Is ctr.

84779

DETECTION OF IRON BACTERIA IN RIVER SEDIMENT, SOIL AND WELL WATER SAMPLES

Barbara J. Butler and Colin I. Mayfield

Waterloo Centre for Groundwater ResearchDepartment of BiologyUniversity of WaterlooWaterloo, Ontario, CanadaN2L3G1

Prepared for: Beak Consultants Limited

June 18 1991

Summary:

A well water sample (GW1) and two soil/river sediment samples (81 and S2) were tested for their ability tocatalyze the formation of oxidized iron precipitate, and analyzed for the presence of microorganismswhich could contribute to iron deposition. Aliquots of S1, S2 and GW1 caused iron deposition in suitablemedia, under conditions where uninoculated media or media amended with autoclaved well water orsoil/river sediment did not precipitate iron. The iron-precipitating activity could also be transferred to freshmedia. The results indicate that iron precipitation was associated with biological activity, and not with thesoil/sediment or well water itself. Microorganisms capable of depositing iron were detected in all 3samples, and included cells with morphologies typical of iron bacteria such as Gallionella, Leptothrix and"Siderocapsa" as well as other rod-shaped, spiral-shaped, filamentous and coccoid bacteria. It is quitepossible that the iron staining observed at the field site from which the samples were taken isbiologically-mediated.

Introduction:

Iron is the fourth most abundant element in the Earth's crust, and a component of almost all types of rocks(Nealson, 1983). Although iron is primarily confined to the oxidized phases of the present-day lithosphere,there are environments conducive to the rapid cycling of iron between its reduced and oxidized forms. Thecycling requires the presence of a reduced zone, such as an anaerobic seepage, an anaerobic orsemianaerobic sedimentary environment, a flooded bog, and so on, where iron reduction and mobilizationcan occur (Nealson, 1983). Once exposed to oxygen, iron oxidation is rapid at neutral pH, although thereaction is pH-dependent and much slower under acidic conditions. Ferric iron tends to hydrolyse insolution to form a multitude of hydrated and oxidized phases. These are frequently interconvertible andmay also interact with various ions in solution, forming polycationic complexes and eventually crystallineforms (Nealson, 1983). Thus in moving from an anaerobic to an aerobic zone, soluble ferrous iron isoxidized to ferric iron which tends to precipitate from solution as various insoluble hydroxides and oxides.However, iron (both ferrous and ferric) is readily chelated by a variety of organic molecules, includingcitrate, humic acids, tannins, and microbially-produced siderophores. The soluble chelates which areformed stabilize the iron in solution, so that the iron content of natural waters may exceed what would beexpected on thermodynamic grounds (Nealson, 1981).

Microbial activity can contribute to the iron cycle through (1) iron scavenging and uptake, (2) ironreduction or solubilization, and (3) iron oxidation or precipitation. The first and second may contribute tothe genesis of the iron-rich groundwaters that seem to be present at the field site, but the first andparticularly the third type of interactions are relevant to what is occurring in the sampled well water and inthe water discharging from the valley slope at the site.

Virtually all organisms require iron as an essential trace nutrient, and many microorganisms producesiderophores to facilitate iron uptake, and possess, high-affinity uptake systems. Common soilmicroorganisms including Pseudomonas, Bacillus, Serratia, Acinetobacter, filamentous fungi, Nocardia,and Streptomyces, precipitate chelated iron from solution as they metabolize the organic chelator(Alexander, 1977). Many microorganisms promote iron oxidation indirectly, simply because they alter theoxidation-reduction potential and/or the pH of their own environment (Ehriich, 1981). In addition, a numberof bacterial species appear to be specifically associated with iron (and often manganese) precipitation atneutral pH. These "iron bacteria", often directly identifiable when iron-bearing water or iron deposits aremicroscopically examined, include the stalked bacterium Gallionella, filamentous, sheathed bacteria of theLeptothrix-Sphaerotilus group, Crenothrix, a spherical bacterium that forms chains of elongated cellswithin a mucilaginous tubular sheath that becomes impregnated with Fe(OH)s, budding bacteria such asHyphomicrobium and Pedomicrobium, the ovoid, iron-encrusted, capsulated "Siderocapsa", andMetallogenium, a bacterium studied for more than 20 years, that may in fact be a nonliving, microscopicartifact (Nealson, 1983; Atlas and Bartha, 1987; Paul and Clark, 1989; Hanert, 1989; Tuovinen et al., 1989;Zavarzin, 1989). Many of the iron bacteria have never been obtained in axenic culture, and have beenstudied only in situ or in mixed culture. Recently it was demonstrated conclusively that Gallionella is a truechemolithotroph using ferrous iron oxidation to obtain energy, and carbon dioxide as a sole source ofcarbon (Hanert, 1989), but whether the other neutral iron bacteria obtain energy for growth from ironoxidation remains controversial. Many iron bacteria probably do not oxidize iron at all, but are commonlyassociated with accumulated iron oxides because of their ability to precipitate or accumulate iron, forexample, from oxidized iron chelates (Nealson, 1983). Iron-oxidizing acidophiles such as Thiobacillusferrooxidans are known to derive energy for growth from the oxidation, indeed, the rate of ferrous ironoxidation at acid pH is very slow in the absence of microbial activity (Singer and Stumm, 1970).

At neutral pH, it is difficult to distinguish unequivocally between abiotic and biotic iron oxidation, becauseboth occur in parallel. Thus, at the study site, if the discharging groundwater is anaerobic, the reduced ironit contains will eventually oxidize in the absence of iron bacteria, although the presence of such bacteriawill contribute to the rapidity of the reaction. Microbial activity may also contribute to the observed irondeposition by precipitating chelated iron from solution. Our strategy in this work has been to demonstratethat microbial activity may be contributing to the production of the observed oxidized iron precipitates atthe site. Various bacteriological media, intended for the growth of some of the iron bacteria describedabove, were inoculated with well water and soil taken from the site, and the subsequent occurrences

observed and compared with occurrences in uninoculated media and media inoculated with autoclavedsoil and well water. Iron oxidation and precipitation was scored on the basis of the appearance of rustyred-brown colour and precipitate in the media. Microscopic examination of the culture fluids and theoriginal well water sample was undertaken to determine if cell morphologies typical of iron bacteria wereobservable.

Materials and Methods:

SamplesThe three supplied samples were stored at 4°C upon receipt, until required. GW1 was a groundwatersample obtained from MB-2, and S1 and S2 were two soil/river sediment samples. The pH of the GW1water was 6.9. Suspensions of soil in distilled water (1 part:2 parts) had reactions of 7.7 (S1) and 7.8 (S2).The S1 sample appeared to be composed of sand and clay, and also contained roots and other plantmaterial. The soil was sticky and plastic, and was interspersed with pockets of rusty-coloured material. Themoisture content of S1 was 30%. In contrast, S2 was composed of much coarser material, mostly sand,had a more uniform rusty, red-brown colouration, and did not contain plant material. Its moisture contentwas 13%. The GW1 water was clear and uncoloured, but rusty, flocculent precipitate was evident at thebottom of the sample bottle. This material was suspended in the water prior to subsampling.

MediaWinogradsky's medium for iron bacteria, described by Rodina (1972), contained 0.5 g NmNOs, 0.5 gNaNOa, 0.5 g KaHPCU, 0.5 g MgSO4-7H2O, 0.2 g CaCl2.2H20,10 g ferric ammonium citrate in 1000 mLdistilled water, adjusted to pH 6.0. 9K medium (Silverman and Lundgren, 1959) contained 0.5 g teHPCX0.01 g Ca(NO3)2.H2O, 3.00 g (NH^SCU, 0.10 g KCI, 0.5 g MgSO4.7H2O, 44.0 g FeSO4-H2O in 1000 mLdistilled water, adjusted to about pH 3 with sulphuric acid. A medium suitable for growth of heterotrophiciron bacteria (Rodina, 1972) contained 1 g iron citrate, 0.05 g KH2PO4, 5 g peptone and 12 g agar in 1000mL tap water. A medium described by Mulder (1989) and previously used for Leptothrix (Rouf and Stokes,1964) contained 5 g peptone, 0.15 g ferric ammonium citrate, 0.2 g MgS04.7H2O, 0.05 g CaCl2-2H2O,0.05 g MnS04.H2O, 0.01 g FeCla.eHaO and 12 g agar in 1000 mL tap water. Modified Wolfe's medium wasprepared as described by Krieg (1981). The overlay consisted of 1 g NHUCI, 0.2 g MgS04.7H2O, 0.1 gCaCl2.2H2O, 0.5 g KhtePCU, 0.016 g bromothymol blue and 0.004 g bromocresol purple in 1000 mLdistilled water. FeS precipitate was prepared by reacting equimolar quantities of ferrous ammoniumsulphate and Na2S'9H2O in freshly boiled distilled water. The resultant precipitate was washed repeatedlywith boiled water before use. The overlay was dispensed in 50-mL aliquots into bottles containing 10 mL ofsolidified FeS agar (1.5 g purified agar (L28, Oxoid) in 100 mL of FeS precipitate), and sterile CO2 wasbubbled through the medium for 10-15 sec to lower the pH.

Liquid media were inoculated with soil (5 g wet wt/50 mL medium), well water (5 mL/50 mL medium) and1/10 dilutions (in 0.1% Na pyrophosphate, pH 7.0) of the soil and water (5 mL dilute suspension/50 mLmedium). Sterile controls were amended with autoclaved soil or well water. Solid media were streaked orspread with the above inocula. A series of sterile, foil-covered beakers containing about 100 mL GW1, 50mL GW1 plus 50 ml Winogradsky's medium, and S1 or S2 soil in 100 mL Winogradsky's medium wasprepared. Sterile microscope slides were immersed in the fluid in each beaker, and later examinedmicroscopically for developing attached microbial populations. All cultures were incubated quiescently atroom temperature, except flasks of 9K medium were shaken (175 rpm).

MicroscopyBoth bright field and phase contrast microscopy were used. Dried smears were treated with 2% potassiumferrocyanide (2-3 min), followed by 5% hydrochloric acid (1-2 min) and then 5% erythrosin A (in 5%phenol) (1-2 min), a procedure to stain microbial cells red and iron deposits blue (Rodina, 1972).

Results and Discussion:

The pH of the supplied samples suggested that acidophilic iron-oxidizers were unlikely to be majorcontributors to production of the oxidized iron stains observed at the field site. No evidence of ferric ironproduction was observed in GW1 -inoculated 9K medium. As well, little red-brown colouring appeared in

medium inoculated with 1/10 soil dilutions. Flasks amended with soil did turn rusty-coloured after 24 and72 h but sterilized soil had the same effect (Table 1). This resulted in part from the neutralizing effect of theadded soil, especially in the case of S2. The observed rust-coloured precipitate in S1-amended flasks didnot arise from biological activity because it occurred also in sterile controls, and the effect was nottransferable. Serial dilution from the primary flasks into fresh 9K medium did not result in iron-oxidizingactivity in the secondary flasks (Table 1). Few microbial cells were observed in either of the primarysoil-amended cultures during microscopic examination. It was concluded the acidophilic iron-oxidizer 7.ferrooxidans was not present in the samples.

TABLE 1 9K medium

inoculum

uninoc

GW1

GW1*

S1

S1"

S2

S2*

S1 1/10

S1 1/10*

S21/10

S2 1/10*

primary cultureobservations

no change (10d)

no change (I0d)

no change (24 h)

rust colour (72 h)

muddy rust-brown (24 h)

rust colour (24 h)

rust colour (24 h)

slight rust (10d)

no change (24 h)

slight rust (10d)

no change (24 h)

secondary culturepH observations

-

no change

-

2.7 (1 0 d) no change (6 d)

3.8 (6 d)

6.2 (1 0 d) no change (6 d)

4.0 (6 d)

no change (6 d)

-

no change (6 d)

-

sterile control, soil or water sterilized by autoclaving.uninoc = uninoculated.(time) is the time at which the observation was made

Iron precipitation was evident after 24 h, and extensive after 72 h in all flasks of Winogradsky mediuminoculated with soil or well water (Table 2). The sterile and uninoculated controls remained clear yellow(Figure 1). In soil or 1/10 soil-amended cultures (Figure 1a, 1b), red-brown scum formed on the surface ofthe primary cultures, the medium became colourless, and a dark grey-yellow precipitate formed. TheGW1-inoculated medium developed a red-brown surface scum and the medium became rusty-coloured(Figure 1c). The iron-precipitating activity of all active primary cultures was transferred to secondarycultures (Table 2, Figure 1) and this, plus the observation that no iron precipitated in the sterile controlsindicates that the precipitation was biologically-mediated. Phase contrast microscopic examination of thecultures in Winogradsky medium showed that all contained large, mixed microbial populations.Rod-shaped cells, some motile, predominated, and some chains of sheathed coccobacilli and occasional

filaments, as well as viable protozoa in the soil-amended cultures, were also observed. Stalked and/orsheathed bacteria were not prevalent in these cultures.

Many colonies appeared on plates of either heterotrophic iron agar (Rodina, 1972) or Rouf and Stokes(1964) agar inoculated with S1, S2 or GW1, although not all precipitated iron (Figures 2, 3). Generalizedoxidation of iron in the medium, and prevalence of red-brown coloured colonies were far morepronounced on the former medium (compare Figure 2 and 3a). A few dark-brown or rusty colouredcolonies were detected on Rouf and Stokes agar (Figure 3a), but they did not resemble the brown-blackLeptothrix colonies described by Mulder (1989).

TABLE 2 Winogradsky's medium

Inoculum primary cultureobservations

secondary cultureobservations

uninoc

GW1

GW1*

S1

S1*

S2

82*

S1 1/10

S1 1/10*

S21/10

no change (10 d)

rusty surface scum, red-brown medium,red-brown precipitate (72 h)

no change (6 d)

rusty surface scum, gas bubbles,colourless medium, grey-yellow ,precipitate (72 h)

no change (6 d)

rusty surface scum, gas bubbles,colourless medium, grey-yellowprecipitate (72 h)

no change (6 d)

rusty surface scum, gas bubbles,colourless medium, grey-yellowprecipitate (72 h)

not tested

rusty surface scum, gas bubbles,colourless medium, grey-yellowprecipitate (72 h)

as in primary

as in primary

as in primary

as in primary

as in primary

S2 1/10 not tested

sterile control, soil or water sterilized by autoclaving.uninoc = uninoculated.(time) is the time at which the observation was made.

Figure 3b shows four iron-precipitating isolates taken from heterotrophic iron agar and restreaked onheterotrophic iron agar and trypticase soy agar (a general purpose bacteriological medium). Colouring ofthe medium and the biomass is evident in the iron agar. The pH of all inoculated bottles of modifiedWolfe's medium increased relative to an uninoculated control, as judged by a change in indicator colour inthe medium (Figure 4a) although the appearance of white, cottony growth typical of Gallionella colonieswas most apparent in bottles inoculated with either S1 or a 1/10 dilution of S1 (Figure 4b). Unlike the other,heterotrophic iron bacteria, Gallionella must occupy a niche where both ferrous iron and oxygen areavailable. It is thus a gradient organism, tending to occupy a microaerophilic environment with a Eh of+ 200 to +320 mV, with available COa but little organic material (Hanert, 1989). Perhaps the area fromwhich the S1 soil sample was taken best satisfied these criteria. Microscopic examination of theS1-inoculated Wolfe's medium revealed a preponderance of bacteria that did not conform to the typicaldescription of Gallionella, i.e., an iron-encrusted stalk attached to a kidney-shaped cell (Hanert et al.,1989). Most cells appeared to be rods or coccobacilli, although a few cells possessing short appendagesthat could be stalks were noted (Figure 5a). Sheathed and capsulated iron bacteria will not grow inmodified Wolfe's medium (Hanert, 1989). Gallionella is a sessile bacterium, and removal of samples fromthe cultures could disrupt the natural orientation of the cells breaking the stalks, and perhaps incubationtime was insufficient for mature iron-encrusted stalks to be produced. Twisted stalks typical of Gallionellagrowth were observed in the GW1 well water sample (Figure 5b, 5c).

Evidence of sheathed bacteria was apparent on microscope slides immersed in GW1 (Figure 6a), andother, stalked cells (Figure 6b) and iron-encrusted cocci (perhaps "Siderocapsa" or a similar species) werealso observed (Figure 6c). Direct microscopic observation of the GW1 sample also revealed cellmorphologies typical of "iron bacteria", as in Figure 5.

In contrast, when Winogradsky's medium was provided, the microbial populations that developed fromGW1, and also from S1 and S2, tended to be dominated by rods, spirals and other "normal-looking"bacteria (Figure 7), rather than iron bacteria. Thus, although iron bacteria are present in the environment,much of the observed iron deposition in the soil/sediment at the field site could result from activity of"normal" bacteria if abundant carbon is available. Soil-amended Winogradsky cultures also containedprotozoa and diatoms, although these were not associated with iron deposits. In stained preparations,blue-coloured (i.e. iron-containing) material was observed in all smears although microbial cells and ironprecipitates were not always in intimate contact. Figure 7d shows a chain of 8 cocci that stained blue,indicating that they were probably iron bacteria, whereas the other rods in the field stained pink and werenot iron-coated.

The flocculent red-brown precipitate in the GW1 sample, presumably common in water from the MB-2 well,is probably derived from activity of sheathed bacteria and other typical "iron bacteria". Since ourexperiments have shown that populations of "normal" bacteria will develop from GW1, if chelated iron andcarbon are available, as in Winograsky's medium, the predominance of iron bacteria in the GW1 samplesuggests that the iron available to these well water bacteria in their natural environment is not associatedwith abundant carbon. The well water is a naturally low nutrient environment dominated by thesemicroorganisms.

From this brief study it can be concluded that microorganisms present in the three samples are capable ofmediating the precipitation of oxidized iron deposits. The known ferrous iron-oxidizer Gallionella ispresent, cell morphologies typical of other iron bacteria were noted, and numerous heterotrophic bacteriaable to precipitate iron from solution at neutral pH were present in all samples, suggesting they arecommon. It is quite possible that biological activity causes the iron staining observed at the site. If thedischarging groundwater is anaerobic, containing ferrous ions in solution, these will oxidize uponexposure to air, but Gallionella and perhaps other microorganisms may enhance the rate at which thisoccurs. Chelated iron, if present in the groundwater, may be precipitated from solution by theheterotrophic bacteria detected in this study. Some of these may be sheathed or encapsulated ironbacteria, others may be common soil isolates.

Alexander, M. 1977. Introduction to Soil Microbiology, 2nd edition. John Wiley & Sons, New York.

Atlas, R.M. and Bartha, R. 1987. Microbial Ecology: Fundamentals and Applications, 2nd edition. TheBenjamin/Cummings Publishing Co., Inc. Menlo Park, Ca.

Ehriich, H.L 1981. Geomicrobiology. Marcel Dekker, Inc., New York.

Hanert, H.H. 1989. Genus Gallionella Ehrenberg. In Staley, J.T., Bryant, M.P., Pfennig, N. and Holt, J.G.(eds), Bergey's Manual of Systematic Bacteriology, volume 3. Williams & Wilkins, Baltimore, pp. 1974-1979.

Krieg, N.R. 1981. Enrichment and isolation. In Gerhardt, P., Murray, R.G.E., Costilow, R.N., Nester, E.W.,Wood, W.A., Krieg, N.R. and Phillips, G.B. (eds). Manual of Methods for General Bacteriology. AmericanSociety for Microbiology, Washington, DC.

Mulder, E.G. 1989. Genus Leptothrix Kutzing. In Staley, J.T., Bryant, M.P., Pfennig, N. and Holt, J.G. (eds),Bergey's Manual of Systematic Bacteriology, volume 3. Williams & Wilkins, Baltimore, pp. 1998-2003.

Nealson, K.H. 1983. The microbial iron cycle. In Krumbein, W.E. (ed), Microbial Geochemistry. BlackwellScientific Publications, Oxford, pp. 159-190.

Paul, E.A. and Clark, F.E. 1989. Soil Microbiology and Biochemistry. Academic Press, Inc., San Diego.

Rodina, A.G. 1972. Methods in Aquatic Microbiology. Colwell, R.R. and Zambruski, M.S. (eds, translation,revision). University Park Press, Baltimore.

Rouf, M.A. and Stokes, J.L 1964. Morphology, nutrition and physiology of Sphaerotilus discophorus.Arch. Mikrobiol. 49:132-149.

Silverman, M.P. and Lundgren, D.G. 1959. Studies on the chemoautotrophic bacterium Ferrobacillusferrooxidans I. An improved medium and harvesting procedure for producing high cell yields. J. Bacteriol.77:642-647.

Singer, P.C. and Stumm, W. 1970. Acidic mine drainage: the rate-determining step. Science167:1121-1123.

Tuovinen, O.H., Hirsch, P. and Zavarzin, G.A. 1989. Family "Siderocapsaceae" Pribham. In Staley, J.T.,Bryant, M.P., Pfennig, N. and Holt, J.G. (eds), Bergey's Manual of Systematic Bacteriology, volume 3.Williams & Wilkins, Baltimore, pp. 1874-1882.

Zavarzin, G.A. 1989. Genus "Metallogenium" Perfil'ev and Gabe. In Staley, J.T., Bryant, M.P., Pfennig, N.and Holt, J.G. (eds), Bergey's Manual of Systematic Bacteriology, volume 3. Williams & Wilkins, Baltimore,pp. 1986-1989.

Figure 1

(a) Winogradsky's medium inoculated with S1. Left to right, uninoculated control; sterile control,inoculated with autoclaved S1; primary culture inoculated with Si; secondary culture, inoculated fromprimary culture.

(b) Winogradsky's medium inoculated with S2. Left to right, sterile control, inoculated with autoclaved S2;primary culture inoculated with S2; primary culture inoculated with a 1/10 dilution of S2; secondary culture,inoculated from S2-inoculated primary culture.

(c) Winogradsky's medium inoculated with GW1. Left to right, sterile control, inoculated with autoclavedGW1; primary culture inoculated with GW1; secondary culture, inoculated from primary culture.

iiiiiii

Figure 2

(a) heterotrophic iron agar spread with 1/10 diluted GW1.

(b) heterotrophic iron agar streaked with S1, 32 and GW1 (left to right).

Figure 3

(a) Rouf and Stokes agar, uninoculated (left) and streaked with 1/10 diluted S1 (right). The colour ofuninoculated heterotrophic iron agar was similar to that of uninoculated Rouf and Stokes agar.

(b) Four isolates from heterotrophic iron agar restreaked on iron agar (left) and trypticase soy agar (right).

Figure 4

(a) Modified Wolfe's medium, uninoculated (left) and inoculated with S1.

(b) a closeup of the S1 -inoculated medium, showing white cottony flecks typical of Gallionella colonies,slightly above the layer FeS-containing agar.

IIIIIIIIII

IIIIIIIII

I

Figure 5

(a) from S1-inoculated modified Wolfe's medium. A cell with a short stalk is visible in the centre of the field,but other cells are rods, spirilla, cocci. 800x.

(b) GW1 well water, dried, stained smear. Twisted stalks typical of Gallionella. Much of thelighter-coloured, amorphous material is blue-stained iron precipitate. The filament at the bottom left of thefield may be a bacterial sheath. 320x.

(c) part of the field in (b), 800x.

Figure 6

(a) from a slide immersed in GW1. Sheaths with associated iron deposition, and free rod-shaped cells.800x.

(b) from a slide immersed in GW1. A stalked cell is visible in the middle of the field. 800x.

(c) from a slide immersed in GW1. Clumps of cells and associated iron deposits on the right, and upperleft, and in the left-centre a single cell surrounded by accumulated iron, and 2 or 3 cells in a chain,surrounded by iron precipitate. 800x.

~

Figure 7

(a) from a primary GW1-amended Winogradsky's culture. Most cells in the field are rod-shaped, although afew cocci are visible. The cells were not iron-encrusted, but the lighter, background material was ironprecipitate, and the dark clumps contained cells and iron. 800x.

(b) from a slide immersed in GW1 plus Winogradsky' medium (1:1, v:v). The long, straight rods did notaccumulate iron, but amorphous iron precipitate was apparent, and is visible as the light-coloured diffusematerial throughout the field. The darker areas at the bottom are heavier deposits of iron. 800x.

(c) from a secondary S1-amended Winogradsky's,culture. The cells stained pink and were not intimatelyassociated with deposited iron. 800x.

(d) from a primary GW1-amended Winogradsky's culture. The cocci in the centre of the field accumulatediron, while the rods in the upper right did not, but were associated with amorphous iron precipitate. 800x.