Boosting biogas yield of anaerobic digesters by utilizing concentrated molasses from 2nd generation bioethanol plant

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I
NTERNATIONAL
J
OURNAL OF

E
NERGY AND
E
NVIRONMENT



Volume 4, Issue 2, 2013 pp.199-210

Journal homepage: www.IJEE.IEEFoundation.org


ISSN 2076-2895 (Print), ISSN 2076-2909 (Online) ©2013 International Energy & Environment Foundation. All rights reserved.
Boosting biogas yield of anaerobic digesters by utilizing
concentrated molasses from 2
nd
generation bioethanol plant


Shiplu Sarker
1
, Henrik Bjarne Møller
2


1
Department of Renewable Energy, Faculty of Engineering and Science, University of Agder, Grimstad-
4879, Norway.
2
Department of Biosystems Engineering, Faculty of Science and Technology, Aarhus University,
Research center Foulum, Blichers Allè, Post Box 50, Tjele-8830, Denmark.


Abstract
Concentrated molasses (C
5
molasses) from 2
nd
generation bioethanol plant has been investigated for
enhancing productivity of manure based digesters. A batch study at mesophilic condition (35±1°C)
showed the maximum methane yield from molasses as 286 LCH
4
/kgVS which was approximately 63%
of the calculated theoretical yield. In addition to the batch study, co-digestion of molasses with cattle
manure in a semi-continuously stirred reactor at thermophilic temperature (50±1°C) was also performed
with a stepwise increase in molasses concentration. The results from this experiment revealed the
maximum average biogas yield of 1.89 L/L/day when 23% VS
molasses
was co-digested with cattle manure.
However, digesters fed with more than 32% VS
molasses
and with short adaptation period resulted in VFA
accumulation and reduced methane productivity indicating that when using molasses as biogas booster
this level should not be exceeded.
Copyright © 2013 International Energy and Environment Foundation - All rights reserved.

Keywords: Molasses; 2
nd
generation bio-ethanol plant; Anaerobic digesters; Biogas yield.



1. Introduction
The overwhelming dependence on fossil fuel and the escalating greenhouse gas emissions are the two
concerns heavily contributing in rearranging most of the energy policies worldwide. In response to that,
European energy council, has set a target of 20% renewable energy in proportion of total energy
consumption and 10% bio-fuels in proportion of total fuel consumption by the year 2020 [1]. Coupled
with policies and regulations, technologies encompassing renewable energy have also been diversified.
Deploying lignocellulosic biomass for the production of bioethanol (2
nd
generation bioethanol) [2], is one
example in that direction.
Conventionally, ethanol as a vehicle fuel is produced from different sources of biomass(sugar cane, corn,
gain, rice etc), predominantly containing lower and higher carbohydrates [3]. Bioethanol plants dealing
with biomass rich in sucrose and starch are termed as first generation plants [2]. Although majority of the
World’s ethanol is processed in first generation bio-ethanol plants, their negative impact to the
environment has recently been brought into serious consideration. Competition with food or feed for
fertile land and thereby increasing food prices is one of the long lasting dilemmas in regards of 1
st

generation ethanol industries [4]. Issues like eutrophication and acidification caused by high energy
fossil fuel input for fertilization of ethanol feedstocks are also believed as the outcome of such ethanol
International Journal of Energy and Environment (IJEE), Volume 4, Issue 2, 2013, pp.199-210
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200
plants [5]. Avoiding these limitations yet maintaining continuously rising ethanol demand is a challenge
to combat for which alternative solution is necessary. Second generation bio-ethanol plant (primarily
based on agricultural and industrial residues), is potentially offering the solution of these burning issues
of food or fuel while providing opportunities for the treatment of low value wastes and therefore is
expected to play a vital role in the coming years.
Rooted in the notion of 2
nd
generation bio-ethanol plant, from the year 2003 and onward Inbicon A/S,
Denmark developed the EU project idea of Integrated biomass utilization system (IBUS) [6] to convert
lignocellulosic biomass into bio-ethanol. Since inception, extensive effort has been paid for the further
improvement of its different aspects and now reached to the edge of a commercial reality [6]. Principally,
the Inbicon A/S plant produces bio-ethanol from wheat straw by five processing steps i) pre-treatment ii)
hydrolysis iii) fermentation iv) distillation and v) separation and uses solely steam and enzyme for the
entire process. Pre-treatment, as an important part of this process itself is divided into two lines where
one line is operating with lower capacity (100 kg biomass/hour) for the purpose of research, in contrast
with the other with a higher capacity (1000 kg biomass/hour) for the purpose of mechanical
development.
C
5
molasses as a by-product resulted from two of the above process streams. It is obtained as a residue
either after pre-treatment or after separation. Characteristically, C
5
molasses is different depending on the
point they are originated and on the qualities of the wheat straw it was derived from. Molasses originated
after the pre-treatment unit was concentrated by the evaporation of water to enrich in dry matter content
and used for this study. Previously, molasses was primarily used for animal feeding. But considering its
storage potential and high degradability, it is recently exploited for anaerobic digestion also. Anaerobic
digestion of molasses with a low dry-matter content of 4.4% that derived from the processing stream as
described by Thomsen et al, 2008 was documented by Kaparaju et al, 2009 [7]. However, biogas
production from molasses with a very high dry matter content (58%) has not been investigated before to
the present knowledge of the authors.
Substrate with high dry-matter content is generally suitable for co-digestion which treats two or more
materials with complementary attributes. Despite several advantages that include higher biogas
production, lesser inhibition as well as higher buffering [8], the successful adoption of co-digestion
strategy is challenged by the issue of scarcity of concentrated biomass that can be stored and utilized all
around the year to meet the seasonal variation in energy demand. Biogas plant connected with CHP
(combined heat and power plant) is typically designed for base load due inadequacy of the material
characterized to boost the energy production when peak load is demanded. Generally, peak load is met
from other source of energy often in fossil fuel nature. However, major effort has strongly been applied
to substitute this concept and by displacing fossil fuel from the fuel renewable in nature. Considering
this, the feasibility of utilizing concentrated C
5
molasses for biogas production and short term boosting of
methane yield was examined in semi-continuously fed reactors. Together, the methane potential of C
5

molasses was measured in batch study.

2. Materials and methods
2.1 C
5
molasses
C
5
molasses, a by-product of a bio-ethanol industry [6], was obtained from a second generation bio-
ethanol demonstration plant (Inbicon A/S, Kalundborg, Denmark) and used as a substrate for co-
digestion in this study. It contains a high amount of oligosaccharides and sugars due to the breakdown of
hemicelluloses during processing of input biomass (wheat straw). The physical and chemical properties
of molasses (C
5
molasses) are given in Table 1.

2.2 Dairy cattle manure
Dairy cattle manure (DCM) was obtained from slurry reception tank at Research Center Foulum,
Denmark, during February until March 2011. The average properties of slurry, collected several times
during the experimental period, were: pH=7.7±0.5; Total Nitrogen =3.6±0.6%; Total Solid (TS) =
8.7±0.6, Volatile Solid (VS) =7.5±0.3 and Total ammonia nitrogen (TAN) =1.91±0.2 respectively.

2.3 Inoculum
Two types of inoculum was used for this study, thermophilic inoculum for the continuous reactors and
mesophilic inoculum for the batch reactors. Effluent from main digester of biogas plant at research center
Foulum (Denmark) was employed as thermophilic inoculum. The main digester operates at thermophilic
International Journal of Energy and Environment (IJEE), Volume 4, Issue 2, 2013, pp.199-210
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201
temperature (50±1.0°C) and treats various materials such as pig manure, cattle manure, maize silage and
industrial wastes together. Average TS, VS, pH and TAN of thermophilic inoculum was measured as
3.6±0.5% , 2.2±0.5%, 8.3 and 0.5g/L respectively. Mesophilic inoculum, on the other hand, was
collected from the same facility however from post digester tank where the digested slurry from the main
reactor had been stored at a temperature of 35±0.5°C for further de-gasification. The properties of
mesophilic inoculum when measured were TS=2.83±0.5%, VS=1.43±0.5%, pH= 8.1±0.4 and
TAN=1.82±0.5 g/L respectively.

Table 1. Properties of C
5
molasses

Properties Amount
Density (L/kg) 1.3
pH 4.2
TS (% w/w) 58.1
VS (% w/w) 43.0
Ash (% of TS)
a
26.0
Ash (% of TS)
b
19.0
Lipids (g/kg TS) 0.197
VFA (g/l) 25.4
Acetate (g/l) 24.8
Propionate (g/l) 0.412
Total Nitrogen (g/kgTS) 5.6
Protein (g/kgTS)
3
35.0
Total P (g/kg TS) 1.3
Klason Lignin (g/kg TS) 4.3
Furfurals (g/l) <0.5
5HMF (g/l) <0.5
Phenols (g/kgTS) 0.88
Dietary fiber (g/kg TS) 54.0
Carbohydratres (mg/kg TS) 286.0
Fructose (g/kg TS) 1 (1
1
, 0
2
)
Arabinose (g/kg TS) 18 (18
1
, 0
2
)
Xylose (g/kgTS) 132 (82
1
, 50
2
)
Glucose (g/kgTS) 111 (56
1
, 51
2
)
Mannose (g/kgTS) 14 (7
1
, 7
2
)
Galactose (g/kgTS) 11 (10
1
, 1
2
)
Potassium (K
+
) (g/kgTS) 32.0
Chloride (Cl
-
) (g/kgTS) 6.3
Sodium (Na
+
) (g/kgTS) 48.2
Magnesium (Mg
2+
) (g/l) 1.5
Calcium (Ca
2+
) (g/l) 4.5
Phosphorous (P
2+
) (g/l) 1.3
Copper (Cu
2+
) (g/l) 0
Manganese (Mn
4+
) (g/l) 0.044
Zinc (Zn
2+)
(g/l) 0.03
Iron (Fe
3+)
(g/l)
0.92
1
the monosaccharides present in sugar analysis
2
the polysaccharides present in sugar analysis
3
protein = 6.25 x total nitrogen
a
incomplete evaporation of water from the sample analysed
b
complete evaporation of water from the sample analysed

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202
2.4 Methane potential of C
5
molasses
The biological methane potential of molasses (C
5
molasses) was estimated by batch assay as described
by Møller et al. [9] and complied with the international standard ISO 11734 (1995) . The triplicate batch
tests were conducted in 500 ml total volume infusion bottles for two different substrate concentrations.
The inoculum to substrate ratio (VS/VS) for those two concentrations was 1:0.67 and 1:1.33 respectively
which was prepared by adding 10 gram of molasses in 200 grams of inoculum for one set of bottles and 5
gram of molasses in 200 grams inoculum for other set of bottles. After inoculation, the glass bottles were
flushed with pure N
2
for 5-10 minutes. The bottles were then closed with butyl rubber stoppers and
sealed with aluminium screw tops and incubated for 90 days at 35±0.5°C. The assays with inoculum,
typically defined as control, were also prepared to determine the biogas production from inoculum alone.
Methane and biogas production from the batch tests were periodically measured, by using water
displacement method, ten times in total during the whole incubation period. Water is acidified in the
water displacement method (Figure 1) to reduce CO
2
solubility. To determine actual potential, produced
methane from the samples was corrected from that produced by the inoculum alone. Theoretical methane
yield (m
3
/kgVS) was calculated on the basis of stoichiometric conversion of organic matter to methane
and carbon dioxide as given below [7]:

()
ropionateAcetateLipidsroteinstesCarbohydra
ropionateAcetateLipidsroteinstesCarbohydra
B
u
Ρ+++Ρ+
Ρ+++Ρ+
=
530.0373.0014.1496.0415.0



Figure 1. Water displacement method and measurement of biogas

2.5 Reactor experiment
The experiment was conducted by parallel running of two continuous reactors (CR), each with a capacity
of 10 liters and 7 liters working volume, operated with 17 days hydraulic retention time (HRT). Reactors
was stirred manually during feeding and collection of effluent and placed in an incubator where the
temperature was maintained at 50±1°C.
Both the reactors were filled with 6.6 kg inoculum and 0.4 kg cattle manure during start-up. R(CM) was
operated as a reference (control) reactor and was run with cattle manure throughout the experimental
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203
period. R(CM+M), on the other hand, was the tested reactor undergone for mixed feeding of C
5
molasses
and cattle manure. The experiment start-up (period 1, days 0-21) where R(CM) & R(CM+M) were fed
with DCM alone so that stable performance between the reactors was achieved (average data is presented
in Table 2). The stabilization period of this experiment is in accordance with other study, exemplified as
maximum 5% variation of biogas production between the reactors [10].
After stabilization, molasses was introduced to R(CM+M) and between day 22 to day 38 (period 2), 10
grams of molasses was added with 390 grams of cattle manure. This, in terms of added VS, corresponded
to the feeding ratio of 13:87 (VS
molasses
: VS
cattle manure
) representing 11% increase (4.3 to 4.8 gVS/L/d) in
total OLR (Table 2). In the following period (day 39-51), the concentration of C
5
molasses was doubled
where 20 grams of molasses was combined with 380 grams of cattle manure so that the feeding ratio in
terms of added VS reached as 23:77 (VS
molasses
: VS
cattle manure
). This consequently raised the total OLR to
23% (5.3 gVS/L/d) from the start. C
5
concentration was further increased and each day in period 4 (day
52-71) 30 grams of molasses was mixed with 370 grams of cattle manure which simultaneously changed
the corresponding feeding ratio to 32:68 (VS
molasses
: VS
cattle manure
) and resulted the increase of total OLR
close to 35% (5.8 gVS/L/d) since the experiment was started. The entire feeding scheme from period 1
until period 4 was maintained for 17 days HRT (Table 2) by keeping total feeding and total extraction of
materials from the digesters at the same volume.
Feeding was carried out once in a day by pouring substrate through the opening of a hollow tube which
extends below the liquid level in order to prevent air trapping in the headspace. The opening was
normally sealed by a rubber stopper before and after the feeding. Effluent was collected from the other
opening at the lower end of the reactor wall which was also kept sealed except the instances when
materials were removed. The Process performance was monitored by analyzing TS, VS, pH, VFA, gas
production and gas composition of effluent and raw-materials on a regular interval.

Table 2. Governing parameters of Continuous reactor experiment (thermophilic, 50°C)

R1 R2 R1 R2 R1 R2 R1 R2
Feed ratio (VS molasses :VS CM)
0:100 0:100 0:100 13:87 0:100 23:77 0:100 32:68
OLR of molasses (gVS/L/d) 0 0 0 0.61 0 1.23 0 1.84
Total influent OLR (gVS/L/d) 4.3 4.3 4.3 4.8 4.3 5.3 4.3 5.8
Biogas production (mL/day) 8230 8640 8954 10201 10754 13261 10164 12584
Biogas production (mL/gVS) 275 282 299 304 358 358 339 309
HRT (days) 17 17 17 17 17 17 17 17
Methane yield (mL/L/d) 694 728 755 845 906 1080 857 971
Methane yield (mL/gVS) 162 164 177 176 212 204 202 165
Methane composition, % 59 59 59 58 59 57 59 54
VFA (g/L) - - 0.42 0.5 0.5 1.22 0.94 3.47
TAN (g/L) 1.9 1.85 1.98 2 2 2.02 2.1 2
pH 8.03 8.05 8.16 8.24 8.02 8.06 8.08 8.05
Days of operation
0-21 22-38 39-52 53-70Parameters


2.6 Analytical methods
TS of cattle manure and C
5
molasses was measured after drying samples for 24 hours at 105°C. The
dried samples were further heated at 550°C for 5±0.5 hours to determine ash content. VS was calculated
by subtracting the amount of ash from the amount of TS [11]. pH was measured by using a glass pH
probe (Knick Portamess, 911 pH, Germany) while total nitrogen was determined by using the standard
Kjeldahl method [12] and a Kjell-Foss 16200 auto analyzer (Foss Electric, Hillerød, Denmark).
For volatile fatty acid (VFA) analysis, 1mL of sample was acidified with 4mL of pivalic acid and then
centrifuged for 10 minutes at 12,000 rpm and afterwards filtered with 0.45µm filter before measuring on
Gas Chromatograph (Hewlett Packard 6850A, USA) equipped with a flame ionization detector (FID) and
HP-INNOWax column with a dimension of 30m x 0.25 mm x 0.25µm. The temperature of the column
was gradually increased from 110°C to 220°C at the rate of 10°C/min. Helium (He) was used as carrier
gas at a flow rate of 10 mL/min. Total ammonia nitrogen was analyzed colorometrically at 690®nm with
Merck spectrophotometer (NOVA 60).
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204
The produced biogas was accrued in aluminium coated plastic bags which were connected with the
reactors through plastic tubes. Each reactor was joined with one aluminium coated plastic bag to
facilitate gas collection and for subsequent volume and composition measurement. Collected gas was
measured (for volume) on a daily basis by using acidified water displacement method. Gas samples were
analysed (for composition) twice a week both for CO
2
and CH
4
content with a gas chromatograph (Perkin
Elmer Clarus 500, USA) equipped with a Thermal Conductivity Detector and a Turbomatrix 16
Headspace auto sampler as described by Møller et al [9]. Methane and carbon dioxide was separated by
using a 12’ x 1/8” Haysep Q 80/100 column. The temperature of the injection port, oven and detector
were 110, 40 and 150°C respectively. Helium (He) was used as a carrier gas with a flow rate of 30
mL/min.
Sugar (monosaccharides, polysaccharides, oligosaccharides etc), dietary fibres and klason lignin was
analysed as described by Knudsen [13] whereas mineral analysis (primarily cations and anions) was
conducted as according to Fang et al [14]. Fat was determined by using Danish standard infrared
spectrometry method (DS/R 209:2006) whereas 5-Hyroxymethylfurfural (5-HMF), furfural and phenol
were measured by adopting inductively coupled plasma (ICP) method.

3. Results and discussions
3.1 Characteristics of C
5
molasses
The physical and chemical characteristics of the studied molasses are presented in Table 1. The molasses
was a by-product of the hydrothermal treatment (in 2
nd
generation bioethanol plant) of wheat straw and
mainly composed with C
5
- and C
6
sugars and alkali chlorides that was resulted from the lignocellulosic
component of biomass. After analysis of molasses, average TS, VS, pH and total VFA content were
recorded as 58%, 43%, 4.4±0.2 and 1.2 g/L respectively.
Sugar analysis revealed that the majority of sugars were xylose followed by glucose and arabinose (Table
1). Although not abundant, small amount of other sugars such as rhamnose (<1g/kgTS), mannose and
galactose were also observed. Furthermore, analysis quantified approximately 0.5 g/L of 5-HMF (5-
Hydroxymethylfurfural) (Table 1) that probably resulted from the degradation of hexose during
hydrothermal pre-treatment [15]. The presence of 5-HMF in molasses was considered inhibitory to
several microbial activities (enteric microorganisms) for fermentation [16] but favourable for anaerobic
digestion when concentration is kept below 3 g/L [17].
Based on the chemical analysis, the concentration level of cations Mg
2+
,Zn
2+
,Fe
2+
or Fe
3+
, P
2+
,Cu
2+
,Mn
4+

(Table 1) were found to be well within the acceptable ranges [14]. However, the concentration of other
cations such as Na
+
, K
+
, Ca
2+
(Table 1) was exhibited to be higher than the values previously published
for normal molasses [14]. Na
+
, Ca
2+
, Mg
2+
together with ammonia was reported [18] to present
antagonistic behaviour for reducing ion inhibition. Fang et al. 2011 [14] however documented that
sodium and potassium concentration of approximately 11 and 28 g/L jointly would be responsible for
50% methane inhibition whereas over 5g/L concentration of Ca
2+
was found [19] as inhibitory for
methanogenesis. These altogether are the potential reasons negatively influencing the choice of molasses
with high dry-matter content as a substrate for mono-anaerobic digestion. However, co-digestion of
molasses with cattle manure avoided some of these limitations, as demonstrated elsewhere in the present
study.

3.2 Biological methane potential
The effect of molasses concentrations on biological methane potential is depicted by Figure 2. Methane
yield was influenced by substrate concentration. For instance, where the maximum methane yield as an
effect of low substrate concentration (24.4 gVS/L) was 286.3 L/kgVS, for the addition of higher
concentration (34.1 gVS/L) it was 278.2 L/kgVS. Although the difference of these two yields was only
about 8 L/kgVS (Figure 1), the pattern by which they developed was surprisingly different and
characterized by the long lag phase due to the higher input of C
5
. As illustrated in Figure 2, batch bottles
fed with lower substrate concentration (24.4 gVS/L) gave approximately 86% of methane between day 0
to day 42 (246.42 L/kgVS) which gradually reached to maximum at day 93 (286.3 L/kgVS). On the
contrary, experiment with higher feeding concentrations yielded merely 14% (39.9 L/kgVS) of methane
between day 10 to 42 which thereafter swiftly rocketed to 278.2 L/kgVS until the end of the experiment
at day 93.
Kaparaju et al [20] observed the similar phenomena, however, for a dissimilar experimental conditions
where diluted wheat straw stillage (10.2% VS) was incubated (55°C) for about 65 days. High
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205
concentration induced inhibition was also reported in several other research works such as by [21]. The
low methane yield at high substrate concentration is possibly because of less dilution of inhibitory
compounds that present in C
5
molasses. The long adaptation period of bacteria, on the other hand, was
presumably responsible for the evolution of greater initial lag phase (Figure 2) when higher
concentration feeding was adopted.
Calculation of experimental methane yield from batch assays revealed that approximately 65.3% of
theoretical yield (438.39 L/kgVS as calculated) was realised, implying incomplete conversion partly due
to the recalcitrant nature of organic contents or partly because of substrate and product inhibition as
according to Mösche et al, 1999 [22].

0
10
20
30
40
50
60
70
80
90
0
50
100
150
200
250
300
0 20406080100
Methane concentration (%)
Cumulative methane, ml/gVS
Time (Days)
I:S=1:1.33
I:S=1:0.67
I:S=1:0.67
I:S=1:1.33


Figure 2. Cumulative methane yield vs methane concentration in batch experiment. Open triangles and
closed circles are for methane concentration while black and gray lines are for cumulative methane yield

3.3 Continuous reactors (CR) experiment
The volumetric and weight based biogas and methane yields are demonstrated by Figure 3(a) & (b) while
the effect of VFA and pH on feeding concentration is illustrated by Figure 4.
Co-digestion started at period 2 (day 22-38) when R (CM+M) was supplemented with molasses. There
was an increase of about 14% daily volumetric yield of biogas (10.2 L/d) due to the addition of
0.61gVS/L/d molasses. The gas yield in terms of added volatile solid however was around 2% lower
(p>0.05, n=17) than that from R(CM) (Figure 3(b)). Similar trend was observed for methane
concentration which dropped approximately 1% (p>0.05, n=7) (from 59% to 58%; Table 2). Some
accumulation of total VFA occurred in molasses reactor which rose about 19% than those from R(CM)
(Figure 3(b)). pH was in the range of 8.16±0.2 (Figure 4) while average TAN was observed as 2±0.2g/L,
indicating no serious inhibition at this stage.
In the following period (period 3) between day 39 to day 52, the concentration of molasses for
R(CM+M) was further increased (0.61 to 1.23 gVS/L/d) to 23% of total added VS (Table 2) which
yielded approximately 23% higher biogas (Figure 3(a)) than that from R(CM), in terms of unit reactor
volume. In terms of volatile solid, however, identical (p>0.05, n=14) mean biogas yields (358 L/
kgVS
added
) was observed. Generally, in this period, the average gas production from both the reactors
increased, compared to the previous period. Although acceptable for R(CM+M), this implied the
unexpected yielding pattern of R(CM), possibly resulted due to the variation in cattle manure properties
that varied every time fresh manure was collected (three instances for this entire experiment) and stored.
Stored cattle manure was reported to impact other parameters of anaerobic digestion also, such as for
VFA [23]. In respect of methane composition, R(CM+M) was still showing the decreasing trend and
resulted approximately 2% lower concentration (p>0.01, n=5) (Table 2) as compared to R(CM). There
was a dramatic increase in total VFA which jumped to approximately 144% (1.22g/L) in contrast with
control. VFA accumulation is tightly linked to OLR and expected to play a critical role in this period, as
it (OLR) was increased close to 24% (Table 2). Noticeably, the major part of this total VFA in
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206
R(CM+M) was acetic acid (0.64 g/L) that followed by propionic acid (0.51g/L) and trace amount of
higher molecular weight VFAs. Total VFA from control, as increased from the previous period, also
dominated by the concentration of acetic acid (0.40 g/L) and propionic acid (0.08 g/L) (Figure 4). This is
not surprising as total VFA for digestion of cattle manure alone can reach to a range of 0.2- 3.8 g/L for a
fair adaptation period of over 100 days [24]. Since VFA rose, pH for the experimental reactor dropped
(8.02±0.6) from the previous period. Average TAN remained nearly unchanged (2.02±1.0 g/L) although
the variation among the observed data (for TAN) was quite high.



(a) Specific volumetric yield. Closed circles: Biogas yield from R (CM+M); Open triangles: Biogas yield
from R (CM); Dotted line: Methane yield from R (CM+M); Solid line: Methane yield from R(CM)



(b) Specific yield in terms of VS. Closed circles: Biogas yield from R (CM+M); Open triangles: Biogas
yield from R (CM); Dotted line: Methane yield from R (CM+M); Solid line: Methane yield from R(CM)

Figure 3. Specific biogas and methane yield from continuous reactors in terms of volume and volatile
solid addition
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207


Figure 4. Effect of VFA and pH on experimental reactor. Columns are for VFA and the symbols are for
pH. Column with black shades: acetic acid from R (CM); Columns with white shades: propionic acid for
R(CM); Column with light grey: acetic acid from R (CM+M); Columns with dark grey: propionic acid
for R(CM+M). Open triangles: pH of R (CM); Closed circles: pH of R (CM+M)

In period 4, between day 53 to 71 the concentration of molasses for reactor R (CM+M) was finally
augmented (1.23 to 1.84 gVS/L/d ) to 32% of total added VS which stimulated 24% growth of biogas
(Figure 3(a)) in terms of volume. In terms of added volatile solid, however, the yield from R(CM+M)
(309 L/kg VS
added
) approximately reduced to 10 % compared to R(CM) (339 L/kg VS
added
). While
volumetric methane yield was still increasing, methane composition declined and reached to a level
(54%), lowest for the whole experiment. Furthermore, there was approximately fourfold increase in total
VFA (table 2) that unlike the previous period was alternated by the concentration of propionic acid (2.28
g/L) followed by acetic acid (0.85 g/L). The rapid climb in propionic acid along with acetic acid was the
serious indication of process stress with a possibility to complete failure. In fact, propionic acid alone is a
very potential candidate to severely trigger process imbalance [25]. There were several other potential
factors played a significant role to characterize such VFA pattern of R(CM+M). One, for instance, the
lignin decomposition, as a consequence of which lower molecular weight VFA forms during
hydrothermal treatment of upstream biomass (wheat straw in this case) [20]. This was expected for C
5
molasses as the type of process (section 1) it was involved to originate. Moreover, there was an issue of
present feeding strategy where instead of slow increase in molasses OLR (will be published later),
R(CM+M) was tested for sudden OLR rise to achieve optimum boosting of biogas which presumably
had a strong influence on VFA rise too. Based on these VFA facts, the feeding of R(CM+M) beyond this
period was decisively stopped. Meanwhile, the average pH and TAN for molasses reactor showed very
little variation from the earlier periods as their corresponding values in this level was 8.08±0.05 and
2.0±0.2 respectively. For R(CM), on the other hand, the total VFA along with acetic acid and other
compounds exhibited no serious implications as they were tended to stabilize in this period (Figure 4).
As discussed above, throughout the experiment, rise in VFA compounds was serious concern while pH
was fairly safe with apparently stable values (Figure 4). This was probably attributed to the fact that co-
digestion of C
5
molasses with cattle manure facilitated buffering by neutralizing pH at varying substrate
concentrations and thus sustained the process for perceivably higher OLR input. Similar phenomena was
observed by Fang et al. [14] who noticed higher VFA but stable pH for high concentration feeding.
International Journal of Energy and Environment (IJEE), Volume 4, Issue 2, 2013, pp.199-210
ISSN 2076-2895 (Print), ISSN 2076-2909 (Online) ©2013 International Energy & Environment Foundation. All rights reserved.

208
Despite the fact, straining the process with even higher loading beyond the present level of maximum
OLR might lead to a process imbalance causing buffering capacity to deplete and eventually
deteriorating system stability.
All in all, C
5
molasses played a significant role in the yielding pattern of biogas from R(CM+M) which
generally decreased in terms of added VS as molasses concentration was increased. Essentially, the
subsequent increase in substrate concentration was purposefully adopted in order to optimize the daily
boosting of biogas which was well achieved (23% extra biogas yield) in the middle part of the
experiment, with a tolerable VFA and other parameters. However, between the last two periods due to
the sudden rise in C
5
concentration from 23% to 32% of added VS, the system was stressed and
imbalanced as evidenced by the higher VFA accumulation, although volumetric yield of biogas
continued to increase. Boosting biogas in conditions of later part of the experiment as a result of high
concentration feeding hence should not be replicated in commercial scale biogas digesters, as indicated
by this work.

4. Conclusions
Improvement in productivity of anaerobic digesters together with sustainable utilization of 2
nd
generation
bioethanol plant product are the two potential benefits the present study revealed. The maximum biogas
yield of 358 L/kgVS (1.3 L/L/d) was obtained for the continuous reactor experiment with a total organic
loading rate of 5.3 gVS/L/day beyond which the process was rather unstable. Utilizing C
5
molasses
above 5.3 gVS/L/day of total OLR, or, in other words above 23% concentration of molasses VS,
therefore, is not recommended when the adaption period is shorter.

Acknowledgements
The authors of this study greatly acknowledge Inbicon (Dong energy A/S), Kalundborg, Denmark for the
delivery of raw-materials throughout the experiment and EUDP for the financial assistance. The
technical supports and the lab equipments from the Foulum Biogas plant was also an essential and
integral part of this research and therefore are appreciated with high note.

References
[1] E. Commision, Biofuels in the European Union- a vison for 2030 and beyond. Director- General
for Reasearch Sustainable Energy Systems (2006), in, 2006.
[2] M.O.S. Dias, T.L. Junqueira, C.D.F. Jesus, C.E.V. Rossell, R. Maciel Filho, A. Bonomi,
Improving second generation ethanol production through optimization of first generation
production process from sugarcane, Energy, 43 (2012) 246-252.
[3] K. Öhgren, A. Rudolf, M. Galbe, G. Zacchi, Fuel ethanol production from steam-pretreated corn
stover using SSF at higher dry matter content, Biomass and Bioenergy, 30 (2006) 863-869.
[4] T.R. Society, Sustainable biofuels: prospects and challenges, in, 2008.
[5] J P. Lange, Lignocellulose conversion: an introduction to chemistry, process and economics,
Biofuels, Bioproducts and Biorefining, 1 (2007) 39-48.
[6] J. Larsen, M. Østergaard Petersen, L. Thirup, H. Wen Li, F. Krogh Iversen, The IBUS Process –
Lignocellulosic Bioethanol Close to a Commercial Reality, Chemical Engineering & Technology,
31, (2008) 765-772.
[7] P. Kaparaju, M. Serrano, A.B. Thomsen, P. Kongjan, I. Angelidaki, Bioethanol, biohydrogen and
biogas production from wheat straw in a biorefinery concept, Bioresource Technology, 100 (2009)
2562-2568.
[8] J. Mata-Alvarez, J. Dosta, S. Macé, S. Astals, Codigestion of solid wastes: A review of its uses
and perspectives including modeling, Critical Reviews in Biotechnology, 31 (2011) 99-111.
[9] H.B. Møller, S.G. Sommer, B.K. Ahring, Methane productivity of manure, straw and solid
fractions of manure, Biomass and Bioenergy, 26 (2004) 485-495.
[10] I.H. Varel VH, Bryant MP., Thermophilic Methane Production from Cattle Waste, Applied and
Environmental Micrology 33 (1977) 298-307.
[11] H.B. Møller, A.M. Nielsen, R. Nakakubo, H.J. Olsen, Process performance of biogas digesters
incorporating pre-separated manure, Livestock Science, 112 (2007) 217-223.
[12] APHA, American Public Health Association (APHA). in, 1995.
[13] K.E.B. Knudsen, Carbohydrate and lignin contents of plant materials used in animal feeding,
Animal Feed Science and Technology, 67 (1997) 319-338.
International Journal of Energy and Environment (IJEE), Volume 4, Issue 2, 2013, pp.199-210
ISSN 2076-2895 (Print), ISSN 2076-2909 (Online) ©2013 International Energy & Environment Foundation. All rights reserved.

209
[14] C. Fang, K. Boe, I. Angelidaki, Anaerobic co-digestion of desugared molasses with cow manure;
focusing on sodium and potassium inhibition, Bioresource Technology, 102 (2011) 1005-1011.
[15] R. Boopathy, H. Bokang, L. Daniels, Biotransformation of furfural and 5-hydroxymethyl furfural
by enteric bacteria, Journal of Industrial Microbiology & Biotechnology, 11 (1993) 147-150.
[16] M. Thomsen, A. Thygesen, H. Jørgensen, J. Larsen, B. Christensen, A. Thomsen, Preliminary
results on optimization of pilot scale pretreatment of wheat straw used in coproduction of
bioethanol and electricity, Applied Biochemistry and Biotechnology, 130 (2006) 448-460.
[17] J H. Park, J J. Yoon, H D. Park, D.J. Lim, S H. Kim, Anaerobic digestibility of algal bioethanol
residue, Bioresource Technology, 113 (2012) 78-82.
[18] Y. Chen, J.J. Cheng, K.S. Creamer, Inhibition of anaerobic digestion process: A review,
Bioresource Technology, 99 (2008) 4044-4064.
[19] J H. Ahn, T.H. Do, S.D. Kim, S. Hwang, The effect of calcium on the anaerobic digestion treating
swine wastewater, Biochemical Engineering Journal, 30 (2006) 33-38.
[20] P. Kaparaju, M. Serrano, I. Angelidaki, Optimization of biogas production from wheat straw
stillage in UASB reactor, Applied Energy, 87 (2010) 3779-3783.
[21] R. Sierra-Alvarez, J.A. Field, S. Kortekaas, G. Lettinga, Overview of the anaerobic toxicity caused
by organic forest industry wastewater pollutants, Proc. 4th IAWQ Symp. Forest industry
wastewaters. Tampere, Finland (1993). Ook: Water Sci. Technol. 29 (1994) 353-363.
[22] M. Mösche, H J. Jördening, Comparison of different models of substrate and product inhibition in
anaerobic digestion, Water Research, 33 (1999) 2545-2554.
[23] N.K. Patni, P.Y. Jui, Volatile fatty acids in stored dairy-cattle slurry, Agricultural Wastes, 13,
(1985), 159-178.
[24] C. Rico, H. García, J.L. Rico, Physical–anaerobic–chemical process for treatment of dairy cattle
manure, Bioresource Technology, 102 (2011) 2143-2150.
[25] H.B. Nielsen, H. Uellendahl, B.K. Ahring, Regulation and optimization of the biogas process:
Propionate as a key parameter, Biomass and Bioenergy, 31 (2007) 820-830.



Shiplu Sarker is a PhD research fellow at the University of Agder and is currently involved in the
research of a small scale Combined Heat and Power (CHP) fuelled by gasified biomass and integrate
d
with a gas engine. He has a master in Sustainable Energy Engineering from The Royal Institute of
Technology (KTH), Sweden. Over the years, he has been engaged in research at different directions that
include Computational Fluid Dynamics (CFD) at the University of Udine, Italy; Biochemical process
Engineering at the University of Aarhus, Denmark. He also has a number of years of experience to wor
k
as a Mechanical Engineer in Bangladesh and is a member of (Institute of Engineers’, Bangladesh) IEB.
E-mail address: shiplu.sarker@uia.no




Henrik Bjarne Møller is head of the biogas research group at department of Engineering Aarhus
University. PhD from Danish Technical University in 2003.Dr. Moller’s main expertise: New designs
of biogas plants to improve performance, pre-treatment of biomass, pre-treatments by thermo-chemical
and enzymatic techniques, utilisation of new biomasses as energy crops and crop residues and process
control by varies indicators especially VFA by GC or titration.
E-mail address: henrikb.moller@agrsci.dk