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愛媛大学農学部紀要（Mem. Fac. Agr., Ehime Univ.）
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Effect of Exposure of Zinc at Low Concentration
to Bacterial Production in Seawater＊

Chui Wei BONG１，２）, Yumiko OBAYASHI１，３） and Satoru SUZUKI１）
ABSTRACTZinc (Zn) is one of the essential elements as an enzyme cofactor, however high concentrations of Zn can be toxic.
In the usual ocean environment, Zn is found at trace levels, whereas pollution of Zn has been sometimes found incoastal waters. Bacteria should express a differential response to various concentrations of Zn. Here we found that Zn
gave a rapid impact on bacterial productivity, whereas proteolytic enzymes were not affected. The results showed thata low concentration of Zn (0.01 μM) decreased the bacterial abundance, growth rate and bacterial production, but notinhibited proteolytic enzymes, suggesting that acute effects of Zn are independent of protein utilization enzymes.
Key words: Zinc, bacterial productivity, protease, protein

Zinc (Zn) is an essential micronutrient and that has
been shown to be essential for growth, development anddifferential of all types of biota including bacteria (29).
The Zn ion plays a role as an essential factor for mostclasses of enzymes; however, Zn is potentially harmful atexcessive levels. In the oceanic environment, Zn is a traceelement in the pelagic ocean (0.00005 to 0.009 μM) (22)but is sometimes artificially released to coastal water aswell as other metals (UNEP, 1996 UNEP. 1996. State of
the Marine and Coastal Environment in the Mediterranean
Region. MAP Technical Report Series No. 100. UNEP,
Athens.UNEP 1996, State of the Marine and Coastal
Environment in the Mediterranean Region. MAP
Technical Report Series No. 100, UNEP, Athens).
Toxicity studies of Zn have mostly focused on higher
trophic level species in the aquatic food web (24, 25),
whereas the effects of metals on phytoplankton andbacteria, which are fundamental lower trophic level
members, are relatively scarce with most having been
carried out in the fresh water environment (23, 30).
Toxicity studies applying laboratory-bioassays or
mesocosms have found severe effects of Zn upon marine
microorganisms (1, 23). Rochelle-Newall et al . (26)reported that even at low concentrations Zn showed a
Received April 13, 2011
Accepted July 14, 2011
１）
Center for Marine Environmental Studies (CMES), Ehime
University, Matsuyama, 790-8577, Japan
２）
Institute of Biological Sciences, Faculty of Science,
University of Malaya, 50603 Kuala Lumpur, Malaysia.
３）
Faculty of Engineering and Graduate School of Engineering,
Yokohama National University, 240-8501, Japan＊
Corresponding author. Mailing address: Center for Marine
Environmental Studies (CMES), Sci. Bld. 2, Ehime
University, Matsuyama 790-8577 Japan. Phone: +81-89-9278552, Fax: +81-89-927-8552. E-mail: ssuzuki@ehime-u.ac.jp

negative effect on the food web at the lower trophic level,
and on carbon transfer in sensitive coastal environments.
On the other hand, bacterial isolates from natural habitats
showed Zn tolerance (4, 11). The mechanism of resistance
may be based on 1) metal retention on the surface of cell,2) intracellular transformation into less toxic forms, 3) the
release of metals from cells accompanied with polymers,
and 4) decreasing of permeability of the cell membrane(14).
Protein is an important source of carbon and nitrogenfor bacteria and major component of high molecularweight DOM accounting for 20-35% of total DOM (5).
As pointed out by Nagata (16), protein can be a model tostudy the major features of the complexity in polymerbacteria interactions in the sea. Hydrolysis of highmolecular weight protein is an initial step in microbial
protein utilization. Proteases act as mechanism of protein
utilization and some of the proteases need Zn for thisactivity (17). However, proteases of marine bacteria being
inhibited by Zn at the 100 μM to 1 mM level are known(15, 21), although effect at lower concentrations is notknown. Thus control of protein degradation by Zn issuspected to have an affect on the bacterial growth and/or
production through proteolytic enzymes.
From this evidence, Zn possibly induces inhibition of
essential processes associated with enzyme reaction (12,27), which might cause drastic ecological consequences.
So far, heavy metal effects at lower than toxic
concentrations focusing on bacterial extracellular enzymes
has not been well documented. Therefore, in this study weare interested in understanding the effect of Zn on
extracellular activities and enzyme production in an
aquatic microbial ecosystem since protein is a major
component of biogenic organic matter. Hence, the aims ofthis study are to evaluate the effect of Zn exposure at low
concentrations by acute exposure on the response of thebacterial assemblages and on extracellular protease４２

Chui Wei BONG, Yumiko OBAYASHI and Satoru SUZUKI

activities. This study will help us to expand ourunderstanding on the role of Zn on bacterial-relating
matter cycling in the ocean.
Seawater samples were taken from Scripps Institutionof Oceanography (SIO) pier (32°52’N, 117°
15.4’W) in
La Jolla, CA (USA) during wintertime in February 2009.
Seawater was sampled and maintained using an acidwashed, thoroughly rinsed 10-L carboy. The seawater wastransported within 1 hr in the dark to the laboratory andsamples were processed within 2 hr. Seawater samples
were filtered using 8-μm pore size polycarbonate filter toremove particles. In order to represent the naturalassemblage of bacteria from the marine environment,
seawater was then filtered through 0.6-μm pore size
polycarbonate filter to exclude essential grazers. The
filtrates were distributed to three 1 L flasks, each ofwhich was filled with to 0.6 L. Each flask was subject toeither no addition (control) or an addition of Zn. Zinc
chloride (ZnCl2) was added to the samples to give a final
concentration of 0.01 μM and 0.1 μM. The concentrations
added were within the ranged previously reported in the
San Diego water (0.02 - 0.2 μM). After the addition of Zn
samples were then incubated in the dark at room
temperature (20-25°C) and sub-samples were taken at 0,2, and 8 hr for measurement of bacterial production,
bacterial growth and protease assay.
The hydrolysis of analog methylcoumarylamide (MCA)
peptide substrates were used to estimate potential
proteolytic enzyme activity as described by Obayashi and
Suzuki (20). Aminopeptidase and trypsin types were
measured (Table 1). All of the substrate was completely
dissolved in dimethy sulfoxide (superior grade; Wako
Chemical) before being added to triplicate seawater
samples to give the final concentration of 100 μM. The
enzymatic reaction (0.3 ml volume) was run in a 96 wellplate and incubated at 25°C for one hour. Fluorescence ofhydrolytic product, 7-amino-4-methylcoumarin (AMC)
was determined using SpectraMax M2/M2e Microplate
Readers (Molecular Devices, Sunnyvale, CA). Excitationand emission wavelengths were 380 nm and 460 nm.
Fluorescence units were converted to units of nmol L−1
h−1 with the aid of a standard curve generated from serial
dilution with a known concentration of standard AMC.
Bacterial abundance (BA) in seawater was enumerated
by nucleic acid staining (SYBR Gold) and epifluorescencemicroscopy according to Noble and Fuhrman (18).
Subsamples were fixed with formalin (final conc. 2%. w/v) and stored at 4°C until analysis. A 2 ml sample wasfiltered through an Anodisc membrane filter (0.02-μm
pore size), with a backing filter (0.8-μm Millipore typeAA filter) and stained under darkness for 15 min. Bacteria
were counted under blue light excitation under x100
magnification on an Olympus BX60 epifluorescene
microscope. More than 200 cells were enumerated foreach sample. Leucine incorporation rate was measured as
an indicator of bacterial production (BP). Samples were
processed according to Kirchman (12). [4, 5-3H]-Leucine
was added to triplicate samples to give a final
concentration of 20 nM in 1.7 ml seawater. Triplicate andone trichloroacetic acid (TCA)-killed blank (89 μl 100%
TCA) were incubated in 2 ml screw cap microcentrifuge
tubes for one hr under dark conditions at 25°
C. Theincubation was terminated by the addition 89 μl of 100%
TCA and then centrifuged at 16,000 x g for 10 min. The
pellet was resuspended and washed by the addition of 1.5
ml of 5% TCA. Samples were centrifuged again andsubsequently aspirated, and a 1 ml of liquid scintillation
cocktail (Ecoscint, scintiverse BD, ultima gold) was
added. The radioactivity was determined by scintillation
counting. BP was calculated according to Kirchman (12).
Cell-specific growth rates were calculated assuming
exponential growth. Bacterial specific growth rates were
calculated from BP/BA assuming a mean C content of 20
fg C cell−1.
Regression and analysis of covariance (ANCOVA) wereused to determine whether there were statisticallysignificant differences in bacterial abundance, bacterial
growth rates, bacterial production and protease activity
between controls and treatments.
Concentration of Zn in the San Diego water has beenreported to be 0.02 μM to 0.2 μM (8, 28), which is much
lower than the EPA chronic WQC (1.2 μM; U.S
Environmental Protection Agency 2002, National Recommended Water Quality Criteria EPA-822-R-02I-047)
indicating the sampling area was relatively uncontaminated. Fig. 1 presents the response of heterotrophic
bacterial abundance, bacterial production and growth rate
against Zn. The initial number of total bacterial abundance
was 4.62 × 105, which were slightly increased along the
8 hr incubation (Fig. 1A). The final bacterial counts in
Zn-added group and control were not significantly
different (p>0.05), indicating that the cell growth was notaffected by low concentrations of Zn.
A marked effect was found in bacterial production
measured by leucine incorporation rates (Fig. 1B).
Bacterial production was lower in the Zn treatment group

Table 1: The list of substrates used in this study. Abbreviations: MCA=4-methyl-coumaryl-7-amide
NameType of substrate Molecular formula Molecular weight

Compound

C16H20N2O3
C13H14N2O3
C33H43N7O8
C31H45N7O7
0C34H52N8O10
C32H49N5O7

L-Leucine MCA
L-Alanine MCA
t-Butyloxycarbonyl-L-Phenylalanyl-L-Seryl-L-Arginine MCA
t-Butyloxycarbonyl-L-Valyl-L-Prolyl-L-Arginine MCA
t-Butyloxycarbonyl-L-Leucyl-L-Seryl-L-Threonyl-L-Arginine MCA
t-Butyloxycarbonyl-L-Valyl-Leucyl-L-Lysine MCA

Aminopeptidase
Leu-MCA
Aminopeptidase
Ala-MCA
Trypsin
Boc-Phe-Ser-Arg-MCA
Trypsin
Boc-Val-Pro-Arg-MCA
Trypsin
Boc-Leu-Ser-Thr-Arg-MCA
Trypsin
Boc-Val-Leu-Lys-MCA

288.34246.26665.74627.73732.82615.76

Effect of Exposure of Zinc at Low Concentration to Bacterial Production in SeawaterFig 1. Temporal analysis of bacterial abundance (A), bacterial
production (B) and bacterial growth rates (C) in 0.6-μm
passing through seawater. Bars represent standard
deviation of the mean from triplicates. * Showed lower
than control (◇) with significant difference (p< 0.001).

than the control, showing reductions of 17% and 24% at
0.01 μM and 0.1 μM Zn, respectively (p<0.001). Zn is an
inhibitor of respiratory electron transport in bacteria (10).
Therefore, it is possible that the inhibition of amino acid(leucine) incorporation is caused by uptake system driven
by ATP energy gained through the respiratory chain.
However, the effect of Zn on substrate utilization needed
for growth is virtually unknown. Since Zn is known to
inhibit proteases in some marine bacteria (15, 21), it is ofinterested to determine the effect of Zn on bacterial
production and protein utilization.
However, bacterial growth rates were observed toincrease over the time as shown in Fig. 1C. The growthrate in Zn-treatment groups was significantly lower than４３

the control (p<0.001) although the difference was nothigh, 8% at 0.01 μM and 10% at 0.1 μM. Theoreticallyspecific growth rate is independent of the population size.
Since the incubation time is short in this study,community structure changing is negligible. The aim of
this experiment was to determine the immediate responseof bacteria to low concentrations of Zn. The growth rates
will be affected by bacterial abundance if the incubation iscontinued.
The effect of Zn on potential protease activities isshown in Fig. 2. The substrates for trypsin andaminopeptidase were hydrolyzed with different rates as
described in Obayashi and Suzuki (20). In most trypsin
substrates, activity was initially higher and subsequently
decreased with time (p<0.01), whereas aminopeptidase
activities slightly increased with time (p<0.001). This
temporal change in enzyme activity was similar to ourprevious observations (19, 20). A significant difference
was not observed in protease activity among Zn-treatedand control groups during the 8 hr incubation. However,another experiment with increasing Zn concentration andincubation time (16 hr) showed dose dependent inhibitionof aminopeptidase activity (6), suggesting that the Zn
effect on proteases depended on the concentration andincubation time. Longer exposure effect on aminopeptidase may be explained by the fact that aminopeptidase isa family of Zn-dependent enzyme and thus it is predicted
to be regulated by Zn (7). Zn at high concentrations may
involve masking the catalytically active subunits of thesubstrate proteins, changing the conformation of enzyme
structure and competing with caution activators connectedwith the formation of a substrate enzyme complex (9). If
the source of enzyme would be various, for example
flagellates and ciliates, it is hypothesized that various
proteases would have different susceptibility to Zn.
Various enzymes might play a role to decompose proteinsin seawater as well as bacterial enzymes.
The results presented here show the addition of Zn
clearly decreased the bacterial production rate (〜24%)compared to the control within the 8 hr incubation (Fig. 1
B), suggesting that the bacterial productivity was more
rapidly respond to Zn stress, which is indicator of total
toxic effect including inhibition of respiratory and enzyme
activities.
Alden Demoling and Baath (2) applied leucine
incorporation to study the toxicity of phenols towardbacterial communities extracted from sediment. Theirresults demonstrated that the leucine incorporation
technique to the bacterial community is a rapid andsensitive method to evaluate toxicity with the comparisonof thymidine incorporation.
Meanwhile our data also indicated that an increase in
the concentration of Zn would have a serious affect on the
carbon metabolism and respiratory activities inheterotrophic bacteria in aquatic ecosystems. Our findings
are in agreement with Rochelle-Newall et al. (26), which
reported that Zn addition at low concentrations could givenegative results on the auto- and heterotrophic
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Chui Wei BONG, Yumiko OBAYASHI and Satoru SUZUKI

Fig 2. Hydrolysis rate of aminopeptidase (a-b) and trypsin (c-f) in 0.6-μm passing through seawater.
Bars represent standard deviation of the mean from triplicates.

compartment and carbon transfer in aquatic ecosystem.
Heterotrophic bacteria are the major degraders of bothparticulate and dissolved organic matter, and the rapid
growth rates and production of bacteria represent an
important link in the carbon cycle (3). The suppression ofbacterial production at low concentrations of Zn or shortperiods might result in major shifts in the relative
abundance of the bacterial community and consequently
affecting the production of specific hydrolytic enzymes.
The pollution in marine environment by heavy metals canaffect the microbial carbon flux. These effects will link tothe changing the quality of dissolved organic matter andbiogeochemical cycle of organic matter especially incontaminated marine ecosystem.
Acknowledgments
We are grateful to the Marine Biology Research
Division, Scripps Institution of Oceanography, Universityof California at San Diego, La Jolla, California especiallyto Professor Farooq Azam and Dr. Francesca Malfatti forthe use of their laboratory facilities for these experiments
and useful advice and comments on our research. We also
thank Dr. Todd W. Miller, CMES, for his critical reviewof this paper. This study was partly supported by Global
COE Program at Ehime University. Chui Wei Bong was
financially supported by the Government of Malaysia.
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摘要

ボン・チュイウェイ・大林由美子・鈴木
細菌生産に及ぼす低濃度亜鉛の影響

聡：海水中

亜鉛は酵素反応の補因子として働くが，高濃度では阻害的に作用する金属で，天然海水中では微量しか存在しないが，時に高濃度汚染が起こることもある．細菌は様々な濃度の亜鉛に対して異なる応答を示す．本研究は，低濃度亜鉛は添加後数時間内に細菌生産性を阻害するが，タンパク質分解酵素には影響がないことを示した．このことは，亜鉛の効果は細菌のタンパク質利用系とは別の系に作用していることを示唆している．