Environ Sci Pollut Res
DOI 10.1007/s11356-015-5116-0
ECO-AQUACULTURE, SUSTAINABLE DEVELOPMENT AND PUBLIC HEALTH
The function of advanced treatment process in a drinking water
treatment plant with organic matter-polluted source water
Huirong Lin 1,2 & Shuting Zhang 1 & Shenghua Zhang 1 & Wenfang Lin 1 & Xin Yu 1
Received: 15 February 2015 / Accepted: 22 July 2015
# Springer-Verlag Berlin Heidelberg 2015
Abstract To understand the relationship between chemical
and microbial treatment at each treatment step, as well as the
relationship between microbial community structure in
biofilms in biofilters and their ecological functions, a drinking
water plant with severe organic matter-polluted source water
was investigated. The bacterial community dynamics of two
drinking water supply systems (traditional and advanced treat-
ment processes) in this plant were studied from the source to
the product water. Analysis by 454 pyrosequencing was con-
ducted to characterize the bacterial diversity in each step of the
treatment processes. The bacterial communities in these two
treatment processes were highly diverse. Proteobacteria,
which mainly consisted of beta-proteobacteria, was the dom-
inant phylum. The two treatment processes used in the plant
could effectively remove organic pollutants and microbial
polution, especially the advanced treatment process.
Significant differences in the detection of the major groups
were observed in the product water samples in the treatment
processes. The treatment processes, particularly the biological
pretreatment and O3–biological activated carbon in the ad-
vanced treatment process, highly influenced the microbial
Responsible editor: Gerald Thouand
Electronic supplementary material The online version of this article
(doi:10.1007/s11356-015-5116-0) contains supplementary material,
which is available to authorized users.
* Xin Yu
xyu@iue.ac.cn
1
Institute of Urban Environment, Chinese Academy of Science,
Xiamen 361021, People’s Republic of China
2
Ningbo Urban Environment Observation and Research
Station—NUEORS, Chinese Academy of Science, Xiamen, People’s
Republic of China
community composition and the water quality. Some oppor-
tunistic pathogens were found in the water. Nitrogen-relative
microorganisms found in the biofilm of filters may perform an
important function on the microbial community composition
and water quality improvement.
Keywords Biofilm . Biological treatment . Drinking water
treatment process . Microbial communities . Organic pollutant
Introduction
Supplying safe drinking water to the public remains a major
concern for municipal water providers and consumers.
Microorganisms are widely present in drinking water treat-
ment systems (DWDSs). Many problems in DWDSs are mi-
crobial based, such as pipe wall biofilm growth, nitrification,
biocorrosion of pipe material, deterioration of taste and odor,
and proliferation of opportunistic pathogenic bacteria
(Szewzyk et al. 2000; Wang et al. 2013). Thus, biological
safety problems that result from microbial pollution have
attracted considerable attention. Strategies aimed at
preventing microbial pollution have been considered to con-
trol drinking water quality.
The conventional water treatment systems widely used in
urban drinking water include coagulation, sedimentation, sand
filtration, and disinfection processes (Abbaszadegan et al.
1997). Certain advanced unit operations, such as biological
pretreatment and O3–biological activated carbon (BAC) fil-
ters, are added to further improve the drinking water quality
by preventing the deterioration of surface water quality.
Biofilters are applied to remove organic matter; taste-, odor-,
and color-causing compounds; and inorganic matter (Volk
et al. 2000). The microorganisms that colonize on biofilter
particles (mainly as biofilm) may perform an important

function on water treatment (Simpson, 2008). Thus, under-
standing the ecological functions of the microbial communi-
ties in the filters is important for the biological processes in the
drinking water treatment (Liao et al. 2012; Velten et al. 2011;
Liao et al. 2015). Comparatively, limited information is
known about the effect of each process on the water microbial
community, specifically on species composition and water
quality. Hence, a thorough understanding of the relationship
between chemical and microbial treatment at each treatment
step, as well as the relationship between microbial community
structure in biofilms and ecological functions performed by
these biofilms may facilitate the management of drinking wa-
ter production system.
Previous studies reported that the origin of raw water has a
significant effect on the bacterial communities in DWDSs
(Wu et al. 2015). Different treatment processes and treatment
efficiency can influence the product water quality. To under-
stand the complex bacterial community diversity and the com-
position of the biofilms in biofilters and in the water in each
step, a drinking water plant with severely organic matter-
polluted source water was chosen for this study. Two drinking
water treatment processes with different treatment steps were
investigated. The complex microbial communities in the water
samples at different stages of treatment and in the biofilms
associated with the key treatment steps in these two processes
were determined using 454 pyrosequencing technique to elu-
cidate the function of the biofilms in the biofilters.
Materials and methods
Water treatment processes
The drinking water plant is located in a city in Yangtze Delta
Area, China. The source water of the plant was polluted with
organic pollutants, with an NH3–N content of 1–5.16 mg/L,
usually above 2 mg/L. The CODMn was also high (7.49–
13.3 mg/L), usually more than 10 mg/L on monthly average.
The total organic carbon (TOC) and total nitrogen (TN) con-
tents were also high (Table 1S). The plant operates two drink-
ing water treatment processes. The first is the traditional pro-
cess built in 1994 with a 25,000 m3/day water supply scale
(Fig. 1a). It was mainly used for drinking water before 2006.
Since then, it has mainly been used for industry water supply
when a new process (Fig. 1b) was built. The disinfection dose
was 4–5 mg/L. The other process is the advanced treatment
technology built in 2006 (Fig. 1b). In this process, a biological
pretreatment and two stages of O3–BAC were added (Fig. 1b)
to improve the product water quality. The water supply scale
was 50,000 m3/day. The product water from this process is
mainly used as drinking water. The disinfection dose in this
process was 3–3.5 mg/L (effective chlorine concentration).
Environ Sci Pollut Res
Sampling and water quality
Water samples were collected from each water treatment step
in the two processes. The sample names and their descriptions
are marked in Fig. 1 and Table 1. In addition, biofilms in the
filter materials were also sampled to analyze the functions of
the filters. The biofilms were collected from the sand filtration
in the traditional treatment process (PSO) and the fillers in the
biological pretreatment (PL), sand filtration (PSN), and acti-
vated carbon (PT) in the advanced treatment process. A scan-
ning electron microscope (Hitachi S-4800, Japan) was used to
visualize the biofilms. The sample descriptions are listed in
Table 1. Water quality was also detected during sampling and
results are listed in Table 2. A redundancy analysis was con-
ducted between the biological factors and main abiotic vari-
ables using SPSS 21.
DNA extraction
Three water samples were collected from each step for DNA
extraction. The microbial biomass was harvested from ap-
proximately 3 L of each water sample. The water samples
were filtered through 0.22-μm millipore membranes using a
vacuum pump. The filtered membranes with microbes were
cut into pieces with a sterilized cutter. The total DNA of each
water and biofilm sample was extracted and purified using a
bead-beating method (FastDNATMSPIN Kit for Soil, Bio101
Inc., USA) following the manufacturer’s instructions.
PCR amplification and 454 pyrosequencing analysis
For the pyrosequencing analysis, the V3 and V4 regions
of 16S ribosmal RNA (rRNA) gene were amplified with
the primers 338F (5′-ACTCCTACGGGAGGCAGCAG-
3′) and 802R (5′-TACNVGGGTATCTAATCC-3′). The
DNA amplification conditions used in this study includ-
ed an initial denaturation at 95 °C for 3 min, followed by
35 cycles at 95 °C for 10 s and at 58 °C for 30 s, 6 s at
72 °C, followed by a final extension at 72 °C for 7 min.
The PCR products were purified and end repaired, A
tailed, PE-adapter ligated, and then utilized for pyrose-
quencing on a 454 Genome Sequencer FLX platform
(Xia et al. 2013).
Sequence analysis and phylogenetic assignment
The raw sequences obtained from the 454 pyrosequencing
were optimized. The redundant tags were deleted by
Mothur v.1.11.0 (Schloss et al. 2009). Mothur was used
to trim barcode and primer sequences and eliminate se-
quences shorter than 200 bp, with one or more ambiguous
bases and quality score inferior to 25. In addition, chi-
meras were identified with the Bchimera.uchime^
Environ Sci Pollut Res

Fig. 1 The drinking water a

treatment processes used in this PWO1 PWO2 PWO3
study and the water samples in Source water Cl2
each step. a Traditional treatment
process. b New advanced PSO PWO4
treatment process

Coagulation Sand filtration Disinfection

b
PWN1 PWN2 PWN3 PWN4 PWN5 PWN6 PWN7
Source water ClO2

O3 O3
PSN PT
PL Tap water
PWN8
Biological pretreatment Coagulation Sand filtration O3-BAC I O3-BAC II Disinfection

command. The results were the average values of three Results

parallels. Species richness, diversity indices (Chao1 esti-
mator, Shannon index, Simpson index, and abundance-
based coverage estimator (ACE)), and rarefaction curves
were obtained using Mothur at a 3 % dissimilarity cutoff
(Chao and Lee 1992). Sequences with similarities greater
than 97 % were grouped in one OTU. Each sequence was
compared with the sequences available in GenBank data-
bases using BLAST. Nucleotide BLAST was used for the
16S rRNA gene analysis. The closest relatives were iden-
tified for phylogenetic analysis. The sequences were
realigned and manually edited with ClustalX alignment,
and the phylogenetic analyses were performed with
MEGA5.0 software package using neighbor-joining and
maximum likelihood methods.
Water quality
The drinking water plant is a typical plant with organic matter-
polluted source water. The operation of this plant was studied
for a few years. The water quality of the source water at dif-
ferent months for a whole year was detected. The COD and
NH3–N content of the source water were usually high. The
water quality in each step is listed in Table 1S. The selective
water quality parameter values during sampling are listed in
Table 2. As described in BWater treatment processes^, the
source water was polluted with organic pollutants. The COD
contents were 7.11 and 10.02 mg/L, respectively. The NH3–N
contents were 1.98 and 2.89 mg/L, respectively. The NH3–N,

Table 1 Analysis of the microbial diversity indices and richness estimators using 454 pyrosequencing

Samples Sample description Reads OTUs ACE Chao 1 Shannon Simpson

PWO1 Traditional treatment process Source water 72,754 1629 3647.812 3568.645 6.586 0.971
PWO2 Coagulation 70,145 1039 2276.938 2297.031 5.425 0.942
PWO3 Sand filtration 69,367 1371 2977.021 3007.578 5.856 0.948
PSO Biofilm collected from 15,399 1089 1933.451 1893.863 8.254 0.992
sand filtration
PWO4 Disinfection (product water) 51,770 1243 2811.172 2532.652 6.227 0.963
PWN1 Advanced treatment process Source water 74,755 1471 3774.116 3589.798 6.259 0.969
PWN 2 Biological pretreatment 35,598 815 2040.558 1933.389 4.458 0.863
PL Biofilm collected from 17,680 1662 3112.194 3062.888 8.486 0.987
fillers in biological pretreatment
PWN 3 Coagulation 84,470 1975 6198.764 5331.58 5.990 0.956
PWN4 Sand filtration 73,762 2710 6596.774 6581.87 7.278 0.972
PSN Biofilm collected from sand filtration 16,571 1029 1739.693 1788.449 7.965907 0.986
PWN5 O3–BAC I 51,036 2602 5620.047 5350.789 7.742 0.970
PT Biofilm collected from activated carbon 12,877 957 1710.839 1782.57 7.930779 0.989
PWN6 O3–BAC II 64,998 2028 4393.696 4379.559 6.569 0.951
PWN7 Disinfection (product water) 52,434 899 1574.041 1590.69 5.074 0.922
 Environ Sci Pollut Res

Table 2 Summary of water quality parameter values for selective water samples

Samples Traditional treatment process Advanced treatment process

Water quality Source water Coagulation Production water Source water Biological pretreatment Sand filtration Product water
(PWO1) (PWO2) (PWO4) (PWN1) (PWN2) (PWN4) (PWN7)

Temperature (°C) 29 29 29 29.1 29.2 29.3 29.1

Turbidity (NTU) 17.8 ND 0.39 136 98 0.39 0.29
Chroma 30 ND ND 30 30 10 <5
pH 7.2 7.0 6.9 7.2 7 6.9 6.9
Hardness 120 ND 136 124 ND ND 138
Alkalinity 100 72 ND 98 ND ND 72
T-Fe (mg/L) 0.86 ND 0.5 1.06 ND ND <0.8
Cl− (mg/L) 40 ND 65 45 ND ND 62
Residual chlorine (mg/L) ND ND 0.75 ND ND ND 0.5
TN (mg/L) 5.53 4.81 4.57 5.81 7.56 7.14 4.75
NH3-N (mg/L) 1.98 ND 0.4 2.89 0.7 <0.002 <0.002
NO2-N (mg/L) 0.19 ND <0.002 0.106 0.097 <0.002 <0.002
TOC (mg/L) 4.71 2.93 2.81 5.51 6.94 5.67 3.01
COD (mg/L) 7.11 ND 2.91 10.02 8.73 7.11 0.58

ND not determined, TOC total organic carbon, TN total nitrogen, HPC total heterotrophic plate count, DO dissolved oxygen

NO2–N, and COD levels were higher in the source water
sample than in the product water sample in both the traditional
and advanced treatment processes. Removal efficiency for
NH3–N was 79.8 % in traditional treatment process, which
was lower than that in advanced treatment process (99.9 %).
In addition, the removal efficiency of COD was 59.1 % tradi-
tional treatment process, which is lower than in advanced
treatment process (94.2 %). The NH3–N, NO2–N, and COD
contents decreased with the treatment process. Compared with
the traditional treatment process, a biological pretreatment and
two stages of O3–BAC filters were added to the advanced
treatment process. They contributed to improving product wa-
ter quality, as evidenced by the lower COD and NH3–N con-
tents in PWN7 than those in PWO4 (Table 2).
PWO1 (source water), which decreased to 2811.172 for
PWO4 (product water). In the advanced treatment process,
the OTUs, ACE, Chao 1, and Shannon and Simpson values
in product water were lower than those in source water. The
ACE, Chao1, and Shannon values of PWN7 were lower than
those of PWO4. These results indicate that sample PWN7 had
a relatively lower level of bacterial richness and diversity than
PWO4, suggesting that the product water quality in the ad-
vanced treatment process was better than that in the traditional
treatment process. These results correspond to the water qual-
ity results (Table 2). A redundancy analysis was conducted
between the biological factors and main abiotic variables
using SPSS 21. It was found that some of the correlation
indexes were more than 0.8 (Table 2S).

Richness and diversity analysis of the samples Taxonomic composition of the samples

The richness and diversity indices were calculated at 3 %
width, as shown in Table 1. The bacterial communities in
the water samples showed a large bacterial diversity with
Shannon diversity indices (3.060–7.278). More than 400 gen-
era were identified from the water samples. A total of 84,278
valid sequences and 7508 OTUs at 97 % similarity level were
obtained from all the water samples through the 454 pyrose-
quencing analysis. Each sample contained valid sequences
between 12,877 and 84,470, with the number of OTUs rang-
ing from 815 to 2710 (Table 1).
In the two treatment processes, the values of ACE and
Chao1 decreased with each step of the process. In the tradi-
tional treatment process, the ACE value was 3647.812 for
The bacterial communities of the samples in both phylum
and class levels are presented in Fig. 2. In total, 28 dif-
ferent bacterial phyla were identified across the water and
biofilm samples. Proteobacteria, which accounted for
23.44–85.20 %, was the dominant phylum in all samples
and mainly consisted of four classes, namely, the alpha-
proteobacteria, beta-proteobacteria, gamma-
proteobacteria, and delta-proteobacteria (Fig. 2a). In addi-
tion to Proteobacteria, Actinobacteria, Firmicutes,
Planctomycetes, Bacteroidetes, and Nitrospirae were also
identified in all water and biofilm samples. Among these,
a larger number of Nitrospirae was found in the biofilm
samples than in the water samples (Table 3S).
Environ Sci Pollut Res

a 100 b 100

90 Other phylums 90
Firmicutes
80 Verrucomicrobia 80
Synergistetes Other

Relative abundance (%)

Caldilineae
Relative abundance (%)
70 Proteobacteria 70
Planctomycetes Nitrospira
60 Nitrospirae 60 Spartobacteria
Gemmatimonadetes Verrucomicrobiae
Fusobacteria Erysipelotrichia
50 50
Deinococcus-Thermus Planctomycetacia
40 Chloroflexi Flavobacteria
40
Chlorobi Actinobacteria
30 Chlamydiae Clostridia
30 Bacilli
Bacteroidetes
Armatimonadetes Sphingobacteria
20 20
Actinobacteria Gammaproteobacteria
Acidobacteria Deltaproteobacteria
10 10 Betaproteobacteria
Alphaproteobacteria
0
0
PWN1 PWN2 PWN3 PWN4 PWN5 PWN6 PWN7 PWN8 PWO1 PWO2 PWO3 PWO4 PWN1 PWN2 PWN3 PWN4 PWN5 PWN6 PWN7 PWO1 PWO2 PWO3 PWO4
Samples Samples

Fig. 2 Proportional composition of microbes in the water samples at the phylum (a) and class level (b)

Major taxonomic groups, such as alpha-proteobacteria, beta- Discussion

proteobacteria, and Bacteroidetes, were found in this study.
Several genus known to carry pathogenic strains were detected Effect of treatment process
in the water samples by the pyrosequencing analysis (Table 3).
Significant differences in the detection of the major groups were Physicochemical treatment
observed in the product water samples of the two treatment pro-
cesses (PWO4 and PWN7) (Fig. 2a, b, Fig. 3). Actinobacteria Different treatment processes and treatment efficiency can in-
accounted for 43.9 % of the product water (PWO4) in the tradi- fluence the product water quality and bacterial communities.
tional treatment process and 2.5 % of the product water (PWN7) Optimized or enhanced coagulation combined with activated
in the advanced treatment process. On the contrary, the percent- carbon adsorption can effectively remove organic matter,
age of Synergistetes was higher in PWN7 than in PWO4. which is the main nutrient for microorganisms. As shown in

Table 3 Abundance of some potential pathogen and sulfur- and nitrogen-relative microorganisms in genus level in different bacterial communities
(count)

Samples PWO1 PWO2 PWO3 PWO4 PWN1 PWN 2 PWN 3 PWN4 PWN5 PWN6 PWN7

Pathogen Escherichia_Shigella 5 0 0 0 9 0 0 1 0 0 0
Legionella 10 10 11 4 10 9 8 29 44 38 112
Pseudomonas 0 1 6 4 106 1 11 6 1 0 0
Enterococcus 1 0 1 0 4 0 1 0 1 0 0
Staphylococcus 0 3 1 0 20 23 23 11 11 4 2
Vibrio 2 0 0 0 0 0 0 1 0 0 0
Mycobacterium 3458 3663 3428 1252 1826 921 4262 6463 4453 6982 326
SOB Thiocapsa 2 0 0 0 0 0 0 0 0 0 0
Thiobacillus 1 0 0 0 0 0 0 0 0 0 0
Thiothrix 0 0 0 0 9 0 0 0 1 0 0
Sulfuritalea 11 5 7 18 9 1 7 4 1 1 1
Sulfurospirillum 0 0 0 1 0 0 0 0 0 0 0
Sulfurimonas 0 0 0 0 0 0 0 3 0 0 0
SRB Desulfobacca 0 0 0 0 1 0 0 0 0 0 0
Desulfomonile 7 1 0 0 3 1 0 0 0 0 0
Geobacter 6 1 0 1 3 0 3 4 2 0 0
Desulfocapsa 2 0 0 0 0 0 0 0 0 0 0
Desulfovibrio 0 4 4 3 0 0 0 0 0 0 1
Nitrobacteria Nitrospira 1 3 16 8 4 66 68 30 7 4 2
Nitrosomonas 2 0 0 0 0 1 0 51 5 10 0
Nitrosospira 0 3 2 4 1 0 2 0 0 0 0
Nitriliruptor 0 0 0 0 0 0 0 2 0 0 0
Denitrifying bacteria Denitratisoma 0 0 0 0 1 0 0 1 1 0 0

a b
PWO2
PWN5
PWN4
PWO4 PWN6
PWO1
PWO3
Fig. 3 Principal coordinate analysis of the samples using weighted-UniFrac from pyrosequencing. a Traditional treatment process. b Advanced
treatment process. c Biofilm samples
Table 1, the microbial diversity indices and richness estima-
tors decreased after coagulation, suggesting that coagulation
contributed to microbe removal. In traditional treatment pro-
cess, the TOC suddenly decreased from 4.71 to 2.93 mg/L
after coagulation (Table 2). The TN also decreased from
5.53 to 4.81 mg/L. However, the microbial diversity indices
and richness estimators increased after sand filtration
(PWO3). This result may be due to the detachment of mi-
crobes on the sand. A large number of Nitrospira (289 counts)
(Table 3S) was found in the sand filter biofilms (PSO), indi-
cating that sand filtration was involved in the removal of or-
ganic contaminants.
An increase in the amount of natural organic matter has
been observed in raw water supplies and has a significant
effect on drinking water treatment. Most of the natural organic
matter can be removed by coagulation (Matilainen et al.
2010). In the advanced treatment process, the samples obtain-
ed from the source water (PWN1) and the biological pretreat-
ment tanks (PWN2) were clearly different from the other sam-
ples (Fig. 3b), thereby indicating that coagulation aided in
removing the microbial communities in the treatment process.
Biological treatment
Upgrading the conventional treatment process has aroused
increasing attention from drinking water producers worldwide
because of the deterioration of surface water quality. The high
COD and NH3–N contents in the source water in the drinking
water plant studied indicated the presence of organic pollut-
ants. Biological pretreatment was added before coagulation in
the advanced process. The microbial diversity of the effluent
after biological pretreatment (PWN2) significantly decreased
compared with the source water (PWN1) (Table 1) because of
the attachment of bacteria on the filter material in the biolog-
ical pretreatment. Further analysis of the biofilms on the filter
material (PL) proved this observation (Tables 1 and 2S). The
microbiota that colonizes on the particles was speculated to
perform crucial functions in the reduction or
Environ Sci Pollut Res
c
PT
PSO
PWN3
PWN7
PWN1 PL
PWN2
PSN
biotransformation of organic contaminants. High levels of mi-
crobial diversity indices and richness estimators were found in
the biofilms collected from the fillers in the biological pre-
treatment, suggesting the richness of microbial community
in the biofilm (Table 1). Correspondingly, the COD, NH3–N,
and NO2–N decreased in the effluent after biological pretreat-
ment (PWN2), as shown in Table 2.
The BAC filtration in the treatment process can usually
perform well in removing biodegradable organic compounds
in drinking waters. The COD and NH3–N contents of the
product water (PWN7) were lower in this process than in the
traditional treatment process, thereby suggesting that adding
biological pretreatment, O3–BAC I, and O3–BAC II was im-
portant in improving the water quality. A stable, thin, and
active biofilm is ideal for BAC filtration. The visualization
of the drinking water biofilm matrix in the filters is shown in
Fig. 4. Pyrosequencing analysis was conducted on the biofilm
samples collected from the three reactors as well as the sand
filtration in the traditional treatment process to study the func-
tion of these reactors. The indigenous microbiota in BAC
filters can perform a crucial function in reducing or
biotransforming contaminants.
Assimilable organic carbon (AOC) was not affected by
coagulation, probably because the AOC fraction was com-
posed of small molecular weight, non-humic compounds that
are not amenable to coagulation (Volk et al. 2000). Thus,
biological treatment may function in the removal of these
organic pollutants. The abundance of sub-phyla of
Proteobacteria in drinking water biofilters can vary greatly,
possibly dependent on the properties and concentrations of
available carbon sources (Liao et al. 2013). Alpha-
proteobacteria was the largest bacterial group in the biofilm
samples (18.9 %) compared with the other sub-phyla of
Proteobacteria (8 % for beta-proteobacteria, 2.8 % for delta-
proteobacteria, 0 % for epsilon-proteobacteria, and 10.5 % for
gamma-proteobacteria). They are well known to occur in
freshwater (Zwart et al. 2002; Huang et al. 2011; Kwon
et al. 2011). This bacterial group may have important links
Environ Sci Pollut Res
Fig. 4 SEM analysis of the a b
drinking water biofilms matrix in
the filters. a Sand filtration in
traditional treatment process
(PSO); b sand filtration (PSN) in
advanced treatment process; c
activated carbon (PT) in O3-BAC
I in advanced treatment process; d
activated carbon in O3-BAC II in
advanced treatment process
c d
with the DOC and AOC removal (Liao et al. 2013). The pres-
ent work may provide new insights into the microbial com-
munity and biological process in drinking water biofilters. All
these results suggested that biofilms performed an important
function in water treatment.
Disinfection
Disinfection is an essential process for ensuring safe
drinking water. Disinfectant residuals are the primary
strategy for limiting microbial regrowth in DWDSs, al-
though it may lead to the formation of disinfection by-
products. In this study, significant differences were found
between water samples PWO3 (sand filtration) and PWO4
(product water) in terms of microbial diversity indices and
richness estimators (Table 1), suggesting the effect of dis-
infection on microbial diversity. Principal coordinate anal-
ysis results also showed the difference (Fig. 3a). The OTU
values of the product water samples (PWO4 and PWN7)
in both treatment processes remarkably decreased
(Table 1), suggesting that disinfectant residuals in
DWDSs performed an important function on bacterial re-
duction. Disinfection also showed significant reduction
efficiency on certain bacteria. For example, the percentage
of Firmicutes suddenly decreased after disinfection in the
advanced treatment process (Fig. 2a).
Opportunistic pathogens present a unique challenge in
drinking water systems. Waterborne opportunistic patho-
gens, including Legionella pneumophila, mycobacteria
(NTM), Pseudomonas aeruginosa, and Acanthamoeba
spp., have become an emerging public health concern
(September et al. 2004). Several genus known to carry
pathogenic strains were detected in the water samples by
the pyrosequencing analysis (Table 3). Detection of
Escherichia/Shigella was found in the source water in
both treatment processes; however, it was removed after
treatment. Some Staphylococcus, Pseudomonas, and
Enterococcus were also detected in the water samples.
Legionella and Mycobacterium were detected in all of
the sam ples in this plant. In previous st udi es,
Legionella and Mycobacterium are most commonly de-
tected in drinking water systems and typically present in
trace quantities (Vaerewijck et al. 2005; Marciano-Cabral
et al. 2010; Holinger et al. 2014; Feazel et al. 2009;
Revetta et al. 2013). Surprisingly, more Mycobacterium
was found in this plant compared with other pathogens.
Mycobacteria are generally resistant to disinfectants be-
cause of their complex cell wall (Hall-Stoodley and
Lappin-Scott 1998; Liu et al. 2012a; Szewzyk et al.
2000). The resistance may be attributed to the high tem-
perature (29 °C) during sampling. Mycobacteria are fre-
quently found in DWDSs (September et al. 2004;
Vaerewijck et al. 2005; Liu et al. 2012b). Thus, eliminat-
ing mycobacteria from DWDSs is more difficult than
other pathogens because of this characteristic. They can
form biofilms on the surface of pipelines, and their
growth in the water samples in further distribution sys-
tems may constitute a risk to consumers.

Sulfur- and nitrogen-relative microorganisms in biofilm
and water samples
The bacterial community information obtained was further
analyzed in terms of their potential functions. Sulfur-relative
microorganisms including sulfur-oxidizing bacteria
(Thiocapsa, Thiobacillus, Thiothrix, Sulfuritalea,
Sulfurospirillum, and Sulfurimonas) and sulfate-reducing bac-
teria (Desulfobacca, Desulfomonile, Geopsychrobacter,
Geobacter, Desulfocapsa, and Desulfovibrio) were found
and may have an important role in sulfur transformation in
water.
Nitrification is a two-step biological process that converts
ammonia into nitrite by ammonia-oxidizing bacteria (AOB)
and further into nitrate by nitrite-oxidizing bacteria (NOB)
(Regan et al. 2002; Zhang et al. 2009). Nitrification occur-
rence is conceptually considered a balance between AOB
growth on free ammonia and the inactivation from residual
monochloramine. Certain nitrobacteria (Nitrospira,
Nitrosomonas, Nitrosospira, and Nitriliruptor) and
denitrifying bacteria (Denitratisoma) were found in the bio-
film samples. Although nitrobacteria are not considered hu-
man pathogens, nitrification can affect drinking water quality
and result in regulatory violations. Therefore, nitrification
control is of practical importance.
In the water plant studied, the source water had high NH3–
N content. The AOB (Nitrosomonas, Nitrosococcus, and
Nitrosospira) and NOB (Nitrococcus and Nitrospira) found
in the water and biofilm samples suggested the nitration in
these biological reactors. The nitrite-oxidizing communities
were composed primarily of Nitrospira, although Nitrococus
was detected in certain samples (Table 3). Higher numbers of
Nitrospira were found in biofilm samples (PSO, PL, PSN, and
PT, biological pretreatment, BAC) than in water samples
(Table 2S). The presence of Nitrospirae species in the biofilm
samples suggested the occurrence of nitrification process in
the biological reactors. The microbial community composi-
tion and water quality was influenced by the biological treat-
ments in the advanced treatment process. All these results
suggested the important role of nitrogen-relative microorgan-
isms in the biofilms on water treatment in the advanced treat-
ment process.
Conclusion
In this study, 454 pyrosequencing was used to characterize the
bacterial diversity in each step of the two treatment processes
in a drinking water plant with organic matter-polluted source
water. The source water was seriously polluted with organic
matter. Both treatment processes used in the plant could effec-
tively remove organic pollutants, especially the advanced
treatment process. High bacterial community diversity was
Environ Sci Pollut Res
found in the two drinking water treatment processes.
Proteobacteria, which mainly consisted of beta-
proteobacteria, was the dominant phylum in all water samples.
The treatment processes, particularly the biological pretreat-
ment and O3–BAC, were highly effective at influencing the
microbial community composition and water quality.
Potentially, pathogens were found. Some functional microor-
ganisms (sulfur- and nitrogen-relative microorganisms) in fil-
ter biofilms might performed an important function on micro-
bial community composition and water quality improvement.
Acknowledgments This work was supported by the Natural Science
Foundation of China (Grant No. 31300109), Fujian Provincial Natural
Science Foundation (Grant No. 2013 J05087), and Ningbo Municipal
Natural Science Foundation (Grant No. 2014A610094).
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