Anaerobic digestion of submerged macrophytes

Anaerobic digestion of submerged macrophytes
—Biochemical approach for enhancing the methane production—
沈水植物のメタン発酵処理
—生物化学的アプローチによるメタン生成量の向上—
13D5701
小山
光彦
指導教員
戸田
龍樹
SYNOPSIS
近年、世界各地で沈水植物などの水草類の過剰繁茂による環境悪化が問題となっており、発生抑制方法ならびに除去した
バイオマスの適正処理・有効利用技術の確立が喫緊の課題となっている。沈水植物のような高含水系有機性廃棄物の処理
には、メタン発酵処理が適していると考えられる。本研究は、琵琶湖に過剰繁茂する沈水植物のメタン発酵プロセスの高
効率化を目的とし、初めに各種沈水植物のメタン生成能と化学組成の関係について検討した。次に、難分解性の沈水植物
を対象に、前処理が化学組成とメタン生成量に与える効果と、前処理の副生成物質によるメタン発酵プロセスの阻害効果
を検討した。最後に、前処理した沈水植物の半連続処理特性を評価した。中温回分処理実験の結果、沈水植物のメタン生
成量は種によって 161－361 mL g-VS-1 と大きく異なり、琵琶湖に最も優占するセンニンモが最も難分解性であった。沈水
植物のメタン生成量は陸上草本類や農作物と同様にリグニン量に依存することが明らかとなった。リグニン組成分析の
結果、沈水植物はアルカリで分解しやすいヒドロキシ桂皮酸類を 27－59%含有していた。これらの結果からセンニンモ
の易分解化にはアルカリ処理が効果的であると考えられた。アルカリ処理により、センニンモのリグニン量は無処理基質
の 207 mg g-TS-1 から 85 mg g-TS-1 に減少した。回分処理におけるメタン変換率は無処理条件の 31%-COD から、アルカリ
処理により 51%-COD に増加した。一方で、アルカリ処理により生成される溶出リグニンにより、メタン発酵の各分解過
程とくに加水分解過程が阻害されることが明らかとなった。しかしながら、アルカリ処理したセンニンモを 120 日間にわ
たり半連続処理した結果、運転 20 日目付近からメタン生成は大きく阻害されたが、運転 42 日目以降は回復し、溶出リグ
ニンに対して長期運転により馴化することが明らかとなった。半連続処理において、アルカリ処理したセンニンモのメタ
ン回収率は無処理条件に比べて約 65%高く、回分処理よりも高い増加率を示した。これらの結果から、沈水植物のメタン
発酵の高効率化にはアルカリ処理が有効であることが示された。本研究においてアルカリ処理によってリグニン量が多
い水草のメタン変換率を大きく向上できたことで、難分解性の水草を含む様々な水草ひいては同様の化学組成を有する
植物バイオマスの効率的な省エネルギー・創エネルギー処理が達成可能であることが示された。
Keywords: Submerged macrophyte, Anaerobic digestion, Lignocellulose, Pre-treatment
INTRODUCTION
In recent decades, aquatic macrophytes including floating,
emergent and submerged macrophytes have been excessively
propagated and causing various environmental problems in lakes,
dams and reservoirs worldwide (e.g. Abassi et al. 1990). In the
case of Japan, submerged macrophytes have been covering more
than 90% of the Southern Basin in Lake Biwa (Haga and Ishikawa
2011), and causing water stagnation, foul odor, fishing
interference and landscape fouling. Every year, approximately
4,000 tons-wet weight (wwt) of macrophytes are removed from
the lake, but effective and low-cost treatments for treating the
macrophytes have not been established yet. Anaerobic digestion
(AD) is noted as one of the most effective and low-cost bioenergy
recovery technologies from organic wastes with high moisture
content, such as aquatic weeds.
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, but lower than labile substrate such as food waste
(364 to 489 mL g-VS-1) (e.g. Heo et al. 2004). In general,
hydrolysis of lignocellulose is a limiting step during AD of plant
materials, since recalcitrant lignin protects cellulose and
hemicellulose against microbial/enzymatic attack by coating
them. Some studies have already reported that the methane
recovery of terrestrial woody and herbaceous plants is limited 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. Submerged
1
macrophytes tend to have more flexible body structure as
compared
with
terrestrial
herbaceous
plants
and
floating/emergent macrophytes, because they are mostly
submerged under water (Asaeda et al. 2005). Thus anaerobic
digestibility of submerged macrophyte may be different from
other plant biomass.
In order to enhance the anaerobic digestibility of plant
biomass, application of pre-treatment or increase of digestion
temperature have widely been attempted for improving feedstock
degradability and microbial metabolic rate, respectively.
Amongst the various pre-treatments, alkali pre-treatment is noted
as one of the most promising methods for rapid delignification
(Fernandes et al. 2009; Xie et al. 2011), and enhancement of CH4
recovery by alkaline pre-treatment is reported for terrestrial
herbaceous plants. On the other hand, it has been pointed out that
toxic compounds such as dissolved lignin can be co-produced
during pre-treatment (Barakat et al. 2012; Quéméneur et al. 2012).
For AD process, inhibitory effect of dissolved lignin on
methanogenesis step has been reported in several studies (SierraAlvarez and Lettinga 1991; Yin et al. 2000; Kayembe et al. 2013),
but the effect on hydrolysis and acidogenesis steps are not taken
into account, although it is crucial to concern. Therefore, alkaline
pre-treatment may cause adverse effect on AD of submerged
macrophytes.
The overall research aim of this Ph.D. thesis is to examine
AD process of submerged macrophytes and to enhance the
anaerobic digestibility. The specific objectives were:
1. To reveal the relationship between chemical composition
and anaerobic digestibility of five submerged macrophyte
species which are excessively propagated and dominant in
Lake Biwa.
2. To examine the effect of alkaline pre-treatment on AD of
recalcitrant submerged macrophytes.
3. To evaluate the effect of alkali pre-treatment on semicontinuous AD of submerged macrophyte and to discuss the
techno-economic feasibility.
to 500 mL medium bottle with 150 mL mesophilic AD sludge.
CH4 yield was monitored online by using automatic methane
potential test system (AMPTS) II. For acidogenesis/hydrolysis
toxicity test, 10 g L-1 cellulose was added to 500 mL flask with
250 mL AD sludge. The biogas was collected in 1.0 L aluminum
gas bag. The solubilization efficiency of cellulose was monitored
by measuring CH4 yield and SCOD in the digestate.
Study 3. Effect of alkaline pre-treatment on semi-continuous
anaerobic digestion at two different digestion temperature
Alkali-treated and un-treated P. maackianus was
anaerobically digested in semi-continuous mode. The pretreatment condition was 80ºC, NaOH 0.2 g g-TSsubstrate-1 for 3.0 h,
followed by neutralization using HCl to pH 7. For AD bioreactor,
completely stirred tank reactor (CSTR) of the working volume of
4.5 L was used. Alkali pre-treated or un-treated P. maackianus
was fed to CSTR every two days for the period of 100-120 days.
Organic loading rate (OLR) was 1.0 g-VS L-1 day-1. CSTR was
operated at 37°C, HRT 40 days, and continuous mixing of 100
rpm. The biogas was collected in 10.0 L aluminum gas bag. pH,
TS, VS, biogas composition, COD, VFAs, lignocellulose were
measured. In addition, in order to clarify the composition of
SCOD, acid precipitation was conducted for fractionation of
SCOD.
MATERIALS AND METHODS
Study 1. Chemical composition and anaerobic digestibility of
five submerged macrophyte species
Batch AD test of five submerged macrophyte species was
performed. For the substrate, Ceratophyllum demersum, Egeria
densa, Elodea nuttallii, Potamogeton maackianus and
Potamogeton malaianus were harvested from the Southern Basin
of Lake Biwa, Shiga Prefecture, Japan. For the inoculum,
mesophilic anaerobic sludge treating domestic sewage was
obtained from Hokubu Sludge Treatment Center, Yokohama,
Japan. The mix ratio of the substrate to the inoculum was adjusted
to 1:2 based on volatile solids (VS) content, and the mixture was
loaded to 500 mL flask. Batch AD was performed at 37 ± 1°C for
14 days. The reactors were constantly agitated at 100 rpm using a
shaker. All experiments were run in triplicate. 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. pH, total solids (TS), VS, chemical
oxygen demand (COD), lignocellulose and biogas composition
(CH4, CO2) were measured. Biogas was monitored using a gas
chromatograph equipped with a thermal conductivity detector.
Lignin composition was analyzed by using the
tetramethylammonium hydroxide (TMAH) derivatization
method proposed by Clifford et al. (1995).
RESULTS AND DISCUSSION
Study 1. Chemical composition and anaerobic digestibility of
five submerged macrophyte species
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 most macrophytes
are feasible for AD due to the high methane recovery, but P.
maackianus was not preferable for anaerobic digestion. The CH4
yield of submerged macrophyte was declined with the increase of
the lignin content, and the lignin-CH4 yield correlation was fitted
well on that of other plant biomass (Fig. 1, y=-11.285x + 441.12,
n=44, R² = 0.562, p<0.001). From these results, it was revealed
that the lignin content of submerged macrophyte greatly varies
Cumulative CH4 yield (mL g-VS-1)
Study 2. Effect of alkaline pre-treatment on anaerobic
digestibility of lignin-rich submerged macrophyte
Thermochemical pre-treatment was conducted on P.
maackianus in order to investigate the optimum pre-treatment
conditions, in terms of hydrolysis efficiency and lignin removal.
10 g-wwt of substrate was added with 10 mL of NaOH solution
to 50 mL corning tubes. The NaOH loading rate over TS content
of the substrate was 0, 0.025, 0.05, 0.1, 0.2 g g-TSsubstrate-1,
respectively. The substrates were soaked in the NaOH solution
and heated at 60 and 80ºC, for 0.5, 1.0, 2.0 and 3.0 h, respectively.
All experiments were conducted in triplicate.
A mesophilic batch AD test was conducted to investigate the
digestibility of P. maackianus after thermochemical pretreatment. The substrates were pre-treated at 80ºC, NaOH loading
rate of 0.1 and 0.2 g g-TSsubstrate-1 for 3.0 h, respectively. The pretreated substrates were neutralized using HCl before digestion.
Un-treated substrates were also anaerobically digested as a
control. The configuration of this batch AD test and analysis
parameters are similar with Study 1.
Methanogenic and acidogenesis/hydrolysis toxicity test were
performed in order to elucidate the AD inhibition process by
dissolved lignin. Dissolved lignin was extracted from alkali pretreatment liquor of P. maackianus and purified by Dioxane
extraction method (Björkman 1956). In both experiments,
extracted dissolved lignin were added in different concentration
of 0.5, 1.0, 2.5, 5.0 g L-1 and 0 g L-1 as control. For methanogenic
toxicity test, 2 g L-1 acetate was used for the substrate and added
600
EN: El. nuttallii
ED: Eg. densa
CD: C. demersum
PML: P. malaianus
PMC: P. maackianus
500
400
300
CD
PML
EN
PMC
200
ED
100
0
0
5
10
15
20
25
Lignin content （%-VS）
30
Fig.1. Relationship between lignin content and the CH4 yield of
submerged macrophytes and other plant biomass under
mesophilic batch anaerobic digestion. (■) =Present study, (◇) =
Aquatic weeds (Cheng et al. 2010; Wang et al. 2010), ( 〇 ) =
Herbaceous plants (Gunaseelan 2007; Triolo et al. 2011; Xie et al.
2011; Frigon et al. 2012), ( △ ) = Fruits and vegetable waste
(Gunaseelan 2007).
2
with species and strongly affects the anaerobic digestibility, as
well as previous reports including terrestrial woody and
herbaceous plants, fruits, and vegetables. Lignin consists of four
different unit forming complex three-dimensional structure;
guaiacyl (G) lignin, syringyl (S) lignin, p-hydroxyphenyl (H)
lignin, and hydroxycinnamic acids (ferulic acid and p-coumaric
acid). Hardwoods dominate S and G lignin, while softwoods
contain few S lignin. Non-woody tissues and herbaceous plants
have abundant H lignin and hydroxycinnamic acids as well as G
and S lignin. In the present study, all five macrophyte species
contained all of four types of lignin. The content of
hydroxycinnamic acids was abundant in all macrophytes,
accounting for 27.2 to 59.4% of all lignin phenols. Among all
lignin phenols, only hydroxycinnamic acids have ester bonds,
which is alkali labile (Grabber et al. 1997). Therefore, alkaline
pre-treatment may have a great potential for effective
delignification of lignin-rich macrophytes such as P. maackianus.
Study 2. Effect of alkaline pre-treatment on anaerobic
digestibility of lignin-rich submerged macrophyte
The hydrolysis efficiency expressed by soluble COD per total
COD (SCOD/TCOD) of P. maackianus was greatly enhanced
with the increase of NaOH loading rate, pre-treatment
temperature and treatment time. Amongst others, the pretreatment condition of NaOH 0.2 g g-TSsubstrate-1, 80˚C, and 3.0 h
performed the highest SCOD/TCOD of 45.4%. Contrary, the
SCOD/TCOD with no alkali addition was considerably low,
demonstrating 12.3% at 80˚C and 3.0 h, suggesting NaOH take a
key role in accelerating the hydrolysis. Reduction of lignin and
increase of CH4 yield of P. maackianus by alkaline pre-treatment
(80°C, 3.0 hrs) was summarized in Fig. 2. The lignin content of
P. maackianus was significantly reduced with the increase of
NaOH and pre-treatment temperature. At the pre-treatment
condition of NaOH 0.2 g g-TSsubstrate-1, 80˚C, 3.0 hrs, the lignin
content declined from 207.0 mg g-TS-1 to 84.7 mg g-TS-1. The
cumulative CH4 yields from P. maackianus of untreated, NaOH
0.1 and 0.2 g g-TSsubstrate-1 were 161.3, 231.3 and 242.8 mL g-VS1. The CH conversion efficiency of P. maackianus increased
4
from 33.6%-COD (untreated) to 50.6%-COD (NaOH 0.2 g gTSsubstrate-1), which was approximately 50% higher than untreated
substrate. The increase of CH4 recovery was probably due to the
high delignification efficiency caused by the cleavage of alkalilabile ester linkage between polysaccharides and ferulic acid
coupled with lignin polymer. From these results, it was indicated
that the alkaline pre-treatment at 80˚C is highly effective for
enhancing anaerobic digestibility of lignin-rich macrophytes.
The cumulative CH4 yield at NaOH 0.2 g g-TSsubstrate-1 was
similar to NaOH 0.1 g g-TSsubstrate-1 (p>0.05), despite the lignin
content of the substrate declined from 126.8 mg g-TS-1 to 84.7 mg
g-TS-1 (Fig. 2). A number of studies have pointed out that the pretreatment of lignocellulosic biomass sometimes lead the
formation of toxic compounds for methanogenesis (Xie et al.
2011; Barakat et al. 2012). During heating and/or chemical pretreatment of lignocellulose, some chemical compounds such as
furans (furfural and 5-hydroxymethylfurfural) and phenolics (e.g.
dissolved lignin and tannin) can be produced, which influence the
microbial and enzymatic activity. Recent study revealed that
furans have no inhibitory effect on AD (Barakat et al. 2012).
Other literatures pointed out the methanogenic inhibition by
phenolic compounds derived from lignin, but no proof has been
confirmed yet.
400
(A)
NaOH 0.2
NaOH 0.1
Un-treated
300
200
100
0
CEL
HEM
LIG
Lignocellulose
Cumulative CH4 yield (mL g-VS-1)
Lignocellulose (mg g-TS-1)
Then, the present study calculated the concentration of
dissolved lignin in the digestate during batch AD test from the
amount of lignin removed from the solid fraction of the substrate
by alkaline pre-treatment. The increase of CH4 yield of all
biomass decreased or ceased with the increase of dissolved lignin
concentration in the digestate. The estimated inhibitory level of
dissolved lignin concentration was 0.9 to 1.2 g L-1. These results
suggest that AD can be inhibited at the condition of high lignin
removal efficiency due to the increase of dissolved lignin in the
digestate.
300
(B)
250
200
150
100
NaOH 0.2
NaOH 0.1
Un-treated
50
0
0
3
6
9 12 15
Operation time（day）
Fig. 2. Effect of alkali pre-treatment on AD of P. maackianus:
(A) Reduction of lignocellulose (CEL: cellulose, HEM:
hemicellulose, LIG: lignin), (B) Time course of cumulative CH4
yield.
Fig. 3 summarizes the toxic effect of dissolved lignin on AD
process during toxicity test. Methanogenic and acidogenic
activities were slightly inhibited at high dissolved lignin
concentration of >2.5 g L-1 up to 15% and 10% reduction to the
control, respectively (Fig. 3A, B). It has been proposed that lignin
can directly inhibit microbial cells, and the toxicity is determined
by hydrophobicity (Kayembe et al. 2013), type of substituents on
aromatic ring (Sierra-Alvarez and Lettinga 1991) and molecular
weight (Yin et al. 2000). Sierra-Alvarez and Lettinga (1991)
found that lignin model compound with aldehyde group shows
higher toxicity on methanogenic activity of 50% IC 1.8 g L-1 as
compared with aromatic carboxylic acids (50% IC >9.2 g L-1). In
the present study, aldehyde-substituting lignin accounted only for
4.7% of the dissolved lignin of pre-treated P. maackianus, while
lignin with carboxylic acid accounted for 53.7%. This could
explain the lower toxicity of dissolved lignin on methanogenesis
and acidogenesis observed in the present study.
(A)
100
90
80
70
60
50
110
NHA (%-control)
(B)
100
90
80
70
60
50
0
1 2 3 4 5 6
Dissolved lignin (g L-1)
0
1 2 3 4 5 6
Dissolved lignin (g L-1)
(C)
100
90
80
70
60
50
0
3
110
NAA (%-control)
NMA (%-control)
110
1 2 3 4 5 6
Dissolved lignin (g L-1)
Fig. 3. Effect of dissolved
lignin on AD process: (A)
normalized methanogenic
activity (NMA), (B) normalized acidogenic activity
(NAA), (C) normalized
hydrolysis activity (NHA).
On the other hand, hydrolysis efficiency dropped by 25%
from dissolved lignin 1.0 g L-1 and reached 35% reduction against
control in dissolved lignin 5.0 g L-1 (Fig. 3C). A number of
previous researches have reported that lignin irreversibly adsorbs
cellulase and inhibits the enzymatic hydrolysis of cellulose (e.g.
Rahikainen et al. 2013). Accordingly, it was suggested that
hydrolysis step is the most susceptible steps against dissolved
lignin in AD process, probably not only by direct toxicity to the
microbial cells but also cellulase adsorption.
Study 3. Effect of alkaline pre-treatment on semi-continuous
anaerobic digestion of submerged macrophytes
During semi-continuous AD of alkali pre-treated and untreated P. maackianus, pre-treated condition exhibited higher
CH4 recovery. The cumulative CH4 yield and the CH4 conversion
efficiency was 219.2 mL g-VS-1 and 45.9%-COD for alkali pretreated P. maackianus, which was 65% higher than those of untreated P. maackianus. This result clearly exhibited that alkaline
pre-treatment is preferable AD process for lignin-rich submerged
macrophyte, in terms of higher CH4 recovery. In alkali pretreated condition, the CH4 production rate started to drop from
day 16 and showed lowest CH4 production rate of 59.4 mL L-1
day-1 in day 30 (Fig. 4). The deterioration of CH4 production rate
was observed when theoretical dissolved lignin concentration
exceeded 3.3 g L-1 (day 16). Simultaneously, VFAs in the
digestate dramatically increased from day 20 and reached 5.1 g L1 in day 36, but the pH was maintained around 7.2 to 7.5. These
results suggest that the AD process was inhibited by dissolved
lignin, not by the pH drop. However, the CH4 production rate
started to recover from day 42 and high CH4 production rate of
241.0 ± 32.1 mL L-1 day-1 was maintained throughout the rest of
the operational period (day 46-120). With the increase of CH4
production rate, VFAs concentration started to decline from day
36, and stabilized to 0.1 to 0.4 g L-1 after day 70, indicating the
AD process was fully recovered. These results clearly exhibited
that the acclimatization against the inhibitors occurred in this
phase. The acclimatization period is comparable to AD of phenol,
which reported that the duration of the acclimatization is reported
to ranges from 6 weeks to 10 months, depending on the nature of
inoculum and the operational strategy (Veeresh et al., 2005). In
addition, it is known that the hydrophobicity (i.e. toxicity to
microorganisms) of lignin is weakened when cellulase adsorption
capacity is saturated by the formation of strongly-bonded “ligninenzyme complex” (Funaoka 1998; Palonen et al. 2004). From
these perspectives, it was suggested that dissolved lignin can
“temporary” inhibit anaerobic digestion especially during startup
phase, but acclimatization to dissolved lignin may be due to the
adaptation of microorganisms and/or saturation of enzyme
adsorption on dissolved lignin can recovers the stable anaerobic
digestion process.
In order to confirm the feasibility of alkaline pre-treatment on
AD of submerged macrophytes, the preliminary energy balance
and economic assessment was conducted by assuming full-scale
treatment in Lake Biwa. Extra energy input for heating versus
increase of gained heat energy by biogas combustion, and
additional costs for chemicals (NaOH and HCl) versus increase
of electricity sales (i.e. benefit), were compared with un-treated
condition. Both the economic and heat balance became positive,
indicating the cost for chemicals can be compensated by the
increase of electricity sale, and no extra thermal energy input is
required for alkaline pre-treatment. In conclusion, the present
400
10
350
9
8
300
7
250
6
200
5
150
4
3
100
Pre-treated
Un-treated
Dissolved lignin (theoretical)
50
0
2
Dissolved lignin（g L-1）
CH4 production rate （mL L-1 day-1）
study indicated that alkaline pre-treatment is highly beneficial for
enhancing anaerobic digestibility of lignin-rich submerged
macrophyte.
1
0
0
10 20 30 40 50 60 70 80 90 100 110 120
Operation time (days)
Fig. 4. Time course of CH4 production rate of alkali pre-treated
and un-treated P. maackianus in semi-continuous treatment and
theoretical dissolved lignin concentration in the digestate of alkali
pre-treated condition.
SUMMARY
The present study conducted anaerobic digestion of submerged
macrophytes and clarified the biomethane potential, effect of
alkaline pre-treatment, and effect of operating temperature. Key
findings are listed below: The digestibility of submerged
macrophytes greatly fluctuated with species and was limited by
the lignin content. Alkaline pre-treatment significantly enhanced
the lignin removal and CH4 recovery of lignin-rich macrophyte.
However, the pre-treatment by-product “dissolved lignin”
inhibited AD especially hydrolysis process at high concentration.
During semi-continuous operation of pre-treated macrophyte,
CH4 production was temporary inhibited by dissolved lignin but
acclimatized in approximately 50 days. All of these results
indicated that alkali pre-treatment is essential for AD of ligninrich macrophyte, and the removal of dissolved lignin would
further improve the CH4 recovery.
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Fernandes et al. (2009) Bioresource Technol 100 (9): 2575–2579. Frigon
et al. (2012) Biomass Bioenerg 36: 1–11. Funaoka (1998) Polym. Int.
47(3): 277–290. Grabber et al. (1997) J Agr Food Chem 45: 2530–2532.
Gunaseelan (2007) Bioresource technol 98 (6): 1270–1277. Haga and
Ishikawa (2011) Jpn J Limnol 72: 81–88. Kayembe et al. (2013) Br.
Microbiol. Res. J. 3(1): 32–41. Palonen et al. (2004) J. Biotechnol. 107(1):
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Triolo et al. (2011) Bioresource technol 102 (20): 9395–9402. Veeresh et
al. (2005) Water Res. 39(1): 154–170. Wang et al. (2010) J Environ Eng
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