Environ Eng Res > Volume 27(4); 2022 > Article
Yang, Deng, Jin, Wu, Jiang, and Shi: Hydrolytic performances of different organic compounds in different lignocellulosic biomass during anaerobic digestion

### Abstract

Hydrolytic performances of different organic compounds in lignocellulosic biomass (LB) during anaerobic digestion (AD) are worth investigating due to the complex and refractory structure of lignocellulose. This study aimed to clarify the hydrolytic performances of different lignocellulosic components (hemicellulose, cellulose and lignin) and other typical organics (saccharides, protein and lipid) in AD process. Furthermore, an in-depth study of different lignocellulosic components mono-/co-digestive performances, as well as their effects on digestive systems were also designed to explain the mechanism. Kinetic models were specially applied to evaluate the hydrolytic process and make comparison among different lignocellulosic components. Results showed that hemicellulose obtained high degradation ratio (77.2–85.0%) during anaerobic digestion, while cellulose was difficult to hydrolyze without sufficient acidity. And organics (saccharides, protein and lipid) were much easier to be hydrolyzed than lignocellulose. Results also depicted that lignocellulose addition could efficiently enhance the volatile solid (VS) removals of digestive systems, while lignin existing in systems reduced the VS removal. The limited hydrolysis of lignocellulose hindered the degradation of total VS in digester. It is quite important to obtain high bioenergy conversion, pretreatments, which can destroy the lignin wrapping in LBs digestion. This study could provide a reference for the AD of LBs.

### 1. Introduction

In recent years, increasing attention has been paid on bioenergy recovery from biomass waste [1, 2]. As a kind of dominating biomass waste, lignocellulosic biomass (LB), commonly includes crop straw, grass and leaves, waste paper pulp, municipal sludge and so on, it was widely applied in anaerobic digestion (AD) process [3]. During LBs’ AD process, macromolecular organic compounds in LBs (lignocellulose, saccharides, protein and lipid) are firstly hydrolyzed to micromolecular organics (such as monosaccharide, amino acid, peptide, glycerin and so on) [4]. Then, acidogenesis and acetogenesis convert these micromolecular organics into acetic acid, H2 and CO2 [1]. Eventually, under the action of methanogens, these materials are further converted into biogas, dominated by methane [5]. In this cycle, hydrolysis is the rate-limiting step, and its duration depends on the properties of the substrates [6]. Among these substrates, lignocellulose is the most abundant organic matter in the world, and it is composed of hemicellulose, cellulose and lignin. These three kinds of substances commonly intertwine with each other and form a combined state. Due to the encapsulation of refractory lignin and the bonding of chemical bonds, constitutionally stable lignocellulose is commonly difficult to be hydrolyzed and utilized by microbes [7]. Previous study [8] found that the hydrolysis of LBs was much slower than other organic solid waste, which might be attributed to the refractory property of lignocellulose in LBs. Besides, lignocellulose structure is another important factor which can affect the digestive performances of LBs [9].
Based on the analysis above, this study attempted to investigate the hydrolytic performances of different organic compounds (hemicellulose, cellulose, lignin, saccharides, protein and lipid) in several LBs (excess sludge (ES) and different crop straws) during the AD process. Kinetic models were applied to evaluate the hydrolytic performances of digestive systems and different lignocellulosic components. In addition, the mono-/co-digestibility of different lignocellulosic components in sludge was further studied to clarify its hydrolysis performance and its impact on the digestive system. The innovation of present study lies in the separation of lignocellulosic monomers, verification in real LBs, and analysis by applying kinetic models. This study tried to provide some ideas and directions for the future researches of LBs’ digestion.

### 2.1. Experimental Materials

In this study, excess sludge (ES), corn cob (CC), corn straw (CS), wheat straw (WS) and rice straw (RS) were chosen to represent different kind of LBs. ES was taken from the secondary sedimentation tank of Tangjiatuo sewage treatment plant, Chongqing. This plant adopts anaerobic-anoxic-oxic (A/A/O) biochemical treatment process with a treatment capacity of 400 thousand tons of sewage per day. After one-day gravity precipitation, ES was immediately characterized by its VS and lignocellulosic content, and the remains were stored at 4°C before using. CC, CS, WS and RS were collected from a farm. They were air-dried and then ground into small particles by a mill. The particles were sieved through 20 and 100 mesh sieves respectively to remove the larger and smaller particles. Xylan (HWG25024, HWRK), α-cellulose (C104841, Aladdin) and lignin (L195713, Aladdin) were respectively applied as representatives of hemicellulose, cellulose and lignin [17]. A kind of synthetic sludge (SS) was derived from the sedimentation tank of an A/O reactor in our laboratory. This reactor was fed with ammonium bicarbonate and glucose solution (COD = 1,600 mg/L) every day. Due to no exogenous lignocellulosic addition, SS contained no lignocellulose component. Inocula were the digested sludge taken from our laboratory (SRT = 25 d, TS = 5.5%). The digested sludge was sampled and stored under anaerobic condition at 37 ± 1°C for one week to remove organics as much as possible. All feedstock was stored in the 4°C condition before used. The characteristics of different feedstock’ components are listed in Table 1.

### 2.2. Experimental Design

This study was divided into two tests. Test I aimed to explore the hydrolytic performances of different kinds of LBs (ES, CC, CS, WS and RS) in batch digester. The total lignocellulose amounts in different digester were controlled in accordance with that in ES. Experimental design of Test I is shown in Table 2. Based on the results of Test I, Test II was designed to explore the hydrolytic performances of different kind of lignocellulosic components in batch digester. The focus of this test was to examine the effects of different lignocellulose components on the hydrolysis of the system. Experimental design of Test II is also shown in Table 2. It could be noticed that the added amounts of xylan, α-cellulose and lignin were in accordance with the measured contents in ES.
Batch digesters were several serum bottles with effective volume of 450 mL. Before experiment, different kind of feedstock was added into digesters following the designed formulas. After addition, the rubber stopper was used to seal the bottle, and nitrogen was bubbled through eyelet in rubber stopper for 5 min to obtain the anaerobic condition. After that, the bottles were heated at 37 ± 1°C and shaken at 140 rpm in a water-bath pot. The experimental period was set at 38 d. All experiments were performed in three replicates.

### 2.3. Analytical Methods

Biogas for different digesters were collected by 500 mL gas bags from the eyelet in rubber stopper. A 250 mL syringe was used to measure biogas production. Digestates of different digesters were sampled at specific time points. Soluble chemical oxygen demand (SCOD), total solid (TS) and volatile solid (VS) were measured according to standard methods [18]. Biogas composition was measured using a gas chromatograph (Jiedao GC1690, Hangzhou) with a thermal conductivity detector (TCD). Specific measuring process was shown in previous study [19]. pH values of different digesters were measured by a pH meter (PHS-3S, Leici). Protein in the feedstock was determined through analyzing the concentration of total Kjeldahl nitrogen by a Kjeldahl apparatus (K1305A, Shanghai). Soxhlet extraction method was applied in measuring lipid. Carbohydrates were calculated by subtracting the amount of protein and lipid from total VS. Lignocellulosic components were analyzed using the method developed by Van Soest et al. [20]. Saccharides were calculated by subtracting the total amount of lignocellulose from carbohydrates.

### 2.4. Kinetic Study

Fitzhugh model and modified Gompertz model were applied to explore the hydrolytic and methanogenic performances of different digesters. Hydrolytic constant k value (d−1), which is fitted by Fitzhugh model, can be applied to represent the hydrolytic rate of digestive system [16]. In general, a higher k value indicates a higher hydrolytic rate of digester system. This model is shown in Eq. (1):
##### (1)
$B=B0·[1-exp(-k·t)]$
Where B represents experimental specific biogas yields (mL biogas/g VS) for digestion time t (d), B0 represents simulative specific biogas yields (mL biogas/g VS), and k (d−1) represents hydrolytic constant.
Modified Gompertz model was used to determine the maximum methane production rate and lag phase for AD process by fitting the experimental specific methane yields [18]. This model is shown in Eq. (2):
##### (2)
$Mt=M0·exp{-exp[Rmax·eM0·(λ-t)+1]}$
Where Mt represents experimental specific methane yield (mL/g VS) for digestion time t (d); M0 is the simulative specific methane yield (mL/g VS); Rmax is the maximum methane production rate (mL/g VS/d); λ is the lag phase (d).
Furthermore, another kinetic model-First-order model was applied to analyze the hydrolytic performances of different lignocellulosic components by fitting their degradation data throughout the AD process [16]. This model is shown in Eq. (3)(4):
##### (3)
$dCdt=-k·C$
##### (4)
$Ct=C0·e-kt$
Where C represents degradation ratio of different kind of lignocellulosic component (%) for digestion time t (d), C0 represents experimental degradation ratio (%), and k (d−1) represents hydrolytic constant of different kinds of lignocellulosic components. A higher k value indicated a higher hydrolytic rate of lignocellulosic component.
The experimental data were analyzed using Office Excel 2016, and kinetics were fitted and analyzed by software Origin 8.0 [21].

### 3.1. Biogas Production

Fig. 1 depicts the experimental specific biogas and methane yield of different digesters throughout the AD process. From this figure, it could be observed that different crop straws (CC, CS, WS and RS)-added digesters presented obviously higher cumulative biogas and methane yields than sludge (SS and ES). WS-added digester (S5) obtained the highest biogas and methane yields (490.4 and 287.0 mL/g VS, respectively) among all digesters due to WS’s organic composition and specific lignocellulosic texture, which has been discussed adequately in previous study [22]. In this study, hydrolysis of substrates was much more focused than biogas yield, thus Fitzhugh model and modified Gompertz model were applied, and the obtained kinetic fitting results were shown in Table 3.
Fitting results of Fitzhugh model were shown in the left column of Table 3 (Test I). Hydrolytic constant (k) values fitted by this Fitzhugh model reflect the hydrolytic rates of the whole digestive system. The k values for S1–S6 were 0.060, 0.044, 0.060, 0.031, 0.049 and 0.037 d−1, respectively. SS and CC-added digesters presented higher hydrolytic rates than other digesters during the AD process. Hydrolytic rates of other LBs-added digesters were lower than digester S1, which was due to the huge amount of lignocellulose containing in substrates. Although there was the same amount of lignocellulose in digester S3, the hydrolytic rate of this digester was relative higher than other LBs-added digesters, which could be attributed to the high hemicellulose content (shown in Table 1 and 2) and the looser wrapping of lignocellulose in CC [22]. The result can also verify that CC is an ideal lignocellulosic substrate applying in digestion for its good hydrolytic performance.
The right column of Table 3 (Test I) shows the fitting results of modified Gomportz model. From the fitting results, Rmax and λ can reflect the response rate of microbes to substrates. A smaller λ value indicates a better adaptability of microbes [23], and a higher Rmax indicates the higher activity of methanogens in the digestive system. From this table, it could be noticed that λ value in digester S2 (ES) was obviously higher than others (as high as 10.8 d), which was due to the long generations of methanogens in single ES system [24]. However, the values of λ were greatly shortened in crop straws (CC, CS, WS and RS)-added digesters (5.6–7.5 d), indicating that these types of crop straws (CC, CS, WS and RS) addition could enhance the gasogenic efficiency and microbial function. The unique structures of crop straws could provide suitable adhesion condition for microbes, which is conducive to enrich microbial abundance and to alleviate the inhibitory effects of the intermediates (such as volatile fatty acids and ammonia) on methanogens [25].
In the Test I, hydrolytic performances of different LBs were investigated during the AD process, and the results showed that different LBs presented different hydrolytic performances due to their different lignocellulosic compositions and structure. In order to further explore the mono-/co-digestive properties of different lignocellulosic monomers and their effects on system hydrolysis, Test II was conducted.
Fig. 1 shows the experimental specific biogas and methane yield of different lignocellulosic monomers-added digesters. From this figure, significant differences in biogas/methane production could be observed among different digesters, indicating that different lignocellulosic monomers presented different effects on system hydrolysis and methane yield. Specific gasogenic performances and kinetic study results are shown in Table 3(Test II).
Fitting results of Fitzhugh model are shown in the left column of Table 3(Test II). From this column, it can be observed that the hydrolytic constants (k) in L1–L8 were 0.060, 0.098, 0.027, 0.046, 0.094, 0.055, 0.042 and 0.104 d−1, respectively. The k values of hemicellulose-added digesters were much higher than others, which could be used to explain why the hydrolytic efficiency of CC (whose hemicellulose content was 41.7%) was the highest among all kinds of LBs in Test I. In addition, it could be observed that the hydrolytic rates of free-state lignocellulosic components were much higher than these in different LBs in Test I, indicating that the digestive efficiency could be enhanced by breaking down the lignocellulose fiber before digestion. The lowest k value was obtained in digester L3 (with solo cellulose addition), verifying that cellulose can hardly be hydrolyzed in limit-acidity condition. Compared to digester L3, digester L5 (hemicellulose and cellulose co-addition) presented a higher hydrolytic rate, which can be ascribed to the acceleration of cellulose hydrolysis caused by hemicellulose addition [26]. However, the hydrolytic constant of digester L7 (cellulose and lignin co-addition) was about 0.042 d−1, which is quite close to that in digester L4 (solo lignin addition), indicating that the presence of lignin can hardly affect the hydrolysis of cellulose.
Fitting results of modified Gompertz model (in right column) showed that hemicellulose-added digesters (L2 and L5) presented higher rates of methane yield. However, Rmax of digester L6 and L8 were not as high as expected, which was due to the acid inhibition caused by the fast hydrolysis and acidification of substrates. Furthermore, long lag phases (λ) were observed in lignin-added digesters (L4, L6, L7 and L8), indicating that delignification is beneficial to obtain better AD operations.

From Table 4, it can be concluded that the hydrolytic rates of these lignocellulosic components depend heavily on their properties [30]. Hemicellulose in these LBs presented higher hydrolytic rates (0.069–0.094 d−1) than both cellulose (0.032–0.050 d−1) and lignin (0.003–0.005 d−1), which was consistent with their higher degradation rate. By horizontally comparing the hydrolytic rates of these LBs, it can be drawn that ES presented the highest hemicellulose hydrolytic rate (0.094 dy1) and the lowest cellulose hydrolytic rate (0.032 d−1) than other LBs, which was in highly consistent with their degradation ratios at the end of the digestion.
Table 4 shows the kinetic fitting results of different lignocellulosic monomers’ degradation. By comparing the fitting results of hemicellulose, cellulose and lignin in digester L2, L3 and L4, it could be observed that the hemicellulose hydrolytic rates (0.133 d−1) were much higher than cellulose (0.025 d−1) and lignin (0.003 d−1). In the co-addition system (L5–L8), the hydrolysis rate of the corresponding lignocellulosic monomers was increased, confirming the synergy between these lignocellulosic components. In addition, hydrolytic rates of total lignocellulose were also investigated. Results found that the hydrolytic rate of cellulose and lignin co-addition (L7, 0.033 d−1) was only a few higher than mono-digesters (0.025 d−1 for cellulose and 0.003 d−1 for lignin), indicating that hemicellulose could contribute more to synergistic effects.

After 38-day digestion, VS removals of different digesters were also recorded. VS removals for digester S1–S6 were 38.5%, 42.3%, 68.9%, 66.3%, 65.3% and 62.4%, respectively. These removed VS can be divided into two parts: lignocellulose (hemicellulose, cellulose and lignin) and other organics (saccharides, protein and lipid). It can be seen from Fig. 3 (Test I), that lignocellulose degradation ratios for S2–S6 were 45.8%, 56.8%, 52.7%, 52.0% and 54.8%, while other organics degradation ratios for S2–S6 were 54.0%, 97.0%, 98.3%, 96.7% and 80.4%, respectively. The highest degradation ratio of saccharide was due to the fact that it is the most susceptible to be hydrolyzed and utilized by microbes. In addition, it can be noticed that the protein and lipid degradation ratios were also higher than previous studies, due that the lignocellulosic fiber in LBs provided attachment points for them, which was conducive to their removals [37, 38]. In addition, owing to the low contents of protein and lipid contents in LBs, they were more liable to be consumed. Comparing the VS degradation ratios, conclusions could be drawn that other organics (saccharides, protein and lipid) were much easier to be hydrolyzed than lignocellulose (particularly cellulose and lignin).Therefore, hydrolysis of cellulose in LBs rather than other organics, is the critical step of LBs digestion. Researchers should pay more attention on the hydrolysis of lignocellulose (especially the cellulose component) because of its limited degradation ratio in AD process..

### 4. Conclusions

This study investigated the hydrolytic performances and kinetic analysis of different kinds of LBs and lignocellulosic components in AD. The results showed that the addition of crop straws (CC, CS, WS and RS) could improve the gas production efficiency and microbial function in the anaerobic digestion process, while the digester with CC added shows the best hydrolysis performance and the highest VS degradation rate (68.9%) owing to its rich hemicellulose content.
However, free-state hemicellulose presented higher degradation ratios than that in combined state (in LBs). Results showed that hemicellulose obtained high degradation ratio (77.2–85.0%) during anaerobic digestion, while cellulose was difficult to hydrolyze without sufficient acidity. Results also depicted that lignocellulose addition could efficiently enhance the VS removals of digestive systems, while lignin existing in systems reduced the VS removal. The limited hydrolysis of lignocellulose hindered the degradation of total VS in digester. Therefore, in order to obtain high bioenergy conversion, some measures (such as pretreatment and inoculum acclimation) should be applied to improve the hydrolysis performance of cellulose. This study could provide some ideas and directions for the future researches of LBs’ digestion.

### Acknowledgments

The authors would like to acknowledge Shiyanjia Lab (www.shiyanjia.com) for providing the tests of carbohydrates, lipid and protein.

### Notes

Author Contributions

H.Y. (M.D.) conducted all the experiments and wrote the manuscript independently. R.D. (Associate Professor), J.J. (M.D.) and Y.W. (B.D.) participated in the coordination of the study and reviewed the manuscript. X.J. (M.D.) and J.S. (M.D.) helped analyze the results and assisted in the experiments. All authors read and approved the final manuscript.

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##### Fig. 1
(a) Kinetic fitting of experimental specific biogas yields (b) and experimental specific methane yields of Test I. (c) Kinetic fitting of experimental specific biogas yields (d) and experimental specific methane yields of Test II.
##### Fig. 2
First-order model fitting of different lignocellulosic components degradation during the AD process ((a)–(d) Test I, (e)–(h) Test II). (a), (e) Hemicellulose; (b), (f) Cellulose; (c), (g) Lignin; (d), (h) Total lignocellulose)
##### Fig. 3
Degradation of VS in different LBs after the AD process ((a)–(c) Test I, (d)–(e) Test II). (a), (d) VS in system; (b) VS in lignocellulose; (c), (e) VS in other organics.
##### Table 1
Characteristics of Different Feedstock’s Components
Parameters Units ES SS CC CS WS RS Xylan α-cellulose Lignin Inocula
TS % 3.8 2.8 88.9 88.6 89.0 91.5 97.9 97.1 97.1 5.4
VS % 2.1 1.5 87.3 82.7 79.5 81.1 97.8 97.0 96.8 2.2
pH - 6.96 8.04 - - - - - - - 7.21
Crude protein %TS 8.3 31.6 3.3 4.5 3.9 4.6 - - - 18.3
Crude fat %TS 6.9 18.3 2.4 2.1 2.8 2.5 - - - 21.4
Carbohydrate %TS 15.3 5.4 91.6 82.7 79.2 76.5 - - - 4.3
Saccharides %TS 0.3 5.4 6.1 12.9 9.0 11.3 - - - 2.6
Hemicellulose %TS 17.5 - 41.7 28.1 23.0 28.4 99.3 - - 0.4
Cellulose %TS 7.3 - 36.9 35.5 38.7 32.0 - 99.6 - 1.2
Lignin %TS 15.0 - 7.8 10.2 11.9 9.8 - - 99.4 0.1
Lignocellulose %TS 39.8 - 86.4 73.8 73.6 70.2 99.3 99.6 99.4 1.7
##### Table 2
Experimental Design of Test I and Test II
Test Digester SS (mL) ES (mL) CC (g) CS (g) WS (g) RS (g) Added representatives VS (g) Hemicellulose content (g) Cellulose content (g) Lignin content (g) Lignocellulose content (g)

Xylan (g) α-cellulose (g) Lignin (g)
I S1* 330 - - - - - - - - 5.0 - - - 0
S2 - 350 - - - - - - - 7.5 2.31 0.97 1.98 5.26
S3 44 - 6.8 - - - - - - 7.5 2.54 2.25 0.47 5.26
S4 56 - - 8.0 - - - - - 7.5 2.00 2.53 0.73 5.26
S5 74 - - - 8.0 - - - - 7.5 1.65 2.76 0.85 5.26
S6 56 - - - - 8.5 - - - 7.5 2.13 2.40 0.73 5.26

II L1* 330 - - - - - - - - 5.0 - - - 0
L2 340 - - - - - 2.37 - - 7.5 2.31 - - 2.31
L3 330 - - - - - - 1.00 - 6.0 - 0.97 - 0.97
L4 330 - - - - - - - 2.05 7.0 - - 1.98 1.98
L5 278 - - - - - 2.37 1.00 - 7.5 2.31 0.97 - 2.28
L6 211 - - - - - 2.37 - 2.05 7.5 2.31 - 1.98 4.29
L7 300 - - - - - - 1.00 2.05 7.5 - 0.97 1.98 2.95
L8 148 - - - - - 2.37 1.00 2.05 7.5 2.31 0.97 1.98 5.26

* Digester S1 and group L1 were in the same substrates and experimental conditions.

##### Table 3
Kinetic Fitting Results of Hydrolysis and Methanogenesis in Different Digesters
Digester Fitzhugh model Modified Gompertz model

B0 (mL/g VS) k (d1) R2 (−) M0 (mL/g VS) Rmax (mL/g VS/d) λ (d) R2 (−)
Test I S1 350.0 0.060 0.997 186.0 7.9 6.2 0.988
S2 407.5 0.044 0.995 140.2 5.1 10.8 0.988
S3 466.2 0.060 0.999 232.3 11.0 7.5 0.995
S4 645.7 0.031 0.997 247.2 10.1 6.9 0.976
S5 591.6 0.049 0.994 271.1 13.1 5.6 0.967
S6 619.2 0.037 0.996 250.0 11.7 5.8 0.968

Test II L1 350.0 0.060 0.997 186.0 7.9 6.2 0.988
L2 331.0 0.098 0.995 179.7 12.4 4.8 0.994
L3 300.5 0.027 0.991 132.3 6.4 7.6 0.975
L4 269.1 0.046 0.989 176.2 4.5 12.6 0.991
L5 339.3 0.094 0.994 191.0 13.0 5.5 0.983
L6 383.4 0.055 0.996 198.5 8.7 7.4 0.983
L7 287.2 0.042 0.989 168.7 6.5 7.6 0.973
1L8 397.0 0.104 0.973 226.9 5.5 15.8 0.988
##### Table 4
Removal Ratios of Different Lignocellulosic Components and Their Hydrolytic Rates
Substrates Parameters S2 S3 S4 S5 S6 L2 L3 L4 L5 L6 L7 L8
Hemicellulose Removal (%) 85 77.2 77.9 82.5 79.6 88.3 - - 85.8 88.6 - 88.7
k (d1) 0.094 0.07 0.072 0.077 0.069 0.133 - - 0.125 0.133 - 0.137
R2 0.992 0.999 0.996 0.997 0.996 0.96 - - 0.956 0.986 - 0.985

Cellulose Removal (%) 29.7 45.4 44.6 46.7 47.6 - 40.4 - 63 - 50.5 68.2
k (d1) 0.032 0.035 0.044 0.05 0.047 - 0.025 - 0.049 - 0.037 0.058
R2 0.98 0.961 0.978 0.985 0.995 - 0.978 - 0.995 - 0.977 0.984

Lignin Removal (%) 4.9 8.7 7.7 7.6 7.1 - - 6.6 - 8.2 6.3 7.8
k (d1) 0.003 0.005 0.005 0.005 0.004 - - 0.003 - 0.005 0.003 0.004
R2 0.992 0.989 0.993 0.993 0.987 - - 0.998 - 0.979 0.997 0.988

Lignocellulose Removal (%) 43.7 57.3 52.2 51.1 54.5 - - - 78.1 51.5 20.9 54.1
k (d1) 0.031 0.043 0.037 0.035 0.036 - - - 0.092 0.044 0.033 0.046
R2 0.985 0.999 0.995 0.993 0.995 - - - 0.969 0.987 0.983 0.985
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