Developing fast enzyme recycling strategy through elucidating enzyme adsorption kinetics on alkali and acid pretreated corn stover
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Although various pre-treatment methods have been developed to disrupt the structure of lignocellulosic biomass, high dosage of cellulases is still required to hydrolyze lignocellulose to fermentable sugars. Enzyme recycling via recycling unhydrolyzed solids after enzymatic hydrolysis is a promising strategy to reduce enzyme loading for production of cellulosic ethanol.
To develop effective enzyme recycling method via recycling unhydrolyzed solids, this work investigated both enzymatic hydrolysis kinetics and enzyme adsorption kinetics on dilute acid and dilute alkali pre-treated corn stover (CS). It was found that most of the hydrolysable biomass was hydrolyzed in the first 24 h and about 40% and 55% of the enzymes were adsorbed on unhydrolyzed solids for dilute alkali-CS and dilute acid-CS, respectively, at 24 h of enzymatic hydrolysis. Lignin played a significant role in such adsorption and lignin materials derived from dilute acid-CS and dilute alkali-CS possessed different enzyme adsorption properties. Enzyme recycling was performed by recycling unhydrolyzed solids after 24 h enzymatic hydrolysis for five successive rounds, and successfully reduced 40% and 50% of the enzyme loadings for hydrolysis of dilute alkali-CS and for hydrolysis of dilute acid-CS, respectively.
This study presents that the enzymes adsorbed on the unhydrolyzed solids after short-time hydrolysis could be recycled effectively for efficient enzymatic hydrolysis. Lignin derived from dilute acid-CS has higher enzyme adsorption capacity than the lignin derived from dilute alkali-CS, which led to more enzymes recycled. By applying the enzyme recycling strategy developed in this study, the enzyme dosage needed for effective cellulose hydrolysis can be significantly reduced.
KeywordsEnzyme recycling Enzymatic hydrolysis Enzyme adsorption Lignin
cellulolytic enzyme lignin
high performance liquid chromatography
bovine serum albumin
attenuated total reflection
Lignocellulosic biomass (including agricultural and forestry residues, municipal wastes and energy crops) is abundant, renewable, and of low cost [1, 2]. Utilization of lignocellulosic biomass for production of fuels and chemicals has attracted much attention , and extensive studies have been performed to develop biorefinery technologies [3, 4, 5]. One major barrier for commercialization of biorefinery technologies related to the biochemical route is the high cost of lignocellulose degrading enzymes (cellulases and hemicellulases) . Lignocellulose, evolved to resist degradation by microbial organisms and enzymes, is highly recalcitrant [3, 7]. Although various pre-treatment methods have been developed to disrupt the structure of lignocellulosic biomass and to change physical and chemical properties of lignocellulosic biomass, high dosage of cellulases is still required to hydrolyze lignocellulose to fermentable sugars [8, 9].
To reduce enzyme dosage, various studies have been carried out to develop enzyme recycling technologies [10, 11, 12, 13, 14]. As after the enzymatic hydrolysis of lignocellulosic biomass, cellulases remain either bound to the residual substrate or be free in the supernatant . Thus, recycling cellulases could be achieved through recovering cellulases from both the liquid and solid phases [16, 17, 18]. Although recycling cellulases from the liquid phase of hydrolysate has been studied extensively, it was reported that the enzymes adsorbed on the solid substrates are the key enzymes that are necessary for effective cellulose hydrolysis [19, 20, 21]. This is because the enzymes adsorbed on the solid substrates included the major cellulase degrading enzymes that have been reported to have high binding affinity towards cellulose  and have been considered as the rate-limiting enzyme component for the overall cellulose hydrolysis . Different from the bounded cellulases, the free enzymes have been reported to include mainly β-glucosidase activities and hemicellulose activity . In addition, it has been found that removing the “free/unabsorbed” enzymes had only a minor to negligible effect on the yield of cellulose hydrolysis over a range of cellulosic and lignocellulosic substrates . Also, it has been reported there was a linear relationship between the amount of adsorbed enzyme and the hydrolysis yield . Thus, recovering enzymes adsorbed on the solid substrates is theoretically more important. Some studies have investigated recovering the adsorbed enzymes through desorption. Nevertheless, enzyme activity loss is significant and greatly limits its application in a biorefinery [12, 22]. Recycling unhydrolyzed solids with adsorbed enzymes is a promising approach. Previously, we have demonstrated that recycling unhydrolyzed solids recycled 50–60% enzymes and reduced around 40% enzyme loading on ammonia pre-treated biomass [23, 24]. However, recycling unhydrolyzed soldis has not been well studied on other pre-treated biomass in a high solid loading process. As pre-treatment has a great impact on the property of lignocellulosic biomass, it is expected that biomass pre-treated differently has a different enzyme adsorption property and thereby different enzyme recycling potential via recycling unhydrolyzed solids. For example, acid catalyzed pre-treatments eliminated mainly hemicellulose from biomass [25, 26] while alkali pre-treatments can remove lignin , which may greatly affect their enzyme adsorption properties and thus affect the effectiveness of enzyme recycling via recycling unhydrolyzed solids.
This work studied the kinetics of cellulose hydrolysis on both dilute acid and dilute alkali pre-treated corn stover, and investigated enzyme adsorption kinetics over the course of cellulose hydrolysis. The role of lignin materials (from dilute acid and dilute alkali pre-treated corn stover) in enzyme adsorption was also studied. Based on the kinetic study, a fast enzyme recycling strategy by recycling unhydrolyzed solids after 24 h hydrolysis was developed in a high solid loading process. Enzyme recycling was performed for 5 rounds and 40%–50% enzyme loading reduction was achieved.
Materials and methods
Corn stover and enzymes
Corn stover used in this study was obtained from Lianyungang, Jiangsu Province, China. The particle size of corn stover was reduced by processing corn stover in a Wiley mill with a mesh screen to achieve a particle size of ~ 4 mm. Cellulase (60 mg protein/mL, 118 FPU/mL) and xylanase (25 mg protein/mL, 100,000 U/mL for beechwood xylan) were provided by Qingdao Vland Biotech Inc.
Corn stover pre-treatments
Dilute acid pre-treatment of corn stover was performed in 2-L pressure reactor (Weihai Chemical Device, Shandong, China). In brief, corn stover was pre-soaked with 1% (w/w) sulfuric acid at 10% (w/w) solid loading. Then, the pre-soaked slurry was transferred into the pressure reactor and pre-treated at 180 °C for 10 min with constant stirring (200 rpm). After pretreatment, the resulting biomass slurry was cooled down, washed with excessive water, oven-dried at 60 °C and stored at 4 °C.
Dilute alkali pre-treatment was conducted in an autoclave. Corn stover was pre-treated at 10% (w/w) solid loading, 2% (w/w) NaOH loading in a 1L flask with total working weight of 500 g at 121 °C for 20 min. After the pretreatment, the pre-treated corn stover was adjusted to neutral pH and washed with excessive water, oven-dried at 60 °C and stored at 4 °C.
Biomass composition and characterization
The composition analysis of biomass was performed by following the National Renewable Energy Laboratory (NREL, Denver, CO, USA) protocol . Sugar concentrations were measured by high performance liquid chromatography (HPLC) equipped with a refractive index detector and a Biorad Aminex HPX-87H column running at 65 °C. A mobile phase of 5 mM sulfuric acid was used at a flow rate of 0.6 mL/min. Each sample was diluted and filtered through a 0.22 μm Nylon syringe filter before analysis.
Cellulolytic enzyme lignin (CEL) isolation and characterization
To obtain the enzymatic lignin, the dilute acid/alkali pre-treated corn stover substrates were hydrolyzed using excessive enzymes as described previously . In brief, the pre-treated substrates were enzymatically hydrolyzed at 10% (w/w) solids loading with excessive cellulases and xylanase added with the ratio of 1:1 (protein basis) to make a total enzyme loading of 60 mg protein/g glucan. After 72 h enzymatic hydrolysis, the residue solids were collected by centrifugation and washed with water to remove unbound enzymes. Then the residue solids were further hydrolyzed for another 72 h using the same enzyme loading. The enzymatic hydrolysis step was repeated until no sugar release was detected. The resulting solids were then washed with deionized water, sonicated for 60 min, and further washed with 10 mM sodium acetate and 5 mM calcium acetate (pH 7.5). Protease from Streptomyces griseus (Type XIV, 1 U/mL, Sigma-Aldrich) was then applied to hydrolyze the cellulases adsorbed on solids. Thereafter, protease was deactivated by incubating at 90 °C for 2 h. The obtained residual solids were washed with 1 M NaCl solution and deionized water. Then, the solids (cellulolytic enzyme lignin) were freeze-dried and kept at 4 °C for future use.
The characterization of CEL was performed on Thermo Scientific™ Nicolet™ iS™5 FT-IR spectroscopy with a universal attenuated total reflection (ATR) accessory, which enables samples to be examined directly in the solid state without further preparation. FT-IR spectra were obtained by averaging 16 scans from 4000 to 400 cm−1.
Enzyme adsorption kinetics and isotherm
To measure adsorption kinetics, the protein content in the supernatant of the enzymatic hydrolysate was determined along the time course of cellulose hydrolysis. The enzyme protein concentration of the supernatant of each sample was measured by a modified Ninhydrin method , with bovine serum albumin (BSA) as the standard (0–800 μg/mL). In detail, 200 μL diluted sample was mixed with 100 μL 13.3 g/L NaBH4 in a pressure resistant glass tube and incubate at room temperature for 1 h, followed by adding 600 μL 9 M HCl and shaking the tube to remove bubbles. Then the tube was heated at 130 °C for 2 h. After cooling to room temperature, 100 μL hydrolysate was transferred into a 2 mL centrifugation tube and neutralized by 100 μL 5 M NaOH. 200 μL Ninhydrin reagent (Sigma-Aldrich) was added and sample was then heated at 100 °C for 10 min. 1 mL 50% (v/v) ethanol was added after the tube cooling down to room temperature. Finally, 200 μL sample was transferred into a 96 well microplate and the absorbance was read at 570 nm.
To determine the adsorption isotherm, different amount of enzymes (25–2000 μg/mL) were incubated with CEL isolated from dilute acid and dilute alkali pre-treated corn stover in centrifugation tubes at 4 °C for 2 h to reach equilibrium. The total volume of the mixture was 2 mL and the lignin was 1% (w/w) in 50 mM sodium citrate buffer (pH 4.8). After incubation, the slurry was centrifuged at 10,000 rpm for 10 min and the supernatant was collected to measure the non-absorbed protein concentration by ninhydrin method mentioned above.
Enzyme recycling strategy
The enzyme recycling was performed by recycling the remaining solids after 24 h hydrolysis to next round hydrolysis to ensure that the enzyme absorbed on the unhydrolyzed solids could be recycled at the same time. The 1st round hydrolysis of acid and alkali pre-treated CS was performed at 7% glucan solid loading with enzyme dosage of 30 and 40 mg protein/g glucan, respectively. The cellulase and xylanase were added with the ratio of 1:1 (protein basis). The enzymatic hydrolysis was performed at 50 °C, 250 rpm. After 24 h, the unhydrolyzed solid was separated by centrifuge at 10,000 rpm for 10 min. Then, the recycled solids with enzyme adsorbed were mixed with fresh biomass and fresh enzymes for the next round of hydrolysis.
Results and discussion
Biomass composition comparison of acid and alkali pre-treated corn stover
Composition of untreated, dilute alkali treated and dilute acid treated corn stover
Lignin and ash (%)
37.2 ± 0.6
19.2 ± 0.1
23.3 ± 0.5
Dilute acid treated
50.8 ± 2.3
40.1 ± 0.5
Dilute alkali treated
54.6 ± 0.6
21.8 ± 0.2
10.9 ± 0.7
Compared with acid pre-treated corn stover, the alkali pre-treated corn stover contained similar glucan content (54.6%), higher xylan content (21.8%), but much lower lignin content (10.9%). It is well known that alkaline-based pre-treatments are efficient in lignin removal [36, 38]. Due to the alkaline hydrolysis, the lignin structure was disrupted and the intermolecular ester bonds that crosslink hemicelluloses and other components were saponified [39, 40, 41], resulting in an increased cellulose accessibility and reduced lignin content . The composition differences of acid and alkali pre-treated corn stover render them excellent substrates for comparison studies of enzyme hydrolysis, enzyme adsorption, and enzyme recycle via recycling unhydrolyzed solids.
Enzymatic hydrolysis of pre-treated corn stover
One might suspect that the hydrolysis rate reduction is due to the accumulation of high sugar concentration at high solid loading (7% glucan loading), which inhibit enzyme activities. This might be a cause. Nevertheless, the enzymatic hydrolysis rates reduction after initial hours was also observed when enzymatic hydrolysis was performed at a low solid loading (1% glucan loading, Fig. 1c, d). Therefore, it seems the pre-treated corn stover materials also contained a portion of biomass that is still relatively recalcitrant to hydrolyze, which might be another cause of hydrolysis rate reduction. In addition, it is also possible that the partial lignin in pre-treated corn stover will interact with cellulose and prevent the enzymes from reaching the active sites of cellulose, leading to the decreased hydrolysis rate.
To recycle adsorbed enzymes through recycling unhydrolyzed solids, it would be wise to end enzymatic hydrolysis at 24 h, if the amount of enzymes adsorbed on unhydrolyzed solid substrates is significant, so that the slow hydrolysis period could be avoided. This approach shortens the enzymatic hydrolysis time and the unhydrolyzed biomass can be further hydrolyzed in the subsequent hydrolysis cycles. In addition, enzyme activities could be largely preserved as the hydrolysis time is short (extended hydrolysis time would lose additional enzyme activities).
Enzyme adsorption during enzymatic hydrolysis
More enzymes absorbed on acid pre-treated biomass residuals compared to alkali pre-treated biomass. This could be caused by the differences in composition and structure of the substrates and the hydrolysis yield. Previous studies reported that the properties of lignin affected the cellulase adsorption profile on pre-treated substrates [44, 45]. As alkali pre-treated corn stover contained less amount of lignin while acid pre-treated corn stover contained much higher lignin content (Table 1), it is likely that lignin is the major factor led to more enzymes bound to acid pre-treated corn stover. Moreover, acid pre-treatment and alkali pre-treatment may modify the lignin in a different way and thus result in different enzyme absorption property of the two kinds of lignin.
Enzyme adsorption comparison on lignin materials derived from acid and alkali pre-treated corn stover
Langmuir adsorption isotherm parameters for cellulase adsorbing onto cellulolytic enzyme lignin (CEL) materials
Signal assignment in FT-IR spectra of lignin derived from acid and alkali pretreamtent
Aromatic or aliphatic O–H stretching vibration
–CH2, –CH3 groups C–H vibration
Carboxylic group C–O stretching
Benzene rings C–C stretching vibration
Benzene rings C–C stretching vibration
Methyl and methylene C–H deformation vibration
Benzene rings C–C stretching vibration
Lignin syringyl unit C–H and C–O vibration
Aromatic methyl ether bridges
Aromatic methyl ether bridges
Aromatic C–H deformation vibration of syringyl unit rings and aromatic C–H in plane deformation of guaicyl and syringyl units of lignin
Lignin syringyl unit C–H and C–O vibration
In addition, one might suspect that the difference (about 10%) of sugar yield between alkali and acid pre-treated CS (Fig. 1) indicated that lignin might play a role in decreasing the efficiency of hydrolysis, which seems not consistent with the result shown in Fig. 5. However, such phenomena may be related with the difference in the accessibility of cellulose due to different level of lignin content in the pre-treated substrates. In the pre-treated substrates, lignin interacts with pre-treated substrates, limiting cellulose accessibility to cellulases . Thus, higher lignin content of acid pre-treated substrates indicates more lignin may interact with cellulose, resulting in lower cellulose accessibility and lower sugar yield compared with that of alkali pre-treated substrate. Differently, during the hydrolysis of avicel in the presence of supplemented lignin, both acid and alkali lignin did not form such interaction with cellulose so that cellulose is still accessible for enzymatic hydrolysis, which might result in their negligible effect on enzymatic hydrolysis of avicel.
Enzyme recycling via recycling unhydrolyzed solids
This work studied both enzymatic hydrolysis kinetics and enzyme adsorption kinetics on pre-treated corn stover, and developed enzyme recycling strategy based on kinetic studies. It was found that most of the hydrolysable biomass was hydrolyzed in the first 24 h and about 40% and 55% of the enzymes were adsorbed on unhydrolyzed solids for dilute alkali pre-treated corn stover and dilute acid pre-treated corn stover, respectively. Lignin played a significant role in such adsorption. Recycling enzymes via recycling unhydrolyzed solids reduced 40% and 50% of the enzyme loadings for hydrolysis of dilute alkali-CS and for hydrolysis of dilute acid-CS, respectively.
YY performed most of the experiments, data acquisition and data interpretation, wrote the manuscript. RZ supervised the execution of the experiments, analyzed the data, assisted in coordinating this study, and wrote the manuscript. YL and XC performed the pre-treatment of corn stover. MJ coordinated this study, designed the experiments, evaluated the data and wrote the manuscript. All authors read and approved the final manuscript.
The authors thank Prof. Qiang Zhang from Nanjing University of Science and Technology for his assistance in FT-IR measurement.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included.
Consent for publication
All authors have approved the manuscript for publication.
Ethics approval and consent to participate
This work was supported by “National Key R&D Program of China”, Grant No. 2016YFE0105400, “National Natural Science Foundation of China”, Grant No. 21606132, “Natural Science Foundation of Jiangsu Province”, Grant No. BK20160823, BK20170037 & BK20170832, “The Fundamental Research Funds for the Central Universities”, Grant No. 30916011202, and the Foundation of Jiangsu Specially-Appointed Professor.
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