The effects of dilute acid prehydrolysate from poplar were investigated and compared in the enzymatic hydrolysis, fermentation, and simultaneous saccharification fermentation (SSF) in this study. The improvement of enzymatic hydrolysis and fermentation with resin adsorption and surfactant addition has also been represented. A total of 16 phenolic alcohols, aldehydes, acids and 3 furan derivatives in the prehydrolysates were identified and quantified by gas chromatography/mass spectrometry (GC/MS). The degree of inhibition from the phenolic compounds (26.55%) in prehydrolysate on the enzymatic hydrolysis was much higher than carbohydrates-derived inhibitors (0.52%-4.64%). Around 40% degree of inhibition was eliminated in Avicel enzymatic hydrolysis when 75% of prehydrolysates phenolic compounds were removed by resin adsorption. This showed distinguishing inhibition degrees of various prehydrolysate phenolic compounds. Inhibition of prehydrolysate on enzymatic hydrolysis was more dosage-dependent while their suppression on the fermentation showed a more complicated mode: fermentation could be terminated by the untreated prehydrolysate, while a small number of prehydrolysate inhibitors even improved the glucose consumption and ethanol production in the fermentation. Correlated with this distinct inhibition modes of prehydrolysate, the improvement of Tween 80 addition in SSF was around 7.10% for the final ethanol yield when the glucose accumulation was promoted by 76.6%.
Research Article
Effect and improvement of dilute acid prehydrolysate inhibitors on the enzymatic hydrolysis and the yeast fermentation
https://doi.org/10.21203/rs.3.rs-4852631/v1
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Enzymatic hydrolysis
Prehydrolysates
Phenolic compounds
Yeast fermentation.
Due to the immoderate consumption of non-renewable fossil fuels in recent years, renewable energy has been considered to mitigate the severe environmental issues like the air pollution and the global warming (Ye et al., 2023). Lignocellulosic biomass is abundant and renewable resources, which has the potential to replace fossil resources in the production of biofuels and bio-based chemicals for the reduction for the dependence on petroleum-based chemicals (Lin & Lu, 2021; Zheng et al., 2023). However, the main contents of lignocelluloses, i.e., cellulose, hemicellulose, and lignin, intermix to form the complex structures, which shows high resistance to the chemical and biological treatment (Machineni, 2020). This recalcitrant structure significantly decreases the accessibility of the carbohydrates for the subsequent conversions. Thus, pretreatment to deconstruct the matrix of lignocellulose is essential for the preparation of high-value products such as fermentable sugars (Beig et al., 2021). Dilute acid pretreatment is widely used in the industrial field due to the simplicity, low cost, and broad applicability for various species of biomass (Yildirim et al., 2021). It is one of the most practical pretreatment strategies for the full degradation of hemicellulose and the partial dissolution of lignins (Hou et al., 2023).
However, the hydrolyzed hemicellulose would not only be degraded into mono-sugars like xylose and glucose in dilute acid pretreatment, but also into carbohydrate-derived yeast fermentation inhibitors like acetic acid (AA), formic acid (FA), levulinic acid (LA), furfural, and 5-hydroxymethylfurfural (HMF) (Jönsson & Martín, 2016). These carbohydrate-derived inhibitors have been suggested to be the important indicator of prehydrolysate toxicity (Chen et al., 2018). Correspondingly, lignin in the acidic pretreatment will be degraded into phenolic compounds, which were also designated as the lignin-derived inhibitors for the following fermentation (He et al., 2020). These fermentation inhibitors, especially phenolic compounds, have been reported to significantly limit the fermentability and ethanol production of the biomass prehydrolysates by hindering bacteria activity (Deshavath et al., 2017). Efforts have been dedicated towards the identification of the phenolic inhibitors to reveal the mechanism behind the toxicity of the prehydrolysates. Llano etc., have used high performance liquid chromatography (HPLC) to identify the major monosaccharides, aliphatic acids, and furans 10 from sulphite liquor (Llano et al., 2017). Other researchers have identified 46 inhibitors by GC/MS to show strong inhibition on the acetone butanol-ethanol fermentation of Clostridium saccharobutylicum (Zhang et al., 2018). The toxicity of various inhibitors has been thoroughly studied by adding different dosages of inhibitors to the sugar control respectively to mimic the inhibitory effect of each inhibitor in the prehydrolysates (Xie et al., 2016). It is essential to reveal the inhibition mechanism of the real prehydrolysate rather than the individual inhibitors, because the complexity of the prehydrolysate composition (Li et al., 2018a).
Prehydrolysates from dilute acid pretreatment have also been suggested to be toxic to the enzymatic hydrolysis (Agrawal et al., 2021; da Silva et al., 2020). It was reported that a layer of spherical pseudo-lignin droplets would be generated on the pretreated substrates and impede the efficiency of enzymatic digestibility (Zhai et al., 2022). Fermentative inhibitors may bind tightly with the amino acid residues of the cellulase enzymes as how they hinder the fermentation and metabolism of yeast and bacteria (Dos Santos et al., 2019; Madadi et al., 2023). It is not clear whether the mechanism of the prehydrolysate inhibition on the enzymatic hydrolysis stays the same with that on the fermentation of the microorganism. Furthermore, the effect of various inhibitors on the enzymatic hydrolysis was still not clearly understood and the method to overcome the inhibition is also not well elucidated. Numerous research studies have been concentrated on developing a cost-effective detoxification approach to overcome the toxicity and improve the fermentability of prehydrolysates, including physical detoxification by resin adsorption (Yu & Christopher, 2017), chemical detoxification via nucleophilic addition of amino acids and proteins (Sheng et al., 2020), biological methods through laccase and fungi (De La Torre et al., 2017), and direct addition of surfactants due to the change of inhibitor hydrophobicity (Zheng et al., 2021). Surfactants e.g., Tween 80, have been proved to show high performance on the improvement of enzymatic hydrolysis by blocking the hydrophobic non-productive interaction between lignin and cellulase, and increase the stability of the enzymes (Kleingesinds et al., 2018).
The objective of this study is to evaluate the effect of prehydrolysate inhibitors on the enzymatic hydrolysis, and compare with their inhibition on the fermentation. The composition, especially the inhibitors of the prehydrolysates, was the first to be identified by HPLC and GC/MS. Based on this, the distinct effects of the prehydrolysates on enzymatic hydrolysis and fermentation were determined, respectively. The effects of phenolic compounds on enzymatic hydrolysis were evaluated when the carbohydrate-derived inhibitors were controlled unchanged and the amount of the phenolic compounds were varied via prehydrolysates treated by various dosages of adsorption resins. Eventually, the practical knowledge of the inhibitory effect of prehydrolysates on the enzymatic hydrolysis and yeast fermentation were applied in the SSF of dilute acid pretreated poplar. Tween 80 was added to the SSF system to show its potential in the improvement of the enzymatic hydrolysis and fermentation.
2.1. Materials
The poplar wood chips in this study were obtained locally, pulverized to 20–80 mesh and sealed at room temperature (9.7% moisture). Cellulase (Cellic CTec2, SAE0020) was purchased from Sigma Shanghai, China (filter paper enzyme activity 200 FPU/g). Saccharomyces cerevisiae used in the fermentation was commercial active dry yeast from Angel Yeast Co., Ltd., and the other chemical reagents were purchased from Nanjing Chemical Reagent Co., Ltd.
2.2. Dilute acid pretreatment
50 g (dry weight) poplar wood chips were soaked in 350 mL of 1% (w/w) H2SO4 with a solid-liquid ratio of 1:7 and then transferred to the reaction tank of oil-bath digester after soaking overnight. The oil bath was heated from room temperature to 180°C and held at 180°C for 60 min. Pretreated materials and the prehydrolysate were separated and collected by filtration after dilute acid pretreatment, and the substrates were washed with distilled water to pH neutrality. The pH of the prehydrolysate was tested to be 2.3. The washed materials and the prehydrolysates were stored under refrigeration at 4°C for further utilization and analysis.
2.3. Enzymatic hydrolysis of Avicel and pretreated substrates with prehydrolysates
The pH of the added prehydrolysates in the enzymatic hydrolysis was adjusted to 4.8 with NaOH and buffered with 0.05 mol/L citrate buffer. The enzymatic hydrolysis was carried out in a 20 mL hydrolysis system with 20 FPU/g cellulase enzyme and prehydrolysates in 150 mL conical flasks. 2% (w/v) glucan of corresponding Avicel or pretreated poplar were applied in the enzymatic hydrolysis. The conical flasks were placed in a 50 ℃ shaker at 150 rpm, and the samples were taken at 0, 3, 6, 12, 24, 48 and 72 h. The samples were centrifuged for solid-liquid separation, and then diluted and filtered by membrane filtration. Each set of experiments was conducted with duplicate and the experiment results were averaged from the parallels.
2.4. Yeast fermentation of prehydrolysates
The yeast fermentation was carried out using bakery yeast (Saccharomyces cerevisiae) with the spiked glucose concentration in deionized water (sugar control) and prehydrolysates (PH, PH-20, PH-50, PH-75) to be around 20 g/L to produce ethanol. The dry yeast was activated in YPD medium (20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract) for 12 h. The pH of the prehydrolysates was adjusted to 6.0 with NaOH before it applied in the fermentation. 6 OD of yeast was applied as the inoculum amount for ethanol fermentation determined by an ultraviolet (UV) spectrophotometer with the wavelength of 600 nm. Fermentation experiments were carried out in a thermostatic air bath shaker at 30 ℃, 150 rpm. Each set of reactions was placed in 150 mL conical flasks with a 30 mL of reaction system. 0.2 ml samples were taken at 0, 3, 6, 9, 12, 24, 48 h and analyzed by HPLC to calculate the consumption of glucose and ethanol production during ethanol fermentation. Experiment results were averaged from the double parallel experiments.
2.5. Determination of total phenols
The total phenol content (TPC) was determined by the Folin-Ciocalteu (FC) method as what has been reported (Allard-Massicotte et al., 2017). Briefly, standard curve was evaluated by the absorbance of supernatant at 745 nm various concentrations of gallic acid solution with FC reagent, 6% Na2CO3, and distilled water for 120 min in a 30°C water bath (80 rpm). The TPC of samples (1 mL) was determined with the same protocol according to the standard curve.
2.6. Resin adsorption of prehydrolysates
As one of the most commonly-used resins applied for phenolic compound adsorption of biomass prehydrolysate (Heinonen et al., 2017; Li et al., 2020), XAD-16N resin was used to remove the inhibitors in prehydrolysate such as phenolic compounds from dilute acid pretreatment. The resins should be washed by deionized water and methanol before use as previous reports (Cao et al., 2020). Based on condition optimization, 10, 40, and 150 g/L (dry weight) of the washed resin were applied to adsorb the inhibitors in a thermostatic water bath at 30°C and 80 rpm for 5 min before solid-liquid separation. HPLC analysis of the separated liquid was conducted to determine the amount of FA, AA, LA, HMF, and furfural. The TPC of the detoxified prehydrolysates was determined as the FC method mentioned above.
2.7. Addition of external inhibitors
Carbohydrates-derived inhibitors including FA, AA, LA, HMF, and furfural were added into the enzymatic hydrolysis and the fermentation with high concentration stock solution prepared with deionized water or ethanol and pure chemical. After the addition of a single or all stock solutions, the volume of was fixed to the required volume for hydrolysis or fermentation. With the addition of external inhibitors, a new concept of “degree of inhibition” was introduced to show the inhibition variance on the enzymatic hydrolysis between different inhibitors. The degree of inhibition was defined as the following equation:
2.8. Analysis of compounds in prehydrolysate
The samples from enzymatic hydrolysis and yeast fermentation were analyzed by Aminex Bio-Rad HPX-87H column and the high-performance liquid chromatography (HPLC) system with 0.6 mL/min 5 mM sulfuric acid as mobile phase for the glucose yield, glucose consumption and ethanol concentration (column and RID detector temperature: 45°C). The inhibitors extracted from prehydrolysates were determined and quantified by GC/MS. A 1:1 extraction of the pretreatment solution and dichloromethane was first performed and repeated twice. The excess water in the extract was removed with anhydrous sodium sulfate, and the dewatered dichloromethane solution was concentrated by nitrogen blowing to obtain a 2 mL concentrated sample. GC/MS analysis was performed on an Agilent 7000B equipped with a quadrupole mass detector with a HP-5MS column (30 m× 0.25 mm internal diameter) (Zhang et al., 2018).
2.9. SSF of pretreated substrates with prehydrolysates
The SSF process of the washed dilute acid pretreated poplar (AP) and the unwashed pretreated poplar with residual prehydrolysates (UNAP) was carried out in 150-mL conical flasks with reaction volume of 30 mL with 2% (w/v) glucan of corresponding pretreated substrates. pH of the SSF reaction was adjusted to 4.8 with NaOH and buffered with 0.05 mol/L citrate buffer. 1.5 g/L of Tween 80 was added to the system right before the addition of cellulase enzyme to eliminate the inhibition of the prehydrolysate on the SSF process as a comparison. The enzyme loading for the SSF was 20 FPU/g glucan in a thermostatic shaker at 50 ℃ with a rotation speed of 150 rpm. The samples were taken at 0, 3, 6, 12 and 24 h, respectively. After the 24th hour of SSF reaction, the temperature of reaction was reduced to 35°C and 6 OD of yeast cells was inoculated into the system to initiate the fermentation process of SSF. The following samples were taken at 27th, 30th, 36th, 48th and 72nd h of SSF. The supernatants of the samples collected by centrifugation and filtration were analyzed by HPLC. Data representing experiment results were all averaged from duplicates.
3.1. Chemical composition analysis of pretreated substrates and prehydrolysates from dilute acid pretreatment
To assess the effect of prehydrolysates (PH) on the enzymatic hydrolysis and yeast fermentation, PH and the corresponding pretreated substrates (AP) were collected from the dilute acid pretreatment of poplar. Their chemical compositions were evaluated and represented in Table 1 (AP) and Table 2 (PH). Compared to raw poplar, the content of ethanol extractive of AP was increased from 3.21–16.79% after dilute acid pretreatment. Extractives of pretreated substrates have been reported to be the decomposed lignin and other minor components of biomass (waxes, starches, tannings, etc.,) after pretreatment (Lai et al., 2014), so the increasement of ethanol extractives showed a potentially good decomposition of lignin. Although the acid insoluble lignin (AIL) did not alter too much (23.49–23.59%) after the pretreatment, the decrease of acid soluble lignin (ASL) from 2.30–0.64% may be related to the reaction during the dilute acid pretreatment as well (Hsu et al., 2010). Based on the content of glucan and xylan in the raw poplar, the glucan in AP increased from 39.83–52.40% while the xylan was decreased from 15.11–2.65%. This may be due to the better thermal stability of cellulose (mainly composed of glucan) than hemicellulose (mainly composed of xylan) in the acidic pretreatment (Shen et al., 2010). With the high recovery rate of 98.87%, the major of glucan was proved to be maintained in the pretreated substrates. However, the content of xylan was decreased with the recovery rate of only 12.65%. This means more than 87% of xylan was released as xylose or further degraded into yeast fermentation inhibitors like furfural and acetic acid in PH under this pretreatment condition (Yoon et al., 2014).
| Biomass | Composition (%) | Recovery rate (%) | |||||
|---|---|---|---|---|---|---|---|
| Ethanol Extractives | Glucan | Xylan | Acid insoluble lignin (AIL) | Acid soluble lignin (ASL) | Glucan | Xylan | |
| Raw material | 3.21 ± 0.09 | 39.83 ± 0.33 | 15.11 ± 0.22 | 23.49 ± 2.71 | 2.30 ± 0.01 | - | - |
| AP | 16.79 ± 0.08 | 52.40 ± 0.06 | 2.65 ± 0.59 | 23.59 ± 0.49 | 0.64 ± 0.01 | 98.87 | 12.65 |
| Prehydrolysates | Monosaccharides (g/L) | Inhibitors (g/L) | TPC (mg/L) | |||||
|---|---|---|---|---|---|---|---|---|
| Glucose | Xylose | FA | AA | LA | HMF | Furfural | ||
| PH | 3.48 | 0.78 | 1.50 | 5.82 | 0.26 | 1.50 | 5.42 | 2678.42 |
To determine the degraded monosaccharides and inhibitors from hemicellulose and cellulose in the prehydrolysates (PH), its chemical composition, including the fermentation inhibitors degraded from the monosaccharides and lignin, has been analyzed by HPLC (Table 2). The amount of total phenolic content (TPC) was analyzed by the method of Folin–Ciocalteu and presented in Table 2 as well. With the high pretreatment temperature of 180°C, the glucose concentration was comparable (3.48 g/L) while the xylose concentration (0.78 g/L) in PH was relatively low, which may be the reason of a comparatively high concentration of furfural (5.42 g/L) compared to what we reported previously (Sheng et al., 2020). High content of acetic acid (5.82 g/L) is another indicator of the severe degradation of hemicellulose under 180°C. Furthermore, FA, AA, LA, and HMF have been revealed as the fermentation inhibitors degraded from glucose (Rackemann et al., 2016). The concentrations of FA, LA, HMF in PH were 1.50 g/L, 0.26 g/L, and 1.50 g/L, respectively, which were relatively high compared to what reported before. Consistently, the decomposition of lignin was intense as well considering the 2678.42 mg/L phenolic compounds (TPC) observed. The high inhibitor content of PH may make it a good candidate to reveal the effect of inhibitors on the enzymatic hydrolysis, which has not been fully elucidated before.
Besides the observation of sugar degradation compounds and lignin degradation compounds analyzed by HPLC, the other chemical components of PH were further analyzed by GC/MS and shown Fig. 1 and Table 3. The concentrations of furfural and HMF were 3117.21 mg/L and 465.61 mg/L, which were divergent from the number examined by HPLC (5417 mg/L and 1494 mg/L, Table 2). This may be related to the content loss during the liquid-liquid extraction process before GC/MS sample injection and the higher sensitivity of HPLC (Hao et al., 2007). In total, 16 potential phenolic inhibitors, including phenol, vanillin, and syringaldehyde, were identified and quantified by GC/MS and represented in the TIC spectrum (Fig. 1). Except the three aromatic acids and phenol, 15 of them are phenolic aldehydes and ketones, which are believed to be the most toxic to the yeast fermentation even with relatively low concentration (Xie et al., 2012). Therefore, PH and AP are good candidates to examine the effect of prehydrolysates and its phenolic compounds on the enzymatic hydrolysis when the tolerably high toxicity of PH and good recovery of cellulose of AP are considered.
| GC peak | Compound name | Retention time (min) | Concentration a (mg/L) |
|---|---|---|---|
| 1 | Furfural | 5.24 | 3117.21 |
| 2 | 5-Methylfurfural | 5.59 | 50.83 |
| 3 | Phenol | 6.07 | 86.91 |
| 4 | 2-Methoxyphenol | 6.92 | NA |
| 5 | Benzoic acid | 8.09 | 18.45 |
| 6 | HMF | 8.63 | 465.61 |
| 7 | 3,4,5-Trihydroxybenzaldehyde | 9.51 | 12.30 |
| 8 | Vanillin | 10.07 | 87.73 |
| 9 | Guaiacylacetone | 10.55 | 21.32 |
| 10 | 1-Hydroxy-3-(4-hydroxy-3-methoxyphenyl)propane-2-one | 11.13 | 17.63 |
| 11 | Hydroxypropiovanillone | 11.58 | 20.91 |
| 12 | 1-(3,4,5-Trihydroxyphenyl)propane-1,2-dione | 12.00 | 5.74 |
| 13 | Syringaldehyde | 12.13 | 184.88 |
| 14 | Homosyringaldehyde | 12.41 | 50.01 |
| 15 | 1-Hydroxy-3-(4-hydroxy-3-methoxyphenyl)-2-propanone | 12.75 | 32.80 |
| 16 | Syringylacetone | 12.88 | 83.63 |
| 17 | 1-(4-Hydroxy-3,5-dimethoxyphenyl)propane-1,2-dione | 13.26 | 41.81 |
| 18 | 2-Hydroxy-1-syringyl-ethanone | 14.42 | 121.75 |
| 19 | (4-Hydroxy-3,5-dimethoxybenzoyl)-acetaldehyde | 14.54 | NA |
| Total amount of inhibitors | 5245.63 | ||
| a The concentrations of the inhibitors were calculated based on the integration area of each compound. | |||
3.2. Effect of prehydrolysates and its phenolic compounds on enzymatic hydrolysis
With the addition of the highly toxic PH into the enzymatic hydrolysis, the digestion of Avicel (Fig. 2a) and pretreated substrates (AP) by the cellulase enzymes were the first to be evaluated (Fig. 2b). The enzymatic hydrolysis of both Avicel and AP was dramatically inhibited by PH: the 72 h hydrolysis yield of Avicel was deduced from 81.75–48.13% when PH was added into the hydrolysis system. Similarly, the glucose yield of digested AP was reduced from 46.62–27.74% with PH. Analogous to what we hypothesized, the phenolic compounds-rich PH was particularly inhibitory to the enzymatic hydrolysis to both pure cellulose (Avicel) and pretreated substrates (AP).
Furthermore, the enzymatic hydrolysis of Avicel with the addition of FA, AA, LA, HMF, and furfural, were investigated separately and together and represented in Fig. 3 to distinguish the effect of sugar degradation compounds and lignin degradation compounds. The addition of PH in the enzymatic hydrolysis of Avicel deducted the final glucose yield from 83.17–48.13%, while the addition of FA, AA, LA, HMF, furfural, and these five carbohydrates-derived inhibitors together (All) brought this number to 80.51%, 81.16%, 82.74%, 82.05%, 79.31%, 59.90%, respectively (Fig. 3a). As shown in Fig. 3b, the addition of FA, AA, LA, HMF, and furfural all showed little degree of inhibition on the 72 h hydrolysis yield of Avicel (FA: 3.20%, AA: 2.42%, LA: 0.52%, HMF: 1.34%, and furfural: 4.64%). This was consistent with the weak inhibition of acid and furan aldehyde on the yeast fermentation reported previously (Wang et al., 2020). These components may poison the crucial proteins of cellulase in the enzymatic hydrolysis due to analogous reasons as they negatively affect the metabolic activity of the micro-organism (Jönsson & Martín, 2016). However, all the above inhibitors together (“All” in Fig. 3) showed an inhibition degree of 15.58%, which was slightly higher than the sum of inhibition degree (12.12%) of the above inhibitors. This may be related to the additive effect of several inhibitors together (Basak et al., 2020). More interestingly, the inhibitory effect of PH was 42.13%, which means the net degree of inhibition of phenolic compounds from prehydrolysates was 26.55% on the enzymatic hydrolysis, when their single concentration (< 184.88 mg/L) and total concentrations (2678.42 mg/L) were both much lower than the carbohydrates-derived inhibitors (0.26–5.82 g/L). This suggested the suppression of phenolic compound on the enzymatic hydrolysis was much stronger than the inhibition degree of carbohydrates-derived inhibitors. This huge variance between the inhibition of PH and all carbohydrates-derived inhibitors together (Fig. 3a) should be due to the phenolic compounds coming from the decomposition of lignin. According to previous reports, phenolic compounds are thought to be the most detrimental factors for the membranes of microbes and the metabolism inside of the microbial cells (Palmqvist & Hahn-Hägerdal, 2000), therefore, their inhibition could be alike when they hydrophobically and chemically interacted with cellulase enzymes.
In order to test the characteristic inhibition of phenolic compounds, different loads of resin were applied to adsorb 20%, 50%, and 75% of TPC in PH (named as PH-20, PH-50, and PH-75) (Table 4), and the corresponding removal rate of the carbohydrates-derived inhibitors in the prehydrolysates were shown in Fig. S1. Extra FA, AA, LA, furfural, and HMF were added back into PH-75 to match the same amount of carbohydrates-derived inhibitors in PH and named as “PH-75 (Add inhibitors)”. The effect of phenolic inhibitors on the enzymatic hydrolysis was further explored when PH, PH-75, PH-75 (Add inhibitors) were added into the enzymatic hydrolysis of Avicel (Fig. 4a) and AP (Fig. 4b), respectively. Compared to the 33.62% (from 81.75–48.13%) inhibition of PH addition in the Avicel, the glucose yield of Avicel with the addition of PH-75 and PH-75 (Add inhibitors) after 72 h enzymatic hydrolysis was decreased from 81.75–66.52% (15.23% inhibited) and 61.65% (20.10% inhibited) (Fig. 4a). Although all 5 carbohydrates-derived inhibitors did not make much difference in the inhibition of enzymatic hydrolysis (4.87% difference between PH-75 and PH-75 (Add inhibitors)), around 40% of degree of inhibition was eliminated when 75% of TPC were removed in Avicel hydrolysis (PH-75 (Add inhibitors)). This indicated various inhibition levels for different phenolic compounds with distinguishing adsorption affinity to the resin (Sun & Zheng, 2021). Phenolic compounds with double bonds and aldehyde/ketone groups have been reported to show stronger inhibition on the microbial organisms (Xie et al., 2016). The reason behind their different inhibition levels on the cellulase enzymes may be similar. Correspondingly, the final glucose yield for enzymatic hydrolysis of AP was deducted from 46.62–36.00% (PH-75) and 33.20% (PH-75 (Add inhibitors)) when PH could decrease this yield to be 27.74%. Only 29% degree of inhibition was eliminated when 75% of TPC were removed in AP hydrolysis with PH. This means similar distinguishing inhibition of different phenolic compounds on the enzymatic hydrolysis. Moreover, the inhibitory effect of PH, PH-75, PH-75 (Add inhibitors) in the enzymatic hydrolysis of pretreated substrates were variant to those in the pure cellulose.
| Prehydrolysates | FA (g/L) | AA (g/L) | LA (g/L) | HMF (g/L) | Furfural (g/L) | TPC (mg/L) |
|---|---|---|---|---|---|---|
| PH | 1.33 | 5.96 | 0.23 | 1.44 | 4.96 | 2395.84 |
| PH-20* | 1.28 | 5.80 | 0.16 | 1.21 | 3.28 | 1924.09 |
| PH-50* | 1.18 | 5.34 | 0.14 | 0.88 | 1.55 | 1116.85 |
| PH-75* | 0.82 | 3.36 | 0 | 0.24 | 0.21 | 667.84 |
| * PH, PH-20, PH-50, and PH-75 indicate prehydrolysates with 0%, 20%, 50%, and 75% TPC removal treated by resin adsorption, respectively. | ||||||
Therefore, the prehydrolysates from dilute acid pretreatment of poplar showed strong inhibition on the enzymatic hydrolysis of pure cellulose and pretreated substrates, which mainly originated from some certain types of phenolic compounds in the prehydrolysates but not the carbohydrates-derived inhibitors including FA, AA, LA, furfural, and HMF.
3.3. Effect of prehydrolysates and its phenolic compounds on the yeast fermentation
To compare the effect of prehydrolysates and its phenolic compounds on yeast fermentation and the enzymatic hydrolysis, the PH and the partially detoxified PH including PH-20, PH-50, and PH-75, were added into the fermentation of sugar control by S. cerevisiae, respectively (Fig. 5). The results showed that PH and PH-20, which contain more than 1924.09 mg/L TPC (Table 4), could completely stop the fermentation of yeast cells. The glucose consumption in both cases was less than 1 g/L and the corresponding ethanol production was less than 0.5 g/L. In comparison to the inhibition of PH in the enzymatic hydrolysis, PH terminated the metabolism of yeast cells and killed the cells while it only partially inhibited the enzymatic hydrolysis by cellulases. This may be because the high toxicity of PH could poison not only the function of intracellular proteins like cellulases, but also other significant metabolisms like ATP and energy consumption, etc (Wang et al., 2018). Under the same condition of prehydrolysate addition, the simpler enzyme assisted hydrolysis reaction would be just hindered but not completely terminated. Interestingly, with higher loading of resin in the prehydrolysate detoxification, PH-50 and PH-75 became not only fermentable with 6 OD of S. cerevisiae, they even showed higher fermentability (higher glucose consumption rate and ethanol productivity) with yeast cells than those in the artificial pure sugar controls. In PH-50, 16.76 g/L of glucose was fully consumed with the ethanol production of 5.07 g/L (fermentation completed after 12 h, ethanol evaporated to 3.82 g/L after 48 h), while the same inoculation of yeast finished its consumption of 15.09 g/L glucose with the ethanol production of 5.74 g/L (fermentation completed after 9 h, ethanol evaporated to 5.27 g/L after 48 h) in PH-75. In both detoxified prehydrolysates, yeast finished their consumption of glucose in 9–12 h when the cells finished 16.28 g/L of glucose with ethanol production of 4.88 g/L in 48 h completely in sugar control. Yeast cells converted similar amount of glucose in a higher rate with detoxified prehydrolysates, which means the promotional effect other than inhibitory effect on the fermentation of S. cerevisiae in PH-50 and PH-75. This stimulation may be related to the promotional effect of the less than 50 mM salts of AA (< 1.440 g/L, Table 4) on the fermentation rate and productivity of S. cerevisiae as reported previously (Cao et al., 2014), the limited amount (< 3.28 g/L as indicated in Table 4) salts of FA and LA may further improve the stimulatory effect on the yeast fermentation. Therefore, because of the complex metabolism of the active cells, prehydrolysates showed distinct inhibition on the enzymatic hydrolysis and fermentation. PH with different amounts of inhibitors hindered the enzymatic hydrolysis all the time. However, PH with high concentration of phenolic inhibitors could deactivate the fermentation of the yeast cells while PH with low concentration of inhibitors stimulated the fermentation.
To distinguish the inhibitory effect of phenolic compounds and the carbohydrates-derived inhibitors, FA, AA, LA, HMF, and furfural were added into the fermentation of sugar controls, respectively and altogether, in comparison to yeast fermentation in the real prehydrolysates (PH). The glucose consumption and ethanol production of yeast cells in the fermentation were evaluated and represented in Fig. 6a and Fig. 6b. Compared to the sugar control, the addition of FA, LA, and HMF, respectively, showed merely no inhibition on the fermentation of yeast cells. Interestingly, the addition of AA even improved the glucose consumption rate and promoted the complete conversion of 19.30 g/L of glucose to only 12 h and improved the final ethanol yield from 6.75 g/L to 7.88 g/L. The addition of FA, HMF and even all inhibitors together showed similar promotion on final ethanol yield (7.49 g/L, 7.64 g/L, 7.68 g/L). Moreover, the addition of FA, LA, and HMF, improved the ethanol production rate of yeast (slope of ethanol production curve). This was consistent with what discussed above about the stimulatory effect of salts of weak acid in the fermentation of sugar control. The fermentation of sugar control with furfural showed the strongest inhibition with the addition of individual inhibitor. It slowed down the completion of 19.14 g/L of glucose by yeast from 24 h to 48 h with the ethanol production of 6.80 g/L, which is similar to that of sugar control (6.75 g/L). Compared to the individual inhibitors, yeast in the sugar control with all carbohydrates-derived inhibitors together (All, including FA, LA, AA, HMF, furfural) even had higher glucose consumption rate (completed in 24 h) than it with furfural only (completed in 48 h). The final ethanol production was 7.68 g/L, which was 1.13 g/L higher than it in the sugar control. This was potentially because the stimulatory effect of salts of weak acids concealed the obvious inhibition of furfural. Importantly, PH was not fermentable even when all the carbohydrates-derived inhibitors only showed limited inhibition together (slower rate but completion of glucose in 24 h still). These results emphasized the strong inhibition of phenolic compounds to the yeast fermentation. In the supplementary information, the fermentability of detoxified prehydrolysate, PH-75, and its fermentation with the addition of all individual inhibitors discussed above (FA, LA, AA, HMF, furfural) was examined and compared with PH. Resin detoxification made the PH actively fermentable to generate 7.51 g/L ethanol with 18.59 g/L of glucose in 9 h. The addition of all carbohydrates-derived inhibitors and around 25% of TPC (PH-75 (Add inhibitors)) decreased the fermentation rate of yeast cells but not the ethanol yield of 7.51 g/L out of the glucose consumption of 19.39 g/L in 24 h (Fig.S2 a and b). Overall, the prehydrolysates from dilute acid pretreatment of poplar showed strong inhibition on yeast fermentation and could even terminate the cell metabolism completely. This inhibitory effect may mainly come from phenolic compounds in the PH. But this strong inhibition could be eliminated and turned into stimulatory effect on the fermentation when the TPC was controlled under 1116 mg/L.
3.4. Effects of prehydrolysates and Tween on the SSF of pretreated substrates
To apply the above understanding of the prehydrolysate inhibition on the enzymatic hydrolysis and yeast fermentation in the practical use, SSF of water washed pretreated substrates (AP) and unwashed pretreated substrates (UNAP) containing prehydrolysates was conducted with the addition of PH (Fig. 7). Since the surfactant of Tween 80 was reported to be beneficial on the enzymatic hydrolysis by the release of non-productive binding between cellulase and lignin (Hou et al., 2022), and also detoxification of the phenolic compounds (Lee et al., 2015; Qin et al., 2016), Tween 80 was added before the initiation of the SSF to overcome the inhibition of prehydrolysates (Fig. 7). For the enzymatic hydrolysis in the first 24 h, the addition of Tween significantly improved the 24 h glucose yield of AP from 7.78 g/L to 14.52 g/L and UNAP with PH from 3.64 g/L to 7.55 g/L before the change of culture temperature and the addition of yeast. This means that the stimulatory effect of Tween 80 worked in both cases of enzymatic hydrolysis even when the pretreated substrates were not washed with the prehydrolysates together. After the enzymatic hydrolysis for 24 h, the culture temperature was dropped down to 30°C and 6 OD of yeast cells were inoculated in the hydrolyzed substrates and prehydrolysates. The glucose from enzymatic hydrolysis in the washed substrates (AP) and the washed substrates with Tween (AP + Tween) was started to be consumed right after the inoculation of yeast, which means the glucose consumption rate of yeast was higher than the glucose accumulated from the following enzymatic hydrolysis of the washed substrates after yeast inoculation. The final ethanol production reached 6.25 g/L for AP with Tween added when 14.52 g/L of accumulated glucose in the enzymatic hydrolysis and some undetected further hydrolyzed glucose were all consumed in 48 h of yeast inoculation. While for AP without any surfactant, the ethanol production was only 2.48 g/L when at least 7.78 g/L of accumulated glucose was consumed in 12 h (Fig. 7a and b). The final ethanol yield was promoted by 152% after Tween 80 was added to the SSF process of AP. This may be the toxicity of the residual lignin on the surface of the washed substrates to the yeast cells as it did to the cellulase in the enzymatic hydrolysis (Zhang et al., 2012). Furthermore, with the addition of Tween 80, the initial glucose consumption rate (6 h) of AP + Tween was also promoted from 0.63 g/L/h (AP) to be 1.34 g/L/h. This indicates the stimulatory effect of surfactant in yeast fermentation by its benefits to overcome the inhibition from PH (Li et al., 2018b). For the unwashed substrates (UNAP + PH, UNAP + PH + Tween) after the inoculation of yeast, glucose continued to be accumulated for 3 h to 5.63 g/L for UNAP with prehydrolysates (UNAP + PH), and continued to be accumulated for 12 h to 10.10 g/L for UNAP with prehydrolysates and Tween 80 (UNAP + PH + Tween) (Fig. 7a). Glucose concentrations were steady for UNAP + PH (5.57 ~ 5.72 g/L) and UNAP + PH + Tween (9.87 ~ 10.10 g/L) after these time points, which means no obvious glucose consumption or accumulation was observed from the 36th h of SSF to the termination of the process (72 h). 2.83 g/L and 2.96 g/L ethanol were produced with UNAP + PH and UNAP + PH + Tween in 6 h and 12 h of yeast inoculation, respectively (Fig. 7b). Ethanol concentrations were steady for UNAP + PH (2.71 ~ 2.82 g/L) and UNAP + PH + Tween (2.82 ~ 3.02 g/L) after the 30th and 36th h of SSF process, which means the fermentations of UNAP + PH and UNAP + PH + Tween were stopped after 6 h and 12 h of yeast inoculation, respectively, even when there was 5.63 g/L and 10.10 g/L glucose left in UNAP + PH and UNAP + PH + Tween. With the addition of highly toxic prehydrolysate, the promotion of Tween 80 in the final ethanol yield was around 7.10% when 76.6% of glucose promoted to be accumulated. This correlates with the previous finding that strong inhibition from prehydrolysates showed more obvious inhibitory effect on yeast fermentation than enzymatic hydrolysis or even terminated its reaction and metabolisms. The potential detoxification effect of Tween 80 on the SSF may be due to the alleviation of the attachment between the prehydrolysate inhibitors, especially the phenolic compounds, and the cellulase enzymes (Alkasrawi et al., 2003). It has also been suggested that surfactant may solubilize the hydrophobic PH inhibitors by embodying them into micelles, and thus weaken their interaction with the cell membrane, therefore, eliminate their inhibition on the yeast cells (Guan et al., 2018). Therefore, Tween 80 could benefit both enzymatic hydrolysis and fermentation of washed substrates without prehydrolysates and promoted the overall ethanol production from the pretreated substrates. It can also benefit the enzymatic hydrolysis of unwashed substrates with prehydrolysates by the stimulation on glucose consumption, however, show no obvious promotion of the subsequent fermentation when the prehydrolysate showed strong inhibition on the fermentation. So, the beneficial effect of Tween 80 in SSF process may be only valid for washed substrates without prehydrolysates, but not for the unwashed substrates with prehydrolysates.
Prehydrolysates and pretreated substrates from dilute acid pretreatment with harsh condition were collected and analyzed with HPLC and GC/MS in this study. 3 aliphatic acids, 3 furan derivatives, and 19 phenolic alcohols, aldehydes, and acids were identified, and their effects on the enzymatic hydrolysis and fermentation were investigated, respectively. Carbohydrates-derived inhibitors including AA, FA, LA, furfural, and HMF showed less than 4.64% degree of inhibition separately and 15.58% of inhibition together. This is much lower than the suppression of phenolic compounds (26.55%) from prehydrolysates on the enzymatic hydrolysis, even when their single concentration (<184.88 mg/L) and total concentrations (2678.42 mg/L) were both much lower than the carbohydrates-derived inhibitors (0.26-5.82 g/L). When different loads of adsorption resin were applied to detoxify the phenolic compounds in the prehydrolysates, 40% of degree of inhibition was eliminated when 75% of total phenolic content was removed in Avicel hydrolysis. This indicated distinct inhibition levels of various phenolic compounds: a small number of inhibitors improved the glucose consumption rate and ethanol production in the fermentation, but the untreated prehydrolysate terminated the fermentation while the prehydrolysates only partially hindered the enzymatic hydrolysis. Correlated with the distinguishing inhibition modes of prehydrolysates on the enzymatic hydrolysis and fermentation, addition of Tween 80 in the SSF reduced the inhibition of prehydrolysates on the enzymatic hydrolysis but showed deficient “detoxification effect” on the fermentation process with prehydrolysates in SSF. The saccharification process was promoted with the surfactant addition, but the fermentation, although stimulated, still terminated half-way with 9.66 g/L glucose remained.
Authorship contribution statement
XT: Conceptualization, Methodology, Formal analysis, Investigation, Data Curation, Writing-Original Draft, Funding acquisition.
LW: Investigation, Resources, Writing-Original Draft.
YL: Software, Validation, Writing-Review & Editing.
QZ: Conceptualization, Methodology, Validation.
ST: Writing-Review & Editing, Software.
YS: Supervision, Funding acquisition, Project administration.
CL: Resources, Visualization.
All authors reviewed the manuscript.
Acknowledgments
This work was supported by Natural Science Foundation of Jiangsu Province (BK20200775), Natural Science Foundation of Anhui Province (2308085QC103), the Natural Science Research Projects of Universities in Anhui Province (2023AH050900), Wuhu Science and Technology Plan Project (2023yf116), Anhui Polytechnic University Research Startup Foundation (2022YQQ069, S022023011).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data Availability
Data will be made available on request.
Ethics approval
This article does not contain any studies with animals or human participants.
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No competing interests reported.