Accepted Manuscript

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

Vivekanand Vivekanand, Daniel Girma Mulat, Vincent G.H. Eijsink, Svein J.

Horn

PII: S0960-8524(17)31734-0
DOI: https://doi.org/10.1016/j.biortech.2017.09.169
Reference: BITE 18997

To appear in: Bioresource Technology

Received Date: 5 July 2017

Revised Date: 22 September 2017
Accepted Date: 23 September 2017

Please cite this article as: Vivekanand, V., Mulat, D.G., Eijsink, V.G.H., Horn, S.J., Synergistic effects of anaerobic
co-digestion of whey, manure and fish ensilage, Bioresource Technology (2017), doi: https://doi.org/10.1016/
j.biortech.2017.09.169

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

fish 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

*Corresponding author. Tel.: + 47 67232488; Fax: + 47 64965901. E-mail address:

svein.horn@nmbu.no

1
Abstract

Biogas production potential of the three feedstocks fish ensilage, manure and whey was

evaluated using biochemical methane potential (BMP) tests. Since anaerobic digestion of

single substrates may be inefficient due to imbalances in the carbon-nitrogen ratio, degree of

biodegradability and/or due to lack of nutrients needed by the microbial community, co-

digestion of these substrates was also assessed, revealing synergistic effects and a particularly

good effect of combining manure with fish ensilage. In this latter case, methane yields were

up to 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.

Keywords: Whey, manure, fish ensilage, biogas, co-digestion.

2
1. Introduction

Anaerobic digestion of biomass requires a coordinated action of a complex microbial

community carrying out processes commonly referred to as hydrolysis, acidogenesis,

acetogenesis and methanogenesis The microbes that take part in this process vary in terms of

their metabolic 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 is 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 & 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 instance, animal manures are generally

considered to combine low organic 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 inhibitory 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 feedstock,

which, however, is characterized by a low nitrogen content and high recalcitrance. For this

3
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 & 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 & 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 Zelic (Hublin & 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. (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 investigated in a few cases,

showing that biogas yield increased when increasing the fraction of the former compared to

the later. Co-digestion of a maximum of 16% (volume basis) of fish ensilage with manure

increased the methane production by 100%, in comparison to the methane production

obtained from cow manure alone (Solli et al., 2014). In all these studies, only a limited

number of substrate mixing ratios were tested. Thus, there is a lack of more comprehensive

4
studies testing many substrate-blending ratios. Moreover, no single study has investigated the

co-digestion of the three substrates manure, fish ensilage and whey.

The objective of this study was to explore the possibility of utilizing blends of the three

important waste streams whey permeate (dairy industrial waste), fish ensilage (aquaculture

waste) and manure for biogas production. This was done by co-digesting manure with either

fish ensilage or whey, whey with fish ensilage and combinations of all three substrates.

Several mixing ratios were studied in order to determine optimum mixing ratios and

synergetic effects. Moreover, our study suggests possible mechanisms for observed

synergetic effects, such as the beneficial combination of substrates with different

biodegradability rates.

2. Materials and Methods

2.1 Biogas inoculum

The microbial inoculum utilized for the biogas experiments was collected from a biogas plant

(Biowaz, Tomb, Norway) running large-scale continuous anaerobic digestion of food waste

and cow manure at mesophilic temperature with a 22 days hydraulic retention time (HRT)

and 3.2 kg VS/m3/day organic loading rate (OLR). It had a total solid (TS) content of 5.2%, a

volatile solid (VS) content of 68.5% (of TS), and a pH of 7.6. Prior to the experiments, this

inoculum was pre-incubated anaerobically at 37 oC for 10 days in order to reduce endogenous

biogas 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 bottles. A total of 69 batch bottle

digesters were prepared and supplied with substrate as described in Table 1. Information on

5
the chemical composition of the inoculum and all feedstocks used in this study is provided in

Tables 2 and 3 (see below).

2.2 Feedstocks

Cow manure was procured from the Department of Animal Sciences, Norwegian University

of Life Sciences (NMBU), Ås, Norway. TINE, the largest Norwegian dairy product

cooperative based in Oslo, Norway, and Biokraft, based in Trondheim, Norway, supplied

whey and fish ensilage, respectively. These substrates were characterized in detail (see Tables

2 and 3) and stored at 4°C until the start of the biogas experiments. Manure, whey and fish

ensilage had TS contents of 15.9, 16.2 and 32.3%, respectively. The C/N ratios of the

feedstocks manure, whey and fish ensilage were 42.4, 96.6 and 8.8, respectively (Table 2). It

should be noted that that C/N ratio of manure is somewhat high due to some in mix of

sawdust.

2.3 Evaluation of the biogas potential of different combinations of raw materials

Anaerobic digestion of different combinations of raw materials (manure, whey and fish

ensilage; Table 1) was performed in sealed batch bottle digesters, in triplicates. Cellulose

(Avicel, Sigma, USA.) was 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 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 incubation, the bottles were purged with nitrogen for 2 min,

to ensure anaerobic conditions, closed with rubber seals and aluminum screw caps, and

transferred to the shaker (Multitron Standard, Infors HT, Switzerland) for incubation (37°C,

90 rpm). All experiments were run in triplicates and the average results are presented with

standard deviations (the standard deviations are not always visible in the Figures because
6
standard deviations were generally small; see Table 4). The methane yields were reported at

standard temperature and pressure (0 °C and 1 atm) after correcting the background biogas

production from inoculum alone (control).

2.4 Gas composition and calculations

Gas composition analysis and calculations were performed as described previously

(Vivekanand et al., 2013). In brief, biogas production was monitored by measuring the

generated pressure in the bottle digesters using a digital pressure transducer (GMH 3161,

Greisinger Electronic, Germany). A gas chromatograph (3000 Micro GC, Agilent

Technologies, USA) equipped with a thermal conductivity detector (TCD) and helium as

carrier gas was used for analyzing the biogas composition. After recording the pressure in the

bottles, the overpressure was released by penetrating the septum with a needle. Using the

measured overpressure, headspace volume of the bottles and measurements of methane

concentrations as input, the ideal gas law was applied for calculating methane production. To

avoid excessive dissolution of CO2 with possible effects on pH the overpressure was always

kept below 200 kPa (Holliger et al., 2016).

2.5 Other analyses

TS and ash content were determined by drying and incinerating the samples at 105°C and

550°C overnight, respectively. The VS content was calculated by subtracting the ash from the

TS. The elemental composition of nitrogen, carbon and hydrogen (Table 2) was determined

by combustion using a Leco CHN-1000 instrument (St. Joseph, Michigan, USA), whereas

ICP-MS was used for the determination of metals (Table 3).

7
2.6 Theoretical methane potential (Bo) and extent of degradation (fd)

Theoretical methane potential (Bo) was calculated using the empirical formula derived from

the elemental composition of individual substrates (Table 2) according to the following

stoichiometric equation (Buswell & Neave, 1930).

Equation 1

The extent of degradation (fd) is the ratio of observed methane to the theoretical methane

(Bo) and presented in percentage as follows:

Equation 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 reported in this study is similar to values found in

a study by Solli et al (Solli et al., 2014). Considering the high TS and VS contents of fish
8
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 ensilage 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 different 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 considered more optimal.
9
The nutrient requirements of a biogas reactor have been estimated based on the general

composition of microbial biomass. For macronutrients 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.(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 concentration 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. Figure 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

10
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 Equation 2 using the theoretical methane potential (Bo)

calculated from Equation 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 contains 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 & 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 overestimated, in particular for manure

(Agger et al, 2014).

Different combinations of the substrates (whey, manure and fish ensilage) were then tested to

map the biogas potential of a wide range of combinations that may be useful in practice (i.e.

adapted to the availability of multiple feedstocks) and, importantly, to identify combinations

yielding synergy in terms of increased biogas yields. Figures 2, 3 and 4 show methane

11
accumulation curves for different co-digestions of whey and manure, whey and fish ensilage,

and fish ensilage and manure, respectively. Accumulated methane levels after 27 days, i.e. at

the point where biogas production had drastically slowed down in all reactors, are

summarized in Table 4.

Methane production data for the different whey and manure blends

(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, whereas the yields were higher than when using

manure alone (Fig. 1 and 2). A blend has a synergistic effect if more methane is produced

relative to an estimate based on the methane yields of single substrate digestions. All whey

and manure blends showed no synergistic effects, or a very small (up to 9 %) which is not

considered significant (see Table 4).Antagonistic effects were not observed in this series of

experiments. The extent of biodegradation decreased with increased manure mixing ratios

(Table 4) as manure is a poorly degradable substrate. Whey contains easy degradable lactose,

which may lead to rapid acid formation and a pH drop in a biogas reactor (Hagen et al., 2014;

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

carbon, has a presumably more favorable (i.e. lower) C/N ratio, and high alkalinity (Traversi

et al., 2013). It should be noted that the pH in all our experiments were stable and similar

(Table 4).

Fig. 3 shows methane production data for blends of whey and fish ensilage. As expected from

the data for the individual substrates (Fig. 1, Table 4), an increasing content of fish ensilage

yielded higher methane production, although there was no significant difference in methane

12
yield for the digestions with 75% and 85% fish ensilage. Synergistic 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 whey to fish ensilage ratio (Table 4). Importantly, clear

antagonist effects were observed at higher contents of fish ensilage (50 % and more). Despite

fish ensilage being the most biodegradable substrate, an increase in proportion of fish

ensilage from 15 to 85 % during co-digestion with whey led to a decrease in the extent of

biodegradation from 91 % to 83 % (Table 4). It thus seems that small amounts of fish

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

have a detrimental effect on the digestion of fish ensilage. The 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 content of whey-dominated digesters. The antagonism observed when

adding small amounts of whey to fish ensilage-dominated digesters is less easy to explain.

Lipid-rich fish ensilage is considered to be easily degradable, albeit not as easy as whey, but

its anaerobic digestion is nevertheless considered challenging due to the accumulation of

long-change fatty acids (LCFA) (Labatut et al., 2011; Long et al., 2012; Traversi et al., 2013).

Recent findings suggest that LCFA accumulation has an inhibitory effect on acetoclastic and

hydrogenotrophic methanogenesis as well as acidogenesis (Long et al., 2012). It is possible

that the combined production of whey-derived VFAs and fish ensilage-derived LCFAs

creates an inhibitory imbalance in the system.

Fig. 4 shows the results of co-digestion of manure and fish ensilage. Again, as expected, the

methane yields increased with the content of fish ensilage. Importantly, in this case, all blend

ratios gave clear synergistic effects, that were larger with increasing content of manure (Table

4). The highest synergistic effects were an impressive 84 % increase in biogas yield at the

13
85:15 blend ratio of manure and fish ensilage. It would this seem that the combination of

manure and fish ensilage is highly favorable.

The extent of biodegradation of co-digestion of manure and fish ensilage ranged between 79

and 109 %, whereas the biodegradability of 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 values

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 composition (Ebner et al., 2016). Sample heterogeneity could affect the accuracy

of the measurements of the samples used for elemental composition analysis (which in turn

affects the calculated Bo values) and the samples used for biogas potential test (which in turn

affects the measured 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.

14
The synergistic effects seen when combining manure and fish ensilage, 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 degradable

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 synergetic 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 & Markt, 2016). Priming is a well-known phenomenon in

other habitats like soils and sediments where enhanced decomposition of organic matter was

observed upon adding easily degradable substrates (Insam & 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 components and consequently provides extra methane production and nutrient release

(Insam & 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 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

15
batch experiments carried out for a relatively short period because high inoculum-to-substrate

ratio was used and the inoculum had a high buffering capacity and was rich in nutrients.

Inspired by the results above, and taking into account the availability of all three tested

feedstocks in the Norwegian countryside, a non-exhaustive analysis of co-digestion of all

three substrates was carried out. In particular; the antagonistic effects observed, when

combining whey and fish ensilage (Fig. 3; Table 4) could be counteracted by 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 observed 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 effects is useful for countries

such as Norway, where all the three feedstocks are available in large parts of the country. Of

course, the implications of the present findings for continuous anaerobic digestion processes,

as they would run in an industrial setting, need to be investigated 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

16
This study shows that combination of feedstocks with different chemical composition and

biodegradability can be combined to yield efficient methane production that in some cases

exceeds the sum of the methane production from the individual feedstocks (synergetic

effects). As discussed above, there are many possible explanations for the observed

synergistic effects, which could be the subject of further studies. The increase 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).

Figure Captions

Fig. 1 Methane yield from anaerobic digestion of cellulose, whey, manure and fish ensilage

alone in batch bioreactors.

Fig. 2 Methane yield from anaerobic co-digestion of whey and manure in batch bioreactors.

17
Fig. 3 Methane yield from anaerobic co-digestion of whey and fish ensilage in batch

bioreactors.

Fig. 4 Methane yield from anaerobic co-digestion of manure and fish ensilage in batch

bioreactors.

Fig. 5 Methane yield from anaerobic co-digestion of whey, manure and fish ensilage in batch

bioreactors.

References

Agger, J.W., Nilsen, P.J., Eijsink, V.G.H., Horn, S.J. 2014. On the Determination of Water Content in
Biomass Processing. BioEnergy Research, 7(1), 442-449.
Amon, T., Amon, B., Kryvoruchko, V., Zollitsch, W., Mayer, K., Gruber, L. 2007. Biogas production
from maize and dairy cattle manure—Influence of biomass composition on the methane
yield. Agriculture, Ecosystems & Environment, 118(1–4), 173-182.
Atandi, E., Rahman, S. 2012. Prospect of anaerobic co-digestion of dairy manure: a review.
Environmental Technology Reviews, 1(1), 127-135.
Björkman, A. 1956. Studies on finely divided wood. Part 1. Extraction of lignin with neutral solvents.
in: Svensk papperstidning Vol. 59, pp. 477-485.
Buswell, A.M., Neave, S.L. 1930. Laboratory studies of sludge digestion. Jeffersons Print. & stationery
Company.
18
Ebner, J.H., Labatut, R.A., Lodge, J.S., Williamson, A.A., Trabold, T.A. 2016. Anaerobic co-digestion of
commercial food waste and dairy manure: Characterizing biochemical parameters and
synergistic effects. Waste Management, 52, 286-294.
Esposito, G., Frunzo, L., Giordano, A., Liotta, F., Panico, A., Pirozzi, F. 2012. Anaerobic co-digestion of
organic wastes. Reviews in Environmental Science and Bio/Technology, 11(4), 325-341.
Fitamo, T., Boldrin, A., Boe, K., Angelidaki, I., Scheutz, C. 2016. Co-digestion of food and garden
waste with mixed sludge from wastewater treatment in continuously stirred tank reactors.
Bioresource Technology, 206, 245-254.
Gelegenis, J., Georgakakis, D., Angelidaki, I., Mavris, V. 2007. Optimization of biogas production by
co-digesting whey with diluted poultry manure. Renewable Energy, 32(13), 2147-2160.
Hagen, L.H., Vivekanand, V., Linjordet, R., Pope, P.B., Eijsink, V.G., Horn, S.J. 2014. Microbial
community structure and dynamics during co-digestion of whey permeate and cow manure
in continuous stirred tank reactor systems. Bioresource Technology, 171, 350-359.
Holliger, C., Alves, M., Andrade, D., Angelidaki, I., Astals, S., Baier, U., Bougrier, C., Buffière, P.,
Carballa, M., de Wilde, V. 2016. Towards a standardization of biomethane potential tests.
Water Science and Technology, 74(11), 2515-2522.
Hublin, A., Zelić, B. 2013. Modelling of the whey and cow manure co-digestion process. Waste
management & research, 31(4), 353-360.
Insam, H., Markt, R. 2016. Comment on "Synergistic co-digestion of solid-organic-waste and
municipal-sewage-sludge: 1 plus 1 equals more than 2 in terms of biogas production and
solids reduction" [Water Research 87, 416-423]. Water Research, 95, 392-3.
Kuzyakov, Y. 2010. Priming effects: Interactions between living and dead organic matter. Soil Biology
and Biochemistry, 42(9), 1363-1371.
Kuzyakov, Y., Friedel, J.K., Stahr, K. 2000. Review of mechanisms and quantification of priming
effects. Soil Biology and Biochemistry, 32(11–12), 1485-1498.
Labatut, R.A., Angenent, L.T., Scott, N.R. 2011. Biochemical methane potential and biodegradability
of complex organic substrates. Bioresource Technology, 102(3), 2255-2264.
Lo, K.V., Liao, P.H. 1989. Anaerobic-aerobic biological treatment of a mixture of cheese whey and
dairy manure. Biological Wastes, 28(2), 91-101.
Long, J.H., Aziz, T.N., Reyes Iii, F.L.d.l., Ducoste, J.J. 2012. Anaerobic co-digestion of fat, oil, and
grease (FOG): A review of gas production and process limitations. Process Safety and
Environmental Protection, 90(3), 231-245.
Mata-Alvarez, J., Dosta, J., Romero-Güiza, M.S., Fonoll, X., Peces, M., Astals, S. 2014. A critical review
on anaerobic co-digestion achievements between 2010 and 2013. Renewable and
Sustainable Energy Reviews, 36, 412-427.
Nges, I.A., Mbatia, B., Björnsson, L. 2012. Improved utilization of fish waste by anaerobic digestion
following omega-3 fatty acids extraction. Journal of Environmental Management, 110, 159-
165.
Nielfa, A., Cano, R., Fdz-Polanco, M. 2015. Theoretical methane production generated by the co-
digestion of organic fraction municipal solid waste and biological sludge. Biotechnology
Reports, 5, 14-21.
Oechsner, H.-W., Lemmer, A., Ramhold, D., Mathies, E., Mayrhuber, E., Preissler, D. 2010. Method
for producing biogas in controlled concentrations of trace elements Vol. US20100304457 A1,
pp. 1-8.
Raposo, F., De la Rubia, M.A., Fernández-Cegrí, V., Borja, R. 2012. Anaerobic digestion of solid
organic substrates in batch mode: An overview relating to methane yields and experimental
procedures. Renewable and Sustainable Energy Reviews, 16(1), 861-877.
Solli, L., Bergersen, O., Sorheim, R., Briseid, T. 2014. Effects of a gradually increased load of fish
waste silage in co-digestion with cow manure on methane production. Waste Manag, 34(8),
1553-9.

19
Sutaryo, S., Ward, A.J., Moller, H.B. 2014. The effect of mixed-enzyme addition in anaerobic
digestion on methane yield of dairy cattle manure. Environ Technol, 35(17-20), 2476-82.
Traversi, D., Bonetta, S., Degan, R., Villa, S., Porfido, A., Bellero, M., Carraro, E., Gilli, G. 2013.
Environmental Advances Due to the Integration of Food Industries and Anaerobic Digestion
for Biogas Production: Perspectives of the Italian Milk and Dairy Product Sector. BioEnergy
Research, 6(3), 851-863.
Vivekanand, V., Olsen, E.F., Eijsink, V.G.H., Horn, S.J. 2013. Effect of different steam explosion
conditions on methane potential and enzymatic saccharification of birch. Bioresource
Technology, 127, 343-349.
Weiland, P. 2010. Biogas production: current state and perspectives. Applied Microbiology And
Biotechnology, 85(4), 849-860.

20
 800 Cellulos
e
700
Whey
600
Methane yield [mL gVS-1]

500

400

300

200

100

0
0 20 40 60
Time [Days]

21
800
Whey +
700 Manure 85:15
Whey +
600 Manure 75:25
Methane yield [mL gVS-1]

Whey +
500 Manure 50:50

400

300

200

100

0
0 20 40 60
Time [Days]

22
 800
Whey + Fish Ensi
85:15
700 Whey + Fish Ensi
75:25
600 Whey + Fish Ensi
Methane yield [mL gVS-1]

50:50
500

400

300

200

100

0
0 10 20 30 40 50 60
Time [Days]

23
 800

700
Methane yield [mL gVS-1]

600

500

400 Manure + Fish

Ensi 85:15
300 Manure + Fish
Ensi 75:25
200

100

0
0 10 20 30 40 50 60
Time [Days]

24
 800
Whey + Manure + Fish
33:33:34
700
Whey + Manure + Fish
20:20:60
600
Methane yield [mL gVS-1]

500

400

300

200

100

0
0 10 20 30 40 50 60
Time [Days]

25
Table 1: Experimental design

Sl. No. Feedstocks Number of Bottles

1 Control 3

2 Cellulose 3

3 Whey 3

4 Manure 3

5 Fish Ensilage 3

6 Whey + Manure 85:15 3

7 Whey + Manure 75:25 3

8 Whey + Manure 50:50 3

9 Whey + Manure 25:75 3

10 Whey + Manure 15:85 3

11 Whey + Fish Ensilage 85:15 3

12 Whey + Fish Ensilage 75:25 3

13 Whey + Fish Ensilage 50:50 3

14 Whey + Fish Ensilage 25:75 3

15 Whey + Fish Ensilage 15:85 3

16 Fish Ensilage + Manure 85:15 3

17 Fish Ensilage + Manure 75:25 3

18 Fish Ensilage + Manure 50:50 3

19 Fish Ensilage + Manure 25:75 3

20 Fish Ensilage + Manure 15:85 3

21 Whey + Manure + Fish Ensilage 33:33:34 3

22 Whey + Manure + Fish Ensilage 20:20:40 3

23 Whey + Manure + Fish Ensilage 10:10:80 3

Table 2: Chemical composition of different feedstocks (wt%)

26
 Substrate Total C Total H Total N Ash Total O* C/N ratio
% % % % %
Fish Ensilage 56.2±1.5 8.7±0.3 6.4±0.1 5.9±0.0 22.8 8.8

Whey 41.1±0.5 5.3±0.1 0.4±0.0 9.2±0.2 44.0 96.6

Manure 45.4±0.0 5.6±0.3 1.1±0.1 14.3±7.3 33.9 42.4

Cellulose 42.5±0.1 6.4±0.1 0.1±0.0 0.6±0.5 50.5 731.9

Inoculum 32.7±0.1 4.4±0.0 2.8±0.0 31.5±0.1 29.0 11.6

*
O was estimated subtracting the other components from 100 %.

Table 3: Elemental composition of different substrate/feed-stocks

Substrate B Na Mg Al P S K Ca V Cr Mn Fe Co Ni Zn Cu As

g/kg g/kg g/kg mg/kg g/kg g/kg g/kg g/kg mg/kg mg/kg g/kg g/kg mg/kg mg/kg ug/L mg/kg mg/kg

Fish 3.25 7.55 1.20 135.00 9.20 4.90 7.95 8.75 0.23 5.75 0.01 0.42 0.06 0.51 98.50 11.00 1.50
Ensilage

Whey 1.50 7.75 1.60 55.50 8.30 1.00 30.50 7.40 0.27 10.00 0.01 1.95 0.30 2.75 1.75 8.20 0.16

Manure 12.00 2.90 3.90 325.00 4.65 2.80 24.50 8.05 1.15 4.60 0.20 1.25 0.85 3.35 140.00 28.00 0.22

Cellulose 0.28 0.01 0.00 2.25 0.00 0.03 0.01 0.00 0.01 0.05 0.00 0.00 0.01 0.00 0.00 0.00 0.00

Inoculum 48.00 18.00 17.00 1600.00 17.00 5.40 110.00 25.00 4.90 26.95 0.44 2.95 3.20 24.00 545.00 85.50 0.48

Table 4: Synergistic effects of blends, C/N ratio, pH and the extent of biodegradation (fd)
Sample Experimental Estimated Differencea fd (%)b C/N ratio pHc
methane methane (mL) including

27
 yield yield inoculum
(mL CH4 g-1 (mL CH4 g-
1
VS) VS)

Cellulose 351±11 - - 89 160 7.27±0.08

Whey (W) 264±9 - - 74 31 7.15±0.04

Manure 28 18 7.19±0.02
147±9 - -
(M)

Fish 99 10 7.21±0.01

ensilage 691±6 - -

(FE)

W+M +20 69 30 7.17±0.01

266±22 246
85:15

W+M +24 (9.3 %) 65 28 7.17±0.02

259±5 235
75:25

W+M +13 50 25 7.17±0.03

219±2 206
50:50

W+M +1 37 22 7.17±0.02
177±7 176
25:75

W+M -1 33 20 7.16±0.03
164±12 165
15:85

W+FE +41 (12.5 91 28 7.16±0.02

370±15 329
85:15 %)

W+FE +16 87 27 7.17±0.03

387±17 371
75:25

W+FE -26 86 22 7.19±0.01

451±1 477
50:50

W+FE 533±8 584 -51 87 16 7.19±0.03

28
25:75

W+FE -93 83 14 7.17±0.05

534±8 627
15:85

M+FE +192 (83.8 76 17 7.22±0.05

421±7 229
85:15 %)

M+FE +185 82 17 7.25±0.03

468±14 283
75:25

M+FE +166 96 15 7.26±0.02

586±10 420
50:50

M+FE +154 108 13 7.25±0.03

709±17 555
25:75

M+FE +120 109 12 7.24±0.01

729±7 609
15:85

W+M + FE +4 71 20 7.19±0.02
375±11 371
33:33:34

W+M + FE -11 82 17 7.19±0.02

487±7 498
20:20:60

W+M + FE -28 88 14 7.18±0.01

566±27 594
10:10:80

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. bThe extent of biodegradation (fd) was calculated

29
according to Equation 2 using the theoretical methane potential (Bo) calculated from Equation

1 and the measured methane yield. cpH values after incubation.

Highlights:

 Co-digestion of fish ensilage (FE), manure (M) and whey (W) was investigated

 Co-digestion resulted in BMP higher than weighted average of individual substrates

 Co-digestion of M+FE gave the highest synergetic effects (84% higher methane yield)

 The extent of biodegradation of substrates explain the observed synergetic effects

30