Abstract
Eukaryotic communities in aerobic wastewater treatment processes are well characterized, but little is known about them in anaerobic processes. In this study, abundance, diversity and morphology of eukaryotes in anaerobic sludge digesters were investigated by quantitative real-time PCR (qPCR), 18S rRNA gene clone library construction and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Samples were taken from four different anaerobic sludge digesters in Japan. Results of qPCR of rRNA genes revealed that Eukarya accounted from 0.1% to 1.4% of the total number of microbial rRNA gene copy numbers. The phylogenetic affiliations of a total of 251 clones were Fungi, Alveolata, Viridiplantae, Amoebozoa, Rhizaria, Stramenopiles and Metazoa. Eighty-five percent of the clones showed less than 97.0% sequence identity to described eukaryotes, indicating most of the eukaryotes in anaerobic sludge digesters are largely unknown. Clones belonging to the uncultured lineage LKM11 in Cryptomycota of Fungi were most abundant in anaerobic sludge, which accounted for 50% of the total clones. The most dominant OTU in each library belonged to either the LKM11 lineage or the uncultured lineage A31 in Alveolata. Principal coordinate analysis indicated that the eukaryotic and prokaryotic community structures were related. The detection of anaerobic eukaryotes, including the members of the LKM11 and A31 lineages in anaerobic sludge digesters, by CARD-FISH revealed their sizes in the range of 2–8 μm. The diverse and uncultured eukaryotes in the LKM11 and the A31 lineages are common and ecologically relevant members in anaerobic sludge digester.
Highlights
Anaerobic sludge digestion is a well-established waste treatment process
Clones belonging to the uncultured fungal lineage LKM11 in Cryptomycota were dominant members in all libraries (N library: 9 operational taxonomic unit (OTU), 48/55 clones, K library: 7 OTUs, 32/51 clones, S13 library: 6 OTUs, 18/52 clones, S14 library: 9 OTUs, 20/48 clones) except for the clone library of the M plant, in which Chlorophyta in Viridiplantae (9 OTUs, 13/45 clones) was the most predominant (S3 Table)
The most predominant OTU in the N, K, and M libraries belonged to the LKM11 lineage (20 clones, 16 clones, and 6 clones, respectively)
Summary
Anaerobic sludge digestion is a well-established waste treatment process. The benefits of the anaerobic sludge digestion are energy production mostly in the form of methane, sludge volume reduction and sludge stabilization. Anaerobic digestion is a complex and multistep process in which various microorganisms are involved in the degradation of organic matter. One of the well-known microbial interactions in the anaerobic environment is the syntrophic association between fatty-acid-degraders and hydrogenotrophic methanogens. Many researchers have focused their investigation on the prokaryotic community structure in PLOS ONE | DOI:10.1371/journal.pone.0172888. Many researchers have focused their investigation on the prokaryotic community structure in PLOS ONE | DOI:10.1371/journal.pone.0172888 March 6, 2017
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