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Anaerobic digestion of maize and cellulose underthermophilic and mesophilic conditions e Acomparative study
Katarzyna Golkowska*, Manfred Greger
University of Luxembourg, Faculty of Science, Technology, and Communication, 6, rue Richard Coudenhove-Kalergi,
1359 Luxembourg, Luxembourg
a r t i c l e i n f o
Article history:
Received 12 September 2011
Received in revised form
18 April 2013
Accepted 28 May 2013
Available online 4 July 2013
Keywords:
Anaerobic digestion
Cellulose
Maize silage
Thermophilic
Mesophilic
Batch
Abbreviations: DS, dry solids; DSK, correctbutyric acid; n-HBu, n-butyric acid; HPr, proloading rate; ORP, oxidation-reduction potenacids; VS, volatile solids; VSK, corrected vola* Corresponding author. Present address: Pu
avenue des Hauts-Fourneaux, L-4362 Esch-sE-mail addresses: katarzyna.golkowska@
0961-9534/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2013.05.
a b s t r a c t
The aim of this study was to advance in understanding of digestion process of energy
crops. Cellulose and maize silage were fermented in batch mode at mesophilic (38 �C) and
thermophilic (55 �C) conditions and corresponding organic loads of 5.5 � 0.2 kgVS/m3,
11.2 � 0.3 kgVS/m3 and 16.7 � 0.4 kgVS/m3.
For both substrates more stable and faster digestion took place at 38 �C. Due to complex
structure maize degradation was characterized by varying digestion rate and longer total
digestion time resulting form breakdown of hard-degradable fractions. The digestion retard at
increasedOLRs of celluloseand lowerdegradation level obtained for all cellulose series confirm
a higher overloading potential for systems dealingwith single-component-substrates but also
the enhanced sensitivity of such systems to any inconvenient digestion conditions.
Based on observed patterns of volatile fatty acids and oxidation-reduction potential,
different fermentation mechanisms can be concluded for cellulose and maize, but also for
different temperature modes. Conversion of maize at highly reductive conditions with
increased concentrations of butyric acid was accompanied by much higher activity of
hydrogenotrophic methanogens than for cellulose digestion.
Two factors showed a strong potential to influence test results: an insufficient VS content
of inoculum, which caused reduced biogas yields, and a high natural biodiversity of maize
silage, resulting in higher biogas yields than calculated based on the maize composition.
ª 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The global energy demand is forecasted to grow by more than
one-third until 2035. However the energy demand of OECD
and thus of most European countries is barely anticipated to
rise. In the coming years a shift from oil, coal (and nuclear)
ed dry solids; DSN, uncorpionic acid; iso-HVa, iso-tial; sGP, specific biogas ptile solids; VSN, uncorrecblic Research Centre Henur-Alzette, Luxembourg.tudor.lu (K. Golkowska), mier Ltd. All rights reserved029
towards natural gas and renewable energy sources is expected
on the European energy market [1]. Such development has
been promoted by the Renewables Directive of the EU [2] and
in some countries (e.g. Germany or Switzerland) additionally
triggered by the Fukushima Daiichi nuclear disaster from
2011. For that reason the enhanced political and financial
rected dry solids; FM, fresh mass; HAc, acetic acid; iso-HBu, iso-valeric acid; n-HVa, n-valeric acid; OL, organic load; OLR, organicroduction; sGPR, specific biogas production rate; VFA, volatile fattyted volatile solids.ri Tudor, Resource Centre for Environmental Technologies, 6A,
Tel.: þ352 42 59 91 4433; fax: þ352 42 59 91 [email protected] (M. Greger)..
b i om a s s an d b i o e n e r g y 5 6 ( 2 0 1 3 ) 5 4 5e5 5 4546
support for renewable energy and in particular for biogas in-
dustry has been provided in many European countries. As a
result the annual primary biogas production in the EU
increased from 8.4 Mtoe in 2009 to 10.1 Mtoe in 2012 [3,4]. In
particular the fermentation and co-fermentation of energy
crops has developed rapidly in recent time. Use of agricultural
feedstock and waste allows constructing more compact and
therefore more efficient installations and improving perfor-
mance of the biogas plants digestingmainlymanure. The new
energy approach shifts the main focus of the agricultural
biogas plants from disposing of manure to energy production.
This change has an additional social dimension: a traditional
farmer can become energy producer and has a possibility to
profit from the new developments on the energy market.
The predominant number of biogas plants nowadays is run
mesophilic (36e38 �C) [5], while thermophilic digestion mode
(55 �C) is widespread mainly in Scandinavian countries. Most
of the findings related to anaerobic digestion are based on the
experiments conducted with waste water (e.g. [6e9]), manure
(e.g. [10e12]) and solid waste (e.g. [13e15]) or refer to co-
digestion of these substrates (e.g. [16e19]). Though, not
many papers have been published on mono-digestion of en-
ergy crops by now [20e28]. Similar to the industrial trends,
mesophilic fermentation was better described in former
publications, while thermophilic digestion has been mainly a
subject of more recent studies. However, hardly any publica-
tions comparing experiments conducted in the similar way
under thermophilic and mesophilic conditions can be found.
Originally it was assumed that the degradation pathways
and the performance of process parameters for all types of
anaerobic digestion must be similar to that observed for
digestion of waste water and manure, independent of the
substrate, temperature mode or the source of the inoculum.
Themethane was believed to bemainly produced via propionic
acid (HPr) and acetic acid (HAc) [29e33]. The last findings reveal
that this is not always the case [24,34,35]. Many aspects of
anaerobic digestion of energy crops, such as impact of micro-
nutrients, the exact microbiological composition of bacterial
biocenosis, or degradation pathways, have not been researched
by now and together with advanced modeling constitute the
core of the current research.
The aim of this study was to advance in understanding of
digestion process of energy crops. Since most of the experi-
ments concentrate on one operating temperature, no studies
comparingperformanceof thebacterial biocenosis fordifferent
temperatures and (OLs) loads under similar experimental
conditions can be found. This study investigating mono-
fermentation of a model substrate (cellulose) and agricultural
substrate (maize silage) under mesophilic and thermophilic
temperature conditions should help to close this research gap.
2. Material and methods
2.1. Inoculum
Inoculum for thermophilic (55 �C) tests was obtained from
continuously operated 50-l thermophilic plug-flow research
fermenter fed with maize and grass silage mix at organic
loading rate (OLR) of 1.5 kgVS/(m3 d). The uptime of the reactor
before inoculum sampling amounted to one year. Mesophilic
inoculum (38 �C) was provided by an industrial biogas plant
from Beckerich (Luxembourg). This unit consists of three
CSTRs with the working volume of 1500 m3 each, which are
under operation since 2004. The reactors are run at OLR of
2.5 kgVS/(m3 d) and fed with manure (25,000 tonnes/a), maize
silage (5400 tonnes/a), grass silage (1000 tonnes/a) as well as
corn whole-crop-silage (800 tonnes/a).
Both inocula were prepared following the guidelines for the
fermentation of organicmatter [36]. Before the beginning of the
first experiments both inocula were adapted to cellulose or
maize respectively. The adaptation process was conducted in
the similar way as the later experiments. The inoculum was
placed in fermenters fed with maize or cellulose at OLs of
4e5 kgVS/m3. After digestion period of ca. 2 weeks (or shorter if
the biogas production ceased) the feeding process was
repeated and the digestion continued for 2e6 weeks to remove
the residual degradable components. The inocula from all
thermophilic and mesophilic reactors were remixed (sepa-
rately for each temperature mode) to receive homogenous
start conditions and filtered through a kitchen strainer (2 mm
mesh size). The homogenous filtrate was used to inoculate all
reactors of the same experimental series. The subsequent
batch series (with increasing OLs) were performed with the
inoculum retrieved from the previous experiment to achieve
better bacterial adaptation to increasedOLs. The step including
inoculum homogenization and filtration was repeated before
every newOLwas investigated. The volatile solids (VS) content
of inoculum ranged between 1.75 and 2.71% of freshmass (FM)
and 54e60% of dry solids (DS), except for thermophilic test
with maize at 5.7 kgVS/m3, in which the VS reached 0.59% of
FM and 59% of DS. The substrate to inoculum VS ratios
measured for all the experimental series are given in Table 1.
The initial pH of the inoculum in different experimental
series, together with the final pH values reached after the
digestion, are summarized in Table 1. For all trials the pHvalues
ranged from neutral to slightly alkali conditions (6.98e8.60).
2.2. Substrate
Microcrystalline cellulose powder of pharmaceutical grade
was purchased from Euro OTC Pharma GmbH (Bonen, Ger-
many). For maize degradation commercial maize silages of
two harvests were applied: MZ I e for thermophilic batches,
MZ II e for mesophilic batches. The ensilaged maize was cut
into w5 mm fibers, stored frozen and defrosted at low tem-
peratures (4 �C) for 24h before charging of the fermenters. Both
silages were characterized by Van Soest andWeende analysis
[37,38]. Further calculation of corrected DS content (DSK) of the
substrate was done according to [39]. The DS content was
increased by the amount of volatile compounds lost through
volatilization during DS determination according to Eq. (1),
where: DSNemeasured DS value and DSKecorrected DS value.
DSK½%� ¼ 2:22þ 0:960 DSN½%� (1)
The compositions of cellulose and maize silages corrected
according to Eq. (1) are presented in Table 2. Lignin was
considered as a non-degradable component of maize. There-
fore the total theoretical biogas yield was calculated according
to [40] based on the substrate composition but excluding the
Table 1 e Summarized experimental data for digestion of maize and cellulose under mesophilic and thermophilicconditions.
Temp. Substrate OL[kgVS/m3]
VSsubstrate/inoculum
Total biogasyield
[lN/kgVS]
Total CH4
yield[lN/kgVS]
% of max.possible
biogas yield
pH ORP [mV]
Startvalue
Finalvalue
Min.valuea
Max.valuea
D
38 �C Cellulose 5.4 0.30 633 � 17 323 � 5 84 7.80 7.39 �322 �181 141
10.9 0.61 680 � 6 326 � 10 90 7.60 7.40 �329 �262 67
16.3 0.91 700 � 8 343 � 3 93 7.62 7.54 �332 �244 88
Maize 5.5 0.28 646 � 12 388 � 6 97 8.22 7.46 �380 �358 22
11.0 0.55 647 � 51 382 � 29 97 8.27 7.35 �361 �340 21
17.1 0.67 690 � 5 380 � 9 103 7.60 7.46 �386 �352 34
55 �C Cellulose 5.7 0.33 605 � 27 333 � 15 80 8.12 8.04 �399 �343 56
11.4 0.47 656 � 36 348 � 19 87 8.27 8.11 e e e
17.1 0.71 654 � 15 340 � 30 87 8.43 7.96 e e e
Maize 5.7 0.97 550 � 17 328 � 15 87 7.70 6.98 �417 �332 85
11.5 0.64 706 � 6 402 � 16 106 8.60 7.61 �417 �369 48
17.3 0.96 680 � 8 381 � 3 102 7.92 7.84 �428 �369 59
a After reaching anaerobic conditions.
b i om a s s a n d b i o e n e r g y 5 6 ( 2 0 1 3 ) 5 4 5e5 5 4 547
lignin content. The calculated theoretical biogas yield from
the silages amounted to 742 lN/kgVS for MZ I, 743 lN/kgVS for
MZ II and 827 lN/kgVS for cellulose.
Similar to [36] and [41] the maximal expected biogas yield
was calculated by reducing the theoretical biogas yield by 10%.
This followed the assumption of biogas loss due to both mi-
crobial growth (ca. 5%) and undigested carbon residues left in
the effluent (ca. 5%). As a result themaximum expected biogas
yields of 752 lN/kgVS and 669 lN/kgVS were calculated for
cellulose and for MZ I and MZ II respectively.
2.3. Experimental set up
The experimental set up similar to [42] was applied in the
study independent of the substrate or temperature mode. For
Table 2 e Composition of maize and cellulose withoutand with corrections of dry solids (DS) and volatile solids(VS) content according to [39] (DSN, VSNenot correctedvalues; DSK, VSKecorrected values).
Component Unit Cellulose Maize
MZ I MZ II
DSN % FM 100 33.0 32.5
VSN % FM 98 31.7 31.3
DSK % FM 100 33.9 33.4
VSK % FM 98 32.6 32.2
VSK % DSK 98 96.0 96.5
Crude ash 2 4.1 3.3
Crude protein e 6.7 7.3
Crude fat e 3.2 2.8
NFC e 43.2 49.4
NDF e 40.1 34.4
*ADF e 26.3 22.3
*Lignin/ADL e 3.5 3.5
*Hemicellulose e 13.8 12.0
*Cellulose 98 22.8 18.8
VFA e 2.7 2.8
* Subfractions of NDF.
each substrate and temperature mode 3 similar OLs were
investigated: 5.5 � 0.2 kgVS/m3, 11.2 � 0.3 kgVS/m3 and
16.7 � 0.4 kgVS/m3. The number of experimental reactors
within one test series (the same temperature, substrate and
OL) ranged between 12 and 18. For higher OLs the digestion
period was longer. Consequently more reactors were neces-
sary to capture all process stages. The experiments were
conducted in 1 l fermenting bottles filled with 700 g inoculum
and fed with a predefined substrate amount. Each reactor was
opened only once for the sampling purposes. Once stopped,
the reactor was not used in the further study to prevent the
disruption of the digestion process by the air inflow.
Depending on the OL 6e13 reactors were sampled and used to
characterize different stages of the process. Based on the
homogenized content of the reactors the following volatile
fatty acids (VFA) were measured: HAc, HPr, iso-butyric acid
(iso-HBu), n-butyric acid (n-HBu), iso-valeric acid (iso-HVa)
and n-valeric acid (n-HVa). Additional reactors were used to
monitor pH and oxidation-reduction potential (ORP). Due to
the equipment unavailability ORP could not be recorded for 2
out of 3 experiments run with cellulose under thermophilic
conditions.
Daily and total mean gas production together with the
statistical significance of these values were calculated based
on biogas production received form 2 biogas reactors.
Measured biogas volumes were converted into standard
temperature and pressure conditions (273.15 K, 1013.25 hPa).
Background biogas production was measured in a separate
reactor run with inoculum only and subsequently subtracted
from the total biogas production of the substrate digesting
fermenter. The total biogas yield given in Table 1 includes this
correction.
3. Results & discussion
3.1. Biogas production
The total specific biogas production (sGP) measured accumu-
lative over the test period is depicted in Fig. 1, while Fig. 2
0
0.2
0.4
0.6
0.8
to
tal s
GP
[lN
/g
VS
]
maize 38°C
5.5 kgVS/m3
11.0 kgVS/m3
17.1 kgVS/m3
0
0.2
0.4
0.6
0.8
to
tal s
GP
[lN
/g
VS
]cellulose 38°C
5.4 kgVS/m3
10.9 kgVS/m3
16.3 kgVS/m3
0
0.2
0.4
0.6
0.8
0 3 6 9 12 15 18
to
tal s
GP
[lN
/g
VS
]
time [d]
cellulose 55°C
5.7 kgVS/m3
11.4 kgVS/m3
17.1 kgVS/m3
0
0.2
0.4
0.6
0.8
0 3 6 9 12 15 18
to
tal s
GP
[lN
/g
VS
]
time [d]
maize 55°C
5.7 kgVS/m3
11.5 kgVS/m3
17.3 kgVS/m3
Fig. 1 e Time progress of specific biogas production (sGP) for all experimental series.
b i om a s s an d b i o e n e r g y 5 6 ( 2 0 1 3 ) 5 4 5e5 5 4548
presents specific biogas production rate versus time (sGPR).
For displaying sGPR daily gas production volumes were used.
The total biogas yields and methane yields are given in Table
2. Both sGP and sGPR proceeded untypical for maize at OL of
5.7 kgVS/m3 in thermophilic mode, which is associated with
too low VS content of the inoculum (s. Section 3.2). This series
is not included in the further trend analysis in this chapter.
3.1.1. Degradation trends linked to the substrate3.1.1.1. Methane content. Methane content in biogas produced
from maize was slightly higher than for cellulose (s. Table 1).
This is due to different chemical composition of maize
0.00
0.05
0.10
0.15
0.20
0.25
sG
PR
[lN/(d
*g
VS
)]
cellulose 38°C
5.4 kg VS/m3
10.9 kg VS/m3
16.3 kg VS/m3
0
0.05
0.1
0.15
0.2
0.25
0 3 6 9 12 15 18
sG
PR
[lN/(d
*g
VS
)]
time [d]
cellulose 55°C
5.7 kgVS/m3
11.4 kgVS/m3
17.1 kgVS/m3
Fig. 2 e Time progress of daily measured specific bioga
containing 10% of ingredients (3% fats and 7% proteins), which
deliver more methane than carbohydrates during digestion
process [36].
3.1.1.2. Degree of substrate degradation. Considering the sta-
tistical uncertainty of the results, nearly equal total biogas
yields were observed at 38 �C and 55 �C for maize and cellu-
lose. However higher theoretical biogas yields have been
calculated for cellulose in comparison tomaize (s. Section 2.2).
This means that a higher fraction of maize than cellulose was
degraded independent of the temperature mode and OLs.
Despite higher substrate complexity and lower (55 �C) or
0.00
0.05
0.10
0.15
0.20
0.25
0 3 6 9 12 15
sG
PR
[lN/(d
*g
VS
)]
time [d]
maize 38°C
5.5 kgVS/m3
11.0 kgVS/m3
17.1 kg VS/m3
0
0.05
0.1
0.15
0.2
0.25
0 3 6 9 12 15 18
sG
PR
[lN/(d
*g
VS
)]
time [d]
maize 55°C
5.7 kgVS/m3
11.5 kg VS/m3
17.3 kgVS/m3
s production rate (sGPR) for all experimental series.
b i om a s s a n d b i o e n e r g y 5 6 ( 2 0 1 3 ) 5 4 5e5 5 4 549
comparable (38 �C) VS content of the inoculum, over 92% of
maize silage was degraded during the tests. In the experi-
ments with microcrystalline cellulose only 77e89% of sub-
strate was digested. Such low degradation level reached for a
one-component homogenous substrate (cellulose) together
with the observed lag phase (not perceivable for maize) could
not be explained by insufficient bacteria adaptation to the
cellulose digestion. Comparable substrate degradation level
and the lag phase were observed in all cellulose batch tests
conducted by the same authors [42], even though similar to
the current study the already well adapted inocula were used
(s. Section 2.1). Since the inocula from different sources where
applied for cellulose digestion depending on the operating
temperature (s. Section 2.1), the lower degradation level was
either not attributed to the characteristics of the inoculum or
both inocula did not fulfill the conditions required for opti-
mum cellulose degradation.
According to the literature bothmicro andmacro nutrients
are particularly important for regulation of the degradation
velocity and bacterial activity in thermophilic mode
[8,20,22,26e28,43]. Since the nutrients were not analyzed in
the experiment, their shortage cannot be excluded as a po-
tential source of suppressed substrate degradation. Such
deficiency would be even more probable for cellulose diges-
tion as this substrate consists of carbon, oxygen and hydrogen
only and, unlike for maize silage, contains no further nutri-
ents. Moreover, due to the inoculum origin and higher micro
element demand, the shortage of nutrients would rather be
expected for thermophilic than mesophilic inoculum.
Since ammonia content was not controlled in the inoc-
ulum the inhibition effect caused by ammonia [44] cannot be
excluded, although it is less probable given that no elevated
pH values were observed. Furthermore it is not clear whether
the inhibitory effects similar to those reported in the above
mentioned literature may already occur if only 6e12 batches
were prepared with the same inoculum. This question cannot
be answered without analyzing nutrient content and
ammonia concentration during such experiment.
3.1.1.3. Degradation rate. The maximum sGPR (s. Fig. 2) was
always measured for maize independent of the fermenting
temperature and was observed on the first day of digestion.
The main digestion activity was measured for maize between
days 1 and 6, while for cellulose between days 2 and 9 inde-
pendent of the temperaturemode andOL. In spite of inoculum
adaptation all the cellulose experiments began with a 1-day
lag phase. For cellulose the sGPR maxima occurred later than
formaize but the enhanced valuesweremeasured for a longer
time period. For maize an additional increase of sGPR on day
12 was observed. The degradation of maize was faster in the
first stage but the end biogas production was reached later
than for cellulose. Changes in sGPRs during digestion ofmaize
were linked to its more complex composition. Different in-
gredients of maize degraded with different rates causing both
prolongation of total degradation time and additional local
sGPR maxima. The first and highest sGPR maximum can be
correlated with the degradation of easily accessible substrates
(non-fibrous carbohydrates), while the last and smallest peak
was linked to decomposition of not easily degradable fractions
such as hemi cellulose [42,45,46]. Moreover, the multi-peak
sGPR trend was less pronounced for mesophilic conditions,
which can be explained by partial overlap of degradation pe-
riods for different maize components.
3.1.1.4. Influence of the organic load. For maize degradation
there were no considerable differences observed between sGP
patterns for different OLs, except for 5.7 kgVS/m3 under
thermophilic conditions. However in this series much lower
total sGP was reached, which influenced the final stage of the
biogas production (s. Fig. 1). For cellulose a clear flattening of
the biogas production curve was observed for the highest OLs
in both temperature modes. Both sGPR and sGP showed a
delay for cellulose degradation at higher OLs. The trend was
pronounced stronger for thermophilic conditions. This means
that the system overloading can be easier reached for single-
component substrates than for more complex ones (consist-
ing of multiply ingredients characterised by different de-
gradability times and patterns). Furthermore the increased
digestion temperature can additionally strengthen the over-
loading effects.
3.1.2. Degradation trends linked to the temperature modeUnlike in the literature [47e52] higher sGPRs were observed at
38 �C (s. Fig. 2). Mesophilic conditions supported faster
degradation, while in thermophilic mode the sGPR curves
flattened. Such effect can be explained by higher concentra-
tion of active mesophilic bacteria. Even if the production of
enzymes in general is faster at 55 �C, the biodiversity of bac-
terial culture included in the thermophilic digestion process is
much lower than for mesophilic conditions and the bacteria
aremuchmore sensible to even small changes in the digestion
conditions. As a result it is possible for mesophilic biocenosis
to reach higher digestion rates in comparison to thermophilic
cultures. Another explanation for lower sGPRs in thermophilic
mode could be a nutrient limitation or possible ammonia in-
hibition (s. Section 3.1.1). No differences in the total CH4 yield
could be linked to different temperature modes.
3.1.3. Excess biogas productionFor certain maize experiments (usually for higher OLs) inde-
pendent whether at 38 �C or 55 �C higher biogas yields were
obtained (102e106%) than calculated maximum possible
values based on the substrate composition. This can be
attributed to: (i) problems with representative maize sam-
pling, (ii) higher degradation level of the substrate, than 90%
assumed according to the literature [36,41] and/or (iii) lower
uptake of substrate for bacterial growth than assumed in the
literature [36,41] (s. Section 2.2).
Similar excess biogas production was observed in the
semi-batch and continuous experimental series conducted by
[53]. In continuous series the higher biogas yields (exceeding
calculated maximum possible values) were achieved, even
though the total organic load during the experiment in semi-
batch mode was similar. The results of [53] suggest that
continuous digestionmay increase the activity of the bacterial
biocenosis which can result in (ii) and (iii). Consequently these
two effects might be the reason for the higher than expected
biogas yields in continuous and semi-batch mode, but rather
not for batch digestion as described in this paper due to lower
bacterial activity than in continuous mode.
b i om a s s an d b i o e n e r g y 5 6 ( 2 0 1 3 ) 5 4 5e5 5 4550
For batch experiments (i) linked to the natural biodiversity
of ensiledmaize harvest are assumed to be responsible for the
higher than expected biogas yields. Calculation of maximum
possiblebiogasyieldswasbasedon the resultsofVanSoestand
Weende analysis conducted for maize silage (twice sampled
and analyzed). However the natural inhomogeneity of maize
silage is very high. Each variation ofmaize composition affects
the maximum expected biogas yield. With the increase of the
substrate volume introduced into system, the increased un-
certainty of maximum expected biogas yield is produced. For
achieving more accurate biogas production results a develop-
ment of substrate homogenizationmethodwould be advisable
e.g. by designing a model substrate similar to composition of
the maize plant [26] or by drying andmilling [54,55]. Such pre-
treatment would help to improve the final representativity of
the results on one hand, but on the other would certainly
reduce the comparability of the results with the data collected
from pilot or industrial installations digesting maize silage.
3.2. Influence of the inoculum
The sGP for maize at 5.7 kgVS/m3 in thermophilic mode pro-
ceeded untypical (s. Fig. 1). Unlike in the other series, 90% of the
biogas production was already reached on the fourth day of
digestion and the final sGP with 585 lN/kgVS (s. Table 2) was
much lower than for all other experimental series. The VS sub-
strate to inoculum ratios of all experimental series with similar
OL of 5.5 � 0.2 kgVS/m3 ranged between 0.28 and 0.33, while for
maize at 5.7 kgVS/m3and55 �C thisvaluewas three timeshigher
(s. Table 2). The changed digestion performance observed for
thermophilicdigestionofmaizeat5.7kgVS/m3wasattributed to
much lower VS content of the inoculum (s. Section 2.1), than
measuredforall otherexperimental seriesandrecommended in
the appliedguideline [36]. A very lowconcentrationofVS caused
a range of serious parameter changes including lower total
biogas production, lower pH range but higher ORP values and
changed sGPR pattern. Similar influence of the inoculum char-
acteristics on the experimental results was also reported in the
literature [36,56,57]. On the other hand [8] suggests that the
characteristics of the inoculum have smaller impact on the
digestion than theavailability of specificnutrients.Since thetest
with5.7kgVS/m3maizeat 55 �Cwastheonlyoneperforming ina
different way and the same inoculum was used for the experi-
ment with higher OLs, the deficiency of nutrients could be
excluded as a reason of lower degradation activity for this trial.
Table 3 e Possible stoichiometric VFA production and degrada
Substrate Product
1 Glucose HAc, CO2, H2 C6H12O
2 Glucose HPr C6H12O
3 Glucose HAc, HPr, CO2 3C6H12
4 Glucose HBu, CO2, H2 C6H12O
5 Palmitic acid HAc, H2 C16H31
6 Acetic acid CH4, CO2 CH3CO
7 Butyric acid HAc, H2 C3H7CO
8 Propionic acid HAc, CO2, H2 C3H7CO
9 CO2, H2 CH4, CO2 4H2 þ
3.3. Reaction pathways
3.3.1. ORP based indicationsThe initial and final ORP conditions togetherwith theminimum
values reached during the tests are summarized in Table 1. For
all experimental series, independent of the applied OL or tem-
perature mode, ORP ranged between �330 mV and �428 mV,
which corresponds to the ideal conditions for acetogenesis and
methanogenesis [58,59]. Only for mesophilic cellulose digestion
the rapid changing ORP between days 0e2 reached even
�181 mV regarded typical for acidogenic and hydrolytic condi-
tions [58]. The final, stable ORP value ranged always between
�300 mV and �360 mV (typical for methanogenesis [47]) inde-
pendent of the substrate and temperature mode.
A difference in the ORP range was observed between cel-
lulose and maize silage digestion. Under mesophilic condi-
tions cellulose was mainly digested within the range higher
than �300 mV. Since ORP values for thermophilic cellulose
digestionweremeasured only for one experimental series, the
comparison with other temperature and substrate series was
impossible. During degradation of maize silage for both tem-
perature modes ORP values between �350 mV and �500 mV
were recorded. Former studies with anaerobic cultures spec-
ified on hydrogen production [60,61] revealed that the ORP
drop from �350 mV to �550 mV was observed in the days of
experiment identified as a period of the highest hydrogen
production. Following these results, it can be assumed that
during digestion of maize such degradation pathways were
chosen, which result in high hydrogen production (Table 3
reactions 1, 4, 5, 7 and 8). The enhanced hydrogen produc-
tion during digestion of maize monomers at mesophilic con-
ditions must have led to the higher activity of the
hydrogenotrophic methanogens. Unlike for maize, the ORP
ranges observed for cellulose lead to the conclusion that the
hydrogen production was not that strong as for maize and
consequently the hydrogenotrophic methanogens must have
also been less active. The analysis of the microbial commu-
nities at different temperatures and for different substrates
would allow confirming the ORP based indications.
3.3.2. VFA based indicationsThe possible stoichiometric reactions of VFA production and
degradation during conversion of carbohydrates and fats
(during digestion of proteins amino acids aremainly degraded
to acetic acid in the complex Stickland reactions [62]) are listed
tion reactions during conversion of carbohydrates and fats.
Reaction Source
6 þ 2H2O / 2CH3COOH þ 2CO2 þ 4H2 [62,63]
6 þ 2H2 / 2C2H5COOH þ 2H2O [62,63]
O6 / 4C2H5COOH þ 2CH3COOH þ 2CO2 þ 2H2O [62]
6 / C3H7COOH þ 2CO2 þ 2H2 [62,63]
COOH þ 14H2O / 8CH3COOH þ 14H2 [62]
OH / CH4 þ CO2 [62,63]
OH þ 2H2O / 2CH3COOH þ 2H2 [12,62,63]
OH þ 2H2O / CH3COOH þ CO2 þ 3H2 [12,62,63]
CO2 / CH4 þ 2H2O [62,63]
b i om a s s a n d b i o e n e r g y 5 6 ( 2 0 1 3 ) 5 4 5e5 5 4 551
in Table 3. As after hydrolysis both cellulose and greater part
of maize are converted into monosaccharides, mainly HAc
andHPrwere expected as the intermediates in the subsequent
digestion paths [12,62,63]. The time progress of selected VFA
acids measured in the experiments is presented in Fig. 3.
3.3.2.1. Degradation of maize. During degradation of maize
iso-HBu, n-HVa and iso-HVa did not exceed 200 mg/l. The
concentration of n-HBu was enhanced simultaneous to iso-
HBu, even if iso-HBu did not exceed 200 mg/l. According to
previous studies of [64e68], iso-HBu serves as equilibrium
storage of n-HBu so the iso form is irrelevant for further
degradation to HAc.
For maize at higher OLs (17.1 kgVS/m3 mesophilic and 11.5
and 17.3 kgVS/m3 thermophilic) HAc attained very high con-
centrations (s. Fig. 3). A considerable increase of the n-HBu
concentration reaching nearly the level typical for HAc or HPr
was observed. This trend was even more pronounced for
thermophilic trials (s. Fig. 3). The concentration of n-HBu was
increased simultaneous to enhanced HAc. However n-HBu
peaks were degraded earlier than those of HAc. HPr raise at
38 �C followed in parallel to HAc, while at 55 �C only after n-
HBu degradation but still simultaneous to enhanced HAc. HPr
concentration dropped only after HAc uptake. The observed
0
1
2
3
4
5
HA
c [ g
/l ]
cellulose
0.00
0.15
0.30
0.45
0.60
0.75
HP
r [
g/l ]
cellulose
0.0
0.4
0.8
1.2
1.6
0 2 4 6 8 10 12 14
n-H
Bu
[ g
/l ]
time [d]
cellulose
5.7 kgVS/m3 thermophilic
11.4 kgVS/m3 thermophilic
17.1 kgVS/m3 thermophilic
5.4 kgVS/m3 mesophilic
10.9 kgVS/m3 mesophilic
16.3 kgVS/m3 mesophilic
Fig. 3 e Concentration changes of acetic (HAc), propionic (HPr) a
mesophilic digestion of cellulose and maize.
sequence of VFA indicates that for maize silage at 38 �C the
parallel HAc and HPr production and degradation (Table 3,
reactions 1, 3, 5 and 8) took place while for the highest OL of
17.1 kgVS/m3 additional shift to the HBu production and
degradation (s. Table 3, reactions 4 and 7) can be assumed. At
55 �C for the initial 2 days the direct monomer to HAc and HBu
digestionwas in favor (s. Table 3, reactions 1 and 4), while later
a shift towards substrate conversion to HPr (Table 3, reaction
2) took place.
The enhanced concentration of HPr following subsequent to
elevated HBu and HAc is regarded as a proof of hydrogen and
HAc-conditioned inhibition of HPr degraders [69e74]. Increased
hydrogen concentration is reported in the literature for the
activity period of HBu to HAc degraders [12,73]. The final
delayed degradation of HPr was in accordance with literature
characterizing HPr as the last VFA to stabilize due to its slow
degradation rate [6,12]. HBu inhibition byHAc higher than 1.5 g/
l similar to those reported in the literature [75,76] was not
observed for maize in thermophilic mode however cannot be
excluded for the highest OL of maize in mesophilic load.
The presence of HBu as degradation intermediate during
maize digestion implied a subsequent high activity of HBu de-
graders producing high H2 concentrations [72]. Therefore
registered lower ORP values (s. Section 3.3.1), typical for
maize
0
1
2
3
4
5
HA
c [ g
/l ]
maize
0.0
0.3
0.6
0.9
1.2
1.5
HP
r [
g/l ]
maize
0.0
0.4
0.8
1.2
1.6
0 2 4 6 8 10 12 14
n-H
Bu
[ g
/l ]
time [d]
maize
5.5 kgVS/m3 mesophilic
11.0 kgVS/m3 mesophilic
5.7 kgVS/m3 thermophilic
11.5 kgVS/m3 thermophilic
17.3 kgVS/m3 thermophilic
17.1 kgVS/m3 mesophilic
nd n-butyric acid (n-HBu) measured for thermophilic and
b i om a s s an d b i o e n e r g y 5 6 ( 2 0 1 3 ) 5 4 5e5 5 4552
hydrogen production, only confirm the assumed reaction
pathways.
3.3.2.2. Degradation of cellulose. VFA concentrations
measured for cellulose degradation were within the ranges
considered by now as typical for undisturbed anaerobic
digestion [12,69e71]. Unlike for maize, HPr degradation peaks
for cellulose were comparable for the OL higher than
5.5 � 0.2 kgVS/m3 independent of the temperature mode (s.
Fig. 3). The typical VFA performance with increased HAc and
only moderately enhanced HPr (s. Fig. 3) was observed for all
investigated OLs and temperatures. Peaks of HAc, being a
direct degradation product of cellulose monomers (s. Table 3,
reactions 1 and 3) and HPr (s. Table 3, reaction 8), were
observed simultaneous to elevated HPr concentrations. Ac-
cording to these VFA trends a simultaneous conversion of
monosaccharides to HAc and HPr can be assumed.
HAc concentrations measured for higher OLs in thermo-
philic mode reached untypically high concentration range:
they were nearly 3 times higher than for mesophilic condi-
tions. This is an indicator that during thermophilic digestion
of cellulose a direct degradation of monomers to HAc is a
preferable digestion pathway (s. Table 3, reaction 1).
For all tests only insignificant amounts of n-HVa were
measured, while the remaining n-HBu, iso-HBu and iso-HVa
did not exceed 50 mg/l for mesophilic and 150 mg/l for ther-
mophilic mode. Presence of n- and iso-HBu might be
explained by enhanced conversion of cellulose via reaction 4
(s. Table 3), while in general presence of HBu and HVa in small
amounts might be also considered as an effect of HPr and HAc
backreactions [12].
4. Conclusions
The digestion retard at increased OLs of cellulose and lower
degradation level obtained for all cellulose series confirm a
higher overloading potential for systems dealing with single-
component-substrates but also the enhanced sensitivity of
such systems to any inconvenient digestion conditions (nu-
trients limitation, ammonia inhibition, etc).
Mesophilic conditions supported faster and more stable
digestion and can be recommended as the optimumoperating
temperature for commercial biogas plants co-digesting maize
silage. Due to its complex structure maize degradation was
characterized by varying digestion rate. The longer break-
down of hard-degradable fractions prolonged the total diges-
tion time. These components may be regarded as problematic
and only partially degradable in large-scale biogas plants due
to continuous fresh supply of new substrate containing easier
degradable fractions.
Two factors showed a strong potential to influence the test
results: an insufficient VS content of inoculum and a high
natural biodiversity of maize silage. To prevent the negative
impact of the first factor, best efforts should be exerted in
order to keep the recommended VS concentration of the
inoculum. The enhanced homogeneity of maize silage can be
reached by diverse pre-treatment steps e.g. drying, milling or
creation of synthetic substrate with similar composition.
However, depending on the focus of the experiment, gains
and losses need to be balanced. The more complex the sub-
strate pre-treatment, the higher the risk of producing results
distant from the daily biogas practice.
Conversion of maize at highly reductive conditions with
increased HBu concentrations was accompanied by much
higher activity of hydrogenotrophic methanogens than for
cellulose digestion. These results should act as a trigger for
further investigations focusing on specific digestion mecha-
nisms and composition of microbial biocenosis activated
during digestion of different substrates or substrate fractions.
Differences in digestion pathways observed for maize and
cellulose, but also for different temperature modes, must
compel an individual approach to each digestion system.
Therefore, the experience of the plant operators in discovering
system instabilities is so valuable and should be given at least
the same priority as to any other system stability indicators.
Acknowledgments
The authors gratefully acknowledge the cooperation with
Constant Kieffer from Biogas Biekerich in terms of inoculum
supplies, Daniel Lucas from laboratory team and would like to
thank Dr. Jindra Agostini for great GC technical support.
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