Improving enzymatic hydrolysis of acid-pretreated bamboo residues using amphiphilic surfactant derived from dehydroabietic acid

Bioresource Technology 293 (2019) 122055

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Improving enzymatic hydrolysis of acid-pretreated bamboo residues using amphiphilic surfactant derived from dehydroabietic acid ⁎

Wenqian Lina,b, Dengfeng Chenb, Qiang Yonga,b, Caoxing Huanga,b, , Shenlin Huangb,

T

⁎

a

Key Laboratory of Forestry Genetics & Biotechnology (Nanjing Forestry University), Ministry of Education, Nanjing Forestry University, Nanjing 210037, People’s Republic of China b Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s Republic of China

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Amphiphilic surfactant Bamboo Acid pretreatment Enzymatic hydrolysis Lignin adsorption site

In this work, amphiphilic surfactant was obtained using dehydroabietic acid from pine rosin and then preadsorbed with acid-pretreated bamboo residues (AP-BR) to block the residual lignin adsorption site, which is expected to improve its enzymatic digestibility. Results from cryogenic-transmission electron microscopy (CryoTEM) indicated amphiphilic surfactant with PEG with polymerization degree of 34 (D-34) aggregated to form worm-like micelles, which improved enzymatic hydrolysis yield of AP-BR from 24.3% to 71.9% by pre-adsorbing with 0.8 g/L. Amphiphilic surfactants pre-adsorbed on AP-BR could reduce hydrophobicity of AP-BR, adsorption affinity and adsorption capacity of lignin for cellulase from 0.51 L/g to 0.48–0.32 L/g, from 2.9 mL/mg to 1.8–1.4 mL/mg, and from 122.3 mg/g to 101.9–21.4 mg/g, respectively. These changed properties showed compelling positive contributions (R2 > 0.9) for free enzymes in the supernatants and sequently for final enzymatic hydrolysis yield, which was caused by blocking non-productively hydrophobic adsorption between lignin and cellulase.

1. Introduction Conversion of lignocellulosic materials into platform fermentable sugars has recent research and industrial interest due to the ability to further convert resultant sugars (e.g. glucose, xylose, mannose, and

⁎

arabinose) into precursors for bio-based materials such as ethanol, lactic acid, furfural, and 5-hydroxymethylfurfural (He et al., 2018; Ren et al., 2018; Huang et al., 2019). However, limited success has been achieved thus far concerning production of biofuels and biochemicals from lignocellulosics by way of a biorefinery operation. This is

Corresponding authors. E-mail addresses: [email protected] (C. Huang), [email protected] (S. Huang).

https://doi.org/10.1016/j.biortech.2019.122055 Received 18 July 2019; Received in revised form 21 August 2019; Accepted 22 August 2019 Available online 24 August 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.

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(PEG) significantly improved enzymatic digestibility (Kapu et al., 2012; Rahikainen et al., 2011). The mechanism for this improvement by nonionic surfactants is believed due to the surfactant’s ability to block lignin adsorption sites, effectively lowering non-productive enzymatic adsorption to lignin surfaces and allowed for greater enzyme availability. However, these works required the non-ionic surfactant dosages up to 5–20 g/L, which is a high enough concentration to begin affecting resultant hydrolysate purity (Kapu et al., 2012; Lou et al., 2013, 2019). In reaction to this, recent studies have aimed to reduce non-ionic surface dosage while continuing to reap the benefits that they impart upon enzymatic hydrolysis. Non-ionic surfactant simultaneously possessing hydrophilic groups and lipophilic groups in its structure, which is called as amphiphilic surfactant, are capable of exhibiting hydrophobic interactions with hydrophobic molecules like lignin (Bhongale and Hsu, 2006; Lei et al., 2017). The hydrophobic portion of the amphiphilic surfactant could be long-chain oil fatty acid, azobenzene, tricyclic phenanthrene, or something similar to enable such interactions (Lei et al., 2017). In addition, presence of a hydrophilic segment such as polyether poly (ethylene glycol) can improve protein dispersion and reduce hydrophobic adsorption on substrate. Furthermore, if the hydrophobic skeleton of amphiphilic surfactant is aromatic, it may preferentially adsorb with lignin-rich surfaces. This occurrence would hereby block nonproductive enzyme adsorption sites on lignin surfaces (Lou et al., 2013, 2019). When the hydrophobic skeleton of amphiphilic surfactant is adsorbed on the surface of lignin, its hydrophilic groups can be the flexible micelle for reducing unproductive binding of cellulolytic enzymes to lignin (Zhai et al., 2018b). Preparation of amphiphilic surfactant from natural rosin and rosin derivatives has become a recent research in green chemistry, in which the main ingredients are rosin acid (dehydroabietic acid) containing a rigid tricyclic non-planar hydrophenanthrene structure (Zhai et al., 2018b,c). The hydrophobicity of hydrophenanthrene enables the dehydroabietic acid to easily to adsorb on lignin surface. It is also possible to attach PEG as a hydrophilic block to the rosin-derived surfactant, an addition which enables its solubility in water in spite of its plentiful hydrophobic portions. There are a few advantages to the use rosin acids as the precursor to prepare amphiphilic surfactant for improving enzymatic hydrolysis of biomass, such as (1) the dehydroabietic moiety in the hydrophobic skeleton makes the surfactant more hydrophobic than many other conventional surfactants, resulting in more attraction towards lignin; (2) the dehydroabietic moiety has demonstrated superior biocompatibility and environmental friendliness, which suggests it will not be a problem if it is present in downstream biorefinery process streams like the fermentable sugar stream; and (3) natural resin acids are affordable bio-derived products, obtainable from the exudation of pines and conifers. Presently, dehydroabietic acid is mostly used to prepare surfactants that are vital for oil exploration, oil drilling, fluids fracture, drag reduction, and industrial washing (Lipshutz et al., 2011; Zhai et al., 2018b,c). To our knowledge, no work reported using rosin acid-based amphiphilic surfactant to enhance enzymatic digestibility of pretreated lignocellulosic materials. In this work, amphiphilic surfactants containing a rigid hydrophobic skeleton as well as a flexible hydrophilic block were synthesized using dehydroabietic acid and PEG. Critical micelle concentration was quantified and cryogenic transmission electron microscopy (Cryo-TEM) technology was carried out to analysis their microstructures. Next, the amphiphilic surfactants were pre-adsorbed onto acid-pretreated bamboo residues, with the resultant substrate then subjected to enzymatic hydrolysis. Analysis of hydrophobicity and adsorption isotherm were performed to better understand the positive effect exerted by the pre-adsorption, with the intention to prove that sustainable amphiphilic surfactants could be effectively utilized in biorefinery processes.

attributable to the cost associated with the enzymes relied upon to hydrolyze biomass into fermentable platform sugars. In addition, reliable supply and cost of feedstock remains prohibitive (Sheldon, 2018). Hence, seeking a low-cost feedstock that can be effectively digested by hydrolytic enzymes is an essential step towards for realization of the industrial biorefinery. One possible feedstock candidate is bamboo residues, which are comprised of bamboo green, bamboo yellow, nodes, and branches. Bamboo residues are chemically composed of cellulose, hemicellulose and lignin, of which the polysaccharides (cellulose and hemicellulose) are ideal for production of fermentable sugars. These residues are a byproduct of the bamboo utilization industry, with an annual production rate of approximately 46 million tons/year in China (Huang et al., 2015). Bamboo is the herbaceous biomass with high-crystalline cellulose and rich lignin, which chemically consists of 25–40% of cellulose, 17–30% of hemicellulose, and 25–35% of lignin in cell wall (Yang et al., 2014; Huang et al., 2015). Both of these components can be valorized into different bio-fuels and bio-materials (Jin et al., 2016; Guo et al., 2018; Lou et al., 2019). At present, most of the residue is simply burned or landfilled, both of which are cost-ineffective usages of a highly sustainable lignocellulosic resource (Ravindran and Jaiswal, 2016; Huang et al., 2018). In light of this practice, improved utilization of bamboo residues has become an important goal for Chinese bamboo industry. However, there has been relatively limited interest in examining the suitability of bamboo residues as a biorefinery feedstock. Within the plant cell walls of lignocellulosic materials exist the complex matrix comprised of inter-twined polysaccharides and lignin, polysaccharide fractions are resistant to be degraded by microorganism or enzyme (Wen et al., 2014; Guo et al., 2018). Hence, a pretreatment step is considered in biorefinery operations in order to disrupt the recalcitrant structure of lignocellulosic biomass. Various established pretreatment methods have been investigated upon bamboo in particular (including dilute acid, dilute alkaline, organosolv, and steam explosion), with the goal being to enhance downstream enzymatic digestibility for eventual fermentable sugar production (Wu et al., 2017; Qi et al., 2019; Huang et al., 2019). Amongst these pretreatment methods, dilute acid pretreatment is considered to be a viable option at industrial scale due on account of several economic considerations that are improved by its effectiveness (Zhang et al., 2017). During acid pretreatment at high temperature, hemicellulose can be easily degraded into xylose or furfural due to its low degree of polymerization and abundant branched structure. The degraded products (e.g. glucose, xylose and xylooligosaccharides) from in acid prehydrolysate can be further used to produce valuable chemicals (Zhu et al., 2018; Zhai et al., 2018a). However, low enzymatic hydrolysis efficiency (< 30%) was always achieved for the acid-pretreated bamboo (Huang et al., 2015, 2019). This may be due to the remaining lignin and generated pseudolignin preventing enzymatic access to the cellulose (He et al., 2018). Another mechanism for enzymatic inhibition could be nonproductive adsorption between enzyme and lignin or enzyme and pseudo-lignin (Shinde et al., 2018; Luo et al., 2019). Based on this, it is believed that negating lignin’s enzymatic inhibition mechanisms will alleviate the negative effects exerting upon enzymatic hydrolysis of dilute acid pretreated bamboo. It is generally considered that adsorption of enzymes onto ligninrich surfaces takes place as a result of some combination of hydrophobic, electrostatic, and hydrogen bonding interactions. To intervene the interaction between lignin and enzyme, modification of lignin can be taken place on both solid surface lignin as well as the lignin that is solubilized (Shinde et al., 2018; Jiang et al., 2018). For example, covalent modification of lignin by sulfonation or carboxylation during pretreatment can enzymatic affinity for lignin-rich surfaces (Lou et al., 2013). Alternatively, blocking the adsorption site of exposed lignin by adsorbing surfactants or proteins can also lower its affinity to enzymes. Previous research has demonstrated that treatment of acid-pretreated biomass with non-ionic surfactants like Tween and polyethylene glycol 2

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2. Materials and methods

buffer(50 mM), 50 °C, and 150 rpm shaking for 48 h. Aliquots were withdrawn at different times (4, 8, 12, 24, 36, and 48 h) and centrifuged for 10 min at 4000 rpm to separate suspended solids. The supernatants were filtered through a 0.22 μm syringe filter and then measured for concentration of glucose and protein (un-bound enzyme).

2.1. Materials Bamboo residues were provided by He Qi Cang Bamboo Processing Factory (Fujian, China), which were composed of 39.2% glucan, 17.3% xylan, and 32.8% lignin (based on oven-dry biomass). Dehydroabietic acid was purified to 98% from disproportionated rosin that was produced by Institute of Chemical Industry of Forest Products (Jiangsu, China). Cellulase (Cellic CTec2) was provided by Novozymes (NA, Franklinton, USA) with a filter paper activity of 250.0 FPU/mL.

2.5. Hydrophobicity of AP-BR with pre-adsorbed surfactants The hydrophobicity of AP-BR and amphiphilic surfactants pre-adsorbed AP-BR were estimated by measuring the distribution of Rose Bengal (hydrophobic dye) in solution and on substrate using a simple adsorption quantification assay. Specifically, different amount of substrate (0.08, 0.16, 0.24, 0.32, and 0.4 g) were added in 10 mL Rose Bengal solution (40 mg/L) with pH 4.8 citrate buffer (50 mM). The suspension was then shaken at 50 °C and 150 rpm for 2 h to allow for the dye to the substrate surface. After incubation, the suspensions were centrifuged at 5000 rpm for 10 min to obtain a supernatant that was used to measure the amount of free Rose Bengal dye (λ = 543 nm). The amount of adsorbed dye on substrate was measured by the difference between initial and supernatant dye concentration. The ratio of the quantity of absorbed dyes over the residual quantity of free dyes was calculated and termed as partition quotient (PQ) The obtained PQ values were linearly plotted against substrate content to obtain a slope (L/ g), which is interpreted as the surface hydrophobicity of the substrates.

2.2. Synthesis of dehydroabietic acid based amphiphilic surfactants Dehydroabietic acid was used to produce the dehydroabietic succinate monoester according to the work of Chen et al., (2018). Then dehydroabietic succinate monoester was synthesized with different types of PEG, which bear differing degrees of polymerization (7, 12, 18, and 34), to obtain dehydroabietic acid based amphiphilic surfactant. Specifically, dehydroabietic succinate monoester (2.02 mmol), PEG (10 mmol), and p-toluenesulfonic acid (0.15 mmol) were mixed with 20 mL methylbenzene and stirred at 130 °C for 5 h in a 100 mL flask with a magnetic rotor and a reflux condenser. After reaction, the flask was soaked in an ice-bath, and NaHCO3 solution was added into the solution to stop further reaction. The solution was extracted by 30 mL dichloromethane three times, and the organic phase was next mixed for following purification. Purification involved sequential extraction and passing through the silica column, which is the same procedure as previously described. The obtained amphiphilic surfactant from PEG with polymerization degree of 7, 12, 18, and 34 were termed as D-7, D12, D-18, and D-34, respectively.

2.6. Hydrophobicity of the lignin in AP-BR with pre-adsorbed surfactants. The lignin on the surface of acid-pretreated bamboo residues was extracted using 96% 1,4-dioxane solution at a solid-to-liquid ratio of 1:20 (g:mL) for 24 h, with this procedure repeated three times with new solvent. Extract solutions were then mixed and evaporated under vacuum at 40 °C to remove 1,4-dioxane. The obtained solids were next resuspended into deionized water and freeze-dried to produce powdered lignin solids (L-APBR). To investigate the effect of amphiphilic surfactants on the hydrophobicity of the lignin, different dosages of amphiphilic surfactants (0.4 g/L and 0.8 g/L) were mixed with the extracted lignin at 1% (w/v) solids in 50 mM citrate buffer solution and shaken at 150 rpm and 25 °C for 12 h. After shaking, the suspensions were centrifuged at 5000 rpm for 10 min to obtain surfactant-adsorbed lignin and freeze-dried to produce powdered lignin solid. The hydrophobicity of the lignin and surfactant-adsorbed lignin were analyzed by contact angle measurement using an Attension Theta contact angle system apparatus. To measure contact angles, a water droplet with a volume of 5 μL at 6 s was loaded onto the lignin films, which was prepared by the pellet method.

2.3. Characterization of the synthesized amphiphilic surfactants Surface tension of the amphiphilic surfactant solutions (10-7–10-1 g/ L) were measured by the Wilhelmy plate method using a tension meter at 25 °C. Two replicates were performed for each sample with averaged results reported. Chemical structures of the synthesized amphiphilic surfactant were investigated by fourier transform infrared spectroscopy (FT-IR) at a spectral width of 400–4000 cm−1 at 4 cm−1 resolution. The morphology of the amphiphilic surfactants were acquired by Cryo-TEM in 50 mM citrate buffer solution. All the surfactants for CryoTEM measurements were prepared by dissolution into buffer solution (0.8 g/L). For each sample, 5 μL of surfactant solution was embedded onto a copper grid with two pieces of filter paper being used to blot excess sample. After setup, the copper grid was immediately plunged into liquid propane at a temperature of −165 °C to vitrify the solution. After vitrifying, the copper grid was moved into a cryogenic sample holder (Gatan 626) and observed using JEM-1400 TEM (120 kV) at −170 °C. The cryo-TEM images were obtained by a Gatan multiscan CCD camera installing a digital micrograph.

2.7. Cellulase adsorption isotherm of lignin in acid-pretreated bamboo residues with pre-adsorbed surfactants. 0.2 g of lignin sample was added in 10 mL 50 mM citrate buffer (pH 4.8) and incubated with cellulase with different concentrations (0.01, 0.02, 0.04, 0.08, 0.16, 0.5, 1.0, and 2.0 mg/mL). Samples were then incubated at 4 °C and 150 rpm shaking for 4 h as described in our previous work. After incubation, the suspensions were centrifuged at 4000 rpm for 10 min to obtain a supernatant which was used to quantify free cellulase (λ = 595 nm). The amount of adsorbed cellulase protein on lignin was measured by the difference between initial and supernatant protein concentration. Cellulase adsorption on lignin preparations was characterized by Langmuir adsorption isotherms, as follows:

2.4. Enzymatic hydrolysis of AP-BR and Avicel with assistance of amphiphilic surfactants Dilute sulfuric acid (1% (w/v)) was used to pretreat bamboo residues at 160 °C for 60 min in a 15 L vertreal digester (solid-to-liquid ratio 1:10) with electrical heating according to our previous work (Huang et al., 2019). Acid-pretreated bamboo residues (AP-BR) or Avicel at 5% (w/v) solids loading were next enzymatically hydrolyzed using cellulase at a dosage of 20 FPU/g glucan. Different dosages of amphiphilic surfactants and PEG-6000 (0.4 g/L and 0.8 g/L) were mixed with AP-BR or Avicel at 5% (w/v) solids and shaken to pre-adsorb the surfactants on the surface of the substrate. After incubation, CTec2 cellulase (20 FPU/g glucan) was added. All enzymatic hydrolysis assays were performed in 250-mL Erlenmeyer flasks at pH 4.8 citrate

Γ=

Γmax KC 1+ KC

R= Γmax K

Γ: The corresponding adsorbed cellulase on the substrate (mg/g lignin); Гmax: The maximum adsorption capacity (mg/g lignin); K: The Langmuir constant (mL/mg); C: The free cellulase in supernatant (mg/ 3

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mL); R: The distribution coefficient (L/g) 70

D-7 D-12 D-18 D-34

Surface Tension (mN/m)

2.8. Analytical methods The cellulase protein content in supernatant was determined according to the Bradford method using bovine serum albumin as the protein standard (Bradford, 1976). Chemical composition of the original bamboo residues and pretreated bamboo residues was measured according to the procedure developed by the National Renewable Energy Laboratory (Sluiter et al., 2011). The concentration of sugars (glucose and xylose) in the acid hydrolyzate from composition analysis and those in enzymatic hydrolyzate were measured using high-performance liquid chromatography (HPLC) with an Aminex HPX-87H column and a refractive index (RI) detector. The mobile phase (5 mM H2SO4 solution) was eluted at a flow rate of 0.6 mL/min. Finally, enzymatic hydrolysis efficiency was calculated according to the following equations:

60

50

40

30 10-7

10-6

10-5

10-4

10-3

10-2

10-1

Concentration (g/mL) Fig. 1. Equilibrium surface tension curves from synthesized amphiphilic surfactant.

glucose in enzymatic hydrolyzate (g) Enzymatic hydrolysis yield (%) = initial glucan in substrate (g) × 1.11

buffer solution with 0.8 g/L (Supplementary data). It can be seen that a large vesicle and spherical micelles aggregates were formed for D-7 surfactant. When PEG with higher DP was introduced to amphiphilic surfactant, thread-like and annular-like micelles tended to form, which can be seen in the Cryo-TEM images of D-12 and D-18. In addition, it was found that the diameter of the spherical micelles was obviously larger than that of the wormlike micelles, which are structures that seldom intertwine. Surprisingly, a number of fine threadlike wormlike micelles were formed in place annular wormlike micelles. These qualitative imagines indicate that the amphiphilic surfactants synthesized with PEG molecules containing higher degrees of polymerization have a strong tendency to aggregate to form the worm-like micelles as opposed to spheres. The enhanced amount of worm-like micelles can make the surfactant act as a ‘‘living polymers’’ in solution, which can improve the interactions between surfactant and substrate (Xie et al., 2013; Zhang et al., 2013).

× 100%

3. Results and discussion 3.1. Characterization of the synthesized amphiphilic surfactant. In this work, dehydroabietic acid from pine rosin was used as a raw material for synthesis of an amphiphilic surfactant. This surfactant was to designed to contain a rigid skeleton comprised of hydrophobic moieties that are adorned with flexible hydrophilic segments. Different amphiphilic surfactants were produced, with each containing a hydrophilic block that varies in terms of its starting material’s (PEG) degree of polymerization. Synthesized surfactants containing PEG segments with degrees of polymerization 7, 12, 18, and 34 were termed D7, D-12, D-18, and D-34. FT-IR was applied to identify the chemical structures of the amphiphilic surfactant (Supplementary data). The peak at 2921 cm−1 and 2862 cm−1 are attributed to the C–H stretching and asymmetric vibrations of CH3 and CH2. It can also be seen that a stretching vibration attributable to a benzene ring skeleton shows peaks at 1348 cm−1 and 1460 cm−1. It should be pointed out that the telescopic vibration absorption peak of carbonyl band group from ester and ether linkages were also identified at 1735 cm−1 and 1100 cm−1, indicating the successful introduction of PEG in dehydroabietic acid skeleton (Cervantes-Uc et al., 2006). Equilibrium surface tension curves rendered by the synthesized amphiphilic surfactants are shown in Fig. 1. It can be seen that the surface tension of D-7, D-12, D-18, and D-34 solutions were 40.5, 39.6, 37.6, and 37.2 mN/m at each respective critical micelle concentration (cmc), indicating that the D-34 surfactant possessed the most surfaceactivity properties. The reduced surface tensions might be due to the introduced flexible alkyl tails of PEG with increased polymerization degree, which can bend or twist during micelle assembly (Feng et al., 2018). Hence, it can be speculated that D-34 surfactant may exhibit the strongest performance towards improving enzymatic digestibility of pretreated biomass due to its ability to decrease the surface tension to help remove/adsorb hydrophobic lignin. It is reported that the aggregate forms of rod- and worm-like micelles, vesicles, and lamellar micelles can be formed during surfactant aggregation (Zhai et al., 2018b,c). Hence, it is important to determine the microstructures produced for the amphiphilic surfactants synthesized in this work. This will suggest the structures to expect when applying these surfactants into enzymatic hydrolysis systems. Cryo-TEM is a technique that can directly illuminate surfactant microstructures by obtaining real-time images in liquid systems. Therefore, Cryo-TEM was applied to obtain direct images of all four surfactants in a 50 mM citrate

3.2. Effect of synthesized amphiphilic surfactant on the enzymatic digestibility of AP-BR. In this work, acid pretreatment with 1% (w/v) sulfuric acid was carried out to treat bamboo residues to improve it enzymatic digestibility. The main chemical components of the acid-pretreated bamboo residue (AP-BR) were 51.6% glucan, 6.4% xylan, and 37.1% lignin. As shown in Fig. 2a, the enzymatic digestibility of AP-BR was disappointed low despite pretreatment, with an enzymatic hydrolysis yield of 24.3% at 48 h. The low enzymatic digestibility of AP-BR may be due to physical hindrance and unproductive enzyme binding caused by the lignin remaining in the bamboo residues. This problematic lignin could be lignin that was unaffected by pretreatment or perhaps newly condensed structures generated by acid catalyzed inter-lignin condensation (Zhai et al., 2018a; Luo et al., 2019) It has been reported that the non-ionic surfactants (Tween and PEG) with concentrations of 0.1–5 g/L can significantly improve the enzymatic hydrolysis of pretreated lignocellulosic materials by blocking non-productive adsorption sites on lignin towards cellulase (Lou et al., 2013; Zhang et al., 2013; Jin et al., 2016). For example, Tween-80 with concentration of 10 g/L is effective to enhance steam-exploded biomass enzymatic saccharification with 49% increasement (Jin et al., 2016). To examine whether the amphiphilic surfactants can behave similarly, each surfactant was pre-adsorbed on acid-pretreated bamboo residue before being subjected to enzymatic hydrolysis. The effects of these amphiphilic surfactants with dosage of 0.4 g/L and 0.8 g/L on enzymatic digestibility of AP-BR are shown in Fig. 2a and b, respectively. It can be seen that all amphiphilic surfactants and PEG-4000 improved 4

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70

70

Enzymatic hydrolysis yield (%)

(b) 80

Enzymatic hydrolysis yield (%)

(a) 80

60 50 40 30 20 AP-BR AP-BR+D-12 AP-BR+D-34

10 0

0

8

16

24

AP-BR+D-7 AP-BR+D-18 AP-BR+PEG4000

32

40

60 50 40 30 20 AP-BR AP-BR+D-12 AP-BR+D-34

10 0

48

0

8

16

(d)

70

Percentage of free enzymes (%)

Enzymatic hydrolysis yield (%)

(c) 80

60 50 40 30 20

0

Avicel Avicel+D-7 Avicel+D-18

0

8

16

24

Avicel+D-12 Avicel+D-34

32

40

Enzymatic hydrolysis yield (%)

40

48

100 AP-BR AP-BR+D-7 AP-BR+D-18

90

AP-BR+D-12 AP-BR+D-34

80 70 60 50 40

48

0

8

16

24

32

40

48

Time (h)

Time (h) (e)

32

Time (h)

Time (h)

10

24

AP-BR+D-7 AP-BR+D-18 AP-BR+PEG4000

80 70 60

R2=0.90

50 40 30 20

40

45

50

55

60

65

70

Percentage of free enzyme in supernatant (%) Fig. 2. The effects of surfactants (amphiphilic surfactants and PEG) on enzymatic hydrolysis of pre-adsorbed AP-BR and Avicel (a): amphiphilic surfactants and PEG4000 with dosage of 0.4 g/L on enzymatic digestibility of AP-BR; (b): amphiphilic surfactants and PEG-4000 with dosage of 0.8 g/L on enzymatic digestibility of APBR; (c): amphiphilic surfactants with dosage of 0.8 g/L on enzymatic digestibility of Avicel; (d) the percentage of free cellulase in enzymatic hydrolysis suspensions of pre-adsorbed (0.8 g/L) AP-BR; (e): relationship between free enzymes percentages in the surfactant and enzymatic digestibility.

AP-BR comparing to the improvement from commercial surfactant (PEG-4000) at same dosage. Comparing to the acid-pretreated biomass with other surfactants, Tween 80 surfactant at a concentration of 5 g/L improved enzymatic hydrolysis of dilute acid pretreated substrates by 30%, lignin-grafted phosphobetaine surfactant at a concentration of 12 g/L improved enzymatic hydrolysis of sulfite pretreated eucalyptus by 40%, PEG4000 surfactant at 2 g/L improve the enzymatic hydrolysis of acid steam-exploded corn straw by 34% (Zhang et al., 2013; Zhou et al., 2015; Li et al., 2019). In our work, D-34 surfactant achieved similar enzymatic hydrolysis yield improvements (47.6%) at a much lower concentration (0.8 g/L) than the reported work. According to the work of Jin et al., (2016), the added Tween-80 additive (10 g/L)

enzymatic hydrolysis yield after 48 h. The extent of yield improvement by amphiphilic surfactants followed the order of D-34 > D-18 > D12 > D-7. Specifically, the enzymatic hydrolysis yields of AP-BR were improved from 24.3% to 32.2%, 39.9%, 40.7%, and 54.8% with the addition of D-7, D-12, D-18, and D-34 (0.4 g/L). While, the PEG-4000 can improve the enzymatic digestibility of AP-BR from 24.3% to 38.4%. As shown in Fig. 2b, increasing the dosage of these surfactants to 0.8 g/ L resulted in better improved enzymatic digestibility of AP-BR with increase to yield ranging from 18.1 to 47.6%, in which an increasement with 29.1% is attributed to the pre-adsorbing of PEG-4000. These results indicated that the synthesized amphiphilic surfactant (D-18 and D34) showed better performances in improving enzymatic digestibility of 5

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hydrophobicity from 0.51 (L/g) to 0.48, 0.39, 0.37, and 0.32 L/g, respectively. This is due to a combined effect of the adsorbed surfactants on lignin blocking the hydrophobic sites as well as the surfactant’s hydrophilic block promoting hydrophilicity (Dey, 2012). It should be pointed out that the amphiphilic surfactants with higher degree of polymerization PEG showed better performance in reducing the AP-BR hydrophobicity, which suggests that the hydrophilic segments are providing positive contribution towards lowering overall hydrophobicity. Less hydrophobic surfaces cause a decrease in the strength of non-productive hydrophobic interactions between residual lignin and cellulase enzymes, which is benefit for enzymatic hydrolysis efficiency (Lou et al., 2013). As shown in Fig. 3a, a positive correlation (R2 = 0.93) can be fitted between hydrophobicity of amphiphilic surfactants pre-adsorbed AP-BR and corresponding enzymatic hydrolysis yield. Therefore, it can be inferred that pre-adsorbing of amphiphilic surfactants with higher degree of polymerization PEG on AP-BR leads to it with lowered hydrophobicity, which may prove the beneficial mechanism of pre-adsorption for reducing non-productive adsorption between lignin and cellulases. In order to understand if the lessened hydrophobicity of AP-BR is caused by the mitigation to residual lignin’s hydrophobicity, AP-BR’s lignin was extracted and then pre-adsorbed with amphiphilic surfactants (0.8 g/L). The residual lignin in AP-BR, and the extracted lignin that was subjected to pre-adsorption with D-7, D-12, D-18, and D-34 were termed as L-APBR, L-APBR-D7, L-APBR-D12, L-APBR-D18, and LAPBR-D34. Water contact angle measurements were carried out on each lignin preparation to estimate its hydrophobicity, with results shown in Fig. 3b. It can be seen that the contact angles decreased from 112.1° to 101.4°, 96.3°, 95.2°, and 89.9° for L-APBR, L-APBR-D7, LAPBR-D12, L-APBR-D18, and L-APBR-D34, respectively. This reveals that pre-adsorption of amphiphilic surfactants with higher degree of polymerization PEG on residual lignin reduced hydrophobicity, in agreement with results from the dye adsorption experiment previously discussed. In fact, water contact angle was strongly correlated with hydrophobicity (L/g) of AP-BR, demonstrating a linear correlation with R2 = 0.92 (Fig. 3b). Lou et al., (2013) also found that the introduction of hydrophilic surfactant onto the surface of pretreated biomass lowered hydrophobicity, thus improving enzymatic digestion. Hence, it can be concluded that the decline of AP-BR hydrophobicity was correctly assigned to the reduced hydrophobicity of residual lignin in AP-BR by pre-adsorbing with amphiphilic surfactants.

remaining in the enzymatic hydrolysate showed none impact on its ethanol fermentation performance. Hence, it is speculated that the synthesized amphiphilic surfactant (0.8 g/L) in enzymatic hydrolysate will not show the negative impact on yeast fermentation. To better investigate what part of these surfactants are active towards, experiments involving enzymatic hydrolysis of lignin-free pure cellulose (Avicel) with pre-adsorbed surfactant was performed. Results show that there was not a significant enhancement to Avicel’s enzymatic digestibility when they were pre-adsorbed with surfactant (Fig. 2c). Specifically, yield improvement of 0.8–8.1% was achieved for the enzymatic hydrolysis yields of Avicel with 0.8 g/L surfactant, in which the addition of D-34 showed best enzymatic saccharification performance (74.9%) than that of the pure Avicel (66.8%). Compared to the enhancements (18.1–47.6%) from amphiphilic surfactants preadsorbed AP-BR, it seems likely that the synthesized amphiphilic surfactants may adsorb onto the lignin in AP-BR during pre-adsorption stage, effectively reducing nonproductive adsorption of cellulase onto lignin and thereby increasing its digestibility by enzymes. To understand the effects of amphiphilic surfactants on adsorption and desorption performances of cellulase on AP-BR, the percentage of free cellulase in enzymatic hydrolysis suspensions of pre-adsorbed (0.8 g/L) AP-BR were measured (Fig. 2d). For AP-BR without pre-adsorption, the free cellulase in solution decreased rapidly over the first 4 h, then gradually increased over the time period of 4 h to 12 h. After 12 h, the amount of free cellulase began to gradually decrease, with the end (48 h) result being 45.9% of the initial enzyme dosage locatable in the liquid phase. When AP-BR was pre-adsorbed by amphiphilic surfactants, the enzyme quantity in solution showed similar trends of adsorption and desorption. Notably, the amount of the free enzyme proteins were higher than in AP-BR. Specifically, the free enzymes in the supernatants of AP-BR pre-adsorbing by D-7, D-12, D-18, and D-34 surfactant were 58.3%, 63.1%, 63.7%, and 66.4% of the initially loaded enzymes, respectively. The reason for these results could be that the added amphiphilic surfactants adsorb onto the lignin in AP-BR through hydrophobic interactions, resulting in less surface area coverage for lignin to non-productively bind cellulases (Huang et al., 2016). Li et al., (2019) also found that adding surfactant comprised of a hydrophobic skeleton grafted with hydrophilic blocks lead to an increase in hydrophilicity of the surface lignin that can similarly bind cellulases. Based on this report and our results, it is likely that hydrophobic adsorption between enzyme and lignin is prevented by introduction of amphiphilic surfactants to the lignin surfaces, which is the cause for the improved AP-BR enzymatic digestibility (Qing et al., 2010; Lin et al., 2014). This hypothesis is supported by the favorable correlation (R2 = 0.9) between free enzymes percentages in the surfactant of samples and their corresponding enzymatic digestibility, as shown in Fig. 2e.

3.4. Effect of synthesized amphiphilic surfactant on the adsorption of cellulase onto lignin in APBR. To investigate if the decreased hydrophobicity of residual lignin induced by the adsorbed amphiphilic surfactants can affect the adsorption of cellulase onto lignin, adsorption isotherms of cellulase on the extracted lignin preparations were obtained and plotted as Langmuir adsorption isotherm models. Maximum adsorption capacity, affinity, and binding strength of cellulase to these lignin preparations were estimated from the adsorption isotherms and shown in Table 1. All enzyme adsorption data was found to follow Langmuir adsorption isotherm models, as indicated by R2 > 0.95. In the Table 1, it can be seen that the residual lignin in AP-BR (L-APBR) showed the greatest values for maximum adsorption capacity and cellulase affinity, which was Гmax = 122.3 mg/g and K = 2.9 mL/mg. When the L-APBR were pre-adsorbed with 0.8 g/L amphiphilic surfactants, the maximum adsorption capacity of cellulase on L-APBR was significantly reduced accompanying the introduction of surfactants. Maximum adsorption capacities decreased to 21.4–101.9 mg/g after the pre-adsorption, with D34 showing the strongest performance. The reduced capability of LAPBR to adsorb cellulase might be due to the pre-adsorbed amphiphilic surfactants on lignin sequentially reduced the surface area and sites of lignin available for cellulase. The adsorption isotherms also revealed that the affinity of cellulase to L-APBR can be weakened by pre-

3.3. Effects of substrate hydrophobicity on its enzymatic digestibility It is generally agreed that the hydrophobicity of lignocellulosic substrate’s lignin is the primary force in governing non-productive cellulase binding and overall enzymatic hydrolysis efficiency (Wang et al., 2015). The amphiphilic surfactant with hydrophobic group skeleton and hydrophilic block can adsorb on lignin by hydrophobic interaction between surfactant’s hydrophobic group and lignin, leaving the surfactant’s hydrophilic block hovering above these problematic surfaces (Zhu et al., 2015). Hence, it is important to understand if the overall hydrophobicity of AP-BR can be changed during the pre-adsorption process using amphiphilic surfactants. In this work, the hydrophobicity (L/g) of all samples was evaluated using an adsorptive assay with the results shown in Fig. 3a. The relationship between hydrophobicity and enzymatic digestibility of pre-adsorbed AP-BR was evaluated by correlations relationship in Fig. 3a. Fig. 3a showed that a notable decrease in substrate hydrophobicity was achieved by pre-adsorption with the amphiphilic surfactants. Preadsorpting with D-7, D-12, D-18, and D-34 surfactant reduced AP-BR’s 6

Bioresource Technology 293 (2019) 122055

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Blank

Blank+D-7

Blank+D-12

Blank+D-18

(b) 0.55

Hydrophobicity of AP-BR (L/g)

(a)

Enzymatic hydrolysis yield (%)

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Blank+D-34

60

R2=0.93

50 40 30 20 0.55

0.50

0.45

0.40

0.35

0.50

0.45

0.40

0.35

0.30 115

0.30

R2=0.91

110

105

100

95

90

85

Contact angle (o)

Hydrophobicity (L/g)

Fig. 3. The effect of amphiphilic surfactants on substrate and lignin hydrophobicity (a): the relationship between pre-adsorbed AP-BR’s hydrophobicity and their enzymatic digestibility; (b): the effects of amphiphilic surfactants on the hydrophobicity of residual lignin in AP-BR.

et al., 2016). This statement is supported by the linear correlation (R2 = 0.82) between lignin’s affinity and percentages of free cellulase in enzymatic hydrolysis solutions (Fig. 4). Hence, it can be conducted that the nonproductive adsorption between cellulase and residual lignin in AP-BR can be reduced by preadsorbing with amphiphilic surfactants containing a rigid hydrophobic skeleton and flexible hydrophilic adornments. In addition, the pre-adsorbed amphiphilic surfactants on lignin surface reduced its affinity for cellulases, imparting it with the ability to resist nonproductive adsorption of cellulase. This effect allowed for more free cellulase, which in turn improved enzymatic hydrolysis of AP-BR (Lou et al., 2013; Lin et al., 2014; Luo et al., 2019).

Table 1 The maximum adsorption capacity (Гmax), Langmuir constant (K), and distribution coefficient (R) of cellulase on AP-BR’s lignin with and without amphiphilic surfactants. Гmax (mg/g)

K (mL/mg)

R (mL/mg)

L-APBR L-APBR-D7 L-APBR-D12 L-APBR-D18 L-APBR-D34

122.3 101.9 44.8 24.8 21.4

2.9 1.8 1.5 1.5 1.4

354.6 183.4 67.2 37.2 29.9

Percentage of free enzyme in supernatant (%)

Sample

70

4. Conclusion 65

In this work, dehydroabietic acid-based amphiphilic surfactants were synthesized with different polymerization degree polyethylene glycol (PEG). Pre-adsorption of these surfactants to acid-pretreated bamboo residues effectively enhanced enzymatic hydrolysis from 24.3% to 42.3–71.9%. Pre-adsorbed amphiphilic surfactants were found to reduce overall hydrophobicity of residual lignin on the substrate, which was beneficial for weakening non-productive hydrophobic adsorption between lignin and cellulase. The changed hydrophobicity of residual lignin was also found to weaken the adsorption affinity and adsorption capacity of lignin for cellulase, which contributed to lower non-productively hydrophobic adsorption between lignin and cellulase.

60 55

R2=0.97

50 45 40 3.0

2.5

2.0

1.5

1.0

Acknowledgements

Affinity of lignin for cellulase (mL/mg)

This work was supported by the National Natural Science Foundation of China (31800501) and Natural Science Foundation of Jiangsu Province (BK20180772). We would thank the graduate student in Professor Shenlin Huang’s group for preparing and characterizing the dehydroabietic acid-based amphiphilic surfactants in this work.

Fig. 4. The relationship between lignin’s affinity for cellulase and percentage of free cellulase in enzymatic hydrolysis solutions.

adsorbing with amphiphilic surfactants, which was lowered from 2.9 mL/mg (L-APBR) to 1.8 mL/mg (L-APBR-D7), 1.5 mL/mg (L-APBRD12), 1.5 mL/mg (L-APBR-D18), and 1.4 mL/mg (L-APBR-D34). This indicates that when higher degree of PEG (hydrophilic substance) was introduced to the substrate, the affinity of cellulase to lignin can be reduced. Lou et al., (2013) also found that the lignosulfonate surfactants with more hydrophilic sulfonic acid groups caused the substrate to become more hydrophilic, thus weakening lignin’s affinity for cellulase. In addition, the decrease in affinity of cellulase to L-APBR was similar to reduced maximum adsorption capacities for cellulase on lignin. This indicated that reducing the lignin’s affinity for cellulase can make contributions that lead to reduction of non-productive cellulase absorption and free enzyme quantities (Sammond et al., 2014; Strobel

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