Microb Ecol (2013) 66:60–72
DOI 10.1007/s00248-013-0207-2
ENVIRONMENTAL MICROBIOLOGY
Effect of Seawater–Sewage Cross-Transplants on Bacterial
Metabolism and Diversity
Jie Xu & Hongmei Jing & Liangliang Kong &
Mingming Sun & Paul J. Harrison & Hongbin Liu
Received: 2 June 2012 / Accepted: 28 February 2013 / Published online: 15 March 2013
# Springer Science+Business Media New York 2013
Abstract Bioassays experiments were conducted to deter-
mine the metabolic and community composition response of
bacteria to transplants between relatively pristine coastal
seawater and sewage-impacted seawater. There were four
treatments: (1) pristine seawater bacteria+pristine seawater
(Pb+Pw), (2) sewage-impacted bacteria+sewage-impacted
water (Sb+Sw), (3) pristine seawater bacteria+sewage-impact-
ed water (Pb+Sw), and (4) sewage-impacted bacteria+pristine
seawater (Sb+Pw). Sewage-derived DOC was more labile and
readily utilized by bacteria, which favored the growth of high
nucleic acid (HNA) bacteria, resulting in high bacterial produc-
tion (BP, 113±4.92 to 130±15.8 μg C l−1 day−1) and low
respiration rate (BR, <67±11.3 μg C l−1 day−1), as well as high
bacterial growth efficiency (BGE, 0.68±0.09 to 0.71±0.05). In
contrast, at the relatively pristine site, bacteria utilized natural
marine-derived dissolved organic matter (DOM) at the expense
of lowering their growth efficiency (BGE, <0.32±0.02) with
low BP (<62±6.3 μg C l−1 day−1) and high BR 133±14.2 μg C
l−1 day−1). Sewage DOM input appeared to alter the partitioning
of carbon between respiration and production of bacteria,
resulting in a shift toward higher BGE, which would not
enhance oxygen consumption. Taxonomic classification based
on 454 pyrosequencing reads of the 16S rRNA gene amplicons
revealed that changes in bacterial community structure oc-
curred when seawater bacteria were transferred to the eutrophic
sewage-impacted water. Sewage DOM fueled the growth of
Gammma-proteobacteria and Epsilson-proteobacteria and
J. Xu (*) : H. Jing : L. Kong : M. Sun : P. J. Harrison : H. Liu
Division of Life Science, Hong Kong University
of Science and Technology, Clear Water Bay,
Hong Kong, China
e-mail: xujie@ust.hk
reduced the bacterial richness, but the changes in the commu-
nity were not apparent when sewage-impacted bacteria were
transferred to pristine seawater.
Introduction
The quality and quantity of dissolved organic carbon (DOC)
can exert significant impacts on bacterial composition and
their function in the microbial loop [2]. In coastal waters,
there are a wide range of variations in the quality and
quantity of nutrients and DOC. Considerable attention has
been paid to bacterial respiration (BR) and changes in the
supply of organic matters in the coastal areas [14, 24].
Kirchman et al. [21] reported that dissolved organic matter
(DOM) can affect bacterial community composition and in
turn the changes in bacterial diversity could have an impact
on DOM hydrolysis. Alpha-proteobacteria are found to be
active in the uptake of amino acids [10] and leucine [1],
while Gamma-proteobacteria are stimulated by high concen-
trations of glucose [1, 33].
Bacterial production (BP) has been extensively used to
infer total C consumption by bacteria in the oceans [7,
15]. However, this link is regulated by bacterial growth
efficiency (BGE) which varies widely with the degree of
environmental hostility [12, 34]. In oligotrophic oceans,
due to energy limitation, a large fraction of the available
carbon is respired for maintenance and turnover func-
tions, rather than cell division [13]. C processing and
bacterial growth should be linked to the underlying C
availability and consumption [8]. As an important pollu-
tion source, sewage delivers a variety of DOM into
coastal waters in some areas such as the upper part of
Effect of Transplants on Bacterial Metabolism and Diversity
the Pearl River estuary where the DOC concentration
was up to 473 μmol C l−1 [18].
Hong Kong waters are subjected to the year round input
of sewage in Victoria Harbour which receives untreated
sewage from many small outfalls, plus 2×106 tonnes of
sewage effluent that is subjected to enhanced primary treat-
ment each day [5]. However, there have been few studies on
the effect of DOM from sewage sources on bacterial metab-
olism and community composition. In our study, a cross-
transplant experiment was conducted, where the bacterial
community from eutrophic sewage-impacted seawater in
Victoria Harbour and the relatively pristine coastal seawater
with no influence of river or sewage discharge were cross-
transplanted, in order to test the metabolic activity of pris-
tine seawater and sewage-impacted bacterial communities
and the partitioning of carbon between respiration and pro-
duction in response to changes in DOM sources (natural vs.
sewage-derived), as well as shifts in bacterial community
composition.
Methods
Sampling Sites
Seawater samples were taken on August 3, 2011, during
high tide, at the surface at two stations, a relatively pristine
coastal station (PM7) with no influence of sewage or Pearl
River freshwater discharge, and at a eutrophic sewage-
impacted station (VM5) (Fig. 1). Samples for analysis of
nutrients, DOC, and bacterial abundance (BA) and viral
abundance (VA) were taken.
Cross-Transplant Experiments
Water samples were passed through a 1-μm polycarbonate
membrane and the filtrate was used as the bacterial inoc-
ulum. Bacteria-free seawater was obtained by double fil-
tration of seawater through a 0.2-μm cartridge. About
600 ml of the bacterial inoculum was mixed with 5.4 l
of 0.2-μm seawater filtrate from each station (i.e., bacteria
and phytoplankton were removed) in acid-washed and
Milli-Q rinsed polycarbonate carboys and then distributed
to three 1-l glass bottles. Four treatments were conducted:
(1) pristine seawater bacteria+pristine seawater (Pb+Pw),
(2) sewage-impacted bacteria + sewage-impacted water
(Sb+Sw), (3) pristine seawater bacteria+sewage-impacted
water (Pb+Sw), and (4) sewage-impacted bacteria+pris-
tine seawater (Sb+Pw). Samples were incubated in tripli-
cate in the dark for 24 h. All the bottles were sealed
during the incubation period. Running seawater was used
to maintain the surface in situ temperature (29±2°C).
Samples for analysis of nutrients, BA, BP, BR, and VA
61
were taken in triplicate at the beginning and the end of the
incubations.
Nutrient and Dissolved Organic Carbon
Inorganic nutrient concentrations (NO

þ
3 ; NO2 ; NH4 ; PO4
3

and Si(OH)4) were determined colorimetrically with a
SKALAR autoanalyser following the protocols described
by Strickland and Parsons [38] and Grasshoff et al. [16].
Dissolved inorganic nitrogen (DIN) was the sum of NO
3;
NO
2 and NH þ
4 . Samples for DOC were filtered through a
0.2-μm filter. DOC concentrations were determined with a
high temperature combustion method using a Shimadzu
TOC-5000 analyzer.
Bacterial and Viral Abundance, Bacterial Production,
Respiration and Cell Size
Samples for bacterial and viral abundance were collected in
microcentrifuge tubes, fixed with buffered paraformalde-
hyde (final concentration 0.5 %), and then stored at −80°C
until analyzed by a flow cytometer (Becton-Dickinson
FACSCalibur). Samples for counting bacteria were stained
with 0.01 % SYBR Green I in the dark at room temperature
for 45 min before analysis [26]. Bacteria were detected on a
plot of green fluorescence vs. side scatter (SSC), which also
provided information on DNA content and cell size by the
mean of the SYBR-green fluorescence and side scatter,
respectively. The values for DNA content and cell size were
normalized to the fluorescence and SSC of beads. Viral
abundance was determined using flow cytometry and SYBR
Green I staining after samples were diluted by Tris-EDTA
buffer according to Marie et al. [27].
BP was measured using 3H leucine following the JGOFS
protocol [23]. 3H leucine (final concentration 23 nM, spe-
cific activity 140 Cimmol−1) was added to 1.8 ml subsam-
ples (triplicate) with one control fixed by 5 % trichloroacetic
acid (TCA). The subsamples were incubated for 1 h, and
then terminated by adding TCA and filtered on a 0.2 μm
polycarbonate membrane. The incorporated 3H was deter-
mined using a Perkin-Elmer Wallac 1414 scintillation coun-
ter. BP was calculated with the empirical conversion factor
of 3 kg C mol leucine−1 [32].
For BR measurements, six 60-ml BOD bottles were filled
for each treatment. Three bottles were fixed with Winkler
reagents and subsequently determined for dissolved oxygen.
The other three bottles, which were sealed throughout the
incubation, were incubated in the dark alongside the large
volume incubations. Dissolved oxygen was titrated using an
automated titration apparatus (716 DMS Titrino Metrohm)
that analyzed samples with a potentiometric detector to deter-
mine the titration end point [31]. BR was presented in carbon
units assuming that the respiratory quotient was 1 [20].
62
Fig. 1 Location of the
sampling stations in Hong
Kong waters. These two
stations are the same as the EPD
monitoring stations. The water
depths for VM5 and PM7 are 22o30’
13 and 17 m, respectively
Latitude (oN)
22o20’
o
22 10
’
113o50’
The growth rates (μ) of bacteria and viruses were calcu-
lated using the following equation:
μ ¼ lnðAf =Ai Þ= Δt;
where Ai and Af are the initial and final abundance of
bacteria or viruses, respectively, and Δt is the time interval
between the beginning and the end of the experiment.
BGE was calculated using the following equation:
BGE ¼ BP=ðBP þ BRÞ
Sample Collection, RNA Extraction and cDNA Synthesis
To investigate the active bacterial composition in the exper-
iment, the bacterial biomass between the 0.2 and 1.0 μm
size fraction was collected on a 0.2 μm Millipore polycar-
bonate (PC) membrane (47 mm). The water in triplicate
bottles in each treatment was mixed together and then 500
to 700 ml water was filtered using a low vacuum. The PC
membrane were immediately immersed in RNAlater
(Ambion) solution and stored at −80 °C until further analy-
sis. As residual RNAlater was found to inhibit the dissoci-
ation of nucleoprotein complexes and finally reduce the
generation of the RNA, before the TRIzol reagent was
added, RNAlater was removed from the PC membrane by
using the following procedure: the PC membrane was trans-
ferred to a new 0.7-ml tube, which had a pinhole on the
bottom. The 0.7 ml tube was set on the top of a 1.5-ml
centrifuge tube, and the residual RNAlater was removed
J. Xu et al.
China
Shenzhen
Hong Kong
Victoria Harbour
PM7
VM5
Hong Kong Island
N
0 5 10km
114o00’ 114o10’ 114o20’ 114o30’
Latitude (oE)
from the PC membrane since it flowed into the 1.5 ml tube
through the pinhole when the two tubes were centrifuged
together. After removing the RNAlater, the PC membrane
was transferred to a new tube and the total RNA was
extracted from the membrane with a TRIzol plus RNA
purification kit (Invitrogen, Carlsbad, CA). RNA was then
purified following the instructions provided with the TRIzol
reagent and finally eluted in 50 μl of elution buffer that
came with the kit.
Before cDNA synthesis, purified RNA was treated with
DNase I to eliminate DNA contamination. Total RNA (up to
400 ng) was then reverse transcribed to cDNA using the
SuperScript III first strand cDNA synthesis kit (Invitrogen).
The reaction consisted of 8 μl DNase I-treated RNA, 50 ng
random hexamers, 1× RT buffer, 5 mM MgCl2, 0.5 mM each
deoxynucleoside triphosphate (dNTP), 10 mM dithiothreitol,
1 U RNaseOUT and 1 U SuperScript III reverse transcriptase
(RT). A parallel reaction without SuperScript III RT was used
as a RT-PCR negative control. RNA was reverse transcribed at
50 °C for 50 min and the reaction was terminated at 85 °C for
5 min. Residual RNA was removed by addition of 2 U RNase
H at 37 °C for 20 min.
16S rRNA Gene Pyrosequencing
Bacterial 16S rRNA gene was amplified using the primer set
of 16S-341F (5′-CCTACGGGAGGCAGCAG-3′) and 16S-
787R (5′-CCTATCCCCTGTGTGCCTTGGCAGTC-3′) that
targets the V3–V4 region of the 16S rRNA gene. The different
Effect of Transplants on Bacterial Metabolism and Diversity
MID sequences for pyrosequecing were added to the forward
primer. Non-RT samples were always used as a negative
control to confirm that there is no DNA contamination. Trip-
licate PCR reactions for each sample were mixed and purified
using the gel-cut method. An amplicon library was
constructed, and emPCR was conducted to generate millions
of identical copies of 16S rRNA sequence linked to each bead,
according to the methods described by the Roche Company
(454 Life Science). The DNA beads linked with unique 16S
rRNA sequence and other sequencing beads were successive-
ly deposited onto the PicoTiterPlater. The plate containing
hundreds of thousands of DNA beads was then sequenced
on a GS Junior system (454 Life Science).
The obtained sequences were checked in the RDP
pyrosequencing pipeline for quality control [9]. The standard
control was defined as a sequence length longer than 300 bp, at
most two mismatches in the primer sequences, quality score
larger than 20, and no ambiguous base (N). The reads that do
not match any of the standards were removed from further
analysis. The remaining reads were analyzed with the Mothur
software package for alignment, distance calculation and the
classification of operational taxonomic units (OTU) [37]. The
Needleman–Wunsch algorithm was applied for the pairwise
sequence alignment against an aligned Silva bacterial reference
database. When calculating the sequence distance, a continuous
gap was only penalized once and an edge gap was not penal-
ized. The OTU was created at the furthest sequence distance
when the sequence dissimilarity between any two sequences
within the OTU was 1 %, 3 %, 5 % and 10 %. Taxonomic
identification of reads was carried out with Mothur against the
Silva bacterial no-gap reference database at a cutoff value of 80.
The OTU numbers, species richness estimators (Chao 1 and
ACE) and diversity indices (Shannon index H′ and Simpson
index D) were calculated at the cutoff of 1 %, 3 %, 5 % and
10 % using the Mothur command summary single.
Statistical Analyses
Statistical analyses were performed using SPSS software. A
one-sample k–s test indicated that variables had a normal
distribution, and then a parametric ANOVA analysis with a
least squares difference (LSD) multiple comparison tech-
nique was conducted to determine any significant difference
between treatments (p<0.05). The error bars represent a
pooled sample standard deviation of the mean.
Results
Nutrients, DOC, Bacterial Abundance, Viral Abundance
DIN, PO3

4 and Si(OH)4 concentrations were 32.0, 1.26 and
22.7 μM at VM5, respectively, and 3.52, 0.21 and 11.7 μM at
63
PM7, respectively. DOC concentrations were 1.02 mg C l−1 at
PM7 and 2.66 mg C l−1 at VM5. Bacterial and viral abundance
at PM7 were ~15 % higher than VM5, with 1.38×109 cells l−1
and 1.29×1010 particles l−1, respectively at PM7 and 1.16×
109 cells l−1 and 1.11×1010 particles l−1, respectively at VM5.
The abundance ratios of viruses to bacteria were 9.3 and 9.6
for PM7 and VM5, respectively (Table 1).
Bacterial and Viral Growth During Incubation
BA (~2.5±0.04×108 cells l−1) in the pristine seawater bacteria
treatments (Pb+Pw and Pb+Sw) was significantly (p<0.001)
higher (∼2.3±0.05×108 cells l−1) than in the sewage-impacted
bacterial treatment (Sb+Sw and Sb+Pw) at the beginning of
the incubation (Fig. 2). At the end of the incubation, BAwas the
highest (~2.3±0.1×109 cells l−1) in the cross-transplant cultures
(Pb+Sw and Sb+Pw), moderate (1.6±0.6×109 cells l−1) in the
Sb+Sw treatment and the lowest (6.8±0.2×108 cells l−1) in the
Pb+Pw treatment (Fig. 2). Bacterial growth rates (μb) differed
significantly (p<0.01) among the four treatments, with the
highest (2.29±0.02 day−1) in the Sb+Pw treatment, moderate
growth rate (1.94±0.04 day−1 for Pb+Sw and 2.23±0.01 day−1
for Sb+Sw) in the sewage-impacted seawater cultures, and the
lowest growth rate (1.00±0.01 day−1) in the Pb+Pw treatment
(Fig. 3).
Viral abundance (~1.4±0.02×1010 particles l−1) in the
pristine seawater treatments (Pb+Pw and Sb+Pw) was sig-
nificantly (p<0.01) higher than that (~1.1±0.14×1010 par-
ticles l−1) in the sewage-impacted bacterial treatment (Sb+
Sw and Pb+Sw) at the beginning of the incubation (Fig. 2).
At the end of the incubation, viral abundance did not differ
significantly (p>0.05) among the four treatments (Fig. 2).
Viral growth rate (μv) was the highest (0.38±0.01 day−1) in
the Pb+Sw treatment, intermediate (0.01±0.05 to 0.05±
0.05 day−1) in the Sb+Sw and Sb+Pw treatments and the
lowest (−0.32±0.34 day−1) in the Pb+Pw treatment (Fig. 3).
Table 1 Initial nutrient (DIN, PO3

4 and Si(OH)4) concentrations, DOC
concentration, bacterial abundance (BA), viral abundance (VA) and virus
to bacteria abundance ratio (V/B) at two stations (PM7 and VM5)
Parameters PM7 VM5
Salinity 30.5 30.7
DIN (μM) 3.52 32.0
PO3

4 (μM) 0.21 1.26
Si(OH)4 (μM) 11.7 22.7
DOC (mgl−1) 1.02 2.66
BA (cells l−1) 1.38×109 1.16×109
VA (particles l−1) 1.29×1010 1.11×1010
V/B (particle/cell) 9.3 9.6
DIN = NO

þ
3 þ NO2 þ NH4
64 J. Xu et al.

Fig. 2 Changes in bacterial 3 25 b b

a a

BAi (x108 cells L )

BAf (x108 cells L-1)

-1
abundance (BA), viral b b 20
abundance (VA) and virus to c
2
bacteria abundance ratio (V/B) 15
at the beginning (i) and end of 10
1 a
the incubation (f) among four
treatments: (1) pristine seawater 5
bacteria+pristine seawater 0 0
(Pb+Pw), (2) sewage-impacted

VAf (x1010 particles L-1)

VAi (x1010 particles L )
2 2

-1
bacteria+sewage-impacted a
a a a
water (Sb+Sw), (3) pristine a
b b a
seawater bacteria+sewage-
impacted water (Pb+Sw), and 1 1
(4) sewage-impacted bacteria+
pristine seawater (Sb+Pw).
Vertical bars indicate ±1 SD and
n=3. Different letters (a, b, c, d) 0 0
denote that the treatment was 70 20
a b a
significantly different (p<0.05) 60
c d 15
and the same letters denote that 50
the treatment was not 40

V/Bf
V/Bi

significantly different 10 b b
30 b
(p>0.05) 20 5
10
0 0
Pw Pw Sw Sw Pw b+Pw b+Sw b+Sw
Pb+ Sb+ Pb+ Sb+ Pb+ S P S
Treatments Treatments

Virus to bacteria abundance ratios were significantly
(p<0.05) different among four treatments at the beginning of
the incubation, with the maximum (61±2.1:1) in the Sb+Pw
treatment, moderate (50±0.61 to 58±0.45:1) in the Pb+Pw
and Sb+Sw treatment and the minimum (47±0.42:1) in the
Pb+Sw treatment. At the end of the incubation, the virus to
bacteria abundance ratio (16±4.9:1) in the Pb+Pw treatment
was significantly higher than the other treatments where the
ratios (6.1±1.1 to 7.2±0.42:1) did not differ significantly
(Fig. 2).
BP in the sewage-impacted water was 130±15.8 and 113±
4.92 μg C l−1 day−1 for Pb+Sw and Sb+Sw, respectively, but
the two treatments were not significantly different. However,
BP in the sewage-impacted water was significantly (p<0.001)
higher than in the pristine coastal water. BP was 14±0.62 and
62±6 μg C l−1 day−1 for Pb+Pw and Sb+Pw, respectively,
and Sb+Pw was significantly (p<0.001) higher than the Pb+
Pw treatment. The cell-specific BP (103±12 and 123±6 fg C
cell−1 day−1) in the sewage-impacted water was significantly
(p<0.01) higher (29±0.94 and 50±0.53 fg C cell−1 day−1)
than in the pristine seawater, with the highest in the Sb+Sw
treatment and the lowest in the Pb+Pw treatment (Fig. 3).
BR showed the opposite pattern to BP, being high
(160±39.7 and 133±14.4 μg C l−1 day−1) in the pristine
coastal water (i.e., Pb+Pw and Sb+Pw) and low (62±1.7
and 67±11 μg C l−1 day−1) in the sewage-impacted water
(i.e., Pb + Sw and Sb + Sw), irrespective of the bacterial
sources. The cell-specific BR followed the BR pattern, being
high (108±12.1 and 339±18.6 fg C cell−1 day−1) in the pris-
tine coastal seawater (i.e., Sb+Pw and Pb+Pw) and low
(49 ± 1.5 and 73 ± 8.9 fg C cell−1 day−1) in the sewage-
impacted water (i.e., Pb+Sw and Sb+Sw) (Fig. 3).
BGE demonstrated the same pattern as BP, being high
(0.68±0.09 and 0.71±0.05) in the sewage-impacted water
(i.e., Sb+Sw and Pb+Sw) and low (0.08±0.01 and 0.32±
0.01) in the pristine seawater (i.e., Pb+Pw and Sb+Pw),
irrespective of bacterial sources (Fig. 3).
The relative value of mean bacterial cell size estimated by
SSC flow cytometry was high (20.9±0.2 and 18.7±0.2) in
the relatively pristine seawater (i.e., Pb+Pw and Sb+Pw)
and low (15.9±0.7 and 16.5±0.1) in sewage-impacted water
(i.e., Pb+Sw and Sb+Sw), similar to the pattern of BR and
cell-specific BR. There was a significant (p<0.01) correla-
tion between cell size and cell-specific BR (Fig. 4). Inverse-
ly, the DNA content of bacteria was generally lower (155 and
197) in the relatively pristine seawater (i.e., Pb+Pw and Sb+
Pw) than (211±10 and 204±3) in sewage-impacted water
(i.e., Pb+Sw and Sb+Sw) (Fig. 3). The DNA content of
bacteria for Pb+Pw was significantly (p<0.001) lower than
other three treatments in which the DNA content for Sb+Pw
was significantly (p<0.001) lower than Pb+Sw, while the
DNA content for Sb + Sw did not differ significantly
(p>0.05) from Sb+Pw and Pb+Sw (Fig. 3). The relative
bacterial cell size was positively significantly (p<0.01) cor-
related with cell-specific BR and negatively significantly
(p<0.01) correlated with BGE (Fig. 4).
Effect of Transplants on Bacterial Metabolism and Diversity 65

Fig. 3 Changes in bacterial 3 150

sBP (fg C cell-1 d-1)

d
growth rate (μb), viral growth b c c
d 120
rate (μv), production (BP), 2

(d-1)
90
respiration (BR), growth b
a 60
efficiency (BGE), cell-specific

b
1 a
bacterial production (sBP), cell- 30
specific bacterial respiration
0 0
(sBR), cellular carbon demand
1 400

sBR (fg C cell-1 d-1)

(sBP+sBR), relative cell size a
and relative DNA content b 300
among four treatments (see a b

(d-1)
a a 150
Fig. 2 for treatment 0 c
100

v
abbreviations). Vertical bars c
indicate ±1 SD and n=3. 50
Different letters (a, b, c, d) -1 0
denote that the treatment was 150
a

BP ( g C L-1 d-1)
significantly different (p<0.05) c 400
c

(fg C cell-1 d-1)

and the same letters denote that 120

sBP+sBR
300
the treatment was not 90
b c
significantly different b b
60 200
(p>0.05)
30 a 100
0 0
250
BR ( g C L-1 d-1)

Relative cell size

24
200
22 a
b
150
c 20 b
100 c
18 c c
50
16
0

Relative DNA content

1.0 240
c c c,d
0.8 b,d d
210
0.6
BGE

180
0.4 b a
0.2 150
a
0.0 120
Pw Pw Sw Sw Pw b+Pw b+Sw b+Sw
Pb+ Sb+ Pb+ Sb+ Pb+ S P S
Treatments Treatments

16S rRNA Pyrosequencing Reads Statistics and OTU-Based
Species Richness and Diversity Estimates
In total, 97,848 raw reads were obtained from a 454 GS Junior
pyrosequencer and subsequently 57,790 (59 %) reads passed
the RDP pyrosequencing pipeline filters defined in the
methods. Generally, 9,600 sequences with average length of
~420 bp were obtained in each sample. All of the 57,790 high-
quality sequences were identified to be close to bacterial 16S
rRNA sequences against the Silva bacterial reference database
at the threshold of 80 % similarity.
The bacterial community in the initial sample (Pi) from the
relatively pristine station PM7 showed higher species richness
at the sequence dissimilarity levels of 0.01, 0.03, 0.05 and
0.10, respectively (Table 2), compared with the initial sample
(Si) at the sewage-impacted station VM5. The richness esti-
mates Chao1 and ACE indicated that the sampling efforts
were not enough to detect all of the richness and potentially

Fig. 4 A significant correlation 400 1.0

sBR (fg C cell-1 d-1)

Y = 54 * X - 835 Y = -014 * X + 3.00

between relative bacterial cell r2 = 0.86 0.8 r2 = 0.99
300
size and cell-specific bacterial p < 0.01 p < 0.01
0.6
BGE

respiration (sBR) and bacterial 200

growth efficiency (BGE) in the 0.4
four treatments (see Fig. 2 for 100
0.2
treatment abbreviations)
0 0.0
16 18 20 22 16 18 20 22
Bacterial cell size Bacterial cell size
66 J. Xu et al.

0.033
0.055
0.076
0.241
0.086
0.150

The OTU, ACE, Chao1, Shannon (H′) and Simpson (D) parameters are presented for a dissimilarity of 1 %, 3 %, 5 % and 10 % between the reads. Si and Pi denote initial samples at VM5 and PM7,
more bacterial OTUs may occur in these environments. After

D
a 1-day incubation in the cross-transplant experiments, all four

4.27
4.01
3.52
2.35
3.63
2.85
treatments indicated a decrease in the species richness and
H’ diversity, among which the decrease in sewage-impacted wa-
1,334
ter treatments (i.e., Pb+Sw and Sb+Sw), especially Sb+Sw,
1,212

1,109
ACE

687
623

301
were more than the pristine seawater treatments (i.e., Pb+Pw
and Sb+Pw) (Table 2).
Chao1
Cutoff=0.10

899
985
570
406
875
288
Taxonomic Classification of Bacterial Communities
OTU

538
613
302
200
517
200
At the bootstrap value of 80 % for the sequence assignment,
more than 94.5 % of the sequences were classified into
0.013
0.017
0.028
0.107
0.034
0.055

known bacterial phyla, including Actinobacteria,

D

Bacteroidetes, Cyanobacteria, Firmicutes, Planctomycetes,

5.40
5.29
4.82
3.42
4.89
4.40

Proteobacteria, Verrucomicrobia and some minor bacterial

H’

groups which accounted for <0.05 % in all of the samples

3,385
3,297
1,678
1,195
3,176
1,235

(Fig. 5). Proteobacteria dominated the bacterial community

ACE

composition at the two stations, making up 81 % of the

abundance at VM5 and 72 % at PM7 (Fig. 5). Bacteroidetes
Chao1

2,121
2,447
1,227

2,143

formed the second largest bacterial group, accounting for

Cutoff=0.05

950

848

18 % and 21 % in abundance at VM5 and PM7, respective-

1,079
1,280

1,140

ly. The contribution of Proteobacteria in all treatments in-

OTU

680
436

471

creased after the incubation, with a higher contribution

(>98 %) in the sewage-impacted water treatments (Pb+Sw
0.007
0.009

0.049
0.015
0.015
0.011

and Sb+Sw). In contrast, the contribution of Bacteroidetes

D

decreased accordingly to <12 %. Furthermore, Gamma-

6.27
6.14
5.94
4.50
5.83
5.56

proteobacteria dominated (>56 %) in the Proteobacterial

H’

sub-population in the initial samples at VM5, but Alpha-

6,398
6,657
3,626
2,364
6,120
2,559

proteobacteria accounted for the largest proportion (38 %) at

ACE

PM7 (Fig. 5). After the incubation, compared to the initial

sample, the proportion of Gamma-proteobacteria increased
Chao1

4,072
4,580
2,413
1,604
4,009
1,797

to 67 % in the Sb+Pw treatment, but decreased to 47 % in

Cutoff=0.03

the Sb+Sw treatment. However, no matter whether sewage-

The numbers of reads after quality control and noise clearance are shown
1,837
2,165
1,272

1,904

impacted bacteria were incubated in pristine seawater or

OTU

766

867

sewage-impacted seawater, after the incubation, Epsilon-

proteobacteria increased considerably from 5 % in the initial
0.001
0.001
0.001
0.007
0.002
0.002

sample to 18 % in the Sb+Pw treatment and 50 % in the

D

Sb+Sw treatment, respectively. The Proteobacteria sub-

Table 2 Similarity-based OTUs and species estimates

8.05
7.98
7.87
6.68
7.73
7.53

population composition remained unchanged after the incu-

H’

bation when pristine seawater bacteria were incubated in

23,099
26,935
15,948
10,453
21,302
12,529

seawater (i.e., Pb+Pw), while the contribution of Gamma-

ACE

proteobacteria was enhanced significantly from 37 % to

93 % after the incubation when pristine seawater bacteria
12,502
15,187

12,567
Chao1

9,008
6,245

6,629

were transferred to sewage-impacted water (i.e., Pb+Sw).

Cutoff=0.01

The eight most abundant bacterial groups at the family

level are listed in Fig. 6. Bacterial species related to
4,592
5,578
3,363
2,276
4,830
2,233
OTU

Alteromonadaceae, Oceanospirillaceae, Rhodobacteraceae,

and Flavobacteriaceae each represented >10 % of the total
10,314
14,563

13,811
Reads

bacterial community in the initial samples of the two stations.

6,967
7,745

4,390

Generally, after the incubation, some major bacterial groups

respectively

became more dominant in all of the treatments, especially in

Sb+Pw
Sb+Sw
Pb+Pw
Pb+Sw
Sample

Sb+Sw and Pb+Sw (Fig. 6). Alteromonadaceae was the only

Si
Pi

group detected with a remarkable increase in all treatments,

Effect of Transplants on Bacterial Metabolism and Diversity 67

especially in the Pb+Sw treatment where they accounted for∼
80 % of the total bacterial community. Interestingly, a signif-
icant increase in Campylobacteraceae was observed from
<4 % in the initial sample to∼50 % in the Sb+Sw treatment.
UPGMA clustering showed that the initial sewage-
impacted bacterial community (Si) was clustered with the
seawater treatment (Sb+Pw), and the same clustering rela-
tionship was observed for the initial seawater bacterial com-
munity (Pi) incubated in seawater (Pb+Pw) (Fig. 5).
Discussion
Bacterial Metabolic Activity and Community Composition
Sewage discharge in Victoria Harbour delivers huge
amounts of nutrients [42] and DOM. The DOC concentra-
tion of 2.66 mg C l−1 at VM5 was comparable to that
observed in the sewage-impacted upper reach of the Pearl
River estuary [18]. In this study, the actual composition of
the DOC was not identified. However, some early studies
showed that total dissolved carbohydrates and amino acid
were the major components of domestic sewage DOC [17,
18], which are among the most labile fractions of bulk
organic matter [3, 28]. Surprisingly, although the DOC
concentration (1.02 mgl−1) at PM7 was over 2-fold lower
than (2.66 mgl−1) that of VM5, BA was slightly higher in
the relatively pristine coastal waters (1.38×109 cells l−1 at
PM7) that was dominated by Alpha-proteobacteria com-
pared to the eutrophic Victoria Harbour waters (1.16×109
cells l−1 at VM5) with Gamma-proteobacteria being domi-
nant (Table 1; Fig. 6). This observation was consistent with
previous findings that Alpha-proteobacteria outcompeted
other bacteria under low nutrient conditions, but Gamma-
proteobacteria dominated under high nutrient concentrations
[10]. Wu et al. [41] also revealed that Gamma-
proteobacteria was the dominant group in the eutrophic
Pearl River estuary near Hong Kong’s western waters.
In our study, bacteria responded significantly to the cross-
transplant in terms of metabolic activity and diversity. The
sewage-derived DOM considerably stimulated pristine seawa-
ter bacterial growth (Pb+Sw). BP increased 10-fold, when

Fig. 5 a UPGMA clustering of

the sequences at the cutoff
value of 0.03 showing the
dissimilarity among six
bacterial communities; b
bacterial community
composition at the phylum level
in each sample; c
proteobacterial community
composition at the class level.
See Fig. 2 for treatment
abbreviations; Si and Pi denote
initial bacteria at the sewage-
influenced station (VM5) and
the more pristine station (PM7)
68
Fig. 6 Relative proportion of
the top eight most abundant
taxonomic groups in each
sample/treatment. See Fig. 4 for
treatment abbreviations
pristine seawater bacteria were transferred to sewage-
impacted seawater (Pb+Sw), compared to that in pristine
seawater (Pb+Pw) (Fig. 3). In contrast, BP decreased by
40 % as sewage-impacted bacteria was transferred to pristine
seawater (Sb+Pw), relative to that in sewage-impacted water
(Sb+Sw) (Fig. 3). The pattern of the relative DNA content
was similar to BP (Fig. 3), corroborating earlier findings that
the high nucleic acid (HNA) bacteria are capable of taking up
leucine much faster than low nucleic acid (LNA) bacteria [25]
and are responsible for the largest proportion of BP in marine
environments [29, 40]. Overall, the pattern of bacterial growth
rate (μb) was similar to BP (Fig. 3). However, the discrepancy
in the highest growth rate and low BP in the Sb+Pw treatment
was more likely attributed to the low quality of DOM in the
relatively pristine seawater which favored the growth of LNA
bacteria, ultimately leading to a shift in the bacterial
community composition. Similarly, the discrepancy be-
tween the two variables was also observed in other studies
due to the difference in the bacterial population [4].
Hence, the number and the relative contribution of HNA
bacteria to the total bacterial population were more reliable
indicators of BP than BA.
Variability in BP and the relative DNA content among
treatments was related to shifts in the bacterial community
structure. Proteobacterial abundance varied with DOM con-
centrations, with the highest Proteobacterial contribution in
the eutrophic sewage-impacted seawater (i.e., Sb+Sw and
Pb+Sw), moderate in the Sb+Pw treatment and the lowest
in the Pb+Pw treatment (Fig. 5), which was consistent with
the relative DNA content and BP. Shifts in the bacterial
community composition have been reported in other exper-
imental studies in response to changes in substrate availabil-
ity [22, 33].
J. Xu et al.
The Proteobacteria subpopulation, Gamma-proteobacteria,
increased in abundance and become dominant (>90 % of total
abundance) instead of Alpha-proteobacteria after the incuba-
tion (Fig. 6), when seawater bacteria were transferred to the
eutrophic sewage-impacted waters (Pb+Sw). Interestingly,
compared to the initial sample (Si) at VM5, the bacterial
community composition shifted in the Sb+Sw treatment,
where Campylobacteriaceae belonging to Epsilon-
proteobacteria was the most abundant (49.5 %) group,
followed by Alteromonadaceae (40.4 %) which was the most
abundant group in the Gamma-proteobacteria subdivision in
all treatments (Fig. 6). In combination with the highest cell-
specific BP in the Sb+Sw treatment, it was speculated that
Epsilon-proteobacteria, such as Campylobacteriaceae, which
had a high capability of incorporating carbon, but vulnerable to
bacterivores that were reduced/eliminated in our experiments,
was more competitive in eutrophic waters than Gammma-
proteobacteria. Under in situ conditions, Campylobacteriaceae
remained less abundant likely due to grazing. Zhang et al. [43]
found that the presence of predators (i.e., flagellates and virus-
es) affected the bacterial community composition in Hong
Kong coastal waters. In this study, the order of dominant
bacterial subgroups along the DOC gradient was Epsilon-
proteobacteria, Gammma-proteobacteria and Alpha-
proteobacteria. The initial bacterial sample (Pi) at PM7 was
clustered with the Pb+Pw treatment, rather than Pb+Sw
(Fig. 5), while the initial sample (Si) at VM5 was clustered
with the Sb+Pw treatment, rather than Sb+Sw (Fig. 5),
suggesting that bacterial community composition was mainly
regulated by DOC (bottom–up control) at the relatively pristine
station, but by microzooplankton (top–down control) and
DOC at the sewage-impacted station, both of which may have
played an equally important role.
Effect of Transplants on Bacterial Metabolism and Diversity
In contrast, BR and cell-specific BR were surprisingly
low in the sewage-impacted seawater, but the highest in the
relatively pristine coastal seawater with no sewage influence
(e.g., Pb+Pw treatment). Contrary to BR and cell-specific
BR, BGE was high (0.68±0.01 to 0.71±0.05) for bacteria
grown on the sewage-derived DOM and significantly lower
(0.08±0.01 to 0.32±0.01) for those grown on seawater
DOM. This was in agreement with the conceptual diagram
proposed by Carlson et al. [6] demonstrating that cell-
specific BR increases and BGE declines as environmental
hostility (i.e., temperature, salinity, pH, substrate availability
and toxins) rises. Our results implied that bacteria utilized
DOM from seawater less efficiently, possibly due to the
need to produce exoenzymes to hydrolyze organic matter,
which lowered their growth efficiency. del Giorgio and Cole
[11] reported that high maintenance respiration rates were
needed in a low substrate treatment since cells must main-
tain a wide array of active transport systems and the corre-
sponding catabolic enzymes. Under conditions of energy
limitation, it is advantageous to maintain the highest possi-
ble energy flow [11, 36]. Consequently, more carbon is
needed for maintenance and less for storage. The situation
at PM7 is somewhat similar to the oligotrophic ocean where
cell-specific maintenance requirements are believed to be
higher due to energy limitation [11].
Bacterial cell size also provides insight into determining
physiological responses of bacteria to changes in substrate
availability [19]. Interestingly, at the end of the incubation,
the cells were largest in the Pb+Pw treatment (low DOC) and
smallest in the Pb+Sw treatment (high DOC). The combined
observations of the low cell-specific BP, low relative DNA
content, high cell-specific BR and large cell size in the Pb+Pw
treatment (Fig. 3), indicated that the larger cell size in the low
substrate treatment was most likely related to low DOM
availability. This was likely due to the low fraction of labile
DOM in the seawater at PM7, since there were still sufficient
inorganic nutrients (e.g., 1.5±0.20 μMN and 0.13±0.03 μM
PO3
4 for Pb+Pw) at the end of incubation and salinity was
almost identical between two stations (Table 1). Furthermore,
a significant correlation was observed between cell size and
cell-specific BR, but there was a negative correlation between
cell size and BGE (Fig. 4). Hence, it was speculated that a
decline in DOM lability limited bacterial cell division,
resulting in larger cells, and hence cell size increased with
decreasing DOM lability. An early study has also shown that
nutrient (i.e., N and P) limitation leads to an increase in cell
size [19]. We speculated that the observed increase in cell size
during our incubation was a consequence of the short-term
response of bacteria to substrate limitation, while a decrease in
cell size along the eutrophic to oligotrophic environmental
gradient was a result of a long-term response (i.e., a shift in the
bacterial community composition to groups that have a small-
er cell size).
69
In contrast, the sewage-derived DOM was highly labile
and utilized by bacteria. As a result, less carbon per unit
organic carbon input was used for maintenance. High BGE
may be associated with the relatively high abundance (up to
30 %) of carbohydrates and amino acids in the DOC pool of
sewage [17, 18]. Furthermore, species richness decreased
more in sewage-impacted water (i.e., Pb+Sw and Sb+Sw)
with high substrate and high BGE than in pristine seawater
with low BGE. This is in agreement with previous observa-
tions that BGE is inversely related to bacterial richness [34],
suggesting that the diversity interacted with the metabolic
activity of the bacterial assemblage. In our study, the win-
ners such as Alteromonadaceae belonging to Gamma-
proteobacteria and Campylobacteriaceae belonging to
Epsilon-proteobacteria, which contained high nucleic acid
content, were fast-growing opportunistic r-strategy-like spe-
cies that become dominant in substrate-rich habitats,
resulting in the concomitant observations of a high BGE
and low species richness.
The cellular carbon demand of bacteria (i.e., sBP+sBR)
was the highest in the Pb+Pw treatment with the lowest
substrate levels (Fig. 3), suggesting that bacteria required
more carbon for their growth in terms of an individual cell in
the oligotrophic waters than the eutrophic waters. The cel-
lular carbon demand was comparable between Sb+Pw and
Pb+Sw, although BGE was considerably different (Fig. 3).
Hence, DOM input from sewage markedly altered the
partitioning of carbon between production and respiration
in seawater which did not necessarily increase cellular car-
bon consumption, since bacteria required less carbon for
catabolism when they utilize the labile sewage-derived
DOC. Similarly, del Giorgio et al. [13] recently reported
that bacterial C consumption is remarkably constant, but
energy limitation increases and BGE decreases from inshore
to offshore. Our results combined with literature findings
indicated that carbon consumption might be independent of
BGE or BP.
Viral Dynamics
Viral abundance and the abundance ratio of viruses to bac-
teria were comparable between the two stations (Table 1).
Higher viral growth rates in two cross-transplant treatments
with high bacterial growth rates (i.e., Pb+Sw and Sb+Pw)
(Fig. 3) suggested that bacteria were vulnerable to exotic
viruses that were different from viruses originating from
bacterial inoculum, while indigenous bacteria were to a
certain extent immune to those indigenous viruses, in agree-
ment with Rodriguez-Brito et al. [35]. In these two cross-
transplant treatments (Pb+Sw and Sb+Pw), high BA due to
high growth rates (μb) (Figs. 2 and 3) provided more hosts
for viruses, resulting in high contact rates, especially for the
Pb+Sw treatment with more abundant HNA bacteria that
70
are believed to be necessary to maintain high viral abun-
dance [40]. As a result, viral growth rate (μv) in the Pb+Sw
treatment was significantly higher than the other three treat-
ments (Fig. 3). In contrast, in the Pb+Pw treatment, viral
abundance decreased after the incubation due to fewer hosts
for viruses. Numerous studies have been conducted on the
effect of indigenous viruses on bacterial metabolism and
diversity [30, 39, 40]. Motegi et al. [30] reported that BGE
depends on the fraction of BP destroyed by viruses, that is,
the viral shunt. In the Pb+Pw treatment, the highest abun-
dance ratio of viruses to bacteria implied that virus-induced
mortality was greater than in other treatments. However,
until now, little attention has been paid to the compositional
and metabolic response of the bacterial assemblage to dif-
ferent sources of viruses [4]. More studies are needed on the
effects of exotic viruses on bacterial growth and diversity.
Evaluation of the Experimental Approach
In this study, a short term (1 day) cross-transplant experi-
ment was adopted to examine the effect of different sources
of DOM on bacterial metabolism and diversity, where sub-
strates and viruses were enriched and grazers were mostly
eliminated at the onset of the experiment. In subtropical
Hong Kong coastal waters, bacterial growth rates were very
high (up to 2.29±0.02 day−1 or 3.4 doublings day−1) due to
high temperatures (29°C) in summer. Therefore, a long-time
(i.e., several days) incubation might deplete substrates dur-
ing the incubation, resulting in an increase in artifacts for
our experiments which focused on the effect of the quality
and quantity of DOM on bacterial metabolism and diversity.
del Giorgio et al. [13] also point out that the long-term
incubations tends to amplify features already present in the
ambient samples such that the magnitude of response may
not be applicable to in situ conditions. Furthermore, an
earlier experiment showed that bacteria grew exponentially
in a 1-day incubation, indicating that there was no lag phase
in the bacterial response (Xu, unpublished data). Hence, a 1-
day incubation might be better than a longer incubation for
these subtropical waters with very high temperatures. Sam-
ples were incubated aerobically, as indicated by DO con-
centrations at the beginning and the end of the incubation
(Table 3).
Large bacteria typically captured on a 1-μm polycarbon-
ate membrane were negligible when the bacterial inoculum
was prepared. Approximately 10 % of the bacteria passed
through a 0.2-μm cartridge when bacteria-free seawater was
prepared, which was comparable to the inoculated bacteria,
likely leading to background contamination especially for
two of the cross-transplant treatments (Sb+Pw and Pb+Sw).
Based on the eight most abundant taxonomic groups from
the pyrosequencing results, the Sb+Pw treatment was clus-
tered with the initial sample (Si) at the eutrophic station
J. Xu et al.
Table 3 Dissolved oxygen (DO) concentrations (mean±SD) at the
beginning and end of the incubation among four treatments (see
Fig. 2 for treatment abbreviations)
Treatments DO concentrations (mgl−1)
Before After
Pb+Pw 6.45±0.038 6.03±0.064
Sb+Pw 6.35±0.036 5.99±0.012
Pb+Sw 6.24±0.003 6.08±0.001
Sb+Sw 6.02±0.060 6.08±0.081
SD standard deviation, n=3
VM5 (Fig. 4), showing that background contamination
was negligible. In the Pb+Sw treatment, the second most
abundant bacterial group was the Oceanospirillaceae that
originated from the sewage-impacted seawater, but they
only accounted for <8 % of the total BA. These results
suggested that background contamination should have a
limited effect on our conclusions. We also need to point
out that the 454-sequencing was done by pooling the three
samples of each treatment and the possible difference
among triplicates was not measured. However, since we
could not distinguish dead from live organisms, it is possible
that some dead organisms contributed somewhat to the
rRNA pools.
A few nanoflagellates might be included in the 1-μm
filtrate and these nanoflagellates might reduce BA over a
few days. However, our incubation time was 1 day and
therefore nanoflagellate grazing would likely be less impor-
tant over 1 day. Further study is needed to address effect of
microzooplankton on bacterial diversity and metabolism in
Hong Kong waters.
Conclusions
Sewage-derived DOC which favored the growth of HNA
bacteria stimulated BP and shifted the bacterial community
composition, with Gamma-proteobacteria becoming domi-
nant over Alpha-proteobacteria, but did not increase BR and
cellular carbon consumption, likely because sewage-derived
DOC was labile and readily utilized by bacteria. We specu-
late that the sewage input could have altered the partitioning
of carbon between BR and production, which would not
enhance oxygen consumption. In contrast, we observed that
bacteria utilized marine-derived DOM in more pristine sea-
water at the expense of lowering their growth efficiency. As
a result, more carbon was needed for maintenance and less
for storage. Changes in the bacterial community structure
were less apparent when sewage-impacted bacteria were
transferred to relatively pristine seawater.
Effect of Transplants on Bacterial Metabolism and Diversity
Acknowledgements Financial support for this research was provided
by the University Grants Committee of Hong Kong AoE project (AoE/
P-04/04-4-II) and Research Grants Council of Hong Kong’s General
Research Fund (661911 and 661610). We thank Ms. Candy Lee for
helping to count viruses with a flow cytometer.
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