BACKGROUND
the activated sludge process is one of the most widely used methods for treatment of wastewater.
an important factor for the operation of activated sludge wastewater treatment plants  is the solid-liquid separation in which flocculation and settling are important processes
<cit> .
poor flocculation and settling of the activated sludge lead to poor effluent quality and can cause environmental problems in the receiving waters.
the sludge characteristics depend on the microbial community composition
<cit> , the microbial activity
<cit>  and the properties of the extra-cellular polymeric substances in the flocs
<cit> .
the bacterial community has been characterized in a number of activated sludge systems
<cit>  but very little is known about archaeal communities in sludge.
the presence of archaea in activated sludge has been shown by fluorescence in situ hybridization , e.g.
<cit> .
methanogens
<cit>  and putative ammonia-oxidizing archaea
<cit>  have been detected by amplification of 16s rrna and archaeal ammonia monooxygenase subunit a genes.
although present, archaea seem to be of minor importance for both nitrogen and carbon removal
<cit> .
however, it is still possible that the archaea have other functions or affect the properties of the activated sludge.
addition of methanogens to the sludge in intermittently aerated bioreactors increased the rates of specific oxygen uptake, denitrification and nitrification suggesting a symbiotic relationship with bacteria  <cit> .
the composition of the methanogenic community in anaerobic sludge has been shown to be crucial for the structure and integrity of granules
<cit>  and if methanogens are present in activated sludge they may contribute to the floc structure.
this study had three aims.
the first was to describe the archaea community in the activated sludge of a full-scale wwtp by cloning and sequencing of 16s rrna genes.
although there are many studies where activated sludge samples have been screened for the presence of aoa , to our knowledge there are only two published studies on the diversity of archaea in activated sludge from a full-scale wwtp
<cit> .
one of the studies investigated two small wwtps
<cit>  and the other a seawater-processing wwtp
<cit> .
the rya wwtp is a large wwtp treating municipal and industrial wastewater, thus different from the wwtps in those two studies.
since little is known about archaea in wwtps and, importantly, sequence coverage for archaea from wwtps is still modest, the 16s rrna sequences we obtained here would indicate if published fish probes were relevant.
if so, the second aim was to quantify the archaea by confocal microscopy and fish and to determine their localization in the flocs.
the third aim was to follow the dynamics of the archaea community for a longer period of time using terminal restriction fragment length polymorphism  analysis.
for the third aim, the samples that were used were collected for previous studies of the dynamics of the floc composition and flocculation and settling properties of the activated sludge at the rya wwtp
<cit> .
analyzing the archaea community in the same samples could give an indication of possible causes or effects of archaea community changes.
RESULTS
observed and estimated richness of the archaea community in the activated sludge
a 16s rrna gene clone library was constructed from a sample of activated sludge collected at the aeration tank of the rya wwtp at a time of normal operating conditions.
there were no atypical process parameter values or extreme events prior to sample collection.
however, the f/m-ratio was higher at the time of the clone library sample collection  compared with the times when samples were collected for fish  and t-rflp analyses  .
cloning and sequencing generated  <dig> archaeal 16s rrna gene sequences of lengths between  <dig> and  <dig> bases.
based on dna similarity the sequences were assigned to operational taxonomic units .
the sequences were assigned to otus corresponding to  <dig> species of  <dig> genera,  <dig> families/classes and  <dig> different phyla.
the archaea community richness was estimated to be at least  <dig> species of  <dig> different genera.
thus, the clone library covered at most 58% of the species and 53% of the genera present in the activated sludge.
accumulation curves  also illustrate that the clone library does not fully cover the archaea community.
a
a the three periods were compared using the kruskal-wallis test.
a statistically significant difference, p <  <dig> , is marked with asterisks .
b average values  from all sample dates and the six days preceding the sample dates.
c data only from sample dates, not including the six preceding days.
d the parameters are water temperature , solids retention time , food to mass ratio , cod going into the activated sludge tanks , nitrite/nitrate levels going in to the activated sludge tanks , standardized sludge volume index  and effluent non-settleable solids .
e samples collected during this period were used for t-rflp analysis.
f a sample collected during this period was used for t-rflp and clone library analysis.
g a sample collected during this period was used for fish analysis.
<cit> .
the accumulation curves show the rate of discovery of new otus at the different sequence similarity thresholds.
evenness and functional organization
figure
<dig> shows a pareto-lorenz evenness curve of the archaea community based on the relative abundances of the  <dig> otus obtained by applying a  <dig> % sequence similarity threshold.
the functional organization  index, the combined relative abundance of 20% of the otus, is 56%, meaning that more than half of the observed sequences belong to only five of the observed otus.
a high fo index is an indication of a specialized community since it means that a big part of the population belongs to a small number of otus and performs a small number of ecological functions.
in a completely even community all otus would have the same number of individuals and it would be possible for a large number of different functions to be equally abundant.
in the clone library, the five most abundant otus, which include 56% of the sequences, all belong to methanosaeta and presumably are all methanogens.
furthermore, the composition of the clone library indicates that the community includes a small number of ecological functions since  <dig> of  <dig> otus, including 77% of the sequences, were identified as methanosaeta .
comparison with available sequences in genbank and silva
searches in genbank using blast
<cit>  and in the silva rrna database
<cit>  found sequences with a sequence similarity of  <dig> % or higher for  <dig> of  <dig> otus, including  <dig> of the  <dig> sequences .
with 100% coverage,  <dig> sequences could only be matched with sequence similarities lower than  <dig> % and may therefore represent new species belonging to the genera methanosaeta  or the thermoplasmatales, cluster b .
the most similar sequences in the databases were from various types of soil environments, water environments and anaerobic bioreactors in north america, europe and asia.
for  <dig> of the  <dig> sequences, the best match came from an anaerobic bioreactor.
a best matching entry in genbank or the silva rrna database with 100% coverage.
b identity in %.
phylogenetic tree analysis
the phylogenetic affiliation of the obtained 16s rrna gene sequences was determined by phylogenetic tree analysis.
a phylogenetic tree for euryarchaea inferred by maximum likelihood analysis is shown in figure
<dig>  a phylogenetic tree for crenarchaea and thaumarchaea inferred by maximum likelihood analysis is shown in figure
<dig>  the majority of the sequences were determined to be of genus methanosaeta .
several sequences also affiliated with divisions of uncultured archaea.
<cit> , grosskopf
<cit>  and chouari
<cit> .
<cit>  and jurgens
<cit> .
dynamics of the archaea community
the community composition, as assessed by t-rflp, changed little throughout the monitored period.
the difference, measured as bray-curtis distance, between the terminal restriction fragment  profile of the first sample in the time series, may  <dig>   <dig>  and all following samples was on average only  <dig>  ±  <dig> % and  <dig>  ±  <dig> % for the alui and rsai analysis, respectively .
on two occasions, in october  <dig> and in january  <dig>  the bray-curtis distance peaked, indicating a deviation from the community composition at the beginning of the time series.
the difference between the trf profiles of may  <dig>   <dig> and may  <dig>   <dig>  four years later, was 10% and 0% for the alui and rsai analysis, respectively.
however, the sensitivity of the t-rflp analysis is limited and trfs of low abundance cannot be detected.
thus, a bray-curtis distance of 0% between the trf profiles of two samples does not indicate identical community composition since there may be differences in the composition of archaea of low abundance.
to get a rough estimate of the sensitivity of the t-rflp method, a comparison was made between the theoretical trf profile of the clone library and the observed trf profile from the same sample.
the comparison showed that only trfs with a relative abundance higher than 20% in the clone library were observed in the trf profile .
thus, the t-rflp analysis shows the dynamics of the major groups of archaea in the activated sludge.
the relative abundances of the observed trfs in all trf profiles in the time series are shown in figures
<dig> and
<dig>
a observed and predicted trf lengths from t-rflp and clone library analysis of a sample from 2007-05- <dig>  b relative abundance based on total fluorescence.
c relative abundance based on frequency in clone library.
d nd indicates cases where a trf could not be predicted or where the predicted trf was outside the detection range.
to identify the trfs the observed trf lengths were compared with the predicted trf lengths of sequences in the clone library.
the predicted trfs from the sequences in the clone library were between  <dig> and  <dig> bases longer than the observed trfs .
such a discrepancy between observed and predicted trf sizes is commonly observed
<cit> .
not all observed trfs in the time series could be matched with the predicted trfs of the clone library sequences.
to explore the possibility that the trfs in the trf profiles from the samples from  <dig> and  <dig> come from sequences other than those found in the clone library a comparison was also made with a database of  <dig> archaeal 16s rrna gene sequences matching the primers used in this study.
the database was checked for sequences that would result in any of the observed combinations of trfs generated by alui and rsai.
the result of the analysis was a number of possible identities for each observed combination of alui and rsai trfs .
although the database comparison may result in false identities of the trfs, it is valuable because it gives an indication about the range of species that could give the observed trf combinations.
by comparison with the clone library sequences the dominating trfs  were determined to represent methanosaeta-like species.
comparisons with the predicted trfs of  <dig> archaea sequences in the database  showed that it is possible that the dominating trfs are from other species of the euryarchaeota than methanosaeta.
however, it is unlikely that the origin of these trfs is species of the crenarchaeota or thaumarchaeota, since all the crenarchaeota or thaumarchaeota sequences in the databases have other predicted trf lengths.
a observed trf combinations that are not included in the table were not found in the database nor in the clone library.
b identification by comparison with predicted trf lengths of clone library sequences.
c identification by comparison with predicted trf lengths of rdp database sequences.
d out of range: the trf in the database or the clone library was either shorter than  <dig> bases or longer than  <dig> bases and would therefore not have been detected.
correlation analysis
several trfs showed a significant correlation with process parameters .
the parameters that correlated with most trfs were water temperature and nitrogen concentration.
there were also significant correlations between several trfs and the sludge and effluent water properties .
the parameter effluent non-settleable solids  and the concentration of carbohydrates in extracted extracellular polymeric substances  correlated with most trfs.
no trf showed a significant correlation with the sludge volume or shear sensitivity.
a the correlations are marked with asterisks corresponding to the level of statistical significance: 95% , 99%  and  <dig> % .
trfs that are not included did not show any statistically significant correlation with any parameter.
the wwtp process parameter data was taken from
<cit> .
b identification by comparison with the rdp database.
c identification by comparison with the clone library.
d the number of times the trf was observed.
e water temperature .
f solids retention time .
g food to mass ratio .
h total cod going into the activated sludge tanks .
i nitrite/nitrate levels going in to the activated sludge tanks .
a the correlations are marked with asterisks corresponding to the level of statistical significance: 95% , 99%  and  <dig> % .
trfs that are not included did not show any statistically significant correlation with any parameter.
the sludge and effluent water parameter data was taken from
<cit> .
b identification by comparison with the rdp database.
c identification by comparison with the clone library.
d the number of times the trf was observed.
e standardized sludge volume index .
f shear sensitivity .
g eps protein .
h eps carbohydrates .
i effluent non-settleable solids .
quantification and localization of archaea in the activated sludge flocs
the 16s rrna gene clone library indicated that published fish probes would cover the archaea at rya wwtp.
archaea could be observed in the activated sludge flocs, both centrally located and close to the edges of the flocs.
fish analyses showed that the average relative abundance of archaea in the activated sludge of the aeration tank was  <dig> % .
in the anaerobic digester and in the water recycled into the activated sludge tanks  there were more archaea than bacteria .
in most images of activated sludge flocs the percentage of archaea was lower than 2% .
occasionally there were larger colonies of archaea  but in most images archaea were either present as individual cells or small colonies .
<cit>  with an applied threshold of  <dig>
discussion
in this study the abundance, localization, composition and dynamics of archaea in the activated sludge of a full-scale wwtp were assessed using fish analysis, 16s rrna gene clone library analysis and t-rflp time series analysis.
these three analyses were all done on samples collected at different times.
however, for most process parameters there were no significant differences between these times .
the wwtp was also operated the same way at all times, except for four months, may  <dig> to september  <dig>   <dig>  when the primary settlers were bypassed.
the samples were therefore considered comparable.
the t-rflp time series analysis showed that the most abundant trfs were the same throughout  <dig> and  <dig> as well as in may  <dig> .
if we assume that the same trf always represent the same group of archaea, then the t-rflp data show that the main part of the archaea community was the same in  <dig>   <dig> and in may  <dig>  and that we can use the clone library data to identify the trfs in the t-rflp time series.
we further assume that the archaea community stayed mainly the same in december  <dig>  which make it possible to use the clone library data to choose appropriate probes for the fish analysis.
the clone library sequences indicated that already published fish probes were relevant for an estimation of the relative abundance of major archaea groups.
the relative abundance of the archaea has been estimated in other investigations to be low, based on activity measurements
<cit> , and up to 8% of bacteria <cit>  or 10% of total cell numbers
<cit> .
in this study archaea was estimated, by fish, to make up  <dig> % of total cell numbers in the activated sludge, a relatively low abundance.
however, the importance of a microbial group cannot be deduced by abundance alone.
putative aoa were 1-10% of total cell numbers in activated sludge, but despite this abundance they did not contribute significantly to nitrification
<cit> , whereas foaming organisms have great impact on floc structure and sludge properties even when present in numbers around 1%
<cit> .
another example is ammonium oxidizers, which at an abundance of 3-5% , could perform the first step in a successful 80% reduction of nitrogen in an activated sludge system
<cit> .
thus, despite their relatively low abundance, a possible contribution of archaea to sludge properties cannot be ruled out.
the composition of the archaea community was investigated by analysis of  <dig> 16s rrna sequences.
the community richness was estimated to be  <dig> species of  <dig> genera.
as expected, the clone library does not fully cover the archaea community .
however, the  <dig> species of  <dig> genera that were observed are assumed to represent the most abundant groups.
the archaea community was functionally specialized  with 77% of the sequences in the clone library being methanosaeta-like organisms .
specialized communities dominated by methanogens, although of other genera than methanosaeta, have also been observed in activated sludge from other wwtps
<cit> .
the t-rflp time series analysis showed that the archaea community was practically the same in most samples , despite variations in environmental conditions such as organic loading rate and temperature
<cit> .
only in a few samples more than two trfs were observed.
however, as shown in table
<dig>  the sensitivity of the t-rflp analysis was low, so it is possible that there were changes in the composition of the less abundant groups of archaea.
a comparison between the observed trf lengths and the predicted trf lengths of the clone library sequences identified the two main trfs as coming from methanosaeta sequences, given the assumption that all trfs represent the same groups of archaea in all samples where they are observed, as discussed above.
an alternative way of identifying the trfs would be to compare the observed alui and rsai trf combinations with the predicted trf combinations from archaea sequences in the rdp database.
a comparison with  <dig> archaea 16s rrna sequences showed that sequences of methanosaeta or other euryarchaea would give the observed alui and rsai trf combinations, but no crenarchaeota or thaumarchaeota sequences.
in the following discussion we therefore assume that the two main trfs come from methanogens.
methanogens are anaerobic and the oxygen concentration in activated sludge is high.
however, in the deeper parts of activated sludge flocs anoxic microenvironments can exist
<cit>  which may allow growth of anaerobic organisms.
in the activated sludge at rya wwtp, methanogens were observed both deep within the flocs and close to the surface .
although exposed to oxygen, the methanogens at the surface are not necessarily inactive since methanogens have been shown to be able to maintain viability
<cit>  and activity
<cit>  in the presence of oxygen.
to avoid washout from the activated sludge, microorganisms need to be active and have a doubling time shorter than the sludge retention time.
pure cultures of methanosaeta concilii have a temperature optimum at 35-40°c
<cit>  and a doubling time of 4-7 days at 37°c
<cit> .
the low water temperature at rya wwtp, 10-20°c, does not necessarily prevent activity since methanosaeta-like species have been shown to grow at 9-14°c in bioreactors
<cit>  and dominate methanogenic cultures from rice field soil at 15°c
<cit> .
the solids retention time  at rya wwtp is typically calculated as 5-7 days and could also allow for growth of methanosaeta-like organisms.
in this study, methanosaeta-like trfs dominated throughout 15 months, and correlation analysis showed that some of the methanosaeta-like trfs increased in abundance with increasing temperature and increasing srts , i.e.
theoretically more favorable conditions.
both the constant dominance in the trf profiles and the response to changed environmental conditions would be explained by an active population of methanosaeta-like organisms.
the detection of methanogens by fish analysis also showed the presence of rrna, which is expressed in active cells.
however, high rrna levels may be maintained despite inactivity.
in conclusion, the activity of the methanosaeta-like organisms is an open question.
if the methanosaeta-like species do not grow fast enough to avoid washout, their constant presence requires that they are constantly added to the sludge.
possible sources are the influent wastewater and recycled water from an anaerobic bioreactor.
by fish analysis, archaea was confirmed to be present in high numbers in both the anaerobic bioreactor and in the reject water .
thus the bioreactor might seed the activated sludge with archaea.
this is supported by the fact that a majority of the detected 16s rrna sequences cluster with sequences from anaerobic sludge .
furthermore, no sequences matched typical methanogens in human fecal matter, such as methanobacter smithii and methanosphaera stadtmanae <cit> , indicating that fecal matter from the influent water was not an important input to the methanogens in the activated sludge.
the second largest group in the clone library was thermoplasmatales-related sequences affiliated with rice cluster iii .
no cultured representative of rc-iii archaea exists, but a study of a methanogenic enrichment culture suggests that rc-iii archaea are mesophilic anaerobes growing heterotrophically on peptides with a doubling time of approximately three days
<cit> .
rc-iii has been detected in soil
<cit> , anaerobic bioreactors
<cit>  and groundwater
<cit> .
this study shows that rc-iii archaea can also be present in activated sludge.
thermoplasmatales-related sequences of cluster b and c were also found in the clone library.
there are currently no cultured representatives or proposed phenotypes for these groups.
cluster b and c sequences have been retrieved from environments with methanogenic communities and complete or partially anoxic zones, such as water
<cit> , landfill leachates
<cit> , sediments
<cit> , bioreactors
<cit>  and the digestive tract of animals
<cit> .
this study adds activated sludge to that list.
one sequence, clone g <dig>  belongs to a yet undescribed lineage of archaea: arc i <cit> .
the arc i lineage is well-represented in anaerobic bioreactors and in reactors with a high abundance of arc i, the abundance of species related to m. concilii is low and vice versa
<cit> , which could indicate a competition for acetate between these two lineages.
the clone library in this study followed the same pattern with low abundance of arc i and high abundance of m. concilii.
the same pattern was also seen in the trf profiles since the only time that the trfs corresponding to sequence g <dig> was observed  the relative abundance of the trfs associated with m. concilii had decreased to around 80%.
archaeal ammonia monooxygenase subunit a  genes have been observed in various activated sludge wwtps
<cit> .
one of our sequences affiliated with crenarchaea cluster  <dig> b, which includes several putative aoa
<cit> .
however, it has recently been shown that not all amoa-carrying thaumarchaeota are ammonia-oxidizing autotrophs
<cit> .
the presence of aoa at the rya wwtp can therefore not be confirmed, and as has been suggested for other wwtps
<cit> , aoa are most likely of minor or no importance for ammonia-oxidation at the rya wwtp.
one clone affiliated with crenarchaea cluster  <dig> .
there are no cultured representatives of cluster  <dig> , but spatial co-localization
<cit>  and a relation between the abundance of cluster  <dig>  and methanosaeta-like species has been reported
<cit> .
in other aggregate structures, such as anaerobic sludge granules, methanosaeta are important for structure and stability and they form dense aggregates which act as nuclei for granule formation
<cit> .
in the activated sludge the methanosaeta did not appear to have this function as they were mostly detected as small colonies or single cells  and there was no apparent difference in structure between flocs with high and low numbers of methanosaeta.
the lowest relative abundances of the methanosaeta-like trfs were observed in january and february  <dig> .
in october  <dig> the two main methanosaeta trfs also decreased in relative abundance but it cannot be ruled out that the trfs that appeared in those samples were also methanosaeta .
the lowest water temperatures of the period were recorded during january and february  <dig>  which could have reduced the survival or proliferation of methanosaeta-like species and allowed other archaea to increase.
in anaerobic sludge, a decrease in methanosaeta abundance has been linked to granule disintegration
<cit> .
although the flocs had high shear sensitivity and a more open structure in january and february  <dig> when the methanosaeta trfs decreased and although there was a significant negative correlation between methanosaeta trfs and effluent non-settleable solids  it cannot be concluded that the archaea are important for the floc structure.
the increased shear sensitivity and changed floc structure in january and february  <dig> could be due to the reduced general microbial activity, which has been shown to decrease floc stability
<cit> .
furthermore, increased shear sensitivity and changed floc structure was also observed from june to august  <dig>  after the primary settlers were bypassed, but during this period the relative abundance of the methanosaeta trfs was 100%.
thus, if the composition of the archaea community has any effect on floc structure or stability it is certainly only one of many other factors.
CONCLUSIONS
by sequencing and t-rflp analysis of 16s rrna genes and fish we showed that archaea were present in the activated sludge of a full-scale wwtp.
the archaea community was dominated by methanosaeta-like species, which probably entered the sludge with a stream of recycled water from an anaerobic bioreactor.
the archaea were present both as colonies and single cells but only in low numbers, estimated as  <dig> % of total cell numbers in the activated sludge.
during 15 months major changes in community composition were observed twice, but in both cases the community returned to the previous composition.
even in samples collected three years apart the main part of the community remained the same according to t-rflp data.
we now know that archaea can constitute a small but constant and integral part of the activated sludge and that it can therefore be useful to include archaea in future studies of sludge or floc properties.

 the activated sludge process is one of the most widely used methods for treatment of wastewater. an important factor for the operation of activated sludge wastewater treatment plants is the solid-liquid separation in which flocculation and settling are important processes. poor flocculation and settling of the activated sludge lead to poor effluent quality and can cause environmental problems in the receiving waters. the sludge characteristics depend on the microbial community composition, the microbial activity and the properties of the extra-cellular polymeric substances in the flocs. the bacterial community has been characterized in a number of activated sludge systems but very little is known about archaeal communities in sludge. the presence of archaea in activated sludge has been shown by fluorescence in situ hybridization, e.g.. methanogens and putative ammonia-oxidizing archaea have been detected by amplification of 16s rrna and archaeal ammonia monooxygenase subunit a genes. although present, archaea seem to be of minor importance for both nitrogen and carbon removal. however, it is still possible that the archaea have other functions or affect the properties of the activated sludge. addition of methanogens to the sludge in intermittently aerated bioreactors increased the rates of specific oxygen uptake, denitrification and nitrification suggesting a symbiotic relationship with bacteria. the composition of the methanogenic community in anaerobic sludge has been shown to be crucial for the structure and integrity of granules and if methanogens are present in activated sludge they may contribute to the floc structure. this study had three aims. the first was to describe the archaea community in the activated sludge of a full-scale wwtp by cloning and sequencing of 16s rrna genes. although there are many studies where activated sludge samples have been screened for the presence of aoa, to our knowledge there are only two published studies on the diversity of archaea in activated sludge from a full-scale wwtp. one of the studies investigated two small wwtps and the other a seawater-processing wwtp. the rya wwtp is a large wwtp treating municipal and industrial wastewater, thus different from the wwtps in those two studies. since little is known about archaea in wwtps and, importantly, sequence coverage for archaea from wwtps is still modest, the 16s rrna sequences we obtained here would indicate if published fish probes were relevant. if so, the second aim was to quantify the archaea by confocal microscopy and fish and to determine their localization in the flocs. the third aim was to follow the dynamics of the archaea community for a longer period of time using terminal restriction fragment length polymorphism analysis. for the third aim, the samples that were used were collected for previous studies of the dynamics of the floc composition and flocculation and settling properties of the activated sludge at the rya wwtp. analyzing the archaea community in the same samples could give an indication of possible causes or effects of archaea community changes.  observed and estimated richness of the archaea community in the activated sludge a 16s rrna gene clone library was constructed from a sample of activated sludge collected at the aeration tank of the rya wwtp at a time of normal operating conditions. there were no atypical process parameter values or extreme events prior to sample collection. however, the f/m-ratio was higher at the time of the clone library sample collection compared with the times when samples were collected for fish and t-rflp analyses. cloning and sequencing generated archaeal 16s rrna gene sequences of lengths between and bases. based on dna similarity the sequences were assigned to operational taxonomic units. the sequences were assigned to otus corresponding to species of genera, families/classes and different phyla. the archaea community richness was estimated to be at least species of different genera. thus, the clone library covered at most 58% of the species and 53% of the genera present in the activated sludge. accumulation curves also illustrate that the clone library does not fully cover the archaea community. a a the three periods were compared using the kruskal-wallis test. a statistically significant difference, p < , is marked with asterisks. b average values from all sample dates and the six days preceding the sample dates. c data only from sample dates, not including the six preceding days. d the parameters are water temperature, solids retention time, food to mass ratio, cod going into the activated sludge tanks, nitrite/nitrate levels going in to the activated sludge tanks, standardized sludge volume index and effluent non-settleable solids. e samples collected during this period were used for t-rflp analysis. f a sample collected during this period was used for t-rflp and clone library analysis. g a sample collected during this period was used for fish analysis.. the accumulation curves show the rate of discovery of new otus at the different sequence similarity thresholds. evenness and functional organization figure  shows a pareto-lorenz evenness curve of the archaea community based on the relative abundances of the otus obtained by applying a % sequence similarity threshold. the functional organization index, the combined relative abundance of 20% of the otus, is 56%, meaning that more than half of the observed sequences belong to only five of the observed otus. a high fo index is an indication of a specialized community since it means that a big part of the population belongs to a small number of otus and performs a small number of ecological functions. in a completely even community all otus would have the same number of individuals and it would be possible for a large number of different functions to be equally abundant. in the clone library, the five most abundant otus, which include 56% of the sequences, all belong to methanosaeta and presumably are all methanogens. furthermore, the composition of the clone library indicates that the community includes a small number of ecological functions since of otus, including 77% of the sequences, were identified as methanosaeta. comparison with available sequences in genbank and silva searches in genbank using blast and in the silva rrna database found sequences with a sequence similarity of % or higher for of otus, including of the sequences. with 100% coverage, sequences could only be matched with sequence similarities lower than % and may therefore represent new species belonging to the genera methanosaeta or the thermoplasmatales, cluster b. the most similar sequences in the databases were from various types of soil environments, water environments and anaerobic bioreactors in north america, europe and asia. for of the sequences, the best match came from an anaerobic bioreactor. a best matching entry in genbank or the silva rrna database with 100% coverage. b identity in %. phylogenetic tree analysis the phylogenetic affiliation of the obtained 16s rrna gene sequences was determined by phylogenetic tree analysis. a phylogenetic tree for euryarchaea inferred by maximum likelihood analysis is shown in figure  a phylogenetic tree for crenarchaea and thaumarchaea inferred by maximum likelihood analysis is shown in figure  the majority of the sequences were determined to be of genus methanosaeta. several sequences also affiliated with divisions of uncultured archaea., grosskopf and chouari. and jurgens. dynamics of the archaea community the community composition, as assessed by t-rflp, changed little throughout the monitored period. the difference, measured as bray-curtis distance, between the terminal restriction fragment profile of the first sample in the time series, may and all following samples was on average only  ±  % and  ±  % for the alui and rsai analysis, respectively. on two occasions, in october and in january the bray-curtis distance peaked, indicating a deviation from the community composition at the beginning of the time series. the difference between the trf profiles of may and may four years later, was 10% and 0% for the alui and rsai analysis, respectively. however, the sensitivity of the t-rflp analysis is limited and trfs of low abundance cannot be detected. thus, a bray-curtis distance of 0% between the trf profiles of two samples does not indicate identical community composition since there may be differences in the composition of archaea of low abundance. to get a rough estimate of the sensitivity of the t-rflp method, a comparison was made between the theoretical trf profile of the clone library and the observed trf profile from the same sample. the comparison showed that only trfs with a relative abundance higher than 20% in the clone library were observed in the trf profile. thus, the t-rflp analysis shows the dynamics of the major groups of archaea in the activated sludge. the relative abundances of the observed trfs in all trf profiles in the time series are shown in figures  and a observed and predicted trf lengths from t-rflp and clone library analysis of a sample from 2007-05- b relative abundance based on total fluorescence. c relative abundance based on frequency in clone library. d nd indicates cases where a trf could not be predicted or where the predicted trf was outside the detection range. to identify the trfs the observed trf lengths were compared with the predicted trf lengths of sequences in the clone library. the predicted trfs from the sequences in the clone library were between and bases longer than the observed trfs. such a discrepancy between observed and predicted trf sizes is commonly observed. not all observed trfs in the time series could be matched with the predicted trfs of the clone library sequences. to explore the possibility that the trfs in the trf profiles from the samples from and come from sequences other than those found in the clone library a comparison was also made with a database of archaeal 16s rrna gene sequences matching the primers used in this study. the database was checked for sequences that would result in any of the observed combinations of trfs generated by alui and rsai. the result of the analysis was a number of possible identities for each observed combination of alui and rsai trfs. although the database comparison may result in false identities of the trfs, it is valuable because it gives an indication about the range of species that could give the observed trf combinations. by comparison with the clone library sequences the dominating trfs were determined to represent methanosaeta-like species. comparisons with the predicted trfs of archaea sequences in the database showed that it is possible that the dominating trfs are from other species of the euryarchaeota than methanosaeta. however, it is unlikely that the origin of these trfs is species of the crenarchaeota or thaumarchaeota, since all the crenarchaeota or thaumarchaeota sequences in the databases have other predicted trf lengths. a observed trf combinations that are not included in the table were not found in the database nor in the clone library. b identification by comparison with predicted trf lengths of clone library sequences. c identification by comparison with predicted trf lengths of rdp database sequences. d out of range: the trf in the database or the clone library was either shorter than bases or longer than bases and would therefore not have been detected. correlation analysis several trfs showed a significant correlation with process parameters. the parameters that correlated with most trfs were water temperature and nitrogen concentration. there were also significant correlations between several trfs and the sludge and effluent water properties. the parameter effluent non-settleable solids and the concentration of carbohydrates in extracted extracellular polymeric substances correlated with most trfs. no trf showed a significant correlation with the sludge volume or shear sensitivity. a the correlations are marked with asterisks corresponding to the level of statistical significance: 95%, 99% and %. trfs that are not included did not show any statistically significant correlation with any parameter. the wwtp process parameter data was taken from. b identification by comparison with the rdp database. c identification by comparison with the clone library. d the number of times the trf was observed. e water temperature. f solids retention time. g food to mass ratio. h total cod going into the activated sludge tanks. i nitrite/nitrate levels going in to the activated sludge tanks. a the correlations are marked with asterisks corresponding to the level of statistical significance: 95%, 99% and %. trfs that are not included did not show any statistically significant correlation with any parameter. the sludge and effluent water parameter data was taken from. b identification by comparison with the rdp database. c identification by comparison with the clone library. d the number of times the trf was observed. e standardized sludge volume index. f shear sensitivity. g eps protein. h eps carbohydrates. i effluent non-settleable solids. quantification and localization of archaea in the activated sludge flocs the 16s rrna gene clone library indicated that published fish probes would cover the archaea at rya wwtp. archaea could be observed in the activated sludge flocs, both centrally located and close to the edges of the flocs. fish analyses showed that the average relative abundance of archaea in the activated sludge of the aeration tank was %. in the anaerobic digester and in the water recycled into the activated sludge tanks there were more archaea than bacteria. in most images of activated sludge flocs the percentage of archaea was lower than 2%. occasionally there were larger colonies of archaea but in most images archaea were either present as individual cells or small colonies. with an applied threshold of discussion in this study the abundance, localization, composition and dynamics of archaea in the activated sludge of a full-scale wwtp were assessed using fish analysis, 16s rrna gene clone library analysis and t-rflp time series analysis. these three analyses were all done on samples collected at different times. however, for most process parameters there were no significant differences between these times. the wwtp was also operated the same way at all times, except for four months, may to september when the primary settlers were bypassed. the samples were therefore considered comparable. the t-rflp time series analysis showed that the most abundant trfs were the same throughout and as well as in may. if we assume that the same trf always represent the same group of archaea, then the t-rflp data show that the main part of the archaea community was the same in and in may and that we can use the clone library data to identify the trfs in the t-rflp time series. we further assume that the archaea community stayed mainly the same in december which make it possible to use the clone library data to choose appropriate probes for the fish analysis. the clone library sequences indicated that already published fish probes were relevant for an estimation of the relative abundance of major archaea groups. the relative abundance of the archaea has been estimated in other investigations to be low, based on activity measurements, and up to 8% of bacteria or 10% of total cell numbers. in this study archaea was estimated, by fish, to make up % of total cell numbers in the activated sludge, a relatively low abundance. however, the importance of a microbial group cannot be deduced by abundance alone. putative aoa were 1-10% of total cell numbers in activated sludge, but despite this abundance they did not contribute significantly to nitrification, whereas foaming organisms have great impact on floc structure and sludge properties even when present in numbers around 1%. another example is ammonium oxidizers, which at an abundance of 3-5%, could perform the first step in a successful 80% reduction of nitrogen in an activated sludge system. thus, despite their relatively low abundance, a possible contribution of archaea to sludge properties cannot be ruled out. the composition of the archaea community was investigated by analysis of 16s rrna sequences. the community richness was estimated to be species of genera. as expected, the clone library does not fully cover the archaea community. however, the species of genera that were observed are assumed to represent the most abundant groups. the archaea community was functionally specialized with 77% of the sequences in the clone library being methanosaeta-like organisms. specialized communities dominated by methanogens, although of other genera than methanosaeta, have also been observed in activated sludge from other wwtps. the t-rflp time series analysis showed that the archaea community was practically the same in most samples, despite variations in environmental conditions such as organic loading rate and temperature. only in a few samples more than two trfs were observed. however, as shown in table the sensitivity of the t-rflp analysis was low, so it is possible that there were changes in the composition of the less abundant groups of archaea. a comparison between the observed trf lengths and the predicted trf lengths of the clone library sequences identified the two main trfs as coming from methanosaeta sequences, given the assumption that all trfs represent the same groups of archaea in all samples where they are observed, as discussed above. an alternative way of identifying the trfs would be to compare the observed alui and rsai trf combinations with the predicted trf combinations from archaea sequences in the rdp database. a comparison with archaea 16s rrna sequences showed that sequences of methanosaeta or other euryarchaea would give the observed alui and rsai trf combinations, but no crenarchaeota or thaumarchaeota sequences. in the following discussion we therefore assume that the two main trfs come from methanogens. methanogens are anaerobic and the oxygen concentration in activated sludge is high. however, in the deeper parts of activated sludge flocs anoxic microenvironments can exist which may allow growth of anaerobic organisms. in the activated sludge at rya wwtp, methanogens were observed both deep within the flocs and close to the surface. although exposed to oxygen, the methanogens at the surface are not necessarily inactive since methanogens have been shown to be able to maintain viability and activity in the presence of oxygen. to avoid washout from the activated sludge, microorganisms need to be active and have a doubling time shorter than the sludge retention time. pure cultures of methanosaeta concilii have a temperature optimum at 35-40°c and a doubling time of 4-7 days at 37°c. the low water temperature at rya wwtp, 10-20°c, does not necessarily prevent activity since methanosaeta-like species have been shown to grow at 9-14°c in bioreactors and dominate methanogenic cultures from rice field soil at 15°c. the solids retention time at rya wwtp is typically calculated as 5-7 days and could also allow for growth of methanosaeta-like organisms. in this study, methanosaeta-like trfs dominated throughout 15 months, and correlation analysis showed that some of the methanosaeta-like trfs increased in abundance with increasing temperature and increasing srts, i.e. theoretically more favorable conditions. both the constant dominance in the trf profiles and the response to changed environmental conditions would be explained by an active population of methanosaeta-like organisms. the detection of methanogens by fish analysis also showed the presence of rrna, which is expressed in active cells. however, high rrna levels may be maintained despite inactivity. in conclusion, the activity of the methanosaeta-like organisms is an open question. if the methanosaeta-like species do not grow fast enough to avoid washout, their constant presence requires that they are constantly added to the sludge. possible sources are the influent wastewater and recycled water from an anaerobic bioreactor. by fish analysis, archaea was confirmed to be present in high numbers in both the anaerobic bioreactor and in the reject water. thus the bioreactor might seed the activated sludge with archaea. this is supported by the fact that a majority of the detected 16s rrna sequences cluster with sequences from anaerobic sludge. furthermore, no sequences matched typical methanogens in human fecal matter, such as methanobacter smithii and methanosphaera stadtmanae, indicating that fecal matter from the influent water was not an important input to the methanogens in the activated sludge. the second largest group in the clone library was thermoplasmatales-related sequences affiliated with rice cluster iii. no cultured representative of rc-iii archaea exists, but a study of a methanogenic enrichment culture suggests that rc-iii archaea are mesophilic anaerobes growing heterotrophically on peptides with a doubling time of approximately three days. rc-iii has been detected in soil, anaerobic bioreactors and groundwater. this study shows that rc-iii archaea can also be present in activated sludge. thermoplasmatales-related sequences of cluster b and c were also found in the clone library. there are currently no cultured representatives or proposed phenotypes for these groups. cluster b and c sequences have been retrieved from environments with methanogenic communities and complete or partially anoxic zones, such as water, landfill leachates, sediments, bioreactors and the digestive tract of animals. this study adds activated sludge to that list. one sequence, clone g belongs to a yet undescribed lineage of archaea: arc i. the arc i lineage is well-represented in anaerobic bioreactors and in reactors with a high abundance of arc i, the abundance of species related to m. concilii is low and vice versa, which could indicate a competition for acetate between these two lineages. the clone library in this study followed the same pattern with low abundance of arc i and high abundance of m. concilii. the same pattern was also seen in the trf profiles since the only time that the trfs corresponding to sequence g was observed the relative abundance of the trfs associated with m. concilii had decreased to around 80%. archaeal ammonia monooxygenase subunit a genes have been observed in various activated sludge wwtps. one of our sequences affiliated with crenarchaea cluster b, which includes several putative aoa. however, it has recently been shown that not all amoa-carrying thaumarchaeota are ammonia-oxidizing autotrophs. the presence of aoa at the rya wwtp can therefore not be confirmed, and as has been suggested for other wwtps, aoa are most likely of minor or no importance for ammonia-oxidation at the rya wwtp. one clone affiliated with crenarchaea cluster. there are no cultured representatives of cluster, but spatial co-localization and a relation between the abundance of cluster and methanosaeta-like species has been reported. in other aggregate structures, such as anaerobic sludge granules, methanosaeta are important for structure and stability and they form dense aggregates which act as nuclei for granule formation. in the activated sludge the methanosaeta did not appear to have this function as they were mostly detected as small colonies or single cells and there was no apparent difference in structure between flocs with high and low numbers of methanosaeta. the lowest relative abundances of the methanosaeta-like trfs were observed in january and february. in october the two main methanosaeta trfs also decreased in relative abundance but it cannot be ruled out that the trfs that appeared in those samples were also methanosaeta. the lowest water temperatures of the period were recorded during january and february which could have reduced the survival or proliferation of methanosaeta-like species and allowed other archaea to increase. in anaerobic sludge, a decrease in methanosaeta abundance has been linked to granule disintegration. although the flocs had high shear sensitivity and a more open structure in january and february when the methanosaeta trfs decreased and although there was a significant negative correlation between methanosaeta trfs and effluent non-settleable solids it cannot be concluded that the archaea are important for the floc structure. the increased shear sensitivity and changed floc structure in january and february could be due to the reduced general microbial activity, which has been shown to decrease floc stability. furthermore, increased shear sensitivity and changed floc structure was also observed from june to august after the primary settlers were bypassed, but during this period the relative abundance of the methanosaeta trfs was 100%. thus, if the composition of the archaea community has any effect on floc structure or stability it is certainly only one of many other factors.  by sequencing and t-rflp analysis of 16s rrna genes and fish we showed that archaea were present in the activated sludge of a full-scale wwtp. the archaea community was dominated by methanosaeta-like species, which probably entered the sludge with a stream of recycled water from an anaerobic bioreactor. the archaea were present both as colonies and single cells but only in low numbers, estimated as % of total cell numbers in the activated sludge. during 15 months major changes in community composition were observed twice, but in both cases the community returned to the previous composition. even in samples collected three years apart the main part of the community remained the same according to t-rflp data. we now know that archaea can constitute a small but constant and integral part of the activated sludge and that it can therefore be useful to include archaea in future studies of sludge or floc properties. 
the results presented here show that archaea can constitute a constant and integral part of the activated sludge and that it can therefore be useful to include archaea in future studies of microbial communities in activated sludge. the activated sludge process is one of the most widely used methods for treatment of wastewater and the microbial community composition in the sludge is important for the process operation. during a 15 month period major changes in the community composition were only observed twice despite seasonal variations in environmental and operating conditions. water temperature appeared to be the process parameter that affected the community composition the most. the diversity and dynamics of the archaea community in a full-scale activated sludge wastewater treatment plant were investigated by fluorescence in situ hybridization, terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16s rrna genes. the archaea community was dominated by methanosaeta-like species. while the bacterial communities have been characterized in various activated sludge systems little is known about archaeal communities in activated sludge. several terminal restriction fragments also showed strong correlations with sludge properties and effluent water properties. the archaea were estimated to make up % of total cell numbers in the activated sludge and were present both as single cells and colonies of varying sizes.
 the activated sludge process is one of the most widely used methods for treatment of wastewater and the microbial community composition in the sludge is important for the process operation. while the bacterial communities have been characterized in various activated sludge systems little is known about archaeal communities in activated sludge. the diversity and dynamics of the archaea community in a full-scale activated sludge wastewater treatment plant were investigated by fluorescence in situ hybridization, terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16s rrna genes.  the archaea community was dominated by methanosaeta-like species. during a 15 month period major changes in the community composition were only observed twice despite seasonal variations in environmental and operating conditions. water temperature appeared to be the process parameter that affected the community composition the most. several terminal restriction fragments also showed strong correlations with sludge properties and effluent water properties. the archaea were estimated to make up % of total cell numbers in the activated sludge and were present both as single cells and colonies of varying sizes.  the results presented here show that archaea can constitute a constant and integral part of the activated sludge and that it can therefore be useful to include archaea in future studies of microbial communities in activated sludge. 