Uptake of redox couples by algae immobilized on porous cellusosic films

Eleetrochimica Aria, Vol . 35,

No.

9, pp . 1377-1381, 1990

0013-4686190 $3.00+0 .00 C 1990. Pergamon Press plc .

Printed in Great Britain .

UPTAKE OF REDOX COUPLES BY ALGAE IMMOBILIZED ON POROUS CELLULOSIC FILMS JOSEPH WANG* and TEDDY MARTINEZ Department of Chemistry, New Mexico State University, Las Cruces, NM 88003, U .S .A . DENNIS DARNALL Biorecovery Systems Inc ., Las Cruces, NM 88003, U .S .A. (Received 19 September 1989 ; in revised form 5 December 1989) Abstract-Cellulose acetate films are used as matrices for the immobilization of algal species at electrodes . Base hydrolysis is shown to alter the collection of ionic reactants by the immobilized microorganisms . The larger network porosities thus obtained offer attractive features, including shorter equilibration times and larger ion-exchange capacities . Non-hydrolysed films, however, offer improved retention of bound complexes . The binding of multiply-charged ions (eg Fe(CN)s , Ru(NH 3 t) is investigated using cyclic voltammetry, as a function of hydrolysis time, immersion time, film composition, reactant concentration, scan rate and other variables . Scanning electron micrographs reveal non-uniform surface microstructures, with aggregates of alga particles covered with a cellulosic layer . Key

words : microorganism, polymeric coating, modified electrode .

INTRODUCTION Chemically modified electrodes are the focus of considerable current research . Several studies have recently described the utility of microorganisms, particularly algae, as surface modifiers[I-3] . The unusual collection of ionic species by surface-bound algae has shown great potential for electrocatalysis[2] and electroanalysis[l, 3] . The cells of algae exhibit high binding capacities for different ionic species . The binding of such species occurs by different mechanisms, including electrostatic or covalent attachments to cell wall constituents[4-6] . Different algae possess different amounts and kinds of surface functionalities (carboxylate, amine, sulthydryl, imidazoles, etc) and thus exhibit different specificity of affinity toward ionic species . In addition, the binding of different ions to a given alga is strongly dependent upon conditions (such as the pH) . To date, fabrication schemes of algae modified electrodes have counted on the incorporation, by mixing, of different algae (eg Eisenia bicyclis, Chlorella pyrenoidosa) into the bulk of carbon paste electrodes . The purpose of the work described here is to demonstrate the immobilization of microorganisms on porous cellulosic films . Cellulose acetate films have been proved to be highly useful for a variety of electrochemical applications[7-13] . In particular, the porosity of cellulose acetate films-and hence their permeability-can be varied by hydrolysing them in alkaline media for different periods[8, 9] . Thus, effective size-exclusion detectors have been obtained . The open structure of cellulosic films can also be advantageous for the incorporation of various reactive moieties. For example, composite films based on the incorporation of the cationic polyelectrolyte poly(4-vinylpyridine) or the catalyst cobalt phthalo-

cyanine into cellulosic matrices exhibit properties superior to those of the individual components alone[12, 13] . Optical pH sensors, with very rapid response times, have been constructed by binding pH indicators at a porous cellulosic film[14] . The incorporation of different (non-living) algae onto hydrolysed cellulosic supports is shown, in the following sections, to offer various attractive features including very rapid equilibration of counterionic reactants and large binding capacities . The immobilized microorganisms retain their binding properties and incorporated redox couples retain their electroactivity. Such properties of algae-containing cellulose acetate-coated electrodes can be manipulated by control of the hydrolysis time .

EXPERIMENTAL Apparatus The electrochemical cell was a 10 ml glass vial (Model VC-2, Bioanalytical Systems (BAS)) . The cell was joined to the working electrode, reference electrode (Ag/AgCI (3M NaCI), Model RE-1, BAS) and platinum wire auxiliary electrode through holes in its Teflon cover . The three electrodes were connected to the EG&G PARC Model 264A polarograph, the output of which was displayed on a Houston Omniscribe x-y recorder . Electrode coating procedure Prior to its coating, the 3 mm dia. glassy carbon electrode (Model MF2012, BAS) was polished with 0.05 pm alumina particles, sonicated for 5 min, and allowed to air-dry. The electrode was coated with 10p1 of the alga-cellulose acetate solution, placed to cover the active disk and its surroundings . The

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solvents were allowed to evaporate with a heat gun . The alga-cellulose acetate solution was prepared by dispersing a certain weight of the alga powder in a given volume of the 2% cellulose acetate solution (usually 0 .013 g ml - '). The 2% cellulosic solution in (1 :I acetone-cyclohexanone) was prepared according to a previously described procedure[8] . The lyophilized algae were first ground in a mortar and pestle to particles of around 100-120µm. The film was hydrolysed in a stirred 0 .07 M KOH solution for the desired time . Alga-Nafion coated electrodes were prepared in a similar manner, using 5µl of the 13 mg ml alga-Nafion suspension . The resulting films were not hydrolysed . Experiments were conducted at room temperature, using unstirred solutions .

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Reagents

All solutions were prepared with doubly distilled water . Cellulose acetate, hexammine ruthenium chloride (Aldrich), and potassium ferricyanide (Fisher) were used without further purification . The 5% Nafion solution was obtained from Solution Technology Inc . The copper ion stock solution (1 x 10 - 'M) was prepared by dissolving Cu(NO3)2 in nitric acid and diluting to volume with doubly distilled water . The various algae were obtained from Biorecovery Systems Inc . The supporting electrolytes were 0 .05 M phosphate buffer (adjusted to the desired pH with phosphoric acid) and 0 .05 M acetate buffer (pH 5 .0) for the collection of metal complexes and copper ions, respectively .

RESULTS AND DISCUSSION The immobilization of redox mediators on electrode sufaces has been a very active research area . Alga] cells function like a mixture of ion-exchange resins and can thus bind both anionic and cationic metal complexes . We have recently demonstrated the ion-exchange incorporation of multiply-charged ions at algae-modified carbon paste electrodes[2] . The incorporation of different algae within the cellulose acetate network and the advantages which accrue from the controlled porosity (permeability) of these surface microstructures are presented below . Figure I compares cyclic voltammograms for Fe(CN)6 - recorded continuously at a glassy carbon electrode coated with Rhodymenia palmata-cellulose acetate, following different hydrolysis times (0(A), 20(B) and 40(C) min) . All electrodes exhibit a facile incorporation of Fe(CN)6 - and a well-defined response . However, clear changes in the rates of incorporation and surface coverages are observed for different durations of the base-hydrolysis . These changes are indicated from the values given in Table L For example, the quantity of bound Fe(CN)6-(f") increases from 1 .75 x 10 -9 to 7 .55 x 10' moi cm - ' and the equilibration time (Q decreases from 47 to 19 min upon hydrolysing the network for 40 min (relative to the non-hydrolysed film) . Such changes are attributed to the greatly reduced barriers to mass transport and the exposure of additional binding sites, associated with the larger film porosity. The electrostatic binding of the anionic complex is accomplished at low pH (where amine or

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Fig . 1 . Cyclic voltammograms demonstrating the collection of Fe(CN)' - at the Rhodymenia palmata-cellulose acetate (13 mg ml-1) coated electrode . Hydrolysis time: 0(A), 20(B) and 40(C) min . Continuous scanning at 50 mV s ' using a phosphate buffer solution (0.05 M, pH 2) containing I x l0 -' M Fe(CN)6 - . Analogous voltammograms at an alga-free (20 min hydrolysed) cellulose acetate coated electrode are shown in D ; (1) indicates the last scan . imidazole groups are protonated) . In the absence of bound alga, the cellulosic film exhibits significantly smaller redox peaks (trace D) . Figure 2 shows the dependence of the charge (Q), for the surface bound Fe(CN)6 - , upon the immersion time of the Rhodymenia palmata-cellulose acetate-coated electrode, for different hydrolysis times . In accordance with observations of Fig. 2 and Table 1, clear changes in the rate of binding and the quantity of anion bound are observed following different hydrolysis periods . The collection cationic metal complexes can also be facilitated by the immobilization of algae at porous cellulosic matrices . Increased pH, and hence deprotonation of surface binding sites, promotes the binding Table 1 . Loading properties of Rhodymenia palmata containing cellulosic films hydrolysed for d ifferent periods* Hydrolysis time/min 0

Q/pC

F)molcm 2t

t,/mint

aEp /V

11 .9 13 .1 51 .4

1 .75 x 10 -9 1 .92 x 10 - ' 7 .55 x 10 -9

47 24 19

0.06

20 0.06 40 0.09 *Conditions : as in Fig. 1 . tThe charge and surface coverage were obtained by integrating the area under the cathodic peak ; glassy carbon area, 0 .07 cm 2 . :Equilibration time .



Uptake of redox couples by algae

Time (min)

Fig . 2 . Time dependence of the attachment of Fe(CN)6 - to Rhodymenia palmata-cellulose-coated electrode . Hydrolysis time : 0(A), 20(B) and 40(C) min. Other conditions, as in Fig . 1 . of such species . Figure 3 shows cyclic voltammograms which result when different algae-containing electrodes are placed in solutions containing Ru(NH 3 )6+ . A very rapid incorporation of the cationic reactant is observed at both electrodes . A faster attainment of equilibrium (13 vs 28 min) is observed upon hydrolysing the polymeric support for Rhodymenia palmata (B). At a Cyanidium caldarium electrode (A), the equilibrium is attained within 15 min for both non-hydrolysed and hydrolysed films . Increased quantities of collected Ru(NH 3 )6+ are also observed at the hydrolysed films (3 .7 x 10 -9 -' and 1 .6 x 10 - 'vs 1 .1 x 10 -9 and 9 x 10 -10 molcm for the non-hydrolysed films, A and B, respectively) . Effective collection of Ru(NH 3 )6+ was also obtained at the Spirulina platensis-cellulose-coated electrode

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(30 min hydrolysis) with a steady-state coverage of 3 .5 x 10 - ' mol cm - ' and equilibration time of 40 min (not shown) . A careful examination of the surface microstructure, using scanning electron microscopy (Fig . 4), reveals that the alga particles are simply covered by the cellulosic layer, which causes their adherence to the carbon surface . Notice also that coverage of the surface by these particles is not complete or uniform, as they form aggregates on the base electrode . The film thickness is not uniform (1-5µm range). As was discussed earlier, it appears that the increased porosity, associated with the base hydrolysis, enhances the permeability of ionic reactants toward the alga cell wall and increases the accessibility of binding sites on these walls . It appears also that the algae remain intact during the hydrolysis process (ie exposure to high pHs do not affect the binding properties) . The quantity of the surface-bound alga has a pronounced effect upon the binding properties of the electrode . Figure 5 shows loading cyclic voltammograms for Fe(CN)6 - at non-hydrolysed films containing different contents of Rhodymenia palmata . Well-defined peaks are observed at different assemblies . As expected, the quantity of incorporated anion increases from 1 .75 x 10 -9-9 .0 x 10 -9 mot cm -2 upon increasing the alga content from 0 .013 to 0.250 g ml - ' cellulose acetate . The rate of accumulation is also affected by the alga loading with faster attainments of steady-state for higher quantities of Rhodvmenia palmata (47 vs 27 min for 0.013, and 0 .250 g ml - ', respectively). A gradual increase in the separation of the peak potentials, from 62 to 150 mV, is also observed upon increasing the alga content . The exact reasons for these changes in the rate of binding or peak-potential separation are still unclear . Peak currents at the Rhodymenia palmata-cellulose acetate electrode assembly, after equilibration from Fe(CN)6 and Ru(NH 3 )6' solutions, were proportional to the square root of the scan rate .

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Fig . 3. Cyclic voltammograms demonstrating the collection of Ru(NH 3 )6+ at the Cyanidium caldarium(A)- and Rhodymenia palmata(B)-cellulose acetate-coated electrodes . Hydrolysis time : 0(A) slid 30(B) min . Continuous scanning at 50 mV s ' using a phosphate buffer solution (0 .05 M, pH 6) containing I x 10 -3 M Ru(NH 3 )6+ EA 3519-E



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Fig. 4 . Scanning electron micrograph of the Rhodymenia palmata cellulose acetate-coated electrodes . Magnification : 320 x . Hydrolysis time: 0 min . Larger slopes of (peak-current) vs (scan rate)' plots were observed upon extending the hydrolysis time, reflecting the larger quantities of bound complexes . Changing the bulk concentration of Fe(CN)6 - , between I x 10'M and 6 x 10 -4 M, resulted in a linear increase of the steady-state coverage (conditions as in Fig. 1, with 15 min hydrolysis) . The fast equilibrium between alga-bound and the solution ions can be further recognized by the pro-

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Potential (V) Fig . 5 . Effect of alga loading on the collection of Fe(CN)s at the Rhodymenia palmata-cellulose acetate-coated electrode . Alga content, 0.013(A), 0.127(B) and 0 .250(C) g ml - ' cellulose acetate . Other conditions, as in Fig . IA .

gressive loss of ionic species from the surface upon transfer of the loaded electrode to a pure supporting electrolyte solution . Because of their higher porosity, hydrolysed coatings offer inferior retention capabilities compared to non-hydrolysed ones . For example, retention ratios (1 45 /15 ) of 0 .06 and 0 .17 were estimated at the 30 min hydrolysed Spirulina platensis and Rhodymenia palmata based surfaces, respectively, after equilibration from an Ru(NH3 )6' solution, compared to 0.32 and 0 .21 at the non-hydrolysed films . (115 11-5 is the ratio of the coverage 45 min after transfer to the blank solution over that immediately after such transfer .) Hence, and in accordance with Fig . 1, the porous matrix facilitates the transport of reactants to and from the binding sites . Lowering of pH can facilitate the removal of bound cationic complexes from the surface (via proton competition) and hence the use of a single surface for multiple "loading"/regeneration cycles . In addition to the binding of multiply-charged ions, with its potential electrocatalytic implications, the immobilization of microorganisms on porous cellulosic films can greatly benefit the area of electroanalysis . In particular, measurements of metal ions can be improved (with respect to selectivity and sensitivity) via the preconcentration/mediumexchange/voltammetric scheme at algae modified electrodes[l] . Figure 6 illustrates such measurements of copper following different preconcentration periods at the Eisenia bicyclis-coated electrode . The copper peak, recorded after accumulation from the stirred sample solution followed by transfer to a blank solution, increases linearly with the preconcentration time . Improved selectivity accrues from the use of Eisenia bicyclis (with its strong affinity toward copper due to high carboxylate content[I]), the choice of solution conditions and the medium-exchange step (that eliminates contributions from non-accumulated electroactive species) . Surface renewal was accomplished by dipping in hydrochloric acid. It is possible to employ other polymeric matrices for supporting algal species on electrode surfaces . For



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Fig . 6. Differential pulse voltammograms demonstrating the collection of copper ion at the Eisenia bicyclis-cellulose acetate-coated electrode . Hydrolysis time: 0 min . Preconcentration from a stirred 5 x 10-° M copper solution for 2(A), 4(B), and 6(C) min . Scan rate : 10 mV s ' . Amplitude : 50 mV. Preconcentration/medium-exchange/voltammetric scheme, as in Ref. [I] .

Fig . 7 . Cyclic voltammograms demonstrating the collection of Fe(CN)6 - at the Rhodymenia palmata-Nafioncoated electrode . Electrode modification was accomplished by transferring 5p1 of the alga-Nafion suspension (13 mg ml - ') to the surface and allowing it to dry . Other conditions, as in Fig . 1 .

schemes, exploiting the advantageous features of the cellulose acetate network, are currently being pursued in this laboratory . financial support provided by the National Institutes of Health (GM 30913-06 and RR0812615) and the National Science Foundation (CBT-8610461) is acknowledged . Acknowledgements-The

example, we have used the perfluorinated ionomer Nafion for supporting Rhodymenia palmata. Repetitive cyclic voltammograms demonstrating the ability of this alga-Nafion electrode assembly to collect Fe(CN)6 - are shown in Fig . 7 . The incorporation attains its final value within 14 min . The voltammograms are substantially broader, with a large peak potential separation (- 300 mV), compared to analogous experiments using the cellulosic support (eg Fig . 1) . An Fe(CN)6 coverage of 2 .83 x 10 -8 mol cm - 'can be estimated from the steady-state voltammogram .

CONCLUSION We have described the preparation and characteristics of algae modified electrodes based on the use of a porous support microstructure . Our data indicate that the binding properties of the immobilized algal species are retained and the incorporated redox couples remain electroactive . This strategy provides a means of rapid mass transport of species from solution into immobilized algal species . Besides potential electrochemical applications, such algalbound porous microstructures may find important applications in removal and recovery of metals from water. Similar applications of algal biomass have been reported[5] . Other reagent immobilization

REFERENCES 1 . J . Gardea-Torresdey, D . Darnall and J. Wang, Anal. Chem . 60, 72 (1988) . 2 . J . Wang, T. Martinez and D. Darnall, J. electroanal. Chem . 259, 295 (1989) . 3 . J . Gardea-Torresdey, D. Darnall and J. Wang, J. electroanal. Chem . 252, 197 (1988) . 4. D . Khummongkol, C. S. Canterford and C. Fryer, Biotechnol. Bioengng 24, 2643 (1982) . 5 . B . Green, M . Henzl, M . Hosea and D . W. Darnall, Biotechnol. Bioengng 28, 764 (1986) . 6. N . Kuyucak and B. Volesky, Biotechnol. Bioengng 33, 809 (1989) . 7. G . Sittampalam and G . S . Wilson, Anal. Chem. 55,1608 (1983) . 8 . J . Wang and L. D . Hutchins, Anal. Chem, 57, 1536 (1985) . 9. J . Wang and P . Tuzhi, Anal. Chem . 58, 3257 (1986). 10. L . S . Kuhn, S . G. Weber and K . Z. Ismail, Anal. Chem. 61, 303 (1989) . II . L . D . Hutchins-Kumar, J, Wang and P . Tuzhi, Anal. Chem . 58, 1019 (1986) . 12. J . Wang and P. Tuzhi, J. electrochem . Soc. 134, 586 (1987) . 13. J . Wang, T. Golden and R. Li, Anal. Chem . 60, 1642 (1988) . 14. T . P . Jones and M . D . Porter, Anal. Chem. 60, 404 (1988) .