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Communication

Effect of Ammonia–Autoclave Pretreatment on the Performance of Corn Straw and Cow Manure Batch Anaerobic Digestion

by
Yonghua Xu
1,†,
Xinrui Xu
2,†,
Xiaohong Su
2,
Wei Liu
2,
Jingbo Qu
1 and
Yong Sun
1,*
1
College of Engineering, Northeast Agricultural University, Harbin 150030, China
2
Energy & Environmental Research Institute of Heilongjiang Province, Harbin 150027, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2023, 9(2), 178; https://doi.org/10.3390/fermentation9020178
Submission received: 25 December 2022 / Revised: 13 February 2023 / Accepted: 14 February 2023 / Published: 16 February 2023
(This article belongs to the Section Industrial Fermentation)
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Abstract

:
Biomass pretreatment is a critical method for improving the anaerobic digestion (AD) performance of lignocellulosic feedstocks. In this study, an effective combined ammonia–autoclave pretreatment method was selected for the pretreatment of corn straw at 90 °C using four ammonia concentrations (7%, 9%, 11%, and 13%). The results showed that the combined pretreatment improved the substrate’s degradation efficiency and the system’s buffer capacity, and significantly improved the hydrolysis and biogas production performance of corn straw. After pretreatment, the lignin removal rate increased by 11.28–39.69%, and the hemicellulose degradation rate increased from 10.12% to 21.23%. Pretreatment of corn straw with 9% ammonia and an autoclave gave the highest methane yield of 257.11 mL/gVS, which was 2.32-fold higher than that of untreated corn straw, making it the optimal pretreatment condition for corn straw. Therefore, the combined ammonia–autoclave pretreatment technology can further improve the AD performance of corn straw.

1. Introduction

AD is considered one of the most suitable ways to effectively use agricultural and livestock waste, which can synchronously realize the treatment of organic waste and the preparation of clean renewable energy, such as the production of green energy from crop straw biogas [1]. As a large agricultural country, China has abundant lignocellulosic biomass resources, with crop straw production of hundreds of millions of tons per year and a wide range of species, with corn straw accounting for the majority of production [2,3]. However, during the AD process of such substrates, the structure of lignin, cellulose, and hemicellulose in the raw material is difficult to crack due to the specificity of the spatial structure that is achieved through the cross-linking of carbon chains, which makes it difficult for fermenting microorganisms to use these substrates efficiently [4,5].
Several pretreatment strategies have been explored to improve the hydrolysis efficiency of AD of raw materials with high cellulose content [6]. The main principle of pretreatment is to disrupt the structure of lignin and the crystallinity of cellulose, thereby increasing the biodegradation rate of the substrate and biogas production [7]. The complex structure of lignin and its water-insoluble nature not only hinder the degradation and utilization of cellulose but also inhibit the enzymatic cellulose and AD processes [8]. Currently, pretreatment methods are divided into physical, chemical, and biological pretreatments and their combined strategies [9]. Physical methods such as autoclave pretreatment have been adopted to shear the particle size of raw materials and increase the effective surface area [10,11]. Through acid–base and other chemical solvents, the chemical bonds of lignocellulose are degraded so as to remove lignin and improve the accessibility of hydrolases [12]. Degradation of lignocellulose has also been performed using specific microorganisms, e.g., white-rot fungus [13].
However, pretreatment strategies are often accompanied by high energy consumption costs, large equipment investments, harmful products, and other problems [14,15]. Therefore, a pretreatment method with high efficiency and low by-product generation is more conducive to improving the biogas production performance of AD and to ensuring the safe treatment of its products.
Free ammonia (FA, NH3) is derived as a by-product of wastewater treatment and is particularly prominent in numerous lignocellulosic pretreatment methods [16]. Ammonia treatment leads to cell lysis and disruption of dense structures, thereby enhancing the enzymatic hydrolysis of lignocellulosic biomass [17].
Ammonia pretreatment of frozen corn straw yielded as much as 261 mL/gVS methane [18]. After pretreating rice straw with 2% ammonia for 5 days, the methane yield was 23.10% higher than that of untreated rice straw [19]. Ammonia pretreatment conditions are relatively mild, the required equipment is simple, and the by-products have little impact on the subsequent fermentation. It has economic benefits and is environmentally friendly, but there is a partial loss of hemicellulose at higher ammonia concentrations [20]. Combined physical and chemical pretreatment is a more efficient method, with alkali-mechanical pretreatment having a positive effect on the destruction of the straw structure and the removal of lignin [21].
Accordingly, this study aimed to investigate the fermentation characteristics of cellulosic feedstock after ammonia–autoclave pretreatment and to explore the optimum conditions for AD of corn straw mixed with cow manure after pretreatment with different concentrations of ammonia. The effect of ammonia pretreatment on biomethane production was revealed by the dynamic changes in the monitoring indicators of the fermentation system. The results of the study provide a comprehensive evaluation of AD following the combined ammonia–autoclave pretreatment of highly cellulosic feedstocks and promote the application of cost-effective pretreatment strategies in AD.

2. Materials and Methods

2.1. Raw Material and Inoculum

The air-dried corn straw was taken from the Acheng experimental base of Northeast Agricultural University, crushed and sieved into 2 mm straw pieces with a medium-sized grinder, sealed in plastic bags, and stored in a −20 °C refrigerator. Fresh cow manure was obtained from the farm of Northeast Agricultural University, Acheng. Non-biodegradable impurities were removed, crushed uniformly using a medium-sized grinder, and stored at −20 °C before use. The inoculum was obtained from the medium-temperature anaerobic digester stably operated in the biogas laboratory of Northeast Agricultural University. The characteristics of the corn straw, cow manure and inoculum were presented in Table 1.

2.2. Experimental Methods

The experiment was carried out using an experimental batch AD device consisting of a 1 L anaerobic digester with a working volume of 0.8 L, equipped with a 1 L gas bag collection device and placed in a 35 ± 1 °C water bath to continuously heat the AD device. Ammonia solutions of 7%, 9%, 11% and 13% by volume fraction (diluted with ammonia of analytical purity) were prepared for pretreatment. The corn straw was mixed with ammonia solution, sealed and soaked at room temperature for 3 days, filtered and washed to neutrality. The ammonia-treated corn straw was placed in the autoclave at 90 °C for 30 min, and then part of the residue was collected for determination of cellulose, hemicellulose, and lignin content. The ammonia–autoclave pretreated corn straw was mixed with cow manure at a dry matter mass ratio of 1:1 for anaerobic fermentation, and untreated corn straw was added to the manure at the same ratio. The ratio of substrate to inoculum addition was adjusted according to the anaerobic fermentation system’s TS of 8% and C/N ratio of 25. A total of five parallel experimental groups were set up with three replicates for each treatment to reduce experimental error, and the error values were the corresponding standard deviations. All reactors were flushed with N2 into the headspace for 5 min to exhaust air and subsequently sealed with rubber plugs to maintain anaerobic conditions.

2.3. Analytical Methods

The elemental content of the organic matter was determined by a Vario EL element analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). Gas volume and composition were measured daily using a 100 mL gas collector. Gas chromatography (GC2014 Shimadzu, Kyoto, Japan) was used to determine the composition of the biogas. Analysis of the composition of corn straw (such as cellulose, hemicellulose and lignin content) was carried out as previously described by Van Soest [22]. The pH was determined by a pH meter (FE28-Meter, Mettler Toledo, Columbus, OH, USA) using the glass electrode method. Volatile fatty acid concentrations (VFAs) were determined by high performance liquid chromatography (Waters 2695 HPLC, Waters Corporation, Milford, MA, USA).

3. Results and Discussion

3.1. Changes in the Composition of Corn Straw after Ammonia–Autoclave Pretreatment

As illustrated in Table 2, ammonia–autoclave pretreatment significantly increased the degradation rate of lignocellulose from corn straw. Compared to the untreated reactor, the combined pretreatment reactor enhanced hemicellulose and lignin degradation rates by 11.28–39.69% and 10.12–21.23%, respectively. This indicated that the combined pretreatment effectively removed the waxy layer from the straw cells, releasing hemicellulose and lignin to be used by hydrolytic microorganisms. It has been reported that lignin, compared to cellulose and hemicellulose, severely limits degradation and utilization by anaerobic bacteria due to its recalcitrant condensed structure, restricting the efficiency of AD [23,24]. The highest lignin removal was achieved at an ammonia concentration of 9%, at which lignin content decreased from 18.46% to 10.53%. This suggested that the combined pretreatment effectively exposed the internal cellulose of the corn straw and increased the porosity of the corn straw, thus accelerating the enzyme contact and degradation efficiency. The increased specific surface area and exposed organic matter structure therefore made the co-pretreated corn straw more susceptible to degradation and utilization by hydrolytic bacteria than the untreated corn straw. This may be because hemicellulose and lignin are alkali-soluble, and they both decrease in solubility with increasing ammonia concentration [18,25]. In addition, the cellulose content of the combined pretreated corn straw increased to varying degrees. This may be due to the decrease in hemicellulose and lignin content, resulting in an increase in the cellulose percentage. Overall, the ammonia–autoclave pretreatment resulted in structural changes in the corn straw that affected the solubility of the cellulose components. It was possible that ammonia, as an alkaline solvent, penetrated the fibrous structure of the straw, increasing the accessible surface area of the biomass and selectively breaking the ester bond and ether bond of the lignin, breaking the chemical bond between the lignin and the carbohydrate [19]. It may also be that the autoclave environment has a positive effect on the rapid disruption of the lignin association process and increases the pore volume [19,26].

3.2. Methane Production

As observed in Figure 1a, the daily methane yield of the pretreatment reactors all showed a fluctuating pattern, beginning with a slow increase and then stabilizing. The differences between the daily methane yield of each reactor were not significant during days 1–2, probably because the easily degradable biomass in the substrate started to degrade gradually at this stage. The methane content increased significantly from day 3 to day 6, with all the different pretreatment reactors showing an advanced peak in daily gas production. Peak methane production rates were 1.47–2.52-fold higher in the pretreated reactors compared to those in the untreated reactors, and daily methane production was significantly higher in the case of 9% ammonia autoclave pretreatment, peaking at day 6 (23.65 mL/gVS). In contrast, daily methane production from the untreated reactor peaked at 9.38 mL/gVS on day 13. This suggested that there may be a mutual synergy between the ammonia–autoclave pretreatment methods, with the combined pretreatment affording easier hydrolyzation and being more successful in improving the biogas production performance of the AD. This may be attributed to the pretreatment altering the microstructure of the corn straw, thereby increasing the accessibility of the enzyme to the straw. The increased lignin removal after pretreatment resulted in significant degradation of exposed cellulose and hemicellulose into small-molecule organic matter by microorganisms and accelerated AD hydrolysis, resulting in an earlier peak in biogas production [14,27]. This result was similar to that obtained in previous research [28].
The cumulative methane production of the reactor pretreated with different concentrations is shown in Figure 1b. The cumulative methane yield of corn straw pretreated with the combined pretreatment was significantly higher than that of the untreated reactor. After day 5 of fermentation, the rate of cumulative methane production increased faster in the pretreatment group and stabilized after 26 days. Differences in cumulative methane production between pretreatments of different concentrations were found but were not significant. The cumulative methane production of the pretreated reactors was 1.65–2.32-fold higher than that of the untreated corn straw, with the 9% pretreatment group having the highest production (257.11 mL/gVS). These results suggested that an ammonia concentration of 9% can effectively improve methane production, which was consistent with the effect of daily methane production. This may be attributed to the pretreatment promoting the degradation of lignin and effectively facilitating the removal of residual lignin, resulting in cellulose and hemicellulose having a larger area accessible to microorganisms [29,30,31]. In addition, the efficiency of AD can be expressed in terms of the T80 time (i.e., 80% of the total cumulative gas production after one cycle of AD) [32,33]; the higher the T80 time, the higher the biogas production efficiency. The T80 times for each reactor were 11 days (7%) and 13 days (9%, 11%, 13%), which were 13.33–26.67% shorter than those for the unpretreated reactor (15 days). This indicated that pretreatment improved digestion efficiency, promoted rapid utilization of the fermentation substrate, shortened the delay period of the reaction, and enhanced the buffering capacity of the system. This result suggested that ammonia–autoclave pretreatment has greater potential in promoting the efficiency of anaerobic hydrolysis and biogas production.

3.3. VFAs

VFAs are an important factor affecting the degradation efficiency and stability of anaerobic fermentation, both as a product of the hydrolysis and acidification stages of AD, and as a substrate used in the methanogenic stage [34,35]. The accumulation of VFAs is generally considered to reduce the pH of the fermentation system, which is detrimental to the growth and reproduction of methanogens, inhibiting methanogenic function and ultimately leading to a decrease in biogas production [36]. The variation in the concentration of VFAs during the AD of each reactor was shown in Figure 2. In the initial stage of AD, VFAs accumulated rapidly in the untreated group, reached a maximum value of 2.83 g/L on day 15, and then began to degrade slowly until reaching complete degradation on day 40. This may be due to the acidification of the untreated corn straw in the early stages of AD, which hindered the growth and proliferation of methanogens, and was also illustrated by the change in methane production. In all pretreatment systems, VFA accumulation was lower compared to that of the control group, demonstrating that the VFAs produced by the pretreatment of corn straw were more readily converted to biogas by the microorganisms, accelerating the utilization of substrate and facilitating the fermentation process. This may be inferred from the increased activity of hydrolytic enzymes as a result of pretreatment, such as cellulase. The pretreated reactors with 9% and 13% ammonia showed a faster degradation of organic acid content, reaching a maximum of 1.43 g/L on day 6 and ending on day 30, indicating that the organic matter after pretreatment was degraded more completely, probably because pretreatment enhanced the hydrolysis rate of the feedstock and alleviated organic acid accumulation. The high VFA concentration inhibited the metabolic activity of methanogens, resulting in a reduction in methane production and in the buffering capacity of the reactor [26,37]. This also explained the higher biogas production in the pretreatment reactor.

3.4. pH

As shown in Figure 3, the trend in pH corresponds to the changes in VFA concentration, as the pH decreases with increasing VFA production and is an important factor in the stability of the fermentation system [38]. The effect of pretreatment on the pH of the anaerobic fermentation system was more pronounced. The pH trend in the pretreatment system was relatively stable compared to that in the control, remaining between 7.44 and 8.54. According to previous studies, the pH after pretreatment (7.96–8.80) is the optimum operating condition for the production of VFAs [39]. The pH range of the control group was 7.20–7.90. The overall increase in the pH of the pretreatment system compared to that of the control group was probably due to the conversion and degradation of organic acids by methanogens. In the initial state of anaerobic fermentation, the pH range of the pretreatment reactor was higher than that of the control, probably due to the addition of ammonia. In the later stages of fermentation, the pH started to gradually increase, probably mainly due to the gradual utilization of VFAs in the later stages. In summary, it was shown that the combined pretreatment improved the buffering capacity of the fermentation system and effectively prevented the pH from decreasing.

4. Conclusions

In this study, the effect of ammonia–autoclave pretreatment on corn straw was evaluated by a batch AD experiment with corn straw and cattle manure. It was demonstrated that combined ammonia and high temperature pretreatment could improve the methane yield of AD. It was found that the combined pretreatment significantly reduced the lignin content of the feedstock, effectively alleviated VFA accumulation and enhanced the stability of the system. The highest cumulative methane yield of 257.11 mL/gVS was obtained for the batch AD at 9% ammonia, which was 2.32-fold higher than that of the untreated corn straw.

Author Contributions

Conceptualization, Y.X.; methodology, X.X.; validation, Y.X. and X.X.; formal analysis, X.S.; resources, Y.X. and X.S.; data curation, Y.X. and J.Q.; writing—original draft preparation, J.Q.; writing—review and editing, Y.X. and Y.S.; supervision, Y.X. and Y.S.; project administration, W.L.; funding acquisition, Y.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Heilongjiang Province “Unveiling Leader” Technology Research Project (2022ZXJ05C01-03), the National Key Research and Development Program Strategic Key Special Subject of International Science and Technology Innovation Cooperation (2018YFE026602), the National Key R&D Program of China (2019YFD1100603), and the Heilongjiang Province Science and Technology Plan, Provincial Academy Science and Technology Cooperation Project (YS20B01).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00178 g001 550
Figure 1. Changes in biogas production with different pretreatment conditions. (a) Daily methane yield; (b) Cumulative methane yield.
Figure 1. Changes in biogas production with different pretreatment conditions. (a) Daily methane yield; (b) Cumulative methane yield.
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Figure 2. The VFA concentration of different pretreatment conditions.
Figure 2. The VFA concentration of different pretreatment conditions.
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Figure 3. The pH value of different pretreatment conditions.
Figure 3. The pH value of different pretreatment conditions.
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Table
Table 1. Characteristics of the corn straw, cow manure and inoculum.
Table 1. Characteristics of the corn straw, cow manure and inoculum.
ParametersTotal Solids (%)Volatile Solids (%TS)C (%TS)N (%TS)H (%TS)C/N
Corn straw94.85 ± 0.2586.20 ± 0.0140.52 ± 0.080.82 ± 0.015.85 ± 0.3149.41 ± 1.20
Cow manure16.35 ± 0.3189.77 ± 0.0535.64 ± 0.831.95 ± 0.055.37 ± 0.1218.28 ± 1.12
Inoculum2.53 ± 0.0367.93 ± 0.26NANANANA
The data represent the mean ± standard deviation of three replicates. NA: not analyzed.
Table
Table 2. Component analysis of corn straw before and after ammonia–mechanical pretreatment.
Table 2. Component analysis of corn straw before and after ammonia–mechanical pretreatment.
SamplesAmmonia ConcentrationCelluloseHemicelluloseLignin
CK0%41.56% ± 1.8128.36% ± 0.4318.46% ± 0.58
R17%42.12% ± 1.6525.49% ± 0.3515.49% ± 0.62
R29%44.45% ± 1.5322.34% ± 0.2910.53% ± 0.41
R311%45.16% ± 1.5123.08% ± 0.3813.64% ± 0.52
R413%45.53% ± 1.4222.65% ± 0.5713.62% ± 0.54
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Xu, Y.; Xu, X.; Su, X.; Liu, W.; Qu, J.; Sun, Y. Effect of Ammonia–Autoclave Pretreatment on the Performance of Corn Straw and Cow Manure Batch Anaerobic Digestion. Fermentation 2023, 9, 178. https://doi.org/10.3390/fermentation9020178

AMA Style

Xu Y, Xu X, Su X, Liu W, Qu J, Sun Y. Effect of Ammonia–Autoclave Pretreatment on the Performance of Corn Straw and Cow Manure Batch Anaerobic Digestion. Fermentation. 2023; 9(2):178. https://doi.org/10.3390/fermentation9020178

Chicago/Turabian Style

Xu, Yonghua, Xinrui Xu, Xiaohong Su, Wei Liu, Jingbo Qu, and Yong Sun. 2023. "Effect of Ammonia–Autoclave Pretreatment on the Performance of Corn Straw and Cow Manure Batch Anaerobic Digestion" Fermentation 9, no. 2: 178. https://doi.org/10.3390/fermentation9020178

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