Eukaryotic Community in UASB Reactor

Treating Domestic Sewage Based on 18S

rRNA Gene Sequencing

Y. Hirakata1(&), M. Hatamoto2, M. Oshiki3, N. Araki3,

and T. Yamaguchi1,2
1
Department of Science of Technology Innovation,
Nagaoka University of Technology, Nagaoka, Japan
2
Department of Environmental Systems Engineering,
Nagaoka University of Technology, Nagaoka, Japan
3
Department of Civil Engineering, National Institute of Technology,
Nagaoka College, Nagaoka, Japan

Abstract. Microbial eukaryotes play important roles in sewage treatment sys-

tems. In an up-flow anaerobic sludge blanket (UASB) reactor fed with domestic
sewage, anaerobic bacteria, archaea, and microbial eukaryotes (protist and fungi)
coexist. To date, bacterial and archaeal communities in the UASB reactor have
been widely studied. However, little is known about the eukaryotic community
structure and function in most of anaerobic treatment systems. In this study, we
analyzed eukaryotic community in the UASB reactor treating domestic sewage
over 2 years operational period based on 18S rRNA gene sequences. In addition,
multivariate statistics were applied to elucidate the correlation between eukary-
otic community and operational conditions of the UASB reactor. The dominant
protist groups observed were from phylum Ciliophora, Apicomplexa, Perkisea
and Amoebozoa in the UASB reactor. Followings were flagellate protist such as
Cercozoa, Sulcozoa, Bicosoecida, Choanomonada, Dinoflagellata and Meta-
monada. The result showed protist phylum and water temperature were not
correlated. Only phylum Sulcozoa positively correlated with COD and suspended
solid (SS), whereas other protist showed low correlation. The dominant fungi
groups were LKM11, LKM15 and phylum Ascomycota. Uncultured LKM15
correlated with sulfide whereas phylum Discicristoidea and Chytridiomycota
showed negative correlation. Phylum Ascomycota seemed most abundant when
sulfide was low, but their association with environmental variables remained
unclear. These results suggested that some protist and fungi groups could be used
as indicator of environmental parameters in the UASB reactor.

Keywords: Eukaryotic community
Anaerobic treatment
Domestic sewage

1 Introduction

Microbial eukaryotes play important roles in sewage treatment systems. Protists are
often used as indicators of treatment performance in activated sludge process treating
domestic sewage. The reduction of sludge production by protist bacterivory is well
known in aerobic treatment systems. Some of fungi are also known to contribute to
© Springer International Publishing AG 2017
G. Mannina (ed.), Frontiers in Wastewater Treatment and Modelling,
Lecture Notes in Civil Engineering 4, DOI 10.1007/978-3-319-58421-8_34
 Eukaryotic Community in UASB Reactor Treating Domestic Sewage 219

denitrification and cellulose degradation. In an up-flow anaerobic sludge blanket

(UASB) reactor fed with domestic sewage, anaerobic bacteria, archaea, and microbial
eukaryotes (protist and fungi) coexist. To date, bacterial and archaeal communities in
the UASB reactor have been widely studied. However, little is known about the
eukaryotic community structure and function in most of anaerobic treatment systems.
The eukaryotes have been commonly identified morphologically by microscopic
observation. However, identifying anaerobic protist by microscopic observation is prone
to error because some protist species are fast-moving protozoa and indiscernibly small.
Furthermore, most fungi are difficult to identify by microscopic observation. Recently,
molecular biological techniques have been applied to analyze a greater diversity of
eukaryotes in wastewater treatment systems and environments (Miyaoka et al. 2016;
Simon et al. 2015). Molecular techniques can detect eukaryotes that cannot be observed
by microscopic observation. In this study, we analyzed eukaryotic community in UASB
reactor treating domestic sewage over 2 years of operational period using 18S rRNA
gene sequencing. In addition, multivariate statistics were applied to elucidate correlation
between eukaryotic community and operational condition of the UASB reactor.

2 Materials and Methods

An UASB reactor with total volume of 1,178 L and 4.7 m height was operated in
domestic sewage treatment center at Nagaoka City, Japan. The hydraulic retention time
of the system was set at 8 h. To activate the microorganisms responsible for sulfur
redox cycles, the system was fed with raw sewage that was supplemented with 50–
150 mg-S L−1 sodium sulfate. Additional details on the UASB reactor were described
previously (Aida et al. 2015). Sludge samples were collected from sampling port at
1.278 m above from the bottom of the reactor. The collected samples were then
immediately stored at −20° C until DNA extraction was performed.
The genomic DNA was extracted from collected samples using a FastDNA SPIN
Kit for Soil (MP Biomedicals, Carlsbad, CA, USA). The DNA concentration was
determined using a NanoDrop Spectrophotometer ND-1000 (Thermo Fisher Scientific,
Waltham, MA, USA).
General eukaryotic primers V4_1F and TAReukREV3 were used to amplify the V4
region of the eukaryotic 18S rRNA gene (Bass et al. 2016). The PCR was performed
using a thermal cycler (Applied Biosystems, Foster City, CA, USA) under the fol-
lowing cycling conditions: 5 min at 94° C; 15 cycles of 30 s at 94° C, 45 s at 53° C,
and 1 min at 72° C; 20 cycles of 30 s at 94° C, 45 s at 48° C, and 1 min at 72° C; and
the final extension step 10 min at 72° C. The amplification of the 18S rRNA gene
region was ascertained by agarose gel electrophoresis using 100 bp DNA ladder
(NIPPON Genetics, Tokyo, Japan). The amplicon was purified using an Agencourt
AMPure XP Kit (Beckman Coulter, Brea, CA, USA) and used for preparation of a
library by means of the MiSeq Reagent Kit v2 nano (Illumina, San Diego, CA, USA)
for sequencing on Illumina MiSeq. Amplicon library concentrations were measured
using BioAnalyzer DNA 1000 (Agilent Technologies, Santa Clara, CA, USA).
Sequence reads were processed using Quantitative Insights Into Microbial Ecology
(QIIME) version 1.8.0 (Caporaso et al. 2012). Sequence reads with a low quality score
220 Y. Hirakata et al.

(Phred quality score  30) were eliminated using the Trimmomatic v0.33 (Bolger
et al. 2014), and paired-end sequence reads were then assembled in the paired-end
assembler for the Illumina sequence software package (PANDAseq) (Masella et al.
2012). Operational taxonomic units (OTUs) clustering at 97% sequences identity were
conducted with the UCLUST algorithm (Edgar et al. 2010). The taxonomic classifi-
cation was determined using SILVA database 123 (Quast et al. 2012). Chimeric
sequences were identified with ChimeraSlayer (Haas et al. 2011). After detrended
correspondence analysis (DCA) was performed to determine the appropriate type of
model for direct gradient analysis, canonical correspondence analysis (CCA) and
redundancy analysis (RDA) were performed to investigate correlations between
eukaryotic communities and environmental factors with R software (Oksanen et al.
2007). These analyses include operational condition of the UASB reactor and protist or
fungi OTUs representing at least 0.3% reads per sample, on average.

3 Results and Discussions

In this study, we analyzed the protist community structures in 10 sludge samples

collected from UASB reactor treating domestic sewage based on 18S rRNA gene
sequencing. A total of 168,317 sequences were obtained. Fungi were predominantly
detected with relative abundance of 84% from total sequences. Relative protist abun-
dance was very low (4% of the total number of sequences). In addition, metazoa and
algae were also detected (8% and 4% respectively). Metazoa and algae most probably
had flowed into the UASB reactor with sewage.
Taxonomic classification of the protist and fungi community structures was shown
in Fig. 1. The dominant protist groups detected were from phylum Ciliophora, Api-
complexa, Perkisea and Amoebozoa, with average relative abundance of 30%, 21%,
12%, and 12% of all protist sequences, respectively. Followings were flagellate protist
such as phylum Cercozoa, Sulcozoa, Bicosoecida, Choanomonada, Dinoflagellata and
Metamonada (>1%, on average). Ciliophora, Amoebozoa and most heterotrophic
flagellate protists are major bacterial consumer in anaerobic environment. On the other
hand, phylum Apicomplexa and Perkisea are well known as parasitic protists. These
parasitic protists present in wastewater were reported capable of forming cyst (Ajonina
et al. 2012). Phylum Ciliophora was also dominantly observed microscopically in the
UASB reactor. On the other hands, phylum Amoebozoa and flagellate protist cannot be
observed by microscopic observation (Hirakata et al. 2016). 18S rRNA gene analysis
showed that existing larger protist diversity, which was recognized by microscopic
observation previously was uncovered in UASB reactor. In particular, phylum Cilio-
phora and Amoebozoa were found frequently from both aerobic and anaerobic envi-
ronment. It is possible that some species of Ciliophora and Amoebozoa present in
effluent sewage.
The dominant fungi groups were LKM11, LKM15 and phylum Ascomycota with
average relative abundance of 39%, 31%, and 19% of all fungi sequences, respectively
(Fig. 2). The member of phylum Ascomycota was capable of degrading cellulose.
Organisms in LKM11 and LKM15 group were detected from anoxic environment such
as lake or pond sediments (Wurzbacher et al. 2016). Additionally, LKM11 group was
 Eukaryotic Community in UASB Reactor Treating Domestic Sewage 221

Fig. 1. Phylum level taxonomic classification of the protist community structures based on 18S
rRNA gene in the UASB reactor treating domestic sewage over 2 years of operation

Fig. 2. Phylum level taxonomic classification of the fungi community structures based on 18S
rRNA gene in the UASB reactor treating domestic sewage over 2 years of operation
222 Y. Hirakata et al.

Fig. 3. Multivariate statistics. CCA plot based on operational condition of the UASB reactor
and protist OTU abundance (A). RDA plot based on operational condition and fungi OTU
abundance (B)
 Eukaryotic Community in UASB Reactor Treating Domestic Sewage 223

also detected from activated sludge treating domestic sewage (Matsunaga et al. 2014).
Thus, their functions of LKM11 and LKM15 group in sewage treatment process are still
largely unknown. Their functions need to be examined in more detail in future study.
Multivariate statistics were performed to see correlations between eukaryotic
communities and environmental parameters. The CCA analysis showed that Sulcozoa
OTUs positively correlated with COD and suspended solid (SS), whereas most OTUs
showed a low correlation (Fig. 3A). Most OTUs and water temperature were not
correlated too. This result suggested that Sulcozoa protist grow under high organic
loading lates while other protists that had low correlation with COD and SS were not
influenced by organic loading lates in the UASB reactor. As shown by RDA analysis,
OTU related uncultured LKM15 correlated with sulfide, whereas Discicristoidea and
Chytridiomycota showed a negative correlation (Fig. 3B). Phylum Ascomycota seemed
most abundant when sulfide was lower, but their association with environmental
variables remained unclear. Therefore, effect of sulfide could be important for these
fungi phylum. These results suggested that some protist and fungi groups could be used
as indicator of environmental parameters such as COD, SS and sulfide.

4 Conclusions

The eukaryotic community analysis based on 18S rRNA gene sequencing revealed
larger diversity in the UASB reactor. The dominant protist groups were phylum Cil-
iophora and Amoebozoa in the UASB reactor. In addition, uncultured LKM11 and
LKM15 groups were also dominated in the UASB reactor. The multivariate statistics
showed that some protist and fungi groups could be used as indicator of environmental
parameters in the UASB reactor. The physiological role of these uncultured eukaryotes
groups should be examined to understand relationship between eukaryotes and envi-
ronmental parameters in the future studies.

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