Chiang Mai J. Sci. 2019; 46(4) : 639-652http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Biogas Production from Single Digestion of Napier Grass Hydrolysate and Co-Digestion of Solid Fraction of Microwave Acid Pretreated Napier Grass with Swine ManurePrawit Kongjan [a], Alissara Reungsang [b,c], Naphatsarnan Phasukarratchai [d] and Sureewan Sittijunda *[d][a] Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000,

Thailand.[b] Biotechnology program, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand.[c] Research Group of Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen

University, Khon Kaen 40002, Thailand.[d] Faculty of Environment and Resource Studies, Mahidol University, Nakhon Pathom 73170, Thailand.*Author for correspondence; e-mail: sureewan.sit@mahidol.ac.th

Received: 16 October 2018Revised: 12 March 2019

Accepted: 21 March 2019

ABSTRACT The biogas production from hydrolysate and solid fraction of microwave acid pretreated

Napier grass was investigate in the batch experiments. Factors influencing methane production including initial pH, inoculum concentration, and carbon to nitrogen ratio (C/N ratio) were investigated. For the hydrolysate of Napier grass, maximum methane production (MP) and methane production rate (MPR) of 621.31 mL-CH4/L and 0.72 mLCH4/L h were obtained at initial pH of 8 and inoculum concentration of 15 g-VSS/L. Using the solid fraction of pretreated Napier grass co-digested with swine manure, maximum MP and MPR of 630.05 mL-CH4/L and 0.74 mLCH4/L h were obtained at C/N ratio of 21.03. Main methanogenic bacteria found in the hydrolysate and solid fraction fermentation of pretreated Napier grass co-digested with swine manure were Methanosarcina sp. Methanoregula sp. Methanospirillum sp. Methanocullues sp. and Methanothrix sp. Overall energy production from Napier grass hydrolysate and the solid fraction of pretreated Napier grass co-digested with swine manure was 7.99 kJ/g-VSadded.

Keywords: methane, anaerobic co-digestion, agricultural residues, energy production, C/N ratio

1. INTRODUCTIONDepletion of non-renewable energy

reserves and greenhouse gas (GHG) emission have greatly impacted on human society [1] and contributed to the development of renewable energy such as wind, solar, hydrothermal, and biomass-based sources. One renewable raw

material used for bioenergy production is lignocellulosic biomass as a source of sugar consisting of pentose (C5) and hexose (C6) which can be further converted to liquid or gas fuels. Thailand is an agricultural country that produces approximately 38,700 million

Chiang Mai J. Sci. 2019; 46(4)640

kilograms of agricultural residues annually [2]. Irresponsible disposal of agricultural wastes causes a greater environmental impact than pollution by organic industrial wastes [3]. Anaerobic digestion (AD) is an efficient method to reduce waste via biogas production and can be used to convert wastes into usable energy [4].

Napier grass is a popular agricultural crop in Thailand and it is promoted by the government as a renewable energy crop [3]. Napier grass is a complex material composed of cellulose, hemicellulose and lignin. Cellulose and hemicellulose primarily contain glucose and xylose, respectively and they can be fermented to produce renewable energy using several microbial processes. Many previous studies showed that pretreatments were capable of significantly enhancing methane yields from Napier grass [5]. Based on previous research, pretreatment is necessary to decompose lignin and allow processing of cellulose and hemicellulose.

The most widely used pretreatments were categorized into three major areas as physical, chemical and biological methods. A combination of physical and chemical methods as microwave acid pretreatment is an interesting option offering simplicity (easy to conduct), speed, economy, and efficiency [6]. Two phase end products as hydrolysate and a solid phase are obtained after the pretreatment. The hydrolysate is comprised of mainly glucose and xylose and a small amount of arabinose [7,8]. These sugars are solubilized from the hemicellulosic fraction [6]. The solid phase consists of the cellulose fraction which can be subsequently hydrolyzed to glucose and further converted to methane by an anaerobic digestion process. Previous research mainly focused on the utilization of hydrolysate as the substrate, with the solid phase discarded as waste. Therefore, here, we focused on both the hydrolysate and solid fraction as the substrate for methane production.

The solid fraction consists of a carbon source and is hard to degrade by microbes; therefore, methane yield obtained from the solid fraction was quite low. Codigestion of mixed substrates for biogas production gave better results than single substrate digestion [9,10].

This process has become increasingly important due to the need for sustainable solutions to handle and recycle waste. Swine and chicken manure are popular raw material for AD since they are inexpensive, high in nitrogen content and abundant. Manure is a good nitrogen source for co-digestion with lignocellulosic biomass and provides buffering capacity and nitrogen, while the lignocellulosic material provides carbon. The mixture has a more balanced C/N ratio and reduced risk of ammonia inhibition and acidification. Additionally, the input of readily biodegradable organic matter into animal manure digesters significantly increases biogas production [7,8,11].

Therefore, the aim of this study was to evaluate the biochemical methane potential (BMP) from single digestion of Napier grass hydrolysate and co-digestion of the solid fraction of pretreated Napier grass and swine manure. To maximize methane production, the influences of initial pH and inoculum concentration on methane production from Napier grass hydrolysate were investigated. Effects of the ratios of microwave acid pretreated Napier grass and swine manure (C/N ratio) on methane production were also investigated. To understand the anaerobic digestion process phenomenon a microbial population analysis was also conducted. Energy generation from methane produced at the optimal scenario using the hydrolysate and solid fraction was also evaluated. The information obtained from this study would pave the way towards continuous biogas production from hydrolysate and solid fraction of Napier grass.

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2. MATERIALS AND METHODS 2.1 Inoculum Preparation

Anaerobic granules obtained from an up-flow anaerobic sludge blanket (UASB) reactor were used as a seed inoculum. This UASB reactor was used to produce biogas from the wastewater of a brewery in Khon Kaen Province, Thailand. Prior to use, the UASB granules were washed twice with tap water and then soaked in distilled water for 7 days, before storage in a refrigerator at 4 °C until required for use. Initial cell concentration was measured and reported in grams of volatile suspended solids (VSS) per gram of UASB dry weight (gvss/gdry weight).

2.2 Napier Grass and Swine ManureNapier grass (Pennisetum purpureum) strain

Pakchong 1 was obtained from Sriviroj Farm

Public Company Limited, Khon Kaen Province, Thailand. Prior to use, the grass was chopped into small pieces, air dried and ground using a blender to a size of 0.30 x 0.30 mm. The grass was stored in plastic bags at room temperature.

Swine manure was obtained from the Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand. The manure was air dried and ground using a blender. Ground swine manure was stored in plastic bags at room temperature until required for use. Compositions of Napier grass and swine manure are presented in Table 1.

2.3 Microwave Acid Pretreatment of Napier Grass

Microwave acid pretreatment of Napier grass was carried out in an LG/MD 2022D microwave. Thirty grams of ground Napier

Table 1. Compositions of heat treated UASB granules, Napier grass, Napier grass hydrolysate, solid fraction of microwave acid pretreated Napier grass and swine manure.

ComponentHeat treated

UASB granules

Napier grass

Napier grass hydrolysate

Solid fraction of microwave

acid pretreated Napier grass

Swine manure

Carbon content (%) NA 49.93 3.40b 42.53a 33.17

Nitrogen content (%) NA 2.02 0.012b 1.37a 2.23

TS (g/g-dry weight) 0.19 0.86 10.67c 0.84 0.93

VS (g/g-dry weight) 0.18 0.86 5.08c 0.53 0.6

Lignin (%) w/w NA 32.04 9.14 22.9 NA

Cellulose (%) w/w NA 34.25 4.35 29.9 NA

Hemicellulose (%) w/w NA 17.36 4.36 13.0 NA

Total sugar (g/L) NA NA 6.36 NA NA

Reducing sugar (g/L) NA NA 2.91 NA NA

Glucose (g/L) NA NA 1.63 NA NA

Xylose (g/L) NA NA 0.95 NA NA

Arabinose (g/L) NA NA 0.19 NA NA

Acetic acid (mg/L) NA NA 0.001 NA NAa : unit in % w/w, b : unit in % w/vc : unit in g/LNA : not analyzed

Chiang Mai J. Sci. 2019; 46(4)642

grass were added to 500 mL of Duran bottle containing 450 mL of 1.56% (v/v) H2SO4 [6]. Microwave assisted H2SO4 condition was 450 Watts for 7.5 min [6]. After pretreatment, the hydrolysate and solid fraction were separated by filtration through a thin cloth layer and used as substrates. The solid fraction was washed with tap water to neutralize the pH, and then air dried and kept in a plastic bag at room temperature. The pH of the hydrolysate fraction was adjusted to 10 with Ca(OH)2 and the resulting solid fraction was removed by centrifugation at 5,000 rpm for 15 min, then re-acidified to pH 7, followed by further centrifugation at the same rpm and centrifugation time [12]. Concentrations of sugars (glucose, xylose, and arabinose)and inhibitors (furfural, hydroxymethylfufural (HMF), and acetic acid) in the supernatant were analyzed high performance liquid chromatography (HPLC). Then storage at -20 °C until required for use.

2.4 Methane Production from Napier Grass Hydrolysate Using Anaerobic Mixed Cultures

Methane production from Napier grass hydrolysate was conducted in 120 ml serum bottles with an 85 ml working volume. The methane production medium was supplemented

with Napier grass hydrolysate at initial total sugar concentration of 5.41 g/L (4.32 g-VS/L), trace elements contained in modified basic anaerobic (BA) medium [13], and inoculum. Inoculum concentration was varied at 10, 15 and 20 g-VSS/L, and initial pH in each experiment was varied at 6, 7 and 8, respectively (Table 2). Serum bottles were capped with rubber stoppers and aluminum caps. Headspaces of the serum bottles were flushed with nitrogen gas to create anaerobic conditions and the serum bottles were incubated at room temperature (35 ± 4 °C). During incubation, volume of biogas was measured using a wetted gas syringe method [14]. All treatments were conducted in triplicate. Measurement of methane production continued until biogas production ceased. A control experiment was performed in the same manner but without the addition of inoculum.

2.5 Methane Production from Co-Digestion of the Solid Fraction of Microwave Acid Pretreated Napier Grass with Swine Manure

Co-digestion of the solid fraction with swine manure for methane production was conducted in serum bottles. Ratios of solid fraction of microwave acid pretreated Napier Grass to swine manure were 0.5:1, 1:1, and

Table 2. MPR, MY and kh from Napier grass hydrolysate at various initial pH and inoculum concentrations.

Initial Inoculum concentration MPR MYkh (d

-1)pH (g-VSS/L) (mL-CH4/L h) (L-CH4/kg-VSadded)

6 10 0.06 + 0.02 12.90 + 2.02 0.020

6 15 0.29 + 0.05 58.00 + 7.12 0.018

6 20 0.18 + 0.01 35.17 + 5.34 0.020

7 10 0.37 + 0.07 74.71 + 6.11 0.061

7 15 0.49 + 0.09 97.27 + 10.54 0.043

7 20 0.40 + 0.01 80.20 + 9.21 0.038

8 10 0.29 + 0.03 58.50 + 5.67 0.056

8 15 0.72 + 0.04 143.82 + 16.12 0.103

8 20 0.24 + 0.07 47.64 + 6.37 0.055

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2:1 (w/w) for C/N ratios of 18.71, 21.03 and 23.79, respectively (Table 3). The methane production medium contained 15 g-VSS/L of UASB granules as the inoculum and different C/N ratios of solid fraction of microwave acid pretreated Napier grass and swine manure. Initial pH of the fermentation broth was adjusted to 8 with addition of either 1N NaOH or 1 N HCl.

Control experiments were performed with solid fraction without inoculum, swine manure without inoculum, and co-digestion of solid fraction with swine manure without inoculum addition. All treatments were conducted in triplicate. The creation of anaerobic conditions, volume of gas produced, and gas composition were conducted as outlined in the previous section.

Table 3. MPR and MY from solid fraction of microwave acid pretreated Napier grass co-digested with swine manure at various C/N ratios.

Ratio (w/w) C/N ratio MPR

(mL-CH4/L h)MY

(L-CH4/kg-VSadded)kh (d

-1)

0.5:1 18.71 0.38 + 0.05 36.90 + 7.32 0.024

1:1 21.03 0.74 + 0.07 55.76 + 9.12 0.014

2:1 23.79 0.14 + 0.02 7.34 + 3.62 0.018

Swine manure only - 0.02 + 0.01 4.35 + 1.12 0.011

2.6 Analytical Methods Biogas compositions including methane,

nitrogen and carbon dioxide were determined using a gas chromatograph (GC, Shimadzu 2014, Japan) equipped with a thermal conductivity detector (TCD) and a column packed with Shin carbon charcoal (50/80 mesh). The GC-TCD conditions were set according to Saraphirom and Reungsang [15]. Concentrations of glucose, xylose,v arabinose and inhibitors were measured using HPLC with conditions set according to Khamtib et al. [6]. The pH was measured using a digital pH meter (Sartorius, Germany). TS and VS concentrations were determined using a hot air oven and furnace at 105 °C for 4 h and 550 °C for 2 h, respectively. Extent of fiber destruction by microwave acid pretreatment was examined using scanning electron microscopy (SEM). Methane production was determined by measurement of the headspace gas composition and total volume of methane produced after each time interval by mass balance [16]. The rate of methane production was calculated by 1storder kinetic equation [17,18].

Fermentation broth obtained from the experimental runs that gave the highest methane production from sections 2.4 and 2.5 were collected. Triplicates fermentation broth from each run were pour together before extracted DNA. Total genomic DNA was extracted using the phenol-chloroform extraction method. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis and sequencing of the extracted DNA were performed according to the method described by Konjan et al. [19]. This method is successfully used to analyze the microbial community in the fermentation process [20-23]. Closet matches of partial 16S rRNA gene sequences were identified through database searches in GenBank using BLAST.

3. RESULTS AND DISCUSSION 3.1 Hydrolysate and Solid Fraction of Microwave Acid Pretreated Napier Grass

The composition of Napier grass hydrolysate is shown in Table 1. Total sugar and reducing sugar concentrations were 6.36 and 2.91 g/L.

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Main sugars found in the hydrolysate fraction were glucose (1.63 g/L), xylose (0.95 g/L) and arabinose (0.19 g/L). Acetic acid (0.001 mg/L) was found as an inhibitor in the hydrolysate. Furfural was not observed due to the short microwave irradiation time. Results revealed that the microwave acid method broke down hemicellulose and some cellulose structures inside Napier grass and glucose, xylose and arabinose were obtained in the hydrolysate fraction.

Compositions of non-pretreated Napier grass and solid fraction of microwave acid pretreated Napier grass are shown in Table 1. Results indicated that the solid fraction had low cellulose (29.90% w/w), hemicellulose (13.00% w/w), and lignin contents (22.90% w/w) compared to non-pretreated Napier grass. Low hemicellulose, lignin, and cellulose contents after pretreatment showed that this method effectively removed amorphous parts of the lignocellulosic materials, i.e., lignin and hemicellulose and also hydrolyzed some microcrystalline cellulose. Moreover, lignin content in microwave acid pretreated Napier grass (22.90% w/w) also decreased compared with non-pretreated. Based on our results, the microwave acid pretreatment method was suitable to break down the lignocellulosic structure and also remove lignin in Napier grass.

Generally, sulfuric acid preferentially breaks down hemicellulose and lignin rather than crystalline cellulose [24,25] by solubilizing the hemicellulosic fraction of the biomass, making the cellulose more accessible.

Structural changes in microwave acid pretreated Napier grass and non-pretreated Napier grass are seen in the SEM images (Figure 1). Results show that the texture of non-pretreated Napier grass was compact and covered by a thin wax layer (Figure 1A). After pretreatment, the surface of Napier grass was quite loose, with a diminished wax layer (Figure 1B). Previous studies indicated that the surface of grass treated with microwave assisted acid became loose and irregular [26]. This suggests that microwave acid pretreatment improved the digestibility and removed silica [26] from the grass sample. In the solid fraction, accessible cellulose remained and was used as the substrate for methane production by anaerobic mixed cultures.

3.2 Methane Production from Napier Grass Hydrolysate Using Anaerobic Mixed Cultures

Methane production from Napier grass hydrolysate at various initial pH and inoculum concentrations is depicted in Figure 2. Results showed that different initial pH and inoculum

BA

Figure 1. Scanning electron microscopy images of Napier grass fibers. (A) Untreated Napier grass; (B) microwave acid pretreated Napier grass (magnification, x 200 and x 500).

Chiang Mai J. Sci. 2019; 46(4) 645

concentration led to variations in cumulative methane production. At inoculum concentrations of 10, 15, and 20 g-VSS/L increase in initial pH from 6 to 8 resulted in increased methane production. Mildly acidic conditions (initial pH of 6.0) did not favor methane-producing bacteria, resulting in low methane production and methane production rate (MPR) (Figure 2 and Table 2). Low methane production and MPR at the acidic condition (pH < 6) resulted from fermentation suppression. The pH was a key factor that determined the dominant microbial population, related with the production pathway between acetoclastic methanogenesis and hydrogenotrophic methanogenesis [23]. Generally, conversion of hydrogen and carbon dioxide (H2 and CO2) to methane by hydrogenotrophic methanogenesis often dominates at low pH (pH < 4), whereas conversion of acetic acid into methane by acetoclastic methanogenesis dominates at a higher pH than 4. Therefore, controlling pH during anaerobic digestion was necessary for the two methanogenesis pathways which impacted on methane production performance. Neutral and mildly alkaline pH values (pH of 7.0 and 8.0), were suitable for growth, resulting in higher methane production and MPR values (Figure 2 and Table 2). This

result was consistent with previous studies, which found that an initial pH between 6.5 and 8.5 was optimal for methane production by anaerobic mixed cultures. At the optimal pH, methane-producing bacteria showed highest efficiency in degradation of organic matter.

The effect of inoculum concentration on methane production is shown in Figure 2. At initial pH 6, 7 and 8, an increase in inoculum concentration from 10 to 15 g-VSS/L resulted in an increase in methane production, methane production rate and methane yield (MY). A further increase in inoculum concentration greater than 15 g-VSS/L resulted in a decrease in methane production, methane production rate, and methane yield (Table 2). Maximum methane yield of 143.82 L-CH4/kg-VSadded was obtained at initial pH of 8 and inoculum concentration of 15 g-VSS/L. Inoculum concentration was an important key that impacted on methane production. Low methane production at inoculum concentrations of 10 and 20 g-VSS/L may be attributed to system overloading. At inoculum concentration of 15 g-VSS/L, the highest methane production was obtained as the substrate and inoculum ratio (S/I ratio) were suitable. This result implies that a suitable S/I ratio enhances substrate degradation efficiency

Time (h)

0 200 400 600 800 1000

Cum

ulat

ive m

etha

ne p

rodu

ctio

n (m

L-CH

4/L)

0

100

200

300

400

500

600

700pH 6, 10 g-VSS/LpH 6, 15 g-VSS/LpH 6, 20 g-VSS/LpH 7, 10 g-VSS/LpH 7, 15 g-VSS/LpH 7, 20 g-VSS/LpH 8, 10 g-VSS/LpH 8, 15 g-VSS/LpH 8, 20 g-VSS/L

Figure 2. Methane production from Napier grass hydrolysate at various initial pH and inoculum concentrations.

Chiang Mai J. Sci. 2019; 46(4)646

during anaerobic digestion. The kinetic constant of methane production from hydrolysate napier grass ranged from 0.018–0.103 d−1 (Table 2). The data obtained in this study were comparable with the values of 0.016-0.125 d−1 reported by Gunaseelan [18]. It appear that the inhibitors founded in hydrolysate napier grass was not inhibit the methane producing bacteria.

3.3 Methane Production from Solid Fraction of Microwave Acid Pretreated Napier Grass Co-Digestion with Swine Manure

Figure 3 shows the effect of C/N ratios (18.71, 21.03, and 23.79) on methane production at the suitable pH of 7. Results indicated that C/N ratio influenced cumulative methane production which increased with increase in C/N ratios from 18.71 to 21.03 and decreased when C/N ratio was greater than 21.03. At incubation time of 0 to 200 h, cumulative methane production slowly increased and after that, it sharply increased. This result might occur because methanogenic bacteria take time to adapt and digest the substrate for conversion to methane. Maximum methane production, MPR and MY of 630.05 mL-CH4/L, 0.74 mL-CH4/L h and 55.76 L-CH4/kg-VSadded were achieved at C/N ratio of 21.03 (Table 3). Based on previous findings, C/N ratio can greatly impact the

efficiency of methane production [27,28]. Previous findings reported optimal C/N ratio for biogas production as between 25 and 30 [29]. Lower or higher C/N ratio than the optimal range results in adverse effects on the methane production process. At low C/N ratio, the process is inhibited by accumulation of NH3

produced from protein degradation [17] with insufficient carbon and nitrogen available for microbial growth. At C/N ratios higher the optimal range, methane production and MPR are inhibited by lack of nitrogen sources for methanogenic bacterial growth. At the optimal C/N ratio, spontaneous co-digestion of the solid fraction and swine manure was performed as a control. In the control experiment, methane production of 46.26 mL-CH4/L was obtained which was 13.75 times lower than the experiment with inoculum addition (630.05 mL-CH4/L) (Figure 3). This result suggested that addition of inoculum enhanced methane production from co-digestion of the solid fraction and swine manure. Based on our results, variations of C/N ratio and initial pH influenced methane production from co-digestion of the solid fraction and swine manure.

Microbes are very sensitive to environmental conditions and different substrates can affect fermentation performance. Changes in methane

Time (h)

0 200 400 600 800 1000

Cum

ulta

ive m

etha

ne p

rodu

ctio

n (m

l-CH 4/L

-sub

stra

te)

0

100

200

300

400

500

600

700C/N ratio 18.71C/N ratio 21.03C/N ratio 23.79

Figure 3. Methane production from the solid fraction of microwave acid pretreated Napier grass co-digested with swine manure at various C/N ratios.

Chiang Mai J. Sci. 2019; 46(4) 647

production performance might result from changes in the microbial community during the fermentation process. Therefore, analysis of the microbial community responsible for anaerobic digestion could increase understanding of the reasons for different microbial performances at similar substrate concentrations.

3.4 Microbiological Analysis PCR-DGGE analyses of bacterial and

archaeal populations at the end of fermentation under optimal condition, using the hydrolysate and solid fraction co-digested with swine manure as the substrate are depicted in Figures 4A and 4B, respectively. Figure 4A depicts the bacterial population whereas Figure 4B depicts the archaea population. Optimal conditions were initial pH and inoculum concentration of 8 and 15 g-VSS/L when using the hydrolysate as substrate, and initial pH of 7 and C/N ratio of

27 590

B

C D

A 40%

70%

27 590

B

C D

A 40%

70%

Figure 4. Bacterial (A) and archaeal populations (B) from the hydrolysate and solid fraction of microwave acid pretreated Napier grass co-digested with swine manure using PCR-DGGE analysis; lane A: bacterial community under optimal condition from the solid fraction of microwave acid pretreated Napier grass co-digested with swine manure; lane B: bacterial community under optimal condition from Napier grass hydrolysate; lane C: archaeal community under the optimal condition from the solid fraction of microwave acid pretreated Napier grass co-digested with swine manure; lane D: archaeal community under optimal condition from Napier grass hydrolysate.

21.03 when using the solid fraction as substrate. Results in lane A show the main bacterial population at optimal condition of co-digested solid fraction and swine manure as Clostridium

sp. (band 1), Clostridium sp. (band 2), Clostridium tyrobutyricum (band 3), Bradyrhizobium sp. (band 4), and Clostridium sp. (band 5) (Figure 4A). Main bacterial populations found in optimal

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condition of Napier grass hydrolysate were Clostridium sp. (band 6), Clostridium bifermentans (band 7), Clostridium sp. (band 8), Alcaligenes faecalis (band 9), Pseudomonas aeruginosa (band 10), and Actinomyces sp. (band 11) (band B, Figure 4A). Clostridium sp., Clostridium tyrobutyricum and Clostridium bifermentans are well known as H2-producing bacteria [6,12]. These microorganisms are capable of degrading macromolecules and converting to them volatile fatty acids (VFAs), ethanol and hydrogen. This coincided with the presence of VFAs and ethanol in the liquid phase and hydrogen in the gas phase. Pseudomonas aeruginosa has been reported as able to degrade lipids to produce fatty acids and glycerol [30]. Alcaligenes faecalis is a hydrogen sulfide degrading bacteria found in wastewater treatment plants [31] while Actinomyces sp. is a well-known cellulolytic bacteria which degrades lignocellulosic material and occurs in anaerobic sludge [32]. Other species, such as Bradyrhizobium sp. have not yet been reported as hydrogen producers.

The archaeal community is shown in Figure 4B. Lane C depicts the archaeal community found in fermentation of the co-digested solid fraction and swine manure, while lane D depicts the archaeal population found in methane production from Napier grass hydrolysate. These results show that archaeal populations were Methanosarcina sp. (band 1) Metanospirillum sp. (band 5), Metahnoculleus sp. (band 2), Methanospirillum sp. (band 6), Methanothrix sp. (band 3), Methanoregula sp. (band 7), and Methanosarcina sp. (band 4). Bands 1-4 were the dominated by archaea in the anaerobic co-digestion of solid fraction and swine manure. Bands 5-7 were the dominated archaea by archaea in the anaerobic digestion of Napier grass hydrolysate. All archaeal populations were methanogens or methanogenic bacteria which used acetate, hydrogen and carbon dioxide as the substrate to produce methane.

In Napier grass hydrolysate, Pseudomonas

aeruginosa and Actinomyces sp. were functional as degrading bacteria at the hydrolysis step because they produced extracellular enzyme to degrade the macromolecules. During the initial fermentation period, Pseudomonas aeruginosa and Actinomyces sp. degraded the complex substrate to monomers and then Clostridium sp. (bands 6, 7, and 8, lane B Figure 4A) further converted the monomers to VFAs, hydrogen and carbon dioxide. Hydrogen and carbon dioxide were further converted to methane using Methanospirillum sp. (bands 5 and 6, lane D, Figure 4B, Eq. 2) and Methanoregula sp. (band 7, Figure 4B). Methanospirillum sp. is a genus of the Methanospirillaceae which use H2/CO2 as a substrate to produce methane. The other VFAs were further converted to methane by Methanoregula sp. (band 7, Figure 4B) [33].

For co-digestion of the solid fraction and swine manure, initially Clostridium sp., (bands 1, 2, 3, and 5) degraded Napier grass and swine manure to short chain VFAs such as acetate, butyrate and propionate, and also hydrogen and carbon dioxide as gas by-products. Methanogenic substrates including acetate, hydrogen and carbon dioxide were directly converted to methane using equations 1 and 2 via methanogenic archaea.

CH3COO- + H2O CH4 + HCO3- ΔG°

= -31.0 kJ/mol (1) Acetoclastic methanogens

4H2 + HCO3- + H+ CH4 + 3H2O ΔG°

= -135.6 kJ/mol (2) Hydrogenotrophic methanogens

CH3COO- + 4H2O 4H2 + 2HCO3- + H+ ΔG°

= + 104.6 kJ/mol (3) Acetate oxidizing bacteria

CH3CH2COO- + 3H2O CH3COO- + 3H2 +

HCO3- +H+ ΔG°

= + 76.1 kJ/mol (4) Propionate oxidizing bacteria

Chiang Mai J. Sci. 2019; 46(4) 649

CH3CH2CH2COO- + 2H2O 2CH3COO- + 2H2 +H+ ΔG°

= + 48.1 kJ/mol (5) Butyrate oxidizing bacteria

The presence of Methanosarcina sp. (bands 1 and 4) and Methanothrix sp. (band 3) in lane C (Figure 4B) confirmed that the methane production processes using microwave acid pretreated Napier grass and swine manure were acetoclastic cleavage pathway (Figure 4B and Eq. 1) [23]. Detection of Methanoculleus sp. (Figure 4B, band 2) suggested that another methane production process followed the hydrogenotrophic cleavage pathway [23]. Other metabolites such as propionate were further cleaved into acetate and hydrogen (Eq. 4), whereas butyrate was cleaved into acetate and hydrogen (Eq. 5). These cleavage products were further converted to methane using the hydrogenotrophic and acetoclastic cleavage pathways [23,34]. Based on our results, the main methanogenic pathways were acetoclastic

and hydrogenotrophic cleavage. Different methane production performance using both the hydrolysate and solid fraction was caused by coordinate matching of antagonistic and symbiotic relationships among diverse species.

3.5 Comparison of Methane Yield and Energy Production from the Hydrolysate and Co-Digested Solid Fraction and Swine Manure

Maximum methane yields of 143.82 and 55.76 L-CH4/kg-VSadded obtained using the hydrolysate and co-digestion of the solid fraction and swine manure as the substrate. Maximum MY of 143.82 and 55.76 L-CH4/g-VSadded were much lower than MY obtained from previous reports [35-37] (Table 4). However, MY values obtained here were comparable with Forster-Carneiro et al. [38] and Mussoline et al. [39]. Yield discrepancies might be due to different types of substrate and operational temperature. Moreover, lower C/N ratio (21.03) than the optimal range (25-30) might inhibit the lack of

Table 4. Comparison of MY with the literature research.

Inoculum Substrate Optimum conditionsMY

(L-CH4/kg-VSadded)

References

Anaerobic granular

Rice straw and autoclaved paper mill sludge

35 ºC, 5:10 (%w/w) of rice straw and paper mill

43 [39]

Anaerobic granular

Rice straw only 35 ºC, 5 (% w/w) of rice straw 46 [39]

Sludge from wastewater treatment plant

Sludge, food waste, grass clipping and garden waste

10:67.5:15.75:6.75 (%VS), (R1), HRTs 15, 20 and 30 days

425 [37]

Sludge from wastewater treatment plant

Sludge, food waste, grass clipping and garden waste

10:45:31.5:13.5 (%VS), (R2), HRTs 15, 20 and 30 days

385 [37]

- Household source-selected OFMSW

Inoculum/Substrate ratio

(ISR = 3), 30 days, 37°C

365 [35]

UASB granule Solid fraction of microwave acid pretreated Napier grass co-digested with swine manure

C/N ratio of 21.03 pH 7, 35 ºC, static condition

55.76 This study

UASB granule Hydrolysate Napier grass Inoculum 15g-VS/L pH 8, 35 ºC, static condition

143.82 This study

Chiang Mai J. Sci. 2019; 46(4)650

carbon sources for methanogens and reduce accumulation of NH3 produced from protein degradation [17,40], resulting in a low MY.

Energy production was calculated based on the MY, density of methane (0.72 mg/ml) and heating value of the methane (55.6 kJ/g). Maximum MY from the hydrolysate and solid fraction of microwave acid pretreated Napier grass co-digested with swine manure were 143.82 and 55.6 L-CH4/kg-VSadded. Therefore, energy production from the hydrolysate and solid fraction of microwave acid pretreated Napier grass co-digested with swine manure were [(143.82 X 0.72 X 55.6)] = 5.76 kJ/g-VSadded and [(55.76 X 0.72 X 55.6)] = 2.23 kJ/g-VSadded, respectively. Overall energy production from the hydrolysate and co-digestion of the solid fraction of microwave acid pretreated Napier grass with swine manure was 7.99 kJ/g-VSadded.

4. CONCLUSIONSOur results show that Napier grass has

high potential for use as a feedstock for biogas production since it is an inexpensive and abundant lignocellulosic material. The microwave acid pretreatment method breaks down lignin and hemicellulose from the Napier grass, resulting in a solid fraction high in cellulose. Both pH values and C/N ratios had an influence on methane production performance, related with coordinate matching of antagonistic and symbiotic relationships among different species during the anaerobic digestion process. Maximum MY values of 143.82 and 55.76 L-CH4/kg-VSadded were obtained using the hydrolysate and solid fraction of microwave acid pretreated Napier grass co-digested with swine manure. Main methanogenic pathways followed hydrogenotrophic and acetoclastic cleavage. Energy production from the hydrolysate and solid fraction of microwave acid pretreated Napier grass co-digested with swine manure were 5.76 and 2.23 kJ/g-TSadded, respectively.

ACKNOWLEDGEMENTS This research project was supported

by Udon Thani Rajabhat University. The authors appreciate the Center of Science and Technology for Research and Community Development, UDRD and the Research Group of Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen University for facilities support. Partially financial support was also received from TRF Senior Research Scholar (Grant No. RTA6280001) and Thailand Research Fund and Office of the Higher Education Commission (Grant No. MRG6180094).

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