Scholarly article on topic 'Metagenome changes in the mesophilic biogas-producing community during fermentation of the green alga Scenedesmus obliquus'

Metagenome changes in the mesophilic biogas-producing community during fermentation of the green alga Scenedesmus obliquus Academic research paper on "Biological sciences"

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{Microalgae / " Scenedesmus obliquus " / Biogas / "Microbial community" / "Next-generation sequencing" / Metagenomics}

Abstract of research paper on Biological sciences, author of scientific article — Roland Wirth, Gergely Lakatos, Tamás Böjti, Gergely Maróti, Zoltán Bagi, et al.

Abstract A microalgal biomass offers a potential alternative to the maize silage commonly used in biogas technology. In this study, photoautotrophically grown Scenedesmus obliquus was used as biogas substrate. This microalga has a low C/N ratio of 8.5 relative to the optimum 20–30. A significant increase in the ammonium ion content was not observed. The methane content of the biogas generated from Sc. obliquus proved to be higher than that from maize silage, but the specific biogas yield was lower. Semi-continuous steady biogas production lasted for 2 months. Because of the thick cell wall of Sc. obliquus, the biomass-degrading microorganisms require additional time to digest its biomass. The methane concentration in the biogas was also high, in co-digestion (i.e., 52–56%) as in alga-fed anaerobic digestion (i.e., 55–62%). These results may be related to the relative predominance of the order Clostridiales in co-digestion and to the more balanced C/N ratio of the mixed algal–maize biomass. Predominance of the order Methanosarcinales was observed in the domain Archaea, which supported the diversity of metabolic pathways in the process.

Academic research paper on topic "Metagenome changes in the mesophilic biogas-producing community during fermentation of the green alga Scenedesmus obliquus"

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journal of Biotechnology xxx (2015) xxx-xxx

ELSEVIER

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Journal of Biotechnology

journal homepage: www.elsevier.com/locate/jbiotec

Metagenome changes in the mesophilic biogas-producing community during fermentation of the green alga Scenedesmus obliquus

Roland Wirtha, Gergely Lakatosb, Tamas Bojtia, Gergely Marotib, Zoltan Bagia, Mihaly Kisc, Attila Kovacsd, Norbert Acs3, Gabor Rakhelyae, Kornel L. Kovacs a'f

a Department of Biotechnology, University of Szeged, Kozep fasor52, H-6726 Szeged, Hungary

b Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62, H-6726 Szeged, Hungary c Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62, H-6726 Szeged, Hungary d Phytoplankton and Macrophyte Research Team, Balaton Limnological Institute, Klebersberg Kuno 3, H-8237 Tihany, Hungary e Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62, H-6726 Szeged, Hungary f Department of Oral Biology and Experimental Dental Research, University of Szeged, Tisza L. krt. 64, H-6720 Szeged, Hungary

ABSTRACT

A microalgal biomass offers a potential alternative to the maize silage commonly used in biogas technology. In this study, photoautotrophically grown Scenedesmus obliquus was used as biogas substrate. This microalga has a low C/N ratio of 8.5 relative to the optimum 20-30. A significant increase in the ammonium ion content was not observed. The methane content of the biogas generated from Sc. obliquus proved to be higher than that from maize silage, but the specific biogas yield was lower. Semi-continuous steady biogas production lasted for 2 months. Because of the thick cell wall of Sc. obliquus, the biomass-degrading microorganisms require additional time to digest its biomass. The methane concentration in the biogas was also high, in co-digestion (i.e., 52-56%) as in alga-fed anaerobic digestion (i.e., 55-62%). These results may be related to the relative predominance of the order Clostridiales in co-digestion and to the more balanced C/N ratio of the mixed algal-maize biomass. Predominance of the order Methanosarcinales was observed in the domain Archaea, which supported the diversity of metabolic pathways in the process.

© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

ARTICLE INFO

Article history:

Received 12 December 2014

Received in revised form 8June 2015

Accepted 12June 2015

Available online xxx

Keywords: Microalgae Scenedesmus obliquus Biogas

Microbial community Next-generation sequencing Metagenomics

1. Introduction

The increasing global demand for energy heavily depends on fossil fuels such as oil, coal, and natural gas. With the anticipation of fossil fuels becoming exhausted in the foreseeable future, novel strategies need to be discovered for alternative energy generation. Photosynthetic biomass-based fuels are widely regarded as sustainable alternatives to fossil fuels. Biofuels and other forms of bioenergy are currently produced from terrestrial plants (Schenk et al., 2008). Microalgae may represent an alternative to terrestrial crops because they have higher photosynthetic efficiencies and higher growth rates, and can be grown in saline waters and marginal land areas (Posten and Schaub, 2009; Debowski et al.,

* Corresponding author at: Department of Biotechnology, University of Szeged, Közep fasor52, H-6726 Szeged, Hungary.

E-mail addresses: roland.w@freemail.hu (R. Wirth), lakatos.gergely@brc.mta.hu (G. Lakatos), tamas.bojti@gmail.com (T. Böjti), maroti.gergely@brc.mta.hu (G. Maroti), bagiz@brc.hu (Z. Bagi), kis.mihaly@brc.mta.hu (M. Kis), kovacs.attila@okologia.mta.hu (A. Kovacs), acsn@brc.hu (N. Acs), rakhely@brc.hu (G. Rakhely), kovacs.kornel@brc.mta.hu (K.L. Kovacs).

2013). Microalgae can be harvested practically all year round, which results in enhanced biomass-production efficacy. Cultivation can be carried out in closed photobioreactors or in open ponds. Open systems are usually considered economical, whereas closed systems are more efficient from the aspects of biomass production and control of the cultivation parameters (Edward, 2009; Edward, 2009), so that either concept may be competitive in the various applications (Guccione et al., 2014).

Microalgal biomass is of potential for anaerobic digestion (AD) as it can have high contents of lipids, carbohydrates, and proteins, and does not contain recalcitrant lignin (Chen et al., 2009; González-Delgado and Kafarov, 2011; Yen et al., 2013; Ward et al., 2014). With regard to the enormous biodiversity of microalgae and the recent developments in genetic engineering, this group of organisms is clearly one of the most promising sources for new-generation biofuels. Research on the AD of algal biomass goes back more than 50 years (Golueke et al., 1957). That early study made a comparison of sewage sludge and green algae (Scenedesmus sp. and Chlorella sp.). Following such pioneering experiments, relatively few investigations dealt with the anaerobic fermentation of microalgae (Uziel et al., 1974; Keenan, 1977; Binot et al., 1977; Samson and LeDuyt,

http://dx.doi.org/10.1016/jjbiotec.2015.06.396

0168-1656/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativec0mm0ns.0rg/licenses/by-nc-nd/4. 0/).

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Fig. 1. The scheme and timeline of the experimental set-up. A. the time course of the various stages of the experiment. First a 2-3 weeks long "incubation" period indicates the set-up phase of the reactors. During the "start-up" phase the reactors, already producing biogas from a mixture of pig slurry and maize silage, were fed with the selected substrates, i.e., algal biomass, maize silage or a 1:1 mixture thereof. This lasted about 4 weeks. In the "biogas measurement" stage biogas yield from the selected substrates and metagenomic changes were monitored. B: The sample preparation steps for metagenomic studies. Note: the individual steps do not correspond to the time scale indicated in A.

1982; Becker, 1983; Hernández and Córdoba, 1993) until recently. Various freshwater and salt water algal strains were compared under mesophilic conditions (Mussgnug et al., 2010) and the biogas potential proved to depend strongly on the species and on the thickness of the cell wall. One noteworthy feature was that the CH4 content of the biogas from the microalgae was 7-13% higher than that from maize silage (Mussgnug et al., 2010).

Intensive studies of the microbial communities of maize silage-fed anaerobic digesters (Schlüter et al., 2008; Krause et al., 2008; Kröber et al., 2009; Jaenicke et al., 2011; Wirth et al., 2012; Stantscheff et al., 2014; Ziganshina et al., 2014) have demonstrated that, although the anaerobic fermentation conditions (fermenter size, feedstock composition, and origin, mixing, inoculum composition, etc.) differed somewhat, but the substrates were essentially the same (maize silage and pig manure) and coherent data sets could be collected. Members of the phyla Firmicutes and Bac-teroidetes played the most important roles in the hydrolysis of the plant biomass and in the secondary fermentation. In particular, many Clostridium species were identified which possess cellu-lolytic and H2-producing activities, both properties probably being essential for the efficient degradation of the biomass. Methanomi-crobiales, the most abundant order in the domain Archaea in large scale AD process, uses CO2 as a carbon source and H2 as an electron donor for methanogenesis. The general features of the community structure in the domain Bacteria appeared similar in the various studies, but alterations were noted in the domain Archaea. The most sensitive element in the microbiological food chain yielding biogas is the methanogenic group, changes in which may be associated with seasonal fluctuations or the variation of specific fermentation conditions (Rastogi et al., 2008; Lee et al., 2009). As an example, acetoclastic tend to predominate in biogas fermenters operated with wastewater sludge, while reactor communities fed with more diverse substrates prefer hydrogenotrophic methano-genesis (Sundberg et al., 2013).

Little is known about the microbial community of an anaerobic digester sustained with algal biomass. Ellis et al. (2012) employed 454 pyrosequencing to study the archaeal community during microalgal fermentation following the PCR amplification of mcrA gene regions. In alga-fed mesophilic AD inoculated with wastewater sludge, the majority of annotated mcrA sequences were assigned to the genus Methanosaeta. That investigation did not extend to the composition of the bacteria in the substrate algal biomass or within the anaerobic digester, although heavy bacterial representation could be expected in algal biomass cultivated in open ponds filled with wastewater. A more recent study (Wirth et al., 2014) analyzed the complete microbial community of a laboratory-scale AD fed with an algal-bacterial co-culture. A large proportion of bacteria

belonging to the genera Rhizobium and Burkholderia lived in apparent syntrophic community together with the microalgal biomass, which changed the bacterial community composition significantly. This effect obscured the changes in the domain Bacteria as a result of the algal feedstock. The pronounced alterations observed in the domain Bacteria did not affect the microbial composition of the domain Archaea (Wirth et al., 2015).

Scenedesmus obliquus is a common freshwater microalga which can accumulate high amount of oil (Breuer et al., 2014; Mandal and Mallick, 2009) and starch (Batista et al., 2014). It can grow in various industrial wastewaters (Mata et al., 2013; Hodaifa et al., 2008) in a relatively wide temperature range (Xu et al., 2012). We report here an AD process involving the use of a photoautotrophically grown Sc. obliquus microalgal biomass with the aim of determining the response of the biogas producing microbial community to the novel substrate. The microbial community was monitored during the process by using high-throughput sequencing technology. The AD parameters and microbial community in an anaerobic reactor fed with Sc. obliquus and a co-digestion of maize silage and algal biomass were compared with the corresponding data on maize silage alone as control.

2. Materials and methods

2.1. Sc. obliquus biomass production

For biomass production, a culture of Sc. obliquus obtained from the culture collection of algae and protozoa (catalog no. CAAP276/72) was cultivated under natural light illumination at ambient temperature in a 4000 L tubular photobioreactor by first Hungarian Algatechnic Ltd. (ELMAT). BG11 medium was used (Stainer et al., 1971; Rippka et al., 1979). The biomass yield was approximately 2 g L-1. The harvested biomass was stored at -20 °C until utilization.

2.2. Anaerobic fermentation and biogas analysis

Anaerobic fermentations were carried out in 5 L continuously-stirred tank reactors (CSTR) (Kovacs et al., 2013a) in fed-batch operational mode. The experimental design and time course are summarized in Fig. 1. The reactors were operated with a pig manure + maize silage mixture (Wirth et al., 2012) until the biogas yield became stable prior to the commencement of feeding, i.e., start-up phase with the algal/maize silage substrates. The three reactors were fed with distinct substrates from the beginning of the start-up phase. One fermenter was fed with Sc. obliquus biomass at a loading rate of 1 g oDML-1 day-1 (oDM = organic dry mat-

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Table 1

Substrates used in the experiments.

Substrate Wet mass N (mg/g) Wet mass C (mg/g) C/N ratio TS(%) oDM (%)

Maize silage 4.35 196.86 45.3:1 41.19 94.59

Sc. obliquus 8.10 72.22 8.9:1 16.99 97.71

N = nitrogen content; C = carbon content; TS = total solid content; oDM = organic dry matter content.

ter), while parallel fermenters were supplied with a mixture of Sc. obliquus + maize silage (each 0.5 g oDML-1 day-1) or with maize silage (1 g oDM L-1 day-1). Temperature was maintained constant at 37 ± 1.0 °C by an electronically heated jacket which surrounded the cylindrical apparatus. The pH was kept between 7 and 8, and the redox potential was less than -500 mV. After the 1-month start-up phase (weeks 1-4), the gas generated and its quality were measured daily. Gas volumes were measured with thermal mass flow devices (DMFC SLA5860S, Brooks) attached to each gas exit port. The composition of the evolved biogas was measured with a gas chromatograph (6890N Network GC System, Agilent Technologies) equipped with a 5 A molecular sieve column (length 30 m, I.D. 0.53 megabore, film 25 |im). Ultrapure N2 was used as carrier gas.

2.3. Determination of fermentation parameters

Organic dry matter (oDM): The dry matter content was determined by drying the biomass at 105 °C overnight and weighing the residue. Further, heating of this residue at 550 °C provided the organic total solids content.

Density measurement: Sample density was measured by an automatic density meter (Grabner Instruments, MINIDENS)

C/N: To determine C/N, an Elementar Analyzer Vario MAX CN was employed. This works on the principle of catalytic tube combustion under a supply of O2 at high temperatures (combustion temperature: 900°C, post-combustion temperature: 900 °C, reduction temperature: 830 °C, column temperature: 250 °C). The desired components were separated from each other with the aid of specific adsorption columns (containing Sicapent, in CN mode) and determined in succession with a thermal conductivity detector. Helium served as flushing and carrier gas.

NH4+-N: For the determination of NH4+ content, the Merck Spectroquant Ammonium test (1.00683.0001) was used. At the beginning of the experiment the samples contained 1400 mg NH4+-NL-1.

VOA/TIC (Volatile organic acids/Total inorganic carbon): 5 g of fermenter sample was taken for analysis and diluted to 20 g with distilled water. The subsequent process was carried out with a Pronova FOS/TAC 2000 Version 812-09.2008 automatic titrator. At the beginning of the experiment the VOA/TIC ratio was 0.2.

2.4. Substrate composition

The characteristics of the algal biomass and maize silage substrates are presented in Table 1.

2.5. DNA isolation for metagenomic studies

2 mL samples from the reactors were used for total-community DNA isolation. The extractions were carried out with a slightly modified version of the Zymo Research kit (Zymo Research, D6010). Parallel samples from each reactor were lysed with three different lysis mixtures (Table 2). After lysation and bead beating, the Zymo Research kit protocol was followed. The quantity of DNA was determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and a Qubit 2.0 Fluorometer (Life Technologies). DNA integrity was tested by agarose gel electrophoresis and with Agilent 2200 Tape Station (Agilent Technologies).

2.6. Next-generation DNA sequencing and data handling

The sample preparation for total metagenome sequencing of the pooled samples was carried out following the recommendations of the Ion Torrent PGM sequencing platform (Life Technologies). Sequencing was performed using Ion Torrent PGM 316 chips. The reads were analyzed and quality values were determined for each nucleotide. An average of 152,909 reads containing more than 31 million bp were identified. The average read length was 201 bp (Table 3). The individual sequences were further analyzed by using the MG-RAST software package (Meyer et al., 2008), which is a modified version of RAST (Rapid annotations based on subsystem technology). The MG-RAST server computes results against several reference datasets (protein and ribosomal databases) (MG-RAST, 2015). The acceptable percentage of identity was set to be >70%, the read length was >50 bp and the e-value cut-off was <10-6. The generated matches of external databases were used to compute the derived data (Wirth et al., 2012; Kovacs et al., 2013b; MG-RAST, 2015). The sequence data have been uploaded on the NCBI database, accession number SRA271138 and can be found on MG.RAST under project name "Scenedesmus fermentation".

3. Results and discussion

3.1. Biogas yield from Sc. obliquus

The amounts of biogas produced from the biomass substrates in the CSTRs were determined after a one-month start-up phase, i.e., in weeks 1-4 of the experiment (Fig. 1). During this preliminary period, the reactors were fed with the chosen substrate to ensure that all the remaining and digestible biomass from the inoculum (containing pig slurry + maize silage) had been degraded and did

Table 2

Lysis conditions for total-community DNA preparation.

Lysozymea 10% CTABb Genomic CTAB lysis bufferc Qiagen bufferd Zymo buffere

(|xL) (|xL) (|XL) (|xL) (|XL)

A - 100 - 100 550

B 250 100 - 100 300

C 250 - 300 200 -

a 100mg/mL(Applychem). b Cetyltrimethylammonium bromide (w/v).

c 1 M Tris-HCl 100 mL, 500 mM EDTA 50 mL, 5 M NaCl 300 mL, 10% CTAB, 20% SDS, pH 8 (Wirth et al., 2012). d ASL buffer from Qiagen QIAamp DNA Stool miniprep kit (Qiagen, 51,504). e From Zymo Research Fecal DNA kit (Zymo Research, D6010).

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Table З

Sequencing statistics.

AD Bases >Q20a Reads Mean read lenght (bp)

Start point 35,892,397 30.596.563 182,567 196

Sc. obliquus week 1 30,457,018 26,703,086 155,532 195

Sc. obliquus week 5 28,436,345 24,543,128 136,052 209

Sc. obliquus week 9 30,357,877 26,253,819 146,689 206

Co-fermentation week 1 39,261,836 33,955,385 194,460 201

Co-fermentation week 5 39,555,893 34,418,139 197,742 200

Co-fermentation week 9 33,463,221 28,819,903 170.369 200

Maize silage week 1 25,257,622 21,699,918 124,637 202

Maize silage week 5 28,438,018 24,579,600 140,986 201

Maize silage week 9 26,403,818 22,626,256 126.530 208

a Predicted quality (Q20): Predicted error rate of one percent.

Sc. obliquus Co-fermentation Maize silage

2. Specific CH4 production from the various biomasses.

not contribute to the biogas yield. The proper length of the startup phase was determined in separate (unpublished) experiments. Gas production data were collected during weeks 5-9. The extent of biogas generation from the Sc. obliquus biomass was compared with that from co-digestion of the algal biomass and maize silage; reactors fed with maize silage alone were used as controls. The CH4 concentration in the gas from the Sc. obliquus substrate proved to be 55-62%, which was comparable with previous findings (Mussgnug et al., 2010), although the average CH4 content was somewhat lower in our 5 L fermenter. The biogas CH4 concentration from the maize silage alone was 50-52%, as found previously (Amon et al., 2010). Co-digestion of the Sc. obliquus biomass + maize silage in a ratio of 1:1 yielded a CH4 content of 52-56%, a value intermediate between those for the maize silage and the algal biomass. The daily average generated biogas volumes were as follows: from the maize silage 3.20 L day-1, from the co-digestion 2.61 L day-1, and from the Sc. obliquus biomass 1.79 L day-1. Fig. 2 shows the specific average CH4 production levels in normal mL (mLN) calculated in (g oDM-1).

In biodiesel production, pure algal cultures are used to avoid contamination, which makes the production process expensive (Singh and Gu, 2010). The cost of the process can be reduced by using the algal residue in AD and the by-product biomass from biodiesel production is suitable for biogas generation (Sialve et al., 2009; Razon and Tan, 2011; Harun et al., 2011). Our results corroborated these findings. It is noteworthy that in biohydrogen production, pure algal cultures are not needed and this reduces the biomass cultivation price (Lakatos et al., 2014), while the algal-bacterial biomass remaining after biohydrogen production can be used for biogas yield (Wirth et al., 2015).

3.2. Process parameters during the AD fermentations

A constant value of VOA/TIC (Volatile organic acids/Total inorganic carbon) is a reliable indicator of a stable fermentation

Fig. 3. Weekly measured VOA/TIC ratios. The area between the dashed lines indicates the optimum range.

Fig.4. Weekly measured NH4+ concentrations. The dashed line indicates the highest value recommended by the various studies.

process (McGhee 1968; Nordmann, 1977). During the different AD processes, the average VOA was 2gL-1 and the average inorganic carbon was 10-12 g CaCO3 L-1 in all cases. Fig. 3 displays the weekly VOA/TIC ratios. Because of the low loading rate, the VOA/TIC ratios were on the low side, which allowed balanced operations.

The amount of NH4+ formed from nitrogen containing compounds present in the aqueous medium is an indicator of a stable biogas-forming process (Alexander, 1985). Theoretically, levels above 3000 mg NH4+ L-1 may have a negative effect on the methanogenic community, which is the most sensitive group of microbes in the AD process (Chen et al., 2008; Nielsen and Angelidaki, 2008).

As a result of the low C/N ratio (8.9:1) of the Sc. obliquus biomass, the NH4+ content steadily increased during the experiments, but remained under the recommended upper limit of 3000 mg NH4+ L-1 (Fig. 4). Sc obliquus develops a thick cell wall (Stainer et al., 1971), and the ammonification of AD may therefore be retarded. In

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the co-digestion, the higher C/N ratio of the maize silage balanced the increasing NH4+ level.

3.3. Microbial community changes during the AD processes

The composition of the microbial community was investigated four times during the AD processes: at the beginning of feeding with the selected substrate (start), one week later (week 1), when the system was working at full capacity (week 5), and at the end of the process (week 9, see Sections 2.5 and 2.6).

3.3.1. Microbiological compositions of the substrates

The microflora of the maize silage consisted primarily of representatives of the genera Lactobacillus and Acetobacter (Fig. 5). Members of the genus Lactobacillus are to be found in the intestinal flora and they also thrive on degrading plant biomass. These microorganisms produce lactic acid from mono- and disaccharides (Makarova et al., 2006). Members of the genus Acetobacter are acetate producers (Yamada and Yukphan, 2008).

Sc. obliquus was cultivated in an industrial-scale tubular photo-bioreactor. The algal biomass was contaminated with a very low amount (1%) of bacterial cells. These bacterial species belong predominantly in the genus Rhizobium. The interactions of Rhizobia and plants are well known and similar mutualism has also been observed in the case of several microalgal species (Keshtacher-Liebso et al., 1995; Watanabe et al., 2005; Nikolaev et al., 2008;

Amin et al., 2009; Kazamia et al., 2012; Xie et al., 2013; Kim et al., 2014; Wirth et al., 2015). These interactions facilitate the growth of algae and improve their resistance to environmental stresses.

3.3.2. Biogas-producing microbial community

The composition of the biogas-producing microbial community at the start of the experiment was very similar to that found in earlier studies in reactors fed with pig manure + maize silage (Wirth et al., 2012); it may therefore be regarded as an internal control with which to validate the metagenome sequencing method. In the detailed discussion of the metagenomic results, the unidentified sequences are disregarded.

3.3.2.1. Microbial community of maize silage fermentation (Bacteria domain). During the fermentation of maize silage, the dominant taxa were preserved and only small changes occurred in the composition of the taxa (Fig. 6). This was not surprising in view of the fact that the fermentation process had been maintained on maize silage supplemented with pig slurry prior to the start of the experiment. The members of the phyla Firmicutes and Bacteroidetes predominated. In the phylum Firmicutes, the order Clostridiales prevailed, followed by Bacillales, while in the phylum Bacteroidetes the order Bacteroidales was found most commonly.

3.3.2.2. Microbial community in the co-digestion (domain Bacteria). The microbial composition of the reactor fed with the Sc. obliquus

Fig. 5. Composition of the microbial community associated with the substrates: A. Maize silage, B. Sc. obliquus biomass. The communities are indicated at domain, phylum, class, and genus levels. The diagram on the right side of Fig. 4B shows the composition of the bacteria (total abundance 1%) in the algal biomass.

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Week 1 Week 5 Week 9

Fig. 6. Changes in the domain Bacteria of the microbial community at a phylum level.

algal biomass and maize silage displayed minor changes relative to that in the case of maize silage alone. The representatives of the phylum Firmicutes predominated in the bacterial community (Fig. 6). Within this taxon, the members of the order Clostridiales were found in great number. Clostridiales are well known as efficient cellulose-degrading bacteria (Schwarz, 2001); their thriving in the co-digestion is justified by their cellulase activity in the case of the presence of the maize silage. It should be noted that polysac-charide degrading metabolisms are significantly increased in the case of co-digestion (Fig. 7). This finding may be related to the observation that the co-digestion of microalgal biomass with waste paper improved the fermentation process in consequence of the higher C/N ratio of the mixed substrate and the induction of cel-lulase biosynthesis (Yen and Brune, 2007). The elevated cellulase activity may have contributed to the faster breakdown of the algal

cell wall and the efficient release of valuable nutrients from the algal biomass, thereby increasing the biogas yield.

3.3.2.3. Microbial community in the Sc. obliquus AD (domain Bacteria). A pronounced shift in the biogas-producing microbial community was seen when the only substrate was the Sc. obliquus biomass. Because of the low bacterium contamination (1%) of the algal biomass the changes could readily be observed. Within the phylum Bacteroidetes, a predominance of the order Bacteroidales developed, with the concomitant decline of the representatives of the Clostridiales (Figs. 6 and 8), because of which the digestion of the microalgal cell wall was probably not as effective as in the co-digestion.This affected the subsequent steps in the biogas microbial food chain, influencing the biogas yield. Although Bacteriodales, which can degrade cellulose have been found in biogas reactors

Fig. 7. Distribution of identified polysaccharide degrading and metabolism functions at week 5. Open column: at "start", light grey: maize silage, darker grey: Sc. obliquus, black: co-digestion.

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Fig. 8. Changes in the domain Bacteria of the microbial community at the order level.

»« V i

0 15 9

Fig. 9. Eukaryotic sequences in the reactors. Black: of Sc. obliquus, Grey: co-digestion, light grey: maize silage.

(Betian et al., 1977; Bjursel et al., 2006; Wirth et al., 2012), this order has primarily been considered to be a major participant in the secondary fermentation (Delbès et al., 2001; Hanreich et al., 2013). The ineffective degradation of the Sc. obliquus biomass is reflected in the abundance of eukaryotic DNA sequences in the reactors (Fig. 9). The eukaryotic DNA content in the samples taken from the algal biomass AD was three times higher than that in the case

of the maize silage-fed digester, suggesting that the algal cell wall was more recalcitrant than the lignocellulosic material of the maize silage to microbial degradation.

3.3.2.4. The domain Archaea. The microbial composition of the domain Archaea was somewhat different from those in several previously studied mesophilic fermenters fed with maize (Schlüter et al., 2008; Krause et al., 2008; Kröber et al., 2009). Seasonal or uncontrolled factors may be involved in the background of this phenomenon (Rastogi et al., 2008; Lee et al., 2009). The order Methanosarcinales predominated in the Archaeal community throughout the entire study, practically independently of the substrate used, and their number even increased in time in the reactors containing the algal biomass. In an earlier study, in which the specific methanogen marker gene mcrA was monitored by next generation sequencing, the order Methanosarcinales was found in greatest abundance, and within this taxon the strictly acetoclastic genus Methanosaeta was predominant (Ellis et al., 2012). Interestingly, this genus was present in our fermentors too, though in less abundance. Within the order Methanosarcinales, the genus Methanosarcina was found in high abundance (Fig. 10). The main difference between the two genera of Methanosarcinales is that the members of the genus Methanosarcina are able to carry out all three pathways of methanogenesis, i.e., hydrogenotrophic, acetoclastic, and methylotrophic (Sirohi et al., 2010), while Methanosaeta can function only in the acetoclastic mode. Similarly, to our previous

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Fig. 10. Changes in the domain Archaea of the microbial community at the order level.

results (Wirth et al., 2015), the feeding with the microalgal biomass did not cause any appreciable changes in the methanogenic community.

4. Conclusions

The diversity of microalgal species allows their use in various ways. With the help of microalgae, biohydrogen, biodiesel, biogas or other valuable products can be produced. For the production ofbiodiesel and other valuable commodities, microbiologically pure algal biomass is needed, which increases the biomass production costs. In a biorefinery approach, the microalgal biomass that

remains after the extraction of various chemicals is also a good substrate for biogas generation (Sialve et al., 2009; Lakaniemi et al., 2011). We have demonstrated in a separate study that biohydro-gen generation is feasible by means of algal-bacterial co-culturing (Lakatos et al., 2014), and the biomass used is also an appropriate substrate for biogas generation in a two-step process (Wirth et al., 2015).

In the present study, a Sc. obliquus algal biomass was tested. The CH4 content of the produced biogas (55-62%) proved to be higher than that from the commonly used maize silage (50-52%), but the biogas yield estimated on the basis of the organic material input was lower than that from maize silage. Stable operation was

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R. Wirth et al. | Journal of Biotechnology xxx (2015) xxx-xxx 9

achieved during the 2-month duration of the experiment. No sign of any upcoming process failure was observed, in spite of the low C/N ratio (8.9:1) of the Sc. obliquus algal substrate. Sc. obliquus has a thick cell wall, and a slow, though steady increase in ammonium ion content was therefore observed. Because of the delay in the attainment of efficient degradation of the algal biomass, a longer retention time is needed than the conventional retention times based on maize silage fermentation. In co-digestion, the maize silage added to the algal biomass increased the C/N ratio considerably and improved the digestibility of the microbial biomass and balanced the operation.

Metagenome analysis demonstrated that the composition of the microbial communities present in the AD reactors fed with the various substrate combinations changed as a result of the microalgal biomass. During the microalgal AD process, members of the order Bacteroidales became predominant. When the algal and maize biomasses were mixed in 1:1 oDM ratio, thereby increasing the C/N ratio of the substrate, the predominance of the order Clostridiales was maintained. The members of this order are noteworthy in the degradation of cellulose-containing materials; and they therefore appear important for microalgal AD processes too. The high cellu-lase activities of the members of the Clostridiales and the balanced C/N ratio led to co-digestion proving an efficient way to use algal biomass. In the control reactors fed with maize silage alone, the microbial taxa belonging to the phyla Firmicutes and Bacteroidetes persisted.

The pronounced changes observed in the domain Bacteria did not take place in the Archaeal community. The order Methanosarci-nales, and within it the representatives of the metabolically versatile genus Methanosarcina predominated, regardless of the substrate composition.

Author contributions

RW, ZB, and NA developed the DNA extraction protocol, designed and performed the experiments and contributed to the evaluation of the metagenomic data. GL and TB took part in the execution of the anaerobic fermentation process and measurements. MK and AK arranged the large-scale algal fermentation with ELMAT Ltd., GM organized and performed the DNA sequencing work and participated in the evaluation of the data. KLK and ZB conceived the project, participated in its design and in the evaluation of the data. RW and KLK compiled the manuscript. GR supervised the operation and participated in the writing of the manuscript. All the authors have read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interest. Acknowledgments

The authors thank Ms. Netta Bozoki for technical assistance and ELMAT Ltd. for the algal biomass cultivation. This work was supported by the domestic grants GOP-1.1.1-11-2012-0128 and PIAC13-1-2013-0145 provided by the Hungarian Government, and financed by the Research and Technology Innovation Fund. Support from the EU project H2020-LCE-2014-2015-646533 BIOSURF is gratefully acknowledged.

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