Appl Microbiol Biotechnol (1994) 41 : 725-728

Springer-Verlag 1994

B. W e h r h e i m M. W e t t e r n

Biosorption of cadmium, copper and lead by isolated mother cell walls and whole cells of Chlorella fusca

Received: 6 October 1993 / Received revision: 21 February 1994 / Accepted: 28 February 1994

Abstract Using a new m e t h o d for the isolation of released m o t h e r cell walls of Chlorella fusca, the biosorption of cadmium, copper and lead by purified cell wall isolates and whole cell suspensions was comparatively characterized. In all cases whole cells accumulated m o r e metal ions than isolated cell walls. Both the Langmuir and Freundlich isotherm models were suitable for describing the short-term adsorption of cadmium, copper and lead by cell walls and the cadmium and copper adsorption by whole cells. However, neither model could sufficiently explain the lead accumulation by whole cells. T h e feasibility of a practical use of whole cells or isolated cell walls as biosorbents is discussed.

intracellular bioaccumulation. While greater amounts of heavy metals can be accumulated by intracellular accumulation, toxicity problems are excluded by passive biosorption on the cell surface ( G a d d 1990). Therefore, biosorption processes could be an alternative or a supp l e m e n t to purification methods currently used. Many physiological studies dealing with heavy metal uptake of Chlorella fusca are available. However, there is a lack of data concerning the feasibility of utilizing whole cells or isolated cell walls as biosorbents. This p a p e r reports the characterization of the biosorption of heavy metals by whole cells and isolated m o t h e r cell walls (mcw) of C. fusca and examines the suitability of isolated mcw as ion exchangers for the detoxification of heavy metal contaminated waste-water.

introduction
The advanced contamination of the environment with heavy metals presents an increasing p r o b l e m for wastewater t r e a t m e n t (Frischmuth et al. 1991). Traditional precipitation methods or the use of synthetic ion exchangers cannot solve all purification problems (Schmiechen et al. 1992). N u m e r o u s studies have shown that micro-organisms are highly capable of accumulating heavy m e t a l ions. Earlier work was concentrated on the examination of u p t a k e mechanisms and toxicological aspects. Only recently studies have b e e n u n d e r t a k e n to evaluate the use of microbial metal accumulation for the purification of heavy-metal-loaded waste-water (ROhricht et al. 1990). H e a v y metal u p t a k e can be divided into a rapid passive biosorption on the cell surface followed by a slower

Materials and methods

Microorganism and cultivation conditions

C. fusca Shihira et Krauss, DSM strain 211-8b, was cultivated in continuous culture at 27C in an inorganic standard nutrient medium (Kuhl 1962) with a final pH of 6.2.

Isolation of mother cell walls Released mcw were isolated from whole cell suspensions in four centrifugation steps. Firstly, cells and cell walls were concentrated by centrifugation at 12,000 g and 20 C. A sedimentation of cells and cell walls was reached by centrifugation at 26,000 g and 20 C for 10 min. The separation between cells and cell walls was achieved using a 1.9 M sucrose cushion and centrifugation at 68,000 g and 4C for 30 min. The isolated cell wall fraction was washed several times at 90,000 g and 4 C for 10 min.

B. Wehrheim 1 M. Wettern ([]) Botanisches Institut und Botanischer Garten, TU Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany

Surface determination One hundred Single cells and cell walls were measured with an ocularmicrometer and the mean surface area (a) was determined using the formula: a=4 p r 2.

Present address:
1 I.N.R.A., Station d'Agronomie, Centre de Recherches de Bordeaux, F - 33883 Villenave d'Ornon Cedex, France

726 Adsorption experiments All experiments were conducted with 100-ml batch cultures in 5 mM PIPES [piperazine-N,N'-bis (2-ethanesulphonic acid)] at pH 6.0 and 20 C with cadmium, copper and lead concentrations of 5, 10, 20, 50 and 100 mmol/1 and biomass concentrations of 10 7 cells/ml and 107 cell walls/ml. Samples were taken 1, 5, 10, 15 and 30 min after metal addition to determine the dry weight, the residual metal concentration in solution and the metal concentration in the biomass. The metal content of the biomass was determined by differential pulse anodic stripping voltammetry. The evaluation of the isotherm data was carried out with the software "Enzfitter" (Version 1.03, Elsevier Biosoft, 68 Hills Road, Cambridge, UK).
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Results

0.4 With an improved isolation m e t h o d the concentration and isolation of free mcw within a whole cell suspension of C. fusca became possible. The purity of the cell wall fractions was examined by transmission electron microscopy. In order to characterize the heavy metal uptake and clarify the role of the isolated cell wall in ion adsorption, a comparable reference figure had to be found. Determination of the specific surface area and of the dry weight of 108 cells or 108 cell walls resulted in mean surface values of 226 cm 2 and 297 cm 2 and mean dry weights of 6.08 mg and 0.225 mg, respectively. This demonstrates that mcw exhibit only a small fraction of the whole cell dry weight in contrast to their similar surface data. For a physicochemical characterization of binding sites, a specific surface area appeared to be a more suitable reference figure. Short-term adsorption kinetics of c a d m i u m , copper and lead at external metal concentrations of 5, 10, 20, 50 and 100 txmol/1 showed no principal difference between whole cells and isolated mcw. M o r e than 90% of the totally bound metal ions were already adsorbed within the first minute after metal addition. However, in any case whole cells showed a higher maximum adsorption than mcw (Wehrheim and Wettern, 1994). For a more detailed characterization of the adsorption equilibrium, the classical adsorption models of Freundlich (1926) and Langmuir (1918) are often used (de R o m e and Gadd 1987; Xue et al. 1988; Ting et al. 1989; Aksu and Kutsal 1991). Figure 1 shows the adsorption behaviour of whole cells and mcw for cadmium and copper, which is similar using either the Freundlich or the Langmuir isotherms. A log-log plot of the Freundlich isotherm and a Ruzic plot of the Langmuir isotherm (Ruzic 1982) gave straight lines with an acceptable correlation coefficient r (Table 1). The characteristic Freundlich constants were determined from the slope and intercept of the linearized form, which are equal to 1/n and Kf at c = 1, where n and K~ are t e m p e r a t u r e - d e p e n d e n t constants and c the total metal concentration in solution ( m o l - L - 1 ) . In contrast, none of the adsorption models could sufficiently describe lead adsorption by whole cells
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Fig. 1 Langmuir ( A, C) and Freundlich ( B, D) plots of cadmium (e), copper (E) and lead (V) adsorption by mother cell walls ( A, B) and whole cells ( C, D) of Chlorellafusca at metal ion concentrations of 5, 10, 20, 50 and 100 txmol/1 (m, amount of metal ions adsorbed per unit cell surface; c, metal ion concentration remaining in solution at equilibrium)

727 Table I Characteristicdata for the Freundlich and Langmuir isotherms and the correlation coefficient (r) of the linearized isotherms for whole cells and isolated mother cell walls of Chlorella fusca (Kf, constant; n, constant; mm~, maximal mass sorbable;/i, binding constant) Metals Freundlich isotherm Kf [nmol/cm2] Whole cells Cadmium 0.277 Lead 0.403 Copper 0.268 Mother cell walls Cadmium 0.019 Lead 0.080 Copper 0.039 /'t log-log r Langmuir isotherm mma x [nmol/cmz] 0.645 2.385 1.280 0.355 0.501 0.531 gI [ml/nmol] 0.518 0.078 0.120 0.024 0.067 0.043 Ruzic r

4.707 2.900 2.830 1.760 2.572 1.857

0.9912 0.9545 0.9914 0.9740 0.9970 0.9650

0.9983 0.8403 0.9974 0.9397 0.9822 0.9746

(Fig. 1 C + D ) also expressed by the lower correlation coefficient r of the linearized isotherms (Table 1). While a definite surface area was more suitable for the characterization of binding sites, :for a practical approach it is customary to refer the metal uptake to the dry weight. As the dry weight of isolated cell walls exhibits only a small fraction of that of whole cells, one gets contrasting results. Isolated mcw accumulated 0.25 ixmol cadmium/mg, 0.58 p,mol copper/mg and 0.61 txmol lead/mg whereas whole cells accumulated 0.03 tzmol cadmium/mg, 0.05 p~mol copper/rag and 0.09 ~mol lead/mg.

Discussion
In order to compare heavy metal adsorption by whole cells and isolated cell walls of C. fusca, classical adsorption models were used to describe the equilibrium between the metal ions in solution and the metal ions adsorbed on the cell surface (Fig. 1). Equilibrium studies on adsorption give information about the capacity of the adsorbent or the amount required to remove a unit mass of pollutant under the system conditions. An adsorption isotherm is characterized by certain constants the values of which express the surface properties and affinity of the sorbent (A1-Duri et al. 1992). Kf and n are indicators of adsorption capacity and adsorption intensity, respectively. The higher the value of n the lower the slope expressed by 1/n and thus the lower the affinity. Moreover, the closer the n value of Freundlich is to zero the more heterogeneous is the system. The data obtained in this study (Table 1) indicate that whole cells represent a greater heterogeneity than isolated mother cell walls. While the Freundlich model does not describe the saturation behaviour of the sotbent, the Langmuir constant (mm~,) is the monolayer saturation at equilibrium. K1 corresponds to the concentration at which a metal ion amount of mmax/2 is bound and indicates the affinity for the binding of metal ions. A high/1 value indicates a.high affinity. As both models were developed for a single-layer metal adsorption the results indicated that short-term ion uptake by both isolated cell walls and whole cells took place as a monomolecular surface coverage. However,

the Freundlich model physically provides a more realistic description of metal adsorption by organic matter because it accounts for different binding sites. In contrast, lead accumulation by whole cells cannot be explained sufficiently by a model for simple monolayer adsorption. Similar observations were described by de Rome and Gadd (1987). They fouri'd a good adaptation for copper adsorption by Rhizopus arrhizus biomass to the multi-layer adsorption model of Brunauer et al. (1938). As this model was conceived for the adsorption of ideal gases, differences in the adsorption behaviour of metal ions on biomass surfaces could be better explained assuming different binding sites (van Cutsem et al. 1985; Xue et al. 1988). This hypothesis was supported by characteristic parameters taken from the tinearized isotherms. In the case of lead adsorption a strong divergence of the model parameters was noted. While there is a greater affinity at lower metal loading status that can still be achieved with the Freundlich isotherm (relatively low n value), the metal binding at maximum saturation was due to ligands with lower affinity as interpreted from the characteristical Langmuir data (relative low/(1 value). Under the assumption that overall lead accumulation takes place within two different saturation ranges with different types of functional groups, the non-adaptation to a monolayer isotherm model can be explained. For practical reasons the metal adsorption of biosorbents is most often referred to the dry weight. Using the dry weight of whole cells or isolated cell walls as reference values instead of a definite surface area, one gets contrasting results. Isolated m o t h e r cell walls showed tenfold higher accumulation rates than whole cells, reaching uptake values of 0.25 ~mol cadmium/mg, 0.58 txmol copper/rag and 0.61 p~mol lead/mg. The uptake values of whole cells (0.03 p,mol cadmium/mg, 0.05 p,mol copper/mg and 0.09 ixmol lead/mg) correspond well to those reported in the literature. Gerhards and Weller (1977) found a maximum cadmium uptake for whole cells of C. fusca of 0.045 p;mol/mg dry mass. Irmer (1982) measured a lead uptake of 0.077 p,mol/mg for whole cells of C. fusca. The high adsorption capacity of isolated cell walls was documented by Christ et al. (1980), who observed a maximum copper accumulation of 0.6 ixmol/mg by dry cell walls of the alga Vaucheria.

728 Isolated cell walls of the fungus Cunninghamella blakeslean'a reached a maximum copper adsorption of 0.86 ~zmol cell wall/mg (Venkateswerlu and Stotzky 1989). Lastly, Walker et al. (1989) reported uptake amounts of 0.53 ~mol Cu, 0.68 ~mol Cd and 0.54 ixmol Pb/mg dry cell wall for isolated cell walls of Bacillus subtilis. A comparison of the accumulation capacities between different biomasses and synthetic ion exchangers reported by R6hricht et al. (1990) showed that biosorbents reached similar or even higher uptake rates than ion exchangers. For Zoogloea ramigera a maximum copper uptake of 2.67 txmol/mg was measured whereas the ion exchangers Lewatite, Chelite and Duolite had exchange capacities of 2.8, 2.1 and 1.75 ixmol/mg dry resin. A l t h o u g h isolated m o t h e r cell walls of ChloreIla fusca possess a lower uptake capacity than the most effective bacterial biomass, their metal accumulation exceeds that of different whole algal cells or other microbial cell wall structures. With respect to the complete remobilization of metal ions from isolated cell walls (Wehrheim and Wettern, 1994) they appeared to be more suitable as biosorbents than whole cells although their preparation is still expensive. sortion of Cu 2+ by an organic ion exchanger -the Nitella flexills cell wall. React Polym 2:31-35 Freundlich H (1926) Colloid and capillary chemistry. Methuen, London Frischmuth A, Weppen P, Deckwer WD (1991) Quecksilberentfernung aus w~ssrigen Medien durch aktive mikrobielle Prozesse. BioEngineering 3: 38-48 Gadd GM (1990) Heavy metal accumulation by bacteria and other micro-organisms. Experientia 46:834-840 Gerhards U, Weller H (1977) Die Aufnahme von Quecksilber, Cadmium und Nickel durch Chlorella pyrenoidosa. Z Pflanzenphysiol 82: 292-300 Irmer U (1982) Die Wirkung der Schwermetalle Blei, Cadmium und Mangan auf die SiisswassergrUnalgen Chlamydomonas reinhardii Dangeard und Chlorella fusca Shihira et Krauss. Dissertation, Universit~it Hamburg Kuhl A (1962) Beitr~ige zur Physiologie und Morphologie der Algen. Fischer Verlag, Stuttgart, pp 157-166 Langmuir I (1918) The adsorption of gases on plane surfaces of glass, mica, platinum. J Am Chem Soc 40:1361-1403 ROhricht M, Weppen P, Deckwer WD (1990) Abtrennung yon Schwermetallen aus AbwasserstrOmen - Biosorption im Vergleich zu herkOmmlichen Verfahren. Chem Ing Tech 62: 582-583 Rome L de, Gadd M (1987) Copper adsorption by Rhizopus arrhizus, Cladosporium resinae and Penicillium italicum. Appl Microbiol Biotechuol 26: 84-90 Ruzic I (1982) Theoretical aspects of the direct titration of natural waters and its information yield for trace metal speciation. Anal Chim Acta 140:99-111 Schmiechen H, Wittig H, Siegfried M (1992) Enffernung yon Schwermetallen aus Abwfissern mittels Sorption am Biopolymer des Mikroorganismus Ectothiorhodospira shaposhnikovii. BioEngineering 1:38--41 Ting YP, Lawson F, Prince IG (1989) Uptake of cadmium and zinc by the alga Chlorella vulgaris. Part I. Individual ion species. Biotechnol Bioeng 34:990-999 Venkateswerlu G, Stotzky G (1989) Binding of heavy metals by cell walls of CunninghameUa blakesleana grown in the presence of copper and cobalt. Appl Microbiol Biotechnol 31: 619-625 Walker SG, Flemming CA, Ferris FG, Beveridge TJ, Bailey GW (1989) Physicochemical interaction of Escherichia coil cell envelopes and Bacillus subtilis cell walls with two clays and ability of the composite to immobilize heavy metals from solution. Appl Environ Microbiol 55:2976-2984 Wehrheim B, Wettern M (1994) Comparative studies of the heavy metal uptake of whole cells and different types of cell walls from Chlorella fusca. Biotech Lett 8:227-232 Xue HB, Stumm W, Sigg L (1988) The binding of heavy metals to algal surfaces. Water Res 22:917-926

Acknowledgements Special thanks to Herbert Boyle and SallyJean Bell for proof-reading this manuscript and to Wolfgang Jarausch for helpful comments.

References
Aksu Z, Kutsal T (1991) A bioseparation process for removing lead (II) ions from waste water using Chlorella vulgar&. J Chem Technol Biotechnol 52:109-118 A1-Duri B, Khader Y, McKay G (1992) Prediction of binary component isotherms for adsorption on heterogeneous surfaces. J Chem Technol Biotechnol 53: 345-352 Brunauer S, Emmett PH, Teller E (1938) Adsorption of gases in multimolecular layer. J Am Chem Soc 60:309-319 Christ RH, Oberholser K, Shank N, Nguyen M (1980) Nature of bonding between metallic ions and algal cell wails. Environ Sci Technol 15:1212-1217 Cutsem P van, Gillet C, Mestdagh MM, Rouxhet PG (1985) Direct probing of the solid-water interface EPR study of the ad-