BACKGROUND
the problems associated with climate change, and the limited supply of fossil fuels has led to an increasing interest in renewable energy sources.
one of these alternative energy sources is biogas  which is obtained through the anaerobic digestion of organic matter  <cit> .
in recent years, energy crops, crops used to produce energy in form of biofuels, have contributed over 50 % of the total biogas production  <cit> .
however, the use of such crops as substrate for biogas production has several drawbacks:  use of arable land;  consumption of large quantities of water and  increased use of fertilizers  <cit> .
an alternative to energy crops could be the use of algal biomass.
this would overcome the main problems mentioned above; algae do not compete for arable land and with algae it is possible to close the water and nutrient balances  <cit> .
anaerobic digestion of the microalga spirulina was studied in the late 80s by several authors .
however, the circumstances at that time, low oil prices and less environmental concerns, led to a loss of interest.
the need to use non-fossil energy sources and the biorefinery concept has brought back the attention to using algal biomass to produce biofuels .
in this context, the use of the microalga spirulina as substrate for the production of biogas has again become an interesting option.
anaerobic digestion is a biological process in which a wide range of anaerobic bacteria hydrolyze and ferment complex organic compounds first into organic acids, then further to acetate, hydrogen and carbon dioxide, which are subsequently transformed into methane by methanogens  <cit> .
a good understanding of the community structure and the functional interactions between the involved microbial populations, can contribute to the optimization of the anaerobic digestion of the desired substrate.
high-throughput dna sequencing technologies and their application for metagenome analysis have greatly enhanced the study of microbial communities of environmental samples.
several metagenome studies both of biogas producing plants and lab scale anaerobic digesters have been performed to date .
moreover, a recent work by wirth et al.,  <dig>  <cit>  studied changes in the metagenome of a mesophilic biogas reactor fed with scenedesmus obliquus green algae.
in the present study we combine the analysis of the anaerobic digestion process of spirulina with the analysis of the metagenome from the microbial community in a laboratory digester.
total dna was extracted from a lab scale bioreactor that converted spirulina into biogas and sequenced using the ion torrent  platform.
sequencing reads were assembled into contigs and these were analyzed with regard to the predicted genes, and by binning to acquire provisional whole genome sequences of abundant community members  <cit> .
in contrast to the cellulose rich substrates commonly used to date in many large scale biogas production plants, spirulina is a protein rich substrate  <cit> .
to determine if the microbial community in the spirulina fed lab-scale digester displays significant adaptation to the substrate, the mg-rast metagenome analyzer  <cit>  was used to compare the gene content of the obtained metagenome to that of a publicly available metagenome from a fully operative biogas plant fed mainly with cellulose rich material  <cit> .
RESULTS
biogas production via the anaerobic digestion of spirulina
the anaerobic digestion of freeze dried spirulina was studied using a  <dig>  l semi continuous stirred tank reactor  operated at ph  <dig> – <dig> , at 37 °c and with a 20-day hydraulic retention time .
after a 71-day start-up period constant daily biogas production , and constant process parameters , volatile solids ) were observed, indicating that the bioreactor had reached a pseudo steady state condition.
starting from this pseudo steady state, five different organic loading rates , from  <dig>  to  <dig>  g spirulina l− <dig> day− <dig>  were studied to determine the optimal olr for freeze dried spirulina.
the biogas production during each period was constant and, as expected, increased when the olr was increased .
the biogas production ranged from 470 ml of biogas day− <dig>  in period i, with an olr of  <dig>  g spirulina l− <dig> day− <dig>  up to 2210 ml biogas day− <dig>  in period v, with an olr of  <dig>  g spirulina l− <dig> day− <dig> table  <dig> biogas production and sludge characteristics
ch4
co2
n2;o2
codt
bod5
nh4
n.d non detected
biogas and methane production, biogas characteristics, specific biogas and methane productions and sludge characteristics from the anaerobic digestion of spirulina obtained with the five organic loading rate tested.
shown are the mean values of each period with their respective standard deviation
the increment in biogas production was not completely proportional to the loading rate .
apparently, at higher loading rates digestion of the algal biomass was no longer complete, which eventually led to substrate overload causing reactor failure .
this was apparent from:  the drop in biogas production at the end of period v ,  the decreasing methane content of the biogas at high loading rate and  the increase in all the parameters related to organic matter, ts, vs, total and soluble chemical oxygen demand  and five day biological oxygen demand  .
this accumulation of organic matter was especially acute during period v with a 30 % increase for ts, 37 % for vs, 50 % for total organic matter , and 163 % for bod <dig> compared to period iv.
soluble organic matter, acetic acid, propionic acid and ammonia also accumulated in period v .
spirulina is a protein rich substrate  <cit> , therefore its nitrogen content is high, which can explain the observed accumulation of total nitrogen .
the degradation of proteins leads to the production of ammonium nitrogen  which increased gradually from  <dig>  g l− <dig> in period p-i, to  <dig>  g l− <dig> in period p-v while, at the same time ammonia nitrogen  increased from  <dig>  g l− <dig> to  <dig>  g l− <dig> .
this increase in nh <dig> can be attributed both to the increase in substrate concentration and, according to the equation of anthonisen et al.
<cit> , to the increase of ph from  <dig>  to  <dig> .
methanogens are sensitive to ammonia  <cit>  and the accumulation of this compound can lead to digester failure.
the reported levels of free ammonia nitrogen in period v are high and comparable to other reported levels that caused reactor inhibition .
the accumulation of non-degraded biomass and vfas along with the relatively high total nitrogen  and free ammonia nitrogen  concentrations led to the reduction of the biogas production from 2651 ml day− <dig> on day  <dig> to 1586 ml day− <dig> on day  <dig>  which is consistent with a substrate overload in the digester.fig.
<dig> biogas, methane production and sludge characteristics.
left axis: daily biogas  and methane  production normalized to standard temperature and pressure conditions ; right axis: soluble cod ; acetic acid ; propionic acid , nh3-n  obtained from the anaerobic digestion of spirulina.
vertical dashed line indicates a change in the organic loading rate from  <dig>  g spirulina l− <dig> day− <dig> to  <dig>  g spirulina l− <dig> day−1
one of the main bottlenecks perceived for the anaerobic digestion of microalgal biomass is low biodegradability, which can result in low methane yields  <cit> .
to overcome this problem long hrt need to be applied in order to increase the residence time allowing the substrate to be further hydrolyzed  <cit> .
however, this on the long run can have a negative effect on the biogas production as accumulation of inhibitory substances such as ammonia can occur  <cit> .
our results show that at 20 days hrt an orl of  <dig>  g spirulina l− <dig> day− <dig>  results in a good compromise between an optimal methane yield and accumulation of toxic compounds.
at this olr, the specific biogas production  per gram of spirulina was 350 ml biogas with 68 % methane content .
moreover, the highest biodegradability, 42 %, was obtained, and no accumulation of inhibitory substances occurred in this period.
both values, spb and biodegradability are similar to those observed in other studies  <cit> .
metagenome analysis of the anaerobic digester community
dna was extracted at the end of period v  from the sludge of the spirulina digester and sequenced on a 318tm chip with the ion torrent personal genome machine  platform.
obtained sequence reads were quality filtered and trimmed .
the remaining reads were either assembled into contigs or analyzed directly with the mg-rast metagenome pipeline.table  <dig> sequencing statistics
assembly aa
assembly ba
assembly ca
dataset- <dig> sequencing data and assemblies statistics
asee material & methods for details about assembly settings
binning and 16s rdna taxonomy analysis of assembled contigs
three different assemblies were used for the detection of ribosomal 16s genes to taxonomically characterize the microbial community .
assembly a produced the largest contig,  <dig>  bp.
due to the stringent settings, assembly b produced a higher number of shorter contigs , while assembly c  produced contigs with the longest mean size and the longest n <dig> value, 4380 bp .
the same 16s rrna gene sequences were all assembled in each of the three assemblies.
however, for some of the dominant taxa, the length of assembled 16s rrna gene fragments were different between the three assemblies.
assembly a produced the longest contig for the methanosarcinales 16s rrna gene, assembly b yielded the longest anaerolineales 16s sequence, and in assembly c the longest 16s sequences for bacteroidales and clostridiales were obtained.
the 16s sequences of methanomicrobiales and thermotogales were assembled to approximately equal lengths in all three assemblies.
the 16s genes of flavobacteriales and lactobacillales were better assembled in assemblies a and c than in assembly b.
although assembly c yielded the longest average 16s sequences, the length of the archaeal 16s sequences was greater in assembly a. an mg-rast analysis of the reads submitted to assemblies a and c, respectively, revealed that the percentage of reads assigned to firmicutes was markedly higher in the reads submitted to assembly c. this suggests that, with the removal of reads shorter than 220 bp from the read set submitted to assembly a in order to obtain the read set for assembly c, firmicutes sequences were enriched in the latter.
contigs from assembly a were considered to better represent the diversity in the biogas reactor and were selected for binning using the metawatt v <dig>  pipeline to investigate the most abundant populations of the microbial consortium in more detail.
from the  <dig> obtained bins, after manual selection and curation,  <dig> remained which displayed characteristic tetranucleotide frequencies, assembly coverages and consistent phylogenetic signature and together accounted for almost 80 % of the total sequence data .
as was observed in other anaerobic digesters, populations affiliated with firmicutes were most abundant, and constituted 38 % of the total community , followed by bacteroidetes , chloroflexi  and thermotogae .
contigs affiliated with euryarchaeota comprised a single bin and represented only  <dig>  % of the total abundance.
two bins of unknown taxonomic origin accounted for 10 % of the sequenced data .table  <dig> characteristics and taxonomical classification of selected microbial bins
un.
tissierella
un.
clostridiales
un.
clostridiales
proteiniphilum
un.
anaerolineaceae
un.
thermotogaceae
unknown
un.
bacteroidetes
unknown
un.
methanomicrobia
cov coverage
abun abundance
un uncultured
characteristics and 16s rdna taxonomical classification of the  <dig> selected bins obtained from the spirulina metagenome
ribosomal rrna gene sequences corresponding to  <dig> of the  <dig> bins were identified among the contigs of the three different assemblies and/or recovered independently by iterative read mapping with emirge .
assembled 16s rrna sequences identical or highly similar to the emirge sequences were not detected.
however, 16s sequences similar to em- <dig>  and em- <dig>  were found among unbinned contigs of assembly a and/or contigs of assemblies b and c .
no assembled 16s sequence similar to em- <dig> was found.
unfortunately, the three 16s sequences obtained by emirge and affiliated with firmicutes  could not be assigned conclusively to any of the three firmicutes bins .
however, based on comparison of sequencing coverage between the recovered emirge 16s sequences and the contigs in the firmicutes bins it was most likely that the dominant organism, bin-a,  <dig>  % abundance,  was most closely related to tissierella praeacuta .
bins b and c were assigned to uncultured clostridiales .
the 16s rdna sequence classified as atopostipes  was assembled well in all three assemblies, yet it could not be linked to any of the bins.
based on the low sequencing coverage of this 16s sequence, the population was probably of relatively low abundance and poorly assembled .fig.
<dig> 16s rdna bacterial and archaeal phylogenetic trees.
phylogenetic trees of  bacterial and  archaeal 16s rdna sequences.
although the binning was only performed with contigs of assembly a, most of the binned 16s rdna sequences were also assembled in assemblies b and c. if so, for the tree the longest assembled sequence was chosen.
16s rdna sequences not assembled in assembly a, but in b or c, or detected by emirge are also included.
colored: sequences obtained from metagenomic reads.
assignment to metawatt bins is indicated if applicable.
reference sequences in bold: top hits in blast search against ncbi non-redundant nucleotide collection, bold + italics: top hits in blast search against ncbi reference rna sequences.
additional reference sequences in the bacteria tree represent genera detected in other anaerobic digesters.
16s rdna sequences of arthrospira platensis and methanopyrus kandleri were chosen as outgroups, respectively.
bootstrap values at nodes are obtained from  <dig> replicates and are only shown for branches with at least 50 % support .
the scale bar represents  <dig>  nucleotide substitutions per site.
accession numbers of reference sequences are available in additional file 2: table s7
as in a previous study of anaerobic digestion in which a proteinaceous substrate was used, clostridiales were the most abundant bacterial order  <cit> .
the genus tissierella has already been detected in other anaerobic digesters , but the specific function of members of this taxon in anaerobic digesters is still not clear.
however, members of this genus are known to require the presence of certain amino acids and formate for growth and they seem to be unable to utilize carbohydrates such as glucose, cellobiose or xylose  <cit>  which is in accordance to the type of substrate used in our experiment.
proteiniphilium,  a member of the bacteroidales that utilizes peptone and is unable to grow on carbohydrates  <cit> , was previously identified in several biogas studies  <cit> .
anaerolineales and thermotogales were also identified in other biogas reactors but in much lower abundance  <cit> .
their function in anaerobic digestion is not clear yet, however, their relatively high abundance when compared with other anaerobic reactors could be explained by the fact that they are known to utilize proteins as substrate  <cit> .
therefore they might play an important role in the degradation of protein-rich spirulina.
the 16s sequence in bin-d  shows  <dig>  % similarity to proteiniphilum acetatigenes str.
tb <dig> , with differing lengths of homopolymer stretches accounting for  <dig>  % of the differences.
the most similar  described sequence to contig_ <dig> is the porphyromonadaceae clone jx <dig> .
this clone sequence shows  <dig>  % similarity to the 16s gene of proteiniphilum acetatigenes, which supports the classification of bin-d as an unknown proteiniphilum.
among the archaea, we identified one bin, bin-j, with  <dig>  % of abundance, for which the 16s rdna fragments were classified as uncultured methanomicrobia .
methanomicrobiales were also identified as the most abundant methanogens in an anaerobic reactor fed exclusively with casein  <cit> .
a closer look at the 16s rdna phylogeny  suggests the possibility that two methanogenic populations may have been binned together, one related to methanomicrobiales and one to methanosarcinales.
members of these orders, which are able use h <dig>  co <dig>  formate and acetate as their c source  <cit> , are frequently encountered in anaerobic digesters  <cit> .
formate and acetate are both fermentation products of clostridiales and anaerolineales  <cit> , both populations present in high abundance in our experiment .
comparison of two metagenomes by short single read analysis
in parallel to this genome-focused analysis of assembled contigs, the effect of substrate on both taxonomic composition and presence of functional genes was also studied at the level of individual reads, by comparing the unassembled reads from our study to a publicly available metagenome from a biogas plant using the mg-rast platform.
sequencing dataset spirulina-s <dig>  was obtained from our anaerobic reactor fed with spirulina, a protein rich substrate, while the second sequencing dataset, maize-rye , originated from a biogas plant fed with a mixture of mainly cellulose rich substrates  <cit> .
effect on the microbial community composition
the general taxonomic composition did not appear to depend on the type of substrate used.
based on the mg-rast m5nr analysis of the metagenomic reads, bacteria clearly dominated in both datasets while archaea represented less than 10 % in the m-r dataset and merely 3 % in the spirulina-s <dig> dataset.
among bacteria, firmicutes, bacteroidetes and proteobacteria dominated in both sets, but differences could be seen in the abundances of other phyla.
for example, hits in thermotogae were more abundant in the protein rich substrate digester data  compared to 2 % in the cellulose rich substrate digester dataset.
a similar result was observed with chloroflexi,  <dig>  % in spirulina-s <dig> when compared to the m-r dataset,  <dig>  %.
on the other hand, within the archaea such a variety at phylum level was not seen, and as expected, euryarchaeota dominated with over 90 % of all archaea in both datasets.table  <dig> comparison of identified cog and specific pfams
ratio aam/cmb
a% of total identified cogs
b“amino acid transport and metabolism”/“carbohydrate transport and metabolism”
c% of total identified pfams
comparison of the cog categories obtained with the mg-rast platform  and the selected pfams obtained with the hmmscan
among the bacteria, clostridiales dominated in both datasets with  <dig> and 30 % of the total assigned reads in the spirulina-s <dig> dataset and the m-r dataset respectively, followed by bacteroidales, thermoanaerobacterales and bacillales .
in both datasets, m-r and spirulina-s <dig>  the genus to which most of the reads were assigned was clostridium  .
furthermore, as was also found by kovács et al.
<cit> , who used casein and pig blood as substrate, a relatively high number of reads in the spirulina-s <dig> dataset  were most similar to members of the genus alkaliphilus, which is in contrast to the m-r dataset, where only about 2 % of the reads were assigned to this genus .
bacteroides, recruiting  <dig>  % of the hits in the m-r dataset accounted for  <dig>  % in the spirulina-s <dig> dataset.
among other substrates, bacteroides are known to utilize cellobiose and xylose  <cit> , both absent in spirulina, which could explain their lower abundance in our reactor.
interestingly, candidatus cloacamonas, which accounted for  <dig>  % of the bacteria m-r reads,  <dig>  hits, recruited less than  <dig>  % of the hits in the spirulina-s <dig> dataset  .
this bacterium was also present in high abundance at the initial adaptation period of two biogas reactors fed with casein and pig blood and its detection was not possible after 12 weeks of substrate adaptation, which might indicate that it cannot survive without a source of carbohydrates  <cit> .
major differences regarding bacterial taxa were also seen for thermotogales and anaerolineales which were considerably more abundant in the dataset from the protein rich substrate digester, with  <dig> and 1 % respectively when compared to the dataset from the cellulose rich substrate biogas plant,  <dig>  and  <dig>  % respectively .
these two orders are known to include bacteria which utilize proteins as substrate  <cit> .fig.
<dig> comparison of the taxonomic classification of spirulina-s <dig> and m-r reads at order level.
percentage of the total taxonomic assigned reads of each dataset obtained with mg-rast m5nr representative hit tool .
a bacterial orders with the  <dig> most abundant assigned read hits and  all archaeal orders with assigned hits from the metagenomic reads
among the archaeal orders, methanomicrobiales clearly dominated in the m-r dataset, recruiting almost 75 % of the archaeal hits.
in the spirulina-s <dig> dataset, the combined presence of methanosarcinales  and methanomicrobiales  was observed , confirming the analysis of the assembled 16s rdna sequences .
methanosarcinales are known to be able to use acetate, h <dig> and co <dig> as substrate  <cit> , which are the main fermentation products of tissierella and other clostridia  <cit> , and they are usually dominant in reactors where vfas and nh <dig> are present in high concentration as in our case   <cit> .
on the other hand, methanomicrobiales do not use acetate but can grow on h <dig>  co <dig> and formate  <cit> , the latter a common fermentation product of bacteria of the phylum chloroflexi  <cit>  which in our dataset represented almost 8 % of the abundance .
the presence of two methanogenic populations in an anaerobic digestor is quite common and has also been observed in several other studies.
for example ziganshin et al.
<cit>  observed the presence of methanoculleus and methanosaeta in reactors fed with cattle manure and dried distillers grains, and of methanosarcina and methanoculleus in reactors treating maize straw and cattle manure, while li et al.
<cit>  also detected the same two groups of methanogens in a reactor treating multiple substrates .
this presence of different methanogens detected in such a broad range of substrates might indicate that, rather than the type of used substrate, the characteristics of the sludge  and the initial type of inoculum  determine which archaea thrive in anaerobic digesters.
in order to investigate whether the diversity of the species community was affected by the used substrates, the species diversities in both datasets, were analyzed by means of lorenz curves and simpson’s diversity index   and compared with each other.
the mixture of substrates used in the biogas plant , might lead to a higher bacterial diversity than in the reactor solely fed with spirulina.
however, lorenz curves, describing population evenness, of both datasets were similar , as well as simpson’s diversity index values .
the similarity in evenness and diversity in both bacterial populations can be explained by the fact that spirulina as such is not a “simple” substrate, as would be glucose, starch or glycerol, and therefore needs a microbial population with a certain complexity to be fully digested.
apparently, the differences between the substrate types  used in the two compared systems did not affect the diversity of the whole population, yet rather the abundance of certain bacterial taxa .
for archaea, on the other hand, lorenz curves and sdi indicated a difference in their diversity in the two studied datasets.
the archaeal population in the spirulina reactor was more even than the archaeal community in the maize-rye biogas plant .
given the same species richness, a more even population is also more diverse.
indeed, by calculating the sdi a higher diversity for the archaeal community in the spirulina reactor was observed  than in the m-r biogas plant .
the lower sdi for the archaea in the m-r dataset is best explained by the clear dominance of methanoculleus in the archaeal reads of this dataset .
taken together, the results suggest that the type of substrate used in anaerobic digestion mainly affects the bacterial composition, to some extent, at low taxonomic levels, especially at genus and species level.
proteolytic bacteria were probably present in all dominant phyla of the spirulina-s <dig> digester, whereas in the maize-rye biogas plant, cellulolytic bacteria were dominant.
regarding the archaea, an influence of the substrate on their presence and composition is not as clear as for the bacteria.
this could be explained by the fact that the archaea perform the final step in the process of anaerobic digestion, and their presence is probably more dependent on the population composition of the primary substrate degraders , of their metabolic products, the presence or absence of inhibitory compounds and the origin of the inoculum, rather than on the substrate itself.
effect on the abundance of functional genes
both datasets, spirulina-s <dig> and m-r were compared at functional level with two approaches, mg-rast’s cog comparison, and identification of specific protein domains  related to the cellulose degradation pathway and to protein and amino acid degradation .
due to the different types of substrates used, it was expected to see differences regarding the cogs related to amino acids and protein metabolism.
however, with respect to the detected functional genes, the differences between both datasets were minor .
the highest difference was observed in category l, “replication, recombination and repair”, which represented  <dig>  % of the hits in the spirulina-s <dig> dataset and  <dig>  % in the m-r dataset and contains cogs related to groups of genes which participate in the replication process of the microbial community.
this difference in abundance was significant  and could be explained by the fact that at the time of sampling of the spirulina-s <dig> dataset, day  <dig>  the reactor was suffering from substrate overload which resulted in an accumulation of toxic compounds  that hindered the correct function of the bacteria and reduced the daily biogas production .
the cogs related to category e, “amino acid transport and metabolism” were slightly more abundant in the protein rich dataset , than in the cellulose rich dataset  .
the higher relative abundance of cogs from category e in the spirulina-s <dig> dataset could be attributed to the type of substrate used.
however, as most of the cogs from this category represent enzymes involved in general metabolic processes, further research must be conducted in order to fully verify this result.
on the other hand, and contrary to what was expected, no significant differences  could be seen for the cogs from “carbohydrate transport and metabolism”  in both datasets .
however, it should be noted that, in category g, important differences could be seen among those cogs directly related to the degradation of cellulose, xylanose and other complex sugars.
for example, cog <dig> a “beta-xylosidase” represented  <dig>  % in the m-r dataset and was not detected in the spirulina-s <dig> dataset.
similarly, cog <dig>  an “l-arabinose isomerase”, was almost absent in the spirulina-s <dig> dataset  but represented almost  <dig>  % in the m-r dataset, likewise cog <dig> a “beta- <dig> -xylanase” and cog <dig> a “glycosidase” were both significantly less abundant in the spirulina-s <dig> dataset.
this difference in the abundance of cogs correlates with the type of substrate used, spirulina, which has a low content of complex sugars  <cit> .fig.
<dig> cog functional hierarchical classifications.
comparison of the cog classified reads of the spirulina-s <dig> and the m-r metagenomes obtained with the mg-rast metagenome analyzer .
only those categories with hits are represented.
x axis:  information storage and processing: j, translation, ribosomal structure and biogenesis; k, transcription; l, replication, recombination and repair;  cellular processes and signaling: d, cell cycle control, cell division, chromosome partitioning; m, cell wall/membrane/envelope biogenesis; n, cell motility; o, posttranslational modification, protein turnover, chaperones; t, signal transduction mechanisms; u, intracellular trafficking, secretion and vesicular transport; v, defense mechanisms;  metabolism: c, energy production and conversion; e, amino acid transport and metabolism; f, nucleotide transport and metabolism; g, carbohydrate transport and metabolism; h, coenzyme transport and metabolism; i, lipid transport and metabolism; p, inorganic ion transport and metabolism; q, secondary metabolites biosynthesis, transport and catabolism;  poorly characterized: r, general function prediction only; s, function unknown
to corroborate the results obtained with the cogs analysis, specific protein domains  related to cellulose degradation and to protein and amino acid degradation were searched for in both datasets .
pfam domains associated with cellulose summed up to  <dig>  % of the total identified pfams in the m-r dataset compared to  <dig>  % in the spirulina-s <dig> dataset .
the proteases related pfams were slightly more abundant in the spirulina-s <dig> dataset with  <dig>  % of all identified pfams compared to  <dig>  % in the m-r dataset .
also the amino acid degradation pfams were more abundant in the spirulina-s <dig> reads  than in the m-r reads .
two pfam ratios, proteases to cellulases ratio , and amino acid to cellulase ratio  were also calculated to determine the relative abundance of each group.
the p/c ratio in the spirulina-s <dig> dataset,  <dig> , was double the p/c ratio in the m-r dataset,  <dig> , and a comparable result was obtained with the aa/c ratios,  <dig>  for the spirulina-s <dig> and  <dig>  for the m-r dataset .
when a cog “amino acid transport and metabolism” to “carbohydrate transport and metabolism” ratio was calculated , the result was similar,  <dig>  for the spirulina-s <dig> dataset and  <dig>  for the m-r dataset .
the differences in the obtained p/c, aa/c and aam/cm ratios, plus the higher abundance of amino acid metabolism related cogs suggest that the microbial community in the spirulina reactor adapted to the type of substrate degraded, protein rich with low presence of cellulose.
however, since substrates from both reactors were not either pure carbohydrates or proteins, but consisted of both in different proportions, further studies are necessary to determine whether the observed differences in the presence of functional genes would become more distinct for longer run times of the spirulina digester, or if the microbial community was already at functional equilibrium when it was sampled for the metagenome analysis presented here.
binning of contigs vs classification of single short reads
in this work, the same metagenome has been analyzed by two different approaches, assembly of reads into contigs followed by a binning strategy combined with a 16s rdna analysis, and blasting of single reads against a general nucleotide database.
despite the differences in methodology, the results obtained with both approaches were consistent .
the first advantage of using the assembly/binning approach is that the obtained bins form a provisional whole genome sequence of the most abundant organisms .
this way, the function of individual populations can be inferred directly.
the second advantage is that taxonomical classification of contigs is more reliable than the classification of short reads.
in contrast, the main advantage of using mg-rast classification of short reads is that it is more sensitive because functions can be inferred from the presence of non-redundant reads originating from minor community members that remain unassembled.
since ~80 % of the sequenced reads could be mapped to the  <dig> bins corresponding to the  <dig> most abundant populations, approximately 20 % of the microbial community was below the detection limit of the assembly/binning approach.
CONCLUSIONS
the results presented in this study suggest that spirulina is a suitable substrate for the production of biogas with a mean production of 350 ml of biogas per gram of substrate.
as in previous studies, the most abundant bacterial populations making up the consortium performing digestion belonged to clostridia and bacteroidetes whereas the most abundant archaeal populations were affiliated with methanomicrobiales and methanosarcinales.
the microbial community present in the anaerobic digester was well adapted to the type of substrate used, based on taxonomic and functional inferences.
taxonomic analysis of assembled contigs with a binning approach produced results consistent with the classification of short single reads.

 the problems associated with climate change, and the limited supply of fossil fuels has led to an increasing interest in renewable energy sources. one of these alternative energy sources is biogas which is obtained through the anaerobic digestion of organic matter. in recent years, energy crops, crops used to produce energy in form of biofuels, have contributed over 50 % of the total biogas production. however, the use of such crops as substrate for biogas production has several drawbacks: use of arable land; consumption of large quantities of water and increased use of fertilizers. an alternative to energy crops could be the use of algal biomass. this would overcome the main problems mentioned above; algae do not compete for arable land and with algae it is possible to close the water and nutrient balances. anaerobic digestion of the microalga spirulina was studied in the late 80s by several authors. however, the circumstances at that time, low oil prices and less environmental concerns, led to a loss of interest. the need to use non-fossil energy sources and the biorefinery concept has brought back the attention to using algal biomass to produce biofuels. in this context, the use of the microalga spirulina as substrate for the production of biogas has again become an interesting option. anaerobic digestion is a biological process in which a wide range of anaerobic bacteria hydrolyze and ferment complex organic compounds first into organic acids, then further to acetate, hydrogen and carbon dioxide, which are subsequently transformed into methane by methanogens. a good understanding of the community structure and the functional interactions between the involved microbial populations, can contribute to the optimization of the anaerobic digestion of the desired substrate. high-throughput dna sequencing technologies and their application for metagenome analysis have greatly enhanced the study of microbial communities of environmental samples. several metagenome studies both of biogas producing plants and lab scale anaerobic digesters have been performed to date. moreover, a recent work by wirth et al., studied changes in the metagenome of a mesophilic biogas reactor fed with scenedesmus obliquus green algae. in the present study we combine the analysis of the anaerobic digestion process of spirulina with the analysis of the metagenome from the microbial community in a laboratory digester. total dna was extracted from a lab scale bioreactor that converted spirulina into biogas and sequenced using the ion torrent platform. sequencing reads were assembled into contigs and these were analyzed with regard to the predicted genes, and by binning to acquire provisional whole genome sequences of abundant community members. in contrast to the cellulose rich substrates commonly used to date in many large scale biogas production plants, spirulina is a protein rich substrate. to determine if the microbial community in the spirulina fed lab-scale digester displays significant adaptation to the substrate, the mg-rast metagenome analyzer was used to compare the gene content of the obtained metagenome to that of a publicly available metagenome from a fully operative biogas plant fed mainly with cellulose rich material.  biogas production via the anaerobic digestion of spirulina the anaerobic digestion of freeze dried spirulina was studied using a  l semi continuous stirred tank reactor operated at ph  –, at 37 °c and with a 20-day hydraulic retention time. after a 71-day start-up period constant daily biogas production, and constant process parameters, volatile solids ) were observed, indicating that the bioreactor had reached a pseudo steady state condition. starting from this pseudo steady state, five different organic loading rates, from to  g spirulina l− day− were studied to determine the optimal olr for freeze dried spirulina. the biogas production during each period was constant and, as expected, increased when the olr was increased. the biogas production ranged from 470 ml of biogas day− in period i, with an olr of  g spirulina l− day− up to 2210 ml biogas day− in period v, with an olr of  g spirulina l− day− table biogas production and sludge characteristics ch4 co2 n2;o2 codt bod5 nh4 n.d non detected biogas and methane production, biogas characteristics, specific biogas and methane productions and sludge characteristics from the anaerobic digestion of spirulina obtained with the five organic loading rate tested. shown are the mean values of each period with their respective standard deviation the increment in biogas production was not completely proportional to the loading rate. apparently, at higher loading rates digestion of the algal biomass was no longer complete, which eventually led to substrate overload causing reactor failure. this was apparent from: the drop in biogas production at the end of period v, the decreasing methane content of the biogas at high loading rate and the increase in all the parameters related to organic matter, ts, vs, total and soluble chemical oxygen demand and five day biological oxygen demand. this accumulation of organic matter was especially acute during period v with a 30 % increase for ts, 37 % for vs, 50 % for total organic matter, and 163 % for bod compared to period iv. soluble organic matter, acetic acid, propionic acid and ammonia also accumulated in period v. spirulina is a protein rich substrate, therefore its nitrogen content is high, which can explain the observed accumulation of total nitrogen. the degradation of proteins leads to the production of ammonium nitrogen which increased gradually from  g l− in period p-i, to  g l− in period p-v while, at the same time ammonia nitrogen increased from  g l− to  g l−. this increase in nh can be attributed both to the increase in substrate concentration and, according to the equation of anthonisen et al., to the increase of ph from to. methanogens are sensitive to ammonia and the accumulation of this compound can lead to digester failure. the reported levels of free ammonia nitrogen in period v are high and comparable to other reported levels that caused reactor inhibition. the accumulation of non-degraded biomass and vfas along with the relatively high total nitrogen and free ammonia nitrogen concentrations led to the reduction of the biogas production from 2651 ml day− on day to 1586 ml day− on day which is consistent with a substrate overload in the digester.fig. biogas, methane production and sludge characteristics. left axis: daily biogas and methane production normalized to standard temperature and pressure conditions; right axis: soluble cod; acetic acid; propionic acid, nh3-n obtained from the anaerobic digestion of spirulina. vertical dashed line indicates a change in the organic loading rate from  g spirulina l− day− to  g spirulina l− day−1 one of the main bottlenecks perceived for the anaerobic digestion of microalgal biomass is low biodegradability, which can result in low methane yields. to overcome this problem long hrt need to be applied in order to increase the residence time allowing the substrate to be further hydrolyzed. however, this on the long run can have a negative effect on the biogas production as accumulation of inhibitory substances such as ammonia can occur. our results show that at 20 days hrt an orl of  g spirulina l− day− results in a good compromise between an optimal methane yield and accumulation of toxic compounds. at this olr, the specific biogas production per gram of spirulina was 350 ml biogas with 68 % methane content. moreover, the highest biodegradability, 42 %, was obtained, and no accumulation of inhibitory substances occurred in this period. both values, spb and biodegradability are similar to those observed in other studies. metagenome analysis of the anaerobic digester community dna was extracted at the end of period v from the sludge of the spirulina digester and sequenced on a 318tm chip with the ion torrent personal genome machine platform. obtained sequence reads were quality filtered and trimmed. the remaining reads were either assembled into contigs or analyzed directly with the mg-rast metagenome pipeline.table sequencing statistics assembly aa assembly ba assembly ca dataset- sequencing data and assemblies statistics asee material & methods for details about assembly settings binning and 16s rdna taxonomy analysis of assembled contigs three different assemblies were used for the detection of ribosomal 16s genes to taxonomically characterize the microbial community. assembly a produced the largest contig,  bp. due to the stringent settings, assembly b produced a higher number of shorter contigs, while assembly c produced contigs with the longest mean size and the longest n value, 4380 bp. the same 16s rrna gene sequences were all assembled in each of the three assemblies. however, for some of the dominant taxa, the length of assembled 16s rrna gene fragments were different between the three assemblies. assembly a produced the longest contig for the methanosarcinales 16s rrna gene, assembly b yielded the longest anaerolineales 16s sequence, and in assembly c the longest 16s sequences for bacteroidales and clostridiales were obtained. the 16s sequences of methanomicrobiales and thermotogales were assembled to approximately equal lengths in all three assemblies. the 16s genes of flavobacteriales and lactobacillales were better assembled in assemblies a and c than in assembly b. although assembly c yielded the longest average 16s sequences, the length of the archaeal 16s sequences was greater in assembly a. an mg-rast analysis of the reads submitted to assemblies a and c, respectively, revealed that the percentage of reads assigned to firmicutes was markedly higher in the reads submitted to assembly c. this suggests that, with the removal of reads shorter than 220 bp from the read set submitted to assembly a in order to obtain the read set for assembly c, firmicutes sequences were enriched in the latter. contigs from assembly a were considered to better represent the diversity in the biogas reactor and were selected for binning using the metawatt v pipeline to investigate the most abundant populations of the microbial consortium in more detail. from the obtained bins, after manual selection and curation, remained which displayed characteristic tetranucleotide frequencies, assembly coverages and consistent phylogenetic signature and together accounted for almost 80 % of the total sequence data. as was observed in other anaerobic digesters, populations affiliated with firmicutes were most abundant, and constituted 38 % of the total community, followed by bacteroidetes, chloroflexi and thermotogae. contigs affiliated with euryarchaeota comprised a single bin and represented only  % of the total abundance. two bins of unknown taxonomic origin accounted for 10 % of the sequenced data.table characteristics and taxonomical classification of selected microbial bins un. tissierella un. clostridiales un. clostridiales proteiniphilum un. anaerolineaceae un. thermotogaceae unknown un. bacteroidetes unknown un. methanomicrobia cov coverage abun abundance un uncultured characteristics and 16s rdna taxonomical classification of the selected bins obtained from the spirulina metagenome ribosomal rrna gene sequences corresponding to of the bins were identified among the contigs of the three different assemblies and/or recovered independently by iterative read mapping with emirge. assembled 16s rrna sequences identical or highly similar to the emirge sequences were not detected. however, 16s sequences similar to em- and em- were found among unbinned contigs of assembly a and/or contigs of assemblies b and c. no assembled 16s sequence similar to em- was found. unfortunately, the three 16s sequences obtained by emirge and affiliated with firmicutes could not be assigned conclusively to any of the three firmicutes bins. however, based on comparison of sequencing coverage between the recovered emirge 16s sequences and the contigs in the firmicutes bins it was most likely that the dominant organism, bin-a,  % abundance, was most closely related to tissierella praeacuta. bins b and c were assigned to uncultured clostridiales. the 16s rdna sequence classified as atopostipes was assembled well in all three assemblies, yet it could not be linked to any of the bins. based on the low sequencing coverage of this 16s sequence, the population was probably of relatively low abundance and poorly assembled.fig. 16s rdna bacterial and archaeal phylogenetic trees. phylogenetic trees of bacterial and archaeal 16s rdna sequences. although the binning was only performed with contigs of assembly a, most of the binned 16s rdna sequences were also assembled in assemblies b and c. if so, for the tree the longest assembled sequence was chosen. 16s rdna sequences not assembled in assembly a, but in b or c, or detected by emirge are also included. colored: sequences obtained from metagenomic reads. assignment to metawatt bins is indicated if applicable. reference sequences in bold: top hits in blast search against ncbi non-redundant nucleotide collection, bold + italics: top hits in blast search against ncbi reference rna sequences. additional reference sequences in the bacteria tree represent genera detected in other anaerobic digesters. 16s rdna sequences of arthrospira platensis and methanopyrus kandleri were chosen as outgroups, respectively. bootstrap values at nodes are obtained from replicates and are only shown for branches with at least 50 % support. the scale bar represents nucleotide substitutions per site. accession numbers of reference sequences are available in additional file 2: table s7 as in a previous study of anaerobic digestion in which a proteinaceous substrate was used, clostridiales were the most abundant bacterial order. the genus tissierella has already been detected in other anaerobic digesters, but the specific function of members of this taxon in anaerobic digesters is still not clear. however, members of this genus are known to require the presence of certain amino acids and formate for growth and they seem to be unable to utilize carbohydrates such as glucose, cellobiose or xylose which is in accordance to the type of substrate used in our experiment. proteiniphilium, a member of the bacteroidales that utilizes peptone and is unable to grow on carbohydrates, was previously identified in several biogas studies. anaerolineales and thermotogales were also identified in other biogas reactors but in much lower abundance. their function in anaerobic digestion is not clear yet, however, their relatively high abundance when compared with other anaerobic reactors could be explained by the fact that they are known to utilize proteins as substrate. therefore they might play an important role in the degradation of protein-rich spirulina. the 16s sequence in bin-d shows  % similarity to proteiniphilum acetatigenes str. tb, with differing lengths of homopolymer stretches accounting for  % of the differences. the most similar described sequence to contig_ is the porphyromonadaceae clone jx. this clone sequence shows  % similarity to the 16s gene of proteiniphilum acetatigenes, which supports the classification of bin-d as an unknown proteiniphilum. among the archaea, we identified one bin, bin-j, with  % of abundance, for which the 16s rdna fragments were classified as uncultured methanomicrobia. methanomicrobiales were also identified as the most abundant methanogens in an anaerobic reactor fed exclusively with casein. a closer look at the 16s rdna phylogeny suggests the possibility that two methanogenic populations may have been binned together, one related to methanomicrobiales and one to methanosarcinales. members of these orders, which are able use h co formate and acetate as their c source, are frequently encountered in anaerobic digesters. formate and acetate are both fermentation products of clostridiales and anaerolineales, both populations present in high abundance in our experiment. comparison of two metagenomes by short single read analysis in parallel to this genome-focused analysis of assembled contigs, the effect of substrate on both taxonomic composition and presence of functional genes was also studied at the level of individual reads, by comparing the unassembled reads from our study to a publicly available metagenome from a biogas plant using the mg-rast platform. sequencing dataset spirulina-s was obtained from our anaerobic reactor fed with spirulina, a protein rich substrate, while the second sequencing dataset, maize-rye, originated from a biogas plant fed with a mixture of mainly cellulose rich substrates. effect on the microbial community composition the general taxonomic composition did not appear to depend on the type of substrate used. based on the mg-rast m5nr analysis of the metagenomic reads, bacteria clearly dominated in both datasets while archaea represented less than 10 % in the m-r dataset and merely 3 % in the spirulina-s dataset. among bacteria, firmicutes, bacteroidetes and proteobacteria dominated in both sets, but differences could be seen in the abundances of other phyla. for example, hits in thermotogae were more abundant in the protein rich substrate digester data compared to 2 % in the cellulose rich substrate digester dataset. a similar result was observed with chloroflexi,  % in spirulina-s when compared to the m-r dataset,  %. on the other hand, within the archaea such a variety at phylum level was not seen, and as expected, euryarchaeota dominated with over 90 % of all archaea in both datasets.table comparison of identified cog and specific pfams ratio aam/cmb a% of total identified cogs b“amino acid transport and metabolism”/“carbohydrate transport and metabolism” c% of total identified pfams comparison of the cog categories obtained with the mg-rast platform and the selected pfams obtained with the hmmscan among the bacteria, clostridiales dominated in both datasets with and 30 % of the total assigned reads in the spirulina-s dataset and the m-r dataset respectively, followed by bacteroidales, thermoanaerobacterales and bacillales. in both datasets, m-r and spirulina-s the genus to which most of the reads were assigned was clostridium. furthermore, as was also found by kovács et al., who used casein and pig blood as substrate, a relatively high number of reads in the spirulina-s dataset were most similar to members of the genus alkaliphilus, which is in contrast to the m-r dataset, where only about 2 % of the reads were assigned to this genus. bacteroides, recruiting  % of the hits in the m-r dataset accounted for  % in the spirulina-s dataset. among other substrates, bacteroides are known to utilize cellobiose and xylose, both absent in spirulina, which could explain their lower abundance in our reactor. interestingly, candidatus cloacamonas, which accounted for  % of the bacteria m-r reads, hits, recruited less than  % of the hits in the spirulina-s dataset. this bacterium was also present in high abundance at the initial adaptation period of two biogas reactors fed with casein and pig blood and its detection was not possible after 12 weeks of substrate adaptation, which might indicate that it cannot survive without a source of carbohydrates. major differences regarding bacterial taxa were also seen for thermotogales and anaerolineales which were considerably more abundant in the dataset from the protein rich substrate digester, with and 1 % respectively when compared to the dataset from the cellulose rich substrate biogas plant, and  % respectively. these two orders are known to include bacteria which utilize proteins as substrate.fig. comparison of the taxonomic classification of spirulina-s and m-r reads at order level. percentage of the total taxonomic assigned reads of each dataset obtained with mg-rast m5nr representative hit tool. a bacterial orders with the most abundant assigned read hits and all archaeal orders with assigned hits from the metagenomic reads among the archaeal orders, methanomicrobiales clearly dominated in the m-r dataset, recruiting almost 75 % of the archaeal hits. in the spirulina-s dataset, the combined presence of methanosarcinales and methanomicrobiales was observed, confirming the analysis of the assembled 16s rdna sequences. methanosarcinales are known to be able to use acetate, h and co as substrate, which are the main fermentation products of tissierella and other clostridia, and they are usually dominant in reactors where vfas and nh are present in high concentration as in our case. on the other hand, methanomicrobiales do not use acetate but can grow on h co and formate, the latter a common fermentation product of bacteria of the phylum chloroflexi which in our dataset represented almost 8 % of the abundance. the presence of two methanogenic populations in an anaerobic digestor is quite common and has also been observed in several other studies. for example ziganshin et al. observed the presence of methanoculleus and methanosaeta in reactors fed with cattle manure and dried distillers grains, and of methanosarcina and methanoculleus in reactors treating maize straw and cattle manure, while li et al. also detected the same two groups of methanogens in a reactor treating multiple substrates. this presence of different methanogens detected in such a broad range of substrates might indicate that, rather than the type of used substrate, the characteristics of the sludge and the initial type of inoculum determine which archaea thrive in anaerobic digesters. in order to investigate whether the diversity of the species community was affected by the used substrates, the species diversities in both datasets, were analyzed by means of lorenz curves and simpson’s diversity index and compared with each other. the mixture of substrates used in the biogas plant, might lead to a higher bacterial diversity than in the reactor solely fed with spirulina. however, lorenz curves, describing population evenness, of both datasets were similar, as well as simpson’s diversity index values. the similarity in evenness and diversity in both bacterial populations can be explained by the fact that spirulina as such is not a “simple” substrate, as would be glucose, starch or glycerol, and therefore needs a microbial population with a certain complexity to be fully digested. apparently, the differences between the substrate types used in the two compared systems did not affect the diversity of the whole population, yet rather the abundance of certain bacterial taxa. for archaea, on the other hand, lorenz curves and sdi indicated a difference in their diversity in the two studied datasets. the archaeal population in the spirulina reactor was more even than the archaeal community in the maize-rye biogas plant. given the same species richness, a more even population is also more diverse. indeed, by calculating the sdi a higher diversity for the archaeal community in the spirulina reactor was observed than in the m-r biogas plant. the lower sdi for the archaea in the m-r dataset is best explained by the clear dominance of methanoculleus in the archaeal reads of this dataset. taken together, the results suggest that the type of substrate used in anaerobic digestion mainly affects the bacterial composition, to some extent, at low taxonomic levels, especially at genus and species level. proteolytic bacteria were probably present in all dominant phyla of the spirulina-s digester, whereas in the maize-rye biogas plant, cellulolytic bacteria were dominant. regarding the archaea, an influence of the substrate on their presence and composition is not as clear as for the bacteria. this could be explained by the fact that the archaea perform the final step in the process of anaerobic digestion, and their presence is probably more dependent on the population composition of the primary substrate degraders, of their metabolic products, the presence or absence of inhibitory compounds and the origin of the inoculum, rather than on the substrate itself. effect on the abundance of functional genes both datasets, spirulina-s and m-r were compared at functional level with two approaches, mg-rast’s cog comparison, and identification of specific protein domains related to the cellulose degradation pathway and to protein and amino acid degradation. due to the different types of substrates used, it was expected to see differences regarding the cogs related to amino acids and protein metabolism. however, with respect to the detected functional genes, the differences between both datasets were minor. the highest difference was observed in category l, “replication, recombination and repair”, which represented  % of the hits in the spirulina-s dataset and  % in the m-r dataset and contains cogs related to groups of genes which participate in the replication process of the microbial community. this difference in abundance was significant and could be explained by the fact that at the time of sampling of the spirulina-s dataset, day the reactor was suffering from substrate overload which resulted in an accumulation of toxic compounds that hindered the correct function of the bacteria and reduced the daily biogas production. the cogs related to category e, “amino acid transport and metabolism” were slightly more abundant in the protein rich dataset, than in the cellulose rich dataset. the higher relative abundance of cogs from category e in the spirulina-s dataset could be attributed to the type of substrate used. however, as most of the cogs from this category represent enzymes involved in general metabolic processes, further research must be conducted in order to fully verify this result. on the other hand, and contrary to what was expected, no significant differences could be seen for the cogs from “carbohydrate transport and metabolism” in both datasets. however, it should be noted that, in category g, important differences could be seen among those cogs directly related to the degradation of cellulose, xylanose and other complex sugars. for example, cog a “beta-xylosidase” represented  % in the m-r dataset and was not detected in the spirulina-s dataset. similarly, cog an “l-arabinose isomerase”, was almost absent in the spirulina-s dataset but represented almost  % in the m-r dataset, likewise cog a “beta- -xylanase” and cog a “glycosidase” were both significantly less abundant in the spirulina-s dataset. this difference in the abundance of cogs correlates with the type of substrate used, spirulina, which has a low content of complex sugars.fig. cog functional hierarchical classifications. comparison of the cog classified reads of the spirulina-s and the m-r metagenomes obtained with the mg-rast metagenome analyzer. only those categories with hits are represented. x axis: information storage and processing: j, translation, ribosomal structure and biogenesis; k, transcription; l, replication, recombination and repair; cellular processes and signaling: d, cell cycle control, cell division, chromosome partitioning; m, cell wall/membrane/envelope biogenesis; n, cell motility; o, posttranslational modification, protein turnover, chaperones; t, signal transduction mechanisms; u, intracellular trafficking, secretion and vesicular transport; v, defense mechanisms; metabolism: c, energy production and conversion; e, amino acid transport and metabolism; f, nucleotide transport and metabolism; g, carbohydrate transport and metabolism; h, coenzyme transport and metabolism; i, lipid transport and metabolism; p, inorganic ion transport and metabolism; q, secondary metabolites biosynthesis, transport and catabolism; poorly characterized: r, general function prediction only; s, function unknown to corroborate the results obtained with the cogs analysis, specific protein domains related to cellulose degradation and to protein and amino acid degradation were searched for in both datasets. pfam domains associated with cellulose summed up to  % of the total identified pfams in the m-r dataset compared to  % in the spirulina-s dataset. the proteases related pfams were slightly more abundant in the spirulina-s dataset with  % of all identified pfams compared to  % in the m-r dataset. also the amino acid degradation pfams were more abundant in the spirulina-s reads than in the m-r reads. two pfam ratios, proteases to cellulases ratio, and amino acid to cellulase ratio were also calculated to determine the relative abundance of each group. the p/c ratio in the spirulina-s dataset,, was double the p/c ratio in the m-r dataset,, and a comparable result was obtained with the aa/c ratios, for the spirulina-s and for the m-r dataset. when a cog “amino acid transport and metabolism” to “carbohydrate transport and metabolism” ratio was calculated, the result was similar, for the spirulina-s dataset and for the m-r dataset. the differences in the obtained p/c, aa/c and aam/cm ratios, plus the higher abundance of amino acid metabolism related cogs suggest that the microbial community in the spirulina reactor adapted to the type of substrate degraded, protein rich with low presence of cellulose. however, since substrates from both reactors were not either pure carbohydrates or proteins, but consisted of both in different proportions, further studies are necessary to determine whether the observed differences in the presence of functional genes would become more distinct for longer run times of the spirulina digester, or if the microbial community was already at functional equilibrium when it was sampled for the metagenome analysis presented here. binning of contigs vs classification of single short reads in this work, the same metagenome has been analyzed by two different approaches, assembly of reads into contigs followed by a binning strategy combined with a 16s rdna analysis, and blasting of single reads against a general nucleotide database. despite the differences in methodology, the results obtained with both approaches were consistent. the first advantage of using the assembly/binning approach is that the obtained bins form a provisional whole genome sequence of the most abundant organisms. this way, the function of individual populations can be inferred directly. the second advantage is that taxonomical classification of contigs is more reliable than the classification of short reads. in contrast, the main advantage of using mg-rast classification of short reads is that it is more sensitive because functions can be inferred from the presence of non-redundant reads originating from minor community members that remain unassembled. since ~80 % of the sequenced reads could be mapped to the bins corresponding to the most abundant populations, approximately 20 % of the microbial community was below the detection limit of the assembly/binning approach.  the results presented in this study suggest that spirulina is a suitable substrate for the production of biogas with a mean production of 350 ml of biogas per gram of substrate. as in previous studies, the most abundant bacterial populations making up the consortium performing digestion belonged to clostridia and bacteroidetes whereas the most abundant archaeal populations were affiliated with methanomicrobiales and methanosarcinales. the microbial community present in the anaerobic digester was well adapted to the type of substrate used, based on taxonomic and functional inferences. taxonomic analysis of assembled contigs with a binning approach produced results consistent with the classification of short single reads. 
compared to the full scale cellulose-fed digesters, pfam domains related to protein degradation were more frequently detected and pfam domains related to cellulose degradation were less frequent in our sample. the optimal organic loading rate for the anaerobic digestion of spirulina was determined to be  g spirulina l− day− with a specific biogas production of 350 ml biogas g spirulina− with a methane content of 68 %. euryarchaeota represented  % of the total abundance. anaerobic digestion is a biological process in which a consortium of microorganisms transforms a complex substrate into methane and carbon dioxide. a good understanding of the interactions between the populations that form this consortium can contribute to a successful anaerobic digestion of the substrate. in this study we combine the analysis of the biogas production in a laboratory anaerobic digester fed with the microalgae spirulina, a protein rich substrate, with the analysis of the metagenome of the consortium responsible for digestion, obtained by high-throughput dna sequencing. the results presented in this study suggest that spirulina is a suitable substrate for the production of biogas. methanomicrobiales and methanosarcinales dominated the archaeal community. the most abundant organism was related to tissierella, a bacterium known to use proteinaceous substrates for growth. the proteinaceous substrate appeared to have a selective impact on the bacterial community that performed anaerobic digestion. firmicutes dominated the microbial consortium at 38 % abundance followed by bacteroidetes, chloroflexi and thermotogae.
 anaerobic digestion is a biological process in which a consortium of microorganisms transforms a complex substrate into methane and carbon dioxide. a good understanding of the interactions between the populations that form this consortium can contribute to a successful anaerobic digestion of the substrate. in this study we combine the analysis of the biogas production in a laboratory anaerobic digester fed with the microalgae spirulina, a protein rich substrate, with the analysis of the metagenome of the consortium responsible for digestion, obtained by high-throughput dna sequencing. the obtained metagenome was also compared with a metagenome from a full scale biogas plant fed with cellulose rich material.  the optimal organic loading rate for the anaerobic digestion of spirulina was determined to be  g spirulina l− day− with a specific biogas production of 350 ml biogas g spirulina− with a methane content of 68 %. firmicutes dominated the microbial consortium at 38 % abundance followed by bacteroidetes, chloroflexi and thermotogae. euryarchaeota represented  % of the total abundance. the most abundant organism was related to tissierella, a bacterium known to use proteinaceous substrates for growth. methanomicrobiales and methanosarcinales dominated the archaeal community. compared to the full scale cellulose-fed digesters, pfam domains related to protein degradation were more frequently detected and pfam domains related to cellulose degradation were less frequent in our sample.  the results presented in this study suggest that spirulina is a suitable substrate for the production of biogas. the proteinaceous substrate appeared to have a selective impact on the bacterial community that performed anaerobic digestion. a direct influence of the substrate on the selection of specific methanogenic populations was not observed. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords metagenomemetagenome comparisonspirulinabiogasbinningmicrobial communityissue-copyright-statement© the author 2015 