Low Cellulase Loading Research Articles

Improving the enzymatic hydrolysis of dilute acid pretreated wheat straw by metal ion blocking of non-productive cellulase adsorption on lignin

Eleven salts were selected to screen the possible metal ions for blocking the non-productive adsorption of cellulase onto the lignin of dilute acid pretreated wheat straw. Mg2+ was screened finally as the promising candidate. The optimal concentration of MgCl2 was 1mM, but the beneficial action was also dependent on pH, hydrolysis time and cellulase loading. Significant improvement of glucan conversion (19.3%) was observed at low cellulase loading (5FPU/g solid). Addition of isolated lignins, tannic acid and lignin model compounds to pure cellulose hydrolysis demonstrated that phenolic hydroxyl group (Ph-OH) was the main active site blocked by Mg2+. The interaction between Mg2+ and Ph-OH of lignin monomeric moieties followed an order of p-hydroxyphenyl (H)>guaiacyl (G)>syringyl (S). Mg2+ blocking made the lignin surface less negatively charged, which might weaken the hydrogen bonding and electrostatically attractive interaction between lignin and cellulase enzymes.

Cell wall disruption in low temperature NaOH/urea solution and its potential application in lignocellulose pretreatment

Pretreatments of wheat straw by NaOH/urea solvent at low temperature were investigated. To understand the cell wall disruption during this low temperature process, and its impacts on enzymatic hydrolysis, morphology, cellulose crystal structure, and chemical properties were investigated by using the following instruments: optical microscopy, confocal laser scanning microscopy, Fourier transform infrared spectra, and X-ray diffraction. The results implied that the deconstruction of plant cell wall at low temperature was attributed to disruption of the hydrogen bonds in cellulose and solubilization of hemicellulose and lignin. Meanwhile, the pretreatment approach resulted in almost full recovery of cellulose, approximately 60 % of lignin and 70 % of xylan removal, respectively. It’s interesting to note that cellulose I crystal structure in the substrate pretreated at a solid loading of 10 % was partially changed to cellulose II structure, while wheat straw pretreated at a higher solid loading of 20 %, retained the cellulose I structure. Almost complete saccharification (>95 %) of cellulose in pretreated substrates was achieved at a relatively low cellulase loading of 10 FPU/g substrates within 48 h. The loss of xylan in pretreated substrate had a negative effect on the total sugar recovery.

Using a combined hydrolysis factor to optimize high titer ethanol production from sulfite-pretreated poplar without detoxification

Sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) was applied to poplar NE222 chips in a range of chemical loadings, temperatures, and times. The combined hydrolysis factor (CHF) as a pretreatment severity accurately predicted xylan dissolution by SPORL. Good correlations between CHF and pretreated solids enzymatic digestibility, sugar yield, and the formations of furfural and acetic acid were obtained. Therefore, CHF was used to balance sugar yield with the formation of fermentation inhibitors for high titer ethanol production without detoxification. The results indicated that optimal sugar yield can be achieved at CHF=3.1, however, fermentation using un-detoxified whole slurries of NE222 pretreated at different severities by SPORL indicated CHF≈2 produced best results. An ethanol titer of 41g/L was achieved at total solids of approximately 20wt% without detoxification with a low cellulase loading of 15FPU/g glucan (27mL/kg untreated wood).

A comparison of fermentation strategies for cellulosic ethanol production from NaOH-soaked sugarcane bagasse at high solid loading with decreased cellulase loading

NaOH-soaked sugarcane bagasse (ASSB) could be converted into monosaccharides by low cellulase loading at a solid loading of 2%. However, different initial solid loading affected the enzymatic convertibility. Therefore, the effects of solid and cellulase loadings were investigated in this work. Four fermentation strategies, simultaneous saccharification and fermentation (SSF), fed-batch SSF, semi-SSF, and fed-batch semi-SSF were conducted and compared. The results showed that cellulose convertibility decreased with increased initial solid loading from 1.9 to 7.5% in a linear manner. However, the decrease was not obvious when 10% of initial solid was loaded compared to that of 7.5%. Based on the enzymatic hydrolysis at a constant cellulose dose, 7.5FPU/g dry matter, the initial solid loading of 10% was selected for the fed-batch fermentations. The final ethanol yields among the fermentations showed no significant differences. Presaccharification increased the ethanol productivity when the presaccharification time was not counted. Glycerol concentration in fermentations with the presaccharification stage was higher than those without this stage.

Enhancing enzymatic hydrolysis of crystalline cellulose and lignocellulose by adding long-chain fatty alcohols

The effects of long-chain fatty alcohols (LFAs) on the enzymatic hydrolysis of crystalline cellulose by two commercial Trichoderma reesei cellulase cocktails (CTec2 and Celluclast 1.5L) were studied. It was found that n-butanol inhibited the enzymatic hydrolysis, but n-octanol, n-decanol and n-dodecanol had strong enhancement on enzymatic hydrolysis of crystalline cellulose in the buffer pH range from 4.0 to 6.0. LFAs can increase the hydrolysis efficiency of crystalline cellulose from 37 to 57 % at Celluclast 1.5L loading of ten filter paper units (FPU)/g glucan. LFAs have similar enhancement on the enzymatic hydrolysis of crystalline cellulose mixed with lignin or xylan. The enhancement of LFAs increased with the decrease of the crystallinity index. LFAs not only enhanced the high-solid enzymatic hydrolysis of lignocellulose, but also improved the rheological properties of high-solid lignocellulosic slurries by decreasing the yield stress and complex viscosity. Meanwhile, LFAs can improve the enzymatic hydrolysis of cellobiose to glucose, especially at low cellulase loading.

Ethanol production from non-detoxified whole slurry of sulfite-pretreated empty fruit bunches at a low cellulase loading.

Sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) was applied to an empty fruit bunches (EFB) for ethanol production. SPORL facilitated delignification through lignin sulfonation and dissolution of xylan to result in a highly digestible substrate. The pretreated whole slurry was enzymatically saccharified at a solids loading of 18% using a relatively low cellulase loading of 15 FPU/g glucan and simultaneously fermented without detoxification using Saccharomyces cerevisiae of YRH400. An ethanol yield of 217 L/tonne EFB was achieved at titer of 32 g/L. Compared with literature studies, SPORL produced high ethanol yield and titer with much lower cellulase loading without detoxification.

Ethanol production from xylan-removed sugarcane bagasse using low loading of commercial cellulase.

Xylan was always extracted as the feedstock for xylooligosaccharides production. The xylan-removed residue may contain high content of cellulose and thus had a possibility to be converted into ethanol. After soaked in 12% of NaOH at room temperature overnight, solubilization of cellulose, xylan, and lignin was 4.64%, 72.06%, and 81.87% respectively. The xylan-removed sugarcane bagasse (XRSB) was enzymatically hydrolyzed by using decreased cellulase loadings. The results showed that 7.5 FPU/g cellulose could obtain a cellulose conversion yield of 82%. Increasing the cellulase loading did not result in higher yield. Based on this, bioethanol production was performed using 7.5 FPU/g cellulose by employing fed-batch fermentation mode. The final ethanol concentration reached 40.59 g/L corresponding to 74.2% of the theoretical maximum. The high titer ethanol and low cellulase loading may reduce the overall cost.

Multiple effects of swelling by sodium bicarbonate after delignification on enzymatic saccharification of rice straw

The multiple effects of pretreatments by chemical delignification using acidified sodium chlorite (ASC) and swelling using sodium bicarbonate (SB) for enzymatic saccharification of rice straw in bioethanol production have been investigated in this study. The treatment with the combination of ASC three times (3× ASC) first and SB later resulted in the significant reduction in Klason lignin content up to 90% (wt./wt.). By the saccharification of the pretreated rice straw with cellulase enzymes, it was confirmed that SB treatment was an important step in the pretreatment process not only to disintegrate the cellulose structure but also to facilitate the amorphization of the crystalline cellulose as well as the extended removal of integrated lignin. Furthermore, FTIR analyses revealed that the crystal type of cellulose appeared to be changed from type I to type II by SB treatment, thereby increasing the cellulose surface area and making it more accessible to the cellulase enzyme. Conversion rate to sugar was remarkably increased when 3× ASC + SB treatments were applied to untreated rice straw, even though the saccharification of the treated rice straw was performed at a low enzyme loading (1/100, wt.-enzymes/wt.-substrate). Conclusively, rice straw could be saccharified at high yield in short time at low cellulase loading, enables the enzymatic saccharification to be more feasible for practical bioethanol production using rice straw as a substrate.

Recombinant EXLX1 from Bacillus subtilis for enhancing enzymatic hydrolysis of corn stover with low cellulase loadings

BsEXLX1 protein from Bacillus subtilis has been proposed to have a structure that is similar to plant expansin. In this study, the recombinant BsEXLX1 protein was successfully expressed and purified in Escherichia coli BL21 (DE3). When the purified BsEXLX1 which contained the thioredoxin (Trx) protein was incubated with low-dose cellulases either simultaneously or sequentially, it showed a significant synergistic activity in corn stover hydrolysis. Furthermore, an even greater increase in the synergistic activity was obtained when cellulose was pretreated with BsEXLX1 followed by cellulase hydrolysis, and the synergistic activity was found as high as 1.5-fold greater than that when cellulose was treated simultaneously with the same concentrations of BsEXLX1 and cellulases. These results provided a feasible way for the potential application of BsEXLX1 in the efficient saccharification of cellulose materials for bioethanol production. Key word: Bacillus subtilis , BsEXLX1, cellulase, cellulose hydrolysis, bioethanol

Accessibility of Enzymatically DelignifiedBambusa bambosfor Efficient Hydrolysis at Minimum Cellulase Loading: An Optimization Study

In the present investigation, Bambusa bambos was used for optimization of enzymatic pretreatment and saccharification. Maximum enzymatic delignification achieved was 84%, after 8 h of incubation time. Highest reducing sugar yield from enzyme-pretreated Bambusa bambos was 818.01 mg/g dry substrate after 8 h of incubation time at a low cellulase loading (endoglucanase, β-glucosidase, exoglucanase, and xylanase were 1.63 IU/mL, 1.28 IU/mL, 0.08 IU/mL, and 47.93 IU/mL, respectively). Enzyme-treated substrate of Bambusa bambos was characterized by analytical techniques such as Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The FTIR spectrum showed that the absorption peaks of several functional groups were decreased after enzymatic pretreatment. XRD analysis indicated that cellulose crystallinity of enzyme-treated samples was increased due to the removal of amorphous lignin and hemicelluloses. SEM image showed that surface structure of Bambusa bambos was distorted after enzymatic pretreatment.

Relatively High-Substrate Consistency Hydrolysis of Steam-Pretreated Sweet Sorghum Bagasse at Relatively Low Cellulase Loading

Sweet sorghum bagasse (SSB) was steam pretreated in the conditions of 190 °C for 5 min to assess its amenability to the pretreatment and enzymatic hydrolysis. Results showed that pretreatment conditions were robust enough to pretreat SSB with maximum of 87% glucan and 72% xylan recovery. Subsequent enzymatic hydrolysis showed that the pretreated SSB at 2% substrate consistency resulted in maximum of 70% glucan-glucose conversion. Increasing substrate consistency from 2% to 16% led to a significant reduction in glucan conversion. However, the decrease ratio of glucan-glucose conversion was the minimum when the consistency increased from 2% to 12%. When the pretreated SSB consistency of 12% was applied for hydrolysis, increase in cellulase loading from 7.5 up to 20 filter paper units (FPU)/g glucan resulted only in 14% increase in glucan-glucose conversion compared to 20% increase with cellulase loading varying from 2.5 to 7.5 FPU/g glucan. More than 10 cellobiase units (CBU)/g glucan β-glucosidase supplementation had no noticeable improvement on glucan-glucose conversion. Additionally, supplementation of xylanase was found to significantly increase glucan-glucose conversion from 50% to 80% with the substrate consistency of 12%, when the cellulase and β-glucosidase loadings were at relatively low enzyme loadings (7.5 FPU/g and 10 CBU/g glucan). It appeared that residual xylan played a critical role in hindering the ease of hydrolysis of SSB. A proper xylanase addition was suggested to achieve a high hydrolysis yield at relatively high substrate consistency with relatively low enzyme loadings.

Evaluation of swollenin from Trichoderma pseudokoningii as a potential synergistic factor in the enzymatic hydrolysis of cellulose with low cellulase loadings

Swollenin is a novel plant expansin-like protein that has been proposed to have a cellulose disruption activity. In this study, the recombinant swollenin (SWO2) from Trichoderma pseudokoningii S38 was successfully produced and purified in Aspergillus niger with a final yield of up to 10 mg of purified protein from 1 l of fermentation supernatant. The recombinant protein was found to exhibit very low level of endoglucanase activity and caused a slight increase in the crystallinity when treating cellulose. Simultaneous incubation of SWO2 with low-dose cellulases resulted in a significant synergistic activity in cellulose hydrolysis. Specifically, an even greater increase in the synergistic activity was obtained when cellulose was pretreated with swollenin followed by cellulase hydrolysis. Our results, therefore, provide a novel approach for the potential application of swollenin in the efficient saccharification of cellulosic materials.

Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis

The efficient enzymatic saccharification of cellulose at low cellulase (protein) loadings continues to be a challenge for commercialization of a process for bioconversion of lignocellulose to ethanol. Currently, effective pretreatment followed by high enzyme loading is needed to overcome several substrate and enzyme factors that limit rapid and complete hydrolysis of the cellulosic fraction of biomass substrates. One of the major barriers faced by cellulase enzymes is their limited access to much of the cellulose that is buried within the highly ordered and tightly packed fibrillar architecture of the cellulose microfibrils. Rather than a sequential 'shaving' or 'planing' of the cellulose fibrils from the outside, it has been suggested that these inaccessible regions are disrupted or loosened by non-hydrolytic proteins, thereby increasing the cellulose surface area and making it more accessible to the cellulase enzyme complex. This initial stage in enzymatic saccharification of cellulose has been termed amorphogenesis. In this review, we describe the various amorphogenesis-inducing agents that have been suggested, and their possible role in enhancing the enzymatic hydrolysis of cellulose.

Saccharification of a Potential Bioenergy Crop, Phragmites australis (Common Reed), by Lignocellulose Fractionation Followed by Enzymatic Hydrolysis at Decreased Cellulase Loadings

Cost-effective biological saccharification of nonfood lignocellulosic biomass is vital to the establishment of a carbohydrate economy. Phragmites australis (common reed) is regarded as an invasive perennial weed with a productivity of up to 18-28 tons of dry weight per acre per year. We applied the cellulose solventand organic solvent-based lignocellulose fractionation (COSLIF) to the stems and leaves of Phragmites and optimized the pretreatment conditions (e.g., temperature, reaction time, and biomass moisture content) through response surface methodology (RSM). The optimal pretreatment conditions were 85% (w/v) H3PO4 ,5 0°C, and 60 min, regardless of the biomass moisture contents from 5-15% (w/w). Glucan digestibility of the COSLIF-pretreated common reed was 90% at hour 24 at a low cellulase loading (5 filter paper units and 10 � -glucosidase units per gram of glucan). Under these conditions, the overall sugar yields were 88% for glucose and 71% for xylose, respectively. Cellulose accessibility to cellulase (CAC) was increased 93.6-fold from 0.14 ( 0.035 to 13.1 ( 1.1 m 2 per gram of biomass with the COSLIF pretreatment. Results showed that cellulase concentrations could be reduced by 3-fold with only a slight reduction in sugar yield. This study suggested that Phragmites could be used as a carbon-neutral bioenergy feedstock, while its harvesting could help control its invasive growth and decrease nutrient pollution in adjacent waterways.

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility

Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF-pretreated corn stover but reached only approximately 60% for the DA-pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were measured for the first time. The COSLIF-pretreated corn stover had a CAC of 11.57 m(2)/g, nearly twice that of the DA-pretreated biomass (5.89 m(2)/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA- and COSLIF-pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility.

Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids

A number of previous studies determined dilute acid pretreatment conditions that maximize xylose yields from pretreatment or glucose yields from subsequent digestion of the pretreated cellulose, but our emphasis was on identifying conditions to realize the highest yields of both sugars from both stages. Thus, individual xylose and glucose yields are reported as a percentage of the total potential yield of both sugars over a range of sulfuric acid concentrations of 0.22%, 0.49% and 0.98% w/w at 140, 160, 180 and 200 °C. Up to 15% of the total potential sugar in the substrate could be released as glucose during pretreatment and between 15% and 90+% of the xylose remaining in the solid residue could be recovered in subsequent enzymatic hydrolysis, depending on the enzyme loading. Glucose yields increased from as high as 56% of total maximum potential glucose plus xylose for just enzymatic digestion to 60% when glucose released in pretreatment was included. Xylose yields similarly increased from as high as 34% of total potential sugars for pretreatment alone to between 35% and 37% when credit was taken for xylose released in digestion. Yields were shown to be much lower if no acid was used. Conditions that maximized individual sugar yields were often not the same as those that maximized total sugar yields, demonstrating the importance of clearly defining pretreatment goals when optimizing the process. Overall, up to about 92.5% of the total sugars originally available in the corn stover used could be recovered for coupled dilute acid pretreatment and enzymatic hydrolysis. These results also suggest that enhanced hemicellulase activity could further improve xylose yields, particularly for low cellulase loadings.