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.     

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.     

Aqueous ammonia pretreatment, saccharification, and fermentation evaluation of oil palm fronds for ethanol production

Oil palm fronds are the most abundant lignocellulosic biomass in Malaysia. In this study, fronds were tested as the potential renewable biomass for ethanol production. The soaking in aqueous ammonia pretreatment was applied, and the fermentability of pretreated fronds was evaluated using simultaneous saccharification and fermentation. The optimal pretreatment conditions were 7?% (w/w) ammonia, 80?°C, 20?h of pretreatment, and 1:12 S/L ratio, where the enzymatic digestibility was 41.4?% with cellulase of 60?FPU/g-glucan. When increasing the cellulase loading in the hydrolysis of pretreated fronds, the enzymatic digestibility increased until the enzyme loading reached 60?FPU/g-glucan. With 3?% glucan loading in the SSF of pretreated fronds, the ethanol concentration and yield based on the theoretical maximum after 12 and 48?h of the SSF were 7.5 and 9.7?g/L and 43.8 and 56.8?%, respectively. The ethanol productivities found at 12 and 24?h from pretreated fronds were 0.62 and 0.36?g/L/h, respectively.     

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     

Aqueous ammonia pretreatment of oil palm empty fruit bunches for ethanol production

Oil palm empty fruit bunches (EFB) were pretreated by aqueous ammonia soaking for ethanol production. Pretreated EFB, which were pretreated at the optimal conditions of 60 °C, 12 h, and 21% (w/w) aqueous ammonia, showed 19.5% and 41.4% glucose yields during an enzymatic digestibility test for 96 h when using 15 and 60 FPU of cellulase, respectively. Using the pretreated EFB, simultaneous saccharification and fermentation for 168 h with 5% (w/v) glucan loading and 60 FPU of cellulase and 30 CBU of β-glucosidase per gram glucan resulted in ethanol production of 18.6 g/L titer, 65.6% of theoretical maximum yield, and 0.11 g/L/h of productivity.     

Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars

Miscanthus x giganteus is a tall perennial grass whose suitability as an energy crop is presently being appraised. There is very little information on the effect of pretreatment and enzymatic saccharification of Miscanthus to produce fermentable sugars. This paper reports sugar yields during enzymatic hydrolysis from ammonia fiber expansion (AFEX) pretreated Miscanthus. Pretreatment conditions including temperature, moisture, ammonia loading, residence time, and enzyme loadings are varied to maximize hydrolysis yields. In addition, further treatments such as soaking the biomass prior to AFEX as well as washing the pretreated material were also attempted to improve sugar yields. The optimal AFEX conditions determined were 160 degrees C, 2:1 (w/w) ammonia to biomass loading, 233% moisture (dry weight basis), and 5 min reaction time for water-soaked Miscanthus. Approximately 96% glucan and 81% xylan conversions were achieved after 168 h enzymatic hydrolysis at 1% glucan loading using 15 FPU/(g of glucan) of cellulase and 64 p-NPGU/(g of glucan) of beta-glucosidase along with xylanase and tween-80 supplementation. A mass balance for the AFEX pretreatment and enzymatic hydrolysis process is presented.     

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.     

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     

Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol

Wheat straw consists of 48.57 ± 0.30% cellulose and 27.70 ± 0.12% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for production of ethanol. Dilute acid pretreatment at varied temperature and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from wheat straw (7.83%, w/v, DS) by dilute H₂SO₄ (0.75%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase and esterase was 565 ± 10 mg/g. Under this condition, no measurable quantities of furfural and hydroxymethyl furfural were produced. The yield of ethanol (per litre) from acid pretreated enzyme saccharified wheat straw (78.3 g) hydrolyzate by recombinant Escherichia coli strain FBR5 was 19 ± 1 g with a yield of 0.24 g/g DS. Detoxification of the acid and enzyme treated wheat straw hydrolyzate by overliming reduced the fermentation time from 118 to 39 h in the case of separate hydrolysis and fermentation (35 °C, pH 6.5), and increased the ethanol yield from 13 ± 2 to 17 ± 0 g/l and decreased the fermentation time from 136 to 112 h in the case of simultaneous saccharification and fermentation (35 °C, pH 6.0).     

Consolidated bioprocessing (CBP) performance of Clostridium phytofermentans on AFEX-treated corn stover for ethanol production

Consolidated bioprocessing (CBP) is believed to be a potentially cost-efficient and commercially viable way to produce cellulosic biofuels. In this study, we have evaluated the performance of the CBP organism Clostridium phytofermentans (ATCC 700394) on AFEX-treated corn stover (AFEX-CS). Fermentation conditions including temperature, inoculation size, nutrients, and initial pH were investigated. At optimal conditions with 0.5% (w/w) glucan loading of AFEX-CS, C. phytofermentans hydrolyzed 76% of glucan and 88.6% of xylan in 10 days. These values reached 87% and 102% of those obtained by simultaneous saccharification and co-fermentation (SSCF) using commercial enzymes and S. cerevisiae 424A. Ethanol titer for CBP was found to be 2.8 g/L which was 71.8% of that yielded by SSCF (3.9 g/L). Decomposition products from AFEX-CS helped to increase ethanol yield somewhat during CBP. Particle size played a crucial role in the enhancement of sugar conversion by CBP.     

Characterization of a bifunctional xylanase/endoglucanase from yak rumen microorganisms

A new gene, RuCelA, encoding a bifunctional xylanase/endoglucanase, was cloned from a metagenomic library of yak rumen microorganisms. RuCelA showed activity against xylan and carboxymethylcellulose (CMC), suggesting bifunctional xylanase/endoglucanase activity. The optimal conditions for xylanase and endoglucanase activities were 65°C, pH 7.0 and 50°C, pH 5.0, respectively. In addition, the presence of Co⁺ and Co²⁺ can greatly improve RuCelA's endoglucanase activity, while inhibits its xylanase activity. Further examination of substrate preference showed a higher activity against barley glucan and lichenin than against xylan and CMC. Using xylan and barley glucan as substrates, RuCelA displayed obvious synergistic effects with β-1,4-xylosidase and β-1,4-glucosidase. Generation of soluble oligosaccharides from lignocellulose is the key step in bioethanol production, and it is greatly notable that RuCelA can produce xylo-oligosaccharides and cello-oligosaccharides in the continuous saccharification of pretreated rice straw, which can be further degraded into fermentable sugars. Therefore, the bifunctional RuCelA distinguishes itself as an ideal candidate for industrial applications.     

Ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (SFS) with Kluyveromyces marxianus CECT 10875

Simultaneous saccharification and fermentation (SSF) process for ethanol production from various lignocellulosic woody (poplar and eucalyptus) and herbaceous (Sorghum sp. bagasse, wheat straw and Brassica carinata residue) materials has been assayed using the thermotolerant yeast strain Kluyveromyces marxianus CECT 10875. Biomass samples were previously treated in a steam explosion pilot plant to provide pretreated biomass with increased cellulose content relative to untreated materials and to enhance cellulase accessibility. SSF experiments were performed in laboratory conditions at 42 °C, 10% (w/v) substrate concentration and 15 FPU/g substrate of commercial cellulase. The results indicate that it is possible to reach SSF yields in the range of 50–72% of the maximum theoretical SSF yield, based on the glucose available in pretreated materials, in 72–82 h. Maximum ethanol contents from 16 to 19 g/l were obtained in fermentation media, depending on the material tested.     

High titer ethanol production from simultaneous enzymatic saccharification and fermentation of aspen at high solids: a comparison between SPORL and dilute acid pretreatments

Native aspen (Populus tremuloides) was pretreated using sulfuric acid and sodium bisulfite (SPORL) and dilute sulfuric acid alone (DA). Simultaneous enzymatic saccharification and fermentation (SSF) was conducted at 18% solids using commercial enzymes with cellulase loadings ranging from 6 to 15 FPU/g glucan and Saccharomyces cerevisiae Y5. Compared with DA pretreatment, the SPORL pretreatment reduced the energy required for wood chip size-reduction, and reduced mixing energy of the resultant substrate for solid liquefaction. Approximately 60% more ethanol was produced from the solid SPORL substrate (211 L/ton wood at 59 g/L with SSF efficiency of 76%) than from the solid DA substrate (133 L/ton wood at 35 g/L with SSF efficiency 47%) at a cellulase loading of 10 FPU/g glucan after 120 h. When the cellulase loading was increased to 15 FPU/g glucan on the DA substrate, the ethanol yield still remained lower than the SPORL substrate at 10 FPU/g glucan.     

Conversion of paper sludge to ethanol in a semicontinuous solids-fed reactor

Conversion of paper sludge to ethanol was investigated with the objective of operating under conditions approaching those expected of an industrial process. Major components of the bleached Kraft sludge studied were glucan (62 wt.%, dry basis), xylan (11.5%), and minerals (17%). Complete recovery of glucose during compositional analysis required two acid hydrolysis treatments rather than one. To avoid the difficulty of mixing unreacted paper sludge, a semicontinuous solids-fed laboratory bioreactor system was developed. The system featured feeding at 12-h intervals, a residence time of 4 days, and cellulase loading of 15 to 20 FPU/g cellulose. Sludge was converted to ethanol using simultaneous saccharification and fermentation (SSF) featuring a -glucosidase-supplemented commercial cellulase preparation and glucose fermentation by Saccharomyces cerevisiea. SSF was carried out for a period of 4 months in a first-generation system, resulting in an average ethanol concentration of 35 g/L. However, steady state was not achieved and operational difficulties were encountered. These difficulties were avoided in a retrofitted design that was operated for two 1-month runs, achieving steady state with good material balance closure. Run 1 with the retrofitted reactor produced 50 g/L ethanol at a cellulose conversion of 74%. Run 2 produced 42 g/L ethanol at a conversion of 92%. For run 2, the ethanol yield was 0.466 g ethanol/g glucose equivalent fermented and >94% of the xylan fed to the reactor was solubilized to a mixture of xylan oligomers and xylose.     

稀碱预处理棕榈残渣制备纤维乙醇

以棕榈残渣(Empty fruit bunch,EFB)为原料,通过预处理、酶解、发酵等过程制备纤维乙醇.首先对比了碱、碱/过氧化氢等预处理条件对棕榈残渣组成及酶解的影响,结果表明稀碱预处理效果较好.适宜的稀碱预处理条件为:NaOH浓度为1％,固液比为1∶10,在40℃浸泡24 h后于121℃下保温30 min,在该条件下,EFB的固体回收率为74.09％,纤维素、半纤维素和木质素的含量分别为44.08％、25.74％和13.89％.对该条件下预处理后的固体样品,以底物浓度5％、酶载量30 FPU/g底物酶解72 h,纤维素和半纤维素的酶解率分别达到84.44％和89.28％.进一步考察了酶载量和底物浓度对酶解的影响以及乙醇批式同步糖化发酵,当酶载量为30 FPU/g底物,底物浓度由5％增加至25％时,利用酿酒酵母Saccharomyces cerevisiae(接种量为5％,VIV)发酵72 h后乙醇的浓度分别为9.76 g/L和35.25 g/L,可分别达到理论得率的79.09％和56.96％.     

Investigation of enzyme formulation on pretreated switchgrass

This work studied the benefits of adding different enzyme cocktails (cellulase, xylanase, β-glucosidase) to pretreated switchgrass. Pretreatment methods included ammonia fiber expansion (AFEX), dilute-acid (DA), liquid hot water (LHW), lime, lime + ball-milling, soaking in aqueous ammonia (SAA), and sulfur dioxide (SO₂). The compositions of the pretreated materials were analyzed and showed a strong correlation between initial xylan composition and the benefits of xylanase addition. Adding xylanase dramatically improved xylan yields for SAA (+8.4%) and AFEX (+6.3%), and showed negligible improvement (0-2%) for the pretreatments with low xylan content (dilute-acid, SO₂). Xylanase addition also improved overall yields with lime + ball-milling and SO₂ achieving the highest overall yields from pretreated biomass (98.3% and 93.2%, respectively). Lime + ball-milling obtained an enzymatic yield of 92.3 kg of sugar digested/kg of protein loaded.     

Pretreatment of corn stover using low-moisture anhydrous ammonia (LMAA) process

A simple pretreatment method using anhydrous ammonia was developed to minimize water and ammonia inputs for cellulosic ethanol production, termed the low moisture anhydrous ammonia (LMAA) pretreatment. In this method, corn stover with 30–70% moisture was contacted with anhydrous ammonia in a reactor under nearly ambient conditions. After the ammoniation step, biomass was subjected to a simple pretreatment step at moderate temperatures (40–120 °C) for 48–144 h. Pretreated biomass was saccharified and fermented without an additional washing step. With 3% glucan loading of LMAA-treated corn stover under best treatment conditions (0.1 g-ammonia + 1.0 g-water per g biomass, 80 °C, and 84 h), simultaneous saccharification and cofermentation test resulted in 24.9 g/l (89% of theoretical ethanol yield based on glucan + xylan in corn stover).     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

Purification and characterization of xylanase from Aspergillus ficuum AF-98

The purification and characterization of xylanase from Aspergillus ficuum AF-98 were investigated in this work. The extracellular xylanase from this fungal was purified 32.6-fold to homogeneity throughout the precipitation with 50–80% (NH₄)₂SO₄, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified xylanase (specific activity at 288.7 U/ mg protein) was a monomeric protein with a molecular mass of 35.0 kDa as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 45 °C and 5.0, respectively. The xylanase was activated by Cu²⁺ up to 115.8% of activity, and was strongly inhibited by Hg²⁺, Pb²⁺ up to 52.8% and 89%, respectively. The xylanase exhibited K_m and V_max values of 3.267 mg/mL, 18.38 M/min/mg for beechwood xylan and 3.747 mg/mL, 11.1 M/min/mg for birchwood xylan, respectively.     

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.