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University of Hawaii N/A>

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11 T,-_ (Inciude Security Classification)

Examination of Chemical Adsorption and Marine Bioifouling on Metal Surfaces UsingRaman Scattering Techniques and Electrochemical Impedance Spectroscopy (U)

12 PE2SONAL AUTHOR(S)Taylor, Gordon T.; Sharma, Shiv K.; Liebert, Bruce E.; Mower, Howard F.

i3a TYPE OF REPORT 13b TIME COvERED 14 DATE OF REPORT (Year Month,ODay) 15s PAGE COUNTAnnual FPO0M _11/87_ TO 10/88 1989 Jan 13 I11

.6 SUPPLEMENTARY NOTATION

17 COSATI CODES 18 SUBJECT TERMS (Continue on reverse of necessary and identify by block number)

VED GROUP SUB-GROUP Biofouling, Chemical Adsorption, Raman Spectroscopy,06 03 Electrochemical Impedance Spectroscopy.

19 A3STRACT (Continue on reverse it necessary and identify by block number) IA System has been developed to simulate bioifouling of metal surfaces undier al range ofphysical, chemical, and biological conditions. Items of equipment to eliminate andconcentrate organic constituents from seawater and to analyze for proteins, glyco-proteins, and carbohydrates have been acquired. Teflon fouling chambers have been..developed to characterize surface properties of metals in seawater, i.e. , adsorption'of organic materials, using optical and electrochemical spectroscopic probes. Pre-liminary studies using these chambers and surface-enhanced Raman spectrosco~ydemonstrated ability to detect thin layers of pyridine, tryptophan, and phenry lalanineadsorbed to silver electrodes. A system to perform waveguide Internal Reflectance RamanSpectroscopy (WIRRS) on thin films adsorbed to a substratum was developed an~d tested.During preliminary studies, an excellent Raman spectrum was obtained from a 1 11m, layerof polystyrene. For Electrochemical Impedance Spectroscopy (EIS), electronic hardware

(over)

20. DISTRIBUTION I AVAILABILITY OF ABSTRACT 21. ABSTRACT SECURITY CLASSIFICAtIONOUNCLASSIFIEDIUNLIMITeoO 0SAME AS RPT. IC cUSERS (U)

22a NAME OP RESPONSIBLE INDIVIDUAL 22b. TELEPHONE (include Area Code) 22C. OFFICE SYMBOLM.- Marron (202) 696-.4760 ONR

DO FORM 1473, 84 MAR 83 APR edition mayv be used uIII exhausted. SECURITY CLASSIFICATION OF THIS PAGEAll other editions are obsolete.

89 1 26 00.

9'was acquired and software was developed to determine the impedanceattributes of fouled metal coupons. Preliminary EIS studies usingthe enzyme, Ribulose Biphosphate Carboxylase, demonstrated theability to detect adsorption of this protein to a silver electrode.,/,',

DATE: 13 January 1989

ANNUAL REPORT

CONTRACT: N00014-88-K-0044 R&T CODE: 441h005

PRINCIPAL INVESTIGATOR: Gordon T. Taylor

CO-INVESTIGATORS: Shiv K. Sharma, Bruce E. Liebert &Howard F. Mower

CONTRACTOR: University of Hawaii at Manoa

CONTRACT TITLE: Examination of Chemical Adsorption and MarineBiofouling on Metal Surfaces using RamanScattering Techniques and ElectrochemicalImpedance Spectroscopy

START DATE: 1 November 1987

RESEARCH OBJECTIVES:

Our primary objective is to describe the initialchemical and microbiological events which occur on virginmetal surfaces immersed in seawater using primarily non-invasive, in sit analytical techniques. To accomplish this,we've defined the following specific objectives.

(1) Develop optimal spectroscopic sampling strategies for insitu detection and identification of low concentrations ofadsorbed organic materials, including spontaneous RamanScattering (SRS), surface-enhanced Raman scattering (SERS),UV-vis Resonance Raman scattering (RRS), Waveguide InternalReflectance Raman spectroscopy (WIRRS), ElectrochemicalImpedance spectroscopy (EIS), and fiber-optic probes.

(2) Obtain spectra and detection limits for putativefouling compounds; proteins, humic and fulvic acids,glycoproteins, polysaccharides, and fatty acids usingpurified compounds adsorbed to metals in organic-freeseawater.

(3) In situ identification of primary adsorbing compoundson metal surfaces in a flowing seawater system in theabsence and presence of planktonic microbial communities.

(4) Compare coupons fouled in the field (nearshore Oahu,Hawaii and open ocean) with those fouled in the laboratory.

(5) Quantify rates of chemical adsorption and microbial U

attachment to selected metal surfaces under varyingnutrient, biomass, temperature, light, and flow regimes.

(6) Characterize spatial heterogeneity of adsorption andmicrobial attachment on individual coupons. '.odes

COP,, ,, i~

PROGRESS (YEAR 1):

Much of our first year has been dedicated to acquisition,engineering, and technique development. Although funds arrivedlate, we have received all major items of equipment, fabricatedand tested several apparati, and have obtained SERS and EISspectra for several materials adsorbed to metal.

We have developed an environmentally-controlled, flowingseawater chemostat system with test metal coupons in Teflon flow-through fouling chambers (Fig. 1). The system is designed sothat the dilution rate, i.e., microbial growth rate, and the flowrate across the test coupons are controlled independently by twoseparate pumps. The system is well-oxygenated to preventanaerobiosis in the bulk phase. The fouling chambers aredesigned such that there is minimal disturbance in the flowfield, i.e., the test coupons are counter-sunk within the flowchamber. The optical flow chambers have 1.5 or 2" quartz windowswhich permit observation and passage of laser excitation andscattered radiation between the test coupon and the microscopeobjectives of the Raman spectrometer. The electrochemical celluses the test coupon as a working electrode and has a matchedplatinum counter electrode in the upper plate, sealed leads toboth electrodes, and a blind duct for the working solution(seawater) leading to a glass frit, electrolyte, and a referenceelectrode. A modified electrochemical cell with window has beendeveloped in order to perform SERS analysis of adsorbed films(described below).

Incubator

INSET

2- LZ_ _ _I34-

Flow-Through Foulin Canber

1- Seawater Resevoir2- Pump3- Growth Reactor4 Overflow5 Manifold6- Fouling Chambers7- Perista~tic Pump

AC Impedance Raman fiber optic. S. Metal Test Coupon

1 micro-probes 9- Ouartz WindowSpectrscope& Sl:ectromete r

Figure 1. Schematic of continuous culture apparatus withmultiple fouling chambers used to simulate and monitormarine biofouling.

We have acquired and developed systems for water collection,

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filtration, isolation of molecular size fractions, and reductionof organic loadings for large volumes of seawater. Forcollection of uncontaminated seawater with minimal disruption ofthe plankton, an all polymer system comprised of a portable DCvacuum pump which evacuates a reservoir and draws water from 50 -100' was developed. The system is easily operated from a smallboat and can collect 100 liters in less than an hour. Forfiltration, we have acquired a 142 mm Teflon filtrator forparticulate removal and an AMICON DC10L Hollow FiberConcentrator/Dialyzer to remove and concentrate particles > 0.1Am and molecular fractions; 100 kDa - 0.1 Am and 10 - 100 kDa.If desired, the 1 - 10 kDa molecular size fraction can beconcentrated using our AMICON Ultrafiltration membrane apparatus.These molecular fraction concentrates will be used in foulingexperiments and will be characterized chemically using Ramanspectroscopy, FT-IR spectrometry, gel electrophorhesis, ionchromatography with PAD for carbohydrate and glycoprotein, andspectrophotometry. To remove residual organics from theseawater, we have acquired a two-stage activated carbon cartridgesystem which is the final step in our water treatment protocol.Efficacy of the entire system will be tested early in Year 2. Wehave acquired a DIONEX Pulsed Amperomteric Detector and theappropriate columns for an existing DIONEX Quaternary GradientIon Chromatograph for the purpose measuring carbohydrates andglycoproteins. We have also purchased a BIO-RAD Mini-Protean IIGel Electrophorhesis system for characterization of proteins.Using non-ONR funds, we have augmented our analyticalcapabilities by acquisition of a Perkin-Elmer Model 1720X FT-IRspectrophotometer, data station, and Multiple InternalReflectance (MIR) attachment with Ge crystals. This permitsinter-comparison of our samples with previous work from otherlabs. We will also soon have a Reflectance-Absorbance attachmentfor the FT-IR for the direct examination of metal surfaces. Wehave also obtained matching funds from the DoD and the Universityof Hawaii to purchase a BOMEM DA3 near-infrared FT-Ramanspectrometer system with a 1.06-Am laser. Using a near-IR probeon our adsorbed films will be advantageous because excitationradiation in this spectral region produces fluorescence-freeRaman spectra and produces much less thermal decomposition thanvisible radiation. We expect delivery of this system in March1989.

As one approach for studying the interactions betweendissolved biomolecules and metal surfaces in seawater, we electedto use Surface Enhanced Raman Spectroscopy, SERS. We designed athree electrode electrochemical cell comprised of a silverworking electrode (1-mm wire insulated with epoxy or 1-cm 2

coupon), a platinum wire counter electrode, and a calomelelectrode separated from the working solution by a capillary(Fig. 2). Teflon was selected as the supporting body for allfouling chambers because of its inert nature and low surfaceenergy and a quartz glass window was fitted forspectrophotometric observation.

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To gain experience, we tested and optimized our cell using awell-known analytical system (pyridine / 0.1N KCl). The workingelectrode's surface was prepared by polishing with successivelyfiner grades of alumina down to 0.05 gm, then sonicated indeaerated, distilled water. Surface roughening was produced by asymmetric double potential step waveform Oxidation-ReductionCycle (ORC). A potentiostat regulates the static potentialwithin the cell and a coulometer integrates the current passedthrough the electrode. The electrochemical cell with polishedelectrode is stabilized at -0.2 V and stepped up to +0.2 V until3.6 mCoulombs have passed through the electrode. Afterconditioning, spectra were acquired at a -0.6 V potential.

Figure 2. Schematic of flow-through SERS and EIS samplingsystems. SERS cell has optical window over test coupon andoffset platinum wire counter-electrode. For SERS, eitherflat metal coupon or wire can be used as the workingelectrode.

To determine how SERS would perform in a marine system, weexamined 50mM pyridine in filtered UV-oxidized sea water as theelectrolyte. On electrochemically-roughened silver, we obtaineda 60-fold enhancement in Raman scattering intensity of the phenylring breathing mode (" 1000 cm"1 ) for pyridine over the bulkphase and 30-fold enhancement over unconditioned silver at itsresting potential (Fig. 3). The lowest detectable signal wasobtained using a 10"" M pyridine solution.

Preliminary SERS studies on amino acids, peptides, andproteins are currently underway. Aromatic amino acids werechosen as target molecules because the amine group is known tointeract with the silver surface and the phenyl ring is a goodRaman scatterer. We have obtained SERS spectra of tryptophan andphenylalanine adsorbed onto a silver surface, but problemsassociated with fluorescence are particularly evident in thephenylalanine spectra (Fig. 4). Future studies will include moreamino acids and synthetic peptides to gain more information onhow protein primary structure interacts with the metal surface.

4

800

C600

ix

400-

200

0

la Bo- 18

6 30 xC

12-

A

60 x

0400 600 800 1000 1400 400

Raman Shift (cm-1)

Figure 3. SERS study of pyridine. Excitation = 488.0 nm, 6mW; 120 scans, 1 sec exposure. A. bulk pyridine (50 mM) inUV-oxidized seawater. B. pyridine adsorbed on polished Agelectrode in seawater, no ORC or applied potential. C.pyridine adsorbed to conditioned Ag electrode (30 cycles -

0.6 to +0.2 V) and measured at a -0.6 V potential.

5

sow

A B45000-

76000-

70000- 5000-

M 60W 110 iO 16 0* 0 0G00 N 0 i0 10 10 w 4 s 601

NOM $hft Raa shift (cs-1)

Figure 4. SERS study of aromatic amino acids adsorbed onconditioned Ag electrodes in 0.1 N KCl. A. tryptophan. B.phenylalanine.

Ribulose biphosphate carboxylase (RuBisCo) has been targetedby other ONR Biosurfaces researchers as a putative foulingcompounds, so we are performing complementary Raman scatteringand EIS studies on this ubiquitous enzyme. In our firstattempts, we were unable to obtain acceptable SERS spectra ofthis enzyme because of fluorescence problems and impurities inthe preparation. We are continuing our studies by purifying theenzyme and modifying our sampling techniques so that we mayexamine non-SERS producing metals as well as SERS-producingmetals. Techniques under investigation are: UV Resonance RamanSpectroscopy (pulsed excitation, gated detection), WIRRS, andnear IR FT-Raman spectroscopy (instrument expected 3/89).

We have adapted WIRRS techniques as developed by polymerscientists at IBM Research Center to examine thin films adsorbedto substrates, such as glass. In principle, the waveguideprovides an extended pathlength for laser excitation light byinternal reflection within the thin film. This is accomplishedby directing the laser light into the film at a critical grazingangle via a high refractive index prism and the light remainstrapped in the film (waveguide) due to mismatch of the refractiveindices of the substrate, film, and overlayer (air) (Fig. 5A).Scattered emissions are then collected over the waveguide bymeans of a lens or linear fiber optic array and focused on thespectrometer slit. Spectra obtained by WIRRS have a much-improved sensitivity and signal-to-noise ratio relative to bulksample analyses. We have successfully developed a WIRRSapparatus in our laboratory and have obtained excellent Ramanspectra of 1 ;im films of polystyrene on Pyrex substrata(waveguides courtesy of J. Rabolt, IBM Almaden Research Center,CA) (Fig. 5b). We are in the process learning how to prepare ourown waveguides on Pyrex substrata with previously-studiedpolymers and will then prepare them with putative fouling

6

molecules. We anticipate being able to apply the same samplingstrategy to metal substrata. Our second generation waveguideassembly will employ an environmental chamber to control humidityand temperature thereby permitting examination of hydratedsurfaces.

Marnan3.42E 04Remanspecuonto I

A -. P

Polarizatw,,Analyze 4

Figre5. vguie nen a Relcac SetoerSubstrate 6S~5

Lasers..Wm~m* AwbI, Position (dwn)

Figure 5. Waveguide Internal Reflectance Spectrometry(WIRRS). A. Schematic of sampling optics. Adjustablemirror 2 and prism (P) determine critical angle of laserbeam needed to maximize internal reflectance the film (fromSchlotter and Rabolt, 1984). B. Raman spectra of a 1-Mmlayer of polystyrene adsorbed on Pyrex obtained by WIRRS.

Despite long delays in shipping equipment required forElectrochemical Impedance Spectroscopy (frequency responseanalyzer, electrochemical interface, and instrumentationcontroller), B. Liebert and students, M. Nullet, and P. Chonghave successfully produced an operational system, includingelectrochemical cell and operating software. Preliminary runswith a silver electrode, UV-oxidized seawater, and a putativefouling protein, Ribulose 1,5 biphosphate carboxylase (RuBisCo)have been completed.

Prior to adding RuBisCo to the system, a series of runs weremade to establish functionality of the cell and systemparameters. The system stabilized before each run atapproximately -70 mY. A Bode plot of a typical run isindicative of an equivalent circuit consisting of a simpleparallel RC circuit in series with an uncompensated resistance(Fig. 6). Although the frequency does not go high enough todetermine the uncompensated resistance, it is of the order of 10ohms or less. There is no indication of any Warburg(diffusional) impedance in this plot as expected since theelectrolyte is flowing. A double layer capacitance of 50-100 gFcm"2 and a polarization resistance of 250-300 kohm cm"2 can becalculated from Fig. 6a.

7

A i n Fiit sw No utr .j tp 3, / /i p g4 6p, .... '4 r ..e .P4:r ' -d j

* -- Ie'='"=-t -,,. " "1-70A a 0004 V " o '

-70 - , -70- • ; -106+"- V 4 E..

4 S*, -50 4 - 50*C " I .

S-30 3- -30

-20 ? , 1-02 - , 4, , i

0 Ib

-4 -3 -2 -1 0 1 2 -' -3 -2 -1 0 2

Log (Freq.) [Hz. ) Log (Freq.) [Hz.

Figure 6. Bode plots for Ag electrode in (A) unamended UV-oxidized seawater, and in (B) UV-oxidized seawater amendedwith RuBisCo (10 mg 1"1) .

After amendment with RuBisCo (10 mg 1-; final conc.), thesystem stabilized at -85 - 100 mV and again indicates a simpleequivalent circuit as above (Fig. 6b). After RuBisCo addition,the polarization resistance increased to about 400 kohm cm 2 .This is expected as a result of formation of an insulating layerof protein on the working electrode.

. Ac in FSW 1.3OV ? n tr /1 8/11/21 -0 ^ n N t W @ 2 C -0 . e 0 8 / : 3 1 "

A -O B l I g Ia t o

. . -70 5 g -70A x

-60 x _ -60

4 %-50 4 -50

E g -40 ,-4o : Uo",-3

3 -30 3 -30

I ) 1

0 0

-10. *" . -.

10 10..4 -3 -2 -1 0 1 2 -4 -3 -2 -1 0 1 2

Log (Freq.) (Hz. ) Log (Freq.) [Hz. I

Figure 7. Bode plots for Ag electrode with a -100 mVoverpotential applied in (A) unamended UV-oxidized seawaterand in (B) UV-oxidized seawater amended with RuBisCo (10 mg

8

We ran both systems (unamended and amended seawater) at anoverpotential of -100 mV (Fig. 7). Approximately the sameequivalent circuit seems to prevail, although the charge-transferresistance is nearly an order of magnitude larger for the amendedseawater sample (Fig. 7b). These results are consistent with theadsorption of the labile polyelectrolytic protein on the surfaceof the working electrode and the lowering of the potential energybarrier by the application of a -100 mV overpotential.

Our preliminary EIS studies have demonstrated that we candetect changes in the low-frequency impedance caused by theadsorption of a single dissolved solute, a protein, onto a silverelectrode immersed in seawater. We have learned the strengthsand weaknesses of our experimental system and currentlymodifications are underway to improve electrode preparation,reproducibility, and extend frequency to 300 kHz. In ourcontinuing studies, we will determine the sensitivity of oursystem, examine other solutes and other metal coupons beforeintegrating the EIS system into the continuous culture simulationsystem.

WORK PLAN (YEAR 2):

We are engaged in adapting several non-SERS methods for theexamination of films on metals other than silver (WIRRS and UV-RRS). Parallel studies will be performed using FT-IRspectroscopy and EIS. Strategies for desorbing material fromcoupons will be evaluated by conventional analytical techniques,e.g., HPLC, fluorometry, electrophorhesis, etc. Optimalsampling techniques will be obtained for several classes offouling compounds under controlled conditions. We are currentlyproducing our own humic acids from phytoplankton cultures whichwe will refine, molecular size fractionate, characterizespectrophotometrically, and use in kinetic fouling experimentswith our Raman probes. In addition to our laboratorysimulations, short deployments of test coupons will also beperformed in nearshore and offshore environments for comparativepurposes as time permits.

INVENTIONS: none

PUBLICATIONS:

G. T. Taylor and S. K. Sharma. 1988. Use of laser spectroscopicprobes for examination of biofouling. Presented at the NationalInstitute of Oceanography, Goa, India, Nov. 1988.

TRAINING ACTIVITIES:

Our project currently employs one graduate student and twoundergraduates. Our graduate student, Paul Troy, will derive hisPh.D. dissertation from this project (topic presently undeclared)and is receiving training in vibrational spectroscopy, marine

9

chemistry, and microbiology. our undergraduates, Eamonn O'Tooleand Patty Fisher, are Biology majors and are receiving firsthandlaboratory experience in chemistry and microbiology. Mr. MichaelNullet, an M.S. student in Mechanical Engineering, iscollaborating with B. Liebert to refine and operate the EISsystem. P. Chong, an undergraduate in Electrical Engineering hasbeen instrumental in developing the software necessary for EISdata acquisition and reduction. We have an M.S. student inMicrobiology who will begin a directed research project thisSpring examining the microbial ecology of our fouled couponsusing light and electron microscopy. A Ph.D. student in Botany,Tina Michaels, interested in biofouling of natural and artificialsubstrata will be collaborating with us starting this Spring.Ms. Michaels already has experience with field deployments oftest coupons, SEM, and FT-IR and is the recipient of a fellowshipfrom Hawaii Natural Energy Institute for biofouling work.

AWARDS / FELLOWSHIPS: none

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