Bioresource Technology 249 (2018) 35–41

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Synergistic effects of anaerobic co-digestion of whey, manure and fish T

ensilage
⁎
Vivekanand Vivekananda,b, Daniel Girma Mulata, Vincent G.H. Eijsinka, Svein J. Horna,
a
Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P. O. Box 5003, N-1432 Ås, Norway
b
Centre for Energy and Environment, Malaviya National Institute of Technology Jaipur, JLN Marg, Jaipur 302 017, Rajasthan, India

A R T I C L E I N F O A B S T R A C T

Keywords: Biogas production potential of the three feedstocks fish ensilage, manure and whey was evaluated using bio-
Whey chemical methane potential (BMP) tests. Since anaerobic digestion of single substrates may be inefficient due to
Manure imbalances in the carbon-nitrogen ratio, degree of biodegradability and/or due to lack of nutrients needed by the
Fish ensilage microbial community, co-digestion of these substrates was also assessed, revealing synergistic effects and a
Biogas
particularly good effect of combining manure with fish ensilage. In this latter case, methane yields were up to
Co-digestion
84% higher than the weighted average of the methane yields obtained with the individual substrates. The type of
substrate was the dominating cause of variation in methane production rates and yields.

1. Introduction
Anaerobic digestion of biomass requires a coordinated action of a
complex microbial community carrying out processes commonly re-
ferred to as hydrolysis, acidogenesis, acetogenesis and methanogenesis
The microbes that take part in this process vary in terms of their me-
tabolic needs and there are numerous interdependencies between the
members of the microbial community (Björkman, 1956). A stable and
optimal anaerobic digestion process requires regular feeding and a
balanced microbial community that is adapted to the substrate. One
parameter often used to roughly describe the substrate and to assess
substrate variation is the C/N ratio. In biogas processes, a C/N ratio
between 20 and 30 is considered optimal (Parkin and Owen, 1986) but
there are indications that wider ranges of C/N ratios are acceptable (e.g.
C/N ratios of sludge are around 9) (Nielfa et al., 2015).
Anaerobic digestion of single substrates may be challenging due to
their chemical composition and/or their physical properties. For in-
stance, animal manures are generally considered to combine low or-
ganic loads with high nitrogen concentrations, which is unfavorable for
methanogenesis. However, depending on the animal feed, manure can
also contain e.g. high amounts of straw that result in a lower N content.
Meat and fish wastes tend to have high contents of fatty acids and
protein, but their degradation products may accumulate and be in-
hibitory for methanogenesis. Whey is an abundant by-product, but
anaerobic mono-digestion of whey could be problematic because whey
is rich in easily degradable sugars (mainly lactose), which could lead to
rapid acidification and process inhibition (Hagen et al., 2014; Traversi
et al., 2013). Furthermore, whey has a high C/N ratio and a low pH
(below 5.0). Lignocellulosic substrates provide another potential feed-
stock, which, however, is characterized by a low nitrogen content and
high recalcitrance. For this latter substrate, the enzymatic hydrolysis
step may become the bottleneck during anaerobic digestion (Sutaryo
et al., 2014).
Several of the challenges related to using single substrates could be
met by digesting two or more substrates in a so-called co-digestion
process. The idea is to mix substrates to obtain an improved nutrient
balance, a favorable C/N ratio and dilution of inhibitory or toxic
compounds. In some cases, co-digestion may lead to more methane
production than the combined methane production of single substrate
digestions, meaning that synergism is achieved (Atandi and Rahman,
2012; Fitamo et al., 2016; Mata-Alvarez et al., 2014).
Several studies have examined the effects of co-digestion on biogas
production, typically using manure as co-substrate (Atandi and
Rahman, 2012; Ebner et al., 2016; Esposito et al., 2012; Labatut et al.,
2011). The co-digestion of easily degradable substrates like whey with
poorly degradable substrates like manure has been shown to increase
the total methane yield (Labatut et al., 2011). Hublin and Zelić (2013)
concluded that co-digestion of 10% whey with 90% manure (volume
basis) was the optimum mixing ratio for biogas production. However, in
another study by Gelegenis et al. (2007), optimum biogas production
was achieved by co-digestion of up to 50% whey (volume basis) with
manure. Co-digestion of fish ensilage and manure has also been in-
vestigated in a few cases, showing that biogas yield increased when
increasing the fraction of the former compared to the later. Co-digestion

⁎
Corresponding author.
E-mail address: svein.horn@nmbu.no (S.J. Horn).

http://dx.doi.org/10.1016/j.biortech.2017.09.169
Received 5 July 2017; Received in revised form 22 September 2017; Accepted 23 September 2017
Available online 27 September 2017
0960-8524/ © 2017 Elsevier Ltd. All rights reserved.
V. Vivekanand et al. Bioresource Technology 249 (2018) 35–41

of a maximum of 16% (volume basis) of fish ensilage with manure in- Norwegian University of Life Sciences (NMBU), Ås, Norway. TINE, the
creased the methane production by 100%, in comparison to the me- largest Norwegian dairy product cooperative based in Oslo, Norway,
thane production obtained from cow manure alone (Solli et al., 2014). and Biokraft, based in Trondheim, Norway, supplied whey and fish
In all these studies, only a limited number of substrate mixing ratios ensilage, respectively. These substrates were characterized in detail (see
were tested. Thus, there is a lack of more comprehensive studies testing Tables 2 and 3) and stored at 4 °C until the start of the biogas experi-
many substrate-blending ratios. Moreover, no single study has in- ments. Manure, whey and fish ensilage had TS contents of 15.9, 16.2
vestigated the co-digestion of the three substrates manure, fish ensilage and 32.3%, respectively. The C/N ratios of the feedstocks manure, whey
and whey. and fish ensilage were 42.4, 96.6 and 8.8, respectively (Table 2). It
The objective of this study was to explore the possibility of utilizing should be noted that C/N ratio of manure is somewhat high due to some
blends of the three important waste streams whey permeate (dairy in- in mix of sawdust.
dustrial waste), fish ensilage (aquaculture waste) and manure for biogas
production. This was done by co-digesting manure with either fish 2.3. Evaluation of the biogas potential of different combinations of raw
ensilage or whey, whey with fish ensilage and combinations of all three materials
substrates. Several mixing ratios were studied in order to determine
optimum mixing ratios and synergetic effects. Moreover, our study Anaerobic digestion of different combinations of raw materials
suggests possible mechanisms for observed synergetic effects, such as (manure, whey and fish ensilage; Table 1) was performed in sealed
the beneficial combination of substrates with different biodegradability batch bottle digesters, in triplicates. Cellulose (Avicel, Sigma, USA.) was
rates. used as a reference substrate while the inoculum alone was used as a
control for endogenous biogas production. In all cases, a total of 0.60 g
2. Materials and methods of substrate on a VS basis was added to the batch bottles digesters. The
different blend ratios of substrates were also based on VS. Prior to in-
2.1. Biogas inoculum cubation, the bottles were purged with nitrogen for 2 min, to ensure
anaerobic conditions, closed with rubber seals and aluminum screw
The microbial inoculum utilized for the biogas experiments was caps, and transferred to the shaker (Multitron Standard, Infors HT,
collected from a biogas plant (Biowaz, Tomb, Norway) running large- Switzerland) for incubation (37 °C, 90 rpm). All experiments were run
scale continuous anaerobic digestion of food waste and cow manure at in triplicates and the average results are presented with standard de-
mesophilic temperature with a 22 days hydraulic retention time (HRT) viations (the standard deviations are not always visible in the Figures
and 3.2 kg VS/m3/day organic loading rate (OLR). It had a total solid because standard deviations were generally small; see Table 4). The
(TS) content of 5.2%, a volatile solid (VS) content of 68.5% (of TS), and methane yields were reported at standard temperature and pressure
a pH of 7.6. Prior to the experiments, this inoculum was pre-incubated (0 °C and 1 atm) after correcting the background biogas production
anaerobically at 37 °C for 10 days in order to reduce endogenous biogas from inoculum alone (control).
production. Furthermore, the inoculum was diluted to a TS content of
1.6% with water and divided into 400 mL aliquots in 555 mL batch 2.4. Gas composition and calculations
bottles. A total of 69 batch bottle digesters were prepared and supplied
with substrate as described in Table 1. Information on the chemical Gas composition analysis and calculations were performed as de-
composition of the inoculum and all feedstocks used in this study is scribed previously (Vivekanand et al., 2013). In brief, biogas produc-
provided in Tables 2 and 3 (see below). tion was monitored by measuring the generated pressure in the bottle
digesters using a digital pressure transducer (GMH 3161, Greisinger
2.2. Feedstocks Electronic, Germany). A gas chromatograph (3000 Micro GC, Agilent
Technologies, USA) equipped with a thermal conductivity detector
Cow manure was procured from the Department of Animal Sciences, (TCD) and helium as carrier gas was used for analyzing the biogas
composition. After recording the pressure in the bottles, the over-
Table 1 pressure was released by penetrating the septum with a needle. Using
Experimental design.
the measured overpressure, headspace volume of the bottles and mea-
Sl. No. Feedstocks Number of Bottles surements of methane concentrations as input, the ideal gas law was
applied for calculating methane production. To avoid excessive dis-
1 Control 3 solution of CO2 with possible effects on pH the overpressure was always
2 Cellulose 3
kept below 200 kPa (Holliger et al., 2016).
3 Whey 3
4 Manure 3
5 Fish Ensilage 3 2.5. Other analyses
6 Whey + Manure 85:15 3
7 Whey + Manure 75:25 3
8 Whey + Manure 50:50 3 TS and ash content were determined by drying and incinerating the
9 Whey + Manure 25:75 3 samples at 105 °C and 550 °C overnight, respectively. The VS content
10 Whey + Manure 15:85 3 was calculated by subtracting the ash from the TS. The elemental
11 Whey + Fish Ensilage 85:15 3 composition of nitrogen, carbon and hydrogen (Table 2) was de-
12 Whey + Fish Ensilage 75:25 3
13 Whey + Fish Ensilage 50:50 3
termined by combustion using a Leco CHN-1000 instrument (St. Jo-
14 Whey + Fish Ensilage 25:75 3 seph, Michigan, USA), whereas ICP-MS was used for the determination
15 Whey + Fish Ensilage 15:85 3 of metals (Table 3).
16 Fish Ensilage + Manure 85:15 3
17 Fish Ensilage + Manure 75:25 3
18 Fish Ensilage + Manure 50:50 3 2.6. Theoretical methane potential (Bo) and extent of degradation (fd)
19 Fish Ensilage + Manure 25:75 3
20 Fish Ensilage + Manure 15:85 3 Theoretical methane potential (Bo) was calculated using the em-
21 Whey + Manure+Fish Ensilage 33:33:34 3
pirical formula derived from the elemental composition of individual
22 Whey + Manure+Fish Ensilage 20:20:40 3
23 Whey + Manure+Fish Ensilage 10:10:80 3 substrates (Table 2) according to the following stoichiometric equation
(Buswell and Neave, 1930).

36
V. Vivekanand et al.
Table 2
Chemical composition of different feedstocks (wt%).
Substrate Total C Total H
% %
Fish Ensilage 56.2 ± 1.5 8.7 ± 0.3
Whey 41.1 ± 0.5 5.3 ± 0.1
Manure 45.4 ± 0.0 5.6 ± 0.3
Cellulose 42.5 ± 0.1 6.4 ± 0.1
Inoculum 32.7 ± 0.1 4.4 ± 0.0
a
O was estimated subtracting the other components from 100%.
Cn Ha Ob Nc + (n−a/4−b/2 + 3c /4) H2 O→ (n/2 + a/8−b/4−3c /8) CH4
+ (n/2−a/8 + b/4 + 3c /8) CO2 + cNH3 Bo
22.4(n/2 + a/8−b/4−3c /8)
= LCH4 g −1 VS
12n + a + 16b + 14c
(1)
The extent of degradation (fd) is the ratio of observed methane to
the theoretical methane (Bo) and presented in percentage as follows:
observed methane
f d (%) = x100
Bo (2)
where the observed methane and Bo were presented on a VS basis (mL
CH4/g VS added).
3. Results and discussion
3.1. Chemical and elemental analysis of raw materials
The TS content of fish ensilage was almost twice of the manure and
whey, while the VS content of fish ensilage is slightly higher than
manure and whey. The TS content for manure is a little higher than
previous reported values (15.9% vs e.g. 12.4) (Labatut et al., 2011)
whereas the TS of cheese whey is higher than in a previous study (16.2
vs 7.1%) (Labatut et al., 2011). Generally, the TS and VS values re-
ported in this study is similar to values found in a study by (Solli et al.
(2014). Considering the high TS and VS contents of fish ensilage, and a
balanced content of major and minor trace minerals, fish ensilage has a
high potential for biogas production. The low ash content in fish en-
silage also indicates this (Table 2).
It should be noted that dry matter in this paper was analyzed by
means of oven drying, assuming that only water evaporates. However,
substrates like manure and fish ensilage contain organic volatiles that
will lead to underestimation of the dry matter content. In a previous
study using Karl Fisher analysis to determined dry matter content, it
was shown that oven drying underestimated dry mater in fish ensilage
and manure by 13% and 49%, respectively (Agger et al., 2014).
The composition of the different biomasses used in this study is
shown in Tables 2 and 3. The elemental composition is one of the
parameter used for determining the suitability of a substrate for biogas
production. Especially the C/N ratio is essential and a balanced C/N
ratio is needed for optimum microbial growth and reducing ammonia
inhibition. The C/N ratios (Table 2) show that whey and cellulose have
a relatively low nitrogen content. The fish ensilage and cow manure
used in this study had more balanced C/N ratios, although the nitrogen
content in fish ensilage would be considered rather high (C/N ratio is
8.8). Although nitrogen is required for microbial growth, anaerobic
digestion of substrates rich in nitrogen (i.e. too low C/N ratio) may be
inhibited by accumulation of free ammonia. The feedstocks assessed
here had varying C/N ratios, meaning that the co-digestions not only
varied in terms of the type of substrate as such, but also had very dif-
ferent C/N ratios. Also, note that the inoculum influenced the real C/N
ratios in the batch bottles (Table 4). Co-digestion of nitrogen-rich fish
ensilage with low and moderate nitrogen-containing substrates such as
whey and manure, respectively, generated C/N ratios that would be
Bioresource Technology 249 (2018) 35–41
Total N Ash Total Oa % C/N ratio
% %
6.4 ± 0.1 5.9 ± 0.0 22.8 8.8
0.4 ± 0.0 9.2 ± 0.2 44.0 96.6
1.1 ± 0.1 14.3 ± 7.3 33.9 42.4
0.1 ± 0.0 0.6 ± 0.5 50.5 731.9
2.8 ± 0.0 31.5 ± 0.1 29.0 11.6
considered more optimal.
The nutrient requirements of a biogas reactor have been estimated
based on the general composition of microbial biomass. For macro-
nutrients a typical ratio found in the literature for C:N:P:S is 600:15:5:1
(Weiland, 2010). Based on Table 2 and Table 3 it can be calculated that
for fish ensilage, whey and manure these ratios are 11.5:1.3:1.9:1.0,
41.1:0.4:8.3:1.0 and 16.1:0.4:1.7:1.0, respectively. These numbers
show that there is considerable variation between the feedstocks and
also show that P and S are clearly not limiting in any of these substrates.
Trace elements known to be important for microbial growth are iron,
nickel, cobalt, selenium, molybdenum, and tungsten (Weiland, 2010).
These elements were identified in most of the feedstocks used in this
study (Table 3; tungsten was not analyzed. According to Oechsner et al.
(2010), fish ensilage is deficient in iron, nickel and cobalt, and its
molybdenum concentration is close to the lowest recommend limit.
Whey is deficient with selenium, and its cobalt and nickel concentration
is close to the lowest recommend limit. Manure had optimum con-
centration of all the trace elements except nickel, which is found in a
concentration close to the lowest recommendation limit. However,
since the inoculum is rich in nutrients (Table 3), the difference in trace
element composition between the substrates less likely to influence the
biogas process.
3.2. Evaluation of the biogas potential of different combinations of raw
materials
Initially, the biological methane potential of the different individual
feedstocks and cellulose were determined. Fig. 1 shows the cumulative
methane production during batch digestion of cellulose, whey, manure
and fish ensilage with final methane yields of 363, 274, 180 and
740 mL gVS−1, respectively. Cellulose was included as a control to
evaluate the quality of the inoculum and gave a methane yield of
352 mL/g VS, indicating that the inoculum used was well functioning
inoculum (Holliger et al., 2016). The lipid-rich fish ensilage showed the
highest biogas potential, while manure had the lowest potential. The
extent of biodegradation (fd) was calculated according to Eq. (2) using
the theoretical methane potential (Bo) calculated from Eq. (1) and the
measured methane yield. The results showed that the lipid-rich fish
ensilage had the highest (99%) and manure (28%) had the lowest
biodegradation extent among the studied mono-substrates. Lipids con-
tains a high number of H per C, and thus have a higher theoretical
methane potential than carbohydrates and proteins (Esposito et al.,
2012). The carbon in manure is much less accessible than fish ensilage
as manure is enriched in slowly degradable fibers, including lignin-rich
material, because most of the easily degradable fibers in cattle feed are
digested in the cow rumen and gut (Amon et al., 2007). The CH4 yields
reported here for the different feedstocks are comparable with values
reported in previous batch experiment studies of dairy manure
(136–296 mL/g VS (Amon et al., 2007); 127–329 mL/g VS (Labatut
et al., 2011), whey (193–273 mL/g VS) (Lo and Liao, 1989) and fish
residues (742–828 mL/g VS) (Nges et al., 2012). However, it should be
noted that TS and VS contents obtained by the oven drying method is
underestimated since fish ensilage and manure contain volatiles. This
means that the estimated extents of biodegradation are somewhat
37
V. Vivekanand et al. Bioresource Technology 249 (2018) 35–41

overestimated, in particular for manure (Agger et al., 2014).

mg/kg

0.03
0.00
0.00
0.00
0.00
Hg Different combinations of the substrates (whey, manure and fish
mg/kg
ensilage) were then tested to map the biogas potential of a wide range
0.25 of combinations that may be useful in practice (i.e. adapted to the
0.05
0.37
0.00
1.60
Pb

availability of multiple feedstocks) and, importantly, to identify com-

binations yielding synergy in terms of increased biogas yields. Figs. 2–4
mg/kg

30.50

48.50
0.69
0.25

0.04
show methane accumulation curves for different co-digestions of whey
Ba

and manure, whey and fish ensilage, and fish ensilage and manure,
respectively. Accumulated methane levels after 27 days, i.e. at the point
mg/kg

0.19
0.07
0.05
0.00
0.22

where biogas production had drastically slowed down in all reactors,

Cs

are summarized in Table 4.

mg/kg

Methane production data for the different whey and manure blends
0.02
0.01
0.09
0.00
0.28
Cd

(85:15;75:15;50:50;25:75;15:85) show that all blends gave a lower

final methane yield than when using whey as a single substrate,
mg/kg

0.10
0.48
1.65
0.00
8.65

whereas the yields were higher than when using manure alone (Figs. 1
Mo

and 2). A blend has a synergistic effect if more methane is produced

mg/kg

relative to an estimate based on the methane yields of single substrate

0.40
0.00
0.56
0.00
0.89

digestions. All whey and manure blends showed no synergistic effects,

Se

or a very small (up to 9%) which is not considered significant (see

mg/kg

Table 4). Antagonistic effects were not observed in this series of ex-
1.50
0.16
0.22
0.00
0.48
As

periments. The extent of biodegradation decreased with increased

manure mixing ratios (Table 4) as manure is a poorly degradable sub-
mg/kg

11.00

28.00

85.50
8.20

0.00

strate. Whey contains easy degradable lactose, which may lead to rapid
Cu

acid formation and a pH drop in a biogas reactor (Hagen et al., 2014;

140.00

545.00

Traversi et al., 2013). On the other hand, manure contains relatively

98.50
ug/L

1.75

0.00

slowly degradable carbon, has a presumably more favorable (i.e. lower)

Zn

C/N ratio, and high alkalinity (Traversi et al., 2013). It should be noted
mg/kg

24.00

that the pH in all our experiments were stable and similar (Table 4).
0.51
2.75
3.35
0.00
Ni

Fig. 3 shows methane production data for blends of whey and fish
ensilage. As expected from the data for the individual substrates (Fig. 1,
mg/kg

Table 4), an increasing content of fish ensilage yielded higher methane

0.06
0.30
0.85
0.01
3.20
Co

production, although there was no significant difference in methane

yield for the digestions with 75% and 85% fish ensilage. Synergistic
g/kg

0.42
1.95
1.25
0.00
2.95
Fe

effects were only observed at low contents of fish ensilage (15 and
25%), the maximum being a 13% increase in biogas yield at an 85:15
g/kg

0.01
0.01
0.20
0.00
0.44
Mn

whey to fish ensilage ratio (Table 4). Importantly, clear antagonist ef-
fects were observed at higher contents of fish ensilage (50% and more).
mg/kg

10.00

26.95
5.75

4.60
0.05

Despite fish ensilage being the most biodegradable substrate, an in-

Cr

crease in proportion of fish ensilage from 15 to 85% during co-digestion

mg/kg

with whey led to a decrease in the extent of biodegradation from 91%

0.23
0.27
1.15
0.01
4.90

to 83% (Table 4). It thus seems that small amounts of fish ensilage have
V

a beneficial effect on the digestion of whey, whereas small amounts of

25.00
g/kg

8.75
7.40
8.05
0.00

whey have a detrimental effect on the digestion of fish ensilage. The

Ca

synergy observed at low fish ensilage fractions is likely due to the large
effect that small amounts of fish ensilage have on the (low) nitrogen
110.00
30.50
24.50
g/kg

7.95

0.01

content of whey-dominated digesters. The antagonism observed when

K

adding small amounts of whey to fish ensilage-dominated digesters is

g/kg

4.90
1.00
2.80
0.03
5.40

less easy to explain. Lipid-rich fish ensilage is considered to be easily

S

degradable, albeit not as easy as whey, but its anaerobic digestion is

17.00

nevertheless considered challenging due to the accumulation of long-

g/kg

9.20
8.30
4.65
0.00
P

change fatty acids (LCFA) (Labatut et al., 2011; Long et al., 2012;
Elemental composition of different substrate/feed-stocks.

Traversi et al., 2013). Recent findings suggest that LCFA accumulation

1600.00
135.00

325.00
mg/kg

55.50

has an inhibitory effect on acetoclastic and hydrogenotrophic metha-

2.25
Al

nogenesis as well as acidogenesis (Long et al., 2012). It is possible that

the combined production of whey-derived VFAs and fish ensilage-de-
17.00
g/kg

1.20
1.60
3.90
0.00
Mg

rived LCFAs creates an inhibitory imbalance in the system.

Fig. 4 shows the results of co-digestion of manure and fish ensilage.
18.00
g/kg

7.55
7.75
2.90
0.01

Again, as expected, the methane yields increased with the content of

Na

fish ensilage. Importantly, in this case, all blend ratios gave clear sy-
nergistic effects, that were larger with increasing content of manure
12.00

48.00
g/kg

3.25
1.50

0.28

(Table 4). The highest synergistic effects were an impressive 84% in-
B

crease in biogas yield at the 85:15 blend ratio of manure and fish en-
Fish Ensilage

silage. It would this seem that the combination of manure and fish
Inoculum
Substrate

Cellulose
Manure

ensilage is highly favorable.

Table 3

Whey

The extent of biodegradation of co-digestion of manure and fish

ensilage ranged between 79 and 109%, whereas the biodegradability of

38
V. Vivekanand et al. Bioresource Technology 249 (2018) 35–41

Table 4
Synergistic effects of blends, C/N ratio, pH and the extent of biodegradation (fd).

Sample Experimental methane yield Estimated methane yield Differencea fd (%)b C/N ratio including inoculum pHc
(mL CH4 g−1 VS) (mL CH4 g−1 VS) (mL)

Cellulose 351 ± 11 – – 89 160 7.27 ± 0.08

Whey (W) 264 ± 9 – – 74 31 7.15 ± 0.04
Manure (M) 147 ± 9 – – 28 18 7.19 ± 0.02
Fish ensilage (FE) 691 ± 6 – – 99 10 7.21 ± 0.01
W + M 85:15 266 ± 22 246 +20 69 30 7.17 ± 0.01
W + M 75:25 259 ± 5 235 +24 (9.3%) 65 28 7.17 ± 0.02
W + M 50:50 219 ± 2 206 +13 50 25 7.17 ± 0.03
W + M 25:75 177 ± 7 176 +1 37 22 7.17 ± 0.02
W + M 15:85 164 ± 12 165 −1 33 20 7.16 ± 0.03
W + FE 85:15 370 ± 15 329 +41 (12.5%) 91 28 7.16 ± 0.02
W + FE 75:25 387 ± 17 371 +16 87 27 7.17 ± 0.03
W + FE 50:50 451 ± 1 477 −26 86 22 7.19 ± 0.01
W + FE 25:75 533 ± 8 584 −51 87 16 7.19 ± 0.03
W + FE 15:85 534 ± 8 627 −93 83 14 7.17 ± 0.05
M + FE 85:15 421 ± 7 229 +192 (83.8%) 76 17 7.22 ± 0.05
M + FE 75:25 468 ± 14 283 +185 82 17 7.25 ± 0.03
M + FE 50:50 586 ± 10 420 +166 96 15 7.26 ± 0.02
M + FE 25:75 709 ± 17 555 +154 108 13 7.25 ± 0.03
M + FE 15:85 729 ± 7 609 +120 109 12 7.24 ± 0.01
W + M+FE 33:33:34 375 ± 11 371 +4 71 20 7.19 ± 0.02
W + M + FE 20:20:60 487 ± 7 498 −11 82 17 7.19 ± 0.02
W + M + FE 10:10:80 566 ± 27 594 −28 88 14 7.18 ± 0.01

a
The experimental methane yields represent the total amount of methane generated after 27 days of digestion (i.e. at a time point where the methane production rate had become
minimal in all reactors). The estimated methane yield is based on adding the contribution of each of the individual feedstocks as they appear in the first four rows of the Table. The
differences between these two values appear in the column labeled “Difference” and give an indication of synergistic or antagonistic effects.
b
The extent of biodegradation (fd) was calculated according to Eq. (2) using the theoretical methane potential (Bo) calculated from Eq. (1) and the measured methane yield.
c
pH values after incubation.

800 800
Cellulose Whey + Fish Ensi 85:15
Whey Whey + Fish Ensi 75:25
700 700
Manure Whey + Fish Ensi 50:50
Methane yield [mL gVS-1]

Fish
Methane yield [mL gVS-1]

600 600 Whey + Fish Ensi 25:75

Whey + Fish Ensi 15:85
500 500
400 400
300
300
200
200
100
100
0
0 10 20 30 40 50 60 0
0 10 20 30 40 50 60
Time [Days]
Time [Days]
Fig. 1. Methane yield from anaerobic digestion of cellulose, whey, manure and fish en-
silage alone in batch bioreactors. Fig. 3. Methane yield from anaerobic co-digestion of whey and fish ensilage in batch
bioreactors.

800
Whey + Manure 85:15 800
700 Whey + Manure 75:25
Methane yield [mL gVS-1]

Whey + Manure 50:50 700

600
Methane yield [mL gVS-1]

Whey + Manure 25:75 600

500 Whey + Manure 15:85
500
400
400 Manure + Fish Ensi 85:15
Manure + Fish Ensi 75:25
300 Manure + Fish Ensi 50:50
300
Manure + Fish Ensi 25:75
200 Manure + Fish Ensi 15:85
200
100
100
0
0
0 10 20 30 40 50 60
0 10 20 30 40 50 60
Time [Days]
Time [Days]
Fig. 2. Methane yield from anaerobic co-digestion of whey and manure in batch bior-
Fig. 4. Methane yield from anaerobic co-digestion of manure and fish ensilage in batch
eactors.
bioreactors.

39
V. Vivekanand et al.
800
Whey + Manure + Fish 33:33:34
700 Whey + Manure + Fish 20:20:60
Whey + Manure + Fish 10:10:80
Methane yield [mL gVS-1]
600
500
400
300
200
100
0
0 10 20 30
Time [Days]
Fig. 5. Methane yield from anaerobic co-digestion of whey, manure and fish ensilage in
batch bioreactors.
manure and fish ensilage mono-digestion was 28 and 99%, respectively
(Table 4). It is not surprising that the extent of biodegradation of co-
digestion of manure and fish ensilage had increased with the contents of
fish ensilage as mono-digestion of fish ensilage gave the highest extent
of biodegradability among all the studied substrates. These results show
that addition of fish ensilage improved the degradability of manure
during the co-digestion. In theory, the extent of biodegradability (fd)
should always be lower than 100% because part of the organic material
may be inaccessible to microorganisms or not biodegradable, and a
fraction of the substrates (3–15%) is used for cellular growth and
maintenance (Raposo et al., 2012). In this study, the calculated fd va-
lues for co-digestion of manure with fish ensilage content of 75% and
more were higher than 100%. The fd values are subject to uncertainty
due to sample heterogeneity and measurement of elemental composi-
tion (Ebner et al., 2016). Sample heterogeneity could affect the accu-
racy of the measurements of the samples used for elemental composi-
tion analysis (which in turn affects the calculated Bo values) and the
samples used for biogas potential test (which in turn affects the mea-
sured biogas yield). Moreover, calculation of Bo based on the elemental
composition of a substrate is an approximation (Ebner et al., 2016).
Nevertheless, the synergetic effects were generally well correlated with
the increase in extent of biodegradation.
The synergistic effects seen when combining manure and fish en-
silage, and the fact that these effects became larger with increasing
contents of manure could be due to one or several reasons. The most
possible explanation is the improved degradation of the slowly de-
gradable fractions of manure by a bacterial community that is fueled by
easily degradable lipid-rich fish ensilage. This is also supported by the
fd results discussed above. Within the time point where most of the
substrates were degraded (day 27), only 28% of manure was degradable
while almost all of the fish ensilage was degraded. Therefore, the sy-
nergetic effects could only be expected from the improved degradability
of manure during co-digestion. Recently Insam & Markt suggested to
adopt the term “priming” for describing synergistic effects in anaerobic
co-digestion (Insam and Markt, 2016). Priming is a well-known phe-
nomenon in other habitats like soils and sediments where enhanced
decomposition of organic matter was observed upon adding easily de-
gradable substrates (Insam and Markt, 2016; Kuzyakov, 2010;
Kuzyakov et al., 2000). In co-digestion, such as in the present study,
addition of easily degradable substrates (i.e. easily available energy
and/or nutrients) would accelerate microbial enzyme production,
which further enhances (co-) degradation of recalcitrant organic com-
ponents and consequently provides extra methane production and nu-
trient release (Insam and Markt, 2016).
The synergetic effects could also be explained by the more stable
and efficient biogas production attained due to the benefits of adding
manure to compensate the low carbon content (i.e. low C/N), the low
alkalinity, and the low levels of some nutrients (Mn, Co, Ni and, to a
Bioresource Technology 249 (2018) 35–41
lesser extent Zn and Cu) of fish ensilage. This is particularly important
for efficient and stable operation of continuously fed reactors such as
continuously stirred tank reactors (CSTRs) run over long periods (Solli
et al., 2014). However, this is not the case in the current batch ex-
periments carried out for a relatively short period because high in-
oculum-to-substrate ratio was used and the inoculum had a high buf-
fering capacity and was rich in nutrients.
Inspired by the results above, and taking into account the avail-
ability of all three tested feedstocks in the Norwegian countryside, a
non-exhaustive analysis of co-digestion of all three substrates was car-
ried out. In particular; the antagonistic effects observed, when com-
bining whey and fish ensilage (Fig. 3; Table 4) could be counteracted by
40 50 60
the beneficial effects of combining fish ensilage with manure. Blending
whey, manure and fish ensilage in different ratios (Fig. 5) did not have
any synergetic effects on methane yield. Antagonistic effects were ob-
served at higher fish ensilage contents (60% and more, Table 4), but
these were less prominent compared to co-digestion of only whey and
fish ensilage (50% or more, Table 4). Although, a wider mapping of
optimal feedstock combinations may be needed, the triple co-digestion
experiments indicate that development of efficient anaerobic digestion
processes based on combining all three feedstocks is feasible.
The possibility to blend feedstocks and even obtain synergistic ef-
fects is useful for countries such as Norway, where all the three feed-
stocks are available in large parts of the country. Of course, the im-
plications of the present findings for continuous anaerobic digestion
processes, as they would run in an industrial setting, need to be in-
vestigated further. The beneficial (synergistic) effects of small amounts
of fish ensilage on anaerobic digestion of manure deserves particular
attention. Perhaps fish ensilage could be used as a “booster” in small
farm-based biogas plants running primarily on manure.
4. Conclusions
This study shows that feedstocks with different chemical composi-
tion and biodegradability can be combined to yield efficient methane
production that in some cases exceeds the sum of the methane pro-
duction from the individual feedstocks (synergetic effects). As discussed
above, there are many possible explanations for the observed sy-
nergistic effects, which could be the subject of further studies. The in-
crease in methane yields and synergetic effects were generally well
correlated with increase in extent of biodegradation. The beneficial
effects of small amounts of fish ensilage on co-digestion with manure
deserves particular attention.
Acknowledgements
This project was financial supported the Norwegian Research
Council, projects no 203402 (RobuBiogas) and 228747 (BiogasFuel).
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