A novel surfactant-assisted dilute phosphoric acid plus steam explosion pretreatment of poplar wood for fermentable sugar production

doi:10.21203/rs.3.rs-836994/v1

Pretreatment is an indispensable process in lignocellulosic bioethanol production. In this work, a surfactant agent JFC was introduced into the dilute phosphoric acid plus steam explosion pretreatment scheme for fermentable sugar production using poplar as substrate. Four crucial factors (phosphoric acid concentration, surfactant concentration, pressure, and residence time) affecting the pretreatment efficiency were optimized using the single factor tests. The optimal parameters obtained were as follows: 1:2.5 solid/liquid rate, 2 h pre-soaking time, 1.5 %(v/v) JFC-M + 2.0 wt% phosphoric acid, 2.0 MPa pressure, and 120 s residence time, resulting in a maximum cellulose recovery rate of 86.33 % and enzymatic saccharification rate of 84.62 %, which was 38.97 % higher than that of control. The morphological and structural characteristics of samples before and after pretreatment, were characterized by the scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) method. The addition of JFC-M was of a notable influence in overcoming biomass recalcitrance and boosting cellulose digestion, showing great application potentials in biomass conversion process.

Environmental Engineering

Environmental Policy

Steam explosion pretreatment

Dilute phosphoric acid

Surfactant

Enzymatic saccharification

Poplar

Lignocellulosic biomass (LCB) is the most abundant and renewable resource on the planet, which can act a low-cost and promising feedstock for production biofuels, biomaterials, and biochemicals, etc., attracting worldwide interests and attentions (Alonso et al. 2017). However, owing to the high biomass recalcitrance, a suitable pretreatment process is required to remove lignin and hemicellulose, depolymerize of cellulose of LCB, and release fermentable sugars for further bioconversion such as bioethanol production (Qi et al. 2018; Tu and Hallett 2019).

To date, several LCB pretreatment techniques have been developed, which are mainly categorized into physical, chemical, physicochemical, biological techniques, and some cocktail of these strategies (Haldar and Purkait 2020). Among them, steam explosion (SE) method is of advantages such as cost-effective and environmentally -friendly supermolecular deconstruction, which is widely applied in LCB especially hardwood and agricultural residual pretreatment (Jacquet et al. 2015). Meanwhile, to improve the efficiency and reduce the severity of SE (e.g., lowering the temperature and pressure, shortening the residence time), some chemical additives, mainly mineral acids or alkalis (such as SO₂, H₂SO₄, H₃PO₄, and NH₄), are introduced into SE process (Zhang et al. 2020).

Dilute phosphoric acid plus steam explosion (DPASE) is a promising alternative LCB pretreatment method (Fockink et al. 2018; Geddes et al. 2011; Oliva et al. 2020), holding the advantages as following: (i) acting as an exogenous weak acid catalyst, dilute H₃PO₄ results in low sugar loss and yield of toxins simultaneous with SE (Pitarelo et al. 2016; Zeng et al. 2014); (ii) H₃PO₄ can not only serve as an additional source of nutrients for microbe, but can also be partly recovered and reused as a fertilizer (Geddes et al. 2011); and (iii) H₃PO₄ is less corrosive to the SE apparatus, which is beneficial for the equipment and efforts at scaling up (Haykiri-Acma and Yaman 2019; Koradiya et al. 2016). However, regarding the distinct components and microstructures of multisourced LCB feedstocks (e.g., dedicated crops, agricultural residues, and short-rotation energy coppices), more researches are needed to expand the application of DPASE.

It has been reported that surfactants, particularly nonionic surfactants (Tween, Triton, and polyethylene glycol, etc.) in the pretreatment process can promote the wettability of LCB, decrease the crystallinity of cellulose, give higher cellulose conversion, improve the delignification rate, maximize the enzymatic convertibility, and enhance the ethanol yields, etc. (Cao and Aita 2013; Jørgensen et al. 2007; Nasirpour et al. 2014; Zheng et al. 2020). A fatty alcohol polyoxyethylene ether based nonionic surfactant agent JFC (R-O(CH₂CH₂O)₅-H, wherein R = C_7 − 9), holds the properties of reducing interfacial tension, enhancing the wettability of materials, improving the capillary effect inside fibers, etc., which has been introduced in textile modification (Gao et al. 2017), coal mining (Shi et al. 2019), porous geopolymers preparation (Yan et al. 2021), mineral flotation (Chen et al. 2018), etc. Considering its other advantages such as strong acid, alkali, and high temperature resistance and no bio-toxicity, the surfactant agent JFC holds potential of assisting the LCB steam explosion pretreatment.

In this work, a novel surfactant JFC-assisted dilute phosphoric acid plus steam explosion pretreatment of poplar wood craft was developed. Four crucial factors (phosphoric acid concentration, surfactant concentration, pressure, and residence time) affecting the pretreatment efficiency were optimized using the single factor tests. The morphological and structural characteristics of samples were characterized for analyzing the mechanism of pretreatment and further optimizing of the process.

2.1 Materials

The dried European black poplar (Populus nigra L.) chips were pulverized by a plant shredder to sawdust (1–2 mm diameter, 2–15 mm length) as feedstocks. The surfactant JFC-M (industrial grade) was purchased from Shandong Yousuo Chemical Technology Co., Ltd. (Zhou et al. 1995). The cellulase was purchased from Qingdao Vland Biotech INC.(Qingdao, China), holding a filter paper activity (FPA) of 191 IU/mL. The chemical agents like phosphoric acid, sulfuric acid, and calcium carbonate, etc., were analytical grade and could be used without further purification.

2.2 Experimental method

2.2.1 DPASE

The poplar sawdust was presoaked in a JFC-M and dilute H₃PO₄ solution at 1:2.5 solid/liquid rate for 2 h, and then pretreated in a SE apparatus QBS-80B (Tsing Gentle Eco-technology (Suzhou) Co., Ltd.) equipped a 400 mL cylinder. The exploded slurry was collected for further study. For detection the removal of cellulose and hemicellulose after DPASE, the exploded slurry was separated by a centrifuge at 1000 r/min and the filtrate was collected for further analysis.

2.2.2 Single factor experiment

The single factor experiment was conducted using four critical factors influencing the pretreatment efficiency such as surfactant JFC-M and H₃PO₄ concentration, pressure of explosion, and residence time.

2.2.3 Enzymatic saccharification

The exploded slurry was dried in a drying oven (Tianjin Taisite Instrument Co., Ltd.) at 60°C for 48h and then added into100 mL Erlenmeyer flasks containing 50 mL citric acid buffer and with 1:50 solid/liquid rate. The enzymatic saccharification was carried out with parameters as following: 30 IU/g dry exploded slurry cellulase loading, 180 rpm/min stirring speed, initial solution pH 4.8, 50°C reaction temperature for 72 h.

The saccharification rate is calculated as follows (Kim 2011):

Wherein, C₀ is the concentration of reducing sugar (RS) in the hydrolysate. V is the volume of hydrolysate. C₁ and C₂ are the cellulose and hemicellulose contents in the substrate after pretreatment. M is the dry mass of the substrate, and “0.9” means that 1.0 g RS is equivalent to the mass of 0.9 g cellulose and hemicellulose.

2.2.4 Composition analysis

The cellulose, hemicellulose and lignin of samples were analyzed according the national renewable energy laboratory (NREL) standard method (Sluiter et al. 2008).

The solid and cellulose recovery rate (or retention rate) is calculated as follows:

Wherein, m₁ and m₂ are the masses of poplar substrate or cellulose before and after pretreatment, respectively.

The hemicellulose removal rate is calculated as follows:

Wherein, m₁ and m₂ are the masses of hemicellulose before and after pretreatment, respectively.

The concentration of RS was determined by a high-performance liquid chromatography (HPLC, Thermofisher U3000, Thermo Fisher Scientific) with a chromatographic column Aminex HPX-87H according the NREL standard method (Sluiter et al. 2008). The analysis conditions were as follows: 5 mmol/L H₂SO₄ mobile phase, 45°C column temperature, 5 mL/min flow rate, 30°C differential refractive index temperature, and 20 µl sample volume. All tested samples were filtered by a 0.22 µm filter membrane. The standard sugar gradient solution was prepared with cellobiose, glucose, xylose, and arabinose, and the standard curve was drawn by the external standard method.

2.2.5 Structure characterization

The samples before and after pretreatment were dried in a constant-temperature drying oven at 105°C to a constant weight for further structure characterization. The SEM was carried out by a Zeiss Sigma 300 scanning electronic microscope. The cellulose crystallinity of samples were analyzed by an X-ray diffraction analyzer (X'Pert PRO, PANalytical, Netherlands), with the following parameters: 0.15406 nm wavelength, 45 kV voltage, 40 mA current, 5°–50° scanning angle 2θ range, and 5°/min scanning rate.

The crystallinity is calculated as follows (Segal et al. 1959):

Wherein, I₀₀₂ represents the maximum intensity of the crystalline part at 22°, and I_am represents the minimum intensity of the non-crystalline part at 18°.

The Fourier transform infrared (FTIR) analysis was conducted by an IRAffinity-1S spectrometer (Shimadzu Corp., Kyoto, Japan) with following parameters: 400–4000 cm^− 1 wave number, 32 scans per second analysis speed, and 4 cm^− 1 resolution ratio, respectively.

3.1 Effect of phosphoric acid on pretreatment

Generally, the content of glucose and xylose in exploded filtrate reflects the removal of cellulose and hemicellulose, respectively. As shown in Fig. 1a, the content of glucose in exploded filtrate increased when the H₃PO₄ was less than 2.0 wt%, and then decreased slightly with the increase of H₃PO₄, holding a maximum value of 3.61 g/L, which was 3.4 times that in the blank group. The content of xylose in exploded filtrate increased with the increase of H₃PO₄, holding a maximum value of 8.79 g/L at 2.5 wt% H₃PO₄, which was 2.5 times that in the blank group. The higher titter of xylose yielded compared with that of glucose could be attributed to two reasons: (i) xylan is more labile than glucan in LCB substrate (Chen et al. 2007); (ii) the exogenous H₃PO₄ catalyst presents higher capacity of solubilizing hemicellulose of LCB (Oliva et al. 2020). According to Fig. 1b, the saccharification rates of samples were positively correlated with the H₃PO₄ concentrations, holding a maximum value of 78.52 % at 2.5 wt% H₃PO₄, which was 27.95 % higher than that in the blank group. The removal of hemicellulose increased the internal porosity of the fiber and provided more enzyme contact sites, boosting the enzymatic saccharification of substrate (Leu and Zhu 2013). The addition of surfactant JFC-M helps to remove hemicellulose and increase the enzymatic saccharification rate. Considering the cost-effectiveness of pretreatment, 2.0 wt% H₃PO₄ was used for further study.

3.2 Effect of surfactant JFC-M on pretreatment

As shown in Fig. 2a, the content of glucose in exploded filtrate increased when the JFC-M concentration was less than 1.0 %(v/v), and then decreased slightly with the increase of JFC-M, holding a maximum value of 3.28 g/L, which was 3.3 times that in the blank group. The content of xylose in exploded filtrate increased when the JFC-M concentration was less than 1.5 %(v/v), and then decreased with the increase of JFC-M, holding a maximum value of 8.56 g/L, which was 2.5 times that in the blank group. When the JFC-M concentration was less than 2.0 %(v/v), the saccharification rate of samples increased with the increase of JFC-M, and then decreased, holding a maximum value of 80.91 % at 2.0 %(v/v) JFC-M, which was 20.02 % higher than that in the blank group (Fig. 2b). The decreased of saccharification rate with the increase of JFC-M might be caused by the negative effect of surfactant and inhibitors generated in the pretreatment process, which should be done further research.The reasons for this may be as follows (Kim 2011; Leu and Zhu 2013; Li et al. 2014): (i) the addition of surfactant JFC-M increased the surface area and porosity of poplar fiber; (ii) alleviation the adhesion of pseudo-lignin to the fiber surface after explosion, and reduced the ineffective adsorption of cellulase on the surface of lignin; (iii) improvement the stability of cellulase activity; and (iv) changes of the substrate structures to further promote enzymatic hydrolysis. Considering the cost-effectiveness of pretreatment, 1.5 % (v/v) surfactant JFC-M was used for further study.

3.3 Effect of pressure on pretreatment

The contents of glucose and xylose increased with the increase of SE pressure, holding maximum values of 3.48 g/L and 8.73 g/L, respectively (Fig. 3a). The saccharification rates of samples increased with the increase of SE pressure, holding a maximum value of 82.96 % at 2.4 MPa SE pressure, which was 22.07 % higher than that in the blank group (Fig. 3b). Pressure is correlated to temperature and has impacts on hemicellulose degradation to soluble mono- and oligosaccharides, and cleavage of lignin carbohydrate complex (Chen and Qiu 2010). The recently study revealed that with the increase of pressure, the inhibitors generated in SE process using poplar as a feedstock basically maintained at the same level (Wang et al. 2020). Considering the increase of pressure would increase the energy consumption, 2.0 MPa SE pressure was used for further study.

3.4 Effect of residence time on pretreatment

The content of glucose in exploded filtrate increased when the residence time was less than 180 s, and then decreased slightly, holding a maximum value of 3.03 g/L, which was 2.8 times that in the blank group (Fig. 4a). The content of xylose in exploded filtrate increased with residence time, holding a maximum value of 8.70 g/L at residence time of 300 s, which was 2.5 times that in the blank group. As shown in Fig. 4b, when the residence time was less than 120 s, the saccharification rates of samples increased with the increase of JFC-M, and then decreased, holding a maximum value of 84.62 % at residence time of 120 s, which was 23.73 % higher than that in the blank group. The hydrolysis of hemicellulose is closely correlated to the residence time of SE, and a high residence time allows the complete hydrolysis of hemicellulose and decrease the solid recovery (Jacquet et al. 2015). The drops of saccharification rate, when the residence time was over 120 s, might be the reason that part of the xylose or glucose are converted into enzymolysis inhibitors,which adversely inhibits the enzymatic hydrolysis (Junhua Zhang et al. 2012). Considering the saccharification rate of poplar, 120 s residence time was selected. In summary, a maximum saccharification rate was obtained under the DPASE parameters as follows: 1:2.5 solid/liquid rate, 2 h pre-soaking time, 1.5 (v/v)% JFC-M + 2.0 wt% H₃PO₄, 2.0 MPa SE pressure, and 120 s residence time.

3.5 Components change of samples

The components of samples before and after pretreatment are shown in Table 1.

The contents of cellulose and lignin in DPASE (55.61 % and 31.05 %) and JFC-M + DPASE (64.80 % and 33.10 %) group were greater than that of the raw material (44.58 % and 28.32 %), respectively. The content of hemicellulose in the two pretreatment groups (6.05 % and 2.97 %) decreased dramatically compared with that of the aw material (12.05 %). As the assistant removal of hemicellulose and lignin using a surfactant (Qing et al. 2010), the solid recovery rate in JFC-M + DPASE (55.97 %) group was lower than the one in DPASE group (73.12 %). The hemicellulose removal rate in JFC-M + DPASE (83.50 %) group was higher than the one in DPASE group (60.47 %). Comparing the results reported in Table 2, the addition of surfactant JFC-M increased the removal of hemicellulose, leading to a relatively high cellulose recovery rate obtained. Considering the SE conditions, sugars resolved in exploded filtrate, recovery rates, and saccharification rate, the surfactant-assisted DPASE pretreatment method performs advantages over other SE method

Table 1

Compositions (%), recovery and removal rates (%) of samples*.
Component (%)	Raw	DPASE	JFC-M + DPASE
Carbohydrate	56.63	55.61	64.80
Cellulose	44.58	49.56	61.83
Hemicellulose	12.05	6.05	2.97
Lignin	28.32	31.05	33.10
Acid-soluble lignin	1.54	1.36	1.29
Acid-insoluble lignin	26.78	29.69	31.81
Solid recovery rate	/	73.12	55.97
Cellulose recovery rate	/	88.85	86.33
Hemicellulose removal rate	/	60.47	83.50
* Note: (1) the alcohol extract and ash of raw poplar feedstock is 1.68 % and 0.84 %, respectively. (2) All the samples were analyzed according the NREL standard method.

Table 2

Compositions (%) and recovery rates (%) of samples in literature*.
SE Conditions	Composition (%)	Sugars in filtrate(%)	Recovery (%)	Reference
1–2 × 2–15 mm particle size, 1:2.5 solid/liquid rate, 2 h pre-soaking time, 1.5 (v/v)% JFC-M + 2.0 wt% H₃PO₄, 2.0 MPa pressure, 120 s residence time.	44.58 % C, 12.05 % H, 28.32 % L.	1.56–3.41 % glucose, 2.15–8.79 % Xylose.	86.33–88.85 % C, 55.97–73.12 % SR.	this work
30×30×5 mm particle size, N₂ added in SE process, 209 ± 0.5°C, 7 min residence time.	44.1 % C, 19.4 % H, 27.8 % L.	1.35–1.39 % glucose, 7.36–8.14 % xylose.	55.1–56.2 % C, 4.9–5.6 % H, 19.5–20.8 % L, 59.5–65.0 SR.	Wang et al. (2020)
10(± 2) mm particle size, 200 or 220°C, 5 min residence time.	35.5–40.4 % C, 18.1–22.9 % H, 30.1–38.2 % L.	1.3–1.9 % glucose, 6.4–9.8 % xylose.	32.0–40.0 acid-insoluble lignin, 62.8–76.5 SR.	Martín-Davison et al. (2015)
30×15×5 mm size, 190–220°C, 7–11 min residence time.	44.3 % C, 16.2 % H, 26.9 % L.	/	62.8–76.5 SR.	Castro et al. (2014)
2.5×2.5 cm and 5.0×5.0 cm size, Alkaline-oxygen impregnated SE: 12 wt % Na₂CO₃ 100–110°C, 100 psig O₂, 135°C, 3 min; SE: 210°C, 10 min residence time.	45.7 % C, 17.4 % H, 34.9 % L.	/	94–97 % C, 54–71 % H.	Chu et al. (2017)
* Note:(1) C, H, L, and SR represents cellulose, hemicellulose, lignin, and solid recovery, respectively. (2) All the samples were analyzed according the NREL standard method.

4.1 SEM analysis

The morphological changes of samples were characterized by the SEM method, and the results were shown in Fig. 5. The raw poplar is of tightly packed and dense structure with the smooth surface morphology (Fig. 5a,b). In DPASE group (Fig. 5c,d), some slight damages occurred at the surface and interior of poplar fiber. The uneven spherical precipitates were formed on the surface of fiber, which is probably the result of the formation of lignin microspheres or pseudo-lignin microspheres at high temperatures (Cheng et al. 2018). In JFC-M + DPASE group (Fig. 5e,f), severe damages were occurred, performing densely wrapped and broken structures of fiber, which greatly increased the specific surface area and porosity of the feedstock and was beneficial for subsequent enzymatic saccharification. The surfactant JFC-M may have a unique mode of action in assisting of the DPASE process by the means of following. Firstly, JFC-M promotes the wetting of the surface of poplar fiber by H₃PO₄ solution and permeation into the interior of the fibers, so as to improve the acid-catalyzed deconstruction of poplar fiber. The results were in line with literature reported that the addition of surfactants quite better option for acid pretreatment (Singh et al. 2015).

4.2 XRD analysis

Crystallinity of LCB is a crucial factor that influences the enzymatic hydrolysis. The crystallinity index (CrI) of samples are determined by the XRD analyse and the results are shown in Fig. 6. The relative crystallinities of DPASE (62.09 %) and JFC-M + DPASE (63.42 %) pretreated samples were greater than that of the unpretreated poplar(53.59 %), which was consist with the results of previous studies (Singh et al. 2015). The increase of crystallinity was mainly caused by the deconstruction of crystalline area of cellulose, removal of hemicellulose and delignification (Jacquet et al. 2015). Once the crystalline areas become small and amorphous, the accessibility of enzyme to the cellulose substrate could be improved. The higher crystallinity of JFC-M + DPASE pretreated sample than that of the DPASE is in line with the results of the single factor test and changes of components. After enzymatic hydrolysis, the CrI (46.72 %) of sample decreased, which might be caused by some destroy of the crystalline area of cellulose during the enzymatic hydrolysis process, resulting in the loose and irregularly arranged area becoming into the amorphous area.

4.3 FTIR analysis

The structural modifications of samples were identified by a FTIR analysis and the results were shown in Fig. 7. The enhancements of C-H and -OH stretching vibrations (3423 cm^− 1 – 2924 cm^− 1) of cellulose after pretreatment (Wang et al. 2020), indicates more loose cellulose are obtained for the subsequent enzymatic hydrolysis process, especially in the JFC-M added group, which is consistent with the results of SEM. The characteristic absorption peak of hemicellulose xylan (1740 cm^− 1) (Bu et al. 2011), decreases after pretreatment, especially in the JFC-M + DPASE group, indicating that the addition of surfactant is more conducive to removing hemicellulose. Here, 1510 ~ cm^− 1 is the C = C stretching vibration of the pure lignin aromatic ring in LCB (Anderson et al. 1991). The C-H and C-O stretching vibration of the syringyl benzene ring in the lignin is noted at 1120 cm^− 1 (Schwanninger et al. 2004). All samples after pretreatment show absorption at 1120 cm^− 1, indicating the existing of syringyl lignin in poplar wood. The stretching vibration at 898 cm^− 1 is the characteristic peak of β-glycosidic bond (Ibrahim et al. 2010), the intensity of which dropped slightly after pretreatment, indicating that a small amount of glucan is degraded during the pretreatment process. This is consistent with the glucose content in exploded filtrate after pretreatment.

In this study, a surfactant JFC-M assisted dilute phosphoric acid plus steam explosion pretreatment of poplar wood was developed. Four crucial factors such as phosphoric acid concentration, surfactant concentration, pressure, and residence time were optimized using the single factor tests. The results showed under the optimized conditions: 1:2.5 solid/liquid rate, 2 h pre-soaking time, 1.5 (v/v)% JFC-M + 2.0 wt% phosphoric acid, 2.0 MPa pressure, and 120 s residence time, a 86.33 % cellulose recovery rate and 84.62 % enzymatic saccharification rate were obtained. The surfactant JFC-M increased the removal of hemicellulose and a high cellulose recovery rate, which in turn booted the enzymatic saccharification of substrate. The morphological and structural characteristics of samples were analyzed for revealing the mechanism of pretreatment and further optimizing of the process. The novel pretreatment process shows great application potentials in biomass conversion process.

Acknowledgments

The authors are grateful for the support provided by the National Key Research and Development Program of China (Grant No. 2019YFB1503801) and the Key Research and Development Program of Hunan Province (Grant No. 2020WK2019, 2019CB1002).

interest statement

There are no conflicts of interest to declare.

Data availability statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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A novel surfactant-assisted dilute phosphoric acid plus steam explosion pretreatment of poplar wood for fermentable sugar production

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Abstract

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1. Introduction

2. Materials And Methods