Advantage Provided by Iron for Escherichia coli Growth and Cultivability in Drinking Water

The presence of iron, used both as a nutrient and as an electronacceptor, was demonstrated to give an advantage to Escherichiacoli bacteria in drinking water. Slight additions of ferroussulfate to water with initial low iron concentrations led toa significant increase in the number of E. coli bacteria. Thepresence of ferric oxide in water under anaerobic conditionsincreased bacterial cultivability.

INTRODUCTION

Top

Introduction

The presence of iron corrosion products is reported to favorbacterial activity in drinking water networks, resulting inincreases of both suspended bacteria and biofilm-associatedbacteria (2, 8, 13, 22) or in increased occurrence and/or cultivabilityof coliform bacteria, usually used as the indicator of waterbacteriological quality (17, 18, 26). The large accumulationsof bacterial cells observed on environmental ferric mineral(9) and on corroded metallic structures (19) may be partiallyattributed to the surface properties of iron oxides in thattheir high surface area and their surface charge may promoteattachment and colonization by microorganisms (1, 24).

Iron may also be considered a nutrient necessary for bacterialgrowth, like other elements, such as carbon, nitrogen, and phosphorus.It is, for example, a constituent of all heme enzymes, whichinclude cytochromes and hydroperoxidase. Iron has been shownto limit microbial growth in aquatic environments (6, 7, 14,16), where bacteria must display particular mechanisms, suchas production of strong iron chelators called siderophores,when faced with extreme iron deficiency (10, 11).

Iron is also an electron acceptor for bacteria that can coupleorganic matter oxidation to Fe(III) reduction in the absenceof oxygen (20). Iron-reducing bacteria are thus able to gainenergy from the reduction of soluble (12) or solid iron species(15, 23).

The present study was conducted to observe the effect of iron,considered both as nutrient and as electron acceptor, on thegrowth and cultivability of Escherichia coli strain SH 702 indrinking water.

To investigate the role of iron as a limiting nutrient, drinkingwater sampled after treatment chain but before distributionwas sterilized by heat (121°C, 2 atm, 20 min) and 0.22-µmfiltration. Concentrations of dissolved constituents were thefollowing (mg liter^–1): Ca, 23.2; Mg, 6.4; K, 2.2; Na,16.5; SO₄, 51.1; Cl, 16.4; NO₃, 4.4; PO₄, <0.05; organiccarbon, 1.56; Fe, <0.002. The water was then placed in 500-mlglass flasks and supplemented with ferrous sulfate as a solublemineral iron source, at concentrations close to those describedas the minimum necessary to support bacterial growth (21). Threeflasks were then prepared for each iron concentration (no addediron and 2, 5, 10, 15, and 20 µg liter^–1), and finalconcentrations were confirmed by inductively coupled plasmamass spectrometry analysis. E. coli bacteria previously isolatedfrom a distribution system and characterized as strain SH 702were grown in Luria-Bertani broth media (Difco 0446-17-3) at25°C. After 48 h, the cell cultures were washed three timesby centrifugation (5 min; 10,000 x g) and suspended in sterile(0.22-µm filtration) commercial mineral water with anorganic carbon concentration of about 0.2 mg liter^–1.To simulate the type of oligotrophic conditions found in drinkingwater, the cells were starved by incubation in the mineral waterat 20°C for 24 h before being subjected to another washingcycle. E. coli bacteria were then harvested and added to drinkingwater in glass flasks, at a final concentration of 1.5 x 10³cells ml^–1. This concentration was supposed to be lowenough to allow bacterial growth in such poor-nutrient media.Flasks were incubated at 25°C, in the dark, with agitationat 350 rpm. Bacterial growth was monitored by epifluorescencemicroscopy counting after cells had been stained with DAPI (4',6'-diamidino-2-phenylindole)(2). Cultivability was monitored by cultivation on agar (platecount agar; Difco 247940) at 30°C for 24 h, as is used fordrinking water quality control.

To investigate iron as an electron acceptor, 12 flasks wereprepared with the same water as previously described. Six weresupplemented with a 50 mM concentration of a solid iron oxide(lepidocrocite; ${gamma}$ -FeOOH) simulating iron corrosion products.For three flasks with iron oxide and three without, anaerobicconditions were generated by purging with nitrogen for 30 minand closing the flasks with butyl stoppers. In the remainingflasks, aerobic conditions were maintained by agitating andsimply closing flasks with sterile cotton. E. coli bacteriawere prepared as described above before being added to flasks,at a final concentration of 5 x 10⁶ cells ml^–1. This concentrationwas deliberately set high, in order to detect cultivable E.coli for a long time (several days) and hence observe a potentialeffect of iron oxide or anaerobic conditions on cultivability.Total and cultivable bacteria were enumerated as described above.To observe iron reduction, ferrous iron measurements were performedby the o-phenanthroline spectrophotometric method (12) in flaskscontaining iron oxide at the beginnings and ends of experiments.Abiotic controls were also analyzed.

Iron as a nutrient.

Top

Iron as a nutrient.

The number of total E. coli bacteria (epifluorescence counts)increased with time and reached steady state after 16 days.At this point, a clear relationship was observed between totalbacteria and iron concentration (r = 0.87, P < 0.001; analysisof variance, F = 4.101, P = 0.021), and the number of totalcells was on average three times higher in flasks with 20 µgFe liter^–1 than in flasks without added iron, where almostno growth was observed (Fig. 1) (the Tukey test was significantfor 20 versus 0 µg Fe liter^–1, and for 20 versus2 µg Fe liter^–1: P = 0.015 and P = 0.042, respectively).Unlike total bacteria, the number of cultivable E. coli bacteriarapidly decreased from the initial concentration of 1.3 x 10³CFU ml^–1 at the beginning of the experiment to valuesfrom 3.3 x 10^–1 to 4.0 x 10¹ CFU ml^–1 for flaskscontaining 2 and 10 µg Fe liter^–1, respectively,after 16 days. No simple relationship between the iron concentrationand the cultivability of E. coli was observed at any time ofthe experiment. No organic carbon consumption was displayedby measurements performed at the beginnings and ends of experiments.

View larger version (14K):
[in this window]
[in a new window]
FIG. 1. Number of total E. coli bacteria (epifluorescence counts) in drinking water flasks after 16 days, according to iron concentration. Each data point represents the result from a single flask.

The increase in total cells observed here must be due to ironutilization, because all assays were carried out under the sameconditions and with similar initial organic carbon and inorganicnutrient concentrations. Bacterial growth is not attributableto prior nutrient storage in cells during their growth, as noincrease in bacterial number was observed without added iron.The apparent absence of organic carbon consumption during theassays is not surprising, due to the very small amount of nutrientsnecessary to produce 2 x 10³ cells ml^–1, as observed inflasks with 20 µg Fe liter^–1. According to an earlierhypothesis based on previous observations (2), bacterial productionin such conditions requires 1 x 10^–12 g of organic carbonper cell. On this basis, the highest organic carbon consumptionneeded to support bacterial growth in the present experimentswould be only 0.2 µg liter^–1 (in flasks supplementedwith 20 µg Fe liter^–1), which is lower than thesensitivity of the apparatus used here. In this experiment,the benefit provided by the presence of iron to bacterial growthis unlikely to be due to the electron acceptor character ofiron, because iron was initially provided as Fe(II), and experimentswere conducted in the presence of oxygen, which is preferentiallyreduced by bacteria.

Iron as an electron acceptor.

Top

Iron as an electron...

In the assays performed under aerobic conditions, E. coli remainedcultivable during the 23-day experiment, with only a slightdecrease over time, and the presence of iron oxide had no effecton cultivable bacteria in such conditions. In the anaerobicflasks without iron oxide, cultivable E. coli decreased 6 ordersof magnitude after only 6 days and was undetectable thereafter.When iron oxide was included under anaerobic conditions, thecultivability of E. coli was comparable to that in the assaysperformed under aerobic conditions (Fig. 2). Epifluorescencecounts after 23 days did not reflect the trend in CFU and were1.6 (±0.1) x 10⁶ and 4.0 (±0.5) x 10⁶ cells/mlin the anaerobic assays (with and without iron oxide, respectively),and 1.4 (±0.1) x 10⁶ and 5.0 (±0.2) x 10⁶ in theaerobic assays (with and without iron oxide, respectively).Cell concentrations reported for assays with iron oxide areprobably slightly underestimated, owing to the difficulty ofenumerating bacteria attached to oxide particles. Fe(II) measurementsrevealed that iron oxide had been partially reduced during theexperiments: the Fe(II) concentration increased from 0.11 ±0.02 mM under initial conditions in assays with lepidocrociteto 1.06 ± 0.09 mM and 0.82 ± 0.05 mM after 23days under aerobic and anaerobic conditions, respectively. Noreduction was observed in similar assays performed under abioticconditions. A repetition of this experiment performed with thesame E. coli in the same type of water led to similar results.

View larger version (22K):
[in this window]
[in a new window]
FIG. 2. Cultivability of E. coli in drinking water flasks, determined under aerobic and anaerobic conditions with or without iron oxide supplementation.

Despite the presence of oxygen in the bulk media of aerobicflasks, ferrous iron is likely to be stabilized by adsorptionon iron oxide particles, the presence of organic or microbialchelators, and local microanaerobic conditions occurring atwater-mineral-bacterium interfaces.

Fe(III) reduction has been observed under such conditions withE. coli and in comparable amounts under anaerobic and aerobicconditions (4, 27). According to different authors, Fe(III)reduction by E. coli would either be a respiration process (25),meaning a dissimilatory reduction of iron, or a constitutiveprocess involving transport within cells before its utilizationas functional constituent (3, 5, 27), meaning an assimilatorymechanism. In the present assays, if increased cultivabilitywas due to iron as a nutrient only, no difference should occurbetween aerobic and anaerobic conditions. The differing behaviorof cultivable E. coli between anaerobic assays with and withoutiron hydroxide must be due to more than the use of iron as anutrient. Another observation suggesting that iron reductionwas not due to bacterial assimilation is that the amount offerrous iron produced in the assays was close to 1 mM (55.8x 10^–3 g liter^–1) in both aerobic and anaerobicassays. According to the E. coli cell concentration in the medium(about 2 x 10⁶ cells ml^–1), and to the hypothesis of acell mass of 0.2 x 10^–12 g cell^–1, the biomass presentwould be around 4 x 10^–5 g of bacterial cells liter^–1.With 3 orders of magnitude between biomass and ferrous iron,the reduced iron is hence definitely unlikely to have been onlyassimilated by bacteria and stored in cells, as described byBriat (3). Rather, the main iron reduction pathway must be dissimilation.

This study demonstrates that iron in drinking water may promoteboth growth and cultivability of E. coli. Results suggest thatiron may be considered a potentially limiting nutrient in drinkingwater and that increasing iron concentration may thus lead toan increase in E. coli growth. We also demonstrate that whileanaerobic conditions may have a negative effect on E. coli cultivability,this can be cancelled by the presence of iron oxide. These findingsconfirm and partially explain numerous observations made ondistribution systems in which corroded pipe material has beenrelated to bacterial growth and highlight the fact that thepresence of iron oxides in drinking water distribution systemsmay lead to actual degradation in microbiological water quality.

ACKNOWLEDGMENTS

This work was carried out as part of a larger research programentitled "Biofilm" and coordinated by the Centre Internationalde l'Eau de Nancy (NanCIE, France). It was funded by CompagnieGénérale des Eaux (CGE, France), Agence de l'EauSeine-Normandie (AESN, France), Anjou-Recherche (VEOLIA, France),Syndicat des Eaux d'Ile de France (SEDIF, France), and the CommunautéUrbaine du Grand Nancy (CUGN).

FOOTNOTES

* Corresponding author. Present address: CRP-Santé, Laboratoire de Toxicologie, Université du Luxembourg, 162A, avenue de la Faïencerie, L-1511 Luxembourg. Phone: (352) 46 66 44 480. Fax: (352) 22 13 31. E-mail: appenzel{at}cu.lu.

REFERENCES

Top