Applied Energy 87 (2010) 37793783

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Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Optimization of biogas production from wheat straw stillage in UASB reactor

Prasad Kaparaju 1, Mara Serrano, Irini Angelidaki *
Institute of Environment & Resources, Technical University of Denmark, Building 115, DK-2800 Kgs. Lyngby, Denmark

a r t i c l e

i n f o

Article history:
Received 17 February 2010
Received in revised form 1 June 2010
Accepted 3 June 2010
Available online 3 July 2010
Keywords:
Bioethanol
Biogas
Lignocellulose
Stillage
Wheat straw

a b s t r a c t
In the present study, thermophilic anaerobic digestion of wheat straw stillage was investigated. Methane
potential of stillage was determined in batch experiments at two different substrate concentrations.
Results showed that higher methane yields of 324 ml/g-(volatile solids) VSadded were obtained at stillage
concentrations of 12.8 g-VS/L than at 25.6 g-VS/l. Continuous anaerobic digestion of stillage was performed in an up-ow anaerobic sludge blanket (UASB) reactor at 55 C with 2 days hydraulic retention
time. Results showed that both substrate concentration and organic loading rate (OLR) inuenced process
performance and methane yields. Maximum methane yield of 155 ml CH4/g-COD was obtained at stillage
mixtures with water of 25% (v/v) in the feed and at an OLR of 17.1 g-COD/(l.d). Soluble chemical oxygen
demand (SCOD) removal at this OLR was 76% (w/w). Increase in OLR to 41.2 g-COD/(l.d) and/or stillage
concentration in the feed to 3350% (v/v) resulted in low methane yields or complete process failure.
The results showed that thermophilic anaerobic digestion of wheat straw stillage alone for methane production is feasible in UASB reactor at an OLR of 17.1 g-COD/(l.d) and at substrate concentration of 25% in
the feed. The produced methane could improve the process energy and economics of a bioethanol plant
and also enable to utilize the stillage in a sustainable manner.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
Bioethanol is the most widely used alternative automotive biofuel in the world. The worlds largest fuel ethanol producers are
Brazil and the USA. Of the global fuel ethanol production of 67 billion liters, USA and Brazil produced approximately 34 and 27 billion liters, respectively in 2008 [1]. Fuel ethanol is produced
mainly from sugar cane in Brazil and from corn in the USA. On
the other hand, EU produced close to 2.8 billion liters of ethanol
for fuel in 2008 [1].
The production of ethanol from biomass, whether from sugar
crops (sugar beets, sugar cane, molasses, etc.), starch crops (corn,
wheat, rice, cassava, etc.), dairy products (whey) or cellulosic materials (straw, energy crops, bagasse, wood, or municipal solid
waste), results in the concurrent production of stillage. Stillage,
also termed as distillery wastewater, distillery slops, distillery
spent wash and thin stillage, is an aqueous by-product from the
distillation of ethanol following fermentation of carbohydrates.
Stillage production volume has been estimated to be 1020 times
that of ethanol produced and has a considerable pollution potential
[2,3]. Depending upon the ethanol process and stillage treatment,

* Corresponding author. Tel.: +45 45251429; fax: +45 45932850.

E-mail addresses: prakapa@jyu. (P. Kaparaju), ria@er.dtu.dk (I. Angelidaki).
1
Present address: Department of Biological and Environmental Science, P.O. Box
35, University of Jyvskyl, FI 40014, Finland. Tel.: +358 142604263; fax: +358
142601021.
0306-2619/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apenergy.2010.06.005

the chemical oxygen demand (COD) load of the liquid phase of stillages can range from 10 to 100 g O2/l [see reviews e.g. 2,4]. Thus,
disposal of the untreated stillage raises serious problems due to
its high pollution potential.
Traditionally, stillage has been used as animal feed either in the
form of distillers dried solubles or distillers dried grains with solubles (DDGS). However, DDGS production consumes a lot of energy
owing to the evaporation and drying processes. For instance, DDGS
production consumes approximately the same amount of energy
as that of the entire bioethanol production process [5]. Besides drying of the stillage, the bioethanol production process has additional
high energy consuming steps, such as distillation. Therefore, reduction of the process energy consumption is of crucial importance
[4,6,7]. Several efforts are underway to improve process economics
and environmental benets of ethanol production. Several solutions for the treatment, utilization and disposal of stillage have
been reviewed [see e.g. 2,4]. Treatment and disposal measures included aerobic treatment, evaporation and neutralization of stillage with alkali, charcoal production, pyrolysis and/or various
means of fertilizer production. Alternatively, anaerobic digestion
is considered as an attractive sustainable environmental technology for simultaneous treatment (COD removal) and renewable energy production from the stillage [4]. The produced biogas can be
used in the bioethanol plant, as heat or electricity for evaporation
and distillation processes and/or also saves the energy required for
aerobic/anaerobic treatment of stillage. However, to qualify the
production of ethanol as a sustainable green energy process, due

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P. Kaparaju et al. / Applied Energy 87 (2010) 37793783

consideration for treatment and utilization of the thin stillage is

essential.
Several studies on anaerobic treatment of ethanol grain or
starch stillage as an effective treatment has been cited in the literature [812]. During anaerobic digestion of stillage, a signicant
portion (>50%) of COD can be converted to biogas [13]. For instance, the anaerobic treatment of wheat grain stillage of an initial
COD ranging from 91 to 107 g O2/l resulted in 90% [14] to 98% [9]
COD removal efciencies. Similarly, 80% COD removal efciency
was reported during the treatment of grain distillation wastewater
with an initial COD level of 2030 g O2/l in an up-ow anaerobic
sludge bed (UASB) reactor [10]. However, high levels of potassium
[15], metals [16] and sulphate [17] have been demonstrated to inhibit anaerobic digestion of stillage. For instance, cane molasses
stillage with a COD of over 100 g/l was found to inhibit stable
digestion and dilution to a COD of around 50 g/l using other waste
streams at the plant improved the digestion [18]. Considerable
amount of research has been conducted on anaerobic digestion
of ethanol stillage from conventional feedstocks, especially grain
and cane molasses [4,8,19]. However, use ethanol stillage produced
from enzymatic fermentation of hydrothermally pretreated wheat
straw [20] for biogas production was never attempted. In the present study, the anaerobic digestion of wheat straw stillage in an
UASB reactor was investigated at 55 C.
2. Materials and methods
2.1. Substrates
2.1.1. Wheat straw stillage
Wheat straw stillage was used as substrate for biogas production. Stillage was obtained from a laboratory-scale bioethanol production process using wheat straw. Wheat straw was
hydrothermally pretreated in a pilot plant (100 kg/h capacity) at
the rate of 120150 kg-dry matter (DM)/h and fed in a countercurrent with a water ow rate of 400600 l/h [20]. Two fractions
resulted from the hydrothermal pre-treatment, a liquid fraction,
mainly consisting of pentoses, and a solid fraction mainly consisting of hexoses [20]. After enzymatic hydrolysis, fermentation of the
solid ber fraction was performed by Bakers yeast (Saccharomyces
cerevisiae) [21]. Ethanol was distilled away by vacuum distillation,
resulting in the stillage used in the successive experiments.
2.1.2. Pig manure
Filtered pig manure was obtained from a centralized biogas
plant (Hegndal Biogas plant, Denmark). The untreated ltered pig
manure had a total solids (TS) and volatile solids (VS) content of
4.8% and 3.6%, respectively. The total COD and soluble COD values
were 70 g/l and 26 g/l, respectively with an ammonium nitrogen
content of 4.8 g/l. Feed was prepared by diluting the ltered pig
manure with water (1:1).
2.1.3. Inoculum
Thermophilic granules from a potato-processing wastewater
treatment plant (Kruiningen, The Netherlands) were used as inoculum (organic matter content of 0.08 g/g sludge). In addition, basic
anaerobic medium containing vitamins, carbonate buffer, phosphate, ammonium and trace metals as described by Angelidaki
et al. [22] was used as synthetic media in UASB reactor.
2.2. Biological methane potential
Biological methane potential assays were performed according
to Angelidaki et al. [23]. The experiments were performed in
118 ml serum glass bottles with working volume of 40 ml. To each

bottle, 30 ml of inoculum and 10 ml of substrate at a substrate concentration of 12.8 or 25.6 g VS/l was added. The headspace in the
bottle was ushed with pure N2 for 35 min, before adding 23
drops of sodium sulde to ensure anaerobic conditions. The prepared bottles were then sealed with rubber stoppers and aluminium crimps, and incubated statically at 55 C. The experiment
was conducted in triplicates. Assays with inoculum alone were
used as controls. Methane produced from inoculum was subtracted
from the sample assays.
2.3. UASB experiments
A lab-scale glass UASB reactor (334 ml) was operated with a
working volume of 255 ml and HRT of 48 h. Reactor temperature
was maintained at 55 C by circulating hot water in the heat jacket.
The prepared feed, stored at 10 C, was fed continuously (24 times
per day, 5.3 ml per time) at a feed rate of 128 ml/d. The efuent
was collected in a separate bottle.
During the start-up, the reactor was inoculated with 100 ml of
anaerobic granular sludge. During the initial run, ltered and diluted pig manure was fed at an OLR of 2.26 g-COD/l d and HRT of
48 h (days 015). After reaching the steady-state, stillage, diluted
with water and BA-Media was carefully introduced. The feed
contained wheat straw stillage diluted with water (1:9 v/v) and basic anaerobic medium (1:1 v/v). OLR during this period was gradually increased in a step-wise manner from the initial 2.4 to nal
19 g-COD/l d (days 1665). When methanogenesis was inhibited,
pH dropped signicantly and the process was recovered by adding
sodium bicarbonate at 5 g/l reactor volume. Each OLR was operated until a steady-state condition, characterized by a constant
gas production rate (+5%), was noticed. Operating conditions are
shown in Table 2. Process performance was followed by analysing
pH, VFA, gas production, gas composition and COD removals. Samples were collected at regular intervals depending upon the load
change.
2.4. Analytical methods
The produced biogas owed to a tube placed at the top of the
reactor and was measured with a 100 ml step gas counters [20].
Gas samples were taken through the sampling port located on
the top of the reactor while efuent samples were collected near
the reactor outlet.
pH was measured using pH meter (PHM92 LAB). TS, VS, and
suspended solids and ammonium nitrogen and total Kjeldahl nitrogen (TKN) were determined according to the Standard Methods
[24]. TCOD and SCOD were analyzed according to the Danish Standards [25]. SCOD samples were ltered through glass ber lter
paper (U90 mm, GF50, Schleicher and Schuell). Lipid extraction
was carried out through Soxhlet Method [26]. Phenolic compounds
were quantied by gas chromatograph (GC) equipped with ame
ionization detector (FID) as described elsewhere [27]. Compounds
were rstly isolated from the liquid fraction at pH 2 by solid-phase
extraction on polystyrene divinylbenzene polymer columns.
Methane content in biogas was determined by GC tted with a
FID and Porapak 60/80 mol sieve column [20]. Volatile fatty acids
(VFAs) and alcohols were determined by GC (Hewlett Packard,
HP 5890 series II) equipped with a FID detector and HP FFAP column [20].
Sugars (glucose, xylose and arabinose) content in raw and solid
fraction fractions were determined by strong acid (72% w/w H2SO4)
hydrolysis and in liquid fraction by weak acid (4% w/w H2SO4) [21].
Sugars were quantied on high performance liquid chromatography (HPLC) system HP 1100 (Agilent 1100) equipped with a BioRad
Aminex HPX-87 H and a refractive index (RI) detector (RID 1362A)
as described elsewhere [27]. Klason lignin in solid fraction was

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P. Kaparaju et al. / Applied Energy 87 (2010) 37793783

determined as the weight of the lter cake (generated during the

strong acid hydrolysis of solid fraction) from subtracted the ash
content. Furfural and 5-hydroxymethyl-2-furaldehyde (5-HMF)
were quantied using a HPLC for sugars analysis tted with ultraviolet (UV) detector (G1314A) as described elsewhere [27].

were below the concentrations reported to cause inhibition on

the microorganisms [30]. Interestingly, 4-hydroxyacetophenone,
reported to affect biogas process [30], was also detected in low concentration. Total sugars analyses revealed that glucose was the major component accounting for almost 60% of sugars.

2.4.1. Calculations
Theoretical methane yield (m3/kg-VS) was calculated based on
the stoichiometric conversion of organic matter to methane and
carbon dioxide as follows [20]:

3.2. Methane potential of wheat straw stillage

Bo:th

The effect of substrate concentration on biological methane potential was shown in Fig. 1. The experiment was carried out for

0:415 Carbohydrates 0:496 Proteins 1:014 Lipids 0:373 Acetate 0:530 Propionate
Carbohydrates Proteins Lipids Acetate Propionate

3. Results and discussion

3.1. Substrate characteristics
The composition of the studied stillage is presented in Table
1. The stillage was collected at the end of fermentation of solid
fraction (28% TS) of the hydrothermally pretreated wheat straw.
Ethanol was extracted through vacuum distillation to 10% ethanol concentration. The ethanol content in the stillage was
2.3 g/l (Table 1). TS and VS contents were 12.0% and 10.2%,
respectively with a pH of 3.6. The low value of pH was of a concern as the optimal range for methanogenesis is between 6.5 and
8 [28]. Acetate, the main VFA, and NH
4 -N levels were below 1
and 2 g/l, respectively. These concentrations of VFA and NH
4 -N
were reported to cause inhibition [29].
The high COD values noticed in the present study were within
the range reported for similar stillages [4]. The concentrations of
furfurals and 5-HMF were below the detection limit while presence
of lignin-related compounds was detected in a concentration of
0.061 g/l. The main phenolic compounds and its precursors in the
stillage were acetovanillone (AcV), ferulic acid and syringic acid
(data not shown) and were detected in concentrations of 0.01,
0.01 and 0.012 g/l, respectively. These concentrations however

Table 1
Characterization of wheat straw stillage. (Standard
deviations on triplicate samples.)
Parameter

Concentration

pH
TS (%)
VS (%)
Ash content (%)
TSS (g/l)
VSS (mg/l)
TCOD (g/l)
SCOD (g/l)
VFA (g/l)
Ethanol (g/l)
TKN (g/l)
NH
4 -N (g/l)
Proteins (g/l)
Lipids (%)
Carbohydrates (g/l)
Furfurals (g/l)
HMF (g/l)
Phenols (g/l)
Lignin (g/l)
Arabinose (g/l)
Xylose (g/l)
Glucose (g/l)

3.6 0.1
12.0 0.03
10.2 0.03
1.8 0.03
1.4 0.2
69.1 2.5
150 3.59
61 4.36
0.18 0.02
2.3 0.13
1.4 0.02
0.16 0.01
7.7 0.09
0.99
84.5
N.D.
N.D.
0.061
75.6
0.00
6.9
10.3

N.D. Not detected.

44 days. Methane production started immediately in all assays and

maximum methane production was noticed after 20 days of incubation (Fig. 1). However, the concentration of substrate affected the
methane production rates and yields. Lower methane production
rates were noticed in assays incubating at 25.6 g-VS/l than at
12.8 g-VS/L (Fig. 1). Maximum methane yield of 324 ml/g-VSadded
was obtained at a substrate concentration of 12.8 g-VS/l. This value
was lower than the theoretical yield of 479 ml/g-VSadded calculated
from the COD content. The low methane yield at high substrate concentration was probably due to inhibition caused by lignin compounds with low molecular weight or other intermediates.
Previous studies have also shown that phenolic compounds and its
intermediates were utilized as a sole substrate at low concentration
while at high concentrations induced inhibition [31,32]. Moreover,
dilution of substrate has reduced the toxicity of potential inhibitors.
In a similar study, anaerobic digestion of cane molasses stillage was
inhibited at high COD level (>100 g/l) while dilution with other
waste streams (50 g/l) did not cause any process inhibition [18].
3.3. Continuous anaerobic digestion of stillage
The results of continuous anaerobic digestion of stillage in UASB
reactor are presented in Table 2 and Fig. 2. The mean biogas production during anaerobic digestion of pig manure alone was
145.3 ml/g-COD with a SCOD removal of 57% (w/w) (Table 2). From
day 15 onwards, reactor was fed with stillage (diluted with BA
medium). Results showed that stillage concentration in the feed
and/or OLR affected the process and methane yields. Maximum
methane yield of 155 ml CH4/g-COD was obtained at a substrate
concentration of 25% (v/v) and an OLR of 17.1 g COD/(l.d) (days

Table 2
Operating conditions and process performance during semi-continuous anaerobic
digestion of wheat straw stillage in UASB reactor at 55 C.
Substrate

Days
Stillage concentration
HRT (h)
OLR (g COD/(l.d))
COD removal (%)
Biogas production
(ml/mlfeed)
Methane yield
(ml-CH4/g COD)
Methane content (%)
VFA (g/l)
pH

Filtered and Steps

diluted pig
1
2
3
manure
Wheat straw stillage

015

48
2.3
56.9
2.1

1638
5%
48
9.7
63.4
2.8

3944
10%
48
12.4
76.8
4.7

4551
25%
48
17.1
75.9
8.3

5255
50%
48
41.2
31.5
7.8

5665
33%
48
19.0
23.5
2.8

145.3

100.0

127.2

154.8

27.0

61.6
0.64
8.1

70.2
0.29
7.2

67.2
0.09
7.2

63.7
0.21
6.8

21.8
1.35
5.4

0
0.52
4.8

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P. Kaparaju et al. / Applied Energy 87 (2010) 37793783

300

200

100

0
0

10

20

30

40

50

60

Time (d)
Fig. 1. Methane yield during anaerobic digestion of wheat straw stillage incubated
at substrate concentrations of 12.8 g-VS/l (h) and 25.6 g-VS/l (s) in assays at 55 C
compared to theoretical methane yield (-).

4551) with a maximum SCOD removal of 76% (w/w). However,

the methane yields in the present study were much lower than
the theoretical methane yield normalized to 350 ml CH4/g-COD.
In a similar study, Laubscher et al. [10] reported 80% COD removal
efciency during the treatment of grain distillation wastewater in
an UASB reactor with an initial COD concentration of 2030 g O2/l.
An increase in OLR to 41.2 g COD/(l.d) or stillage concentration to
50% in the feed resulted in a corresponding decrease in biogas production and methane yield (days 5255). The SCOD removals were
less than 30% and resulted in a very low methane production. Decrease in OLR to 19 g COD/(l.d) but at a slightly high stillage concentration in the feed (33%) however resulted in complete
process failure (days 5665). This was evident from the accumulation of VFA and drop in pH (Fig. 2). The possible reason for process

failure at an OLR of 19 g COD/(l.d) was due to accumulation of compounds of low molecular weight, originating from lignin decomposition formed during pre-treatment. A similar process failure due
to accumulation of lignin-related compounds in UASB reactor
was reported during anaerobic digestion of bioethanol efuents
[30].
Several Laboratory investigations have reported COD removal
efciencies of 7095% during anaerobic digestion of various stillages including wheat stillage [14], barley stillage and sweet potato
stillage [19], sweet potato stillage [9], potato stillage [8] and sugar
beet stillage [4]. The higher COD removal efciencies without any
process inhibition in the above studies compared to present study
is due to the fact that these studies were performed with the stillage obtained from the fermentation of starch or grain and did not
involved any high temperature and/or pressure pre-treatment of
biomass. On the other hand, the stillage in the present study was
obtained after fermentation of hydrothermally pretreated lignocellulosic waste (wheat straw). The pretreated biomass showed to
contain inhibitory compounds such as lignin-related phenolic
compounds and sugar-related HMF and furfurals [20,30,33]. In
addition, high levels of potassium [15], metals [16] and sulphate
[17], not determined in the present study, were shown to inhibit
anaerobic digestion of stillage.
The results of the present study in practice suggest that anaerobic digestion of wheat straw stillage in UASB reactor is feasible.
However, recirculation of reactor efuent would provide good mixing and thereby facilitate good contact of substrate and nutrients
to microbes and thereby facilitate the degradation of such compounds. Furthermore, an increase in HRT may provide sufcient
time for methanogens to mineralize the organic matter to methane
and carbon dioxide. For instance, accumulation of hydrogen in the
reactor was detected at the end of the experiment as hydrogen was
not utilized by the hydrogenotrophic methanogens. The produced
energy could provide additional energy within a biorenery process, where biomass is used for multi-product generation. For instance, lignin in the solids fraction of stillage is traditionally used
as solid biofuel in a bioethanol plant [20]. Moreover, the energy re-

Stillage

Control

10%

Methane Yield (mL/g-COD)

5%

25%

50%

33%

50

300

40
200

30
20

100

10
0

12

OLR (g-COD/L.d)

Methane yield (ml/gVS)

400

pH

VFA (g/L)

10
2

6
4

10

20

30

40

50

60

70

Time (days)

Fig. 2. Process performance during anaerobic digestion of wheat straw stillage in UASB reactor at 55 C. (s) Methane yield; (-) Organic loading rate; (s) VFA; ( ) pH.

P. Kaparaju et al. / Applied Energy 87 (2010) 37793783

quired for pre-treatment of lignocellulosic material such as straw

in a 2nd generation bioethanol production process could also be
supplemented. If this technology is applied on full-scale plants
then, the efciency of 2nd generation bioethanol plant no longer
depends on the market for DDGS but on the energy market, where
the prices are expected to keep increasing.
4. Conclusions
The present study indicates that anaerobic digestion of wheat
straw stillage alone for biogas production is feasible in UASB reactor at an OLR of 17.1 g COD/(l.d) and at substrate concentration of
25% in the feed. Maximum methane yield of 154.8 ml CH4/g-COD
could be obtained with a SCOD removal of 76% (w/w). Increase in
OLR to 41.2 g-COD/(l.d) and/or substrate concentration to 3350%
in feed would lead to lower methane yields or complete process
failure.
Acknowledgements
Authors thank Hector Garca, Jens Schaarup Srensen and Asst.
Prof. Markus Emili from Technical University of Denmark for setting up the experiments and furfural analyses. Tomas Fernqvist
and Ingelis Larsen from Ris National Laboratories (Denmark) are
greatly acknowledged for the help rendered during sugars and
phenols analyses. The study was supported by the Danish Innovation and Research Council, DSF project No. 2104-06-0004.
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