Anaerobic digestion of submerged macrophytes: Chemical composition and anaerobic digestibility

Ecological Engineering 69 (2014) 304–309

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Anaerobic digestion of submerged macrophytes: Chemical composition and anaerobic digestibility Mitsuhiko Koyama a,∗ , Shuichi Yamamoto a , Kanako Ishikawa b , Syuhei Ban c , Tatsuki Toda a a b c

Graduate School of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan Lake Biwa Environmental Research Institute, 5-34 Yanagasaki, Otsu, Shiga 520-0022, Japan School of Environmental Science, University of Shiga Prefecture, 2500 Hassaka-cho, Hikone, Shiga 522-8533, Japan

a r t i c l e

i n f o

Article history: Received 30 December 2013 Received in revised form 16 April 2014 Accepted 18 May 2014 Available online 12 June 2014 Keywords: Aquatic weeds Submerged macrophytes Anaerobic digestion Lignocellulose

a b s t r a c t Aquatic weeds including submerged macrophyte have been excessively propagated and causing environmental issues in freshwater environment of many countries, and the sustainable treatments have been investigated. In the present study, five submerged macrophyte species dominant in Lake Biwa, Japan, Ceratophyllum demersum, Egeria densa, Elodea nuttallii, Potamogeton maackianus and Potamogeton malaianus were used as a substrate for anaerobic digestion to investigate the chemical composition and the anaerobic digestibility. The lignin content of the submerged macrophyte widely ranged from 3.2 to 20.7%-TS depending on species. The lignin of all macrophytes contained 27.2–59.4% of hydroxycinnamic acids, suggesting they are relatively alkali-labile as compared with woody plants. The total CH4 yield of submerged macrophytes greatly varied from 161.2 to 360.8 mL g-VS−1 depending on species. The CH4 conversion efficiency of C. demersum, El. nuttallii, Eg. densa, P. maackianus and P. malaianus was 57.1, 61.4, 60.6, 33.9 and 72.2%, respectively. The results showed that C. demersum, El. nuttallii, Eg. densa and P. malaianus are feasible for anaerobic digestion due to the high methane recovery, whereas P. maackianus was not preferable for anaerobic digestion. The present study revealed that the methane recovery of submerged macrophytes is regulated by the lignin content, as well as other lignocellulosic biomass. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The vascular aquatic weeds including floating, emergent and submerged macrophytes have been excessively grown and causing various environmental problems in lakes, dams and reservoirs worldwide (Abbasi et al., 1990; Moorhead and Nordstedt, 1993; ˜ Escobar et al., 2011; Haga and O’Sullivan et al., 2010; Munoz Ishikawa, 2011). For instance, the invasion of submerged macrophyte Elodea nuttallii, originated in North America, is serious in ˜ many countries of Europe (Munoz Escobar et al., 2011). In the case of Japan, Lake Biwa, which is the largest lake in the country (674 km2 ), submerged macrophytes have been excessively propagated, covering approximately 90% of the Southern Basin since 1994 (Haga and Ishikawa, 2011). The large quantity of phytomass has been causing water stagnation, foul odor, fishing interference,

∗ Corresponding author. Tel.: +81 426919455; fax: +81 426914086. E-mail address: [email protected] (M. Koyama). http://dx.doi.org/10.1016/j.ecoleng.2014.05.013 0925-8574/© 2014 Elsevier B.V. All rights reserved.

ecosystem change and landscape fouling (Haga et al., 2006a,b). Every year, more than 2600 tons (wet weight: wwt) of the submerged macrophytes are removed from the lake but the harvesting cost reaches more than USD 2.0 million per annum (Kawanabe et al., 2012). Effective and low-cost treatments are needed for treating the excessively propagating macrophytes. Anaerobic digestion is one of the effective and low-cost bioenergy recovery technology from the harvested aquatic weeds (Abbasi et al., 1990; O’Sullivan et al., 2010). Anaerobic digestion generates methane-rich biogas from organic wastes with high moisture content like submerged macrophytes (80–95%-wwt), and the nutrient-rich digested fluid can be used for liquid fertilizer. Furthermore, the operation of anaerobic digester is very simple, and it requires low energy input and cost. A number of previous studies reported that the methane yields of floating aquatic weeds, emergent plants and submerged macrophytes greatly varied depending on the species from 38 to 333 mL g-VS−1 (Table 1), but lower than labile substrate such as food waste (364–489 mL g-VS−1 ) (Verrier et al., 1987; Heo et al.,

M. Koyama et al. / Ecological Engineering 69 (2014) 304–309

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Table 1 CH4 yields from floating, emergent and submerged macrophytes. All data was obtained by batch anaerobic digestion. Aquatic weeds

Type

CH4 yield (mL g-VS−1 )

CH4 yield (mL g-TS−1 )

CH4 yield (mL g-wwt−1 )

Azolla pinnata Cabomba caroliniana Ceratopteris sp. Cyperus sp. Eichhornia crassipes

Floating Floating Floating Emergent Floating

Elodea nuttallii Hydrilla verticillata Salvinia molesta Scirpas sp. Utricularia reticulata Ceratophyllum demersum Elodea nuttallii Egeria densa Potamogeton maackianus Potamogeton malaianus

Submerged Submerged Floating Emergent Emergent Submerged Submerged Submerged Submerged Submerged

132 173 204 38 209 140–180 190 182–193 333 81 242 66 132 249 361 287 161 278

107 – 164 30 – 120–154 – – – 66 204 53 108 191 299 234 136 156

6.3 – 6.7 4.8 – 8.4–10.8 – – – 5.3 11.9 7.4 4.2 12.2 20.9 11.5 13.2 14.2

CH4 conversion efficiency (%-COD) – – – – – –

– – – – – 57.1 61.4 60.6 33.9 72.2

Country

Literature

India Australia India India India USA Australia India Germany India India India India Japan Japan Japan Japan Japan

Abbasi et al. (1990) O’Sullivan et al. (2010) Abbasi et al. (1990) Abbasi et al. (1990) Chanakya et al. (1993) Moorhead and Nordstedt, 1993 O’Sullivan et al. (2010) Patel et al. (1993) ˜ Escobar et al. (2011) Munoz Abbasi et al. (1990) Abbasi et al. (1990) Abbasi et al. (1990) Abbasi et al. (1990) The present study The present study The present study The present study The present study

Some literature values were missing due to the lack of data (TS, VS, and/or COD values) from the literature.

2004). In general, hydrolysis of lignocellulose is a limiting step during anaerobic digestion of plant materials, since recalcitrant lignin protects cellulose and hemicellulose against microbial/enzymatic attack by coating them (Taherzadeh and Karimi, 2008). Lignin is scarcely degraded under anaerobic digestion, demonstrating only 2–17% of methane conversion efficiency even after 300 days of digestion (Benner et al., 1984; Tuomela et al., 2000; Barakat et al., 2012). Previous studies have already reported that the methane recovery of terrestrial woody and herbaceous plants is regulated by the lignin content (Gunaseelan, 2007; Triolo et al., 2011; Frigon et al. 2012). However, the chemical composition in relation to the methane recovery of aquatic weeds, especially submerged macrophytes, has not been investigated yet. The body rigidity and structure of submerged macrophytes is different from other plants. Total phenolic content of submerged macrophytes is lower than that of other aquatic weeds (Smolders et al., 2000). Thus submerged macrophytes have more flexible and softer body structure in order to adapt to the water flow, while terrestrial herbaceous plants and floating and emergent macrophytes normally have more rigid body, since they are predominantly emerged from water (Asaeda et al., 2005). Therefore, the objective of the present study was to investigate the relationship between chemical composition and anaerobic digestibility of five dominant submerged macrophyte species Ceratophyllum demersum, Egeria densa, Elodea nuttallii, Potamogeton maackianus and Potamogeton malaianus corrected from Lake Biwa. Eg. densa and El. nuttallii are widely known as invasive species in ˜ Escobar et al., 2011), many countries (Yarrow et al., 2009; Munoz and accounting for 13 and 6% of the total phytomass in Lake Biwa, respectively (Haga and Ishikawa, 2011). C. demersum, P. maackianus and P. malaianus are native species in Japan and accounts for 6, 56 and 4% of the total phytomass, respectively. In the present study, batch anaerobic digestion was conducted using these five macrophyte species. 2. Materials and methods 2.1. Substrates and inoculum For the substrate, five dominant submerged macrophyte species C. demersum, Eg. densa, El. nuttallii, P. maackianus and P. malaianus were harvested from the Southern Basin of Lake Biwa,

Shiga Prefecture, in Japan. Fresh samples were roughly shredded to the particle size of approximately 0.5–1.5 cm and preserved at −20 ◦ C for the experiment. Before the batch anaerobic digestion and chemical component analysis, substrates were defrosted at room temperature. For the inoculum of batch anaerobic digestion, mesophilic anaerobic sludge treating domestic sewage was used. After we obtained the anaerobic sludge, we preserved the sludge at 37 ◦ C for 2 days before batch anaerobic digestion, in order to digest the sewage sludge left in the anaerobic sludge. The anaerobic sludge was obtained from Hokubu Sludge Treatment Center, Yokohama, Japan. 2.2. Batch anaerobic digestion of submerged macrophytes Batch anaerobic digestion was performed at mesophilic temperature of 37 ± 1 ◦ C in a temperature controlled laboratory for 14 days. The mix ratio of the substrate to the inoculum was adjusted to 1:2 based on volatile solids (VS) contents, and the mixture was loaded to 500 mL Erlenmeyer flask. The batch reactors were sealed by silicon stopper with two sampling ports to allow gas and slurry samples to be collected. 1-L aluminum gas bag (GL Sciences, AAK2, Japan) was attached for the biogas collection. The batch reactors were purged with N2 to make anaerobic environment in the reactor. pH was not controlled throughout the operational period. The reactors were constantly agitated at 100 rpm using a shaker (Taitec, NR-150, Japan). All experiments were conducted in triplicate for El. nuttallii, Eg. densa, P. maackianus, P. malaianus and controls, and duplicate for C. demersum. The methane yield of inoculum was also measured as control, and subtracted from that of each reactor to determine the methane yield from submerged macrophytes. 2.3. Analysis parameters pH, total solids (TS), VS, chemical oxygen demand (COD), lignocellulose and biogas (CH4 , CO2 ) were measured. The pH of the samples was measured using a pH meter (Horiba, B-212, Japan). Standard methods from APHA (1998) were applied to the analysis of TS, VS and COD. Lignocellulose (cellulose, hemicellulose and lignin) content was measured by detergent system (Van Soest et al., 1991) using fiber analyzer (Ankom, A-200, USA). Lignin composition was analyzed using GC/MS by following the method of Clifford et al. (1995). Lignin phenols were derivatized

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Table 2 Chemical composition of submerged macrophytes used in the experiment. Parameter

Unit

Ceratophyllum demersum

Elodea nuttallii

Egeria densa

Potamogeton maackianus

Potamogeton malaianus

Total solids (TS) Volatile solids (VS) VS/TS Total COD Cellulose Hemicellulose Lignin

%-wwt %-wwt % g kg-wwt−1 %-TS %-TS %-TS

6.4 4.9 76.6 90.8 22.3 6.9 15.8

7.0 5.8 82.9 116.7 35.9 N.D. 3.2

4.9 4.0 81.6 50.4 36.2 1.9 4.4

9.7 8.2 84.5 110.3 36.2 11.4 20.7

9.1 5.1 56.0 113.9 22.3 0.4 12.2

N.D., not detected.

from the dried samples using tetramethylammonium hydroxide (TMAH). The present study measured 11 lignin phenols: vanillin, acetovanillin, vanillic acid, syringaldehyde, acetosyringone, syringic acid, p-hydroxybenzaldehyde, p-hydroxyacetophenone, p-hydroxybenzoic acid, p-coumaric acid and ferulic acid. For a standard solution, each lignin phenol was dissolved in methanol to be 200 ␮g mL−1 . Standard solution or 3–5 mg of dried sample was mixed with 150 ␮L TMAH reagent (25%, w/w, in methanol) in a 10 mL ampule. For an internal standard, 100 ␮g mL−1 of n-C19 FA (D) was added to each ampule. After removal of methanol by N2 stream, the ampule was vacuumed and sealed, and heated at 300 ◦ C for 30 min. Ampules were cooled to room temperature and the TMAH derivatives were extracted with 1 mL ethyl acetate. The ethyl acetate was evaporated under reduced pressure and dissolved in 100 ␮L ethyl acetate. The lignin phenol contents were identified by GC/MS (Agilent Technologies, 6890N GC/5973MS, USA). The GC was fitted with a DB-5MS capillary column (30 m long, 0.25 mm i.d., 0.25 ␮m film thickness). The temperature of injector and ion source were maintained at 310 and 230 ◦ C, respectively. The oven temperature was gradually increased from 60 to 310 ◦ C. The temperature of ion source and quadrupole in the mass spectrometry were maintained at 230 and 150 ◦ C, respectively. Helium was used as carrier gas with the flow rate of 1.0 mL min−1 . Biogas collected in the gas bag was quantified using 50 mL disposable syringe under room temperature. Biogas was monitored using a gas chromatograph (Shimadzu, GC-2014, Japan) equipped with a packed column (Shimadzu, Shincarbon ST, Japan) and a thermal conductivity detector. For the calibration, standard gases (H2 , O2 , N2 , CH4 , CO2 : purity >99.9%, Taiyo Nippon Sanso Corp., Japan) were used. The temperature of injector and detector were maintained at 120 and 260 ◦ C, respectively. The column temperature was gradually increased from 40 to 250 ◦ C. Helium was used as carrier gas with the flow rate of 40 mL min−1 .

2.4. Calculation A COD-based methane conversion efficiency of the submerged macrophyte after batch anaerobic digestion was calculated as follows; Methane conversion efficiency(% − COD) =

CODCH4

(1)

CODtotal

where CODCH4 (g-COD) is the COD converted to methane from submerged macrophytes, and CODtotal (g-COD) is the total COD loaded to the batch reactor as a substrate. The methane yield can be expressed as the loss of COD, which is calculated as follows; 1 g-COD = 350 N mL-CH4 (Speece, 1996). 3. Results and discussion 3.1. Chemical composition of submerged macrophytes The organic solid and lignocellulose content of submerged macrophyte significantly varied with species (Table 2). The lignin content of P. maackianus was the highest (20.7%-TS) among five species, which is 1.3 times, 1.7 times, 5.3 times and 6.5 times higher than that of C. demersum (15.8%-TS), P. malaianus (12.2%-TS) Eg. densa (4.4%-TS) and El. nuttallii (3.2%-TS), respectively. Interestingly, the lignin content of native species (P. maackianus, P. malaianus, C. demersum) was considerably higher than that of invasive species (Eg. densa, El. nuttallii). Eg. densa and El. nuttallii are widely known to have strong invasive capacity by its fast growth ˜ Escobar et al., 2011). Poorter rate (Yarrow et al., 2009; Munoz and Bergkotte (1992) investigated the chemical composition of 24 wild plant species and found that fast-growing species had lower lignin and hemicellulose content as compared with slow-growing species. Since body rigidity of plant biomass is determined mainly

Table 3 Lignin phenol composition of submerged macrophytes. Lignin phenol (lignin type)

Unit

Ceratophyllum demersum

Egeria densa

Elodea nuttallii

Potamogeton maackianus

Potamogeton malaianus

Vanillin (G) Acetovanillin (G) Vanillic acid (G) Syringaldehyde (S) Acetosyringone (S) Syringic acid (S) p-hydroxybenzaldehyde (H) p-hydroxy acetophenone (H) p-hydroxybenzoic acid (H) p-coumaric acid (hydroxycinnamic) Ferulic acid (hydroxycinnamic)

%-lignin %-lignin %-lignin %-lignin %-lignin %-lignin %-lignin %-lignin %-lignin %-lignin %-lignin

6.2 4.1 10.6 2.2 4.7 8.5 13.9 0.9 20.4 15.4 13.1

1.7 2.9 6.2 0.0 2.3 3.0 12.7 0.9 25.6 25.0 19.8

2.6 5.0 6.6 0.9 1.1 1.8 10.8 0.7 11.0 17.5 41.9

1.9 2.6 5.3 0.8 4.1 3.7 3.8 0.3 50.3 5.7 21.5

5.5 2.7 13.6 1.4 0.9 3.0 16.4 0.9 14.7 16.4 24.4

20.9 15.5 35.2 28.5

10.8 5.2 39.2 44.8

14.2 3.9 22.5 59.4

9.8 8.6 54.4 27.2

21.9 5.4 32.0 40.8

0.7

0.5

0.3

0.9

0.2

G total S total H total Hydroxycinnamic total S/G ratio

M. Koyama et al. / Ecological Engineering 69 (2014) 304–309

by the lignin content, it was indicated that Eg. densa and El. nuttallii have more flexible body structure than three other native species possibly due to their fast growing characteristic. The composition of lignin significantly varies with plant types and/or tissues (Hedges and Mann, 1979; del Rio et al., 2007). Lignin is amorphous polyphenol consists of three different phenylpropane unit forming extremely complex three-dimensional structure; guaiacyl (G) lignin, syringyl (S) lignin, and p-hydroxyphenyl (H) lignin. Generally, hardwoods are characterized by the dominance of S and G lignin, while softwoods contain relatively few S lignin. Within the hardwood, non-woody tissues such as leaf and pollen, and herbaceous plants are known to have abundant H lignin as well as G and S lignin. In addition to these, recent studies revealed that lignin in herbaceous plant and/or non-woody portion of softwood and hardwood contains hydroxycinnamic acids (ferulic acid and p-coumaric acid), which has ester bonds and are cross-linked to polysaccharides and lignin polymer (Buranov and Mazza, 2008; Sonoda et al., 2010). del Rio et al. (2007) conducted GC/MS analysis for determination of lignin composition of five non-woody plants and reported that the lignin composition greatly varied with species. Table 3 shows the TMAH-derivatized lignin composition of each submerged macrophyte species. All five macrophyte species contained G, S and H lignin and hydroxycinnamic acids, which are characteristic of herbaceous plant and/or non-woody tissue of vascular plants. Rabemanolontsoa and Saka (2012) also confirmed that submerged macrophytes contain these four types of lignin, although the derivatization method and lignin classification is different. In the present study, the S/G lignin ratio of submerged macrophytes ranged from 0.2 to 0.9 depending on species. These S/G ratio are similar with flax (0.4) and hemp (0.8), but these values are considered to be fairly low, as compared with other plant biomass such as jute (1.7), abaca (2.9) and sisal (3.4) (del Rio et al., 2007). In general, low S/G lignin ratio implies lower delignification rate and higher alkali consumption during alkaline delignification process, suggesting lignin polymers of submerged macrophytes are relatively resistant against chemicals. In contrast, the content of hydroxycinnamic acids was abundant in all submerged macrophyte species, accounting for 27.2–59.4% of all lignin phenols (Table 3). Ferulic acid is attached to lignin polymer with alkali-stable ether bonds and to hemicellulose with alkali-labile ester bonds (Ralph et al., 1995). In contrast, p-coumaric acid is ester-linked and ether-linked with lignin polymer. Since alkali can easily cleave ester linkage (Hartley and Morrison, 1991), application of alkali can induce the removal of lignin polymer-ferulic acid complex from the surface of polysaccharides. Accordingly, it is suggested that alkaline pre-treatment may have a great potential for effective delignification of lignin-rich submerged macrophyte species such as P. maackianus. 3.2. Methane production rate and methane yield Fig. 1A shows the methane production rate of roughly-shredded submerged macrophytes. The methane production of C. demersum, El. nuttallii, Eg. densa, P. maackianus and P. malaianus rapidly occurred from day 1. The methane production rate peaked on day 3 for El. nuttallii (116.0 ± 2.8 mL g-VS−1 day−1 ), day 2 for Eg. densa (79.9 ± 13.3 mL g-VS−1 day−1 ) and day 1 for C. demersum (80.4 mL g-VS−1 day−1 ), P. maackianus (37.2 ± 1.4 mL g-VS−1 day−1 ) and P. malaianus (73.6 ± 16.0 mL g-VS−1 day−1 ), respectively. After day 3, the methane production rate of all macrophytes rapidly decreased and the generation of methane mostly completed in 7–14 days. The rapid biogasification was demonstrated probably by the biodegradation of intracellular soluble organic matter (e.g. cytosols) and/or the accessible cellulose and hemicellulose exists in the cell wall.

307

Fig. 1. CH4 production rate (A) and cumulative CH4 yield (B) of submerged macrophytes.

The VS-based cumulative methane yield and COD-based methane conversion efficiency greatly varied with submerged macrophyte species (Fig. 1B and Table 1). The methane yield and methane conversion efficiency of El. nuttallii was 360.8 mL g-VS−1 ˜ and 61.4%-COD, indicating this species was relatively labile. Munoz Escobar et al. (2011) obtained the similar results of approximately 333 mL g-VS−1 . This result confirms El. nuttallii is highly feasible for anaerobic digestion. Eg. densa, P. malaianus and C. demersum were also labile, yielding 287.1 mL g-VS−1 (60.6%-COD), 277.5 mL g-VS−1 (72.2%-COD) and 249.2 mL g-VS−1 (57.1%-COD), respectively. The VS-based methane yield of Eg. densa, C. demersum and P. malaianus was relatively high as compared to most of other aquatic weeds conducted in the previous studies. In addition, TS-based and wet weight based methane yields of these four macrophyte species were higher than the reported aquatic weeds (Table 1). These results indicated that the four macrophyte species were also labile and applicable to anaerobic digestion. Contrary to other four macrophytes, P. maackianus was relatively recalcitrant, showing the total methane yield and methane conversion efficiency of 161.2 mL g-VS−1 and 33.9%-COD, respectively. This result showed that P. maackianus is not preferable substrate for anaerobic digestion without the application of some pre-treatments. 3.3. Methane recovery and lignin content Fig. 2A demonstrates the relationship between lignin content of the submerged macrophytes and the cumulative methane yields

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M. Koyama et al. / Ecological Engineering 69 (2014) 304–309

species and strongly affects the anaerobic digestibility, as well as other terrestrial woody and herbaceous plants, fruits, and vegetables. Thus it was suggested that the lignin content can be used for the prediction of anaerobic digestibility of submerged macrophytes. The present study indicated that El. nuttallii, Eg. densa, P. malaianus and C. demersum are feasible for anaerobic digestion owing to the high methane recovery, whereas P. maackianus is not preferable for anaerobic digestion due to high content of lignin. A number of studies have reported that lignin is scarcely degraded under anaerobic digestion, demonstrating only 2–17% of methane conversion efficiency even after 300 days of digestion (Benner et al., 1984; Tuomela et al., 2000; Barakat et al., 2012). Accordingly, it is assumed that delignification pre-treatment such as alkaline pre-treatment may be effective for P. maackianus in order to obtain the sufficient methane recovery. 4. Conclusions In the present study, the chemical composition and the anaerobic digestibility of five submerged macrophytes species were investigated. The lignin content of the submerged macrophyte widely ranged depending on species from 3.2 to 20.7%-TS. All macrophytes had G, S and H lignin and hydroxycinnamic acids, which indicates the lignin of submerged macrophytes was similar to herbaceous plant and/or non-woody tissue of vascular plants. The total methane yield of submerged macrophytes greatly varied from 161.2 to 360.8 mL g-VS−1 depending on species (El. nuttallii > Eg. densa > P. malaianus > C. demersum > P. maackianus). The present study revealed that the methane recovery of submerged macrophytes is regulated by the lignin content, as well as other lignocellulosic biomass. For the lignin-rich macrophyte (i.e. P. maackianus), the application of delignification pre-treatments is recommended to make this species feasible for anaerobic digestion.

Fig. 2. Relationship between lignin content and the CH4 yield of submerged macrophyte and other plant biomass under mesophilic batch anaerobic digestion. (A) The present study (Y = 368.7e−0.023x, n = 5, R2 = 0.6057); (B) Sum of the present study and other literatures (Y = 473.1e−0.043x, n = 44, R2 = 0.67101). () = Present study, ( ) = aquatic weeds (Cheng et al., 2010; Wang et al., 2010), ( ) = herbaceous plants (Gunaseelan, 2007; Triolo et al., 2011; Xie et al., 2011; Frigon et al., 2012), () = fruits and vegetable waste (Gunaseelan, 2007).

obtained in the present study. It was clearly demonstrated that the higher methane yield is obtained from submerged macrophytes when the lignin content is low, and vice versa (Y = 368.7e−0.023x , n = 5, R2 = 0.6057). From this result, it was revealed that the anaerobic digestibility of submerged macrophytes is regulated by the lignin content of the inherent nature of the substrate. Fig. 2B summarizes the relationship between lignin content of the submerged macrophytes and the total methane yields obtained in the present study and others from the literature. The results of the present study were fitted well on the lignin-methane yield correlation of other plant biomass (Y = 473.1e−0.043x , n = 44, R2 = 0.67101). Previous studies have reported that lignin content of the substrate influences the methane recovery (Gunaseelan, 2007; Triolo et al., 2011; Frigon et al. 2012). Triolo et al. (2011) examined the correlation between total methane yield and several different chemical components of various energy crops and manure, and reported that lignin content was most significantly correlated with methane yield, as compared with cellulose, neutral detergent fiber (NDF: cellulose + hemicellulose + lignin + ash) and acid detergent fiber (ADF: cellulose + lignin + ash) contents. The present study confirmed that the lignin content of submerged macrophyte greatly varies with

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