Microbial communities within

7
biological wastewater
treatment reactors
Rushita Lal1, Anupama Srivastava1 and
Sourish Bhattacharya2
1
Department of Microbiology, Parul Institute of Applied Sciences, Parul
University, Vadodara, India 2Process Design and Engineering Cell, CSIR-
Central Salt and Marine Chemicals Research Institute, Bhavnagar, India

7.1 Introduction
For the ecosystem to function, bacteria are crucial and play
vital roles in carbon, nitrogen, and sulfur cycles. To protect the
water environment by removing contaminants (including
organics, nitrogen, and phosphorus) from wastewater treat-
ment (WWT), processes such as activated sludge (AS) or mem-
brane biological reactors (MBRs) are used (Horner-Devine and
Martiny, 2008; Ahmed et al., 2008). The domestic sewage and
other wastewater undergoes primary sedimentation which is
received by the WWT plant. The effluent (from the primary
sedimentation) flows to aeration basins where the diverse
microorganisms consume the soluble organic matter. Then
effluent flows to clarifiers from the aeration basins, which sed-
iment the microorganisms from the water. The clarifier efflu-
ent is either released or further processed for reuse. To
maintain a high cell density of microorganisms and removal
rates of soluble organic matter, the clarifier sediment or “acti-
vated sludge,” is recycled back to the aeration basins, while
the net growth of microorganisms is sent to the anaerobic
digester. The aeration basins carry a complex and high-density
microbial community, which could be a hot spot for horizontal
gene transfer. The process has a very large-scale application
represented by bioprocess engineering (B105 m3/d).

Wastewater Treatment Reactors. DOI: https://doi.org/10.1016/B978-0-12-823991-9.00006-X

© 2021 Elsevier B.V. All rights reserved. 141
142 Chapter 7 Microbial communities within biological wastewater treatment reactors

There is an incomplete knowledge of microbial communities

for utilizing microbial cultures for biological WWT reactors due to
the limitations of traditional culture-based techniques. (Fuhrman,
2009). By using a variety of molecular methods, microbial com-
munities have been distinguished in natural and engineered eco-
systems. For example, terminal restriction fragment length
polymorphism (t-RFLP) (Smith et al., 2003), denaturing gradient
gel electrophoresis (DGGE) (Eschenhagen et al., 2003), ribosomal
spacer analysis (RISA) (Whiteley and Bailey, 2000), 16S rRNA clone
libraries (Duan et al., 2009), and fluorescence in situ hybridization
(FISH) (Luxmy et al., 2000) were applied not just to evaluate bac-
terial community structure in biological reactors from a WWT
plant but also applied in other ecosystems such as marine,
edaphic, and atmospheric (Monson et al., 2006; Webster et al.,
2003; Maron et al., 2006) microbial systems. However, as they
don’t capture the whole complexity of microbial communities,
there will be incomplete results obtained from these methods
(Sanz and Köchling, 2007).
To study microbial communities, the microarray-based
genomics is an emerging technology (Zhou, 2003). Microarray-
based genomics techniques are powerful tools for viewing thou-
sands of genes expression in a single experiment (Wilson et al.,
2002), it makes them a specific, sensitive, quantitative, and
high-throughput tool for microbial detection, recognition, and
characterization in natural environments. This is a major tech-
nical advantage over other taxonomic nucleic acid-based
assays, especially in complex samples, which are limited by the
rate at which sequences can be analyzed.
With no subsequent DNA isolation and sequencing, large
numbers of microorganisms can be taxonomically identified
through high-density microarrays that target specifically 16S
rRNA genes. In recent years, their application has been signifi-
cantly extended to environmental systems (Brodie et al., 2006;
Huyghe et al., 2008). However, there are no records of using a
high-density universal 16S rRNA microarray to compare micro-
bial communities between various WWT bioreactors operated
at different geographic locations.
A suggestion of aerobic biological treatment processes have
stable and similar bacterial community structures primarily
influenced by the commonality of the influent wastewater. To
evaluate this hypothesis and to analyze the composition of bac-
terial communities from different WWT bioreactors there is a
use of high-density microarrays containing 506,944 probes
targeting 8935 clusters of 16S rRNA genes (PhyloChips).
 Chapter 7 Microbial communities within biological wastewater treatment reactors 143

7.2 Description of wastewater treatment

(WWT) reactors and samples
All bioreactors vary significantly in their size, process configu-
ration, and operational parameters. Bench-scale communities
had stable microbial communities prior to study and were sam-
pled several months after their start. The pilot plants were worked
for at least 1 year prior to analysis. Full-scale plants have been
working for many years. Biomass samples were collected with
sampling date, flow rate, influents, effluents, and operational
parameters of the WWTPs. The samples for the microbial analysis
were stored at
40 C and were sent to the laboratory for DNA
extraction, PCR amplification, and Illumina high-throughput
sequencing (Gentry et al., 2006) (Fig. 7.1).
Characteristics of bioreactors:
• Types of bioreactors—CAS (Conventional AS), MBRs, A/O-
MBR (anaerobic/oxic membrane biological reactors)
• Types of influent water characteristic—domestic and synthetic
• Scale—plant scale, bench, and pilot scale
• Different NH41 concentrations

Figure 7.1 (A) Membrane bioreactor process. (B) Conventional activated sludge.
144 Chapter 7 Microbial communities within biological wastewater treatment reactors

• Different COD (chemical oxygen demand) levels

• Different TSS (total suspended solids)
• Wastewater taken from different locations: Shanghai (China),
San Francisco (USA), Berkeley (USA)
• Each type of bioreactors has different flow rates (m3/day)
and has different temperatures.

7.3 DNA extraction, PCR amplification, and

illumina sequencing
Microbial DNA was extracted by using a Fast Spin Kit
(Qiagen, CA) following the protocol. PCR amplification of
full-length 16S rRNA genes from the total DNA extracted from
the microbial samples with Taq DNA hot- start polymerase by
using primers (where F stands for forward and R-reverse
strand): 27F(50 AGRGTTYGATYMTGGCTCAG-30 ) and 1492R
(50 -RGYTACCTTGTTACGACTT
30 ). PCRs for each sample were
run in triplicate and coimbined before analysis. The PCR mix-
ture contained 1.25 U of Taq polymerase, 1 3 PCR buffer
(Promega, WI), 0.5 μmol of each primer, 2 mM MgCl2, 200 μM
deoxynucleotide triphosphate, and 40 ng of template DNA. To
increase bacterial diversity, PCR amplifications were carried out
in a total volume of 50 μL in 0.2 mL tubes using a DNA gradient
thermocycler (Mastercycles, Eppendorf). To minimize nonspe-
cific amplification a hot-start PCR program was used for all
amplification. The cycling program was as follows: 2 min of ini-
tial denaturation at 95 C, 25 cycles of 95 C for 30 s and 72 C for
60 s, and a final elongation at 72 C for 10 min.
Amplicons were combined from the 12 different annealing
temperatures. By using DNA 7500 chips (Aglient Technologies,
USA) the size of PCR products were estimated by an Aglient 2100
Bioanalyzer. By using PicoGreen assay for dsDNA (Invitrogen, CA)
the amplicon concentrations were measured with a Nano Drop
3300 Flurospectrometer (ThermoSci, DE). Then by using Micron
YM100 spin filter (Micron, MA) the bacterial PCR products were
concentrated. By using PhyloChip, a total of 500 ng of PCR
products was analyzed (Fig. 7.2).

7.4 Application of PhyloChip

G2 PhyloChips were used with probes outlined in the
Lawrence Berkeley National Laboratory (DeSantis et al., 2003).
Eight thousand nine hundred and thirty-five clusters of 16S rRNA
 Chapter 7 Microbial communities within biological wastewater treatment reactors 145

Figure 7.2 Molecular-based techniques for microbial community analysis.

gene sequences were targeted by each PhyloChip probes. By using

additional targeted regions of the chromosomes, the remaining
probes were used for pathogen-specific signature amplicon detec-
tion, normalization controls, and image orientation (DeSantis et al.,
2007). Approximately 3% sequence division contained by each of
the 8935 clusters was considered as OTU (operational taxonomic
unit), representing all 121 distinguished prokaryotic orders.
According to the placement of its member organisms in Bergey’s
Taxonomic Outline, the taxonomic family of each OTU was desig-
nated (Snaidr et al., 1997). The taxonomic outline referred to phylo-
genetic classes containing uncultured environmental organisms or
unclassified families connected to named higher taxa. According to
previously described methods, the OTUs comprising each family
were clustered into 842 subfamilies by transitive sequence identity.
To create the PhyloChips, the selected oligonucleotides were
synthesized by a photolithographic method at Affymetrix Inc.
(Santa Clara, CA) directly onto a 1.28 3 1.28 cm glass surface at
an approximate density of 10,000 molecules/μm2. The probes
features were arranged as a grid of 712 columns and rows. Thus
146 Chapter 7 Microbial communities within biological wastewater treatment reactors

each unique probe sequence on the array had a copy number

of roughly 3.2 3 106 and occupied a square with an 18 μm side.
By using DNase 1 (0.02 U/mg DNA; Invitrogen), the fragmenta-
tion of amplicon pools consisted of 50
200 bp and were then ter-
minally labeled with biotin. After that, the labeled DNA was
denatured (at 99 C for 5 min) and hybridized to the DNA microar-
rays at 60 rpm at 48 C overnight. According to Affymetrix proto-
cols (as described previously) PhyloChip washing and staining
were performed.
As a pixel image, each PhyloChip can be scanned and recov-
ered, and individual signal values and intensities were completed
using standard Affymetrix software (GeneChip Microarray Analysis
Suite, version 5.1). For accuracy control every OTU was interro-
gated by 24 replicate probes. The number of positive probe pairs
(supporting information (SI) materials and methods) divided by
the total number of probe pairs in each probe set (i.e., OTU) was
calculated for the pf value (positive fraction value). By an OTU
when its pf value was greater than 0.9, the “present” value was
evaluated (Fig. 7.3).

7.5 Quantitative analysis of microbial

communities
The OTU profusion (measured by fluorescence intensity)
was normalized by a particular sample’s fluorescence maxima
to yield the relative OUT abundance x (0 , x , 1). Based on their
relative abundances x yielding the frequency distributions P(x),
the OTUs were then separated (divided) into classes. By using
the colinearity analysis, the similarities between the populations
were quantified. In the OTU space for this purpose, the compo-
sition of each sample is represented by an N-dimensional vec-
tor. Such vectors have coordinates (i.e., OTU1, OTU2, OTUN)
where N is the number of OTUs detected in each sample and
OTUi is the relative abundance of i-th OTU in the sample.
To analyze the relationship between the relative abundance
of bacteria (genus level) and environmental variables, redun-
dancy analysis (RDA) was used. This analysis was performed by
using the R-programming language software. The degree of sim-
ilarity can be expressed as the angle between two vectors, each
representing a particular community. The commonly used
method is comparing the species diversity of the ecosystem
using the rarefraction curves. The samples A and B have very
close compositions represented by a small angle α, whereas
samples A and C have diverse compositions since angle β is
 Chapter 7 Microbial communities within biological wastewater treatment reactors 147

Figure 7.3 Application of PhyloChip.

148 Chapter 7 Microbial communities within biological wastewater treatment reactors

much larger. To evaluate the significance of the results, 500

Monte Carlo simulations were performed in which the angles
between the random vectors were calculated. Each vector was
constituted of 1000 elements (N 5 1000). The standard value of
the angle was 41.43 (0.723 rad), between the random vectors as
compared with the standard deviation of 0.89 (0.016 rad).
Data analysis can be performed by the i-sanger platform
(http://www.i-sanger.com/) provided by Major Bio-Pharm
Technology Co. Ltd (Shangai, China). By using the Chao/Ace
estimator and the Shannon diversity index the microbial pheno-
type richness levels were calculated (Knapp et al., 2008).

7.6 Bacterial community composition

At least in one of the samples, by using the PhyloChip, 2199
distinct OTUs were detected. Each of the samples contained
between 53% and 82% of the total OTU accumulated. While in
the individual samples, the number of OTUs varied, all the sam-
ples contained a core of 859 OTUs, representing a large degree
of similarities among the samples.
On the absence or presence of specific operational taxonomic
unit (OTUs), the predominant phyllum identified was the
Proteobacteria, representing between 50% and 62% of all detected
OTUs. The subdominant groups were the Firmicutes, Bacteriodetes,
and Actinobacteria, each representing between 5% and 18% of the
detections. These four bacterial groups constituting approx 80% of
bacteria were detected with the samples. Based on the presence/
absence of OTUs, the similarities extended down to more specific
taxa. For example, the largest group was the γ-subdivision (31%

38%) with Proteobacteria, closely followed by α-Proteobacteria
(from 30% to 35%). Twenty-two taxa were identified within the
γ-Proteobacteria with Enterobacteriales, it is the dominant group
within a narrow range of 20%
25% of all the samples. They were
followed in dominance by Pseudomonadales and Alteromondales,
in each population (14%
19% and 15%
20%, respectively), each of
them also representing a similar fraction. In all the samples the
seven other detected groups (1
3 OTUs) had many fewer detec-
tions (aquatic clone group, Ellin 307/WD2124, uranium waste
clones, Shewanella, Vibrionales, Pasteurellales, SAR86) and consti-
tuted about 7% of γ-Proteobacteria. Rather than bacterial consortia,
more importance are given to axenic bacterial groups. Mostly, each
bacterial consortia contains more than one species (DeSantis et al.,
2007; Snaidr et al., 1997). Even at a more definite level of taxa, the
samples had similar composition, yet the samples were from
 Chapter 7 Microbial communities within biological wastewater treatment reactors 149

different bioreactors. For example, Acidobacteria were pointed out

by 36
51 OTUs (carrying 2.8%
3.6% of the detected OTUs), while
in the samples only eight or nine OTUs of Gemmatimonadetes
were found (0.5%
0.8% of total OTUs).
The remaining Proteobacteria subdivisions (α, β, δ, ε, unclas-
sified) had few detections but were also present in all samples
in very likely proportions. It is interesting that most of the
bacterial cultures present in wastewater reactors are
β-Proteobacteria comprising between 18%
20% of total micro-
bial population or in other words in most of the cases
β-Proteobacteria is being used in WWT bioreactors (Knapp
et al., 2008). Possibly this variation can be explained by the fact
that the PhyloChips are able to evaluate a much larger variety
of dominant microorganisms for a more complete characteriza-
tion of a complex population (Sakano and Kerkhof, 1998).
In more general terms, for characterizing microbial community
composition, the molecular tools are extremely useful, such as
t-RFLP, RISA, DGGE, FISH, and 16S rRNA clone libraries, but for
detecting the depth of the structure of highly complex communi-
ties they are generally not effective. For example, single bands
sometimes do not correspond to single bacterial species, although
PCR-DGGE can generally detect bacterial groups larger than 1%
of the population. The T-RFLP results are typically limited to only
50 or so of the most profuse organisms (Dunbar et al., 2000), so in
extremely complex communities it cannot effectively determine
phylogenetic richness (Hudson, 2008). Finally, 16S rRNA clone
libraries resulting in low-sensitivity are also subject to limitations
of low clone libraries, with even libraries of greater than 1000
clones exhibiting only moderate sensitivity in complex communi-
ties that miss many rare taxa (DeSantis et al., 2007).
To sequence the metagenome of complex communities 454-
pyrosequencing can be used and is more specific and has sig-
nificantly higher throughput than PhyloChip. However, it has
the disadvantage that among most of the alternative technolo-
gies, this is much more expensive. For understanding the com-
plex ecology of WWT bioreactors, the current cost and
complications linked with pyrosequencing complex metagen-
omes at appropriate read levels are likely to limit its use
(Sanapareddy et al., 2009; Nadarajah and Gupta, 2004). Previous
researchers with an average read length of 250.4 bp obtained
378,601 sequences from the AS basin of a WWT plant in
Charlotte using 454-pyrosequencing, but were able to collect
only 0.3% of the sequences into significant contigs (a set of
interesting DNA sequences and to overlapping physical seg-
ments (fragments) accommodated in clones depending on the
150 Chapter 7 Microbial communities within biological wastewater treatment reactors

significantly restricting data interpretation) (Nadarajah and

Gupta, 2004).
As well as the distinct advantages of the PhyloChip for pro-
viding inexpensive and rapid microbial community profiling, it
also has some disadvantages. For example, only targeted
(already known) OTUs are detected on the microarray. In addi-
tion by using bacterial primers with amplification of DNA, there
is a capability for PCR bias (including the 12 different annealing
temperatures) and only the bacterial community is examined,
whereas the archeal and fungal populations are ignored.
Accordingly, in this study, there is no knowledge that if the bio-
logical treatment systems from different locations maintain
similar archeal and fungal populations.
Using quantitative analysis of the OTUs (as opposed to sim-
ply presence/absence) in each sample, the average hybridiza-
tion (fluorescence) intensity was measured. From sample to
sample, to control for intensity variations by the maximum
intensity value in each sample the measured intensities were
normalized and supposed to be equal. A fraction of the corre-
sponding DNA was extracted from the population. As expected
for normalized variables, the frequency distributions P(x)
approximately followed beta probability distribution (Ge et al.,
2018). The only significant divergence from the theoretical dis-
tributions (“fat tails”) were for a few of the most abundant
OTUs (top 1%
3% of the population, 10
50 OTUs) which were
overrepresented compared to the beta distribution.
The Shannon diversity index H0 values were calculated by
assessing the internal (within sample) complexity of individual
microbial populations. The values of H0 were quite close across
the given samples ranging from 7.0
7.4. For diverse microbial
populations without a few strongly dominant taxa, these H0
values are typical. To quantify the similarity between the micro-
bial population in the samples further analysis was performed.
A colinearity analysis was used, for this purpose. For the motive
of the analysis, the vector representing each of the populations
(samples) has to be rectified in the N-dimensional space.
There are two choices of N. To use the common core of
N 5 8590 OTUs present in all samples was the first choice. A
measure of “relatedness/similarity” is represented by the angle
between the vectors such that, by a zero angle perfectly identi-
cal populations would be described, while completely different
populations would be expressed by orthogonal (90 ) vectors.
Therefore, populations with 1 angle would be considered
almost identical, whereas an 85 angle would be proportional to
highly dissimilar populations. Among the population samples
 Chapter 7 Microbial communities within biological wastewater treatment reactors 151

the calculated values were quite small, in all samples between

4.7 and 13.8 (certainly particularly different from random vec-
tors), indicating a very strong similarity of microbial popula-
tions. To further evaluate the effects of dominating OTUs, for
each pairwise combination of populations a similar analysis
was performed, but only with a set of the most abundant OTUs
selected from the common core. For these subsets between
10 and 400 OTUs were chosen and the angles calculated. The
results indicate that the similarity (almost colinear vectors and
characterized by very small angles) of the composition was not
dominated only by the most abundant members but expanded
also to less abundant members in the core. As an example, the
top 20 OTUs in one of the samples and the complementary
ranks and relative abundance of these OTUs in other samples
are generally being observed. However, the ranks do not match
to perfection, the members that are abundant in one sample
are also abundant in other samples.
The second choice of N was that at least in one sample the
total group of N 5 2119 OTUs was detected. In a sample if a par-
ticular OTU was not detected its abundance was assumed to be
zero with the complementary vectors coordinates also set to zero.
Obviously because of larger intersample diversity, this approach
resulted in much larger pairwise angles between the vectors. The
angles varied from 25 to 40 (between two different samples).
Although, from the random vectors, even these larger angles were
significantly lower (and statistically different).
There is a comparison of each pair from the definite popula-
tions (samples), to analyze further the effects of undetected
OTUs on the diversity among the populations. There is a use of
the largest set of OTUs common to each pair, for each pairwise
comparison. The N values were smaller than the total number
of N 5 2119 of detected OTUs, evidently larger than the N 5 859
for the core and varied between 926 and 1378. Those based on
the common core of 859 OTUs the resulting angles in this pair-
wise comparison were very small and virtually the same, with-
out affecting the involvement of up to 60% more OTUs.
Thus the diversity between microbial populations sampled
in the following bioreactors is almost completely due to taxa
that were available in some samples but were not detected in
others. When the populations are differentiated based upon any
subset of taxa available in more than one sample, their compo-
sitions were virtually identical. With a very similar composition
it appears that the microbial populations in these bioreactors’
samples consist of a common core of taxa (one-half to two-
thirds of OTUs), with the remaining portions of diverse taxa
152 Chapter 7 Microbial communities within biological wastewater treatment reactors

detected only in one specific population. There are 10 most

abundant taxa unique to each sample.
For the relative abundance of microbial taxa in each we fur-
ther analyzed the core and the remainder of the populations. In
each sample we found that no core taxa were present in the
least abundant quintile (referring to any of five equal groups
into which a population can be divided according to the distri-
bution of values of a definite variables). However, up to 80% by
abundance the OTUs unique to each sample were also found at
higher levels. From this point of view, each population con-
tained (approximately) the bottom quintile that contained only
unique taxa, the top quintile contained only core taxa, and both
the core and unique taxa were contained by the remaining
middle three quintiles (20%
28%)
The diversity among the samples can be credited only to a
group of unique OTUs that were detected only in specific sam-
ples. In terms of abundance (bottom half), typically, these taxa
ranked somewhat lower, but a few were present in much higher
proportions. In each sample it is not clear at this time which
are responsible for the unique sets of taxa in each sample and
which of the factors contributes to the formation of the com-
mon population core. It looks that there was stronger common-
ality between the samples from China than between the
Chinese and US samples, however, the difference is not very
large. All seven full-scale municipal WWT plants showed similar
community structures, but only for nitrifying bacteria. It is pos-
sible that the origin of inoculum and the characteristics of
wastewater may lead to commonalities in the population.

7.7 Inflow versus effluent samples and

dominant OTUs
Principal coordinate analysis defined the two main clusters
that are inflow and effluent samples. Within the effluent cluster,
the samples were clustered more closely together, whereas in the
inflow cluster the samples were most similar to each other.
At the phylum level there were two well-defined clusters based
on inflow- and effluent-specific bacterial communities, which
represented only minor temporal differences. Abiotic parameters
were very likely responsible for the observed differences in bacte-
rial community composition in the WWT plant inflow versus the
effluent (such as oxygen concentrations as well as different
bacterial species with different metabolic characteristics) [28].
 Chapter 7 Microbial communities within biological wastewater treatment reactors 153

7.8 Identification of microbial communities

16S rRNA gene sequencing
7.8.1 The 16S rRNA gene sequencing advantages
Even to the species level, with some restrictions, more puri-
fied and reliable taxonomic assignment was possible. While
most of the existing studies used only the information of certain
hypervariable regions of the 16S rRNA, we were able to use all
phylogenetically relevant sites of the whole 16S rRNA gene.
Huse et al. compared a full-length sequence with V3 and V6
hypervariable regions and found both methods could rectify the
taxonomy similarly at the level of genus.
1. Universally distributed.
2. Low cost.
3. Capability of measuring phylogenetic relationships across
different taxa.
4. Easily have horizontal gene transfer.

7.8.2 Disadvantages of 16S rRNA gene sequences

1. Relative abundance measurements are undesirable.
2. Copy numbers per genome can vary.
3. Unable to differentiate between closely related species.
4. Diversity of the genes tends to be overinflated.

7.8.3 Protocol for amplification of 16S rRNA gene

sequences
1. The DNA extracted is used as the template for PCR to
amplify a segment of about 500 or 1500 bp of the 16S rRNA
gene sequences.
2. The products of PCR are purified to eliminate excess primers
and nucleotides.
3. The PCR amplification results in multiple copies of the target
DNA sequences being produced.
4. This resulting sequence is then used as a template for the
next step of the process known as cycle sequencing.
5. It is similar to PCR as it uses DNA (purified products of the
first PCR cycle) as the template. Both the forward and
reverse primers are used.
6. Cycle sequencing also differs from PCR in that no new tem-
plate is formed and the same template is reused for as many
cycles as programmed, usually 25 cycles, and the product is
154 Chapter 7 Microbial communities within biological wastewater treatment reactors

a mixture of various lengths of DNA. This is achieved by add-

ing specially labeled bases, which, when they randomly are
incorporated in the second cycle, terminate the sequences.
7. Thus fragments of every size are generated. As each of the
four added labeled terminator bases has a different fluores-
cence dye, each of which absorbs at a different wavelength,
the terminal base of each fragment can be determined by a
fluorometer.
8. The products are purified to remove excess incorporated dye
terminator, and the length of each is determined using
capillary electrophoresis or gel electrophoresis (Fig. 7.4).

7.9 Future perspective

Due to the time-effective, cost-effective and informative fea-
tures of 16S rRNA amplicon sequencing, it is popular. But it is also
limited due to several disadvantages. For multiple cases primarily
16S is well studied, but provides limited taxonomic and functional
information. Secondarily, for the 16S rRNA gene the PCR amplifi-
cation of different regions may generate contradictory results due
not only to the definite binding affinities for corresponding flank-
ing conserved regions, but also due to resolution of each variable
regions across taxa. Therefore shotgun metagenomics or full-
length 16S rRNA gene sequences may be sometimes more favor-
able, especially the latter.

7.10 Conclusion and recommendations

WWT is of great importance to environmental hygiene in urban
environments. Wastewater treatment plants (WWTPs) collect
organic matter, chemicals, and microorganisms including multire-
sistant bacteria and pathogens which may be potentially released
into the environment via WWT effluent. For the better characteriza-
tion of potential pathogenic taxa and other harmful bacteria,
which is required to determine health risks, the full-length 16S
rRNA genes were allowed taxonomic resolution. For a single
wastewater type the WWT plants with low temperature were
found to have the lowest microbial diversity. The WWT plants
with mixed wastewater types were found to have the highest
microbial diversity. The most abundant phyla in all the samples
were Proteobacteria (26.7%
48.9%), Bacteriodetes (19.3%
37.3%),
Chloroflexi (2.9%
17.1%), and Acidobacteria (1.5%
13.8%), all are
considered to be 79.6%
91.2% of the classified sequences. Fify-five
genera, which are considered to be 60.6%
82.7% of the classified
 Chapter 7 Microbial communities within biological wastewater treatment reactors 155

Figure 7.4 Protocol for amplification of 16S rRNA gene sequences.

156 Chapter 7 Microbial communities within biological wastewater treatment reactors

sequences and included unclassified were of Comamonadaceae,

Flavobacterium, Dokdonella, Terrimonas, Tetrasphaera, Simplicispira,
and Nitrospira genera. RDA indicated that influent ammonia con-
centration, water temperature, and oxygen concentrations had a
considerable impact on bacterial community compositions, whereas
the least influence was from pH.

Acronyms
• WWT—wastewater treatment
• WWTPs—wastewater treatment plants
• AS—activated sludge
• MBRs—membrane biological reactors
• 16S rRNA gene—16S (ribosomal) ribonucleic acid
• DNA—deoxyribonucleic acid
• RISA—ribosomal spacer analysis
• T-RFLP—terminal restriction fragment length polymorphism
• DGGE—denaturing gradient gel electrophoresis
• FISH—fluorescence in situ hybridization
• SSCP—single -strand confirmation polymorphism
• TGGE—temperature gradient gel electrophoresis
• ARDRA—restriction analysis of the amplified 16S rRNA gene
or amplified rRNA restriction analysis
• PCR—polymerase chain reaction
• OTUs—operational taxonomic units

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