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Applied and Environmental Microbiology, May 1999, p. 2032-2034, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A New Sensitive Bioassay for Determination of
Microbially Available Phosphorus in Water
Markku J.
Lehtola,1,*
Ilkka T.
Miettinen,1
Terttu
Vartiainen,2,3 and
Pertti J.
Martikainen1,3
Laboratory of Environmental
Microbiology1 and Laboratory of
Environmental Chemistry,2 National Public
Health Institute, FIN-70701 Kuopio, and Department of
Environmental Sciences, University of Kuopio, FIN-70211
Kuopio,3 Finland
Received 20 April 1998/Accepted 2 February 1999
 |
ABSTRACT |
The content of assimilable organic carbon has been proposed to
control the growth of microbes in drinking water. However, recent
results have shown that there are regions where it is predominantly phosphorus which determines the extent of microbial growth in drinking
waters. Even a very low concentration of phosphorus (below 1 µg of P
liter
1) can promote extensive microbial growth. We
present here a new sensitive method to determine microbially available
phosphorus concentrations in water down to 0.08 µg of P
liter1. The method is a bioassay in which the analysis of
phosphorus in a water sample is based on maximum growth of
Pseudomonas fluorescens P17 when the energy supply
and inorganic nutrients, with the exception of phosphorus, do not limit
bacterial growth. Maximum growth (CFU) in the water sample is related
to the concentration of phosphorus with the factor
373,200 ± 9,400 CFU/µg of PO4-P. A linear
relationship was found between cell growth and phosphorus concentration
between 0.05 to 10 µg of PO4-P liter1. The
content of microbially available phosphorus in Finnish drinking waters
varied from 0.1 to 10.2 µg of P liter1 (median, 0.60 µg of P liter1).
| |
INTRODUCTION |
The growth of heterotrophic microbes
is a source of hygienic and aesthetic problems in drinking water
distribution systems. Microbes can be inactivated in the water plant by
disinfection, but microbial growth begins when the residual content of
disinfectants, often chlorine, disappears in drinking water
distribution systems (7). Disinfection with high doses of
chlorine is undesirable, since it can lead to formation of mutagenic
chlorinated by-products (1, 9, 18, 22). High doses of
chlorine can also cause taste and odor problems in the drinking water.
Therefore, microbial growth should be limited by eliminating their
growth factors (7). The amount of organic carbon, especially
the content of assimilable organic carbon (AOC), has been considered to
be the main chemical factor controlling microbial growth in drinking
water distribution systems (8, 21).
The content of assimilable organic carbon does not always correlate
with the extent of heterotrophic microbial growth in distribution networks (3, 13). There are regions where microbial growth in drinking waters is regulated by the content of phosphorus, rather
than organic carbon (11, 12, 19). For example, drinking waters in Finland contain high contents of organic carbon (20, 23) and assimilable organic carbon (AOC) (13). In
these drinking waters, microbial growth is typically limited by
phosphorus; similar results were also obtained in Japan
(19). Therefore, even a very minor change in the phosphorus
concentration can have a major influence on the growth of microbes
(12). Phosphorus concentrations in Finnish drinking waters
are generally low, being less than 2 µg of total P
liter1, but are still high enough to allow remarkable
microbial growth (12).
There are sensitive chemical methods for analyzing total phosphorus
or phosphate. By the automatic ascorbic acid method,
PO4-P can be analyzed down to 1 µg liter1
(4). With capillary electrophoresis detection, the
limit for phosphate is 10 µg of PO4-P
liter1 in clean lake water (16). The most
sensitive chemical method, the magnesium-induced coprecipitation
procedure has sensitivity down to 31 ng of P liter1
(6). However, all of the chemical methods, even
sensitive ones, are unable to show the fractions of phosphorus
supporting microbial growth. For limnic ecosystems, there are
methods for analyzing the fraction of phosphorus available for
algal growth (2). In this paper, we present a new method
for determination of submicrogram concentrations of microbially
available phosphorus (MAP) in water. This bacterial bioassay was used
to analyze the concentrations of MAP in several drinking waters in Finland.
| |
MATERIALS AND METHODS |
Glassware.
All pieces of glassware (Pasteur pipettes, tubes,
Erlenmeyer flasks with glass stoppers) and plastic pipette tips were
first washed with phosphate-free detergent (Deconex; Borer Chemie AG, Zuchwil, Switzerland), immersed in 2% HCl solution for 2 h, and then rinsed with deionized water (Millipore, Molsheim, France). Finally, clean glassware was heated for 8 h at 250°C.
Inocula.
Pseudomonas fluorescens P17, biotype 7.2 (ATCC 49642), was used in the bioassay. Strain P17 has phosphatase
activity. This was tested by a fluorometric method with
4-methylumbelliferylphosphate-Na2 salt (Fluka) as a
substrate according to the method of Miettinen et al.
(10). Bacteria were cultured and stored in spring water (Fons; Valio, Ltd., Kouvola, Finland). This spring water contains 38.6 mg of Ca liter1, 4.0 mg of Mg liter1, and
1.8 mg of K liter1. An organic carbon source,
CH3COONa, was added to attain a final concentration of
1,000 µg of C liter1. The cultures were stored at
+4°C.
Standardization.
Phosphorus standards were made in
acid-washed Erlenmeyer flasks in deionized water (Millipore). Six
milliliters of inorganic nutrient solution was added to the 94-ml water
sample. Nutrients were added to ensure that only the phosphorus of the
inorganic nutrients is limiting for growth. Addition of inorganic salts to the deionized water ensured the same electric conductivity level
(ca. 100 µS/cm) as in drinking water in general. The salt addition
prevents osmotic shock and a possible destruction of bacterial cells in
standard water. The salt solution consisted of
(NH4)2NO3, MgSO4
· 7H2O, CaCl2 · 2H2O, KCl,
and NaCl (Merck). After addition of salt solution, the standard water
had 15 mg of N liter1, 0.6 mg of Mg liter1,
1.6 mg of Ca liter1, 3.2 mg of K liter1,
2.4 mg of Na liter1, and 8.9 mg of Cl
liter1. In order to ensure that standard water is not
carbon limited, CH3COONa was added to reach final
concentration of 2,000 µg of C liter1.
Standardization was made with addition of different amounts of
phosphorus (Na2HPO4; Merck) into the standard
(described above) water. The concentrations of phosphorus ranged from
0.05 to 10 µg of PO4-P liter1 (see Fig. 2).
Standardization was repeated with four standard series with different
phosphorus concentrations. Every standard set contained three to six
different concentrations of phosphorus and a blank sample.
After addition of nutrients and carbon, standards were pasteurized at
60°C in a water bath for 35 min. After cooling, the samples were
inoculated with P. fluorescens P17. The cell density of
P. fluorescens P17 in inoculated samples was
approximately 1,000 CFU/ml. Inoculated water samples were incubated at
15°C to obtain the maximum cell numbers at the PO4-P
concentrations tested. The bacterial cells in water were enumerated
daily by spread plating on R2A agar (17). The plates were
incubated at room temperature (22 ± 2°C) for 3 days before enumeration.
A linear regression model was calculated by using the maximum growth of
P. fluorescens (plate counts in CFU per milliliter)
at
different phosphorus concentrations. The model was calculated
by using
combined results of four experiments. Calculations and
drawings of
figures were done with the Microcal Origin 4.1
program.
Analyses for MAP.
Water samples were taken into
acid-washed glass flasks and kept at +4°C before analysis. The
analysis was started within 24 h after sampling. Residual chlorine
in water samples (100 ml) was removed with the addition of 50 µl of
0.02 M sodium thiosulfate. Inorganic salts and organic carbon (see the
standardization procedure described above) were added to the samples to
ensure that inorganic nutrients (except phosphorus) or organic carbon
did not restrict microbial growth. The final concentrations of added
nutrients in samples were 250 µg of N liter1, 53 µg
of K liter1, 10 µg of Mg liter1, 27 µg
of Ca liter1, 40 µg of Na liter1, and 149 µg of Cl liter1. Sodium acetate was added as described
above. After addition of nutrients and thiosulfate, samples were
pasteurized and inoculated with P. fluorescens P17.
Water samples were incubated at 15°C. The growth of bacteria in water
samples was enumerated after 4, 5, 6, 7, and 8 days
from the
inoculation by spread plating on R2A agar (
17).
Spread-plated
R2A agar was incubated for 3 days at room temperature
(22 ± 2°C)
before enumeration. The maximum plate counts of
P. fluorescens P17 were transformed with a conversion
factor (from standardization)
into the amount of
phosphorus.
Water samples.
Drinking water samples were collected from
three groundwater works, four artificial groundwater works, and
three surface waterworks in Finland (Table
1). Water samples were analyzed for
MAP.
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TABLE 1.
Water treatment techniques and concentrations of
microbially available phosphorus in drinking waters of 10 different
waterworks (n = 1)a
|
|
| |
RESULTS |
Standardization.
Maximum cell count was generally reached
after an incubation time of 4 to 6 days (Fig.
1).
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FIG. 1.
Growth of P. fluorescens P17 with
different phosphorus concentrations (<0.05 to 2 µg of
PO4-P liter1) in one standardization test.
The maximum cell counts used for the determination of the conversion
factor are shown by arrows.
|
|
There was a linear relationship between maximum cell count of
P. fluorescens P17 and phosphorus concentration in the
range
0.05 to 10 µg of PO
4-P liter
1 (Fig.
2). Above 10 µg of PO
4-P liter
1 there was
no longer any linear relationship between the phosphorus
concentration and bacterial growth (data not shown). Microbial
counts
in blank water samples varied from 2.8 × 10
4 to
7 × 10
4 CFU/ml. The arithmetical mean of microbial
counts in blank water
was 4.7 × 10
4 CFU/ml. The
detection limit of the bioassay was 0.08 µg of PO
4-P
liter
1 (Fig.
2) and was
calculated by the mathematical technique of
Hubaux and Vos
(
5).
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|
FIG. 2.
Relationship between maximum colony counts of
P. fluorescens P17 and concentration of phosphorus. The
regression lines and confidence limits are based on four separate
standardization experiments. The inset figure shows regression line and
confidence limits with phosphorus concentrations from 0 to 0.5 µg
liter1; XD is the detection limit
(7). The regression equation there is y = 378,800 (±35,500)x + 53,800 (±7,700); n = 14. The regression
equation with all phosphorus concentrations is y = 373,200 (±9,400)x  10,000 (±31,700); n = 25. x is measured
in micrograms of P per liter, and y is measured in CFU per
milliliter. Standard errors are in parentheses.
|
|
The yield factor was derived from the slope of the line when cell
growth was plotted against PO
4-P concentration. The yield
factor was 373,200 ± 9,400 CFU/µg of PO
4-P.
MAP in Finnish drinking waters.
The MAP analysis gave a
positive result in every drinking water sample studied. The
concentration of MAP varied from 0.1 to 10.2 µg PO4-P
liter1 (Table 1) and was highest in drinking water
produced in groundwaterworks. In drinking waters purified from
surface waters, or produced in artificial recharge plants with slow
sand or bank filtration, the concentration of MAP was invariably below
1 µg liter1 (Table 1).
| |
DISCUSSION |
It is often necessary to analyze microbially available phosphorus,
since extremely small changes in the concentration of phosphorus can
have profound effects on microbial growth. With phosphorus standardization, 1 µg of PO4-P corresponded to 3.73 × 108 CFU of P. fluorescens, while 1 µg
of AOC (acetate) results in 2.04 × 106 CFU
of P. fluorescens (14). Thus 1 µg of
phosphorus accounts for 180 times higher cell counts than 1 µg
of acetate carbon. This is also the basis for the high sensitivity
of the present MAP bioassay. Our results showed that the bioassay can
be applied for assaying very low phosphorus concentrations. There
was a linear response from 0.05 up to 10 µg of PO4-P
liter1. This range was adequate, because in
drinking waters, the maximum microbial growth occurs in the range of 5 to 10 µg of PO4-P liter1 (12,
19).
There are also some sensitive chemical methods for analyzing very
low concentrations of phosphorus in waters (4, 6), but the
chemical analyses do not represent the amount of phosphorus available
for microbial growth. The present MAP analysis is sensitive enough to
also show low concentrations of phosphorus supporting microbial growth.
The standardization of the MAP analyses is based on inorganic
phosphate. Because P. fluorescens P17 has phosphatase activity, the MAP analyzed from water samples also includes organic phosphorus compounds. Presently, we do not know the ratio of
microbially available inorganic phosphorus to microbially available
organic phosphorus in drinking water.
There already exist water purification techniques that effectively can
remove phosphorus from water. The generally used method of chemical
coagulation with polyaluminum chloride effectively removes phosphorus
(15). Our study substantiates these results; the MAP
concentration was low in the chemically treated waters. However,
artificially recharged groundwaters also contained low levels of MAP,
suggesting that biological filtration can also remove phosphorus.
Surprisingly, untreated groundwater contained the highest levels of
MAP. This might increase the risk for microbial regrowth in
distribution systems using untreated groundwater, especially if
drinking water contains a high concentration of organic carbon and is
not initially disinfected. Drinking water produced from groundwater
is usually not disinfected in Finland. Therefore, it might be
necessary to reduce the phosphorus concentration in plants distributing groundwater.
This novel bioassay offers a useful tool when the efficiency of the
present drinking water purification technology is being evaluated or
new technologies for drinking water processes are being developed in
order to improve the chemical and microbiological quality of drinking water.
| |
ACKNOWLEDGMENTS |
The study was supported by the Finnish Research Programme on
Environmental Health (project 42676) and Academy of Finland
(project 34538).
We give special thanks to the staff of the Laboratory of Environmental
Microbiology in the National Public Health Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: National Public
Health Institute, Laboratory of Environmental Microbiology, P.O.
Box 95, 70701 Kuopio, Finland. Phone: 358 17 201371. Fax: 358 17 201155. E-mail: Markku.Lehtola{at}ktl.fi.
| |
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Applied and Environmental Microbiology, May 1999, p. 2032-2034, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
