Geomicrobiology Journal 1863ndash76 2001Copyright Cdeg 2001 Taylor amp Francis0149-045101 $1200 + 00

Dynamics of the MineralndashMicrobe Interface Useof Biological Force Microscopy in Biogeochemistry

and Geomicrobiology

STEVEN K LOWERCHRISTOPHER J TADANIERMICHAEL F HOCHELLA JR

MineralndashMicrobe GroupNanoGeoscience and Technology LaboratoryDepartment of Geological SciencesVirginia Polytechnic Institute and State UniversityBlacksburg Virginia USA

At the most fundamental level inter- and intramolecular forces delineate the interfacebetween a microorganism and a mineral surface A new technique termed biologicalforce microscopy (BFM) is described that can be used to directly probe the dynamicsof the mineralndashmicrobe interface BFM quanties attractive and repulsive forces in thenano-Newton range between living microbial cells and mineral surfaces in aqueoussolutionNative bacterial cells are linked to a force-sensorthat is used in a force micro-scope to measurebacteriandashmineral interactionsas a functionof the distancebetween themineral surface and the cells on the sensor The magnitudes and ranges of the measuredforces re ect the chemical and structural intricacies of the mineralndashmicrobe interfaceBFM is presentedwith potentialapplicationsto studies assessing the role that microbesor biomolecules play in geochemical and mineralogicalprocesses

Keywords adhesionatomic forcemicroscopybacteriaforcegeomicrobiologyinter-face intermolecular intramolecularmicroorganismmineral surface nanotechnology

Mineralndashmicrobe interactions have been occurring for at least 3 billion years on and withinthe earth Thousands of mineral species with enormous variability in surface chemistry andstructure may interact with any of millions of bacterial species that display diverse surfacemosaics and physiologies Minerals and microorganisms are intimately linked such thatone often cannot exist without the other in nature Microbial processes play roles in thecycling of elements and sorption of metals (Stotzky 1986 Ehrlich 1990 Marshall 1996Fein Daughney Yee and Davis 1997 Langley and Beveridge 1999) the dissolution ofminerals (Lovley and Phillips 1986 Robert and Berthelin 1986 Lovley and Phillips 1987Myers and Nealson 1988 Krumbein Urzi and Gehrmann 1991 Welch and Ullman 1993Welch and Vandevivere 1994 Hersman Lloyd and Sposito 1995 Hersman Maurice andSposito 1996 Roden and Zachara 1996 Ban eld and Hamers 1997 Grantham Dove

Received 28 July 2000 accepted 25 September 2000The presentation of this research bene ted from the comments of two anonymous reviewers SKL appre-

ciates the support of J Tak Funding was provided by American Chemical Society grant ACS-PRF 34326-AC2and Department of Energy grant DE-FG02-99ER 15002

Address correspondence to Steven K Lower Department of Geological Sciences Virginia PolytechnicInstitute Blacksburg VA 24061 USA

63

64 S K Lower et al

and DiChristina 1997 Stone 1997 Barker Welch Chu and Ban eld 1998 EdwardsGoebel Rodgers et al 1998 Edwards Schrenk Hamers et al 1998 Forsythe Mauriceand Hersman 1998 Schrenk Edwards Goodman Hamers and Ban eld 1998) and min-eral crystallization (Pentecost and Bauld 1988 Beveridge and Doyle 1989 Schultze-LamHarauz and Beveridge 1992 Schultze-Lam Fortin Davis and Beveridge 1996 Fortinand Beveridge 1997 Fortin Ferris and Beveridge 1997 Warren and Ferris 1998) Con-versely mineralogical processes in uence the distribution activity and diversity of mi-crobes (Stotzky 1986 Fredrickson McKinley Nierzwicki-Bauer et al 1995 BennettHiebert and Choi 1996 Fletcher 1996a Marshall 1996 Barker Welch and Ban eld1997Rogers Bennett and Choi 1998SchrenkEdwards GoodmanHamers and Ban eld1998) the expressionof genes (ArredondoGarcia and Jerez 1994Fletcher 1996aDziurlaAchouak Lam Heulin and Berthelin 1998 Gehrke Telegdi Dominigue and Sand 1998)community structure and development (Lawrence Korber Hoyle Costerton and Caldwell1991 Wolfaardt Lawrence Roberts Caldwell and Caldwell 1994 Thorseth TorsvikFurnes and Muehlenbachs 1995 Brown Sherriff and Sawicki 1997 Kennedy and Gewin1997) and transfer of genetic material (Holben 1997 Trevors and van Elsas 1997)

The thread linking these unimaginably complex interactions is the fact that mineralndashmicrobe processes are dependent on the intimate juxtaposition of a living and nonlivingentity that is the interface between a microbial cell and a mineral surface A fundamentalappreciation of this interface is dependent on our understanding and characterization of thesymphony of inter- and intramolecular forces between microbes and mineral surfaces innature Despite the vast amount of work on microbial affects on mineralogical processesand vice versa the interface remains largely unexplored primarily because it is dif cult todirectly probe this minute and dynamic space

Atomic force microscopymdashand variations thereofmdashis an elegant tool for measuringinter- and intramolecular forces between organic and inorganic surfaces (see Figure 1)For review see Butt Jaschke and Ducker 1995 Cappella and Dietler 1999 Lower andMaurice in preparation Recently we created biological force microscopy (BFM) as amethod to directly probe interfacial and adhesion forces between bacteria and mineralsurfaces in aqueous solution (Lower Tadanier and Hochella 2000) Living cells are linkedto a sensor that is used to quantitatively measure attractive and repulsive forces in thenano-Newton range between bacteria and mineral surfaces at distances between 0 sup1m (iecontact) and 2 sup1m Measured forces re ected the complex interactions of structural andchemical functionalities on the bacteria and mineral surfaces Here we demonstrate anddiscuss this technique and suggest potential applications of BFM to studies assessing therole that microbes or biomolecules play in geochemical and mineralogical processes

Materials and Methods

Mineral and Bacteria

Freshly cleaved muscovite (KAl2(AlSi3O10)(OH)2) and a gram-negative soil bacteria wereused for all experimentsThe bacteria strain was obtained from a chemostat inoculatedwithan iron-oxide rich soil from Pandapas Pond Jefferson National Forest Virginia (TadanierLittle Berry and Hochella in press) Through comparison of 500 bp of 16S rRNA se-quence this bacterium has been aligned at the species level (067 difference) with thepseudomonad Burkholderia cepacia (MIDI Labs Newark DE) Burkholderia sp werecultured on agar plates under oligotrophic (glucose concentrationD 06 mM) or eutrophicconditions (glucose concentration D 28 mM) and used in BFM experiments as describednext (see Tadanier et al in press for complete description of growth media)

Biological Force Microscopy and Interfaces 65

FIGURE 1 Schematic diagram showing the key components of a force microscope Themineral and force sensor are within a uid cell (not shown) containing aqueous solutionForce measurements are made by recording the de ection of a sensor (ie cantilever) in re-sponse to attractiveor repulsive forces between itself and the sample (a mineral in this case)The sample mounted on a piezoelectric scanner indexes toward makes contact with andretracts from the sensor De ection of the sensor is detected by re ecting a laser off the topof the sensor and into a split segment photodiodeIn this study bacteria (shown as a sphere)havebeen linkedto the sensor therebycreatinga biologicallyactive force probe(BAFP) usedto measure interfacial and adhesive forces between bacteria and mineral surfaces in situ

Force Sensor Preparation and Characterization

Biologicallyactive force probes (BAFPs) can be createdby linkingbacteria either directly toa siliconor siliconnitride force sensor (ie cantilever) or indirectly by linking a monolayerof bacteria to a small glass bead that is then xed to the end of a cantilever Direct linkage ofbacteria to the cantilever was accomplished by rst placing a cantilever in a 1ndash5 solutionof the polycationic molecule poly-D-lysine (135 to 150 kDa pH near neutral) Bacteriawere linked to the polylysine functionalized cantilever by lowering it into a colony of livebacteria with the aid of a microscope (Nikon 200x magni cation) and a micromanipulatorto translate the cantilever For the indirect-linkagemethod small glass beads (Polysciencesor Duke Scienti c radii 3ndash7 sup1m cleaned with a solution of 1 hydro uoric acid or 10sodium hydroxide) were activated with a 1ndash5 solution of polylysine The activated beadswere placed in a suspension of Burkholderia sp and centrifuged at 8000 pound g for 5 minA single bacteria-coated bead was attached to a cantilever using a small quantity of epoxyresin which has previously been found to be inert in aqueous solutions (Pincet 1995 YoonFlinn and Rabinovich 1997) This attachment procedure was conducted in solution usinga micromanipulator

Prior to BFM measurements scanning laser confocal microscopy (Zeiss LSM 510)was used to characterize the three dimensional nature of a BAFP A probe was placed inthe uid cell used for force measurements and imaged with a 100x 14 NA objective

66 S K Lower et al

This imaging procedure was facilitated by transforming bacteria with a plasmid (pGLOBio-Rad or pSMC2 provided by G A OrsquoToole Dartmouth University) that encoded anintracellular green uorescent protein The bacteria uoresced when excited by light at458 nm or 488 nm Fluorescence emitted by the epoxy resin revealed that it was con nedto the region between the cantilever and glass bead (ie resin was not in a position thatwould alter the interaction between the bacteria and mineral during BFM experiments)

Cantilever spring constants (N miexcl1) essential for measuring force magnitudes with aforce microscope can vary substantially from the nominal value listed by the manufacturer(ClevelandManne Bocek and Hansma 1993 Senden and Ducker 1994) Spring constantswere determined by attaching known masses to the end of a cantilever and recording thechange in cantilever resonant frequency (Cleveland et al 1993) A linear relationship wasobserved between added mass and resonant frequency with the slope being the springconstant (017 N miexcl1) These measurements were accomplished using 10 cantilevers fromthe same wafer as that used to create BAFPs The variability of spring constants fromcantilever to cantileverwithin the same wafer has been determined to be very small (Sendenand Ducker 1994) This was con rmed by the linear t of our data and the reproducibilityof the resonant frequency of unloaded cantilevers (137 kHz sect 02)

Force Measurements

BFM measurements were performed using a NanoScope IIIa Multimode SPM (DigitalInstruments Santa Barbara CA) in aqueous solution (pH 6 ionic strength 10iexcl5 M 25plusmnC)A piezoelectric scanner was used to translate the mineral toward and away from bacteriaon a BAFP at rates of 01 to 3 sup1m seciexcl1 For comparison this is within the range ofvelocitiesof motilebacteria(Marshall 1976) Interfacial forces were measured as themineralapproached the bacteria on the probe whereas adhesion forces were measured upon contactand subsequent retraction of the mineral from the bacteria Mineral samples were drivento the same contact force to normalize the effect that loading can have on adhesion forcesduring retraction (Weisenhorn Maivald Butt and Hansma 1992)

Force measurement data are collected as cantilever de ection (diode voltage) and cor-responding piezo displacement typically termed a force curve (Figure 2A) This data mustbe manipulatedto produce the familiar forcendashdistance curvewhich describes interfacial andadhesion forces (Figure 2B) Distance (ie separation between bacteria and mineral) is cal-culatedbycorrecting the recordedpiezoposition(ie displacement) by themeasured de ec-tion of the cantileverFor example if the mineral attached to the piezo scanner moves 10 nmtoward bacteria attached to the cantilever and the bacteria are repelled 2 nm due to repul-sive forces then the actual mineralndashbacteria distance (or separation) changes by only 8 nmThe distance axis origin is chosen as the point on the force curve where sensor de ectionbecomes a linear function of piezo displacement (the sensor is in contact with the sample)

Force (F ) is determinedusing Hookersquos Law F D kspd where d is cantileverde ection(meters) and ksp (N miexcl1) is the cantilever spring constant In order to use Hookersquos Lawcantilever de ection measured by the photodiode in volts must be converted to metersA diodedisplacement conversion factor (also called ldquooptical lever sensitivityrdquo) is de nedfrom the slope of the force curve region where the cantilever is in contact with the sampleon the piezo (Figure 2Aiii called the ldquoregion of constant compliancerdquo) The reciprocal ofthis conversion factor (in nm Viexcl1) can be used to convert measured cantilever de ection involts to meters A zero-force reference value is determined as the force curve region wheresensor de ection is independent of piezo displacement (Figure 2Ai the sensor and sampleare not interacting because they are far apart)

It is important to note that using the constant compliance region of the force curveto convert photodiode response into force will overestimate the force of interaction if thebacteria are more compliant than the cantilever Recent measurements of the elasticity

Biological Force Microscopy and Interfaces 67

FIGURE 2 Schematic diagrams of a force curve (A) and force-distance curve (B) Whenthe sensor and sample are far apart (i) they exhibit no interaction (region of zero force) Asthe sample approaches the sensor intermolecular forces between the bacteria and mineralcause the sensor to de ect upward (ii) due to repulsive forces shown here Eventually theprobe makes contact with the sample (iii) and their movement becomes coupled (regionof constant compliance) The sample is then retracted from the probe (iv) until the sensorand sample return to their original positions thereby completing one cycle (an entire cyclerequires nano- to milliseconds) Hysteresis shown here may occur upon retraction dueto adhesion forces See text for discussion on converting cantilever de ection and piezodisplacement into force and distance respectively Interfacial forces are measured on ap-proach and adhesion forces are measured upon retraction repulsive forces are positive andattractive forces are negative

68 S K Lower et al

FIGURE 3 Variation of photodiode shift voltage as a function of optical lever sensitivityusing a J-type piezoelectric scanner and eight silicon nitride cantilevers (200 sup1m in length)All measurements were performed on muscovite and hematite in a uid cell containingaqueous solutions ranging in ionic strength from 0 (ie Milli-Q water) to 10iexcl2M Theslope of the regression line is 642 nm and the intercept is 13 V (R2 D 095) With thiscorrelation the optical lever sensitivity can be determined in a matter of seconds by simplyusing the photodiode shift voltage Furthermore this method is valid regardless of whetherthe cantilever is the most compliant component of the system

of bacterial surface macromolecules suggest that bacteria are less compliant (ie stiffer)than cantilevers having spring constants smaller than 10 N miexcl1 (Xu Mulhern BlackfordJericho Firtel and Beveridge 1996 Yao Jericho Pink and Beveridge 1999)

In instances where bacteria (or biomolecules) linked to the cantilever are more com-pliant than the cantilever or for cells with fragile appendages other methods must be usedto accurately convert the measured de ection of the cantilever (in volts) into a force (inNewtons) of interaction (DrsquoCosta and Hoh 1995 Sader Chon and Mulvaney 1999) Forexample because the optical lever sensitivity is strongly dependenton the shape of the laserspot on the photodiodedetector the ldquophotodiode shift voltagerdquo can be used to convert voltsof cantileverde ection into meters of de ection (DrsquoCosta and Hoh 1995) Photodiode shiftvoltage is measured as the difference in output voltage when the photodiode detector isshifted approximately 318 sup1m (one full turn of the positioning screw) on either side of thezero setting Figure 3 illustrates the strong correlation between photodiode shift voltagesand optical lever sensitivities measured by directing the laser to different positions on thecantileverOnce this correlation is established for a given instrument piezoelectric scanner uid cell and cantilever (eg 200-sup1m long V-shaped silicon nitride cantilevers) the op-tical lever sensitivity can be accurately determined without pressing the cantilever againstany other surface This method ensures that forces can be determined regardless of thecompliance of the cantilever relative to any microorganism attached to it and also ensuresthe preservation of fragile macromolecules on microorganisms attached to the cantilever

Results and Discussion

Characterization of Biologically Active Force Probes

BAFPs were created by either attaching a single bacterium to a force sensor (Figure 4A)or by attaching a single bacteria-coated-bead (Figure 4B) to a force sensor Fluorescent

FIGURE 4 (A) Scanning laser micrograph of a BAFP created by directly linking a singlecell of Burkholderia sp to a force-sensing cantilever (B) Confocal micrograph focused onthe midplane of bacteria-coated beads revealing a layer of cells Focusing up and downon the beads reveal that many are evenly coated with cells Beads showing the most uni-form coverage are attached to cantilevers thereby creating BAFPs These cells have beentransformed with a plasmid encoding a green uorescent protein and are emitting ldquonaturalrdquo uorescence This uorescent protein being intracellular together with the confocal abili-ties of the laser scanning microscope allow noninvasive characterization of the orientationand distribution of cells on the force sensor in situ without affecting the surface chemistryof the microbes Plating experiments in which BAFPs were used as the inoculum revealedthat the bacteria on the sensors are viableand presumably activeduring force measurementsScale bars D 10 sup1m

69

70 S K Lower et al

dyes (ViaGram Molecular Probes Eugene OR) and plating techniques revealed that cellmembranes on these bacteria are intact and the cells are viable Both the direct and indirectmethod of attaching bacteria to a cantileverpreserves the orientationand structural integrityof macromolecules on the cell surface This is important because the native conformationof macromolecules on a bacteria surface is critical to their function activity and role innature By using whole bacteria expressing macromolecules in their natural state ratherthan individual biomolecules (eg exopolysaccharides proteins) puri ed from bacterialsurfaces we avoid situations in which the linkage procedure modi es the conformation ofbiomolecules such that they are no longer in a natural state (Stotzky 1986 Ellen and Burne1996 Turner Peek Wertz Archibald Geer and Gaber 1996 Ingersoll and Bright 1997Turner Testoff Conrad and Gaber 1997)

As both gram-negative and gram-positive bacteria are typically negatively charged atmost pH polylysine with its high pKa is a more-or-less universal linker molecule How-ever due to the large diversity in bacterial surfaces situations will likely arise in whichother linker molecules are necessary The versatility of our linkage protocol allows formodi cation on a case-by-case basis For example we have also fabricated BAFPs usingaminopropyltriethoxysilane and polyethyleneimine with species of Shewanella and Pseu-domonas Additionallymethods used to attach proteins or antibodies to small glass spheresor sensors (eg Florin Moy and Gaub 1994 Frey and Corn 1996 Turner et al 1997Caruso Caruso and Mohwald 1998 Rezania Johnson Lefkow and Healy 1999) could beapplied to our protocol For example lysozyme which has been genetically altered suchthat it possesses an active binding site but an inactive catalytic site (Voet and Voet 1995) isan attractive choice because of its high af nity for bacterial cell walls Antibodies speci cfor cell surface receptors may also be effective linkers

A critical component to consider when designing BAFPs is the relative difference be-tween thoseforces bindingthecells to thesensor and interfacial and adhesiveforces thatwillbeprobedby thecellson the sensor Forces holdingbacteria to the sensormustbe greater thanthe forces that are actuallyprobedotherwise the bacteriawill be stripped from the cantileverduring force measurements Electrostatic attachment of bacteria to sensors via polylysineis very strong Approximations using Coulombrsquos law and molecular dynamic calculationsshow that the energy associated with aminendashsilanol (ie the polylysine glass bead linkageor polylysine cantilever linkage) and aminendashcarboxylic pairs (ie the polylysine bacterialinkage) (Voet and Voet 1995 West Latour and Hench 1997 Pagac Prieve and Tilton1998 Pagac Tilton and Prieve 1998) are at least one to four orders of magnitude strongerthan potential intermolecular forces between a bacteria and mineral (Israelachvili 1992)Organosilane linkages such as aminopropyltriethoxysilane form covalentbondswith silanolgroups (Plueddemann1991) and should therefore be even stronger than polylysinelinkages

Comparison of the direct and indirect linkage protocols suggests that the latter ismore desirable Direct linkage of a single cell to a cantilever (Figure 4A) is very dif cultTechniques such as optical tweezers (Svoboda and Block 1994) or nanotweezers (Kim andLieber 1999) could signi cantly enhance the linkage of a bacterium to a cantilever butsuch techniques are not at present easily applicable to such procedures Another practicalreason for selecting the indirect protocol is the need to use cantilevers with appropriatespring constants in order to probe the entire range of potential forces between microbes andminerals Cantilevers of different composition (eg glass silicon silicon nitride and gold-coated cantilevers) are often used to vary the spring constant Gold-coated and uncoatedsilicon nitride cantilevers for example have very different surface properties and siliconnitride itself can vary from Si3N4 to Si15N4 (WeisenhornMaivaldButt and Hansma 1992)

Biological Force Microscopy and Interfaces 71

These compositional differences would require use of more than one linker molecule todirectly attach one particular strain of bacteria to different cantilevers Conversely indirectlinkage allows the use of one protocol to attach bacteria to glass (or latex) beads of uniformcomposition which can then be attached to cantilevers of different compositions

Forces of Interaction Between Bacteria and Mineral Surfaces in situ

The interactions between muscovite and polylysine (Figure 5A) and muscovite and the soilBurkholderia sp (Figures 5B and 5C) were studied with BFM at pH 6 ionic strength 10iexcl5

M 25plusmnC As expected the positively charged polylysine exhibited a strong attraction to-wards the (001) surface of muscovite which is negatively charged (Figure 5A) Hysteresiswas not observed for the interaction between muscovite and polylysine In contrast theBurkholderia sp exhibited a repulsive interaction with muscovite beginning at raquo80 nm(Figure 5B approach) However once contact was made this repulsive force was over-come and the bacteria exhibited an attractive adhesion toward the mineral (Figure 5Bretraction)

The repulsive interfacial forces between the Burkholderia sp and muscovite illustratedin Figure 5B (approach) are consistent with the negative surface charges of both the bac-teria and mineral at circum-neutral pH The distance at which repulsion occurs is slightlysmaller than that expectedbased solely on electrostatic interactions(Debye length thicknessis raquo100 nm at this ionic strength) Other forces associated with hydrophobic steric andentropic effects likely play a key role in bacteriandashmineral interactions due to the relativelylongpolymerson bacterial surfaces (Israelachviliand McGuiggan1988 Israelachvili1992)

The adhesive behavior between Burkholderia sp and muscovite is drawn out for hun-dreds of nanometers (Figure 5B retraction) This is likely due to stretching and brillationof biomolecules (eg lipopolysaccharides or agella) as a result of attractive forces suchas hydrogen bonds between polymers on the bacteria and surface hydroxyls or structuredwater molecules on muscovite (Jucker Harms Hug and Zehnder 1997)

When the same Burkholderia sp was grown in nutrient poor media it exhibited verystrong af nity for muscovite (Figure 5C) Interfacial repulsion was not detected (Figure 5Capproach) and attractive adhesion forces were very large and long range (Figure 5Cretraction) This phenomenais consistentwith theobservationthatmany bacterial speciesal-ter their surface propertiesunderoligotrophicversus eutropicenvironments(eg Bengtsson1991) In fact many bacteria show greater af nity for mineral surfaces under oligotrophicrather than eutropic conditions (Fletcher 1996b Marshall 1996) This cycling between at-tached and planktonic states is believed to be a strategy bacteria use to proliferate in naturewhere many environments have eutropicoligotropic sequences

Potential Applications of Biological Force Microscopy to Geomicrobiology Studies

BFM could providea uniqueperspectiveinto several aspects of geomicrobiology(egmin-eral dissolution crystal growth and bio lm formation) particularly if used to complementother techniques Next is a list of relevant questions considered as possible applications ofBFM to the eld of geomicrobiology and their answers

1 Do bacteria ldquorecognizerdquo particular minerals or crystallographic orientations Probe dif-ferent minerals or different faces on the same mineral with one BAFP

72 S K Lower et al

FIGURE 5 Biological force microcopy (BFM) force-distance curves between the (001)surface of muscovite and a polylysine functionalized cantilever (A) versus Burkholderiasp grown in a eutropic environment (B) versus Burkholderia sp grown in an oligotropicenvironment (C) Solution conditionswere pH 6 ionic strength 10iexcl5M 25plusmnC The force ofinteraction is plotted as a function of the distance between the two surfaces (ie separationbetween polylysine or bacteria on the sensor and the mineral surface) Note difference inx-axis scale Curves begin on the right and proceed toward the left as the mineral is broughttoward the sensor (solid circles approach curve) The sensor makes contactwith the mineralsurface and subsequently withdraws from the muscovite (open squares retraction curve) tocomplete one cycle

2 Do bacteria show an increased af nity for particular minerals under oligotrophic oranaerobicconditionsChangesolutionconditionswithin uid cell while collectingBFMmeasurements

3 How does inter- and intraspecies communication affect bio lm development Use aBAFP to measure interactions with various species of bacteria that are immobilized ona substrate

4 What intermolecular forces (eg van der Waals electrostatics hydration hydrophobicentropic) are involvedin bacterial attachmentand detachmentprocessesCompare forcesmeasured with BFM to those calculated from theoretical models (eg DLVO theory)

5 What cell surface macromoleculesmediatebacterial attachment to mineral surfaces Areconditioning lms required for attachment Create BAFPs using various mutants that

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

64 S K Lower et al

and DiChristina 1997 Stone 1997 Barker Welch Chu and Ban eld 1998 EdwardsGoebel Rodgers et al 1998 Edwards Schrenk Hamers et al 1998 Forsythe Mauriceand Hersman 1998 Schrenk Edwards Goodman Hamers and Ban eld 1998) and min-eral crystallization (Pentecost and Bauld 1988 Beveridge and Doyle 1989 Schultze-LamHarauz and Beveridge 1992 Schultze-Lam Fortin Davis and Beveridge 1996 Fortinand Beveridge 1997 Fortin Ferris and Beveridge 1997 Warren and Ferris 1998) Con-versely mineralogical processes in uence the distribution activity and diversity of mi-crobes (Stotzky 1986 Fredrickson McKinley Nierzwicki-Bauer et al 1995 BennettHiebert and Choi 1996 Fletcher 1996a Marshall 1996 Barker Welch and Ban eld1997Rogers Bennett and Choi 1998SchrenkEdwards GoodmanHamers and Ban eld1998) the expressionof genes (ArredondoGarcia and Jerez 1994Fletcher 1996aDziurlaAchouak Lam Heulin and Berthelin 1998 Gehrke Telegdi Dominigue and Sand 1998)community structure and development (Lawrence Korber Hoyle Costerton and Caldwell1991 Wolfaardt Lawrence Roberts Caldwell and Caldwell 1994 Thorseth TorsvikFurnes and Muehlenbachs 1995 Brown Sherriff and Sawicki 1997 Kennedy and Gewin1997) and transfer of genetic material (Holben 1997 Trevors and van Elsas 1997)

The thread linking these unimaginably complex interactions is the fact that mineralndashmicrobe processes are dependent on the intimate juxtaposition of a living and nonlivingentity that is the interface between a microbial cell and a mineral surface A fundamentalappreciation of this interface is dependent on our understanding and characterization of thesymphony of inter- and intramolecular forces between microbes and mineral surfaces innature Despite the vast amount of work on microbial affects on mineralogical processesand vice versa the interface remains largely unexplored primarily because it is dif cult todirectly probe this minute and dynamic space

Atomic force microscopymdashand variations thereofmdashis an elegant tool for measuringinter- and intramolecular forces between organic and inorganic surfaces (see Figure 1)For review see Butt Jaschke and Ducker 1995 Cappella and Dietler 1999 Lower andMaurice in preparation Recently we created biological force microscopy (BFM) as amethod to directly probe interfacial and adhesion forces between bacteria and mineralsurfaces in aqueous solution (Lower Tadanier and Hochella 2000) Living cells are linkedto a sensor that is used to quantitatively measure attractive and repulsive forces in thenano-Newton range between bacteria and mineral surfaces at distances between 0 sup1m (iecontact) and 2 sup1m Measured forces re ected the complex interactions of structural andchemical functionalities on the bacteria and mineral surfaces Here we demonstrate anddiscuss this technique and suggest potential applications of BFM to studies assessing therole that microbes or biomolecules play in geochemical and mineralogical processes

Materials and Methods

Mineral and Bacteria

Freshly cleaved muscovite (KAl2(AlSi3O10)(OH)2) and a gram-negative soil bacteria wereused for all experimentsThe bacteria strain was obtained from a chemostat inoculatedwithan iron-oxide rich soil from Pandapas Pond Jefferson National Forest Virginia (TadanierLittle Berry and Hochella in press) Through comparison of 500 bp of 16S rRNA se-quence this bacterium has been aligned at the species level (067 difference) with thepseudomonad Burkholderia cepacia (MIDI Labs Newark DE) Burkholderia sp werecultured on agar plates under oligotrophic (glucose concentrationD 06 mM) or eutrophicconditions (glucose concentration D 28 mM) and used in BFM experiments as describednext (see Tadanier et al in press for complete description of growth media)

Biological Force Microscopy and Interfaces 65

FIGURE 1 Schematic diagram showing the key components of a force microscope Themineral and force sensor are within a uid cell (not shown) containing aqueous solutionForce measurements are made by recording the de ection of a sensor (ie cantilever) in re-sponse to attractiveor repulsive forces between itself and the sample (a mineral in this case)The sample mounted on a piezoelectric scanner indexes toward makes contact with andretracts from the sensor De ection of the sensor is detected by re ecting a laser off the topof the sensor and into a split segment photodiodeIn this study bacteria (shown as a sphere)havebeen linkedto the sensor therebycreatinga biologicallyactive force probe(BAFP) usedto measure interfacial and adhesive forces between bacteria and mineral surfaces in situ

Force Sensor Preparation and Characterization

Biologicallyactive force probes (BAFPs) can be createdby linkingbacteria either directly toa siliconor siliconnitride force sensor (ie cantilever) or indirectly by linking a monolayerof bacteria to a small glass bead that is then xed to the end of a cantilever Direct linkage ofbacteria to the cantilever was accomplished by rst placing a cantilever in a 1ndash5 solutionof the polycationic molecule poly-D-lysine (135 to 150 kDa pH near neutral) Bacteriawere linked to the polylysine functionalized cantilever by lowering it into a colony of livebacteria with the aid of a microscope (Nikon 200x magni cation) and a micromanipulatorto translate the cantilever For the indirect-linkagemethod small glass beads (Polysciencesor Duke Scienti c radii 3ndash7 sup1m cleaned with a solution of 1 hydro uoric acid or 10sodium hydroxide) were activated with a 1ndash5 solution of polylysine The activated beadswere placed in a suspension of Burkholderia sp and centrifuged at 8000 pound g for 5 minA single bacteria-coated bead was attached to a cantilever using a small quantity of epoxyresin which has previously been found to be inert in aqueous solutions (Pincet 1995 YoonFlinn and Rabinovich 1997) This attachment procedure was conducted in solution usinga micromanipulator

Prior to BFM measurements scanning laser confocal microscopy (Zeiss LSM 510)was used to characterize the three dimensional nature of a BAFP A probe was placed inthe uid cell used for force measurements and imaged with a 100x 14 NA objective

66 S K Lower et al

This imaging procedure was facilitated by transforming bacteria with a plasmid (pGLOBio-Rad or pSMC2 provided by G A OrsquoToole Dartmouth University) that encoded anintracellular green uorescent protein The bacteria uoresced when excited by light at458 nm or 488 nm Fluorescence emitted by the epoxy resin revealed that it was con nedto the region between the cantilever and glass bead (ie resin was not in a position thatwould alter the interaction between the bacteria and mineral during BFM experiments)

Cantilever spring constants (N miexcl1) essential for measuring force magnitudes with aforce microscope can vary substantially from the nominal value listed by the manufacturer(ClevelandManne Bocek and Hansma 1993 Senden and Ducker 1994) Spring constantswere determined by attaching known masses to the end of a cantilever and recording thechange in cantilever resonant frequency (Cleveland et al 1993) A linear relationship wasobserved between added mass and resonant frequency with the slope being the springconstant (017 N miexcl1) These measurements were accomplished using 10 cantilevers fromthe same wafer as that used to create BAFPs The variability of spring constants fromcantilever to cantileverwithin the same wafer has been determined to be very small (Sendenand Ducker 1994) This was con rmed by the linear t of our data and the reproducibilityof the resonant frequency of unloaded cantilevers (137 kHz sect 02)

Force Measurements

BFM measurements were performed using a NanoScope IIIa Multimode SPM (DigitalInstruments Santa Barbara CA) in aqueous solution (pH 6 ionic strength 10iexcl5 M 25plusmnC)A piezoelectric scanner was used to translate the mineral toward and away from bacteriaon a BAFP at rates of 01 to 3 sup1m seciexcl1 For comparison this is within the range ofvelocitiesof motilebacteria(Marshall 1976) Interfacial forces were measured as themineralapproached the bacteria on the probe whereas adhesion forces were measured upon contactand subsequent retraction of the mineral from the bacteria Mineral samples were drivento the same contact force to normalize the effect that loading can have on adhesion forcesduring retraction (Weisenhorn Maivald Butt and Hansma 1992)

Force measurement data are collected as cantilever de ection (diode voltage) and cor-responding piezo displacement typically termed a force curve (Figure 2A) This data mustbe manipulatedto produce the familiar forcendashdistance curvewhich describes interfacial andadhesion forces (Figure 2B) Distance (ie separation between bacteria and mineral) is cal-culatedbycorrecting the recordedpiezoposition(ie displacement) by themeasured de ec-tion of the cantileverFor example if the mineral attached to the piezo scanner moves 10 nmtoward bacteria attached to the cantilever and the bacteria are repelled 2 nm due to repul-sive forces then the actual mineralndashbacteria distance (or separation) changes by only 8 nmThe distance axis origin is chosen as the point on the force curve where sensor de ectionbecomes a linear function of piezo displacement (the sensor is in contact with the sample)

Force (F ) is determinedusing Hookersquos Law F D kspd where d is cantileverde ection(meters) and ksp (N miexcl1) is the cantilever spring constant In order to use Hookersquos Lawcantilever de ection measured by the photodiode in volts must be converted to metersA diodedisplacement conversion factor (also called ldquooptical lever sensitivityrdquo) is de nedfrom the slope of the force curve region where the cantilever is in contact with the sampleon the piezo (Figure 2Aiii called the ldquoregion of constant compliancerdquo) The reciprocal ofthis conversion factor (in nm Viexcl1) can be used to convert measured cantilever de ection involts to meters A zero-force reference value is determined as the force curve region wheresensor de ection is independent of piezo displacement (Figure 2Ai the sensor and sampleare not interacting because they are far apart)

It is important to note that using the constant compliance region of the force curveto convert photodiode response into force will overestimate the force of interaction if thebacteria are more compliant than the cantilever Recent measurements of the elasticity

Biological Force Microscopy and Interfaces 67

FIGURE 2 Schematic diagrams of a force curve (A) and force-distance curve (B) Whenthe sensor and sample are far apart (i) they exhibit no interaction (region of zero force) Asthe sample approaches the sensor intermolecular forces between the bacteria and mineralcause the sensor to de ect upward (ii) due to repulsive forces shown here Eventually theprobe makes contact with the sample (iii) and their movement becomes coupled (regionof constant compliance) The sample is then retracted from the probe (iv) until the sensorand sample return to their original positions thereby completing one cycle (an entire cyclerequires nano- to milliseconds) Hysteresis shown here may occur upon retraction dueto adhesion forces See text for discussion on converting cantilever de ection and piezodisplacement into force and distance respectively Interfacial forces are measured on ap-proach and adhesion forces are measured upon retraction repulsive forces are positive andattractive forces are negative

68 S K Lower et al

FIGURE 3 Variation of photodiode shift voltage as a function of optical lever sensitivityusing a J-type piezoelectric scanner and eight silicon nitride cantilevers (200 sup1m in length)All measurements were performed on muscovite and hematite in a uid cell containingaqueous solutions ranging in ionic strength from 0 (ie Milli-Q water) to 10iexcl2M Theslope of the regression line is 642 nm and the intercept is 13 V (R2 D 095) With thiscorrelation the optical lever sensitivity can be determined in a matter of seconds by simplyusing the photodiode shift voltage Furthermore this method is valid regardless of whetherthe cantilever is the most compliant component of the system

of bacterial surface macromolecules suggest that bacteria are less compliant (ie stiffer)than cantilevers having spring constants smaller than 10 N miexcl1 (Xu Mulhern BlackfordJericho Firtel and Beveridge 1996 Yao Jericho Pink and Beveridge 1999)

In instances where bacteria (or biomolecules) linked to the cantilever are more com-pliant than the cantilever or for cells with fragile appendages other methods must be usedto accurately convert the measured de ection of the cantilever (in volts) into a force (inNewtons) of interaction (DrsquoCosta and Hoh 1995 Sader Chon and Mulvaney 1999) Forexample because the optical lever sensitivity is strongly dependenton the shape of the laserspot on the photodiodedetector the ldquophotodiode shift voltagerdquo can be used to convert voltsof cantileverde ection into meters of de ection (DrsquoCosta and Hoh 1995) Photodiode shiftvoltage is measured as the difference in output voltage when the photodiode detector isshifted approximately 318 sup1m (one full turn of the positioning screw) on either side of thezero setting Figure 3 illustrates the strong correlation between photodiode shift voltagesand optical lever sensitivities measured by directing the laser to different positions on thecantileverOnce this correlation is established for a given instrument piezoelectric scanner uid cell and cantilever (eg 200-sup1m long V-shaped silicon nitride cantilevers) the op-tical lever sensitivity can be accurately determined without pressing the cantilever againstany other surface This method ensures that forces can be determined regardless of thecompliance of the cantilever relative to any microorganism attached to it and also ensuresthe preservation of fragile macromolecules on microorganisms attached to the cantilever

Results and Discussion

Characterization of Biologically Active Force Probes

BAFPs were created by either attaching a single bacterium to a force sensor (Figure 4A)or by attaching a single bacteria-coated-bead (Figure 4B) to a force sensor Fluorescent

FIGURE 4 (A) Scanning laser micrograph of a BAFP created by directly linking a singlecell of Burkholderia sp to a force-sensing cantilever (B) Confocal micrograph focused onthe midplane of bacteria-coated beads revealing a layer of cells Focusing up and downon the beads reveal that many are evenly coated with cells Beads showing the most uni-form coverage are attached to cantilevers thereby creating BAFPs These cells have beentransformed with a plasmid encoding a green uorescent protein and are emitting ldquonaturalrdquo uorescence This uorescent protein being intracellular together with the confocal abili-ties of the laser scanning microscope allow noninvasive characterization of the orientationand distribution of cells on the force sensor in situ without affecting the surface chemistryof the microbes Plating experiments in which BAFPs were used as the inoculum revealedthat the bacteria on the sensors are viableand presumably activeduring force measurementsScale bars D 10 sup1m

69

70 S K Lower et al

dyes (ViaGram Molecular Probes Eugene OR) and plating techniques revealed that cellmembranes on these bacteria are intact and the cells are viable Both the direct and indirectmethod of attaching bacteria to a cantileverpreserves the orientationand structural integrityof macromolecules on the cell surface This is important because the native conformationof macromolecules on a bacteria surface is critical to their function activity and role innature By using whole bacteria expressing macromolecules in their natural state ratherthan individual biomolecules (eg exopolysaccharides proteins) puri ed from bacterialsurfaces we avoid situations in which the linkage procedure modi es the conformation ofbiomolecules such that they are no longer in a natural state (Stotzky 1986 Ellen and Burne1996 Turner Peek Wertz Archibald Geer and Gaber 1996 Ingersoll and Bright 1997Turner Testoff Conrad and Gaber 1997)

As both gram-negative and gram-positive bacteria are typically negatively charged atmost pH polylysine with its high pKa is a more-or-less universal linker molecule How-ever due to the large diversity in bacterial surfaces situations will likely arise in whichother linker molecules are necessary The versatility of our linkage protocol allows formodi cation on a case-by-case basis For example we have also fabricated BAFPs usingaminopropyltriethoxysilane and polyethyleneimine with species of Shewanella and Pseu-domonas Additionallymethods used to attach proteins or antibodies to small glass spheresor sensors (eg Florin Moy and Gaub 1994 Frey and Corn 1996 Turner et al 1997Caruso Caruso and Mohwald 1998 Rezania Johnson Lefkow and Healy 1999) could beapplied to our protocol For example lysozyme which has been genetically altered suchthat it possesses an active binding site but an inactive catalytic site (Voet and Voet 1995) isan attractive choice because of its high af nity for bacterial cell walls Antibodies speci cfor cell surface receptors may also be effective linkers

A critical component to consider when designing BAFPs is the relative difference be-tween thoseforces bindingthecells to thesensor and interfacial and adhesiveforces thatwillbeprobedby thecellson the sensor Forces holdingbacteria to the sensormustbe greater thanthe forces that are actuallyprobedotherwise the bacteriawill be stripped from the cantileverduring force measurements Electrostatic attachment of bacteria to sensors via polylysineis very strong Approximations using Coulombrsquos law and molecular dynamic calculationsshow that the energy associated with aminendashsilanol (ie the polylysine glass bead linkageor polylysine cantilever linkage) and aminendashcarboxylic pairs (ie the polylysine bacterialinkage) (Voet and Voet 1995 West Latour and Hench 1997 Pagac Prieve and Tilton1998 Pagac Tilton and Prieve 1998) are at least one to four orders of magnitude strongerthan potential intermolecular forces between a bacteria and mineral (Israelachvili 1992)Organosilane linkages such as aminopropyltriethoxysilane form covalentbondswith silanolgroups (Plueddemann1991) and should therefore be even stronger than polylysinelinkages

Comparison of the direct and indirect linkage protocols suggests that the latter ismore desirable Direct linkage of a single cell to a cantilever (Figure 4A) is very dif cultTechniques such as optical tweezers (Svoboda and Block 1994) or nanotweezers (Kim andLieber 1999) could signi cantly enhance the linkage of a bacterium to a cantilever butsuch techniques are not at present easily applicable to such procedures Another practicalreason for selecting the indirect protocol is the need to use cantilevers with appropriatespring constants in order to probe the entire range of potential forces between microbes andminerals Cantilevers of different composition (eg glass silicon silicon nitride and gold-coated cantilevers) are often used to vary the spring constant Gold-coated and uncoatedsilicon nitride cantilevers for example have very different surface properties and siliconnitride itself can vary from Si3N4 to Si15N4 (WeisenhornMaivaldButt and Hansma 1992)

Biological Force Microscopy and Interfaces 71

These compositional differences would require use of more than one linker molecule todirectly attach one particular strain of bacteria to different cantilevers Conversely indirectlinkage allows the use of one protocol to attach bacteria to glass (or latex) beads of uniformcomposition which can then be attached to cantilevers of different compositions

Forces of Interaction Between Bacteria and Mineral Surfaces in situ

The interactions between muscovite and polylysine (Figure 5A) and muscovite and the soilBurkholderia sp (Figures 5B and 5C) were studied with BFM at pH 6 ionic strength 10iexcl5

M 25plusmnC As expected the positively charged polylysine exhibited a strong attraction to-wards the (001) surface of muscovite which is negatively charged (Figure 5A) Hysteresiswas not observed for the interaction between muscovite and polylysine In contrast theBurkholderia sp exhibited a repulsive interaction with muscovite beginning at raquo80 nm(Figure 5B approach) However once contact was made this repulsive force was over-come and the bacteria exhibited an attractive adhesion toward the mineral (Figure 5Bretraction)

The repulsive interfacial forces between the Burkholderia sp and muscovite illustratedin Figure 5B (approach) are consistent with the negative surface charges of both the bac-teria and mineral at circum-neutral pH The distance at which repulsion occurs is slightlysmaller than that expectedbased solely on electrostatic interactions(Debye length thicknessis raquo100 nm at this ionic strength) Other forces associated with hydrophobic steric andentropic effects likely play a key role in bacteriandashmineral interactions due to the relativelylongpolymerson bacterial surfaces (Israelachviliand McGuiggan1988 Israelachvili1992)

The adhesive behavior between Burkholderia sp and muscovite is drawn out for hun-dreds of nanometers (Figure 5B retraction) This is likely due to stretching and brillationof biomolecules (eg lipopolysaccharides or agella) as a result of attractive forces suchas hydrogen bonds between polymers on the bacteria and surface hydroxyls or structuredwater molecules on muscovite (Jucker Harms Hug and Zehnder 1997)

When the same Burkholderia sp was grown in nutrient poor media it exhibited verystrong af nity for muscovite (Figure 5C) Interfacial repulsion was not detected (Figure 5Capproach) and attractive adhesion forces were very large and long range (Figure 5Cretraction) This phenomenais consistentwith theobservationthatmany bacterial speciesal-ter their surface propertiesunderoligotrophicversus eutropicenvironments(eg Bengtsson1991) In fact many bacteria show greater af nity for mineral surfaces under oligotrophicrather than eutropic conditions (Fletcher 1996b Marshall 1996) This cycling between at-tached and planktonic states is believed to be a strategy bacteria use to proliferate in naturewhere many environments have eutropicoligotropic sequences

Potential Applications of Biological Force Microscopy to Geomicrobiology Studies

BFM could providea uniqueperspectiveinto several aspects of geomicrobiology(egmin-eral dissolution crystal growth and bio lm formation) particularly if used to complementother techniques Next is a list of relevant questions considered as possible applications ofBFM to the eld of geomicrobiology and their answers

1 Do bacteria ldquorecognizerdquo particular minerals or crystallographic orientations Probe dif-ferent minerals or different faces on the same mineral with one BAFP

72 S K Lower et al

FIGURE 5 Biological force microcopy (BFM) force-distance curves between the (001)surface of muscovite and a polylysine functionalized cantilever (A) versus Burkholderiasp grown in a eutropic environment (B) versus Burkholderia sp grown in an oligotropicenvironment (C) Solution conditionswere pH 6 ionic strength 10iexcl5M 25plusmnC The force ofinteraction is plotted as a function of the distance between the two surfaces (ie separationbetween polylysine or bacteria on the sensor and the mineral surface) Note difference inx-axis scale Curves begin on the right and proceed toward the left as the mineral is broughttoward the sensor (solid circles approach curve) The sensor makes contactwith the mineralsurface and subsequently withdraws from the muscovite (open squares retraction curve) tocomplete one cycle

2 Do bacteria show an increased af nity for particular minerals under oligotrophic oranaerobicconditionsChangesolutionconditionswithin uid cell while collectingBFMmeasurements

3 How does inter- and intraspecies communication affect bio lm development Use aBAFP to measure interactions with various species of bacteria that are immobilized ona substrate

4 What intermolecular forces (eg van der Waals electrostatics hydration hydrophobicentropic) are involvedin bacterial attachmentand detachmentprocessesCompare forcesmeasured with BFM to those calculated from theoretical models (eg DLVO theory)

5 What cell surface macromoleculesmediatebacterial attachment to mineral surfaces Areconditioning lms required for attachment Create BAFPs using various mutants that

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

Biological Force Microscopy and Interfaces 65

FIGURE 1 Schematic diagram showing the key components of a force microscope Themineral and force sensor are within a uid cell (not shown) containing aqueous solutionForce measurements are made by recording the de ection of a sensor (ie cantilever) in re-sponse to attractiveor repulsive forces between itself and the sample (a mineral in this case)The sample mounted on a piezoelectric scanner indexes toward makes contact with andretracts from the sensor De ection of the sensor is detected by re ecting a laser off the topof the sensor and into a split segment photodiodeIn this study bacteria (shown as a sphere)havebeen linkedto the sensor therebycreatinga biologicallyactive force probe(BAFP) usedto measure interfacial and adhesive forces between bacteria and mineral surfaces in situ

Force Sensor Preparation and Characterization

Biologicallyactive force probes (BAFPs) can be createdby linkingbacteria either directly toa siliconor siliconnitride force sensor (ie cantilever) or indirectly by linking a monolayerof bacteria to a small glass bead that is then xed to the end of a cantilever Direct linkage ofbacteria to the cantilever was accomplished by rst placing a cantilever in a 1ndash5 solutionof the polycationic molecule poly-D-lysine (135 to 150 kDa pH near neutral) Bacteriawere linked to the polylysine functionalized cantilever by lowering it into a colony of livebacteria with the aid of a microscope (Nikon 200x magni cation) and a micromanipulatorto translate the cantilever For the indirect-linkagemethod small glass beads (Polysciencesor Duke Scienti c radii 3ndash7 sup1m cleaned with a solution of 1 hydro uoric acid or 10sodium hydroxide) were activated with a 1ndash5 solution of polylysine The activated beadswere placed in a suspension of Burkholderia sp and centrifuged at 8000 pound g for 5 minA single bacteria-coated bead was attached to a cantilever using a small quantity of epoxyresin which has previously been found to be inert in aqueous solutions (Pincet 1995 YoonFlinn and Rabinovich 1997) This attachment procedure was conducted in solution usinga micromanipulator

Prior to BFM measurements scanning laser confocal microscopy (Zeiss LSM 510)was used to characterize the three dimensional nature of a BAFP A probe was placed inthe uid cell used for force measurements and imaged with a 100x 14 NA objective

66 S K Lower et al

This imaging procedure was facilitated by transforming bacteria with a plasmid (pGLOBio-Rad or pSMC2 provided by G A OrsquoToole Dartmouth University) that encoded anintracellular green uorescent protein The bacteria uoresced when excited by light at458 nm or 488 nm Fluorescence emitted by the epoxy resin revealed that it was con nedto the region between the cantilever and glass bead (ie resin was not in a position thatwould alter the interaction between the bacteria and mineral during BFM experiments)

Cantilever spring constants (N miexcl1) essential for measuring force magnitudes with aforce microscope can vary substantially from the nominal value listed by the manufacturer(ClevelandManne Bocek and Hansma 1993 Senden and Ducker 1994) Spring constantswere determined by attaching known masses to the end of a cantilever and recording thechange in cantilever resonant frequency (Cleveland et al 1993) A linear relationship wasobserved between added mass and resonant frequency with the slope being the springconstant (017 N miexcl1) These measurements were accomplished using 10 cantilevers fromthe same wafer as that used to create BAFPs The variability of spring constants fromcantilever to cantileverwithin the same wafer has been determined to be very small (Sendenand Ducker 1994) This was con rmed by the linear t of our data and the reproducibilityof the resonant frequency of unloaded cantilevers (137 kHz sect 02)

Force Measurements

BFM measurements were performed using a NanoScope IIIa Multimode SPM (DigitalInstruments Santa Barbara CA) in aqueous solution (pH 6 ionic strength 10iexcl5 M 25plusmnC)A piezoelectric scanner was used to translate the mineral toward and away from bacteriaon a BAFP at rates of 01 to 3 sup1m seciexcl1 For comparison this is within the range ofvelocitiesof motilebacteria(Marshall 1976) Interfacial forces were measured as themineralapproached the bacteria on the probe whereas adhesion forces were measured upon contactand subsequent retraction of the mineral from the bacteria Mineral samples were drivento the same contact force to normalize the effect that loading can have on adhesion forcesduring retraction (Weisenhorn Maivald Butt and Hansma 1992)

Force measurement data are collected as cantilever de ection (diode voltage) and cor-responding piezo displacement typically termed a force curve (Figure 2A) This data mustbe manipulatedto produce the familiar forcendashdistance curvewhich describes interfacial andadhesion forces (Figure 2B) Distance (ie separation between bacteria and mineral) is cal-culatedbycorrecting the recordedpiezoposition(ie displacement) by themeasured de ec-tion of the cantileverFor example if the mineral attached to the piezo scanner moves 10 nmtoward bacteria attached to the cantilever and the bacteria are repelled 2 nm due to repul-sive forces then the actual mineralndashbacteria distance (or separation) changes by only 8 nmThe distance axis origin is chosen as the point on the force curve where sensor de ectionbecomes a linear function of piezo displacement (the sensor is in contact with the sample)

Force (F ) is determinedusing Hookersquos Law F D kspd where d is cantileverde ection(meters) and ksp (N miexcl1) is the cantilever spring constant In order to use Hookersquos Lawcantilever de ection measured by the photodiode in volts must be converted to metersA diodedisplacement conversion factor (also called ldquooptical lever sensitivityrdquo) is de nedfrom the slope of the force curve region where the cantilever is in contact with the sampleon the piezo (Figure 2Aiii called the ldquoregion of constant compliancerdquo) The reciprocal ofthis conversion factor (in nm Viexcl1) can be used to convert measured cantilever de ection involts to meters A zero-force reference value is determined as the force curve region wheresensor de ection is independent of piezo displacement (Figure 2Ai the sensor and sampleare not interacting because they are far apart)

It is important to note that using the constant compliance region of the force curveto convert photodiode response into force will overestimate the force of interaction if thebacteria are more compliant than the cantilever Recent measurements of the elasticity

Biological Force Microscopy and Interfaces 67

FIGURE 2 Schematic diagrams of a force curve (A) and force-distance curve (B) Whenthe sensor and sample are far apart (i) they exhibit no interaction (region of zero force) Asthe sample approaches the sensor intermolecular forces between the bacteria and mineralcause the sensor to de ect upward (ii) due to repulsive forces shown here Eventually theprobe makes contact with the sample (iii) and their movement becomes coupled (regionof constant compliance) The sample is then retracted from the probe (iv) until the sensorand sample return to their original positions thereby completing one cycle (an entire cyclerequires nano- to milliseconds) Hysteresis shown here may occur upon retraction dueto adhesion forces See text for discussion on converting cantilever de ection and piezodisplacement into force and distance respectively Interfacial forces are measured on ap-proach and adhesion forces are measured upon retraction repulsive forces are positive andattractive forces are negative

68 S K Lower et al

FIGURE 3 Variation of photodiode shift voltage as a function of optical lever sensitivityusing a J-type piezoelectric scanner and eight silicon nitride cantilevers (200 sup1m in length)All measurements were performed on muscovite and hematite in a uid cell containingaqueous solutions ranging in ionic strength from 0 (ie Milli-Q water) to 10iexcl2M Theslope of the regression line is 642 nm and the intercept is 13 V (R2 D 095) With thiscorrelation the optical lever sensitivity can be determined in a matter of seconds by simplyusing the photodiode shift voltage Furthermore this method is valid regardless of whetherthe cantilever is the most compliant component of the system

of bacterial surface macromolecules suggest that bacteria are less compliant (ie stiffer)than cantilevers having spring constants smaller than 10 N miexcl1 (Xu Mulhern BlackfordJericho Firtel and Beveridge 1996 Yao Jericho Pink and Beveridge 1999)

In instances where bacteria (or biomolecules) linked to the cantilever are more com-pliant than the cantilever or for cells with fragile appendages other methods must be usedto accurately convert the measured de ection of the cantilever (in volts) into a force (inNewtons) of interaction (DrsquoCosta and Hoh 1995 Sader Chon and Mulvaney 1999) Forexample because the optical lever sensitivity is strongly dependenton the shape of the laserspot on the photodiodedetector the ldquophotodiode shift voltagerdquo can be used to convert voltsof cantileverde ection into meters of de ection (DrsquoCosta and Hoh 1995) Photodiode shiftvoltage is measured as the difference in output voltage when the photodiode detector isshifted approximately 318 sup1m (one full turn of the positioning screw) on either side of thezero setting Figure 3 illustrates the strong correlation between photodiode shift voltagesand optical lever sensitivities measured by directing the laser to different positions on thecantileverOnce this correlation is established for a given instrument piezoelectric scanner uid cell and cantilever (eg 200-sup1m long V-shaped silicon nitride cantilevers) the op-tical lever sensitivity can be accurately determined without pressing the cantilever againstany other surface This method ensures that forces can be determined regardless of thecompliance of the cantilever relative to any microorganism attached to it and also ensuresthe preservation of fragile macromolecules on microorganisms attached to the cantilever

Results and Discussion

Characterization of Biologically Active Force Probes

BAFPs were created by either attaching a single bacterium to a force sensor (Figure 4A)or by attaching a single bacteria-coated-bead (Figure 4B) to a force sensor Fluorescent

FIGURE 4 (A) Scanning laser micrograph of a BAFP created by directly linking a singlecell of Burkholderia sp to a force-sensing cantilever (B) Confocal micrograph focused onthe midplane of bacteria-coated beads revealing a layer of cells Focusing up and downon the beads reveal that many are evenly coated with cells Beads showing the most uni-form coverage are attached to cantilevers thereby creating BAFPs These cells have beentransformed with a plasmid encoding a green uorescent protein and are emitting ldquonaturalrdquo uorescence This uorescent protein being intracellular together with the confocal abili-ties of the laser scanning microscope allow noninvasive characterization of the orientationand distribution of cells on the force sensor in situ without affecting the surface chemistryof the microbes Plating experiments in which BAFPs were used as the inoculum revealedthat the bacteria on the sensors are viableand presumably activeduring force measurementsScale bars D 10 sup1m

69

70 S K Lower et al

dyes (ViaGram Molecular Probes Eugene OR) and plating techniques revealed that cellmembranes on these bacteria are intact and the cells are viable Both the direct and indirectmethod of attaching bacteria to a cantileverpreserves the orientationand structural integrityof macromolecules on the cell surface This is important because the native conformationof macromolecules on a bacteria surface is critical to their function activity and role innature By using whole bacteria expressing macromolecules in their natural state ratherthan individual biomolecules (eg exopolysaccharides proteins) puri ed from bacterialsurfaces we avoid situations in which the linkage procedure modi es the conformation ofbiomolecules such that they are no longer in a natural state (Stotzky 1986 Ellen and Burne1996 Turner Peek Wertz Archibald Geer and Gaber 1996 Ingersoll and Bright 1997Turner Testoff Conrad and Gaber 1997)

As both gram-negative and gram-positive bacteria are typically negatively charged atmost pH polylysine with its high pKa is a more-or-less universal linker molecule How-ever due to the large diversity in bacterial surfaces situations will likely arise in whichother linker molecules are necessary The versatility of our linkage protocol allows formodi cation on a case-by-case basis For example we have also fabricated BAFPs usingaminopropyltriethoxysilane and polyethyleneimine with species of Shewanella and Pseu-domonas Additionallymethods used to attach proteins or antibodies to small glass spheresor sensors (eg Florin Moy and Gaub 1994 Frey and Corn 1996 Turner et al 1997Caruso Caruso and Mohwald 1998 Rezania Johnson Lefkow and Healy 1999) could beapplied to our protocol For example lysozyme which has been genetically altered suchthat it possesses an active binding site but an inactive catalytic site (Voet and Voet 1995) isan attractive choice because of its high af nity for bacterial cell walls Antibodies speci cfor cell surface receptors may also be effective linkers

A critical component to consider when designing BAFPs is the relative difference be-tween thoseforces bindingthecells to thesensor and interfacial and adhesiveforces thatwillbeprobedby thecellson the sensor Forces holdingbacteria to the sensormustbe greater thanthe forces that are actuallyprobedotherwise the bacteriawill be stripped from the cantileverduring force measurements Electrostatic attachment of bacteria to sensors via polylysineis very strong Approximations using Coulombrsquos law and molecular dynamic calculationsshow that the energy associated with aminendashsilanol (ie the polylysine glass bead linkageor polylysine cantilever linkage) and aminendashcarboxylic pairs (ie the polylysine bacterialinkage) (Voet and Voet 1995 West Latour and Hench 1997 Pagac Prieve and Tilton1998 Pagac Tilton and Prieve 1998) are at least one to four orders of magnitude strongerthan potential intermolecular forces between a bacteria and mineral (Israelachvili 1992)Organosilane linkages such as aminopropyltriethoxysilane form covalentbondswith silanolgroups (Plueddemann1991) and should therefore be even stronger than polylysinelinkages

Comparison of the direct and indirect linkage protocols suggests that the latter ismore desirable Direct linkage of a single cell to a cantilever (Figure 4A) is very dif cultTechniques such as optical tweezers (Svoboda and Block 1994) or nanotweezers (Kim andLieber 1999) could signi cantly enhance the linkage of a bacterium to a cantilever butsuch techniques are not at present easily applicable to such procedures Another practicalreason for selecting the indirect protocol is the need to use cantilevers with appropriatespring constants in order to probe the entire range of potential forces between microbes andminerals Cantilevers of different composition (eg glass silicon silicon nitride and gold-coated cantilevers) are often used to vary the spring constant Gold-coated and uncoatedsilicon nitride cantilevers for example have very different surface properties and siliconnitride itself can vary from Si3N4 to Si15N4 (WeisenhornMaivaldButt and Hansma 1992)

Biological Force Microscopy and Interfaces 71

These compositional differences would require use of more than one linker molecule todirectly attach one particular strain of bacteria to different cantilevers Conversely indirectlinkage allows the use of one protocol to attach bacteria to glass (or latex) beads of uniformcomposition which can then be attached to cantilevers of different compositions

Forces of Interaction Between Bacteria and Mineral Surfaces in situ

The interactions between muscovite and polylysine (Figure 5A) and muscovite and the soilBurkholderia sp (Figures 5B and 5C) were studied with BFM at pH 6 ionic strength 10iexcl5

M 25plusmnC As expected the positively charged polylysine exhibited a strong attraction to-wards the (001) surface of muscovite which is negatively charged (Figure 5A) Hysteresiswas not observed for the interaction between muscovite and polylysine In contrast theBurkholderia sp exhibited a repulsive interaction with muscovite beginning at raquo80 nm(Figure 5B approach) However once contact was made this repulsive force was over-come and the bacteria exhibited an attractive adhesion toward the mineral (Figure 5Bretraction)

The repulsive interfacial forces between the Burkholderia sp and muscovite illustratedin Figure 5B (approach) are consistent with the negative surface charges of both the bac-teria and mineral at circum-neutral pH The distance at which repulsion occurs is slightlysmaller than that expectedbased solely on electrostatic interactions(Debye length thicknessis raquo100 nm at this ionic strength) Other forces associated with hydrophobic steric andentropic effects likely play a key role in bacteriandashmineral interactions due to the relativelylongpolymerson bacterial surfaces (Israelachviliand McGuiggan1988 Israelachvili1992)

The adhesive behavior between Burkholderia sp and muscovite is drawn out for hun-dreds of nanometers (Figure 5B retraction) This is likely due to stretching and brillationof biomolecules (eg lipopolysaccharides or agella) as a result of attractive forces suchas hydrogen bonds between polymers on the bacteria and surface hydroxyls or structuredwater molecules on muscovite (Jucker Harms Hug and Zehnder 1997)

When the same Burkholderia sp was grown in nutrient poor media it exhibited verystrong af nity for muscovite (Figure 5C) Interfacial repulsion was not detected (Figure 5Capproach) and attractive adhesion forces were very large and long range (Figure 5Cretraction) This phenomenais consistentwith theobservationthatmany bacterial speciesal-ter their surface propertiesunderoligotrophicversus eutropicenvironments(eg Bengtsson1991) In fact many bacteria show greater af nity for mineral surfaces under oligotrophicrather than eutropic conditions (Fletcher 1996b Marshall 1996) This cycling between at-tached and planktonic states is believed to be a strategy bacteria use to proliferate in naturewhere many environments have eutropicoligotropic sequences

Potential Applications of Biological Force Microscopy to Geomicrobiology Studies

BFM could providea uniqueperspectiveinto several aspects of geomicrobiology(egmin-eral dissolution crystal growth and bio lm formation) particularly if used to complementother techniques Next is a list of relevant questions considered as possible applications ofBFM to the eld of geomicrobiology and their answers

1 Do bacteria ldquorecognizerdquo particular minerals or crystallographic orientations Probe dif-ferent minerals or different faces on the same mineral with one BAFP

72 S K Lower et al

FIGURE 5 Biological force microcopy (BFM) force-distance curves between the (001)surface of muscovite and a polylysine functionalized cantilever (A) versus Burkholderiasp grown in a eutropic environment (B) versus Burkholderia sp grown in an oligotropicenvironment (C) Solution conditionswere pH 6 ionic strength 10iexcl5M 25plusmnC The force ofinteraction is plotted as a function of the distance between the two surfaces (ie separationbetween polylysine or bacteria on the sensor and the mineral surface) Note difference inx-axis scale Curves begin on the right and proceed toward the left as the mineral is broughttoward the sensor (solid circles approach curve) The sensor makes contactwith the mineralsurface and subsequently withdraws from the muscovite (open squares retraction curve) tocomplete one cycle

2 Do bacteria show an increased af nity for particular minerals under oligotrophic oranaerobicconditionsChangesolutionconditionswithin uid cell while collectingBFMmeasurements

3 How does inter- and intraspecies communication affect bio lm development Use aBAFP to measure interactions with various species of bacteria that are immobilized ona substrate

4 What intermolecular forces (eg van der Waals electrostatics hydration hydrophobicentropic) are involvedin bacterial attachmentand detachmentprocessesCompare forcesmeasured with BFM to those calculated from theoretical models (eg DLVO theory)

5 What cell surface macromoleculesmediatebacterial attachment to mineral surfaces Areconditioning lms required for attachment Create BAFPs using various mutants that

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

66 S K Lower et al

This imaging procedure was facilitated by transforming bacteria with a plasmid (pGLOBio-Rad or pSMC2 provided by G A OrsquoToole Dartmouth University) that encoded anintracellular green uorescent protein The bacteria uoresced when excited by light at458 nm or 488 nm Fluorescence emitted by the epoxy resin revealed that it was con nedto the region between the cantilever and glass bead (ie resin was not in a position thatwould alter the interaction between the bacteria and mineral during BFM experiments)

Cantilever spring constants (N miexcl1) essential for measuring force magnitudes with aforce microscope can vary substantially from the nominal value listed by the manufacturer(ClevelandManne Bocek and Hansma 1993 Senden and Ducker 1994) Spring constantswere determined by attaching known masses to the end of a cantilever and recording thechange in cantilever resonant frequency (Cleveland et al 1993) A linear relationship wasobserved between added mass and resonant frequency with the slope being the springconstant (017 N miexcl1) These measurements were accomplished using 10 cantilevers fromthe same wafer as that used to create BAFPs The variability of spring constants fromcantilever to cantileverwithin the same wafer has been determined to be very small (Sendenand Ducker 1994) This was con rmed by the linear t of our data and the reproducibilityof the resonant frequency of unloaded cantilevers (137 kHz sect 02)

Force Measurements

BFM measurements were performed using a NanoScope IIIa Multimode SPM (DigitalInstruments Santa Barbara CA) in aqueous solution (pH 6 ionic strength 10iexcl5 M 25plusmnC)A piezoelectric scanner was used to translate the mineral toward and away from bacteriaon a BAFP at rates of 01 to 3 sup1m seciexcl1 For comparison this is within the range ofvelocitiesof motilebacteria(Marshall 1976) Interfacial forces were measured as themineralapproached the bacteria on the probe whereas adhesion forces were measured upon contactand subsequent retraction of the mineral from the bacteria Mineral samples were drivento the same contact force to normalize the effect that loading can have on adhesion forcesduring retraction (Weisenhorn Maivald Butt and Hansma 1992)

Force measurement data are collected as cantilever de ection (diode voltage) and cor-responding piezo displacement typically termed a force curve (Figure 2A) This data mustbe manipulatedto produce the familiar forcendashdistance curvewhich describes interfacial andadhesion forces (Figure 2B) Distance (ie separation between bacteria and mineral) is cal-culatedbycorrecting the recordedpiezoposition(ie displacement) by themeasured de ec-tion of the cantileverFor example if the mineral attached to the piezo scanner moves 10 nmtoward bacteria attached to the cantilever and the bacteria are repelled 2 nm due to repul-sive forces then the actual mineralndashbacteria distance (or separation) changes by only 8 nmThe distance axis origin is chosen as the point on the force curve where sensor de ectionbecomes a linear function of piezo displacement (the sensor is in contact with the sample)

Force (F ) is determinedusing Hookersquos Law F D kspd where d is cantileverde ection(meters) and ksp (N miexcl1) is the cantilever spring constant In order to use Hookersquos Lawcantilever de ection measured by the photodiode in volts must be converted to metersA diodedisplacement conversion factor (also called ldquooptical lever sensitivityrdquo) is de nedfrom the slope of the force curve region where the cantilever is in contact with the sampleon the piezo (Figure 2Aiii called the ldquoregion of constant compliancerdquo) The reciprocal ofthis conversion factor (in nm Viexcl1) can be used to convert measured cantilever de ection involts to meters A zero-force reference value is determined as the force curve region wheresensor de ection is independent of piezo displacement (Figure 2Ai the sensor and sampleare not interacting because they are far apart)

It is important to note that using the constant compliance region of the force curveto convert photodiode response into force will overestimate the force of interaction if thebacteria are more compliant than the cantilever Recent measurements of the elasticity

Biological Force Microscopy and Interfaces 67

FIGURE 2 Schematic diagrams of a force curve (A) and force-distance curve (B) Whenthe sensor and sample are far apart (i) they exhibit no interaction (region of zero force) Asthe sample approaches the sensor intermolecular forces between the bacteria and mineralcause the sensor to de ect upward (ii) due to repulsive forces shown here Eventually theprobe makes contact with the sample (iii) and their movement becomes coupled (regionof constant compliance) The sample is then retracted from the probe (iv) until the sensorand sample return to their original positions thereby completing one cycle (an entire cyclerequires nano- to milliseconds) Hysteresis shown here may occur upon retraction dueto adhesion forces See text for discussion on converting cantilever de ection and piezodisplacement into force and distance respectively Interfacial forces are measured on ap-proach and adhesion forces are measured upon retraction repulsive forces are positive andattractive forces are negative

68 S K Lower et al

FIGURE 3 Variation of photodiode shift voltage as a function of optical lever sensitivityusing a J-type piezoelectric scanner and eight silicon nitride cantilevers (200 sup1m in length)All measurements were performed on muscovite and hematite in a uid cell containingaqueous solutions ranging in ionic strength from 0 (ie Milli-Q water) to 10iexcl2M Theslope of the regression line is 642 nm and the intercept is 13 V (R2 D 095) With thiscorrelation the optical lever sensitivity can be determined in a matter of seconds by simplyusing the photodiode shift voltage Furthermore this method is valid regardless of whetherthe cantilever is the most compliant component of the system

of bacterial surface macromolecules suggest that bacteria are less compliant (ie stiffer)than cantilevers having spring constants smaller than 10 N miexcl1 (Xu Mulhern BlackfordJericho Firtel and Beveridge 1996 Yao Jericho Pink and Beveridge 1999)

In instances where bacteria (or biomolecules) linked to the cantilever are more com-pliant than the cantilever or for cells with fragile appendages other methods must be usedto accurately convert the measured de ection of the cantilever (in volts) into a force (inNewtons) of interaction (DrsquoCosta and Hoh 1995 Sader Chon and Mulvaney 1999) Forexample because the optical lever sensitivity is strongly dependenton the shape of the laserspot on the photodiodedetector the ldquophotodiode shift voltagerdquo can be used to convert voltsof cantileverde ection into meters of de ection (DrsquoCosta and Hoh 1995) Photodiode shiftvoltage is measured as the difference in output voltage when the photodiode detector isshifted approximately 318 sup1m (one full turn of the positioning screw) on either side of thezero setting Figure 3 illustrates the strong correlation between photodiode shift voltagesand optical lever sensitivities measured by directing the laser to different positions on thecantileverOnce this correlation is established for a given instrument piezoelectric scanner uid cell and cantilever (eg 200-sup1m long V-shaped silicon nitride cantilevers) the op-tical lever sensitivity can be accurately determined without pressing the cantilever againstany other surface This method ensures that forces can be determined regardless of thecompliance of the cantilever relative to any microorganism attached to it and also ensuresthe preservation of fragile macromolecules on microorganisms attached to the cantilever

Results and Discussion

Characterization of Biologically Active Force Probes

BAFPs were created by either attaching a single bacterium to a force sensor (Figure 4A)or by attaching a single bacteria-coated-bead (Figure 4B) to a force sensor Fluorescent

FIGURE 4 (A) Scanning laser micrograph of a BAFP created by directly linking a singlecell of Burkholderia sp to a force-sensing cantilever (B) Confocal micrograph focused onthe midplane of bacteria-coated beads revealing a layer of cells Focusing up and downon the beads reveal that many are evenly coated with cells Beads showing the most uni-form coverage are attached to cantilevers thereby creating BAFPs These cells have beentransformed with a plasmid encoding a green uorescent protein and are emitting ldquonaturalrdquo uorescence This uorescent protein being intracellular together with the confocal abili-ties of the laser scanning microscope allow noninvasive characterization of the orientationand distribution of cells on the force sensor in situ without affecting the surface chemistryof the microbes Plating experiments in which BAFPs were used as the inoculum revealedthat the bacteria on the sensors are viableand presumably activeduring force measurementsScale bars D 10 sup1m

69

70 S K Lower et al

dyes (ViaGram Molecular Probes Eugene OR) and plating techniques revealed that cellmembranes on these bacteria are intact and the cells are viable Both the direct and indirectmethod of attaching bacteria to a cantileverpreserves the orientationand structural integrityof macromolecules on the cell surface This is important because the native conformationof macromolecules on a bacteria surface is critical to their function activity and role innature By using whole bacteria expressing macromolecules in their natural state ratherthan individual biomolecules (eg exopolysaccharides proteins) puri ed from bacterialsurfaces we avoid situations in which the linkage procedure modi es the conformation ofbiomolecules such that they are no longer in a natural state (Stotzky 1986 Ellen and Burne1996 Turner Peek Wertz Archibald Geer and Gaber 1996 Ingersoll and Bright 1997Turner Testoff Conrad and Gaber 1997)

As both gram-negative and gram-positive bacteria are typically negatively charged atmost pH polylysine with its high pKa is a more-or-less universal linker molecule How-ever due to the large diversity in bacterial surfaces situations will likely arise in whichother linker molecules are necessary The versatility of our linkage protocol allows formodi cation on a case-by-case basis For example we have also fabricated BAFPs usingaminopropyltriethoxysilane and polyethyleneimine with species of Shewanella and Pseu-domonas Additionallymethods used to attach proteins or antibodies to small glass spheresor sensors (eg Florin Moy and Gaub 1994 Frey and Corn 1996 Turner et al 1997Caruso Caruso and Mohwald 1998 Rezania Johnson Lefkow and Healy 1999) could beapplied to our protocol For example lysozyme which has been genetically altered suchthat it possesses an active binding site but an inactive catalytic site (Voet and Voet 1995) isan attractive choice because of its high af nity for bacterial cell walls Antibodies speci cfor cell surface receptors may also be effective linkers

A critical component to consider when designing BAFPs is the relative difference be-tween thoseforces bindingthecells to thesensor and interfacial and adhesiveforces thatwillbeprobedby thecellson the sensor Forces holdingbacteria to the sensormustbe greater thanthe forces that are actuallyprobedotherwise the bacteriawill be stripped from the cantileverduring force measurements Electrostatic attachment of bacteria to sensors via polylysineis very strong Approximations using Coulombrsquos law and molecular dynamic calculationsshow that the energy associated with aminendashsilanol (ie the polylysine glass bead linkageor polylysine cantilever linkage) and aminendashcarboxylic pairs (ie the polylysine bacterialinkage) (Voet and Voet 1995 West Latour and Hench 1997 Pagac Prieve and Tilton1998 Pagac Tilton and Prieve 1998) are at least one to four orders of magnitude strongerthan potential intermolecular forces between a bacteria and mineral (Israelachvili 1992)Organosilane linkages such as aminopropyltriethoxysilane form covalentbondswith silanolgroups (Plueddemann1991) and should therefore be even stronger than polylysinelinkages

Comparison of the direct and indirect linkage protocols suggests that the latter ismore desirable Direct linkage of a single cell to a cantilever (Figure 4A) is very dif cultTechniques such as optical tweezers (Svoboda and Block 1994) or nanotweezers (Kim andLieber 1999) could signi cantly enhance the linkage of a bacterium to a cantilever butsuch techniques are not at present easily applicable to such procedures Another practicalreason for selecting the indirect protocol is the need to use cantilevers with appropriatespring constants in order to probe the entire range of potential forces between microbes andminerals Cantilevers of different composition (eg glass silicon silicon nitride and gold-coated cantilevers) are often used to vary the spring constant Gold-coated and uncoatedsilicon nitride cantilevers for example have very different surface properties and siliconnitride itself can vary from Si3N4 to Si15N4 (WeisenhornMaivaldButt and Hansma 1992)

Biological Force Microscopy and Interfaces 71

These compositional differences would require use of more than one linker molecule todirectly attach one particular strain of bacteria to different cantilevers Conversely indirectlinkage allows the use of one protocol to attach bacteria to glass (or latex) beads of uniformcomposition which can then be attached to cantilevers of different compositions

Forces of Interaction Between Bacteria and Mineral Surfaces in situ

The interactions between muscovite and polylysine (Figure 5A) and muscovite and the soilBurkholderia sp (Figures 5B and 5C) were studied with BFM at pH 6 ionic strength 10iexcl5

M 25plusmnC As expected the positively charged polylysine exhibited a strong attraction to-wards the (001) surface of muscovite which is negatively charged (Figure 5A) Hysteresiswas not observed for the interaction between muscovite and polylysine In contrast theBurkholderia sp exhibited a repulsive interaction with muscovite beginning at raquo80 nm(Figure 5B approach) However once contact was made this repulsive force was over-come and the bacteria exhibited an attractive adhesion toward the mineral (Figure 5Bretraction)

The repulsive interfacial forces between the Burkholderia sp and muscovite illustratedin Figure 5B (approach) are consistent with the negative surface charges of both the bac-teria and mineral at circum-neutral pH The distance at which repulsion occurs is slightlysmaller than that expectedbased solely on electrostatic interactions(Debye length thicknessis raquo100 nm at this ionic strength) Other forces associated with hydrophobic steric andentropic effects likely play a key role in bacteriandashmineral interactions due to the relativelylongpolymerson bacterial surfaces (Israelachviliand McGuiggan1988 Israelachvili1992)

The adhesive behavior between Burkholderia sp and muscovite is drawn out for hun-dreds of nanometers (Figure 5B retraction) This is likely due to stretching and brillationof biomolecules (eg lipopolysaccharides or agella) as a result of attractive forces suchas hydrogen bonds between polymers on the bacteria and surface hydroxyls or structuredwater molecules on muscovite (Jucker Harms Hug and Zehnder 1997)

When the same Burkholderia sp was grown in nutrient poor media it exhibited verystrong af nity for muscovite (Figure 5C) Interfacial repulsion was not detected (Figure 5Capproach) and attractive adhesion forces were very large and long range (Figure 5Cretraction) This phenomenais consistentwith theobservationthatmany bacterial speciesal-ter their surface propertiesunderoligotrophicversus eutropicenvironments(eg Bengtsson1991) In fact many bacteria show greater af nity for mineral surfaces under oligotrophicrather than eutropic conditions (Fletcher 1996b Marshall 1996) This cycling between at-tached and planktonic states is believed to be a strategy bacteria use to proliferate in naturewhere many environments have eutropicoligotropic sequences

Potential Applications of Biological Force Microscopy to Geomicrobiology Studies

BFM could providea uniqueperspectiveinto several aspects of geomicrobiology(egmin-eral dissolution crystal growth and bio lm formation) particularly if used to complementother techniques Next is a list of relevant questions considered as possible applications ofBFM to the eld of geomicrobiology and their answers

1 Do bacteria ldquorecognizerdquo particular minerals or crystallographic orientations Probe dif-ferent minerals or different faces on the same mineral with one BAFP

72 S K Lower et al

FIGURE 5 Biological force microcopy (BFM) force-distance curves between the (001)surface of muscovite and a polylysine functionalized cantilever (A) versus Burkholderiasp grown in a eutropic environment (B) versus Burkholderia sp grown in an oligotropicenvironment (C) Solution conditionswere pH 6 ionic strength 10iexcl5M 25plusmnC The force ofinteraction is plotted as a function of the distance between the two surfaces (ie separationbetween polylysine or bacteria on the sensor and the mineral surface) Note difference inx-axis scale Curves begin on the right and proceed toward the left as the mineral is broughttoward the sensor (solid circles approach curve) The sensor makes contactwith the mineralsurface and subsequently withdraws from the muscovite (open squares retraction curve) tocomplete one cycle

2 Do bacteria show an increased af nity for particular minerals under oligotrophic oranaerobicconditionsChangesolutionconditionswithin uid cell while collectingBFMmeasurements

3 How does inter- and intraspecies communication affect bio lm development Use aBAFP to measure interactions with various species of bacteria that are immobilized ona substrate

4 What intermolecular forces (eg van der Waals electrostatics hydration hydrophobicentropic) are involvedin bacterial attachmentand detachmentprocessesCompare forcesmeasured with BFM to those calculated from theoretical models (eg DLVO theory)

5 What cell surface macromoleculesmediatebacterial attachment to mineral surfaces Areconditioning lms required for attachment Create BAFPs using various mutants that

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

Biological Force Microscopy and Interfaces 67

FIGURE 2 Schematic diagrams of a force curve (A) and force-distance curve (B) Whenthe sensor and sample are far apart (i) they exhibit no interaction (region of zero force) Asthe sample approaches the sensor intermolecular forces between the bacteria and mineralcause the sensor to de ect upward (ii) due to repulsive forces shown here Eventually theprobe makes contact with the sample (iii) and their movement becomes coupled (regionof constant compliance) The sample is then retracted from the probe (iv) until the sensorand sample return to their original positions thereby completing one cycle (an entire cyclerequires nano- to milliseconds) Hysteresis shown here may occur upon retraction dueto adhesion forces See text for discussion on converting cantilever de ection and piezodisplacement into force and distance respectively Interfacial forces are measured on ap-proach and adhesion forces are measured upon retraction repulsive forces are positive andattractive forces are negative

68 S K Lower et al

FIGURE 3 Variation of photodiode shift voltage as a function of optical lever sensitivityusing a J-type piezoelectric scanner and eight silicon nitride cantilevers (200 sup1m in length)All measurements were performed on muscovite and hematite in a uid cell containingaqueous solutions ranging in ionic strength from 0 (ie Milli-Q water) to 10iexcl2M Theslope of the regression line is 642 nm and the intercept is 13 V (R2 D 095) With thiscorrelation the optical lever sensitivity can be determined in a matter of seconds by simplyusing the photodiode shift voltage Furthermore this method is valid regardless of whetherthe cantilever is the most compliant component of the system

of bacterial surface macromolecules suggest that bacteria are less compliant (ie stiffer)than cantilevers having spring constants smaller than 10 N miexcl1 (Xu Mulhern BlackfordJericho Firtel and Beveridge 1996 Yao Jericho Pink and Beveridge 1999)

In instances where bacteria (or biomolecules) linked to the cantilever are more com-pliant than the cantilever or for cells with fragile appendages other methods must be usedto accurately convert the measured de ection of the cantilever (in volts) into a force (inNewtons) of interaction (DrsquoCosta and Hoh 1995 Sader Chon and Mulvaney 1999) Forexample because the optical lever sensitivity is strongly dependenton the shape of the laserspot on the photodiodedetector the ldquophotodiode shift voltagerdquo can be used to convert voltsof cantileverde ection into meters of de ection (DrsquoCosta and Hoh 1995) Photodiode shiftvoltage is measured as the difference in output voltage when the photodiode detector isshifted approximately 318 sup1m (one full turn of the positioning screw) on either side of thezero setting Figure 3 illustrates the strong correlation between photodiode shift voltagesand optical lever sensitivities measured by directing the laser to different positions on thecantileverOnce this correlation is established for a given instrument piezoelectric scanner uid cell and cantilever (eg 200-sup1m long V-shaped silicon nitride cantilevers) the op-tical lever sensitivity can be accurately determined without pressing the cantilever againstany other surface This method ensures that forces can be determined regardless of thecompliance of the cantilever relative to any microorganism attached to it and also ensuresthe preservation of fragile macromolecules on microorganisms attached to the cantilever

Results and Discussion

Characterization of Biologically Active Force Probes

BAFPs were created by either attaching a single bacterium to a force sensor (Figure 4A)or by attaching a single bacteria-coated-bead (Figure 4B) to a force sensor Fluorescent

FIGURE 4 (A) Scanning laser micrograph of a BAFP created by directly linking a singlecell of Burkholderia sp to a force-sensing cantilever (B) Confocal micrograph focused onthe midplane of bacteria-coated beads revealing a layer of cells Focusing up and downon the beads reveal that many are evenly coated with cells Beads showing the most uni-form coverage are attached to cantilevers thereby creating BAFPs These cells have beentransformed with a plasmid encoding a green uorescent protein and are emitting ldquonaturalrdquo uorescence This uorescent protein being intracellular together with the confocal abili-ties of the laser scanning microscope allow noninvasive characterization of the orientationand distribution of cells on the force sensor in situ without affecting the surface chemistryof the microbes Plating experiments in which BAFPs were used as the inoculum revealedthat the bacteria on the sensors are viableand presumably activeduring force measurementsScale bars D 10 sup1m

69

70 S K Lower et al

dyes (ViaGram Molecular Probes Eugene OR) and plating techniques revealed that cellmembranes on these bacteria are intact and the cells are viable Both the direct and indirectmethod of attaching bacteria to a cantileverpreserves the orientationand structural integrityof macromolecules on the cell surface This is important because the native conformationof macromolecules on a bacteria surface is critical to their function activity and role innature By using whole bacteria expressing macromolecules in their natural state ratherthan individual biomolecules (eg exopolysaccharides proteins) puri ed from bacterialsurfaces we avoid situations in which the linkage procedure modi es the conformation ofbiomolecules such that they are no longer in a natural state (Stotzky 1986 Ellen and Burne1996 Turner Peek Wertz Archibald Geer and Gaber 1996 Ingersoll and Bright 1997Turner Testoff Conrad and Gaber 1997)

As both gram-negative and gram-positive bacteria are typically negatively charged atmost pH polylysine with its high pKa is a more-or-less universal linker molecule How-ever due to the large diversity in bacterial surfaces situations will likely arise in whichother linker molecules are necessary The versatility of our linkage protocol allows formodi cation on a case-by-case basis For example we have also fabricated BAFPs usingaminopropyltriethoxysilane and polyethyleneimine with species of Shewanella and Pseu-domonas Additionallymethods used to attach proteins or antibodies to small glass spheresor sensors (eg Florin Moy and Gaub 1994 Frey and Corn 1996 Turner et al 1997Caruso Caruso and Mohwald 1998 Rezania Johnson Lefkow and Healy 1999) could beapplied to our protocol For example lysozyme which has been genetically altered suchthat it possesses an active binding site but an inactive catalytic site (Voet and Voet 1995) isan attractive choice because of its high af nity for bacterial cell walls Antibodies speci cfor cell surface receptors may also be effective linkers

A critical component to consider when designing BAFPs is the relative difference be-tween thoseforces bindingthecells to thesensor and interfacial and adhesiveforces thatwillbeprobedby thecellson the sensor Forces holdingbacteria to the sensormustbe greater thanthe forces that are actuallyprobedotherwise the bacteriawill be stripped from the cantileverduring force measurements Electrostatic attachment of bacteria to sensors via polylysineis very strong Approximations using Coulombrsquos law and molecular dynamic calculationsshow that the energy associated with aminendashsilanol (ie the polylysine glass bead linkageor polylysine cantilever linkage) and aminendashcarboxylic pairs (ie the polylysine bacterialinkage) (Voet and Voet 1995 West Latour and Hench 1997 Pagac Prieve and Tilton1998 Pagac Tilton and Prieve 1998) are at least one to four orders of magnitude strongerthan potential intermolecular forces between a bacteria and mineral (Israelachvili 1992)Organosilane linkages such as aminopropyltriethoxysilane form covalentbondswith silanolgroups (Plueddemann1991) and should therefore be even stronger than polylysinelinkages

Comparison of the direct and indirect linkage protocols suggests that the latter ismore desirable Direct linkage of a single cell to a cantilever (Figure 4A) is very dif cultTechniques such as optical tweezers (Svoboda and Block 1994) or nanotweezers (Kim andLieber 1999) could signi cantly enhance the linkage of a bacterium to a cantilever butsuch techniques are not at present easily applicable to such procedures Another practicalreason for selecting the indirect protocol is the need to use cantilevers with appropriatespring constants in order to probe the entire range of potential forces between microbes andminerals Cantilevers of different composition (eg glass silicon silicon nitride and gold-coated cantilevers) are often used to vary the spring constant Gold-coated and uncoatedsilicon nitride cantilevers for example have very different surface properties and siliconnitride itself can vary from Si3N4 to Si15N4 (WeisenhornMaivaldButt and Hansma 1992)

Biological Force Microscopy and Interfaces 71

These compositional differences would require use of more than one linker molecule todirectly attach one particular strain of bacteria to different cantilevers Conversely indirectlinkage allows the use of one protocol to attach bacteria to glass (or latex) beads of uniformcomposition which can then be attached to cantilevers of different compositions

Forces of Interaction Between Bacteria and Mineral Surfaces in situ

The interactions between muscovite and polylysine (Figure 5A) and muscovite and the soilBurkholderia sp (Figures 5B and 5C) were studied with BFM at pH 6 ionic strength 10iexcl5

M 25plusmnC As expected the positively charged polylysine exhibited a strong attraction to-wards the (001) surface of muscovite which is negatively charged (Figure 5A) Hysteresiswas not observed for the interaction between muscovite and polylysine In contrast theBurkholderia sp exhibited a repulsive interaction with muscovite beginning at raquo80 nm(Figure 5B approach) However once contact was made this repulsive force was over-come and the bacteria exhibited an attractive adhesion toward the mineral (Figure 5Bretraction)

The repulsive interfacial forces between the Burkholderia sp and muscovite illustratedin Figure 5B (approach) are consistent with the negative surface charges of both the bac-teria and mineral at circum-neutral pH The distance at which repulsion occurs is slightlysmaller than that expectedbased solely on electrostatic interactions(Debye length thicknessis raquo100 nm at this ionic strength) Other forces associated with hydrophobic steric andentropic effects likely play a key role in bacteriandashmineral interactions due to the relativelylongpolymerson bacterial surfaces (Israelachviliand McGuiggan1988 Israelachvili1992)

The adhesive behavior between Burkholderia sp and muscovite is drawn out for hun-dreds of nanometers (Figure 5B retraction) This is likely due to stretching and brillationof biomolecules (eg lipopolysaccharides or agella) as a result of attractive forces suchas hydrogen bonds between polymers on the bacteria and surface hydroxyls or structuredwater molecules on muscovite (Jucker Harms Hug and Zehnder 1997)

When the same Burkholderia sp was grown in nutrient poor media it exhibited verystrong af nity for muscovite (Figure 5C) Interfacial repulsion was not detected (Figure 5Capproach) and attractive adhesion forces were very large and long range (Figure 5Cretraction) This phenomenais consistentwith theobservationthatmany bacterial speciesal-ter their surface propertiesunderoligotrophicversus eutropicenvironments(eg Bengtsson1991) In fact many bacteria show greater af nity for mineral surfaces under oligotrophicrather than eutropic conditions (Fletcher 1996b Marshall 1996) This cycling between at-tached and planktonic states is believed to be a strategy bacteria use to proliferate in naturewhere many environments have eutropicoligotropic sequences

Potential Applications of Biological Force Microscopy to Geomicrobiology Studies

BFM could providea uniqueperspectiveinto several aspects of geomicrobiology(egmin-eral dissolution crystal growth and bio lm formation) particularly if used to complementother techniques Next is a list of relevant questions considered as possible applications ofBFM to the eld of geomicrobiology and their answers

1 Do bacteria ldquorecognizerdquo particular minerals or crystallographic orientations Probe dif-ferent minerals or different faces on the same mineral with one BAFP

72 S K Lower et al

FIGURE 5 Biological force microcopy (BFM) force-distance curves between the (001)surface of muscovite and a polylysine functionalized cantilever (A) versus Burkholderiasp grown in a eutropic environment (B) versus Burkholderia sp grown in an oligotropicenvironment (C) Solution conditionswere pH 6 ionic strength 10iexcl5M 25plusmnC The force ofinteraction is plotted as a function of the distance between the two surfaces (ie separationbetween polylysine or bacteria on the sensor and the mineral surface) Note difference inx-axis scale Curves begin on the right and proceed toward the left as the mineral is broughttoward the sensor (solid circles approach curve) The sensor makes contactwith the mineralsurface and subsequently withdraws from the muscovite (open squares retraction curve) tocomplete one cycle

2 Do bacteria show an increased af nity for particular minerals under oligotrophic oranaerobicconditionsChangesolutionconditionswithin uid cell while collectingBFMmeasurements

3 How does inter- and intraspecies communication affect bio lm development Use aBAFP to measure interactions with various species of bacteria that are immobilized ona substrate

4 What intermolecular forces (eg van der Waals electrostatics hydration hydrophobicentropic) are involvedin bacterial attachmentand detachmentprocessesCompare forcesmeasured with BFM to those calculated from theoretical models (eg DLVO theory)

5 What cell surface macromoleculesmediatebacterial attachment to mineral surfaces Areconditioning lms required for attachment Create BAFPs using various mutants that

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

68 S K Lower et al

FIGURE 3 Variation of photodiode shift voltage as a function of optical lever sensitivityusing a J-type piezoelectric scanner and eight silicon nitride cantilevers (200 sup1m in length)All measurements were performed on muscovite and hematite in a uid cell containingaqueous solutions ranging in ionic strength from 0 (ie Milli-Q water) to 10iexcl2M Theslope of the regression line is 642 nm and the intercept is 13 V (R2 D 095) With thiscorrelation the optical lever sensitivity can be determined in a matter of seconds by simplyusing the photodiode shift voltage Furthermore this method is valid regardless of whetherthe cantilever is the most compliant component of the system

of bacterial surface macromolecules suggest that bacteria are less compliant (ie stiffer)than cantilevers having spring constants smaller than 10 N miexcl1 (Xu Mulhern BlackfordJericho Firtel and Beveridge 1996 Yao Jericho Pink and Beveridge 1999)

In instances where bacteria (or biomolecules) linked to the cantilever are more com-pliant than the cantilever or for cells with fragile appendages other methods must be usedto accurately convert the measured de ection of the cantilever (in volts) into a force (inNewtons) of interaction (DrsquoCosta and Hoh 1995 Sader Chon and Mulvaney 1999) Forexample because the optical lever sensitivity is strongly dependenton the shape of the laserspot on the photodiodedetector the ldquophotodiode shift voltagerdquo can be used to convert voltsof cantileverde ection into meters of de ection (DrsquoCosta and Hoh 1995) Photodiode shiftvoltage is measured as the difference in output voltage when the photodiode detector isshifted approximately 318 sup1m (one full turn of the positioning screw) on either side of thezero setting Figure 3 illustrates the strong correlation between photodiode shift voltagesand optical lever sensitivities measured by directing the laser to different positions on thecantileverOnce this correlation is established for a given instrument piezoelectric scanner uid cell and cantilever (eg 200-sup1m long V-shaped silicon nitride cantilevers) the op-tical lever sensitivity can be accurately determined without pressing the cantilever againstany other surface This method ensures that forces can be determined regardless of thecompliance of the cantilever relative to any microorganism attached to it and also ensuresthe preservation of fragile macromolecules on microorganisms attached to the cantilever

Results and Discussion

Characterization of Biologically Active Force Probes

BAFPs were created by either attaching a single bacterium to a force sensor (Figure 4A)or by attaching a single bacteria-coated-bead (Figure 4B) to a force sensor Fluorescent

FIGURE 4 (A) Scanning laser micrograph of a BAFP created by directly linking a singlecell of Burkholderia sp to a force-sensing cantilever (B) Confocal micrograph focused onthe midplane of bacteria-coated beads revealing a layer of cells Focusing up and downon the beads reveal that many are evenly coated with cells Beads showing the most uni-form coverage are attached to cantilevers thereby creating BAFPs These cells have beentransformed with a plasmid encoding a green uorescent protein and are emitting ldquonaturalrdquo uorescence This uorescent protein being intracellular together with the confocal abili-ties of the laser scanning microscope allow noninvasive characterization of the orientationand distribution of cells on the force sensor in situ without affecting the surface chemistryof the microbes Plating experiments in which BAFPs were used as the inoculum revealedthat the bacteria on the sensors are viableand presumably activeduring force measurementsScale bars D 10 sup1m

69

70 S K Lower et al

dyes (ViaGram Molecular Probes Eugene OR) and plating techniques revealed that cellmembranes on these bacteria are intact and the cells are viable Both the direct and indirectmethod of attaching bacteria to a cantileverpreserves the orientationand structural integrityof macromolecules on the cell surface This is important because the native conformationof macromolecules on a bacteria surface is critical to their function activity and role innature By using whole bacteria expressing macromolecules in their natural state ratherthan individual biomolecules (eg exopolysaccharides proteins) puri ed from bacterialsurfaces we avoid situations in which the linkage procedure modi es the conformation ofbiomolecules such that they are no longer in a natural state (Stotzky 1986 Ellen and Burne1996 Turner Peek Wertz Archibald Geer and Gaber 1996 Ingersoll and Bright 1997Turner Testoff Conrad and Gaber 1997)

As both gram-negative and gram-positive bacteria are typically negatively charged atmost pH polylysine with its high pKa is a more-or-less universal linker molecule How-ever due to the large diversity in bacterial surfaces situations will likely arise in whichother linker molecules are necessary The versatility of our linkage protocol allows formodi cation on a case-by-case basis For example we have also fabricated BAFPs usingaminopropyltriethoxysilane and polyethyleneimine with species of Shewanella and Pseu-domonas Additionallymethods used to attach proteins or antibodies to small glass spheresor sensors (eg Florin Moy and Gaub 1994 Frey and Corn 1996 Turner et al 1997Caruso Caruso and Mohwald 1998 Rezania Johnson Lefkow and Healy 1999) could beapplied to our protocol For example lysozyme which has been genetically altered suchthat it possesses an active binding site but an inactive catalytic site (Voet and Voet 1995) isan attractive choice because of its high af nity for bacterial cell walls Antibodies speci cfor cell surface receptors may also be effective linkers

A critical component to consider when designing BAFPs is the relative difference be-tween thoseforces bindingthecells to thesensor and interfacial and adhesiveforces thatwillbeprobedby thecellson the sensor Forces holdingbacteria to the sensormustbe greater thanthe forces that are actuallyprobedotherwise the bacteriawill be stripped from the cantileverduring force measurements Electrostatic attachment of bacteria to sensors via polylysineis very strong Approximations using Coulombrsquos law and molecular dynamic calculationsshow that the energy associated with aminendashsilanol (ie the polylysine glass bead linkageor polylysine cantilever linkage) and aminendashcarboxylic pairs (ie the polylysine bacterialinkage) (Voet and Voet 1995 West Latour and Hench 1997 Pagac Prieve and Tilton1998 Pagac Tilton and Prieve 1998) are at least one to four orders of magnitude strongerthan potential intermolecular forces between a bacteria and mineral (Israelachvili 1992)Organosilane linkages such as aminopropyltriethoxysilane form covalentbondswith silanolgroups (Plueddemann1991) and should therefore be even stronger than polylysinelinkages

Comparison of the direct and indirect linkage protocols suggests that the latter ismore desirable Direct linkage of a single cell to a cantilever (Figure 4A) is very dif cultTechniques such as optical tweezers (Svoboda and Block 1994) or nanotweezers (Kim andLieber 1999) could signi cantly enhance the linkage of a bacterium to a cantilever butsuch techniques are not at present easily applicable to such procedures Another practicalreason for selecting the indirect protocol is the need to use cantilevers with appropriatespring constants in order to probe the entire range of potential forces between microbes andminerals Cantilevers of different composition (eg glass silicon silicon nitride and gold-coated cantilevers) are often used to vary the spring constant Gold-coated and uncoatedsilicon nitride cantilevers for example have very different surface properties and siliconnitride itself can vary from Si3N4 to Si15N4 (WeisenhornMaivaldButt and Hansma 1992)

Biological Force Microscopy and Interfaces 71

These compositional differences would require use of more than one linker molecule todirectly attach one particular strain of bacteria to different cantilevers Conversely indirectlinkage allows the use of one protocol to attach bacteria to glass (or latex) beads of uniformcomposition which can then be attached to cantilevers of different compositions

Forces of Interaction Between Bacteria and Mineral Surfaces in situ

The interactions between muscovite and polylysine (Figure 5A) and muscovite and the soilBurkholderia sp (Figures 5B and 5C) were studied with BFM at pH 6 ionic strength 10iexcl5

M 25plusmnC As expected the positively charged polylysine exhibited a strong attraction to-wards the (001) surface of muscovite which is negatively charged (Figure 5A) Hysteresiswas not observed for the interaction between muscovite and polylysine In contrast theBurkholderia sp exhibited a repulsive interaction with muscovite beginning at raquo80 nm(Figure 5B approach) However once contact was made this repulsive force was over-come and the bacteria exhibited an attractive adhesion toward the mineral (Figure 5Bretraction)

The repulsive interfacial forces between the Burkholderia sp and muscovite illustratedin Figure 5B (approach) are consistent with the negative surface charges of both the bac-teria and mineral at circum-neutral pH The distance at which repulsion occurs is slightlysmaller than that expectedbased solely on electrostatic interactions(Debye length thicknessis raquo100 nm at this ionic strength) Other forces associated with hydrophobic steric andentropic effects likely play a key role in bacteriandashmineral interactions due to the relativelylongpolymerson bacterial surfaces (Israelachviliand McGuiggan1988 Israelachvili1992)

The adhesive behavior between Burkholderia sp and muscovite is drawn out for hun-dreds of nanometers (Figure 5B retraction) This is likely due to stretching and brillationof biomolecules (eg lipopolysaccharides or agella) as a result of attractive forces suchas hydrogen bonds between polymers on the bacteria and surface hydroxyls or structuredwater molecules on muscovite (Jucker Harms Hug and Zehnder 1997)

When the same Burkholderia sp was grown in nutrient poor media it exhibited verystrong af nity for muscovite (Figure 5C) Interfacial repulsion was not detected (Figure 5Capproach) and attractive adhesion forces were very large and long range (Figure 5Cretraction) This phenomenais consistentwith theobservationthatmany bacterial speciesal-ter their surface propertiesunderoligotrophicversus eutropicenvironments(eg Bengtsson1991) In fact many bacteria show greater af nity for mineral surfaces under oligotrophicrather than eutropic conditions (Fletcher 1996b Marshall 1996) This cycling between at-tached and planktonic states is believed to be a strategy bacteria use to proliferate in naturewhere many environments have eutropicoligotropic sequences

Potential Applications of Biological Force Microscopy to Geomicrobiology Studies

BFM could providea uniqueperspectiveinto several aspects of geomicrobiology(egmin-eral dissolution crystal growth and bio lm formation) particularly if used to complementother techniques Next is a list of relevant questions considered as possible applications ofBFM to the eld of geomicrobiology and their answers

1 Do bacteria ldquorecognizerdquo particular minerals or crystallographic orientations Probe dif-ferent minerals or different faces on the same mineral with one BAFP

72 S K Lower et al

FIGURE 5 Biological force microcopy (BFM) force-distance curves between the (001)surface of muscovite and a polylysine functionalized cantilever (A) versus Burkholderiasp grown in a eutropic environment (B) versus Burkholderia sp grown in an oligotropicenvironment (C) Solution conditionswere pH 6 ionic strength 10iexcl5M 25plusmnC The force ofinteraction is plotted as a function of the distance between the two surfaces (ie separationbetween polylysine or bacteria on the sensor and the mineral surface) Note difference inx-axis scale Curves begin on the right and proceed toward the left as the mineral is broughttoward the sensor (solid circles approach curve) The sensor makes contactwith the mineralsurface and subsequently withdraws from the muscovite (open squares retraction curve) tocomplete one cycle

2 Do bacteria show an increased af nity for particular minerals under oligotrophic oranaerobicconditionsChangesolutionconditionswithin uid cell while collectingBFMmeasurements

3 How does inter- and intraspecies communication affect bio lm development Use aBAFP to measure interactions with various species of bacteria that are immobilized ona substrate

4 What intermolecular forces (eg van der Waals electrostatics hydration hydrophobicentropic) are involvedin bacterial attachmentand detachmentprocessesCompare forcesmeasured with BFM to those calculated from theoretical models (eg DLVO theory)

5 What cell surface macromoleculesmediatebacterial attachment to mineral surfaces Areconditioning lms required for attachment Create BAFPs using various mutants that

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

FIGURE 4 (A) Scanning laser micrograph of a BAFP created by directly linking a singlecell of Burkholderia sp to a force-sensing cantilever (B) Confocal micrograph focused onthe midplane of bacteria-coated beads revealing a layer of cells Focusing up and downon the beads reveal that many are evenly coated with cells Beads showing the most uni-form coverage are attached to cantilevers thereby creating BAFPs These cells have beentransformed with a plasmid encoding a green uorescent protein and are emitting ldquonaturalrdquo uorescence This uorescent protein being intracellular together with the confocal abili-ties of the laser scanning microscope allow noninvasive characterization of the orientationand distribution of cells on the force sensor in situ without affecting the surface chemistryof the microbes Plating experiments in which BAFPs were used as the inoculum revealedthat the bacteria on the sensors are viableand presumably activeduring force measurementsScale bars D 10 sup1m

69

70 S K Lower et al

dyes (ViaGram Molecular Probes Eugene OR) and plating techniques revealed that cellmembranes on these bacteria are intact and the cells are viable Both the direct and indirectmethod of attaching bacteria to a cantileverpreserves the orientationand structural integrityof macromolecules on the cell surface This is important because the native conformationof macromolecules on a bacteria surface is critical to their function activity and role innature By using whole bacteria expressing macromolecules in their natural state ratherthan individual biomolecules (eg exopolysaccharides proteins) puri ed from bacterialsurfaces we avoid situations in which the linkage procedure modi es the conformation ofbiomolecules such that they are no longer in a natural state (Stotzky 1986 Ellen and Burne1996 Turner Peek Wertz Archibald Geer and Gaber 1996 Ingersoll and Bright 1997Turner Testoff Conrad and Gaber 1997)

As both gram-negative and gram-positive bacteria are typically negatively charged atmost pH polylysine with its high pKa is a more-or-less universal linker molecule How-ever due to the large diversity in bacterial surfaces situations will likely arise in whichother linker molecules are necessary The versatility of our linkage protocol allows formodi cation on a case-by-case basis For example we have also fabricated BAFPs usingaminopropyltriethoxysilane and polyethyleneimine with species of Shewanella and Pseu-domonas Additionallymethods used to attach proteins or antibodies to small glass spheresor sensors (eg Florin Moy and Gaub 1994 Frey and Corn 1996 Turner et al 1997Caruso Caruso and Mohwald 1998 Rezania Johnson Lefkow and Healy 1999) could beapplied to our protocol For example lysozyme which has been genetically altered suchthat it possesses an active binding site but an inactive catalytic site (Voet and Voet 1995) isan attractive choice because of its high af nity for bacterial cell walls Antibodies speci cfor cell surface receptors may also be effective linkers

A critical component to consider when designing BAFPs is the relative difference be-tween thoseforces bindingthecells to thesensor and interfacial and adhesiveforces thatwillbeprobedby thecellson the sensor Forces holdingbacteria to the sensormustbe greater thanthe forces that are actuallyprobedotherwise the bacteriawill be stripped from the cantileverduring force measurements Electrostatic attachment of bacteria to sensors via polylysineis very strong Approximations using Coulombrsquos law and molecular dynamic calculationsshow that the energy associated with aminendashsilanol (ie the polylysine glass bead linkageor polylysine cantilever linkage) and aminendashcarboxylic pairs (ie the polylysine bacterialinkage) (Voet and Voet 1995 West Latour and Hench 1997 Pagac Prieve and Tilton1998 Pagac Tilton and Prieve 1998) are at least one to four orders of magnitude strongerthan potential intermolecular forces between a bacteria and mineral (Israelachvili 1992)Organosilane linkages such as aminopropyltriethoxysilane form covalentbondswith silanolgroups (Plueddemann1991) and should therefore be even stronger than polylysinelinkages

Comparison of the direct and indirect linkage protocols suggests that the latter ismore desirable Direct linkage of a single cell to a cantilever (Figure 4A) is very dif cultTechniques such as optical tweezers (Svoboda and Block 1994) or nanotweezers (Kim andLieber 1999) could signi cantly enhance the linkage of a bacterium to a cantilever butsuch techniques are not at present easily applicable to such procedures Another practicalreason for selecting the indirect protocol is the need to use cantilevers with appropriatespring constants in order to probe the entire range of potential forces between microbes andminerals Cantilevers of different composition (eg glass silicon silicon nitride and gold-coated cantilevers) are often used to vary the spring constant Gold-coated and uncoatedsilicon nitride cantilevers for example have very different surface properties and siliconnitride itself can vary from Si3N4 to Si15N4 (WeisenhornMaivaldButt and Hansma 1992)

Biological Force Microscopy and Interfaces 71

These compositional differences would require use of more than one linker molecule todirectly attach one particular strain of bacteria to different cantilevers Conversely indirectlinkage allows the use of one protocol to attach bacteria to glass (or latex) beads of uniformcomposition which can then be attached to cantilevers of different compositions

Forces of Interaction Between Bacteria and Mineral Surfaces in situ

The interactions between muscovite and polylysine (Figure 5A) and muscovite and the soilBurkholderia sp (Figures 5B and 5C) were studied with BFM at pH 6 ionic strength 10iexcl5

M 25plusmnC As expected the positively charged polylysine exhibited a strong attraction to-wards the (001) surface of muscovite which is negatively charged (Figure 5A) Hysteresiswas not observed for the interaction between muscovite and polylysine In contrast theBurkholderia sp exhibited a repulsive interaction with muscovite beginning at raquo80 nm(Figure 5B approach) However once contact was made this repulsive force was over-come and the bacteria exhibited an attractive adhesion toward the mineral (Figure 5Bretraction)

The repulsive interfacial forces between the Burkholderia sp and muscovite illustratedin Figure 5B (approach) are consistent with the negative surface charges of both the bac-teria and mineral at circum-neutral pH The distance at which repulsion occurs is slightlysmaller than that expectedbased solely on electrostatic interactions(Debye length thicknessis raquo100 nm at this ionic strength) Other forces associated with hydrophobic steric andentropic effects likely play a key role in bacteriandashmineral interactions due to the relativelylongpolymerson bacterial surfaces (Israelachviliand McGuiggan1988 Israelachvili1992)

The adhesive behavior between Burkholderia sp and muscovite is drawn out for hun-dreds of nanometers (Figure 5B retraction) This is likely due to stretching and brillationof biomolecules (eg lipopolysaccharides or agella) as a result of attractive forces suchas hydrogen bonds between polymers on the bacteria and surface hydroxyls or structuredwater molecules on muscovite (Jucker Harms Hug and Zehnder 1997)

When the same Burkholderia sp was grown in nutrient poor media it exhibited verystrong af nity for muscovite (Figure 5C) Interfacial repulsion was not detected (Figure 5Capproach) and attractive adhesion forces were very large and long range (Figure 5Cretraction) This phenomenais consistentwith theobservationthatmany bacterial speciesal-ter their surface propertiesunderoligotrophicversus eutropicenvironments(eg Bengtsson1991) In fact many bacteria show greater af nity for mineral surfaces under oligotrophicrather than eutropic conditions (Fletcher 1996b Marshall 1996) This cycling between at-tached and planktonic states is believed to be a strategy bacteria use to proliferate in naturewhere many environments have eutropicoligotropic sequences

Potential Applications of Biological Force Microscopy to Geomicrobiology Studies

BFM could providea uniqueperspectiveinto several aspects of geomicrobiology(egmin-eral dissolution crystal growth and bio lm formation) particularly if used to complementother techniques Next is a list of relevant questions considered as possible applications ofBFM to the eld of geomicrobiology and their answers

1 Do bacteria ldquorecognizerdquo particular minerals or crystallographic orientations Probe dif-ferent minerals or different faces on the same mineral with one BAFP

72 S K Lower et al

FIGURE 5 Biological force microcopy (BFM) force-distance curves between the (001)surface of muscovite and a polylysine functionalized cantilever (A) versus Burkholderiasp grown in a eutropic environment (B) versus Burkholderia sp grown in an oligotropicenvironment (C) Solution conditionswere pH 6 ionic strength 10iexcl5M 25plusmnC The force ofinteraction is plotted as a function of the distance between the two surfaces (ie separationbetween polylysine or bacteria on the sensor and the mineral surface) Note difference inx-axis scale Curves begin on the right and proceed toward the left as the mineral is broughttoward the sensor (solid circles approach curve) The sensor makes contactwith the mineralsurface and subsequently withdraws from the muscovite (open squares retraction curve) tocomplete one cycle

2 Do bacteria show an increased af nity for particular minerals under oligotrophic oranaerobicconditionsChangesolutionconditionswithin uid cell while collectingBFMmeasurements

3 How does inter- and intraspecies communication affect bio lm development Use aBAFP to measure interactions with various species of bacteria that are immobilized ona substrate

4 What intermolecular forces (eg van der Waals electrostatics hydration hydrophobicentropic) are involvedin bacterial attachmentand detachmentprocessesCompare forcesmeasured with BFM to those calculated from theoretical models (eg DLVO theory)

5 What cell surface macromoleculesmediatebacterial attachment to mineral surfaces Areconditioning lms required for attachment Create BAFPs using various mutants that

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

70 S K Lower et al

dyes (ViaGram Molecular Probes Eugene OR) and plating techniques revealed that cellmembranes on these bacteria are intact and the cells are viable Both the direct and indirectmethod of attaching bacteria to a cantileverpreserves the orientationand structural integrityof macromolecules on the cell surface This is important because the native conformationof macromolecules on a bacteria surface is critical to their function activity and role innature By using whole bacteria expressing macromolecules in their natural state ratherthan individual biomolecules (eg exopolysaccharides proteins) puri ed from bacterialsurfaces we avoid situations in which the linkage procedure modi es the conformation ofbiomolecules such that they are no longer in a natural state (Stotzky 1986 Ellen and Burne1996 Turner Peek Wertz Archibald Geer and Gaber 1996 Ingersoll and Bright 1997Turner Testoff Conrad and Gaber 1997)

As both gram-negative and gram-positive bacteria are typically negatively charged atmost pH polylysine with its high pKa is a more-or-less universal linker molecule How-ever due to the large diversity in bacterial surfaces situations will likely arise in whichother linker molecules are necessary The versatility of our linkage protocol allows formodi cation on a case-by-case basis For example we have also fabricated BAFPs usingaminopropyltriethoxysilane and polyethyleneimine with species of Shewanella and Pseu-domonas Additionallymethods used to attach proteins or antibodies to small glass spheresor sensors (eg Florin Moy and Gaub 1994 Frey and Corn 1996 Turner et al 1997Caruso Caruso and Mohwald 1998 Rezania Johnson Lefkow and Healy 1999) could beapplied to our protocol For example lysozyme which has been genetically altered suchthat it possesses an active binding site but an inactive catalytic site (Voet and Voet 1995) isan attractive choice because of its high af nity for bacterial cell walls Antibodies speci cfor cell surface receptors may also be effective linkers

A critical component to consider when designing BAFPs is the relative difference be-tween thoseforces bindingthecells to thesensor and interfacial and adhesiveforces thatwillbeprobedby thecellson the sensor Forces holdingbacteria to the sensormustbe greater thanthe forces that are actuallyprobedotherwise the bacteriawill be stripped from the cantileverduring force measurements Electrostatic attachment of bacteria to sensors via polylysineis very strong Approximations using Coulombrsquos law and molecular dynamic calculationsshow that the energy associated with aminendashsilanol (ie the polylysine glass bead linkageor polylysine cantilever linkage) and aminendashcarboxylic pairs (ie the polylysine bacterialinkage) (Voet and Voet 1995 West Latour and Hench 1997 Pagac Prieve and Tilton1998 Pagac Tilton and Prieve 1998) are at least one to four orders of magnitude strongerthan potential intermolecular forces between a bacteria and mineral (Israelachvili 1992)Organosilane linkages such as aminopropyltriethoxysilane form covalentbondswith silanolgroups (Plueddemann1991) and should therefore be even stronger than polylysinelinkages

Comparison of the direct and indirect linkage protocols suggests that the latter ismore desirable Direct linkage of a single cell to a cantilever (Figure 4A) is very dif cultTechniques such as optical tweezers (Svoboda and Block 1994) or nanotweezers (Kim andLieber 1999) could signi cantly enhance the linkage of a bacterium to a cantilever butsuch techniques are not at present easily applicable to such procedures Another practicalreason for selecting the indirect protocol is the need to use cantilevers with appropriatespring constants in order to probe the entire range of potential forces between microbes andminerals Cantilevers of different composition (eg glass silicon silicon nitride and gold-coated cantilevers) are often used to vary the spring constant Gold-coated and uncoatedsilicon nitride cantilevers for example have very different surface properties and siliconnitride itself can vary from Si3N4 to Si15N4 (WeisenhornMaivaldButt and Hansma 1992)

Biological Force Microscopy and Interfaces 71

These compositional differences would require use of more than one linker molecule todirectly attach one particular strain of bacteria to different cantilevers Conversely indirectlinkage allows the use of one protocol to attach bacteria to glass (or latex) beads of uniformcomposition which can then be attached to cantilevers of different compositions

Forces of Interaction Between Bacteria and Mineral Surfaces in situ

The interactions between muscovite and polylysine (Figure 5A) and muscovite and the soilBurkholderia sp (Figures 5B and 5C) were studied with BFM at pH 6 ionic strength 10iexcl5

M 25plusmnC As expected the positively charged polylysine exhibited a strong attraction to-wards the (001) surface of muscovite which is negatively charged (Figure 5A) Hysteresiswas not observed for the interaction between muscovite and polylysine In contrast theBurkholderia sp exhibited a repulsive interaction with muscovite beginning at raquo80 nm(Figure 5B approach) However once contact was made this repulsive force was over-come and the bacteria exhibited an attractive adhesion toward the mineral (Figure 5Bretraction)

The repulsive interfacial forces between the Burkholderia sp and muscovite illustratedin Figure 5B (approach) are consistent with the negative surface charges of both the bac-teria and mineral at circum-neutral pH The distance at which repulsion occurs is slightlysmaller than that expectedbased solely on electrostatic interactions(Debye length thicknessis raquo100 nm at this ionic strength) Other forces associated with hydrophobic steric andentropic effects likely play a key role in bacteriandashmineral interactions due to the relativelylongpolymerson bacterial surfaces (Israelachviliand McGuiggan1988 Israelachvili1992)

The adhesive behavior between Burkholderia sp and muscovite is drawn out for hun-dreds of nanometers (Figure 5B retraction) This is likely due to stretching and brillationof biomolecules (eg lipopolysaccharides or agella) as a result of attractive forces suchas hydrogen bonds between polymers on the bacteria and surface hydroxyls or structuredwater molecules on muscovite (Jucker Harms Hug and Zehnder 1997)

When the same Burkholderia sp was grown in nutrient poor media it exhibited verystrong af nity for muscovite (Figure 5C) Interfacial repulsion was not detected (Figure 5Capproach) and attractive adhesion forces were very large and long range (Figure 5Cretraction) This phenomenais consistentwith theobservationthatmany bacterial speciesal-ter their surface propertiesunderoligotrophicversus eutropicenvironments(eg Bengtsson1991) In fact many bacteria show greater af nity for mineral surfaces under oligotrophicrather than eutropic conditions (Fletcher 1996b Marshall 1996) This cycling between at-tached and planktonic states is believed to be a strategy bacteria use to proliferate in naturewhere many environments have eutropicoligotropic sequences

Potential Applications of Biological Force Microscopy to Geomicrobiology Studies

BFM could providea uniqueperspectiveinto several aspects of geomicrobiology(egmin-eral dissolution crystal growth and bio lm formation) particularly if used to complementother techniques Next is a list of relevant questions considered as possible applications ofBFM to the eld of geomicrobiology and their answers

1 Do bacteria ldquorecognizerdquo particular minerals or crystallographic orientations Probe dif-ferent minerals or different faces on the same mineral with one BAFP

72 S K Lower et al

FIGURE 5 Biological force microcopy (BFM) force-distance curves between the (001)surface of muscovite and a polylysine functionalized cantilever (A) versus Burkholderiasp grown in a eutropic environment (B) versus Burkholderia sp grown in an oligotropicenvironment (C) Solution conditionswere pH 6 ionic strength 10iexcl5M 25plusmnC The force ofinteraction is plotted as a function of the distance between the two surfaces (ie separationbetween polylysine or bacteria on the sensor and the mineral surface) Note difference inx-axis scale Curves begin on the right and proceed toward the left as the mineral is broughttoward the sensor (solid circles approach curve) The sensor makes contactwith the mineralsurface and subsequently withdraws from the muscovite (open squares retraction curve) tocomplete one cycle

2 Do bacteria show an increased af nity for particular minerals under oligotrophic oranaerobicconditionsChangesolutionconditionswithin uid cell while collectingBFMmeasurements

3 How does inter- and intraspecies communication affect bio lm development Use aBAFP to measure interactions with various species of bacteria that are immobilized ona substrate

4 What intermolecular forces (eg van der Waals electrostatics hydration hydrophobicentropic) are involvedin bacterial attachmentand detachmentprocessesCompare forcesmeasured with BFM to those calculated from theoretical models (eg DLVO theory)

5 What cell surface macromoleculesmediatebacterial attachment to mineral surfaces Areconditioning lms required for attachment Create BAFPs using various mutants that

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

Biological Force Microscopy and Interfaces 71

These compositional differences would require use of more than one linker molecule todirectly attach one particular strain of bacteria to different cantilevers Conversely indirectlinkage allows the use of one protocol to attach bacteria to glass (or latex) beads of uniformcomposition which can then be attached to cantilevers of different compositions

Forces of Interaction Between Bacteria and Mineral Surfaces in situ

The interactions between muscovite and polylysine (Figure 5A) and muscovite and the soilBurkholderia sp (Figures 5B and 5C) were studied with BFM at pH 6 ionic strength 10iexcl5

M 25plusmnC As expected the positively charged polylysine exhibited a strong attraction to-wards the (001) surface of muscovite which is negatively charged (Figure 5A) Hysteresiswas not observed for the interaction between muscovite and polylysine In contrast theBurkholderia sp exhibited a repulsive interaction with muscovite beginning at raquo80 nm(Figure 5B approach) However once contact was made this repulsive force was over-come and the bacteria exhibited an attractive adhesion toward the mineral (Figure 5Bretraction)

The repulsive interfacial forces between the Burkholderia sp and muscovite illustratedin Figure 5B (approach) are consistent with the negative surface charges of both the bac-teria and mineral at circum-neutral pH The distance at which repulsion occurs is slightlysmaller than that expectedbased solely on electrostatic interactions(Debye length thicknessis raquo100 nm at this ionic strength) Other forces associated with hydrophobic steric andentropic effects likely play a key role in bacteriandashmineral interactions due to the relativelylongpolymerson bacterial surfaces (Israelachviliand McGuiggan1988 Israelachvili1992)

The adhesive behavior between Burkholderia sp and muscovite is drawn out for hun-dreds of nanometers (Figure 5B retraction) This is likely due to stretching and brillationof biomolecules (eg lipopolysaccharides or agella) as a result of attractive forces suchas hydrogen bonds between polymers on the bacteria and surface hydroxyls or structuredwater molecules on muscovite (Jucker Harms Hug and Zehnder 1997)

When the same Burkholderia sp was grown in nutrient poor media it exhibited verystrong af nity for muscovite (Figure 5C) Interfacial repulsion was not detected (Figure 5Capproach) and attractive adhesion forces were very large and long range (Figure 5Cretraction) This phenomenais consistentwith theobservationthatmany bacterial speciesal-ter their surface propertiesunderoligotrophicversus eutropicenvironments(eg Bengtsson1991) In fact many bacteria show greater af nity for mineral surfaces under oligotrophicrather than eutropic conditions (Fletcher 1996b Marshall 1996) This cycling between at-tached and planktonic states is believed to be a strategy bacteria use to proliferate in naturewhere many environments have eutropicoligotropic sequences

Potential Applications of Biological Force Microscopy to Geomicrobiology Studies

BFM could providea uniqueperspectiveinto several aspects of geomicrobiology(egmin-eral dissolution crystal growth and bio lm formation) particularly if used to complementother techniques Next is a list of relevant questions considered as possible applications ofBFM to the eld of geomicrobiology and their answers

1 Do bacteria ldquorecognizerdquo particular minerals or crystallographic orientations Probe dif-ferent minerals or different faces on the same mineral with one BAFP

72 S K Lower et al

FIGURE 5 Biological force microcopy (BFM) force-distance curves between the (001)surface of muscovite and a polylysine functionalized cantilever (A) versus Burkholderiasp grown in a eutropic environment (B) versus Burkholderia sp grown in an oligotropicenvironment (C) Solution conditionswere pH 6 ionic strength 10iexcl5M 25plusmnC The force ofinteraction is plotted as a function of the distance between the two surfaces (ie separationbetween polylysine or bacteria on the sensor and the mineral surface) Note difference inx-axis scale Curves begin on the right and proceed toward the left as the mineral is broughttoward the sensor (solid circles approach curve) The sensor makes contactwith the mineralsurface and subsequently withdraws from the muscovite (open squares retraction curve) tocomplete one cycle

2 Do bacteria show an increased af nity for particular minerals under oligotrophic oranaerobicconditionsChangesolutionconditionswithin uid cell while collectingBFMmeasurements

3 How does inter- and intraspecies communication affect bio lm development Use aBAFP to measure interactions with various species of bacteria that are immobilized ona substrate

4 What intermolecular forces (eg van der Waals electrostatics hydration hydrophobicentropic) are involvedin bacterial attachmentand detachmentprocessesCompare forcesmeasured with BFM to those calculated from theoretical models (eg DLVO theory)

5 What cell surface macromoleculesmediatebacterial attachment to mineral surfaces Areconditioning lms required for attachment Create BAFPs using various mutants that

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

72 S K Lower et al

FIGURE 5 Biological force microcopy (BFM) force-distance curves between the (001)surface of muscovite and a polylysine functionalized cantilever (A) versus Burkholderiasp grown in a eutropic environment (B) versus Burkholderia sp grown in an oligotropicenvironment (C) Solution conditionswere pH 6 ionic strength 10iexcl5M 25plusmnC The force ofinteraction is plotted as a function of the distance between the two surfaces (ie separationbetween polylysine or bacteria on the sensor and the mineral surface) Note difference inx-axis scale Curves begin on the right and proceed toward the left as the mineral is broughttoward the sensor (solid circles approach curve) The sensor makes contactwith the mineralsurface and subsequently withdraws from the muscovite (open squares retraction curve) tocomplete one cycle

2 Do bacteria show an increased af nity for particular minerals under oligotrophic oranaerobicconditionsChangesolutionconditionswithin uid cell while collectingBFMmeasurements

3 How does inter- and intraspecies communication affect bio lm development Use aBAFP to measure interactions with various species of bacteria that are immobilized ona substrate

4 What intermolecular forces (eg van der Waals electrostatics hydration hydrophobicentropic) are involvedin bacterial attachmentand detachmentprocessesCompare forcesmeasured with BFM to those calculated from theoretical models (eg DLVO theory)

5 What cell surface macromoleculesmediatebacterial attachment to mineral surfaces Areconditioning lms required for attachment Create BAFPs using various mutants that

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

Biological Force Microscopy and Interfaces 73

differ in surface macromolecules and use these in BFM on minerals with and withoutsorbed organic molecules

6 How do bacteria alter their cell surface in response to changing environmental condi-tions Do mineral surfaces induce genetic expression of particular macromolecules ona cell surface If so how much time is necessary for production of a protein or ex-tracellular polysaccharide What is the distribution of a particular macromolecule onthe cell surface Combine BFM measurements with biomolecule sensitive uorophoresscanning laser confocal microscopy andor scanning near- eld optical microscopy

7 Is direct bacteriandashmineral contact necessary for oxidative or reductive dissolution ofsul des iron oxides or manganese oxides Use BFM to measure bacterial af nity forvarious minerals under aerobic versus anaerobic conditionsCombineBFM with surfacesensitive spectroscopies

8 How does the conformation of a protein change when electrons are shuttled tofroma mineral surface (eg in the case of Shewanellandashiron oxide interactions) CombineBFM measurementswith uorescenceresonanceenergytransfer scanninglaser confocalmicroscopy andor proteins labeled with uorescent tags

9 Do organic acids exhibit an increased af nity for particular mineral faces How strongare the bonds between a siderophore or organic ligand and mineral surface A forcesensor could be functionalized with speci c biomolecules and used to probe differentfaces on the same mineral

Tools such as BFM offer biochemistsbiophysicistsgeochemists environmentalengineersmicrobiologists and mineralogists a unique insight into the fundamental intricacies of theubiquitous interfaces between microbes and minerals

References

Arredondo R Garcia A Jerez CA 1994 Partial removal of lipopolysaccharide from Thiobacillusferrooxidansaffects its adhesion to solids Appl Envir Microbiol 602846ndash2851

Ban eld JF Hamers RJ 1997 Processes at minerals and surfaces with relevance to microorganismsand prebiotic synthesis Rev Mineral 3581ndash122

Barker WW Welch SA Ban eld JF 1997 Biogeochemical weathering of silicate minerals RevMineral 35391ndash428

Barker WW Welch SA Chu S Ban eld JF 1998Experimentalobservationsof the effectsof bacteriaon aluminosilicateweathering Amer Mineral 831551ndash1563

Bengtsson G 1991 Bacterial exopolymer and PHB production in uctuating groundwater habitatsFEMS Microbiol Ecol 8615ndash24

Bennett PC Hiebert FK Choi WJ 1996 Microbial colonization and weathering of silicates inpetroleum-contaminated groundwater Chem Geol 13245ndash53

Beveridge TJ Doyle RJ 1989 Metal ions and bacteria New York Wiley p 461Brown DA Sherriff BL Sawicki JA 1997 Microbial transformation of magnetite to hematite

Geochim Cosmochim Acta 613341ndash3348Butt HJ Jaschke M Ducker W 1995 Measuring surface forces in aqueous electrolyte solution with

the atomic force microscope BioelectrochemBioenergetics 38191ndash201Cappella B Dietler G 1999 Forcendashdistance curves by atomic force microscopy Surf Sci Reports

341ndash104Caruso F Caruso RA Mohwald H 1998 Nanoengineering of inorganic and hybrid hollow spheres

by colloidal templating Science 2821111ndash1114Cleveland JP Manne S Bocek D Hansma PK 1993 A nondestructive method for determining

the spring constant of cantilevers for scanning force microscopy Rev Sci Instrum 64403ndash

405DrsquoCosta NP Hoh JH 1995 Calibration of optical lever sensitivity for atomic force microscopyRev

Sci Instrum 665096ndash5097

74 S K Lower et al

Dziurla M-A Achouak W Lam B-T Heulin T Berthelin J 1998 Enzyme-linked immuno ltrationassay to estimate attachment of Thiobacilli to pyrite Appl Envir Microbiol 642937ndash2942

Edwards KJ Goebel BM Rodgers TM Schrenk MO Gihring TM Cardona MM Hu B McGuireMM Hamers RJ Pace NR Ban eld JF 1998 Geomicrobiologyof pyrite (FeS2 ) dissolution acase study at Iron Mountain California Geomicrobiol 161ndash25

Edwards KJ Schrenk MO Hamers R Ban eld JF 1998 Microbial oxidation of pyrite experimentsusing microorganisms from an extreme acidic environmentAmer Mineral 831444ndash1453

Ehrlich HL 1990 GeomicrobiologyNew York Marcel Dekker p 646EllenRPBurneRA 1996Conceptualadvancesin researchon theadhesionof bacteriato oral surfaces

In Fletcher M editor Bacterial adhesion molecular and ecological diversity WashingtonDCAmerican Society for Microbiologyp 201ndash247

Fein JB Daughney CJ Yee N Davis T 1997 A chemical equilibrium model for metal adsorptiononto bacterial surfaces Geochim Cosmochim Acta 613319ndash3328

Fletcher M 1996a Bacterial adhesion molecular and ecological diversity New York Wiley-Lissp 361

Fletcher M 1996b Bacterial attachment in aquatic environments a diversity of surfaces and adhe-sion strategies In Fletcher M editor Bacterial adhesion molecular and ecological diversityWashington DC American Society for Microbiology p 1ndash24

Florin E-L Moy VT Gaub HE 1994 Adhesion forces between individual ligandndashreceptor pairsScience 264415ndash417

ForsytheJH MauricePA Hersman LE 1998Attachmentof a Pseudomonassp to Fe(III)-(hydr)oxidesurfaces Geomicrobiol 15293ndash308

Fortin D Beveridge TJ 1997 Role of the bacterium Thiobacillus in the formation of silicates inacidic mine tailings Chem Geol 141235ndash250

Fortin D Ferris FG Beveridge TJ 1997 Surface-mediated mineral development by bacteria RevMineral 35161ndash180

FredricksonJK McKinleyJPNierzwicki-BauerSA WhiteDC RingelbergDB RawsonSA Shu-MeiL Brockman FJ Bjornstad BN 1995 Microbial community structure and biogeochemistry ofMiocene subsurface sediments implications for long-term microbial survival Molecular Ecol4619ndash626

Frey BL Corn RM 1996 Covalent attachment and derivatization of poly(L-lysine) monolayerson gold surfaces as characterized by polarization-modulation FTndashIR spectroscopy Anal Chem683187ndash3193

Gehrke T Telegdi J Dominique T Sand W 1998 Importance of extracellular polymeric sub-stances from Thiobacillus ferrooxidans for bioleaching Appl Environ Microbiol 642743ndash

2747Grantham MC Dove PM DiChristina TJ 1997 Microbially catalyzed dissolution of iron and alu-

minum oxyhydroxidemineral surface coatings Geochim Cosmochim Acta 614467ndash4477Hersman L Lloyd T Sposito G 1995 Siderophore-promoted dissolution of hematite Geochim

Cosmochim Acta 593327ndash3330Hersman L Maurice PA Sposito G 1996 Iron acquisition from hydrous Fe(III)-oxides by an aerobic

Pseudomonas sp Chem Geol 13225ndash31Holben WE 1997 Isolation and puri cation of bacterial community DNA from environmental sam-

ples In Hurst CJ editor Manual of environmental microbiology Washington DC AmericanSociety for Microbiology p 431ndash436

Ingersoll CM Bright FV 1997 Using uorescence to probe biosensor interfacial dynamics AnalChem 69403Andash408A

Israelachvili J 1992 Intermolecular and surface forces London Academic Press p 450Israelachvili JN McGuiggan PM 1988 Forces between surfaces in liquids Science 241795ndash

800Jucker BA Harms H Hug SJ Zehnder AJB 1997 Adsorption of bacterial surface polysaccharides

on mineral oxides is mediated by hydrogen bonds Colloids Surf B 9331ndash343Kennedy AC Gewin VL 1997 Soil microbial diversity Present and future considerationsSoil Sci

162607ndash617

Biological Force Microscopy and Interfaces 75

Kim P Lieber CM 1999 Nanotube nanotweezers Science 2862148ndash2150Krumbein WE Urzi CE Gehrmann C 1991 Biocorresion and biodeteriorationof antique and me-

dieval glass Geomicrobiol 9139ndash160LangleyS BeveridgeTJ 1999EffectofO-side-chain-lipopolysaccharidechemistryonmetal binding

Appl Envir Microbiol 65489ndash498Lawrence JR Korber DR Hoyle BD Costerton JW Caldwell DE 1991 Optical sectioning of mi-

crobial bio lms J Bacteriol 1736558ndash6567Lovley DR PhillipsEJP 1986Organicmatter mineralizationwith reductionof ferric iron in anaerobic

sediments Appl Envir Microbiol 51683ndash689Lovley DR Phillips EJP 1987 Competitive mechanisms for inhibition of sulfate reduction and

methane production in the zone of ferric iron reduction in sediments Appl Envir Microbiol532636ndash2641

Lower SK Maurice PM in preparation From minerals and crystal lattices to microorganisms andnucleic acids the use of atomic force microscopy in geomicrobiologyEarth Sci Rev

Lower SK Tadanier CJ Hochella MF Jr 2000 Measuring interfacial and adhesion forces betweenbacteria and mineral surfaces with biological force microscopy Geochim Cosmochim Acta643133ndash3139

Marshall KC 1976 Interfaces in microbial ecology Cambridge MA Harvard University Pressp 156

Marshall KC 1996 Adhesion as a strategy for access to nutrients In Fletcher M editor Bacterialadhesion molecular and ecological diversity Washington DC American Society for Microbi-ology p 59ndash87

Myers C Nealson KH 1988 Bacterial manganese reduction and growth with manganese oxide asthe sole electron acceptor Science 2401319ndash1321

Pagac ES Prieve DC Tilton RD 1998 Kinetics and mechanisms of cationic surfactant adsorptionand coadsorptionwith cationicpolyelectrolytesat the silicandashwater interfaceLangmuir 142333ndash

2342Pagac ES Tilton RD Prieve DC 1998 Depletion attraction caused by unadsorbed polyelectrolytes

Langmuir 145106ndash5112Pentecost A Bauld J 1988 Nucleation of calcite on the sheaths of cyanobacteria using a simple

diffusion cell Geomicrobiol 6129ndash135Pincet F Perez E Wolfe J 1995 Does glue contaminate the surface forces apparatus Langmuir

11373ndash374Plueddemann EP 1991 Silane coupling agents New York Plenum p 253Rezania A Johnson R Lefkow A Healy KE 1999 Bioactivationof metal oxide surfaces1 Surface

characterizationand cell response Langmuir 156931ndash6939Robert M Berthelin J 1986 Role of biological and biochemical factors in soil mineral weathering

In Huang PM Schnitzer M editors Interactions of soil minerals with natural organics andmicrobes Madison WI Soil Science Society of America p 453ndash495

Roden EE Zachara JM 1996 Microbial reduction of crystalline iron(III) oxides in uence of oxidesurface area and potential for cell growth Envir Sci Technol 301618ndash1628

Rogers JR Bennett PC Choi WJ 1998 Feldspars as a source of nutrients for microorganismsAmerMineral 831532ndash1540

Sader JE Chon JWM Mulvaney P 1999 Calibration of rectangular atomic force microscope can-tilevers Rev Sci Instrum 703967ndash3969

Schrenk MO Edwards KJ Goodman RM Hamers RJ Ban eld JF 1998 Distributionof Thiobacillusferrooxidansand Leptospirillumferrooxidansimplicationsfor generationof acid mine drainageScience 2791519ndash1522

Schultze-Lam S Fortin D Davis BS BeveridgeTJ 1996 Mineralizationof bacterial surfacesChemGeol 132171ndash181

Schultze-LamS Harauz G BeveridgeTJ 1992Participationof a cyanobacterialS-layer in ne-grainmineral formation J Bacteriol 1747971ndash7981

Senden TJ Ducker WA 1994 Experimental determination of spring constants in atomic force mi-croscopy Langmuir 101003ndash1004

76 S K Lower et al

Stone AT 1997 Reactions of extracellular organic ligands with dissolved metal ions and mineralsurface Rev Mineral 35309ndash341

Stotzky G 1986 In uence of soil mineral colloids on metabolic processes growth adhesion andecologyofmicrobesandvirusesInHuangPM SchnitzerM editorsInteractionsof soilmineralswith natural organics and microbes Madison WI Soil Science Society of America p 305ndash

428Svoboda K Block SM 1994 Biological applications of optical forces Ann Rev Biophys Biomol

Struct 23247ndash285Tadanier CJ Little JC Berry DF Hochella MF Jr 2000 Energetics of microbial nutrient acquisi-

tion from mineral surfaces In Hollmann R Wood SA editors Geochemical Society SpecialPublicationmdashDavid A Crerar Honorary Volume Washington DC Geochemical Society ofAmerica

Thorseth IH Torsvik T Furnes H Muehlenbachs K 1995 Microbes play an important role in thealteration of oceanic crust Chem Geol 126137ndash146

Trevors JT van Elsas JD 1997 Quanti cation of gene transfer in soil and the rhizosphere InHurst CJ editor Manual of environmental microbiology Washington DC American Societyfor Microbiologyp 500ndash508

Turner DC Peek BM Wertz TE Archibald DD Geer RE Gaber BP 1996 Enzymatic modi cationof a chemisorbed lipid monolayer Langmuir 124411ndash4416

Turner DC TestoffMA ConradDW Gaber BP 1997Enzymatichydrolysisof a chemisorbedpeptide lm using beads activated with covalently bound chymotrypsinLangmuir 134855ndash4860

Voet D Voet J 1995 Biochemistry New York Wiley p 1361Warren LA Ferris FG 1998 Continuum between sorption and precipitationof Fe(III) on microbial

surfaces Envir Sci Technol 322331ndash2337WeisenhornAL MaivaldPButt H-J HansmaPK 1992Measuringadhesionattractionand repulsion

between surfaces in liquids with an atomic-force microscope Phys Rev B 4511226ndash11232Welch SA Ullman WJ 1993 The effect of organic acids on plagioclase dissolution rates and stoi-

chiometry Geochim Cosmochim Acta 572725ndash2736Welch SA VandevivereP 1994Effectof microbialand other naturallyoccurringpolymerson mineral

dissolutionGeomicrobiol 12227ndash238West JK Latour R Jr Hench LL 1997Molecularmodeling study of adsorptionof poly-L-lysineonto

silica glass J Biomed Materials Res 37585ndash591WolfaardtGMLawrence JR RobertsRD CaldwellSJCaldwellDE 1994Multicellularorganization

in a degradative bio lm community Appl Envir Microbiol 60434ndash446Xu W Mulhern PJ BlackfordBL JerichoMH Firtel M BeveridgeT 1996Modeling and measuring

the elastic properties of an Archaeal surface the sheath of Methanospirillumhungatei and theimplications for methane production J Bacteriol 1783106ndash3112

Yao X Jericho M Pink D Beveridge T 1999 Thickness and elasticity of Gram-negative mureinsacculi measured by atomic force microscopy J Bacteriol 1816865ndash6875

Yoon RH Flinn DH Rabinovich YI 1997 Hydrophobic interactions between dissimilar surfacesJ Colloid Interface Sci 185363ndash370