Microenvironments of pH in biofilms grown on dissolving silicate surfaces

Chemical Geology 171 Ž2000. 1–16 www.elsevier.comrlocaterchemgeo

Microenvironments of pH in biofilms grown on dissolving silicate surfaces Laura J. Liermann a,) , Amy S. Barnes b, Birgitta E. Kalinowski a , Xiangyang Zhou c , Susan L. Brantley a a

Department of Geosciences, The PennsylÕania State UniÕersity, UniÕersity Park, PA 16802, USA Department of Materials Science and Engineering, The PennsylÕania State UniÕersity, UniÕersity Park, PA 16802, USA Department of Energy and GeoenÕironmental Engineering, The PennsylÕania State UniÕersity, UniÕersity Park, PA 16802, USA b

c

Received 3 March 1999; accepted 3 February 2000

Abstract Differences in pH between silicate–biofilm interfaces and bulk medium Ž D pH s pH interface y pH bulk . were detectable with commercial microelectrodes in cultures grown in unbuffered medium Ž< D pH < s 0.27–1.08. for an arthrobacter species, but were generally beneath detection Ž D pH - 0.04. for a streptomyces species. Biofilm half-thicknesses developed by Arthrobacter ranged from 1.2 to 11.5 mm, and were highly variable even for replicates. In buffered medium, neither bacterium produced a measurable D pH across the biofilms grown on silicates. The silicates consisted of polished hornblende, synthetic Fe-rich glass similar to hornblende in bulk composition, and two commercially available Afloat glasses,B one low-Fe and one high-Fe. The two species of soil bacteria investigated are both known to accelerate release of Fe from hornblende. For the Arthrobacter, values of < D pH < developed on hornblende crystal or glass substrates were generally larger than those developed on either float glass. Differences in D pH developed on different substrates could not be related simply to relative rates of dissolution of substrates. Differences between the two bacterial species are probably related to differences in Ž1. rates of growth, Ž2. production of low-molecular-weight organic acids, Ž3. physical characteristics of polysaccharide slimes excreted andror Ž4. production of siderophores. Although values of < D pH < developed at mineral–water interfaces in natural systems may not be as large as those measured here except for water-saturated systems, it is probable that significant values of D pH ŽF pH unit. develop where fast-growing, acid-producing microbes colonize slow-dissolving phases in the presence of unbuffered solutions. q 2000 Elsevier Science B.V. All rights reserved.

1. Introduction Microbial weathering of mineral surfaces associated with decreases in pH by production of organic acids is well documented Že.g. Duff et al., 1963; Keil )

Corresponding author. Fax: q1-814-863-8724. E-mail address: [email protected] ŽL.J. Liermann..

and Schwartz, 1980; Barman et al., 1992; Bosecker, 1993; Welch and Ullman, 1993; Barker et al., 1997.. However, most pH changes in laboratory experiments have been detected in the bulk fluid of the culture medium. If a growing culture is agitated, this may represent an average pH change for the culture as a whole. In static natural systems, it is likely that a pH gradient exists because of metabolic products

0009-2541r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 5 4 1 Ž 0 0 . 0 0 2 0 2 - 3

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L.J. Liermann et al.r Chemical Geology 171 (2000) 1–16

and biofilm materials excreted into the medium surrounding the bacterial cells, and this gradient should be preserved in an undisturbed culture. Moreover, the effects of these chemical microenvironments may be important in understanding bulk observations of natural systems Že.g. Hochella and Banfield, 1995; Nugent et al., 1998.. We have conducted a study on pH gradients in microbial biofilm–mineral cultures in buffered and unbuffered medium using two different bacteria and four different silicate surfaces: hornblende, hornblende glass, and high-Fe and low-Fe AfloatB glasses. The two bacteria are both known to accelerate release of Fe from a hornblende substrate. In these experiments, the effects of bacterial type and substrate reactivity were investigated with respect to biofilm environments. Our specific goals were to Ž1. determine whether pH microenvironments developed in biofilms on silicates could be measured using commercial microelectrodes, Ž2. measure the values of pH differences across biofilms as a function of bacterial species, Ž3. determine whether these microenvironments correlated with changes in surface chemistry or topography during biofilm growth, and Ž4. determine whether the solid substrate was important in the development of microenvironments of pH.

been conducted Žsee review by Little et al., 1991.. Techniques employed in these studies include use of microelectrodes for measuring dissolved oxygen ŽDO. and pH. Many of these microelectrodes are hand-made by specialists with tip radii as small as 10 mm. Changes in DO, and pH to a lesser extent, have been used to document microbial activity Že.g. Bungay et al., 1969; Revsbech and Jorgensen, 1986; Revsbech and Ward, 1988; Lewandowski et al., 1989, 1991; Wimpenny et al., 1993; Costerton et al., 1994; Villaverde and Fernandez, 1997. and photosynthesis ŽRevsbech and Ward, 1984; Revsbech et al., 1983; Jorgensen et al., 1983, 1985.. Pang and Zhang Ž1998. recently constructed a microelectrode to measure redox potential within vegetated contaminated soils. Indicator dyes have also been used to monitor pH microenvironments in microbial biofilms ŽCosterton et al., 1994; Barker et al., 1998., and in rhizosphere systems ŽRuiz and Arvieu, 1990.. However, microenvironments of pH have rarely been quantified as a function of distance from the mineral surface in weathering systems, and it was our intent to investigate several simple systems with commercially available electrodes.

1.1. Biofilm formation and effects

The bacterial species used in these studies were isolated from hornblende-containing soil from Gore Mountain in the Adirondacks, New York, USA ŽLiermann et al., 2000.. The 16S rRNA gene sequence of neither organism matched sequences in the Ribosomal Database Project database ŽMichigan State University., suggesting that these are new isolates. However, the two isolates were found to be a streptomycete of the genus Streptomyces, most closely related to S. liÕidans, and an arthrobacter, most likely of the genus Arthrobacter. The two species were isolated in Fe-limited growth experiments and were chosen because of their observed ability to mobilize Fe from a hornblende surface ŽKalinowski et al., 2000; Liermann et al., 2000.. The two microbes differ significantly in appearance and growth. Streptomycetes are fungus-like, growing by filamentous extensions, or hyphae, from the vegetative cell bodies, and sporulating within the hyphae when nutrients are depleted. The spores germinate into new cells when conditions for growth are

Biofilm formation on mineral surfaces is a multistep process influenced by many factors, including the specific mineralogy of the rock, solution chemistry Že.g. pH, ionic strength., and characteristics of the microorganisms Že.g. hydrophobicity, surface charge. Žsee reviews by Characklis, 1989; Banfield and Hamers, 1997; Little et al., 1997.. Biofilm formation involves the excretion by microbes of extracellular polymeric substances ŽEPS. containing various polysaccharides; the composition and chemical reactivity of the EPS depends on the microorganisms involved. Malinovskaya et al. Ž1990. reported that acid polysaccharide bacterial slimes promoted silicate solubilization, while studies by Welch and Vandevivere Ž1994. showed that microbial EPS both increased and inhibited dissolution of feldspars under different conditions. Numerous studies on the corrosive effects of biofilms on solid surfaces, especially metals, have

1.2. Soil bacteria isolates

L.J. Liermann et al.r Chemical Geology 171 (2000) 1–16

favorable. Because of the hyphal growth, collectively termed mycelia, colonies of streptomycetes appear as interwoven filamentous mats. The Arthrobacter sp. exhibits more typical bacterial growth, reproducing by binary fission of individual cells, resulting in chains of cells as well as individual cells within a colony. As shown in previous studies ŽKalinowski et al., 2000; Liermann et al., 2000., both of these bacteria attach to hornblende surfaces, extract Fe from hornblende, produce catecholate siderophores Žhigh-affinity FeŽIII. ligands., and produce lowmolecular-weight organic acids. Both species of bacteria formed biofilms on the surface of the silicate samples when incubated in culture medium.

2. Materials and methods 2.1. Preparation of mineral and glass samples Hornblende used in these studies was obtained from Gore Mountain, New York, USA. Hornblende samples were cut into slabs ; 2 mm thick; one

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surface of each slab was polished to 0.25 mm, with acetone cleaning of the slabs between each polishing stage. After polishing, ; 8-mm diameter discs were cored from the slabs and cleaned with ultrapure acetone. The discs were placed in a desiccator until use. Bulk composition of the hornblende, determined by X-ray fluorescence ŽXRF, XRAL Activation Services, Ann Arbor, MI. is reported in Table 1. Wet chemistry analysis of FeŽII. ŽXRAL Activation Services., was used with the XRF data for total Fe to calculate the FeŽII.rFeŽIII. ratio Žs 3.6.. Compositional data ŽTable 1. compares favorably with published results for Gore Mountain hornblende chosen from museum specimens Že.g. Brabander and Giletti, 1995.. However, electron microprobe ŽEM. analysis of polished hornblende reveals the presence of some secondary phases in our material that were not present in the museum specimens, including a sometimes pervasive phyllosilicate Žinferred to be chlorite., plus occasional biotite, and minor plagioclase and garnet. Composition of chlorite as determined semi-quantitatively by EM analysis is summarized ŽTable 1..

Table 1 Composition of hornblende, high-Fe, and low-Fe glassesa Oxide

Hornblendeb

Chlorite c

Hornblende glassd

Low-Fe float glassd

High-Fe float glassd

35.1 10.7 21.5 ND 0.3 18.7 0.04 0.01 ND ND ND ND 86.4 ND

44.4 20.0 11.4 9.6 8.9 2.9 2.4 0.5 0.1 0.1 0.01 ND 100.3 0.3 f

72.4 ND 4.0 NA 8.3 NA 13.4 NA NA NA NA ND 98.1 ND

71.9 0.1 3.5 1.2 8.2 0.4 13.8 0.1 NA NA 0.2 ND 99.4 ND

wt. % SiO 2 Al 2 O 3 MgO Fe 2 O 3 CaO FeO Na 2 O K 2O TiO 2 P2 O5 MnO H 2O Total wt. % FeŽII.rFeŽIII. a

44.3 16.2 12.2 11.1 8.9 ND 2.8 0.5 0.8 - 0.1 - 0.1 1.2 97.9 3.6 e

Abbreviations: NA — not applicable; ND — not determined. Determined by XRF Žoxide wt.% values.. Analyses were completed by XRAL analysis on two samples of hornblende from Gore Mountain, New York. c Semi-quantitative analytical chemical composition determined by EM Žone sample of chlorite.. d Determined by lithium metaborate fusion and ICP-AES in the Materials Characterization Laboratory, Penn State. e FeŽII.rFeŽIII. ratio determined from analysis of Fe Žtotal. from ICP-AES analysis and FeŽII. analysis from wet chemistry, as completed by XRAL analysis on two samples. f FeŽII.rFeŽIII. ratio determined in the Materials Characterization Laboratory, Penn State, as described in text Žglass. and by wet chemistry ŽXRAL analysis, hornblende crystal.. b

L.J. Liermann et al.r Chemical Geology 171 (2000) 1–16

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A glass of similar composition to hornblende was synthesized for study in recognition of problems related to secondary phases in the natural mineral. No attempt was made to match the FeŽII.rFeŽIII. ratio of the glass with the Gore Mountain crystal. The components ŽFeSO4 P 7H 2 O, CaCO 3 , Na 2 CO 3 , K 2 CO 3 . of the hornblende-like glass were melted in air at 13508C in a kyanite crucible, holding at temperature for 6 h before pouring into preheated graphite molds. The glass was annealed for 6 h at 5508C. The glass was sectioned into ; 8 mm = 8 mm squares, 2 mm thick. The samples were polished to 0.25 mm using SiC grit paper and then oil-based diamond slurries to avoid aqueous attack of the glass surface. The recipe and final composition of the hornblende glass, determined by the lithium metaborate fusion technique ŽSuhr and Ingamells, 1966; Medlin et al., 1969. followed by ICP-AES, is shown in Table 1. The iron valence of the glass was found

by the following method: the ferrous content was first determined by dissolving the sample and titrating the reduced iron from 2q to 3q with a potassium dichromate standard, with sodium diphenylamine sulfonate as the indicator ŽGoldich, 1984.. This information was used to calculate the ferric iron equivalent and subtracted from the total iron content determined instrumentally. A low-Fe AfloatB glass substrate was also used to determine whether biofilms would develop even without Fe in the solid. A float glass of similar composition, but containing Fe Žtermed high-Fe float glass in this paper., was also used for comparison with the low-Fe float glass. Both float glasses are commercially available alkali glasses ŽGlass, Inc., State College, PA.. The low-Fe glass contains only trace Al, Fe, or S; the compositions of both glasses are in Table 1. The surface was not polished as the manufactured surface is more pristine than can be

Table 2 Summary of pH changes in unbuffered hornblende and glass culturesa Sample

Initial pH b

Incub.c Ždays. Bulk pH

Interface pH

D pH d

Rep. a

Rep. a

D Le Žmm.

Rep. a

Rep. a

1

2

1

2

1

2

1

2

Rep. a 1

2

HB control HB q Stm HB q Arth

6.81 6.81 6.70

9 9 9

9 9 9

6.76 5.48 6.16

6.75 5.47 6.20

6.76 5.47 5.23

6.75 5.46 5.35

BD BD y0.93

BD BD y0.85

– – 11.5

– – 6.2

HB glass control HB glass q Stm HB glass q Arth

6.68 6.76 6.82

9 9 9

9 9 9

6.74 5.77 5.75

6.72 5.76 6.15

6.74 5.75 5.35

6.72 5.73 5.07

BD BD y0.40

BD BD y1.08

– – 1.2

– – 5.5

Low-Fe float glass control Low-Fe float glass q Stm Low-Fe float glass q Arth

6.67

9

9

6.77

6.77

6.77

6.77

BD

BD

–

–

6.78

9

9

5.89

6.10

5.87

6.04

BD

y0.07

–

2.0

6.78

9

9

5.62

5.62

5.29

5.35

y0.33

y0.27

4.5

8.0

High-Fe float glass control High-Fe float glass q Stm High-Fe float glass q Arth

6.75

9

9

6.73

6.70

6.73

6.70

BD

BD

–

–

6.75

9

9

5.79

5.86

5.78

5.84

BD

BD

–

–

6.67

9

9

5.80

5.70

5.15

5.19

y0.65

y0.51

1.8

8.8

a

Abbreviations: HB s hornblende; Stm s Streptomyces sp.; Arth s Arthrobacter sp.; BD s below detection Ž< D pH < - 0.042.. Initial pH s pH of medium at experimental set-up, before substrate Žand bacteria, where applicable. were added. c Incub.s time duration in which substrateq mediumq bacteria were incubated together before measurement. d D pH s pH interface y pH bulk . e D L s half-thickness of biofilm; distance from substrate surface such that pH s pH interface q <1r2D pH <. b

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Blanks of all silicate samples were prepared in an identical manner as described for samples but were not placed in media. Controls are samples prepared identically, but placed in medium without bacteria for incubation. 2.2. Culture media

Fig. 1. Diagrammatic representation of pH microelectrode instrumentation and mineralrbiofilm culture set-up. Not drawn to scale.

obtained by polishing. Since the glass was poured with no subsequent polishing, there were no polishing scratches or similar surface damage. The glass was sectioned into pieces the same size as the hornblende glass pieces Ž8 = 8 = 2 mm3 ..

The unbuffered medium used in these experiments contained 0.5% Žwrv. glucose, 0.5% Žwrv. casamino acids, 2 ml ly1 of a B-vitamin solution, and 10 ml ly1 of Wolfe’s Mineral Elixir ŽWolin et al., 1963., prepared without the chelating agent nitrilotriacetate. The buffered medium was a modified minimal medium, designated MM9 ŽManiatis et al., 1982; Schwyn and Neilands, 1987., with the following composition: 6.0 g ly1 Na 2 HPO4 , 0.3 g ly1 KH 2 PO4 , 0.5 g ly1 NaCl, 1.0 g ly1 NH 4 Cl, and 6.06 g ly1 Ž50 mM. TRIS base, pH 7.4. This solution was sterilized by autoclaving, allowed to cool to

Fig. 2. Measured pH vs. distance from surface for two replicate hornblende glass q Arthrobacter sp. experiments Žreplicates a1 and a2.. After each measurement equilibrated, the pH electrode was moved before the next measurement was made. For each replicate, measurements were made twice as shown. Note differences in pH measurements at similar distances but at different times, due to disturbance of the biofilm from the first measurement.

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Fig. 3. Measured D pH vs. distance from Ža. hornblende, Žb. hornblende glass, Žc. low-Fe float glass, and Žd. high-Fe float glass surfaces in unbuffered medium where x s distance from interface. Note that x s 0 indicates the solid–biofilm interface. Error Ž"0.042. is approximately the same size as symbols. Open symbols Žincluding dotted center. indicate replicate runs with bacteria as indicated and solid symbols indicate runs with medium only.

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Fig. 3 Ž continued ..

room temperature, and supplemented with 2% Žvrv. casamino acids ŽDifco Laboratories. from a 10% Žwrv. stock solution, 0.2% Žvrv. 1 M MgSO4 , 1%

Žvrv. filter-sterilized 20% Žwrv. glucose, and 0.01% Žvrv. 1 M CaCl 2 . These solutions were prepared and sterilized separately.

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2.3. Preparation, growth, and analysis of cultures Cultures in buffered medium were prepared by adding 10 ml of sterile medium to test tubes pre-sterilized with hornblende discs. These cultures were inoculated with either Streptomyces sp. or Arthrobacter sp. and incubated at ambient temperature for 4 weeks. Cultures in unbuffered medium were prepared by adding 50 ml of sterilized medium to 100 ml glass beakers, which had been previously autoclaved with hornblende discs or glass. The cultures were then inoculated with 0.5 ml of late log to stationary phase tube cultures of either Streptomyces sp. or Arthrobacter sp. Control experiments were not inoculated. All experiments were incubated undisturbed at room temperature for 9 days. For all experiments, two replicates were completed ŽTable 2.. The pHs of biofilms at the silicate–medium interface and in bulk solution were measured with a pH microelectrode ŽMicroelectrodes., illustrated in Fig. 1. The tip of the microelectrode is a hemispheric glass membrane with an outer diameter of 1.2 mm. An AgrAgCl reference electrode is built into the pH microelectrode. The microelectrode is moved with a manipulator that can move the electrode in three directions: vertical, leftrright, and forwardrbackward. The minimum step in the vertical direction is 0.005 mm. The microelectrode was connected to a Chemcadet pH meter with a precision of "0.01 pH unit and an input impedance of 10 12 V. Before the pH microelectrode was installed with the manipulator, it was calibrated using two standard buffer solutions. Buffers with pH 7.00 and 4.01 were used for the acidic and neutral samples, and buffers with pH 7.00 and 10.00 were used for the basic samples. After a measurement was completed, the pH microelectrode was put into the buffer solutions again to determine the drift of the electrode. It was found that the maximum drift was "0.03 pH unit. The error in values of D pH Žs pH interface y pH bulk . was therefore "0.042. In each experiment, the microelectrode was first installed to measure the pH of the bulk solution at a position 10–20 mm above the surface of the disc. The electrode tip was then placed on the surface of the disc, and moved up step by step in increments of 0.5–2.0 mm. Normally, the time between two consecutive movements was 5 min. If

the pH value fluctuated, the electrode was left for longer periods of time until the pH value remained stable. After pH measurements, aliquots of media were removed, filtered through 0.2 mm SFCA syringe filters ŽNalgene., and frozen until analysis for lowmolecular-weight organic acids by ion chromatography using an IonPac AG11 Ž4 = 50 mm2 . guard column and an ion-exchange IonPac AS11 Ž4 = 250 mm2 . column on a Dionex Series 4000I ŽDionex, Sunnyvale, CA.. An anion micromembrane ASRSUltra self-regenerating suppressor was used for suppression of conductivity. The mobile phase was 0.1 M NaOH solution run at a flow rate of 1 mlrmin. Data collection was completed at 30 minrsample. Organic analysis was completed only for low-Fe float glass and hornblende glass experiments. Control experiment solution samples were also analyzed by ICP-AES ŽMaterials Characterization Laboratory, The Pennsylvania State University. to compare dissolution of the different substrates. To analyze silicate surfaces under scanning electron microscopy ŽSEM., separately prepared streptomyces and arthrobacter cultures with two pieces each of hornblende, low-Fe float glass, and hornblende glass were incubated for 5–18 days, then autoclaved for 20 min to kill the bacteria. The solid substrates Žglass or crystal. were removed after autoclaving, rinsed with dI water and held in dI water for 5 days; they were then treated for 20 min with the enzyme lysozyme Ž0.15 mg mly1 ., which cleaves specific polysaccharide bonds in bacterial cell walls, to remove adherent bacteria. The samples were then cleaned for 20 min with ultrapure acetone and placed in a desiccator. One sample from each experiment was gold-coated and examined in a Philips LX-20 scanning electron microscope equipped with an energy dispersive spectrometer.

3. Results 3.1. pH gradients across biofilms In all experiments inoculated with bacteria, cloudy white biofilms on the substrate surface and the bottom of the beaker were visible within 3–4 days and

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grew until the cultures were terminated. The streptomyces biofilms were more clumpy and uneven than the more uniform biofilms of the arthrobacter. Buffered cultures were analyzed using a pH microelectrode to ascertain whether buffered medium would prevent pH changes at the microbe–mineral surface. The changes in pH are expressed as D pH s pH interface y pH bulk . After 4 weeks of growth, we found an insignificant pH change across the biofilm Ž< D pH < F 0.04. in buffered cultures containing either strain of bacteria. In these experiments, the microelectrode was moved up and down to measure both surface and bulk pH at least twice, with reproducible results. In most replicates with unbuffered medium inoculated with bacteria, both bulk and interface pH values were measured twice. Most measurements made at the same distance were within error for control and streptomyces cultures, but not always for arthrobacter cultures ŽFig. 2.. For comparison purposes, therefore, pH values and corresponding values for D pH between bulk and interface were determined based on initial bulk pH measurements, as these pH values represent the least-disturbed state. Analysis of pH in unbuffered cultures at various distances from the mineralrglass surfaces revealed pH changes across biofilms ŽTable 2, Fig. 3.. As stated above, D pH - 0 implies pH of biofilm- pH of bulk medium. D pH was beneath detection Ž0.04. for all controls, and for most cultures with streptomyces ŽTable 2, Fig. 3.. In the presence of the arthrobacter, D pH was reproducible Ž"0.042; Table 2, hornblende or low-Fe float glass, Fig. 3a and c., or generally not reproducible Žhornblende glass or high-Fe float glass, Table 2, Fig. 3b and d.. Some trends in < D pH < were reproducible however: < D pH < increased for Arthrobacter biofilms in the order low-Fe float glass - high-Fe float glass - samples of hornblende composition. It was also observed that, even where D pH was reproducible, pH interface and pH bulk were not consistently reproducible ŽTable 2.. The biofilm half-thickness was defined as D L, the distance over which pH varies from its lowest value to that value q1r2 < D pH < ŽTable 2.. Since D pH is below detection limits for most streptomyces experiments, D L is considered only for the arthrobacter experiments. Values of D L for the arthrobacter varied from 1.2 to 11.5 mm.

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3.2. SEM analyses 3.2.1. Hornblende samples Slightly enhanced weathering of hornblende along polishing lines was observed by SEM for experiments where bacteria were present as compared to uninoculated controls ŽFig. 4.. No significant differences were detected between discs incubated with the two different bacteria. Under the scanning electron microscope, both control ŽFig. 4a. and bacteriaincubated ŽFig. 4b. hornblende discs exhibited dark and light areas that differ compositionally, as determined by energy dispersive spectroscopy ŽEDS. and EM analyses. These light and dark patterns were not observed in blank discs Ždiscs not exposed to medium or bacteria.. The controls showed the same approxi-

Fig. 4. SEM photomicrograph of hornblende surface after incubation in: Ža. unbuffered medium without bacteria Žcontrol.; and Žb. unbuffered medium incubated with Arthrobacter sp. Note light ŽL. and dark ŽD. areas, presence of increased weathering along polishing scratches ŽP. in sample Žb. as compared to control sample Ža.. Scale bar is 20 mm in both photos.

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mate Fe concentration in light and dark areas, but increased Ca and Mg and decreased Si and Al concentrations in light compared to dark areas. In contrast, the light areas on the arthrobacter-incubated disc had less Fe and Mg and more Al compared to the dark areas. 3.2.2. Hornblende glass samples The hornblende glass control ŽFig. 5a. and sample exposed to the arthrobacter looked similar under the scanning electron microscope. The hornblende glass exposed to streptomycetes ŽFig. 5b. had patches of biofilm remaining on much of the surface, even after treatment with lysozyme. Previous studies with

Fig. 5. SEM photomicrograph of hornblende glass surface after incubation in: Ža. unbuffered medium without bacteria Žcontrol.; Žb. unbuffered medium incubated with Streptomyces sp. Note the presence of biofilm patches and small bacterial particles on the surface of the glass incubated with Streptomyces sp. ŽS.. Scale bar is 2 mm in Ža. and 5 mm in Žb..

Fig. 6. SEM analysis of low-Fe float glass surface after incubation in Ža. unbuffered medium without bacteria Žcontrol.; Žb. unbuffered medium incubated with Streptomyces sp.; Žc. unbuffered medium incubated with Arthrobacter sp. Note streptomycetes remnants ŽS. in sample incubated with Streptomyces sp., and Ž2. raised areas with pitting ŽR. on the surface of the glass incubated with Arthrobacter sp. Such pitting was observed on this sample and, occasionally, on the control. Scale bars are 2 mm for Ža. and Žb., and 1 mm for Žc..

this organism have shown it to be extremely adherent and difficult to remove from surfaces by non-

L.J. Liermann et al.r Chemical Geology 171 (2000) 1–16

destructive methods, including lysozyme ŽLiermann et al., 2000.. In the exposed areas between the biofilm patches, the surface looked similar to the

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control and the arthrobacter-incubated sample. Blanks also examined by SEM appeared similar to the experimental samples.

Fig. 7. Results from ion chromatographic analysis of aliquots of medium after incubation with hornblende glass or low-Fe float glass Žas indicated.. ŽA. Experiments with Arthrobacter sp. ŽB. Experiments with Streptomyces sp. For both graphs, abiotic data refer to the average of concentrations observed in the abiotic controls Žsubstrates placed in media without bacteria. run for each glass. Detection limits for malonate, phthalate, and citrate were 0.43, 0.7, and 0.4 mmol, respectively.

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3.2.3. Low-Fe float glass samples The surface of the low-Fe float glass control ŽFig. 6a. showed no significant features with SEM analysis, except for occasional raised patches. These raised patches were between 2 and 10 mm in diameter with pits on the order of 100 nm in diameter, and were compositionally identical to other areas. The streptomycete-incubated low-Fe float glass ŽFig. 6b. did not reveal an organic layer covering as in the hornblende glass sample, but only small, scattered pieces of streptomycete. The remaining surface of the streptomycete-incubated sample was similar to that seen in blank samples. In the arthrobacter-incubated sample ŽFig. 6c., most of the surface was again similar to blank and control samples. Similar to the control sample, however, the arthrobacter-incubated sample showed small raised patches scattered over the surface.

Little if any difference in dissolution rates were observed among samples: solution concentrations of Fe and Si Ž0.26 ppm Fe and 1.5 ppm Si. were slightly higher during hornblende crystal dissolution as compared to values for the glass substrates Ž0.14– 0.20 ppm Fe and 1.1–1.5 ppm Si..

4. Discussion 4.1. Detection of D pH

3.3. Solution chemistry

4.1.1. Reproducibility Differences in two pH measurements taken at or near the same distances at different times in one experiment were seen primarily in cultures incubated with the arthrobacter. Presumably, measurements disturbed the structure of the biofilm. Therefore, such measurements can only be made once for each biofilm.

Changes in pH occurred not only in the biofilms but also in the bulk medium for all samples from the initial pH measured before introduction of the substrate to day 9, when bulk and interface pH values were determined ŽTable 2.. For many samples incubated for 9 days, bulk pH decreased more in the presence of Streptomyces sp. than Arthrobacter sp., although differences between bulk and interface pH values were larger for Arthrobacter sp. ŽTable 2.. Analysis of organic acid production by ion chromatography ŽIC. was accomplished using the Dionex Peaknet program. Only the following acids were investigated: formic, acetic, oxalic, malonic, citric, and phthalic acid. In general, the total organic carbon of samples Ž) 3000 mgrl. was comprised of longer chain organic molecules than those identified by IC. Many of the small chain acids were present in abiotic controls. However, the IC showed that both species of bacteria produced acetic acid ŽFig. 7.. The arthrobacter also produced small amounts of oxalic and citric acids, and larger amounts of formic acid; more acetic and formic acids Žfound at ; 1000 mMrl each. were produced in the presence of the low-Fe float glass than with the hornblende glass by the arthrobacter ŽFig. 7.. To compare the dissolution of the substrates, samples of control solutions were analyzed by ICP-AES.

4.1.2. Magnitude of < D pH < Õalues We have shown that commercial microelectrodes can detect pH microenvironments in biofilms on mineral surfaces incubated in unbuffered medium without agitation for Arthrobacter sp. Absolute values of D pH as large as ; 1.1 between biofilm and bulk medium were measured. To place our observations in context, we review a few published studies of biofilm pH measurements. Published microelectrode studies of photosynthetic microbial mats revealed < D pH < s 1.9–2.2 within the microbial mat and < D pH < s 0.4 below the mat during a diurnal cycle, < D pH < s 1 between water above the mat and within the mat, and < D pH < s 0.9 between waters above and below the mats ŽJorgensen et al., 1983; Revsbech and Ward, 1984; Revsbech et al., 1983.. These values are thus in the same range as many of our measured values of D pH. In contrast, studies by Barker et al. Ž1998. using pH-sensitive fluorescent dyes detected decreases of 3–4 pH units between exterior surfaces as compared to microcolonies within confined interior spaces within minerals. However, these workers failed to detect pH microgradients in exterior microcolonies. Although such D pH values are larger than those observed here, they do not strictly reflect pH gradients from one interface to the bulk, as we have measured. In

L.J. Liermann et al.r Chemical Geology 171 (2000) 1–16

other studies more similar to our measurements, symbiotic photosynthesis in a planktonic foraminiferan revealed a D pH s y0.08 between the surrounding water and the shell surface in the dark; in the light, D pH was q0.39 ŽJorgensen et al., 1985.. Based on these and our own studies, D pH values from bulk to interface may range up to a pH unit, reflecting substantial differences in pH between the bulk and the interface. Of course, our measurements were performed in media rather than in natural solutions, so investigations of biofilms grown in more natural media are needed. 4.1.3. Biofilm half-thicknesses The pH differences between bulk and interface Ž D pH. were used to calculate D L, the half-thickness of the biofilms. Values of D L were variable, even for replicates. Differences in D L are most likely because of differences in polysaccharide and acid production by the bacteria, differing growth rates, as well as, perhaps, differences in reactivity of the substrates. Biofilm thicknesses varied for all substrates from 1.2 to 11.5 mm. 4.2. Acid production Biofilms of Streptomyces sp. created pH gradients below the limits of detection for all substrates. The larger differences seen in Arthrobacter sp. experiments could be due to the fact that the arthrobacter grows faster than the streptomycetes, producing more acidic metabolites during experiments. However, in many cases, the bulk pH of the medium was lower after growth with the streptomycetes as compared to the arthrobacter. Therefore, it is more likely that, since the streptomycetes grows in uneven clumps, the streptomyces biofilm may not maintain a pH gradient as efficiently as the biofilm produced by the arthrobacter. Previous studies ŽKalinowski et al., 2000; Liermann et al., 2000. have shown that both the arthrobacter and the streptomycete produce catecholate siderophores in Fe-limited buffered medium; concentrations of Fe in the unbuffered medium used in these studies Ž; 0.20 ppm. is low enough that siderophore production is likely to have occurred here as well. We have previously demonstrated ŽKalinowski et al., 2000; Liermann et al., 2000. that

13

Arthrobacter sp. accelerated the Fe release from hornblende more than Streptomyces sp. in batch experiments in buffered media. Faster leaching of Fe by the arthrobacter may suggest that siderophore production is more rapid or more efficient in scavenging Fe for Arthrobacter sp. as compared to Streptomyces sp. Because siderophore–Fe complexation releases Hq from the siderophore ligand, siderophore chemistry may contribute to larger D pH values for the arthrobacter. The greater D pH values seen for the arthrobacter are also consistent with the IC data showing more variety of organic acids secreted by this organism Žformic, acetic, oxalic, citric. as compared to the streptomycete Žacetic.. Other organic acids may also have been produced in these experiments. For example, preliminary data from our laboratory suggests production of benzoic and butanoic acids by the streptomycete used in these studies. Organic acids commonly produced by soil bacteria and fungi include 2-ketogluconic, lactic, acetic, citric, oxalic, pyruvic, and succinic ŽDuff et al., 1963; Rozycki and Strzelczyk, 1986; Palmer et al., 1991; Urzi et al., 1991; Vandevivere et al., 1994.. Urzi et al. Ž1991. reported production of gluconic, lactic, pyruvic, and succinic acids by a strain of Micrococcus growing on marble, and Rozycki and Strzelczyk Ž1986. reported production of pyruvic, a-ketoglutaric, lactic, citric, succinic and oxalic acids by a soil streptomycetes. 4.3. Surface morphology Hornblende blanks do not show the same patterns of light and dark as seen under the scanning electron microscopy in the experimental samples, suggesting that the medium dissolves two phases differently ŽFig. 4a.. However, the differences in Fe composition of light Žpresumed chlorite. and dark Žpresumed hornblende. areas between control and arthrobacter samples are consistent with increased leaching of Fe into the medium in the arthrobacter-incubated sample as compared to controls. Increased etching along polishing lines of the hornblende incubated with bacteria also supports increased weathering of this sample compared to the control and as compared to the glass compositions. Interpretation of the surface morphology of the other samples was equivocal. For example, for low-Fe float glass, the raised patches

L.J. Liermann et al.r Chemical Geology 171 (2000) 1–16

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seen under the scanning electron microscope may be the result of either dissolution or precipitation. For surface changes of a significant magnitude to be observed under the scanning electron microscope, incubation times would most likely need to be longer than those used in these experiments. For example, Staudigel et al. Ž1995. observed small corrosion pits on surfaces of polished glass incubated with a marine cyanobacterium in pre-sterilized seawater for 142 days; larger grooves and pits Žup to 10 mm long and 0.5 mm wide. were observed in continuous-flow experiments with unsterilized seawater containing a natural mixed-microbe population. 4.4. Effect of substrates Several phenomena are expected to control bulk solution pH in these experiments: Ž1. CO 2 dissolution in medium, Ž2. silicate dissolution, Ž3. production of organic acids and chelates by bacteria. Because media were exposed to the atmosphere for similar amounts of time for each experiment, we assume the effects of Ž1. were equivalent between experiments. Such effects probably partially explain the decrease in pH observed for controls ŽTable 2.. In contrast, the effect of silicate dissolution Ž2. should be to increase the pH of solution. For example, congruent dissolution of low-Fe float glass raises pH by 2.24 mol OHy per mole of Si released Žfor simplicity, the presence of CO 2 is ignored.:

Ž 0.82MgO P 0.12CaO P 0.18Na 2 O P SiO 2 . Ž glass . q3.12H 2 OŽ1.

™ H SiO 4

2q 4Žaq . q 0.82Mg Žaq .

q y q0.12Ca2q Žaq .q 0.36Na Žaq .q 2.24OH Žaq .

Ž 1.

The similar values of pH of the bulk solution of the hornblende and glass controls are consistent with roughly identical extents of dissolution of CO 2 and silicate into solutions. As mentioned earlier, solution chemistry also documented little to no evidence for differences in dissolution rates of the four substrates. Rates of dissolution of the substrates may not, therefore, explain the differences in D pH observed. Why then are the observed < D pH < values larger for the hornblende crystalrglass as compared to the float glass? Possibilities include the effects of chemical composition Že.g. Fe, Na, Si, Al content. or initial

surface topography Žsurfaces of both float glasses were smoother than surfaces of either hornblende phase..

5. Conclusions In this work, we made the following observations: Ž1. Biofilms developed by common genera of soil bacteria Žarthrobacter. produce pH microenvironments that can be measured by commercial microelectrodes. Ž2. pH drops across the biofilms as high as 1.1 pH units were observed. Ž3. Values of D pH varied with substrate such that the largest < D pH < values were observed for substrates of hornblende composition. Microbe–surface interactions play a significant role in weathering and corrosion processes, and understanding the chemistry of the biofilm has important implications in phenomena ranging from bioremediation to biofouling. Although values of < D pH < developed at mineral–water interfaces in natural systems may only be as large as those measured here for water-saturated systems, it is probable that significant pH gradients Žup to a pH unit. probably develop in some cases, especially where fast-growing, acid-producing microbes colonize slow-dissolving phases in the presence of unbuffered solutions. Such large values of < D pH < would be expected to affect mineral dissolution significantly ŽBrantley and Chen, 1995.. Microelectrodes can be used to measure pH, dissolved O 2 , CO 2 , NH 4 , redox potential, and H 2 S. Improvements in microelectrode technology will undoubtedly allow for more precise measurements of biofilm microniche characteristics. Further investigations such as those outlined here should be pursued in order to define the importance of the chemistry, reactivity, and roughness of the substrate and the characteristics of the bacteria species.

Acknowledgements The authors thank the following for their contributions to these studies: Tom Rusnak for SEM assistance, Sharon Givens and Don Voigt for hornblende

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preparation and technical assistance, Jon Chorover and Mingxin Guo for organic acids analyses, and Serguei Lvov for use of lab facilities for microelectrode pH measurements. This manuscript was greatly improved by the comments of Hubert Staudigel and Jeremy Fein. Birgitta E. Kalinowski is grateful to The Swedish Foundation for International Cooperation in Research and Higher Education ŽSTINT. for partial support. The project was funded by NSF grant CHE 9631528 and Liermann is partially funded by NSF DGE-9972759 to S.L. Brantley and the Penn State Biogeochemical Research Initiative for Education ŽBRIE..

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