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Fish waste: an efficient alternative to biogas and methane production in an

anaerobic mono-digestion system

Francielle Bücker, Munique Marder, Marina Regina Peiter, Daniel Neutzling Lehn,
Vanessa Mendonça Esquerdo, Luiz Antonio de Almeida Pinto, Odorico Konrad

PII: S0960-1481(19)31327-8
DOI: https://doi.org/10.1016/j.renene.2019.08.140
Reference: RENE 12219

To appear in: Renewable Energy

Received Date: 05 October 2018

Accepted Date: 30 August 2019

Please cite this article as: Francielle Bücker, Munique Marder, Marina Regina Peiter, Daniel
Neutzling Lehn, Vanessa Mendonça Esquerdo, Luiz Antonio de Almeida Pinto, Odorico Konrad,
Fish waste: an efficient alternative to biogas and methane production in an anaerobic mono-
digestion system, Renewable Energy (2019), https://doi.org/10.1016/j.renene.2019.08.140

This is a PDF file of an article that has undergone enhancements after acceptance, such as the
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Fish waste: an efficient alternative to biogas and methane production in an

anaerobic mono-digestion system

'Declarations of interest: none

Francielle Bückera*, Munique Mardera, Marina Regina Peitera, Daniel Neutzling Lehnb,

Vanessa Mendonça Esquerdoc, Luiz Antonio de Almeida Pintoc, Odorico Konradd

aLaboratório de Biorreatores, Universidade do Vale do Taquari, 171 Avelino Talini St.,

Universitário, Lajeado, RS, Brazil. Email: franbucker@gmail.com,

mmarder@univates.br , marina.peiter@univates.br

bCentro de Ciências Exatas e Tecnológicas - CETEC, Universidade do Vale do Taquari,

171 Avelino Talini St., Universitário, Lajeado, RS, 95914-014, Brazil. Email:

lehn@univates.br

cEscola de Química e Alimentos - Universidade Federal do Rio Grande - FURG, Italia

Avenue km 8, Rio Grande, RS, 96203-900, Brazil. Email: nessafurg@gmail.com;

dqmpinto@furg.br

dPrograma de Pós-graduação em Ambiente e Desenvolvimento, Laboratório de

Biorreatores, Universidade do Vale do Taquari, 171 Avelino Talini St., Universitário,

Lajeado, RS, 95914-014 Brazil. Email: okonrad@univates.br

*corresponding author. Tel: +55 51 37147000. E-mail: franbucker@gmail.com

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Highlights

>Biogas can be successfully produced through the mono-digestion of fish waste.

>The waste from the first fish processing step produced the highest volume of biogas.

> Fungi, Bacteria, and Archaea were capable of using the fish waste to perform

methanogenesis.

>The fungal community diversity was very similar in both fish waste experiments.

Abstract

The use of industrial fish processing waste as the only substrate to produce biogas may

be an efficient procedure due to its characteristic lipid content. The relationship between

the microbial community and the biogas/methane production was evaluated during the

anaerobic digestion at 35°C of two waste types derived from the fish processing industry.

The experiments showed that fish waste (FW) and fish crude oil waste (FCOW) produced

methane at 540.5 CH4 mL gVS−1 and 426.3 CH4 mL gVS− 1, respectively. Clostridia,

Synergistia were the predominant bacterial classes and the Methanomicrobia archeal

class at the end of the anaerobic digestion in both substrates. The fungal community was

similar in both treatments. The fungal diversity included orders of the Ascomycota

phylum: Eurotiales, Sordariales, Saccharomycetales, Sporidiales, and Capnodiales

Microascales. Representatives of Basidiomycota included Wallemiales and Tremellales.

This research demonstrated that industrial fish processing waste can be efficiently

converted to methane in a mono-digestion process.

Keywords: fish waste; biogas; biomethane

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1. Introduction

Presently, maintaining the global energy supply security is a challenge. The current

scenario of traditional fossil fuels is unstable. Biogas has been considered as a viable

alternative to replace conventional fuels, especially when they are linked to

environmental issues [1]. Biogas is an alternative biofuel that can be produced from

several organic sources and wastes during anaerobic digestion. Compared with fossil

fuels, the production of biogas using locally available substrates and renewable resources

as raw materials is an energy-efficient and environmentally friendly technology since it

contributes to reduce greenhouse gases (GHG) emissions [1–3]. Moreover, Leme and

Seabra [4] conducted a technical-economic study about the biomethane production in the

Brazilian bioethanol industry and concluded that biomethane is a cost-competitive fuel

with natural gas, and biodiesel.

Anaerobic digestion (AD) is a biochemical process involving a large variety of

microorganisms and is regarded as an engineered ecosystem in which organic molecules

undergo biodegradation. This process can be used for the generation of bioenergy in the

form of biomethane, for example [5,6]. All reactions are performed by different microbial

populations that include a wide range of Fungi, Bacteria, and Archaea [7–10]. In the last

few years, high-throughput sequencing technologies have been used to analyze the

structure of communities involved in anaerobic digestion processes [5]. However, there

is a lack of comprehensive studies evaluating the microbial community composition in

reactors using fish waste as substrates for biogas production.

The challenges of using a mono-substrate in AD are associated with the stability of the

process. When using several substrates, in a co-digestion process, AD becomes more

stable [11]. Therefore, more studies are needed to infer the feasibility of AD when using

a mono- substrate. Vivekanand et al. [12] compared the biogas production using the waste
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streams: whey permeate, fish ensilage (aquaculture waste), and manure. Fish ensilage,

used as an AD mono-substrate, produced the highest amount of biogas (~690 mL CH4 g−1

VS). Whey and manure produced nearly 265 and 145 mL CH4 g−1 VS, respectively.

Moreover, the co-digestion of fish ensilage and manure (85:15) resulted in ~730 mL CH4

g−1 VS biogas, i.e., an increment of only 5%. The transport of two or more substrates to

perform the anaerobic digestion could make the biogas production financially unfeasible.

The shipping costs from the point of generation of residues to the anaerobic digestion

plant should be considered [11,12] since these costs increase as more co-substrates are

added.

The latest Brazilian statistics indicate that aquaculture and fisheries produced about 472

and 765 t of fish in 2016, respectively. Depending on the processing method, a large part

(up to 50%) of the fish mass (viscera, head, spine, and skin) ends up being discarded as

residue [13]. However, these residues could have other purposes (including biogas

production) since they contain materials potentially biodegradable by anaerobic digestion

[13]. There are few studies that considered the biogas production as a solution for waste

treatment in the fishery industry. However, other findings have shown that the fish waste

has an interesting potential for biogas production [12,14–20]. The anaerobic digestion is

a promising tool for biogas production and has been increasingly used in the Brazilian

bioenergy scenario. Besides using renewable biomass sources and mitigating

environmental problems, AD can contribute to the local generation of electric energy

[21].

This paper discusses the potential biogas production using fishery processing wastes as a

single carbon source. Biogas and methane production were evaluated under mesophilic

conditions via anaerobic digestion using the VDI 4630 as the reference normative. Some

chemical characteristics of the substrates were also determined. The potential biogas

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production using these substrates was then compared with the microbial community

established in the reactor at the end of AD.

2. Material and Methods

2.1 Anaerobic digestion (AD)

The anaerobic digestion was conducted using 2,000-mL glass reactors (1,500-g working

weight) at mesophilic temperature (35°C). The experiments were conducted in triplicates.

To estimate the biogas production, the reactors were connected to tubes coupled to an

automated biogas flow measurement system, which reads the standard biogas production

at the standard temperature and pressure conditions set up according Konrad et al. [22].

To perform the AD experiments, reference was made to the VDI 4630 standard, which

establishes rules and equipment requirements for conducting fermentation tests on

organic materials. The reactors with the different substrates were prepared according to

the VDI 4630 restrictions, where the amount of substrate should not exceed the amount

of inoculum VSsubstrate/VSinoculum < 0.5 and the solids concentration in the batch test should

not exceed 10%, ensuring adequate mass transfer during the test [23]. Aliquots (20 mL)

of biogas produced in each reactor were extracted with plastic syringes to perform the

percentage of methane in the biogas in a specific sensor (Advanced Gasmitter). The

Biochemical Potential of Biogas (BMP) was estimated as the total biogas production

divided by the respective volatile solid sample content. The results were normalized to

the total biogas production from the negative controls (inoculum-only) [23].

Microcrystalline cellulose U.S.P. (thin powder, Synth) was used as a positive internal

control. The reactors, which had only the inoculum (henceforth referred to as negative

control) were used as negative controls according to VDI 4630 recommendations [23].

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Total solids (TS) and volatile solids (VS) were estimated by drying the samples at 105°C

for 24 h and at 550°C for 1 h, respectively, according to the standard method 2540 [24].

Fish waste (FW) and fish crude oil extraction waste (FCOW) from common carp

(Cyprinus carpio) viscera were used as substrates in the AD experiment. The carp viscera

were provided by a commercial fishery processing plant from Roca Sales, RS, Brazil and

submitted to a thermomechanical process according to Crexi et al. [25]. The carp viscera

were initially cooked (Rochedo – Idealclave, Brazil) for 30 min at 98 ± 2°C. The retained

material was sifted and pressed, resulting in press liquor and solids. The solids were stored

at -20°C for the AD experiments and denominated as fish waste (FW). The separation of

fish crude oil from the press liquor was performed by centrifugation (7,000 × g at 25°C

for 20 min) (Sigma 6-15, D-37250, Germany). The centrifugation resulted in a bottom

solid cake, an aqueous middle layer, and an oily upper layer [25]. The bottom solid cake

was separated and stored at -20°C for the AD experiments. The fish crude oil waste

(FCOW) was used as substrate.

The inoculum used in the experiments came from an anaerobic microbiota of a biogas

plant sludge mixture from the Centro de Estudos de Biogás e Energias Renováveis, at

Universidade do Vale do Taquari, south of Brazil. The inoculum was acclimated to the

operating conditions at 35°C for 21 days prior to the beginning of the experiments. During

this period, the amount of biogas was estimated, and, at the end of 21 days, it had the

appropriate biogas production rate and constant composition to be used in the AD

experiments.

The characteristics and volumes of substrates, microcrystalline cellulose, and inoculum

added to each experiment are shown in Table 1.

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Table 1 Volume of substrates, microcrystalline cellulose, and inoculum added to each

experimental reactor, according to VDI 4630 [23].

Experiment Inoculum Substrate TS VS gVS gVS

(g) (g) (%) (%) (Inoculum) (Sample)

Fish Waste (FW) 1457.4 42.6 29.1 89.0 21.5 11.0

Fish Crude Oil 1453.3 46.7 28.8 82.0 21.4 11.0

Waste (FCOW)

Microcrystalline 1488.3 11.7 94.4 99.8 21.9 11.0

Cellulose

Negative Control 1500.0 0.0 3.4 43.8 22.1 0.0

Additionaly, the carbon, nitrogen, and hydrogen contents of both FW and FCOW were

determined by combustion using a PerkinElmer® 2400 Series II CHNS/O Elemental

Analyzer (PerkinElmer, Michigan, USA), and the pH was measured using a digital pH

meter (DIGIMED).

2.2 Microbial community

To analyze the microbial community, at the end of AD, 10 mL of the final biomass of

each triplicate were retained in a 50 mL sterile tube and centrifuged at 6,000 rpm for 10

min. The supernatant was discarded, and 10 mL of the pellet was stored in an icebox with

CO2 dry ice and sent to Neoprospecta Microbiome Technologies (Florianópolis, SC,

Brazil). The DNA extraction was performed with PowerSoil DNA Isolation Kit (MO BIO

Inc., Laboratories, USA), according to the manufacturer’s instructions. The extracted

genomic DNA was quantified and standardized prior the amplification by PCR according

to Christoff et al. [27].

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The DNA analyses were performed using high-throughput sequencing on the MiSeq

Sequencing platform (Illumina, Inc., USA), according to Christoff et al. [27]. Bacteria

and Archaea primers were used to amplify the V3-V4 regions of the 16S rRNA gene [28,

29]. In the first PCR (PCR 1), the primers were analyzed based on Illumina sequences

using the TruSeq structure adapter (Illumina, San Diego, CA). The DNA was denatured

at 95°C for 5 min, followed by 25 cycles of 45 s at 95°C, 30 s at 55°C, and 45 s at 72°C,

and a final extension at 72°C for 2 min. Then, a second PCR (PCR 2) with indexing

sequences was performed. In PCR 2, the DNA was denatured at 95°C for 5 min, followed

by 10 cycles of 45 s at 95°C, 30 s at 66°C, 45 s at 72°C, and a final extension at 72°C for

2 min. The Illumina 16S protocol was used (Illumina Technical Note 15044223 Rev. B)

to compare the PCR assays. The fungal community was accessed through PCR

amplification of the ITS region using the primers ITS1 [30] and ITS2 [31], according to

Schmidt et al. [30]. The final PCR reaction was cleaned up using AMPureXP beads

(Beckman Coulter, Brea, CA) and samples were pooled in sequencing libraries for

quantification [27].

Amplicon estimates were performed with Picogreen dsDNA assays (Invitrogen, USA),

and libraries were diluted for an accurate qPCR quantification using KAPA Library

Quantification Kit for Illumina platforms (KAPA Biosystems, Woburn, MA). The

identification of the taxonomic groups was based on the 16S rRNA database of the

Neoprospecta Microbiome Technologies database (NeoRefdb). Sequences with at least

99% identity in the reference database were taxonomically assigned [27].

2.3 Statistical analysis

The biogas production and BMP values of the two fish waste experiments were compared

with Tukey’s HSD test of differences of means using SISVAR [32]. The 5% significance

level was considered.

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3. Results and Discussion

3.1 Fish processing wastes

Identifying the characteristics of the substrates used in anaerobic digestion leads to a

better understanding of the relationship between biomass and biogas production. Under

anaerobic conditions, fats, proteins, and carbohydrates are biologically degradable and

can be converted to biogas; therefore, the biogas yield depends on the content of these

organic compounds [30]. The composition of the FW and FCOW used in the experiments

are shown in Tables 1 and 2. Although the nitrogen content of FW and FCOW were

slightly high, the C/N ratios were quite balanced, as also pointed by Vivekanand et al.

[12]. These authors inferred that the inoculum influences the real C/N ratios, improving

the conditions for AD. Although nitrogen is extremely necessary for the microbial

growth, the AD can be inhibited when there is an accumulation of free ammonia due to a

large amount of nitrogen in the substrate. Even though FW and FCOW are rich in

nitrogen, this issue was not observed during our experiments, the balancing may have

been provided by the acclimated added inoculum. The pH was measured at the beginning

and at the end of AD. It was verified that in the beginning the values were negative control

7.90 + 0.14; microcrystalline cellulose 7.40 + 0.03; FCOW 7.85 + 0.01; e, FW 7.70 +

0.02. pH measurements inform the development of AD. The values obtained show that

AD occurred adequately, since at the end of AD, it was verified that negative control pH

was 8.07 + 0.02; microcrystalline cellulose 8.12 + 0.02; FCOW 7.89 + 0.01; e, FW 7.97

+ 0.02.

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Table 2: Chemical composition of carbon, nitrogen, and hydrogen in fish waste (FW)
and fish crude oil waste (FCOW).

Substrate Total C Total N Total H C/N ratio

(%wt) (%wt) (%wt)
FW 52.6 + 0.1 8.1 + 0.1 9.8 + 0.1 6.5

FCOW 54.8 + 0.2 8.5 + 0.1 9.7 + 0.2 6.4

3.2 Potential biogas production

The methodology used in the study was the German Standard VDI 4630 (2006), which

says that the investigated substrate must be combined with inoculum and at the end of the

experiment, the inoculum production should be subtracted, indicating the potential of the

sample. The BMP value is calculated from the biogas production and the volatile solids

inserted in the reactor corresponding to the sample, as stated in the standard VDI. The

cumulative biogas production during the AD of microcrystalline cellulose, fish waste

(FW), fish crude oil waste (FCOW), and negative control (inoculum-only) are shown in

Fig. 1. The final biogas yield from each substrate was 657.38 mL gVS−1 (microcrystalline

cellulose), 791.78 mL gVS−1 (FW), 733.93 mL gVS−1 (FCOW) and 37.66 mL gVS−1

(negative control). The positive control (microcrystalline cellulose) allowed the

evaluation of the inoculum quality, and the obtained volume indicates that it was well-

functioning [33]. Aziz et al. 2018 [30] showed that fish waste is rich in fat and protein.

The decomposition of fats to produce biogas is a result of the high number of C and H

atoms in the molecules [30,32]. As confirmed by our results, substrates rich in proteins

frequently produce biogas with a high methane content [33]. By using a mono-substrate

we could obtain biogas with high levels of methane (Fig. 2) without using co-digestion.

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Biochemical potential biogas (mL gVS−1)

800

600

400

200

0
1 4 7 10 13 16 19
Time (Day)

Negative Control Fish Waste (FW) Fish Crude Oil Waste (FCOW) Microcrystalline cellulose

Fig. 1. Cumulative biogas production curves of microcrystalline cellulose, fish waste

(FW), fish crude oil waste (FCOW) and negative control using an acclimated inoculum

1,000

900
Biochemical potential (mL gVS−1)

800

700

600

500

400

300

200

100

0
Negative Control FW FCOW Microcrystalline cellulose

Biochemical potential of Biogas Biochemical potential of Methane

Fig. 2. Biochemical potential of biogas and methane of fish waste (FW), fish crude oil

waste (FCOW) and negative control using an acclimated inoculum.

The daily methane production during the batch digestion of cellulose, FW, FCOW, and

negative control in the bioreactors are shown in Fig. 3. The final methane yields

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(accumulated) were 336.53 mL gVS−1 (cellulose) 540.45 mL gVS−1 (FW), 426.36 mL

gVS−1 (FCOW), and 3.63 mL gVS−1 (negative control). The analysis of BMP retention

time depended on the biogas production rate, as predicted by the VDI 4630. The

experiment was considered finished when the methane production rate was undetectable

or less than 1% of the total amount during the three subsequent days. The total duration

of digestion in the two experiments varied from 14 (FW) to 17 days (FCOW) (Fig. 3)

200
Biochemical potential methane (mL gVS−1)

180
160
140
120
100
80
60
40
20
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Time (Day)

Negative Control FW FCOW Microcrystalline cellulose

Fig. 3. Daily methane yield from anaerobic digestion in batch bioreactors of cellulose,

fish waste (FW), fish crude oil waste (FCOW) and negative control.

The methane content in the daily biogas production in all treatments is shown in Fig. 4.

It is possible to observe that both substrates had, at many points of the biogas production

curve, a high methane content (~70%). The fish waste had the best daily methane

production performance and produced biogas with higher methane content than FCOW

in less time. By the end of the experiments, FW produced more methane than FCOW (p

< 0.05).

The volume of biogas and methane produced in our experiments support previous data

that indicated that the mono-digestion of fishery processing waste can be a viable

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alternative for biogas production. Kafle et al. [15] evaluated the use of fish waste ensilage

for biogas production and mixed the fish waste with bread waste and brewery grain waste.

After 96 days, the biogas and methane yields from the fish waste ensilages were 671–763

mL gVS−1 and 441–482 mL gVS−1, respectively [15]. The authors used a modified

Gompertz model and found digestion values similar to ours. The production of biogas

from three feedstocks (fish ensilage, manure, and whey) was evaluated using biochemical

methane potential tests by Vivekanand et al. [12] who verified that the mono-digestion of

fish ensilage had the highest biodegradability extent among all the studied substrates and

produced nearly 690 mL CH4 g-1 VS biogas [12]. Nges et al. [19] found biogas volumes

produced by fish waste and fish sludge (residue after the enzymatic treatment used to

extract fish oil and fish protein hydrolysate) similar to ours: 828.15 mL CH4 g-1 VS and

742.17 mL CH4 g-1 VS, respectively.

100%
Methane conteint in biogas (%)

90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Time (Day)

Negative Control FW FCOW Microcrystalline cellulose

Fig. 4. Methane production (%) from anaerobic digestion in batch bioreactors of

cellulose, fish waste (FW), fish crude oil waste (FCOW) and negative control.

Fish waste is the residue of the first step of oil extraction and it is probably composed of

a higher percentage of lipids and proteins than the fish crude oil waste. In our experiments,

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FW remained rich in lipids (fatty acids) and proteins and showed the highest biogas

production potential (p < 0.05), in comparison with FCOW. The main fatty acids of carp

oil are C18:1–w9 (oleic), C16:0 (palmitic), C16:1 (palmitoleic), C18:2–w6 (linoleic), and

C18:3 w3 (linolenic) [19]. The remaining lipids can be metabolized and degraded to

acetate and hydrogen through the β-oxidation by the microbial community and syntrophy

with hydrogenotrophic or acetoclastic methanogens [34].

Many studies have shown that biogas production using animal waste as a mono-substrate

generates little amount of biogas with low levels of methane, which may not always be

economically viable [35]. However, our results reinforce the technical and economic

viability of fish residues, corroborating other authors which used fishery processing

residues as the only substrate in AD [21,34,35].

Bioethanol is produced from sugarcane and is part of the Brazilian energy matrix that

successfully contributes to the outstanding positioning of the country in the global trend

of using new renewable energy sources [21]. However, one of the problems of producing

bioethanol is the treatment of vinasse wastewater [36] and the energy production from

the AD of vinasse has been seen as an alternative solution. According to Parsaee et al.

(2019) [36], the AD of 1 m3 of vinasse produces 10 m3 of biogas (with 60% methane).

The vinasse production is approximately 8–20 L per liter of ethanol and its carbon-

nitrogen ratio demands additional substrates (such as animal manure and organic

industrial waste) to improve the biogas yield [36]. Our results showed that FW and FCOW

can produce, respectively, around 205.0 and 181.0 m3 of biogas per ton of the respective

residue (containing ~70% methane). In our experiments, the production of FW from the

fishery industry was around 98 g per kg of fish and the production of FCOW was around

35 g per 100 g of FW. These residues did not require any addition to generate a high

methane content in the biogas production during anaerobic digestion.

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3.2 Microbial community composition

3.2.1 Bacteria and Archaea structure

Understanding the changes and mechanisms of the microbial community during the AD

of potential renewable substrates (biomasses) for the biogas production can improve the

efficiency of the process. The microbial community in the digestate at the end of the

anaerobic digestion of FCOW and FW is shown in Fig. 5. The samples were collected

when the daily volume of produced biogas was below 1% of the total accumulated after

three consecutive days [25]. This indicates a low microbial activity, a condition in which

the rates of DNA replication are low. However, the molecular analysis is not a limiting

factor in this condition, since the DNA may persist in the environment even after a cell

ceases its metabolic activity [34].

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Trichococcus
Pseudomonas
Proteiniclasticum
Enterococcus
Dietzia
Cryptanaerobacter
Clostridium
Brevundimonas
Aminobacterium
Alcaligenes

FCOW FW

Fig. 5. Genus-level bacterial and archaeal composition from fish crude oil waste (FCOW)

and fish waste (FW).

Amha et al. [34] reviewed the structure of the microbial community in the AD involved

in the digestion process and identified four main microbial classes in the microbiome:

Methanobacteria, Clostridia, Bacteroidia, and Synergistia. We identified Clostridia,

Synergistia, at the end of AD of both experiments, and the methanogenics

Methanosarcina and Methanosaeta (Methanomicrobia class) at the end of AD in of

FCOW and FW substrates. However, Gammaproteobacteria, represented by a single

species of the genus Pseudomonas was the predominant class in FCOW (∿40%),

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followed by Clostridia (27.5%) and Synergistia (∿15%). In FW, Clostridia was the

predominant class (67.5%), represented by four species of the genus Clostridium (one

uncultured Clostridium sp.), Cryptanaerobacter and Proteiniclasticum species, followed

by Synergistia class (∿20%).

In comparison with other microorganisms, both substrates had a low proportion of

methanogenic microorganisms, which may be related to the sampling time. Since the

DNA extraction was performed at the end of AD, i.e., a period of low methane production

(> 1%), the low abundance of methanogenic microorganisms at this period is

unsurprising. The methanogenic microorganisms was represented by Methanosaeta

concilii (∿1.5%) in FW and Methanosarcina acetivorans (∿2.5%) in FCOW.

Methanosaeta is an obligate acetoclastic methanogen that uses acetate as a substrate for

methanogenesis [36]. The limited presence of Methanosaeta concilii in the fish waste

reactor could have been associated with the abundance of Aminobacterium colombiense

(∿20%), as pointed by Lee et al. [39]. These authors examined changes in the microbial

community structure during cow grass digestion over three months and demonstrated that

the increase in Aminobacterium abundance was accompanied by a decrease in

Methanosaeta. Aminobacterium is a protein-degrading bacterium that produces acetate,

succinate, and propionate. Propionate accumulation, in turn, could interfere with the

development of Methanosaeta, decreasing its abundance. In the fish waste reactor, nearly

36% of sequences were identified as Cryptanaerobacter phenolicus, previously reported

as a phenol-degrading bacterium isolated from methanogenic environments [38]. Phenol

compounds have been found in the digestate from bioreactors degrading swine manure,

municipal solid waste, and plant materials, and are associated with the biodegradation of

aromatic amino acid such as tyrosine [39–41]. Proteiniclasticum ruminis (∿8%), detected

in FW, is a strictly anaerobic proteolytic bacteria [42]. Species of the genus Clostridium

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comprised 23.5% of the relative abundance of microorganisms at the end of the AD in

the FW experiment. The presence of species of the genus Clostridium (and other

microorganisms such as Synergistetes spp., Bacteroidetes spp., and Bacillus spp.) in

biogas reactors is considered suitable for the biogas production and could be involved in

the acetogenic phase [43].

Methanosarcina acetivorans is a methanogen that can metabolize acetate, hydrogen, and

C1 compounds. Depending on the reactor conditions, methanogens of the Methanosaeta

group may be inhibited and mixotrophic microorganisms might appear (i.e., organisms

able to consume acetate and hydrogen to produce methane). Methanosarcina is a

mixotrophic organism that became predominant in the AD of the FCOW reactor and

would explain the absence of Methanosaeta. The evaluation of the AD experiment

occurred after a period of starvation of the anaerobic digester system and compounds such

as total ammoniacal nitrogen could accumulate in the system. This could generate a stress

factor for the microbiota, leading to changes in the microbial structure [44]. Thus, the

presence of denitrifying bacteria capable of transforming nitrate and ammonia into N2

[45], such as Pseudomonas caeni (∿40% relative abundance in the AD reactor), is

acceptable.

At the end of AD, the differences observed between the microbial communities of FW

and FCOW could be attributed to the individual adaptation of microorganisms to the

substrate of each reactor system. Jonge et al. [44] found similar results when studying the

evolution and population dynamics of microbial communities in anaerobic digesters

during stable operation and recovery after a prolonged starvation.

3.1.2 Fungal community structure

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Both reactors had the same fungal diversity, identified based on the ITS1-ITS2 rRNA

gene sequencing (Fig. 6).

Xeromyces
Wallemia
Thermomyces
Scedosporium
Rhodotorula
Pichia
Penicillium
Humicola
Galactomyces
Fusarium
Cystobasidium
Cryptococcus
Acremonium
Cladosporium
Candida
Aspergillus
FCOW FW
Fig. 6. Genus-level composition of the fungal community from fish crude oil waste

(FCOW) and fish waste (FW).

At the end of AD, the same genera were found in both substrates with slight variations in

their abundances depending on the reactor. The origin of these fungi is probably

associated with the added microbial inoculum, which was composed of a stable fungal

community. Bengelsdorf et al. [46] studied a mesophilic, continuously operating, biogas

plant and found that the resident microbiota used food residues, stale bread, and other

waste co-substrates together with pig manure and maize silage to produce biogas The

authors observed that the fungal community was kept constant over a 1-year period [46].

Microscopic fungi were predominant in our experiments. Ascomycota was the most

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abundant phylum in both substrates, mainly represented by the orders Eurotiales

(Aspergillus spp., Penicillium spp., and Thermomyces sp.), Sordariales (Humicola sp.),

Saccharomycetales (Galactomyces spp., Candida sp., and Pichia sp.), Sporidiales

(Rhodotorula sp.), Capnodiales (Cladosporium sp.), Microascales (Scedosporium sp.),

and the genus Xeromyces sp. (undefined order — Incertae sedis).

Facultatively anaerobic fungi were found among the genera identified in the reactors.

Several reports indicated that besides yeasts (such as Galactomyces, Candida, Pichia,

Rhodotorula sp.), mycelial fungi (such as Aspergillus and Penicillium ) are also able to

grow under anaerobic conditions [46–51]. Some fungi, such as Ascomycota yeasts, have

a fermentative metabolism. Yeasts are known to have proteolytic and lipolytic activities

(in both aerobic and anaerobic conditions) and probably participate in the biodegradation

of the evaluated fish processing residues composed basically of residual lipids and protein

[52]. Besides participating in the biomass hydrolysis, these organisms can produce

volatile fatty acids and play an important role in the production of biomethane. For

instance, species of the genus Rhodotorula and Cryptococcus are associated with lipid

production [53]. Fisgativa et al. [20] recorded the possible participation of yeasts in the

anaerobic and aerobic degradation process of food waste and identified Ascomycota as

the predominant yeast in the studied reactor.

Our molecular analysis did not detect strictly anaerobic fungi at the end of AD. Currently,

eight genera of anaerobic fungi from the phylum Neocallimastigomycota are known:

Neocallimastix, Orpinomyces, Caecomyces, Cyllamyces, Piromyces, Anaeromyces,

Oontomyces, and Buwchfawromyces [54]. These fungi are associated with the digestive

tract of large mammalian herbivores, where they play an important role as primary

colonizers of the ingested food [55,56]. Fungi can be transported into biogas plants

attached to the animal waste used as substrate [56,57]. According to Dollhofer et al. [58],

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anaerobic fungi can survive only for a short period in conventional biogas plants and they

are strongly dependent on the process conditions. The close association between

anaerobic fungi and methanogens archaea is well known and has been widely reported

[17, 59–63]. Therefore, according to these studies, it is acceptable that no anaerobic fungi

were identified in the reactors of our experiments.

The phylum Basidiomycota was represented by the orders Wallemiales (Wallemia sp.)

and Tremellales (Cryptococcus sp.). Chen et al. [61] conducted an AD experiment in a

bioreactor designed for methanogenic degradation of cellulose-containing sewage. The

authors found out that Basidiomycota fungi and the methanogen Methanoregula

apparently established a syntrophic association during the process [64]. Ravella et al. [48]

were able to isolate different viable fungal strains, including a possible new species of the

genus Cryptococcus, and showed that the biogas reactor contained adequate conditions

even for the growth of facultative anaerobic fungi.

4. Conclusions

The biogas processing resulted in a highly methane yield, suggesting that fish residues

are a promising alternative for biogas production in mono-digestion processes. High-

throughput sequencing identified the main microorganisms in both substrates, which

belonged to Bacteria, Fungi, and Archaea. The dominant Bacteria were Clostridia,

Synergistia, and Gammaproteobacteria. The Archaea group was represented by

Methanosaeta and Methanosarcina. Ascomycota was the prevalent phylum in FCOW

and FW, represented by Eurotiales, Sordariales Saccharomycetales, Sporidiales,

Capnodiales, and Microascales. The phylum Basidiomycota was represented by the

orders Wallemiales and Tremellales. Therefore, both wastes used in our experiments can

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be recommended as substrates to produce biogas through anaerobic digestion yielding a

high methane content.

Acknowledgements

The authors would like to thank the University of Vale do Taquari (UNIVATES) and the

Univates Technology and Scientific Park (TECNOVATES) for their collaboration and

financial support during the development of this research. We also thank the Council of

Scientific and Technological Development (CNPq) for the financial support and a

scholarship

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