibility

Biomass contains cellulose, xylan and lignin in a complex interwoven structure that hinders enzymatic hydrolysis of the cellulose. To separate these components in yellow poplar biomass, we sequentially pretreated with dilute sulfuric acid and enzymatically-generated peracetic acid. In the first step, the dilute acid with microwave heating (140°C, 5 min) hydrolyzed 90% of xylan. The xylose yield in hydrolysate after dilute acid pretreatment was 83.1%. In the second step, peracetic acid (60°C, 6 h) removed up to 80% of lignin. This sequential pretreatment fractionated biomass into xylan and lignin, leaving a solid residue enriched in cellulose (~80%). The sequential pretreatment enhanced enzymatic digestibility of the cellulase by removal of the other components in biomass. The glucose yield after enzymatic hydrolysis was 90.5% at a low cellulase loading (5 FPU/g of glucan), which is 1.6 and 18 times higher than for dilute acid-pretreated biomass and raw biomass, respectively. This novel sequential pretreatment with dilute acid and peracetic acid efficiently separates the three major components of yellow poplar biomass, and reduces the amount of cellulase needed.