SYTTY
MICROBIALLY AVAILABLE PHOSPHORUS IN DRINKING WATER
Project leader: Pertti Martikainen, University of Kuopio, Department
of Environmental Sciences, Bioteknia 2, P.O.Box 1627, FIN-70211, Kuopio,
Finland, tel. +358-17-163586, e-mail: Pertti.Martikainen@uku.fi.
| PUBLICATIONS |
| TIIVISTELMÄ SUOMEKSI |
Researchers:
National Public Health Institute
Markku Lehtola, tel. +358-17-201371, e-mail Markku.Lehtola@ktl.fi
Ilkka Miettinen, tel. +358-17-201371, e-mail Ilkka.Miettinen@ktl.fi
Terttu Vartiainen, tel. +358-17-201346, e-mail Terttu.Vartiainen@ktl.fi
Tiia Myllykangas, tel. +358-17-201181, e-mail Tiia.Myllykangas@ktl.fi
Tarja Pitkänen, tel. +358-17-201153, e-mail Tarja.Pitkanen@ktl.fi
Minna Keinänen, tel. +358-17-201369, e-mail Minna.Keinanen@ktl.fi
Panu Rantakokko, tel. +358-17-201181, e-mail Panu.Rantakokko@ktl.fi
Consortium: Drinking water and health
Financing SYTTY organisation: The Academy of Finland
Funding from SYTTY / Total funding of project (€): 139932
/ 231006
Person-months of work funded by SYTTY / Total person-months of work:
58 / 93
KEY WORDS: phosphorus, drinking water, microbes, water treatment
EXTENDED ABSTRACT
1 Introduction
Microbial growth in drinking water distribution networks is a remarkable hygienic and economical problem. Microbial growth in drinking water is affected by many factors like retention time in distribution network, temperature, disinfection, biofilms and availability of microbial nutrients. The microbes can be destroyed effectively by disinfection, e.g. by chlorination. However, chlorine reacts with organic matter, and low doses might not be able to prevent microbial growth in the periphery of pipeline networks. On the other hand, high doses of chlorine cause taste and odor problems, and formation of mutagenic byproducts, especially if the content of humus compounds in water is high. Another way to prevent microbial growth in water is to remove essential nutrients supporting microbial growth. It has earlier been suggested that the main limiting nutrient for microbial growth is organic carbon, especially assimilable organic carbon (AOC) (van der Kooij 1982). In 1996 it was found that in boreal regions like in Finland, phosphorus is often the nutrient limiting microbial growth (Miettinen 1996). Similar results were later reported for drinking waters in Japan (Sathasivan 1997). These studies showed that the concentration of phosphorus needed to stimulate microbial growth was very low (<2 µg P/l). Present standard chemical methods for analysing phosphorus in water are able to detect soluble reactive phosphorus or total phosphorus concentrations down to 2 µg P/l. However, microbes can use for their growth both soluble inorganic phosphate phosphorus and phosphorus released from organic phosphorus compounds by phosphatase enzymes. Therefore, standard methods are not sensitive enough to analyse the phosphorus concentrations supporting microbial growth, and they do not distinguish between the total and the phosphorus available for microbes.
The main goals of this project were: 1) to develop a method for analysing low concentrations of microbially available phosphorus (MAP) in water, 2) to study the effect of water purification processes on phosphorus concentrations and microbial growth in distributed water, 3) to study the effects of phosphorus availability in water on the development of biofilms.
2 Methods
Analyses of MAP in water samples. Water samples (100 ml) were treated by inorganic (except phosphorus) and organic nutrients to ensure phosphorus limitation. Natural microbial population in samples was destroyed by pasteurization at 60 °C in a water bath for 30 minutes. After cooling, the test strain Pseudomonas fluorescens was inoculated to samples, and the growth was followed for 4-8 days at 15 oC (in standardization it was found that maximum growth always occurred within 4-8 days). Growth of the test bacteria was analysed every day by spread plating on R2A-agar. Plates were incubated at 22oC for three days before counting the colonies. The maximum number obtained was converted to MAP concentration by the regression coefficient of the standardization experiments.
Other analyses. Heterotrophic plate counts (CFU, colony forming units) of water were analysed by spread plating on R2A-agar. Plates were incubated for 7 days at 22oC before counting the colonies. Heterotrophic growth potential (HGR) was analysed by incubating water samples (100 ml) at 15oC temperature in the dark for 21 days. Microbial growth in water was analysed every second day by spread plating technique on R2A-agar. Phosphorus limitation was tested by adding to water 20 µg/l phosphorus as Na2HPO4 and by analysing microbial growth as in HGR. The maximum number of microbes during the incubation of water is used in the results. AOC was analysed by a modification of the Van der Kooij method (Van der Kooij et al. 1982, Miettinen et al. 1999) which included the addition of inorganic nutrients to ensure carbon limitation of the water. In UV study the molecular size fractions of the organic matter were determined with a high performance size exclusion chromatography.
Water samples were taken from 21 different waterworks using groundwater or surfacewater as raw water. Samples were taken after every separate step of the purification process. For ozonation study water samples were taken from 5 waterworks using ozonation, samples were taken before and after ozonation. Effects of UV-disinfection were studied from samples taken from 3 waterworks using UV-disinfection, also UV-irradiation experiments were done in laboratory.
Biofilms. The effect of phosphorus on the formation of biofilms was studied in laboratory with water where phosphorus was the limiting nutrient for microbial growth. Phosphorus was added as Na2HPO4 giving extra phosphorus concentrations of 1, 2 and 5 µg P/l. Biofilm development was studied on the PVC slides (area 15.9 cm2), which were placed into PVC chambers. Water was pumped with flow velocity of 1 ml/min. Formation of biofilm was analysed as viable counts of heterotrophic bacteria, total number of bacteria and the content of adenosine triphosphate (ATP). Microbial communities in drinking waters and biofilms were characterized by phospholipid fatty acids (PLFA) and lipopolysaccharide hydroxy fatty acids.
3 Results and Discussion
MAP bioassay. There was a linear relationship between the maximum cell count of P. fluorescens and phosphorus concentration from 0.05 to 10 µg P/l. Based on the standardization 1 µg of PO4-P corresponded to 3.73 x 108 CFU of P. fluorescens. High yield of bacterial cells by phosphorus enables very low detection limit in the analyses (0.08 µg P/l).
MAP in Finnish drinking waters. Waterworks using surface water as a raw water had the lowest MAP concentrations in purified drinking water, on average 0.41 µg/l (range 0.06 – 1.15 µg P/l). In waterworks using groundwater the MAP concentrations were on average 2.08 µg/l (range 1.23 –10.20 µg P/l) and in artificially recharged groundwaters MAP concentration was on average 0.94 µg/l (0.10-2.42 µg P/l).
Chemical coagulation reduced the contents of MAP and AOC from water, which was also seen in decreasing microbial growth potential of water. Also activated carbon filtration and infitration through soil decreased these nutrients. Ozonation and liming increased the content of MAP. In ozonation, also AOCpotential increased strongly. In ozonated waters, 1 µg P/l increase in MAP concentration corresponded to 109 CFU increase in microbial growth potential. UV-irradiation decreased the AOC concentration and the sum of molecular size fractions, but MAP was not changed with the UV-doses used in waterworks.
In waterworks using surface water as raw water chemical purification process removed effectively phosphorus, 90 % of MAP was removed. Total organic carbon decreased on average 59 %, but AOCpotential increased during the process. Because of the efficient removal of phosphorus and high levels of AOC, microbial growth in chemically purified drinking waters was usually limited by phosphours. Infiltration through soil decreased the content of MAP on average 64 %, the content of AOC decreased on average 53 %. In ground waterworks changes in content of AOC were minor, but the content of MAP increased in liming of the water.
Biofilms. Very low additions (1 µg/l) of phosphorus increased
microbial biofilm growth. Additions of more than 1 µg/l had minor
effect on the viable counts of heterotrophic bacteria and total bacterial
numbers, but content of ATP increased with increasing content of phosphorus
(up to 5 µg/l), showing the phosphorus limitation in biofilms. Also,
there were changes in microbial population analysed with PLFA. The addition
of phosphorus increased the proportion of gram-negative bacteria in biofilms
and also changed the community structure of gram-negative bacteria. However,
the largest difference in community structures was between waters and biofilms.
Within this SYTTY project we have obtained new knowledge on the importance
of phosphorus in regulating microbial growth in drinking waters in Finland,
and on the effects of various water treatment techniques on microbially
available phosphorus and associated microbial growth. In studied waterworks
the concentration of MAP correlated with the microbial growth potential
(p=0.90, p=0.000) of the water. Also, in phosphorus limiting water the
formation of biofilms was affected by the content of phosphorus. Therefore,
in drinking water distribution systems the content of MAP has importance
in regulating the microbial quality of the water, especially if disinfectant
(chlorine) is applied not at all or the content of disinfectant is too
low to prevent microbial growth.
4 Conclusions
We have now a new sensitive method to analyze microbially available phosphorus in waters. This method enables us to study MAP concentrations in drinking waters, and the changes in MAP by various water treatment processes. Chemically purified water had the lowest MAP concentrations and ground waters had highest concentration of MAP. Phosphorus enhanced also the microbial growth of biofilms.
5 References
Miettinen, I. T., T. Vartiainen, and P. J. Martikainen. 1996. Contamination of drinking water. Nature 381:654-655.
Miettinen, I.T., T. Vartiainen, and P. J. Martikainen. 1999. Determination of assimilable organic carbon (AOC) in humus-rich waters. Wat. Res. 33:2277-2282.
Sathasivan, A., S. Ohgaki, K. Yamamoto, and N. Kamiko. 1997. Role of inorganic phosphorus in controlling regrowth in water distribution system. Wat. Sci. Tech. 35:37-44.
Van der Kooij D, Hijnen WAM and Visser A. 1982. Determining the concentration
of easily assimilable organic carbon. J. AWWA. 74:540-545.