Vol. 51, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1986, p. 163-1700099-2240/86/010163-08$02.00/0Copyright © 1986, American Society for Microbiology

Metabolism and Metal Binding by Surface-Colonizing Bacteria:Results of Microgradient Measurements

P. E. KEPKAY,1* P. SCHWINGHAMER,' T. WILLAR,1 AND A. J. BOWEN2Department of Fisheries & Oceans, Marine Ecology Laboratory, Bedford Institute of Oceanography, Dartmouth,Nova Scotia, Canada B2 Y 4A2,' and Department of Oceanography, Dalhousie University, Halifax, Nova Scotia,

Canada B3H 4A22

Received 22 July 1985/Accepted 30 September 1985

Short-term (65-h) bacterial colonization of 0.2-,um (pore size) filters submerged in water from LakeCharlotte, Nova Scotia, was characterized by a well-defined succession of cell types, in which small cocci gave

way to larger, rod-shaped cells. This succession agrees with the concept of attachment as a strategy for survival,in which inactive cocci can attach to a surface and grow into larger, rod-shaped cells by using endogenousnutrients and the nutrients accumulated at the solid-liquid interface. Analyses of oxygen and CO2microgradients above colonized surfaces indicated that a peak of respiration accompanied the succession ofrods from cocci. CO2 fixation then became apparent as the rods began to bind manganese and iron to theirsurfaces. This means that survival by attachment may not be just the province of heterotrophs. It could alsobe a strategy adopted by metal-oxidizing chemoautotrophs. Long-term (34-day) colonization of similar filtersindicated that, while a succession of attached cell types may indeed be a natural occurrence, other factors (suchas the selective grazing of larger cells) tend to obscure the development of this succession.

While manganese-oxidizing bacteria can often determinethe fate of metals in a natural environment, the same bacteriacannot be directly associated with the formation of fer-romanganese concretions. Since the first isolates ofBeijerinck (4), many authors have shown that pure culturescan oxidize manganese in the laboratory (summary in refer-ence 19), but it is still not clear whether cultures can harnessenergy from oxidation. Despite these limitations, Emersonet al. (9, 10) and Kepkay (17, 18) have found that manganeseoxidizers can be an important natural catalyst of oxidationand metal binding outside the laboratory.To produce a concretion, however, bacteria must first

attach to the surface of a nucleus (12), and the ecologicalimportance of this attachment may lie in its value as astrategy for survival. Dawson et al. (8) and Kjelleberg et al.(24) have isolated bacteria which are a specific type ofcopiotroph. They need relatively high nutrient concentra-tions to grow and are also capable of attaching to surfaces.When nutrients decrease, their cells fragment with no in-crease in biomass (27-29), and the small cocci produced byfragmentation are capable of surviving for long periods (28).In some cases, the cocci can attach to a surface and grow byusing endogenous nutrients (15, 16) and the nutrients accu-mulated at the solid-liquid interface (24, 25).

If these copiotrophs are to both survive and flourish afterattachment, their metabolism must increase as the small,starved cells begin to grow. There is no evidence fromnatural environments to support this contention, but thetools to collect the evidence have recently become available.Very small (micro) electrodes have been developed to mea-sure dissolved oxygen (32, 33), and larger (mini) electrodesare commercially available to measure dissolved CO2. Theseelectrodes make it possible to measure the microgradients ofoxygen and CO2 produced by respiration and CO2 fixation ata surface. We report here a technique which allows themetabolic rate of attached bacteria to be expressed in termsof microgradients. It was applied to the case of surface

* Corresponding author.

colonization in water from an area rich in ferromanganeseconcretions, where bacteria are involved in the accumula-tion of manganese and iron (17).

MATERIALS AND METHODS

Sampling site. All experiments were carried out in a regionof Lake Charlotte, Nova Scotia, referred to as "ConcretionCove" by Kindle (22). The location chosen for in situexperiments and for the collection of lake water correspondsto site S of Kepkay (17). At this site, a hard, sandy bottomunder 1 m of water is covered with disklike ferromanganeseconcretions 5 to 15 cm in diameter. The distribution,composition, and structure of the concretions have beendescribed in detail by Kindle (21, 22), Beals (3), and Harrissand Troup (12). The water chemistry of Lake Charlotte istypical of many Nova Scotian brown-water lakes (30), withhigh concentrations of dissolved metals associated withhumic and fulvic acids. These metals and organic acids areleached into the lake from adjacent bogs and streams (22),forming organic metal complexes which give the lake waterits characteristic brown color. While humic and fulvic acidsare responsible for the relatively large amounts of dissolvedorganic carbon in the lake water (17), their refractory naturemeans that the lake is oligotrophic (30), with most of thecarbon remaining unavailable to organisms. Water tempera-ture varied between 17 and 22°C, and pH was 6.9 to 7.1 forthe duration of the experiments.

General experimental procedure. Two types of experimentwere conducted with water from site S as a source ofbacteria for surface colonization. Sterile surfaces were ini-tially deployed in situ for different lengths of time. Thesurfaces, which were Nuclepore membrane filters 2.5 cm indiameter with a 0.2-,um pore size, were stretched over andattached to the plastic snap-caps of 20-ml glass vials. Toensure that the filters were stored in contact with a sterileliquid, the vials were prefilled with bottom water that hadbeen filter sterilized through a 0.22-,um (pore size) membranefilter (Millipore Corp.) and autoclaved at 120°C for 15 min.

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164 KEPKAY ET AL.

Fourteen of the sterile vials were attached by their caps to anenamel tray which was 60 by 30 cm and had legs 5 cm long.The tray, complete with caps, filters, and vials, was thenplaced by hand at site S so that the filter surfaces were 3.5cm above bottom. At each sampling time during a 34-dayexperiment, two vials were removed from their respectivecaps, exposing the filter surfaces to lake water. The endresult of seven duplicate exposures was a series of surfacesthat had been subject to colonization for 1 to 34 days.A second type of experiment was conducted in the labo-

ratory, where a flow-through system was used to inducecolonization of filter surfaces in the absence of light and overmuch shorter periods. Lake water was collected from site Sby immersing a 20-liter acid-washed polypropylene carboy atapproximately 10 cm above the bottom. The carboy of waterwas kept well aerated and at 20°C in the laboratory to act asa reservoir for five glass surface colonization chambers.Each 90-ml chamber was 10 cm high, had a 6-cm insidediameter, and was fitted with a 65-mm, 0.2-,m membrane(Nuclepore) on a solid, ground-glass base to act as a sub-stratum for colonization. All of the chambers wereautoclaved at 120°C for 15 min with filters installed and wereconnected by silicone rubber tubing to a peristaltic pump andthe reservoir. The pump ensured a slow and constant flow ofwater over the filter in each closed chamber. The averageflow rate (12.7 + 1.3 ml h-1) was calculated from theoverflow of each chamber during a given period.The oxygen and carbon dioxide gradients above five

separate colonized surfaces were measured at predeter-mined intervals over 65 h. Time intervals were chosen sothat the filter surface in each succeeding chamber wasexposed to colonization for a longer period than the filtersampled previously. The water in each chamber was sam-pled for dissolved metals and bacterial counts before gradi-ents were measured. The chamber was then drained andcarefully refilled with lake water that have been filter steril-ized through a 0.22-,um Millipore filter so that the bacteriaattached to the surface remained undisturbed. The presenceof stationary, bacteria-free water above the surface ensuredthat gradients were caused only by activity on the surfacerather than by additional bacteria in the water, and pilotstudies were used to determine the rate at which filter-sterilized water could be introduced above a filter withoutdisturbing attached bacteria. A disk, which was 2.5 cm indiameter, was removed from the center of the filter immedi-ately after the gradients for bacterial counts on the colonizedsurface.

Cell counts and frequency of dividing cells. Total bacteria,the number of rods and cocci which made up the total, andeucaryote cells were counted in water samples and onsurfaces as described by Hobbie et al. (14). Three 1-mlsamples of water from each chamber were filtered through1.3-cm, 0.2-pum membranes (Nuclepore) and acidified with50 ,ul of 1.0 N HCl to be stored at 5°C in acid-washed vialsfor metal analysis. The remainder of each 5-ml water samplewas filtered through 2.5-cm, 0.2-p,m membranes (Nuclepore)which had been prestained with 2% (wt/vol) Irgalan blackdissolved in 2% (vol/vol) acetic acid. The cells collected oneach filter were stained in 1.5 ml of 0.01% (wt/vol) acridineorange (Becton Dickinson and Co.; no. 4940) for 2 min,destained for 30 s in 0.5 ml of 90% ethanol followed by anequal volume of deionized distilled water, and mounted inimmersion oil for counting by epifluorescence microscopy.The ethanol, distilled water, and solution of acridine orangewere filtered through 0.2-,um membranes (Nuclepore) beforeuse. The only modification which was necessary for counting

the colonized filters was the omission of Irgalan blackprestaining.

Bacterial counts were performed with a Zeiss ICM 405microscope equipped with an HBO 50 mercury vapor lamp,a sharp-cutoff (450 to 490 nm) excitation filter, a longwavepass barrier filter (520 nm), and a Zeiss 10Ox Neofluar oilimmersion objective. At least 400 cells were counted todetermine bacterial numbers and the number of dividingcells to ± 10% at the 95% confidence level (6). If less than 30dividing cells were present on each field, a further 20 fieldswere counted, as recommended by Hagstrom et al. (11). Thesmaller number of eucaryote cells, which were primarilymicroflagellates (probably cryptomonads), required thecounting of at least 200 fields with a 40x Neofluar objective.The average volumes of microflagellate and bacterial cellswere also determined from the measurement of at least 100cells from each sample, and the frequency of dividing cellswas calculated by dividing the number of bacteria undergo-ing cell division by the total number of bacteria (11).

Dissolved metals. Triplicate 20-pul water samples wereanalyzed by direct injection for dissolved manganese andiron on a Varian 975 flameless atomic absorption unitequipped with an autosampler and a pyrolytic graphitefurnace. The high concentrations of the two dissolved metalsmade extraction procedures unnecessary, and water sampleshad to be diluted 50 to 150 times before analysis. Distilled,deionized water (Millipore super Q) was used for blanks andas the diluent for both samples and standards (made up fromFisher atomic absorption stocks). All glassware and thepolypropylene vials used for storage and analysis werepresoaked in 6 N HCl (Aristar) and rinsed in distilled,deionized water. The detection limit for the injection ofdiluted Mn or Fe samples was 0.1 FM and the standarddeviations between triplicate analyses were all less than2.7% of the mean.

Metals on surfaces. When filters exposed to colonization atsite S for 1 to 34 days were prepared for bacterial counts,small sections of each filter (about 1 cm2) were mounted onscanning electron microscope stubs and carbon coated. Thecoated filters were viewed at either x30,000 or x100,000magnification in a scanning electron microscope (CambridgeStereoscan 180) equipped with an ORTEC EEDS-II energy-dispersive electron microprobe. The probe allowed circularareas with diameters of about 1 ,um to be scanned for themanganese and iron accumulated on a surface. The dimen-sions of many of the bacterial cells (see Fig. 5) were smallerthat the areas scanned, and background correction had to beapplied to the electron energy spectra received from individ-ual cells by scanning adjacent attachment surfaces for man-ganese and iron. In addition, the ability of the probe to detectthe electron energy spectra of a large number of metals wasnot accompanied by the ability to measure an absoluteamount of metal per unit of surface area. The magnitude ofa spectral peak for a particular metal was not only dependenton the amount of metal present but was governed by othervariables, such as the orientation of the area scanned toincident electrons. This means that only a relative idea of theamount of metals on a surface could be obtained for ele-ments with adjacent spectra (such as manganese and iron). Atotal of 306 cells from filters colonized for 1 to 34 days wereexamined to estimate the percentage which had detectablequantities of the two metals on their surfaces.Oxygen consumption and carbon dioxide production. When

oxygen is consumed by the surface population, a gradient ofdissolved oxygen concentration is created in the stationary,bacteria-free water above the surface. The degree to which

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METALS AND MICROGRADIENTS 165

this gradient extends upward into the water is controlled bythe rate at which oxygen travels downward by moleculardiffusion. The gradient is therefore a balance between thediffusive supply of oxygen and the consumption producingthe gradient. When this balance is defined quantitatively, atheoretical gradient can be generated to compare with mea-surements. A rate of oxygen consumption by the communitycan then be calculated from the balance and used, in turn, togenerate a theoretical carbon dioxide gradient for compari-son with measurements.

Gradient measurement. Two electrodes were used to mea-sure the gradients of dissolved oxygen and CO2 above eachof five colonized surfaces. Oxygen was measured with apolarographic oxygen sensor manufactured by Micro-electrodes Inc. (type MI 730). The sensor has been designedas a combination electrode with a Pt cathode and Ag-AgClanode in an electrolyte solution. The cathode is situatedabout 20,um behind a teflon membrane which allows oxygento diffuse into the electrolyte. The size and response of thiscombination electrode are similar to those of thenoncombination minielectrode of Helder and Bakker (13). Itis 0.7 mm in diameter and has a 90% response time to achange in oxygen concentration of between 25 and 30 s. Theoutput current in response to a stabilized polarization volt-age of -0.75 V is approximately 2,500 pA in air-saturated,filter-sterilized lake water. Electrode drift is less than 0.5%min-'. Current was measured on a picoammeter (Keithly;model 485), and double-shielded coaxial cables completedthe circuit between cathode, picoammeter, polarization volt-age, and anode. Dissolved CO2 was measured with a carbondioxide minielectrode (Microelectrodes Inc.; type MI 720).This is a combination pH electrode which is situated about20,um behind a Teflon membrane and is immersed in anNaCl-NaHCO3 buffer. Carbon dioxide is free to diffusethrough the membrane and into the buffer solution. Thediameter of the electrode is 0.7 mm, and the 90% responsetime is typically between 25 and 30 s. Electrode drift is lessthan 1% min-1. The change in potential produced by anincrease of CO2 in the buffer solution was measured inmillivolts on a pH meter (Metrohm; model 605) and double-shielded coaxial cable connected the electrode to the pHmeter.The recommendations of Revsbech (32) and Reimers et al.

(31) were followed during the calibration of the oxygenelectrode in filter-sterilized lake water. Electrode responsewas linear as the water was bubbled first with nitrogen, thenwith air, and finally with oxygen. When nitrogen wasreflushed through the water, the output of the electrodereturned to a background current of 200 pA. When the waterwas bubbled with air, a 5-ml sample was taken for themeasurement of dissolved oxygen by a micro-Winklermethod described by Kepkay and Novitsky (20). This al-lowed the background-corrected output current to be ex-pressed as oxygen concentration. The CO2 electrode wascalibrated in filter-sterilized lake water which was flushedwith air, followed by a calibrated gas mixture (Matheson) of0.13% CO2 in nitrogen, and finally by a similar gas mixture of0.53% of CO2 in nitrogen. Electrode response was linearwithin this rangeof CO2 concentrations but became far lesssensitive when CO2 was increased. Dissolved CO2, or theCO2(aq) of Stumm and Morgan (35), was calculated on thebasis that the air flushed through the water contained CO2 at320 ppm and that the Henry's Law constant (Kh) for CO2dissolution in fresh water is 3.388 x 10-2 mol liter-' atm-1 at200C.As soon as the calibrations were completed, the electrodes

00x

0

0i.E

t-

0

x

z

JillJ00 - 0 - -.- -2-00 10 20 30

TIME (h)40 50 60

FIG. 1. Short-term (65-h), bacterial colonization of five filtersurfaces (-.---) exposed in the laboratory to a bacterial popula-tion of cocci ( ) in water from Concretion Cove in LakeCharlotte, Nova Scotia.

were mounted in a micromanipulator (Stoelting) so that thetips were in vertical alignment at a distance of 0.3 cm tip totip. They were then positioned in each surface colonizationchamber to determine oxygen and CO2 gradients, first at 40min and then at 2 h after the addition of filter-sterilized lakewater. The electrodes were lowered through the water untilthe tips just touched the surface, causing distortion of themembrane which was visible under a dissecting microscope.They were immediately retracted to 10 + 0.02 mm above thesurface and left in this position for 3 min. During this time,the outputs of the picoammeter and pH meter were moni-tored on a two-channel strip-chart recorder to ensure thatthe signals did not drift due to the acquisition of materialfrom the surface. The electrodes were then lowered to 5 mmabove the surface, and their outputs were noted when flattraces were recorded. Oxygen and CO2 measurements werethen taken at 10 or 11 horizons between 5 and 0.05 mm abovethe surface. The time taken for the outputs of both elec-trodes to reach apparent equilibrium or steady state (definedas flat traces) was usually between 25 and 35 s. This meantthat the measurement of a pair of gradients required between5 and 7 min. When the gradients were completed, theelectrodes were immediately returned from 0.05 to 10 mmabove the surface. At this point, the performance of theelectrodes was acceptable if their outputs differed by lessthan 5% from the outputs originally recorded at 10 mm (31).An additional test was applied, in which outputs at 10 mmhad to be within 5% of the values recorded during theprevious calibrations. A 5-ml water sample was taken at 10mm to complete the test and ensure that air saturation wasmaintained at this horizon throughout the measurements.

Gradient analysis. When the downward flux of oxygen inthe system is equal to the rate of oxygen consumption at asurface, the gradient, as a balance between flux and con-sumption, can be expressed in terms of Fick's second law

(1)

where C is the concentration of dissolved oxygen, z isincreasing distance above the surface (defined as z = 0), andD0x is the molecular diffusion coefficient for dissolved oxy-gen. Our experiments dictate that equation 1 must be subjectto the following boundary conditions: (i) C = C(oo) at t = 0;

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percentages of the cells colonizing each of five surfaces over theshort term (65 h).

(ii) C = C(oo) as z -- * for all times; and (iii) flux = -kC(O)at z = 0, t > 0. C(oo) is the oxygen concentration produced byair saturation, C(0) is the oxygen concentration at z = 0, andk is the rate constant for oxygen consumption at z = 0 (whichshould not change during the development of a particulargradient). The solution to equation 1 within these boundaryconditions can then be obtained by a Laplace transformtechnique (7) and is

C(t, z) Z

= erf + exp(Z) exp(T2) erfcT + -2 (2)Q00o) 2T 2T)

where T = kV\(tIDOx), Z = kz/Dox, erf(x) = 2/\/fxexp(-y2)dy, and erfc(x) = 1-erf(x). The oxygen concentrationat a given distance from the surface at any given time hasbeen expressed in equation 2 as a fraction of C(oo). Theaeration of the bacteria-free water added to each coloniza-tion chamber ensured that the oxygen concentration at 10mm remained at C(oo) or the concentration produced by airsaturation at 20°C.

Gradients of oxygen concentration were initially gener-ated from equation 2 with a Dox of 2.06 x 10-5 cm2 s-1 at20°C in fresh water (5), a time (t) for gradient development of40 min, and a rational approximation of erf(x) fromAbramowitz and Stegun (1). The rate constant (k) for oxygenconsumption at the surface (z = 0) was varied until thepredicted curve fit the measurements. The fitting procedurewas based on the assumption that k remained constantduring the development of the gradients over a time span of

40 min. This assumption was tested by fitting curves tomeasured gradients that were allowed to develop for a longerperiod (2 h). Gradients could not be determined over shortertimes, because 5 to 7 min was necessary to acquire acomplete set of measurements. The oxygen consumed perunit of colonized surface area was calculated by multiplyingthe best-fit k by the oxygen concentration at z = 0 on thebest-fit curve, and this was then used to generate a CO2gradient.

Gradients of carbon dioxide concentration are also gov-erned by equation 1 subject to boundary conditions (i)through (iii), but in this case C(0) represents the concentra-tion of dissolved CO2 at z = 0, and k is set equal and oppositeto the rate constant for oxygen consumption (so that it isnow -k). The solution to equation 1 can then be obtained bya Green's function technique and is

C aF(t',O) ( _Z2_)_ t'(3C(t, z) exp 7 dt' (3)

,rrVD,t t') 4Dc(t

0,

where Dc is the molecular diffusion coefficient for dissolvedCO2 and F is the input flux at z = 0 (which is equal andopposite to the rate of oxygen consumption). A proportion-ality factor (a) has also been included to account for thepossibility that a one-to-one relationship did not exist be-tween oxygen consumption and CO2 production. Gradientsof dissolved CO2 were generated for a time (t) of 40 min byintegrating equation 3 numerically, with time steps of 2 min,distance (z) steps of 0.1 mm, a Dc of 1.64 x 10-5 cm2 s-1 at20°C in fresh water (5), and t' as the dummy variable.The gradients produced by the numerical solution were

fitted to the CO2 measurements, and the factor a was used asa fitting parameter in cases in which CO2 production did notmatch oxygen consumption (i.e., when a did not equal 1).The bacterial counts from each surface allowed the con-sumption of 02 and the production of CO2 to be expressedper cell as the community developed. It was assumed that allcells contributed equally to the measured metabolic rates,irrespective of shape or size.

RESULTS

Short-term colonization and dissolved metals. The numberof bacterial cells attached to each surface increased (Fig. 1)during the short-term (65-h) experiment in the laboratory.Cell number decreased in the water above each surface atthe beginning of the experiment. This was followed by an

increase in cell number (Fig. 1) which leveled off after 20 h.Cells in the water remained as cocci throughout the experi-ment and were in excess of the cells found on surfaces by a

TABLE 1. Short-term bacterial colonization of sterile filters in water from site S in Lake Charlotte, Nova Scotia. Rate of oxygenconsumption and CO, production by the surface community are presented alongside the colonization parameters

Bacterial colonization 0, consumption CO, production Dissolved metal

Time of Flux orcolonization Cell no Cell Frequency consumption Consumption Rate Production Proport-

(h) (10' cells cm-2) carbon of dividing per cm2 (fmol cell- constant pk (frol cell ionality (nmol ml-') (nmol ml-')(,ug cm-) cells (%) (nmol cm - h'" (mm h- ) hf c' factor, ah - ')"

11.75 4.42 0.013 0 10.16 ± 1.48 22.99 ± 3.36 0.41 ± 0.06 22.99 ± 3.36 1.00 4.21 22.3916.92 3.25 0.018 0.91 10.13 ± 2.01 30.85 ± 6.17 0.40 ± 0.08 30.85 ± 6.17 1.00 4.19 20.3223.00 3.37 0.038 2.03 18.12 ± 1.99 53.12 ± 5.91 0.82 + 0.09 39.25 ± 5.91 0.73 2.94 13.5940.16 9.70 0.123 0.61 21.34 ± 2.11 22.00 ± 2.18 1.01 ± 0.10 16.28 ± 2.18 0.74 3.07 13.6661.67 14.00 0.161 0 22.64 ± 2.04 16.18 ± 1.46 1.11 ± 0.10 11.65 ± 1.46 0.72 2.37 12.52

'The errors were calculated from the errors involved in the determination of the rate constants (k) for oxygen consumption.

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METALS AND MICROGRADIENTS 167

OXYGEN ( % OF SATURATION )

0 12 24 36 48

CARBON DIOXIDE ( n moles ml-' )

FIG. 3. (A) Curves fitted to oxygen measurements taken aftergradients had developed for 40 min above surfaces colonized for11.75 and 61.67 h. The rate constants for oxygen consumption (k),which were used to fit curves to the measurements, were 0.41 + 0.06and 1.11 + 0.10 mm h-' (Table 1). The error envelope around eachbest-fit curve is delineated by a shaded region. (B) Curves fitted tooxygen measurements above a filter surface colonized for 11.75 h.

T c0f-

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-

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0 00N0

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TIME (h)

FIG. 4. Oxygen consumption per cm2 ( ), oxygen con-sumption per cell (.), and the frequency of dividing cells

.) at each of five surfaces colonized over the short term (65 h).

factor of 1.6 to 10.8. Cocci were the predominant type of cellon the surfaces between 0 and 16.9 h (Fig. 2); larger,rod-shaped cells then became dominant by 23 h. Dissolvedmanganese decreased by 1.25 nmol ml-', and dissolved irondecreased by 6.73 nmol ml-1 as the rods became predomi-nant between 16.9 and 23 h (Table 1).The number of microflagellates in the water or on a surface

remained at less than 2.05 x 102 cells cm-2 or cells ml-' anddid not increase throughout the short-term experiment. Inaddition, the microflagellates accounted for only 0.02 to0.05% of the surface population compared with the bacterialnumbers in Table 1. Even when cell numbers were con-verted to cell carbon, bacteria were still by far the mostcommon component of the surface population. Assumingthat wet-cell density was 1.1 g ml-1 and the ratios of wetweight to dry weight and dry weight to cell carbon were 0.23and 0.50, respectively (26), an average coccoidal cell with adiameter of 0.75 p.m and a volume of 0.22 ,um3 contained 2.80x 10-8 ,ug of C cell-'. With the same conversion factors, anaverage rod-shaped cell 0.78 p.m in diameter and 2.62 p.mlong had a volume of 1.25 p.m3 and contained 1.58 x 10-7 p.gof C cell-'. The volume of an average large cell (32.1 ,um3)was equivalent to 4.06 x 10-6 pg of C cell-' when the sameconversion factors were used again, and the maximumnumber of large cells on a surface was therefore equivalentto 8.32 x 10-4 p.g of C cm-2. This was 15.6 to 193.5 timesless than the bacterial carbon per cm2 which was calculatedfrom appropriate conversion factors and the cell numbers inTable 1.

Dissolved oxygen was measured after gradients had developed for 40min and 2 h. Curves were fitted to the measurements with a rateconstant for oxygen consumption (k) of 0.41 0.06 mm h-i (Table1). The error envelope around each best-fit curve is delineated by ashaded region. (C) Curves fitted to CO2 measurements taken aftergradients had developed for 40 min above surfaces colonized for11.75 and 61.67 h. At 11.75 h, a value for CO2 production of 10.16 +1.48 nmol cm-2 h-' and a proportionality factor (a) of 1.00 were usedto fit the curve to the measurements. At 61.67 h, a value for CO2production of 22.64 + 2.04 nmol cm2 h-' and an aL of0.72 were usedto fit the curve to the measurements. The gradient produced whenCO2 production remained at 22.64 nmol cm-2 h-', but with a = 1.00,is shown as a broken line for reference. The error envelope (shadedregion) around each best-fit curve (Table 1) was ultimately derivedfrom the error apparent in the oxygen consumption rate used toproduce the curve.

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Metabolism on surfaces. At no time during the measure-ment of gradients was the oxygen concentration at a surfacebelow 70% saturation. Gradients were also in good agree-ment with oxygen measurements taken after 40 min or 2 hwhen the same rate constant for consumption (k) was used asa fitting parameter (Fig. 3A). These two observations indi-cate that cells on the surfaces were not oxygen limited, andthe rate of oxygen consumption was constant over at leastthe time periods chosen. An error envelope was establishedwhen each theoretical gradient was fitted to the measure-ments. The envelope was chosen to be symmetrical aroundeach best-fit curve and encompassed all of the measureddata. The magnitudes of the errors involved in the determi-nation of rate constants for oxygen consumption are re-corded in Table 1.The uniformity of oxygen consumption during the mea-

L.....I surement of any one set of gradients was not maintained5 (Fig. 3B; Table 1) as the number of bacteria increased on the

surfaces (Fig. 1). The rate constants for consumption in-creased from 0.41 to 1.11 mm h-1 as the number of bacteriaincreased from 4.4 x 105 to 14.0 x 105 cells cm-2. Theutilization of oxygen at the surface is expressed in Fig. 4 asboth consumption per cm2 and consumption per cell. Nor-malization of the data resulted in a peak of consumption percell at 23 h, which was accompanied by a peak in thefrequency of dividing cells (Fig. 4; Table 1).When oxygen consumption was used to determine CO2

production, the gradients predicted for surfaces colonizedbefore 23 h were in good agreement with CO2 measurements(Table 1; Fig. 3C). This means that when cocci were pre-dominant (Fig. 2), the consumption of oxygen was matchedby the production of C02, and the proportionality factor (a)was 1.00. In contrast, when the rods became dominant at 23h (Fig. 2), a had to be decreased to fit a gradient to themeasurements (Fig. 3C), and an additional process appearedto be in operation, reducing CO2 production by as much as28% when a was equal to 0.72 (Table 1).Long-term colonization and metals on bacterial surfaces.

The results from long-term colonization in the field for 34days were very different from the short-term results. After 1day of colonization, there were 3.1 x 105 bacterial cellscm-2, and from 1 to 34 days there was no further increase incell number on the filter surfaces. This means that between1.4 and 4.5 times fewer cells were apparent on surfacescolonized for the long term when these results were com-pared with the short-term data in Table 1. In addition, thefrequency of dividing cells remained at 2.75% from 1 to 34days-near the maximum frequency of dividing cells foundover the short term (Fig. 4)-and there was no clear predom-inance of either rods or cocci (with 46% rods and 54% cocciat 34 days). The rods were not only capable of attaching to afilter surface over the long term but could also attach to thesurface of diatom frustules on the filter (Fig. 5A). Bacterialcells appeared to accumulate significant quantities of man-ganese and iron (Fig. 5B), but it was also found that diatomfrustules could accumulate the two metals (Fig. 5C). Thishad to be subtracted from the total spectrum to give an

8.8KeV

FIG. 5. (A) Scanning electron micrograph of rod-shaped bacte-rial cells attached to the surface of a pennate diatom after 22 days ofcolonization. Two circular areas (approximately 1 p.m in diameter)which were scanned by the ORTEC EEDS-11 microprobe are

indicated by arrows. One of the areas included the surface of a

bacterial cell, and the other was located only on the surface of the

diatom frustule. (B) Total manganese and iron (in counts of electronsreturned per second) detected by the microprobe on the area

including the bacterial surface. (C) Manganese and iron detected bythe microprobe on the surface of the diatom. (D) Corrected spectrumfrom the bacterial surface after the signal from the diatom frustulewas subtracted from the total spectrum by the ORTEC EEDS-11computer system.

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METALS AND MICROGRADIENTS 169

accurate indication of the metals accumulated by any onebacterial cell (Fig. 5D). When 306 bacteria were scanned forthe presence of manganese and iron, 17.6% of the cells hadaccumulated detectable amounts of Mn, and 21.1% hadaccumulated detectable amounts of Fe. When a two-samplet test was applied at a 95% confidence level, there was nosignificant change in these percentages from 1 to 34 days.

DISCUSSION

A well-defined succession of bacterial cell types wasapparent on filters exposed to short-term colonization. Smallcocci were the first to attach and were followed by thedevelopment of larger, rod-shaped cells (Fig. 2). Given thatcocci were the only type of cell found in the water supplyingthe bacteria for colonization (Fig. 1), it is likely that rodsdeveloped from cocci on the surfaces. This is supported bythe first predominance of the rods at 23 h (Fig. 1 and 2) inassociation with maxima for growth and cell division, asindicated by the frequency of dividing cells (Fig. 4).

Since the cocci are capable of dividing and growing toform large rods, it can then be argued that these bacteria arenaturally occurring copiotrophs who can survive and flourishby attachment to surfaces. The average cell volume of thecocci (0.22 ,um3) was small-near the low end of the sizerange proposed by Kjelleberg et al. (24)-and could meanthat they had survived in the water column for long periods(23). The average volume of a rod (1.25 ,um3) was 5.7 timesthe volume of an average coccoidal cell (0.22 p.m3), which iswell within the range of size differentials established byNovitsky and Morita (27), Dawson et al. (8), and Kjelleberget al. (24, 25). The rapid colonization of surfaces by the cocciand the development of rods within a day are also notunusual when compared with the results obtained in thelaboratory by Novitsky and Morita (28), Kjelleberg et al.(24), and Amy et al. (2). It must be stressed, however, thatour simple picture of short-term colonization is not main-tained over the long term. Compared with the results fromshort-term colonization in the laboratory, 1.4 to 4.5 timesfewer cells were found on filters colonized in the field for 34days. This could have been the result of predation byprotozoans observed on the filters (P. Schwinghamer andP. E. Kepkay, unpublished data), or some other factorwhich inhibited bacterial colonization and was accompaniedby a relatively high frequency of dividing cells (2.75%) andno discernable dominance of either rods of cocci. Together,our laboratory and field observations suggest that, whilecopiotrophy associated with attachment can exist in nature,selective grazing of larger bacterial cells may prevent thedevelopment of a recognizable succession of cell types.The consumption of oxygen at each surface appears to

reflect the short-term development of a bacterial population.When the rods became dominant at 23 h, the oxygenconsumed per cell reached a maximum and was accompa-nied by a peak in the frequency of dividing cells (Fig. 4). Itthus appears that a specific bacterial population has activelyadapted to life on a surface. This agrees with Kjelleberg's(23) and Humphrey and Marshall's (16) contention thatcopiotrophic survival is not just a passive process. Instead,energy must be expended to colonize a surface. Whether thisenergy was obtained primarily from nutrients on the surfaceor from endogenous nutrients within the bacteria was notdetermined in our experiments.

Respiration may not have been the only process producingenergy for colonization and growth, although it seemed to be

dominant during the first 17 h, when the consumption ofoxygen was matched by the production of CO2 (Table 1; Fig.3C). The production of CO2 was lower than oxygen con-

sumption by as much as 28% from 23 to 62 h (Table 1; Fig.3C). This decrease in production was presumably due to theconsumption of CO2 and was coincident with both theenhanced removal of manganese and iron from solution(Table 1) and the predominance of rods rather than cocci(Fig. 2). The consumption of CO2 could be the result ofaerobic heterotrophy giving way, at least in part, tochemoautotrophy as nutrients run low on a surface and cellsturn to manganese oxidation as a source of energy (19).While speculative, this conclusion agrees with data fromsediments in the same area (17, 18), but our experiments donot rule out the possibility that anaplerotic CO2 fixation or a

change in heterotrophic metabolism was responsible for theconsumption of CO2.

Results of scanning electron microscopy (Fig. 5) alsoindicate that 17.6% of a bacterial population from site S canaccumulate detectable quantities of manganese. These dataprovide no information on the oxidation state of the metaland certainly cannot be used to demonstrate that energy wasgenerated for growth. Nevertheless, a first attempt can bemade at establishing a budget for binding and cell growth byapplying a crude stoichiometry to the relationship betweenmanganese uptake from solution and bacterial cell carbon onthe surfaces. Dissolved manganese decreased by 1.25 nmolml-1 from 17 to 23 h and was accompanied by an increase of2.0 x 10-2 jig of cell C cm-2 of surface (Table 1). Ifmanganese oxidation generates 18.7 kcal (78,240.8 J) ofMn2+ oxidized mol-1 (35), and there are 7.3 kcal (30,543.2 J)of ATP mol-1 and 7.9 mol of ATP mol-1 of carbon (34), a

decrease of 1.25 nmol of Mn2+ ml-' would be equivalent toan increase of S x 10-3 ,g of cell C cm-2 if all the manganesewas oxidized. This is 25% of the observed increase in cellcarbon and agrees with the results from CO2 gradients (Table1), in which 26 to 28% of the CO2 produced by respirationwas consumed (resulting in proportionality factors of be-tween 0.72 and 0.74). Together, these data suggest thatmanganese oxidation can only supplement the energy pro-vided by respiration. In addition, it is still not obvious why a

bacterial population would just survive in the water columnand oxidize manganese only when attached to a surface.However, since surfaces in Lake Charlotte are excellentscavengers of metals by adsorption (17) and because metalscomplexed with organic compounds often remain unavail-able in solution (30), it makes sense that these bacteria wouldbind metals after attachment.Two conclusions can be drawn from our examination of

surface colonization. Oxygen consumption by a microscopicsurface community can now be determined without nutrientenrichment and under the most natural conditions possible.More important, the type of metabolism can be monitoredduring colonization. When our results are considered alongwith the possibility that cells may obtain energy from man-

ganese oxidation on a surface, attachment becomes more

than just the province of heterotrophs. It could also be a

strategy adopted by metal-oxidizing bacteria and even in-clude a fundamental change from aerobic heterotrophy to

chemoautotrophy as the surface community matures.

ACKNOWLEDGMENTSWe thank D. E. Willis for developing the computer programs to

solve the microgradient equations. We also thank J. A. Novitsky,B. T. Hargrave, B. P. Boudreau, and two anonymous reviewers fortheir many useful criticisms of the manuscript.

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170 KEPKAY ET AL.

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