Improving the Anaerobic Digestion of Switchgrass via Cofermentation

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Biofuels and Biomass

Improving the anaerobic digestion of switchgrass via the cofermentation of rumen microorganisms (rumen bacteria, protozoa, and fungi) and a biogas slurry Yulong Zheng, Xuekai Wang, and Fuyu Yang Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.8b03496 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

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Improving the anaerobic digestion of switchgrass via the cofermentation of rumen microorganisms (rumen bacteria, protozoa, and fungi) and a biogas slurry Yulong Zheng, Xuekai Wang, Fuyu Yang* Department of Grassland Science, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China

KEYWORDS: Anaerobic digestion; Rumen microorganisms, Cofermentation; Methane ABSTRACT: Rumen microorganisms can effectively digest lignocellulosic biomass. Various rumen microbial groups (bacteria, fungi and protozoa) make different contributions to the fermentation of biomass material. In this study, physical, chemical and antibiotic methods were used to treat rumen fluid to obtain the following groups: whole rumen fluid (WRF), protozoa + bacteria (PB), fungi + bacteria (FB), fungi + protozoa (FP), bacteria (B), protozoa (P), fungi (F), and negative control (CON). Subsequently, a biogas slurry was added to each rumen microbial group (at ratios of 1:1, 3:1, 1:3, 5:1 and 1:5 v/v), and the mixtures were used to ferment switchgrass. These mixtures were labeled based on their inoculum and ratio; for example, the WRF and biogas slurry at ratios of 1:1, 3:1, 1:3, 5:1, and

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1:5 (v/v) were abbreviated WRF1, WRF2, WRF3, WRF4, and WRF5, respectively. All mixtures were compared to mono-digestion with the biogas slurry (CK). The results showed that rumen bacteria play an important role in improving anaerobic digestion in the cofermentation system. The FB2,WRF3, FB5 and PB5 groups of cofermentation achieved methane production of 189.5, 180.7, 176.9 and 174.5 mL/g of volatile solids (VS), respectively, which were all significantly higher than that of CK (137.9 mL/g VS), with increases of 37.37%, 31%, 28.24% and 26.5%, respectively. The total solids (TS), glucans and hemicellulose degradation rate in CK was almost the same as or higher than that in the mixed inoculum groups, but CK achieved lower biogas and methane production than did FB2,WRF3, FB5 and PB5. Therefore, cofermentation can improve the efficiency of anaerobic digestion and enhance the efficiency of methane conversion. 1. INTRODUCTION Lignocellulosic biomass, a renewable and abundant material in the renewable energy industry, has aroused great interest due to the low cost and large quantities of raw material available.1 Switchgrass (Panicum virgatum L.), a perennial C4 grass, is a bioenergy resource with great application potential that is composed of cellulose, hemicellulose and lignin, which can be used as feedstock and converted to biogas, ethanol and other bioenergy products.2,3 The anaerobic digestion of biomass is an attractive avenue for methane production for renewable energy or green fertilizer.4 However, the crystallinity of cellulose and the association of cellulose and 2


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hemicellulose with lignin (lignin-carbohydrate complexes) hamper their biological conversion.1,5 Therefore, the relatively refractory structure of switchgrass needs to be broken down to improve its fermentation efficiency. To resolve the above problems, various methods have been implemented to improve the efficiency of anaerobic digestion. In general, these methods can be divided into the following types: physical, chemical and biological methods.6 Among the three kinds of methods, a range of effective methods have been studied in many recent investigations. For example, research on the co-composting of sewage sludge and agricultural waste treated with silver nanoparticles showed that this method could not only degrade the lignocellulosic biomass but also affect the quality of final composts.7,8 A biological pretreatment using rumen fluid, which has complex microbial populations with considerable ability to degrade lignocellulosic biomass, could be promising for effective production of methane from rice straw.9 Augmentation may be another effective method in which adding different microorganisms to the fermentation system can significantly degrade the recalcitrant matter in cellulose and improve the methane production of anaerobic digestion.10,11 Various inocula can also be used to improve fermentation, such as rumen fluid, manure, digester sludge and biogas slurry, and mixed inocula are another potential approach.12,13 Rumen fluid is a common inoculum source, and there are also reports regarding fermentation with rumen fluid mixed with other inocula. Seaweed was digested by cofermentation of rumen fluid and anaerobically digested sludge at

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±0.5 ℃, which achieved higher methane production than digested sludge as the sole 3


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inoculum after 18 days fermentation, and rumen fluid at an inoculum ratio of 6% enhanced methane production by 31.14% in comparison with the blank test.14 Another study also found that anaerobic palm oil mill effluent mixed with rumen fluid was more effective than the mono-digestion that was used to treat supernatant palm oil mill effluent at 37±3 ℃.15 However, the application of rumen bacteria, protozoa, and fungi with biogas slurry has not been reported as a cofermentation system to improve the hydrolysis efficiency of lignocellulosic biomass and methane production. Rumen fluid, which contains archaea, bacteria, protists and fungi, is found in the rumen.16 Ruminal bacteria, protozoa, and fungi are all associated with the degradation of plants in the rumen.17 Cellulolytic bacteria, such as Ruminococcus albums, Ruminococcus flavefaciens and Fibrobacter succinogenes, are the dominant plant-degrading microbes in the rumen due to their numerical predominance and metabolic diversity.18 However, protozoa and fungi also play important roles in plant degradation. For example, protozoa digest 25-30% of the total ingested fiber,17 and rumen fungi help decompose plants mechanically via the penetration and degradation of lignin-containing plant cell walls by mycelia.19 In addition to the roles of individual groups of rumen microorganisms, the interactions between the microbial groups are responsible for the complexity of the microecosystem and are thus the subject of substantial research. The interactions between the three microbial groups (bacteria, protozoa and fungi) can be synergistic or antagonistic, and the relative contributions of each group are receiving increasing attention. A synergistic interaction between the rumen bacteria and fungi exists, likely because fungal hyphae penetrate plant cell 4


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walls and produce hydrolases, resulting in the reduction in plant particle size as well as the degradation of lignin and plant tissue, which increases the substrate surface for bacteria.17,19 Negative effects are also observed in the bacteria + protozoa and fungi + protozoa systems, mostly because protozoa can digest bacteria with fungi, while fungal sporangia can be degraded by protozoal chitinolytic enzymes.17 In this study, rumen microorganisms (bacteria, protozoa, and fungi, alone and in combination) mixed with a biogas slurry were used as inoculum resources to increase the hydrolysis efficiency and methane production of switchgrass. The objectives of this work are (1) to investigate improvements in the hydrolysis efficiency and methane production of switchgrass through cofermentation with various ruminal microbial groups and a biogas slurry and (2) to explore the optimum combination ratio of rumen microorganisms and the biogas slurry. Our study lays a foundation for future research on microbial community composition that could employ rumen microorganisms in the treatment of switchgrass. 2. MATERIALS AND METHODS 2.1 Substrate and Inoculum Resources. The switchgrass substrate was grown at the Shangzhuang Experiment Field, Beijing, China. The plants were harvested at the reproductive growth stage, and the plant biomass was air dried, sheared, ground and then passed through a 1-mm screen.

Table 1. Characteristics of the Switchgrass, Biogas Slurry and Rumen Fluid

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TC

TN

(%)

(%)

Switchgrass

41.67

0.99

Biogas slurry

0.62

Rumen fluid

0.71

Samples

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Glucan

HC

Lignin

TS

VS

(%)

(%)

(%)

(%)

(%)

42.1

42.5

31.7

8.41

94.09

90.01

0.16

3.88

ND

ND

ND

1.07

0.21

3.38

ND

ND

ND

1.51

C/N

AA

PA

BA

(mg/L)

(mg/L)

(mg/L)

5.89

ND

ND

ND

0.64

7.33

502.32

203.25

353.21

0.81

6.89

1103.1

313.5

345.24

pH

Note: TC, total carbon; TN, total nitrogen; TS, total solids; VS, volatile solids; ND, not determined. HC, hemicellulose; AA, acetic acid; PA, propionic acid; BA, butyric acid.

One inoculum for the anaerobic digestion was biogas slurry collected from the Doudian biogas station in Hebei Province. The other inoculum, rumen fluid, was obtained from three fistulated dairy cows fed thrice with a main diet of silage. The rumen fluid was collected from the bottom of the rumen 3 h after the morning feeding and was stored in a thermos bottle, which was brought to the laboratory as soon as possible. Both inoculum sources were subsequently squeezed through four layers of gauze. The characteristics of the switchgrass and inoculum resources were analyzed using the methods described in the next section and are listed in Table 1. 2.2 Preparation of Rumen Microorganisms. To evaluate the relative contributions of the rumen microbial community and their interactions to the anaerobic digestion of switchgrass, eight microbial groups were prepared from the strained rumen fluid according to the method described by Lee et al. (2000) and Zhang et al. (2007)17,20 with minor modifications:

(1) Whole rumen fluid (WRF): the rumen contents from the cow were strained through four layers of cheesecloth to remove the small feed particles and were then pooled together. 6


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(2) Protozoa + bacteria (PB): antifungal agents (cycloheximide [0.05 mg/mL] and nystatin [200 U/mL]) were added to the WRF. (3) Fungi + bacteria (FB): the WRF was centrifuged at 500 g for 5 min to remove the protozoa, yielding the fungi + bacteria system in the supernatant. (4) Fungi + protozoa (FP): antibacterial agents (streptomycin sulfate, penicillin G, potassium, and chloramphenicol [0.100 mg/mL each]) were added to the WRF. (5) Bacteria (B): the WRF was centrifuged at 500 g for 5 min, and antifungal agents were added to the supernatant. (6) Protozoa (P): antibacterial and antifungal agents were added to the WRF. (7) Fungi (F): the WRF was centrifuged at 500 g for 5 min, and antibacterial agents were added to the supernatant. (8) Negative control (CON): the WRF was centrifuged at 500 g for 5 min, and antibacterial and antifungal agents were added to the supernatant. All antibiotics used were of scientific research grade. The average temperature of the rumen is 39 ℃ when it reached a relatively stable state. Therefore, all samples of the eight rumen microorganism groups were maintained under an N2 environment at 39 ℃ before use.20 2.3 Biochemical Methane Potential (BMP) Assays. In this experiment, different groups and proportions of rumen microorganisms were used to improve methane production. Previous studies have shown that the proportion of rumen fluid in 7


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cofermentation is low (approximately 2%-15%).14,15,21 However, the function and fermentation effects of adding a high proportion of rumen fluid to the fermentation system is unclear; thus, this study explored the effects of high proportions of rumen fluid (1:1, 3:1 and 5:1). Previous research has found that codigestion (10% rumen fluid + 90% digested sludge) has a better effect than 5% rumen fluid inoculum.15 Another study also found that a high proportion of rumen fluid (15%) has a better performance in the fermentation process than proportions of 10% and 5%.21 Therefore, our research applied the ratio of 1:5 for the cofermentation of rumen fluid and biogas slurry, which was set as the lowest proportion of rumen fluid. The ratios of rumen microorganisms (WRF, FB, PB, FP, B, F, P, and CON) to the biogas slurry were 1:1, 3:1, 1:3, 5:1, and 1:5 (v/v), respectively, and the mixtures were labeled based on their inoculum and ratio; for example, the WRF and biogas slurry mixtures at ratios of 1:1, 3:1, 1:3, 5:1, and 1:5 (v/v) were abbreviated WRF1, WRF2, WRF3, WRF4 and WRF5, respectively. Each mixture was used as an inoculum for anaerobic digestion. As a control for the above treatments, switchgrass was inoculated with the biogas slurry alone (CK). The experiments were conducted in a 200 mL reaction device (serum glass bottles). Each bottle contained a total of 42 mL inoculum, 2 g switchgrass material and distilled water, resulting in a total working volume of 140 mL. The switchgrass and the inoculum were mixed thoroughly. Urea was used as a nitrogen source to adjust the carbon/nitrogen ratio (C/N) to 25 for the normal growth of anaerobic microorganisms.22 The pH was adjusted to between 7.0 and 7.5 before fermentation. Subsequently, highly pure nitrogen was flushed into the reactors to establish anaerobic conditions. Finally, the reaction devices were placed into a fermentation bath at 37 °C and mixed manually each day just before the gas volume measurement 8


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to degas the fermentation system and prevent the formation of a dry layer.23 At the initial stage of fermentation, gas production and composition were measured every other day as their changes were obvious. Gas production was monitored using the saturated brine displacement method.24 Liquid samples were collected every few days and were kept at -20 °C to test other indicators. 2.4 Analytical Methods. TS and VS were determined by the weight loss method.25 The hemicellulose, glucans and lignin contents were estimated as described by Van Soest et al. (1991).26 The pH of switchgrass according the method described by Chen et al.( 2015) and Pan et al. (2019) with minor modifications: the switchgrass (2 g) was blended with 18 ml distilled water extracted for 24 h, then filtered through 4 layers of sterilized cheesecloth. Afterwards, pH value in the filtrate was measured.27,28 The collected liquid was used to measure the pH and ammonia nitrogen (NH3-N) and organic acid contents. The pH of the liquid was measured using a pH meter (PHS-3C, INESA Scientific Instrument Co., Ltd, Shanghai, China). The NH3-N concentration was determined using the method of Broderick and Kang (1980).29 The volatile fatty acid(VFA) content, including acetic acid, propionic acid and butyric acid, was determined by high-performance liquid chromatography ( LC-20A; Shimazu, Japan) which equipped with a diode array detector ( SPD-20A, Shimadzu, Japan). The analytical conditions were as follows: column, Shodex RSPak KC-811-DVB gel C( Shimadzu, Japan); oven temperature, 50 °C; mobile eluent, 3 mmol/L HClO4; flow rate, 1.0 mL/min; injection volum, 5 uL. The biogas was analyzed using a gas chromatograph (GC-8600 (Beijing Beifen Tianpu Instrument Tech. Co., Ltd., Beijing, China)) equipped with a Porapak Q stainless-steel packed column and a thermal conductivity detector (TCD). The temperatures of the injector, detector, and column were kept at 150, 100 and 50 °C, 9


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respectively. The concentrations of gas standards were CH4, 60.1%; CO2, 34.92%; and N2, 4.98% (Beijing Shougang Oxygen Plant)). The carrier gas was hydrogen, and it was provided by a nitrogen/hydrogen air generator (Beijing Huijia Precision Instrument Co., Ltd.) at a flow rate of 50 mL/min. 2.5 Theoretical Calculations. The degradation rates of the TS and the chemical composition of the switchgrass were calculated using Equations (1) - (2):30

TS Degradation Rate  (TS ini - TS fin )/TS ini

Ei 

X i ,ini  TS ini  X i , fin  TS fin X i ,ini  TS ini

(1)

(2)

where Ei is the degradation rate of switchgrass with composition i (glucans and hemicellulose); Xi,ini and Xi,fin are the initial and final compositions of i, respectively; and TSini and TSfin are the initial and final contents of the TS substrate, respectively. 2.6 Statistical Analysis. One-way analysis of variance (ANOVA; significance: P < 0.05) followed by Duncan’s multiple range test was performed for mean separation (SPSS Inc., Chicago, version 16.0). 3. RESULTS AND DISCUSSION 3.1 Biogas and Methane Production with Different Inocula. Biogas production is an important parameter for understanding the performance of anaerobic digestion with different inocula. The biogas production trends, shown in Figure 1, were similar to those of the methane production. Biogas production by F1 (343.023 mL/g VS), F3 (329.894 mL/g VS), F5 (296.239 mL/g VS) and FP5 (287.547 mL/g VS) was significantly higher than that of CK (276.822 mL/g VS), suggesting that 10


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rumen fungi can improve the fermentation of switchgrass. However, the cumulative methane production of CK was higher than those of these groups (Figure 3), likely because all ratios of the FP, P, F, and CON groups lack rumen bacteria, including methanogens, indicating the key role of rumen bacteria in the formation of methane.

Figure 1. Biogas production of the switchgrass with different inocula. Values are averages from three trials. a, b, c, d and e represent the combination ratios 3:1, 1:3, 5:1, 1:5 and 1:1 of the rumen microorganisms and biogas slurry, respectively.

Figure 2 shows the methane content of the biogas produced by the fermentation of switchgrass with different inocula. For all five ratios of the cofermentation groups, the methane content increased with fermentation time at first and then remained relatively stable. In the first 15 days, the methane content of CK was significantly higher than those of the inoculum groups with rumen microorganisms and the biogas 11


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slurry at ratios of 1:1, 3:1 and 5:1; however, for the 1:3 and 1:5 ratios, no differences were detected between these groups, consistent with the methane production results (Figure 3). The principal gases that are produced in the rumen are CO2 (60%), CH4 (30-40%), and small amounts of N2, H2S, H2 and O2.15 The activity of the methanogens in the biogas slurry was higher than that of the methanogens in rumen fluid. In addition, the low pH and high VFA content (Figure 4 and Figure 5) of these groups limited the activity of methanogens. Therefore, less methane was produced by the mixed inocula (at ratios of 1:1, 1:3 and 1:5) because of the high proportion of rumen microorganisms. Methane is produced via the reduction in CO2 and is formed by methanogenic bacteria. At early stages of fermentation, the main biogas products are CO2 and H2. Methane is produced later by methanogens that utilize H2 with a carbon source and acetic acid. Furthermore, methanogens grow more slowly than other microorganisms in anaerobic digestion systems.31 Therefore, the methane content of all the groups, especially the cofermentation groups with high proportions of rumen microorganisms, was very low initially. The methane content then increased during fermentation, ultimately reaching over 65% (in the FB group at all five ratios). Previous grass digestion studies have yielded methane contents in the range of 50– 53% (v/v) with food waste and manure inocula.32 In the codigestion of palm oil mill effluent with small quantities of rumen fluid (5-10% by volume and < 1 cm particle size) at 37 °C, the methane composition of the biogas increased to concentrations of 50-55%; however, in this study, the methane content of the group with no added rumen fluid remained low and did not exceed 50% (v/v),33 indicating that the methane 12


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concentrations were lower than those obtained in this study.

Figure 2. Methane content of the switchgrass with different inocula. Values are averages from three trials. a, b, c, d and e represent the combination ratios 3:1, 1:3, 5:1, 1:5 and 1:1 of the rumen microorganisms and biogas slurry, respectively.

The structure of the microbial community in the rumen fluid and slurry system is complex. However, the experimental results show that affecting the interactions of the microbial community in the biogas slurry anaerobic system and enhancing the methane production by adding rumen microorganisms are possible. As shown in Figure 2, after the initial stage of anaerobic digestion, the mean methane concentrations of the cofermentation groups reached over 60% (WRF2 and FB1-5) or 65% (WRF2 and FB4), higher levels than those of CK (54-56%). Cofermentation with rumen fluid and sludge was shown to increase the relative abundances of some 13


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genera of Christensenellaceae, Rikenellaceae and Prevotella, which are known to utilize various sugars and produce VFAs as fermentation end products.9,34-35 In addition, Methanobrevibacter increased to 29.1% and 12.2%, respectively, during cofermentation with rumen fluid and anaerobic sludge and during cofermentation with a rumen fluid pretreatment system, indicating that the strategy of adding rumen fluid significantly improves the methane production capacity of a system.14 Therefore, the cofermentation system can be speculated to increase the density of these bacteria, enhancing the hydrolysis of switchgrass and consequently improving the methane production by promoting the formation of methane by methanogens. The next step of our research is to perform a microbial community analysis via high-throughput sequencing to explore changes in the microbial community in the cofermentation system. The cumulative methane production from the anaerobic digestion of switchgrass with different inoculation ratios of rumen microorganisms and a biogas slurry is shown in Figure 3. The mixed inoculum groups FB2, WRF3, FB5, PB5, WRF2, WRF4, B5, B3, WRF5, FB3 and FB4 achieved significantly higher methane production than the biogas slurry alone (CK) (P < 0.05), suggesting that the rumen fluid effectively enhanced the methane production during the fermentation of switchgrass. According to the above results, the groups with significantly increased methane production all had rumen bacteria, indicating the superiority of bacteria among all rumen microorganisms in improving the anaerobic digestion of switchgrass. The numerical predominance and metabolic diversity of cellulolytic bacteria18 and the presence of methane-forming bacteria (methanogens)15 in the rumen 14


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as well as the interactions between the rumen bacteria and slurry microorganisms could all explain these findings. A similar study found that gas production by WRF, PB and FB was significantly higher than that of other groups after 72 h of fermentation at 39 °C with rumen microorganisms.20 Compared with CK (137.939 mL/g VS), cofermentation in FB2 WRF3, FB5, PB5 and B5 achieved methane production of 189.494, 180.661, 176.892, 174.465 and 169.748 mL/g VS, which were significantly increased by 37.37%, 30.972%, 28.24%, 25.03% and 23.06%, respectively. These results are consistent with another report of rumen fluid and sludge cofermentation, which indicated that rumen fluid can enhance methane emission. This study showed that rumen fluid at an inoculum ratio of 6% enhanced the highest methane potential by approximately 76 L CH4·kg VS−1 added, a 31.1% increase compared with that of seaweed digested by sludge alone at a temperature of 38 ± 0.5 °C after 18 days of fermentation.15 However, the methane production of the above groups was higher than those of other results. For example, the reported methane production of switchgrass leaves and stems was 134.81 and 99.35 mL/g VS, respectively, when they were fermented by municipal sludge at 37°C for 60 days.24 Another study using a biogas slurry as the sole inoculum achieved methane production of 135.31 mL/g VS at 35 ±1 °C after 44 days of fermentation from switchgrass.36

The methane production trends for all the fermentations with the different inocula are shown in Figure 3. A similar trend was observed for the digesters with inocula containing rumen microorganisms and the biogas slurry at ratios of 1:1, 3:1 and 5:1. At the initial fermentation stage, the methane production of the mixture groups were lower than that of the sole inoculum, but the methane levels in the FB2, 15


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WRF2, WRF4 and FB4 groups eventually exceeded those in the control group. The initial methane production results were probably due to the high content of rumen microorganisms, which caused large amounts of VFAs to accumulate and decreased the pH when the switchgrass was hydrolyzed (Figure 4 and Figure 5), probably limiting the formation of methane by methanogens.15,37 Another reason for these results may be a lower quantity of methanogens in these digesters, which resulted in low methane content, as shown in Figure 2. However, as shown in Figure 3e, at a ratio of 1:1, the methane production of some of the mixed inoculum groups was lower than or similar to that of CK. However, at ratios of 1:3 and 1:5 (Figure 3b and 3d), the cofermentation groups had similar trends to that of the biogas slurry inoculum alone throughout the fermentation period—methane production increased as time passed and then remained relatively stable. Moreover, during the first 10 days of fermentation, the methane production of all the mixed groups was almost the same as that of CK. Subsequently, methane production in the cofermentation systems containing rumen bacteria surpassed that of the biogas slurry inoculum and ultimately reached significantly higher levels than those of CK after 40 days of fermentation. The best ratios of rumen microorganisms to the biogas slurry were 1:3 and 1:5 as the WRF, FB, PB and B groups all had higher methane production than did CK (Figure 3b and 3d). However, the methane production of the other cofermentation systems (FP, P, F and CON) was either not significantly different or was lower than that of CK. These results also show the important role of rumen bacteria in the cofermentation system. Figure 3 show that interactions between the microbes in the 16


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FB group occurred because the methane produced by this group was significantly higher than that of either constituent group alone at a given proportion (3:1 and 5:1). Several studies have observed the same phenomenon; fungi produce hydrolases and their hyphae penetrate plant cell walls, leading to decreases in plant particle size as well as cell wall and lignin degradation, which increases the substrate surface for bacterial hydrolases and promotes the function of bacteria.17,19-20 However, at ratios of 1:1, 1:3 and 1:5, the FB group showed no differences from the B group, mostly due to interactions between rumen microorganisms and slurry microbes, in addition to higher contents of the biogas slurry.

Figure 3. Cumulative methane production, expressed as mL/g VS, obtained from the fermentation of switchgrass under different inoculation conditions. The values are averages from three trials. a, b, c, d and e represent the combination ratios 3:1, 1:3, 5:1, 1:5 and 1:1 of the rumen microorganisms and biogas slurry, respectively. 17


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3.2 Characterization of the Liquid Fraction during the Fermentation Process. The pH value is an important indicator for monitoring the performance of the anaerobic digestion process as it reflects the VFA content.15 Optimal pH is vital to the fermentation process since free ammonia (NH3) has been suggested to be the actual toxic agent responsible for anaerobic digestion,38 and high pH in the reactor would result in increased toxicity39 because of the shift to a higher free ammonia to ionized ammonia (NH4+) ratio at higher pH. For the low pH of the reactors, pH decreases to the greatest extent because of the accumulation of VFAs, and high concentrations of VFAs are toxic to methanogens and inhibit hydrolysis rates.37 Therefore, research has suggested that optimal pH values for anaerobic digestion range from 6.5 to 7.5.37,40 The pH trends during the fermentation process for all treatments are shown in Figure 4. For the digestor inoculated with the biogas slurry alone as well as the mixtures inoculated at ratios of 1:1, 1:3 and 1:5, the pH increased to the greatest extent during the early stage due to the large amount of biogas slurry inoculum and the consumption by methanogens, but the pH decreased due to their subsequent accumulation of fatty acids. However, at ratios of 3:1 and 5:1 (Figure 4a and 4c), the pH value of all the mixed inoculum groups decreased during the first 10 days. This finding can be attributed to increases in the relative abundances of certain rumen microorganisms from the inocula, which utilize various sugars to produce VFAs as well as their cofermentation,14 leading to the accumulation of VFAs.41 The very low pH values observed after 10 days, such as that of WRF4 (5.84), which was the lowest 18


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pH value recorded, could hinder volatile solid hydrolysis and methane formation, as shown in Figure 7 and Figure 3.37,42 At 10 days, the pH value of CK increased to 8.71, and those of PB1 (8.497) and FB3 (8.617), though slightly lower than that of CK, were considered to be higher than the acceptable pH range. These increases in the pH might have been due to high ammonia fermentation because of the degradation of proteins, and the alkaline substance of ammonium is toxic to bacteria, especially methane-forming bacteria.38,40 When the pH value of a reactor was greater than the optimum range, the ammonium nitrogen content was found to be increased considerably (Figure 6). However, although the ammonium nitrogen content in the digestor with ratios of 1:3 and 1:5 was higher than that with other ratios (Figure 6b and 6d), the content was still within the normal range.43

Figure 4. pH variation in the samples during the anaerobic digestion process with different 19


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inocula. a, b, c, d and e represent the combination ratios 3:1, 1:3, 5:1, 1:5 and 1:1 of the rumen microorganisms and biogas slurry, respectively.

The content of VFAs (acetic acid, propionic acid and butyric acid) is shown in Figure 5. The accumulation of VFAs leads to a change in the pH, thus affecting the growth of methanogens during the anaerobic digestion process.44 Moreover, high concentrations of VFAs are toxic to methanogens and inhibit hydrolysis in reactors.42 The trend of total VFAs and acetic acid production seemed to depend on the proportion of rumen microorganisms, and the total VFA production and acetic acid at the ratios of 1:3 and 1:5 were higher than 3:1 and 5:1 after 10 days; that is, a higher rate of acidogenensis was observed in the group with a high proportion of rumen microorganisms. The result showed that there were a higher utilization efficiency of VFAs at the ratios of 1:3 and 1:5, which contributed to a higher methane production (Figure 3). A very high content of total VFAs because of the addition of rumen fluid was also found in a previous study.45 In addition, considering that propionic acid is the best indicator of process instability, a decrease in the accumulation of propionic acid after day 10 in systems with ratios of 1:3 and 1:5 resulted in a stable process for these digesters, but other ratios of the fermentation system did not exhibit this phenomenon.46 There was a high concentration of butyric acid during the whole process of fermentation, but the acetic acid and propionic acid concentrations decreased to very low levels at the end of fermentation.

20


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Figure 5. Variation in VFA (acetic acid, propionic acid and butyric acid) contents of the samples during the fermentation process with different inocula. a, b, c, d, e, f, g and h represent the inoculum of WRF, FB, PB, B, FP, P, F and CON, respectively. R1, R2, R3, R4 and R5 represent the combination ratios 1:1, 3:1, 1:3, 5:1, and 1:5 of the rumen microorganisms and biogas slurry, respectively.

The ammonium nitrogen content is shown in Figure 6. The ammonia produced by the biological degradation of proteins and urea often results in VFA accumulation.44 Moreover, ammonia is the common inhibitor of anaerobic digestion due to several mechanisms, such as a change in the intracellular pH, increased maintenance energy requirement, and inhibition of a specific enzyme reaction.38 In all the treatments, the content of ammonium nitrogen decreased at the early stage of the fermentation, followed by a further increase during the fermentation process due to 21


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the degradation of proteins and urea leading to the accumulation of ammonium nitrogen (NH4-N). The ammonium nitrogen content decreased again at 40 days due to the cessation of fermentation. In the CK group, the ammonium nitrogen concentration increased considerably at 15 days due to the degradation of proteins during the anaerobic digestion, and the pH also increased considerably, as shown in Figure 4, and these parameters were higher than those of most cofermentation systems. These results indicate that adding rumen microorganisms improved the balance of the fermentation system. Moreover, the ammonium nitrogen content in all the treatments was below the methanogenic activity inhibitory level.47

Figure 6. Variation in ammonium nitrogen contents of the samples during the anaerobic digestion process with different inocula. a, b, c, d and e represent the combination ratios 3:1, 1:3, 5:1, 1:5 and 1:1 of the rumen microorganisms and biogas slurry, respectively. 22


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3.3 Degradation of the TS, Glucans and Hemicellulose from Switchgrass at the End of Fermentation. The degradation of the TS, glucans and hemicellulose from switchgrass under different treatment conditions and after 40 days of fermentation are shown in Figure 7. During the anaerobic digestion of lignocellulosic biomass, substrate loss is due to the consumption of easily dissolved organic matter and the decomposition of lignocellulose into small molecules by cellulolytic microorganisms, converting it to VFAs and biogas.48 Biogas and methane are generated from biological conversion of the substrate, which can be represented by the changes in the biomass material components (TS, glucans and hemicellulose). As shown in Figure 7a, TS degradation was ranked in the order: 1:5 > 1:3 > 1:1 for the WRF, FB, PB and B groups, which all showed higher TS degradation did than the 3:1 and 5:1 ratios for these groups. These results indicate that a higher proportion of rumen microorganisms achieved lower degradation rates of TS. However, in mammals, the rumen is a natural cellulose-degrading system, the microorganisms of which can effectively digest lignocellulosic biomass.16 Moreover, rumen microorganisms exhibit a greater ability to degrade lignocellulosic biomass, such as the organic fraction of municipal waste and grass, than other common anaerobic microorganisms.21 In general, the microbial cellulolytic activity of rumen fluid is higher than that of biogas slurry. Increasing the proportion of rumen microorganisms led to less TS degradation, possibly because more interactions among the rumen microorganisms and the biogas slurry occurred with a low proportion of rumen microbes. Moreover, the lack of a methanogenic process led to the accumulation of VFAs (Figure 5), and high concentrations of VFAs are toxic to methanogens and 23


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hinder substrate hydrolysis in the anaerobic digestion system.37 The optimum ratio of the mixed inoculum for substrate degradation was 1:5. The TS degradation rates for the WRF, FB, PB and B groups were significantly higher than those of the other mixed inoculum groups (FP, P, F, CON) at the same ratios (P < 0.05), suggesting that the rumen bacterial community was predominately responsible for substrate hydrolysis. However, a previous study found that the relative contributions of three microbial fractions to cell wall digestion were in the following order: fungal fraction > bacterial fraction > protozoal fraction.17 However, in our research, rumen bacteria played an important role in the hydrolysis of switchgrass. Especially for the B groups, TS degradation rates were significantly higher than those in the F groups. Therefore, our results are likely due to the synergistic interactions between the rumen bacteria and the slurry community, which enhanced the hydrolysis efficiency of the switchgrass. The TS degradation rates of B5, WRF5, CK, PB5, B3 and FB5 were 52.78%, 50.48%, 49.45%, 47.97%, 47.76% and 47.47%, respectively, over a period of 40 days of anaerobic digestion, higher than those of the other mixed inoculum groups. The increases in biogas and methane production could be attributed to the improved TS degradation. The above groups (except for the CK group) achieved high biogas and methane production (Figures 1 and 3). This phenomenon was also found for FB2, which achieved higher biogas and methane production even with low TS degradation rates. Therefore, adding rumen microorganisms to the inoculum probably enhanced the biogas and methane conversion efficiency. 24


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Switchgrass is mainly composed of hemicellulose, cellulose and lignin, and anaerobic digestion mostly utilizes cellulose and part of the hemicellulose. Therefore, the hemicellulose and glucans contents were calculated at the end of fermentation (Figure 7b and 7c) to examine the functions of the microorganisms. As shown in Figure 7b and 7c, the degradation rates of hemicellulose (except for the WRF group ) and glucans under all treatments had a similar trend to that of TS degradation. For glucans, the degradation rates of B5 (52.78%), CK (52.40%), PB5 (52.31%) and FB5 (51.73%) were higher than those of other groups. Approximately 51.21%, 49.25%, 49.02% and 47.11% of the hemicellulose was degraded in the B5, WRF5, CK and PB5 groups, respectively, which were higher rates than those of other treatments. The hemicellulose and glucans degradation rates were consistent with the changes in the TS degradation rates. The above phenomenon indicates that the hydrolysis efficiency of hemicellulose and glucans was highest at a rumen microorganism:biogas slurry ratio of 1:5 in all the groups, indicating that the optimum ratio of rumen microorganisms to biogas slurry for the hydrolysis of switchgrass is 1:5.

25


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Figure 7. Degradation of the (a) TS, (b) glucans, and (c) hemicellulose of switchgrass after anaerobic digestion under different treatment conditions. The 1:1, 3:1, 1:3, 5:1 and 1:5 ratios represent the ratio of rumen microorganisms to biogas slurry. Data are the means of three replicates.

Based on the above results, rumen microorganisms enhance the production of methane from switchgrass. A high proportion of rumen microorganisms may lead to a lower pH in the cofermentation system as well as lower methane production (except for the WRF2, WRF4 and FB2 groups in this study) and a lower hydrolysis rate. The ratio of 1:5 rumen microorganisms to the biogas slurry led to high methane production and content, making it the optimum ratio. As shown in Figure 5, methane production by FB2 (189.494 mL/g VS) was significantly higher than that of FB5 (176.892 mL/g VS) and WRF5 (159.679 mL/g VS), and the methane production of FB5 was significantly higher than that of FB1 (148.573 mL/g VS) and CK (137.939 mL/g VS).The FB5 and WRF5 groups were chosen to compare the effects of the 26


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protozoa, which may have a negative influence on anaerobic digestion. The FB2, FB5 and FB1 groups were selected to compare the changes in the microbial community at different proportions of rumen microorganisms, as well as to investigate the microbial community of the fermentation system. 4. CONCLUSIONS In this research, the feasibility of enhancing the hydrolysis efficiency and methane production of switchgrass by cofermentation with rumen microorganisms was investigated through anaerobic digestion. The main conclusions of this study are as follows:

① Both the hydrolysis efficiency and methane production of switchgrass were improved through cofermentation with rumen microorganisms and a biogas slurry. ② Among the microorganisms present in the rumen (bacteria, protozoa, and fungi), bacteria play a prominent role in the cofermentation.

③ Considering the hydrolysis efficiency and methane production of switchgrass, the optimum ratio of rumen microorganism to the biogas slurry was 1:5.

④ The next step of our research is to explore changes in the microbial communities of the FB2, FB5, WRF5 and FB1 cofermentation systems to identify hydrolytic bacteria as well as their methanogen community structures.

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Funding Sources: This project was supported by funding from the National Key Technology Research and Development Program [No. 2012AA101802]. Notes The authors declare no competing financial interest. Abbreviations WRF whole rumen fluid PB

protozoa + bacteria

FB

fungi + bacteria

FP

fungi + protozoa

B

bacteria

P

protozoa

F

fungi

CON negative control R1

rumen microorganisms : biogas slurry 1:1 28


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R2

rumen microorganisms : biogas slurry 3:1

R3


R4


R5


REFERENCES: (1) Sawatdeenarunat, C.; Surendra, K. C.; Takara, D.; Oechsner, H.; Khanal, S. K. Anaerobic Digestion of Lignocellulosic Biomass: Challenges and Opportunities. Bioresour. Technol. 2015, 178,178-186. (2) Hendriks, A.T. W. M.; Zeeman, G. Pretreatments to Enhance the Digestibility of Lignocellulosic Biomass. Bioresour. Technol. 2009, 100 (1), 10-18. (3) Xu, J.; Chen, Y.; Cheng, J. J.; Sharma-Shivappa, R. R.; Burns, J. C. Delignification of Switchgrass Cultivars for Bioethanol Production. Bioresources. 2011, 6(1),707-720. (4) Thanakoses, P.; Black, A. S.; Holtzapple, M. T. Fermentation of Corn Stover to Carboxylic Acids. Biotechnol. Bioeng. 2003, 83, 191-200. (5) Op den Camp, H. J. M.; Verhagen F. J. M.; Kivaisi, A. K.; de Windt, F. E.; Lubberding, H. J.; Gijzen, H. J.; Vogels, G. D. Effects of Lignin on the Anaerobic Degradation of(Lingo) Cellulosic Wastes by Rumen Microorganisms. Appl. Microbiol. Biot. 1988, 29, 408-412. 29


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(6) de Oliveira Gorgulho Silva, C.; Filho, E. X. F. A Review of Holocellulase Production Using Pretreated Lignocellulosic Substrates. Bioenerg. Res. 2017, 10, 592-602. (7) Zhang, L. H.; Zeng, G. M.; Dong, H. R.; Chen, Y. N.; Zhang, J. C.; Yan, M.; Zhu, Y.; Yuan,Y. J.; Xie, Y. K.; Huang, Z. Z. The Impact of Silver Nanoparticles on the Co-Composting of Sewage Sludge and Agricultural Waste: Evolutions of Organic Matter and Nitrogen. Bioresour. Technol. 2017, 230,132-139. (8) Zeng, G. M.; Zhang, L. H.; Dong, H. R.; Chen, Y. N.; Zhang, J. C.; Zhu, Y.; Yuan,Y. J.; Xie, Y. K.; Fang, W. Pathway and Mechanism of Nitrogen Transformation during Composting: Functional Enzymes and Genes under Different Concentrations of PVP-AgNPs. Bioresour. Technol. 2018, 253, 112-120. (9) Zhang, H. B.; Zhang, P.; Ye, J.; Wu, Y.; Fang, W.; Gou, X.; Zeng, G. M. Improvement of Methane Production from Rice Straw with Rumen Fluid Pretreatment: A Feasibility Study. Int. Biodeter. Biodegr. 2016, 113, 9-16. (10) Ho, C.; Chang J. J.; Lin, J. J.; Chin, T. Y.; , Mathew, G. M.; Huang, C. C. Establishment of Functional Rumen Bacterial Consortia (FRBC) for Simultaneous Biohydrogen and Bioethanol Production from Lignocellulose. Inte. J. Hydrogen Energ. 2011, 36(19), 12168-12176.

30


Page 30 of 43

Page 31 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(11) Kovács, K. L.; Ács, N.; Kovács, E.; Wirth, R.; Rákhely, G.; Strang, O.; Herbel, Z.; Bagi, Z. Improvement of Biogas Production by Bioaugmentation. BioMed. Research. International. 2013, 1-7. (12) Sonakya, V.; Raizada, N.; Dalhoff, R.; Wilderer, P. A. Elucidation Mechanism of Organic Acids Production from Organic Matter (Grass) Using Digested and Partially Digested Cattle Feed. Water. Sci. Technol. 2003, 48(8), 255e9. (13) Hu, Z. H; Liu, S. Y.; Yue, Z. B.; Yan, L. F.; Yang, M. T.; Yu, H. Q. Microscale Analysis of in Vitro Anaerobic Degradation of Lignocellulosic Wastes by Rumen Microorganisms. Environ. Sci. Technol. 2008, 42(1), 276e81. (14) Zou, Y.; Xu, X. C.; Li, L.; Yang, F. L.; Zhang, S. S. Enhancing Methane Production from U. lactuca Using Combined Anaerobically Digested Sludge (ADS) and Rumen Fluid Pre-Treatment and the Effect on the Solubilization of Microbial Community Structures. Bioresour. Technol. 2018, 254, 83-90. (15) Alrawi, R.A.; Ahmad, A.; Ismail, N.; Kadir, M. O. A. Anaerobic Co-Digestion of Palm Oil Mill Effluent with Rumen Fluid as a Co-Substrate. Desalination. 2011, 269(1-3), 50-57. (16) Yue, Z. B.; Li, W. W.; Yu, H. Q. Application of Rumen Microorganisms for Anaerobic Bioconversion of Lignocellulosic Biomass. Bioresour. Technol. 2013, 128,738-744.

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Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(17) Lee, S. S.; Ha, J. K.; Cheng, K. J. Relative Contributions of Bacteria, Protozoa, and Fungi to in Vitro Degradation of Orchard Grass Cell Walls and Their Interactions. Appl. Environ.Micro. 2000, 66(9), 3807-3813. (18) Tsuda, T.; Sasaki,Y.; Kawashima, R. Physiological Aspects of Digestion and Metabolism in Ruminants. Academic Press: Ontario, Canada,1991; pp 595-624; DOI: 10.1016/B978-0-12-702290-1.50031-X. (19) Prochazka, J.; Mrazek, J.; Strosova, L.; Strosova, L.; Fliegerova, K.; Zabranska, J.; Dohanyos, M. Enhanced Biogas Yield from Energy Crops with Rumen Anaerobic Fungi. Eng. Life Sci. 2012, 12(3), 343-351. (20) Zhang, Y.; Gao, W.; Meng, Q. Fermentation of Plant Cell Walls by Ruminal Bacteria, Protozoa and Fungi and Their Interaction with Fibre Particle Size. Arch. Anim. Nutr. 2007, 61(2),114-125. (21) Lopes, W. S.; Leite, V. D.; Prasad, S. Influence of Inoculum on Performance of Anaerobic Reactors for Treating Municipal Solid Waste. Bioresour. Technol. 2004, 94, 261-266. (22) Zhang, R. H.; Zhang, Z. Q. Biogasification of Rice Straw with an Anaerobic -Phased System. Bioresour. Technol. 1999, 68(3), 235-245. (23) Kandel, T. P.; Sutaryo, S.; Moller, H. B.; Jorgensen, U.; Laerke, P. E. Chemical Composition and Methane Yield of Reed Canary Grass as Influenced by Harvesting Time and Harvest Frequency. Bioresour. Technol. 2013, 130, 659-666. 32


Page 32 of 43

Page 33 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

(24) Wu, C. H.; Zhou, H.; Yang, F.Y. Microwave Pretreatments of Switchgrass Leaf and Stem Fractions to Increase Methane Production. Bioresources. 2015, 10(3), 3922-3933. (25) Pei, P.; Zhang, C.; Li, J.; Chang, S.; Li, S.; Wang, J.; Zhao, M.; Jiang, L.; Yu, M.; Chen, X. Optimization of NaOH Pretreatment for Enhancement of Biogas Production of Banana Pseudo-Stem Fiber Using Response Surface Methodology. Bioresources. 2014, 9(3), 5073-5087. (26) Van Soest, P. J.; Robertsom, J. B.; Lewis, B. A. Methods for Dietary Fiber, Neutral Detergent Fiber and Nonstarch Polysaccharides in Relation to Animal Nutrition. J. Dairy. Sci.1991, 74, 3583-3597. (27) Chen, L.; Guo, G.; Yu, C. Q.; Zhang, J.; Shimojo, M.; Shao, T. The Effects of Replacement of Whole-Plant Corn with Oat and Common Vetch on the Fermentation Quality, Chemical Composition and Aerobic Stability of Total Mixed Ration Silage in Tibet. Anim. Sci. J. 2015, 86,69-76. (28) Pan, T.; Xiang, H. Y.; Diao, T. T.; Ma, W.; Shi, C.; Xu, Y.; Xie, Q. H. Effects of Probiotics and Nutrients Addition on the Microbial Community and Fermentation Quality of Peanut Hull. Bioresour. Technol. 2019, 273,144-152. (29) Broderick, G. A.; Kang, J. H. Automated Simultaneous Determination of Ammonia and Total Amino Acid in Ruminal Fluid and in Vitro Media. J. Dairy. Sci. 1980, 63, 64-75. 33


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(30) Yue, Z. B.; Yu, H. Q.; Harada, H.; Li, Y. Y. Optimization of Anaerobic Acidogenesis of an Aquatic Plant Canna indica L., by Rumen Cultures. Water. Res. 2007, 41 (11), 2361-2370. (31) Masse, D. I.; Masse, L.; Verville, A.; Bilodeau, S. The Start-up of Anaerobic Sequencing Batch Reactors at 20℃ and 25℃ for the Treatment of Slaughterhouse Wastewater. J. Chem. Technol. Biot. 2001, 76, 393-400. (32) Wall, D. M.; Allen, E.; Straccialini, B.; O’Kiely, P.; Murphy, J. D. Optimisation of Digester Performance with Increasing Organic Loading Rate for Mono- and Co-Digestion of Grass Silage and Dairy Slurry. Bioresour. Technol. 2014, 173, 422-428. (33) Wall, D. M.; Straccialini, B.; Allen, E.; Nolan, P.; Herrmann, C.; O Kiely, P.; Murphy, J. D. Investigation of Effect of Particle Size and Rumen Fluid Addition on Specific Methane Yields of High Lignocellulose Grass Silage. Bioresour. Technol. 2015, 192, 266-271.. (34) Han, G. S; Shin, S. G.; Joonyeob, L. S.; Shin, J.; Hwang, S. H. A Comparative Study on the Process Efficiencies and Microbial Community Structures of Six Full-Scale Wet and Semi-Dry Anaerobic Digesters Treating Food Wastes. Bioresour. Technol. 2017, 245, 869-875.

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Page 35 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

(35) Krishna, K. V.; Mohan, S. V. Selective Enrichment of Electrogenic Bacteria for Fuel Cell Application: Enumerating Microbial Dynamics Using MiSeq Platform. Bioresour. Technol. 2016, 213, 146-153. (36) Niu, H. Z.; Kong, X. Y.; Li, L. H.; Sun, Y. M.;Yuan, Z. H.; Zhou, X. Y.; Analysis of Biogas Produced from Switchgrass by Anaerobic Digestion. Bioresources. 2015, 10(4), 7178-7187. (37) Zhang, T.; Liu, L. L.; Song, Z. L.; Ren, G. X.; Feng, Y. Z.; Han, X. H.; Yang, G. H. Biogas Production by Co-Digestion of Goat Manure with Three Crop Residues PLoS One. 2013, 8, 1-7. (38) Chen,Y.; Cheng, J. J.; Creamer, K. S. Inhibition of Anaerobic Digestion Process: A Review. Bioresour. Technol. 2008, 99,4044-4064.

(39) Borja, R.; Sanchéz, E.; Weiland, P. Influence of Ammonia Concentration on Thermophilic Anaerobic Digestion of Cattle Manure in Upflow Anaerobic Sludge Blanket (UASB) Reactors. Process Biochem. 1996, 31(5), 477-483. (40) Weiland, P. Biogas Production: Current State and Perspectives. Appl. Microbiol. Biot, 2010, 85(4),849-860. (41) Nkemka, V. N.; Gilroyed, B.; Yanke, J.; Gruninger, R.; Vedres, D.; McAllister, T.; Hao, X. Y. Bioaugmentation with an Anaerobic Fungus in a Two-Stage Process for Biohydrogen and Biogas Production Using Corn Silage and Cattail. Bioresour. Technol. 2015, 185, 79-88. 35


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(42) Veeken A.; Hamelers, B. Effect of Temperature on Hydrolysis Rates of Selected Biowaste Components. Bioresour. Technol. 1999, 69, 249-254. (43) Cheng, X.; Zhong, C. Effects of Feed to Inoculum Ratio, Co-Digestion, and Pretreatment on Biogas Production from Anaerobic Digestion of Cotton Stalk. Energ. Fuel. 2014, 28(5), 3157-3166. (44) El-Mashad, H. M.; Zhang, R. Biogas Production from Co-Digestion of Dairy Manure and Food Waste. Bioresour. Technol. 2010, 101(40), 4021-4028. (45) Wall, D. M.; Allen, E.; O'Shea, R.; O'Kiely, P.; Murphy, J. D. Investigating Two-Phase Digestion of Grass Silage for Demand-Driven Biogas Applications: Effect of Particle Size and Rumen Fluid Addition. Renew. Energ. 2016, 86, 1215-1223. (46) Angelidaki, I.; Ahring, B. K. Methods for Increasing the Biogas Potential from the Recalcitrant Organic Matter Contained in Manure. Water. Sci. Technol. 2000, 41 189-194. (47) Cheng, X.; Zhong, C. Effects of Feed to Inoculum Ratio, Co-Digestion, and Pretreatment on Biogas Production from Anaerobic Digestion of Cotton Stalk. Energ. Fuel. 2014, 28(5), 3157-3166. (48) Anjum, M.; Khalid, A.; Mahmood, T.; Aziz, I. Anaerobic Co-Digestion of Catering Waste with Partially Pretreated Lignocellulosic Crop Residues. J. Clean.Prod. 2016, 117, 56-63.

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Table 1. Characteristics of the Switchgrass, Biogas Slurry and Rumen Fluid

TC

TN

(%)

(%)

Switchgrass

41.67

0.99

Biogas slurry

0.62

Rumen fluid

0.71

Samples

Glucan

HC

Lignin

TS

VS

(%)

(%)

(%)

(%)

(%)

42.1

42.5

31.7

8.41

94.09

90.01

0.16

3.88

ND

ND

ND

1.07

0.21

3.38

ND

ND

ND

1.51

C/N

AA

PA

BA

(mg/L)

(mg/L)

(mg/L)

5.89

ND

ND

ND

0.64

7.33

502.32

203.25

353.21

0.81

6.89

1103.1

313.5

345.24

pH

Note: TC, total carbon; TN, total nitrogen; TS, total solids; VS, volatile solids; ND, not determined. HC, hemicellulose; AA, acetic acid; PA, propionic acid; BA, butyric acid.

Figure 1. Biogas production of the switchgrass with different inocula. Values are averages from three trials. a, b, c, d and e represent the combination ratios 3:1, 1:3, 5:1, 1:5 and 1:1 of the rumen microorganisms and biogas slurry, respectively.

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Figure 2. Methane content of the switchgrass with different inocula. Values are averages from three trials. a, b, c, d and e represent the combination ratios 3:1, 1:3, 5:1, 1:5 and 1:1 of the rumen microorganisms and biogas slurry, respectively.

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Figure 3. Cumulative methane production, expressed as mL/g VS, obtained from the fermentation of switchgrass under different inoculation conditions. The values are averages from three trials. a, b, c, d and e represent the combination ratios 3:1, 1:3, 5:1, 1:5 and 1:1 of the rumen microorganisms and biogas slurry, respectively.

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Figure 4. pH variation in the samples during the anaerobic digestion process with different inocula. a, b, c, d and e represent the combination ratios 3:1, 1:3, 5:1, 1:5 and 1:1 of the rumen microorganisms and biogas slurry, respectively.

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Figure 5. Variation in VFA (acetic acid, propionic acid and butyric acid) contents of the samples during the anaerobic digestion process with different inocula. a, b, c, d, e, f, g and h represent the inoculum of WRF, FB, PB, B, FP, P, F and CON, respectively. R1, R2, R3, R4 and R5 represent the combination ratios 1:1, 3:1, 1:3, 5:1, and 1:5 of the rumen microorganisms and biogas slurry, respectively.

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Figure 6. Variation in ammonium nitrogen contents of the samples during the anaerobic digestion process with different inocula. a, b, c, d and e represent the combination ratios 3:1, 1:3, 5:1, 1:5 and 1:1 of the rumen microorganisms and biogas slurry, respectively.

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Figure 7. Degradation of the (a) TS, (b) glucans, and (c) hemicellulose of switchgrass after anaerobic digestion under different treatment conditions. The 1:1, 3:1, 1:3, 5:1 and 1:5 ratios represent the ratio of rumen microorganisms to biogas slurry. Data are the means of three replicate.

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Improving the Anaerobic Digestion of Switchgrass via Cofermentation

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