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Sorption sites in biofilms
e:>
Pergamon
WQl. ScL T~ch. Vol. 32, No.8, pp. 27-33, 1995.
PII:S0273-1223(96)OOO04-2
Copyright 01996 IAWQ Printed in Great Britain.AU rights reserved. 0273-1223I9S S9'SO + lHlO
SORPTION SITES IN BIOFILMS Hans-Curt Flemming Lehrstuhl fur WassergUteund Abjallwirtschaft der TU MiUlchen Am Coulombwall;
D-85748 Garching, Germany
ABSTRACT The distribution of pollutants in water is strongly dependent on the processes at the solid-liquid interface, such as sorption. The solid phase is represented by many different materials, mostly minerals but also metals and organics. In technical and natural water systems, biofilms occur to a greater or lesser extent, They will cover the underlying material (substratum) at least partially and represent a gel-type layer, mainly consisting of extracellular polymeric substances (EPS), formed by biofilm organisms, in which the latter are embedded . Thus, the sorption properties of biofilms will significantly determine the overall process. This effect may have been almost completely overseen, e.g., in the assessment of the fate of pollutants in waters, in the process of sediment diagenesis, and in chemical sorption studies. Sorption data can differ considerably between sterile and non-sterile assays; this aspect puts in question many results gained in biologically uncharacterized systems. Sorption in biofllms can be undesired, if pollutants accumulate in biomass such as activated sludge. The same effect, however, can be bioteehnologica1lyexploited, e.g. in biosorption reactors. Biofilms can sorb water, inorganic and organic solutes and particles. As sorption sites can serve: EPS, cell walls, cell membranes and cell cytoplasm. These sites display different sorption preferences, capacities and properties. The situation becomes even more complex as biofilms may respond physiologically to sorbed substances. For example: the uptake of toluene can lead to the formation of uronic acids in the EPS and, thus, to an increased sorption capacity for cations. When decomposing, biofilms will release sorbed substances. This can be of significance if trickling deposition of sewage water on soil is finished , The biomass will decompose, sorbed pollutants are remobilized and can contaminate the ground water if not retained abiotically by other soil components. A substantial research demand is identified regarding the following questions: (i) wbat do biofilms sorb; (ii) what are the binding sites: (iii) what are the sorption mechanisms and capacity, and (iv) what is the remobi1ization potential?
KEYWORDS Bioftlms; extracellularpolymersubstances (EPS);remobilization; sorption. INTRODUCTION The distribution and fate of pollutants in soils, sediment and water is highly influenced by sorption processes. It is well known that "organic matter" plays an important role in both sorption preference and capacity. However, the term is mostly used to describe a black box. Organic matter is composed of humic substances, debris, and microbial biomass. The latter is organized in an immobilized form in biofilms. This is the most dynamic component of organic matter, as it contains living organisms which can respond to enVironmental changes, by the formation of EPS or other metabolites. 27
28
H.-C.FLEMMING
Biofilms are very diverse and one of their major characteristics is heterogeneity. They consist of various microorganisms, develop on various surfaces and under various conditions. They are ubiquitous in natural and most technical water systems. Surfaces will be at least partially covered by biofilms. Dissolved substances will sorb first to biofilms until they may reach the original mineral surface and possibly sorb there. The sorption properties of this "insulating layer" are therefore of importance for the sorption kinetics and mechanisms. For example, the sorption of the pesticide chlortoluron to sediment material was 40% less under sterile than under non-sterile conditions (Klaas and Hemming, in press). The ability of microorganisms to undergo chemical exchanges with their aqueous environment, involving both metal sorption and mineral precipitation, has been largely overseen as an important factor in determining the chemistry of world rivers (Konhauser et al.; 1993). Leinfelder et al. (1993) demonstrated the role of biofilms in the genesis of microbial crusts of the late Jurassic - there are many more examples for the participation of biofilms in mineral genesis. The degree to which the resulting effects can be quantified and predicted depends upon the extent to which the fundamental aspects of sorption are understood, and upon the accuracy with which these phenomena can be characterized and modelled in complex subsurface and water systems. Accounting for biofilm sorption properties is a challenge, but the information will be incomplete and misleading if this one neglects to do so. Information about nature and physico-chemical properties of biofilms is available, but it must be gleaned from very diverse fields. For example, publications on the technical approaches to remove heavy metals and organic pollutants from water streams by immobilized organisms in bioreactors contain a large body of data about biofilms as sorbents. Unfortunately, most of these studies do not distinguish between different sorption sites in the biomass; usually assuming but not verifying that sorption occurs at cell walls. Even in thorough studies such as the one performed on the sorption of organochlorines to biomass by Yan and Allen (1994), there is no indication of a concept of a structured matrix which includes the diverse compartments with different sorption properties. COMPosmON OF BIOFILMS
In general, five regions in a biofilm can be roughly distinguished; they are depicted schematically in Fig. 1: (i) EPS (including capsules) with cationic groups in amino sugars and proteins (e.g.: -NH+-) anionic groups in uronic acids and proteins (e.g.: -COO"; -HP04"- ) apolar groups from proteins (such as in aromatic amino acids) groups with a high hydrogen bonding potential, such as polysaccharides; (ii) Outer membrane and lipopolysaccharides of gram-negative cells with their lipid membrane, and the lipoteichoic acids in gram positive cells; (iii) Cell wall consisting of N-acetylglucosamine and N-acetylmuramic acid, offering cationic and anionic sites; (iv) Cytoplasmic membrane, offering a lipophilic region; (v) Cytoplasm, as a water phase separated from the surrounding water. EXTRACELLULAR POLYMER SUBSTANCES (EPS) EPS consist of polysaccharides, proteins and lipids. In gram-negative cells, the EPS are composed of lipopolysaccharides, capsule polysaccharides, and other excreted polysaccharides and proteins which are less firmly bound to the cell surface. In gram-positive cells, lipoteichoic acids as well as polysaccharides and proteins which are not anchored in the cell wall con tribute to the EPS. Many microorganisms produce EPS, whether grown in suspended cultures or in biofilms. A considerable part of the protein moiety of the EPS may be extracellular enzymes. It seems that the composition of EPS is not constant but influenced by growth conditions and environmental stress (Schmitt et al.. 1995). Many strains form more than one strain-specific type of EPS, and the composition may change during their life cycle (Uhlinger and White, 1983). The EPS molecules provide the forces responsible for cohesion of the biofilm and adhesion to the substratum. This is performed by weak interactions such as van der Waals forces, electrostatic interactions and hydrogen bonds.
29
Sorption sites in biofilms
- - Cy
EPS
Cy
LPS C
Figure I. Different compartments in a biofilm, including a gram-negative (left) and a gram-positive organism (right>. CY =cytoplasm; CM =cytoplasmic membrane; M =murein ; OM =outer membrane; LPS = lipopolysaccbaride; C =capsule; LTA =Iipoteicboic acid.
Water. A common feature of the EPS is that they are highly hydrated; in biofilms a ratio of 1-2% (w/w)
EPS and 98% water is not unusual (Christensen and Characklis, 1990). This affinity to water gives a slimy consistency to biomass and serves as protection against desiccation (Roberson and Firestone, 1992); colanic acid was identified as the dominant water binding component in certain organisms (Ophir and Gutnick, 1994). It is the water bound in biofilms which has to be removed under considerable efforts when sewage sludge is dewatered. In countries with a cold climate, water bound by biofilms in pores and crevices of concrete can cause severe damage if the temperature falls below -10 to -15'C. Then, the water will freeze eventually which leads to frost cracking (Blaschke, 1987). An effective method to weaken the binding force for water could be economically very interesting.
Cations. Because EPS may contain anionic groups such as carboxyl. phosphoryl. and sulphate groups
(Sutherland. 1984). they represent a cation exchange potential. A survey of a wide variety of marine and freshwater bacteria by Kennedy and Sutherland (1987) has shown that bacterial EPS typically contained 20• 50% of their polysaccharides as uronic acids. A wide variety of metal ions is reportedly being bound to EPS (Flemming et al.• in press). Theoretical predictions of metal binding capacities. based on estimated numbers of available carboxyl and hydroxyl groups. suggest a very high capacity, provided in particular by the acidic polysaccharides. Harvey (1981) found a binding capacity for lead of 0.13 IlM Ilg-1 of EPS. He calculated that if EPS represented only a very small proportion of the organic matter in sediments. they could still complex all available Pb2+ in the surface layer sediments of a Palo Alto salt marsh. Adsorption densities as high as 22 ng Ilg-1 have been reported for copper (Kaplan et al., 1987). The stability constants for Ni2+, Cu2+. Pb2+. Cd2+ and Zn2+ complexes with EPS range between 105 and 109 (Kaplan et al., 1987; Geesey et al., 1988). As there exists a competition between H+ and metal ions, the stability constants strongly depend upon the pH value. In studies of freshwater lakes. microbial biofilrns under near-neutral pH scavenged metals up to 12 orders of magnitude higher than biofilms under lower pH (acidic) conditions (Ferris et al., 1989). EPS have been shown to accumulate up to 25% their weight as metal ions (Dugan and Pickrurn, 1972). Alginate has been proposed in biotechnological applications for sorbing dissolved copper from aqueous media (lang et al.• 1990).
H.-C.FLEMMING
30
Anions. In general, there is only very little information available concerning the binding of anions in biofilms. However, sorption of anions must occur, as amino groups in sugars, sugar acids and proteins provide positive charges which can act as anion binding sites. A biofilm which caused irreversible fouling on a reverse osmosis membrane contained accumulated sulphate at levels tenfold higher than the water it was in contact with (Flemming and Schaule, 1989). A biofilm of Pseudomonas diminuta accumulated sulphate, phosphate and nitrate from the nutrient broth (Schaule, unpublished observation). Polar organic molecules. Amino acids are obviously sequestered from aqueous streams by biofilms because they are utilized as substrates (Decho, 1990). The mechanism must involve the EPS as these have to be crossed by a molecule before it can be metabolized in the interior of a cell. Detailed data about the affinity of the EPS for these molecules and the binding capacity could not be found in the literature. Apolar and hydrophobic molecules. Some EPS exhibit surface activity , such as emulsan (Rosenberg and Kaplan, 1986), in particular during growth on hydrophobic nutrients, e.g., oil. grease and fat (Cameron et al.; 1988). In return, surface activity will change the sorption and transport properties of a biofilm in regard to traces of dissolved hydrophobic substances, such as some pesticides. Dohse and Lion (1994) coated sand in a column with EPS of phenanthrene-degrading organisms which caused a significant enhancement of phenanthrene transport. The sorption of apolar substances by EPS is largely unexplored although it must play an important role - not only in trapping these substances in biofilrns but also in the process of adhesion of microorganisms to hydrophobic surfaces, which can be compared to sorption. The flrst cellular components in contact with such a surface are the hydrated, hydrophilic EPS. Still, they provide the adhesion force for the cells. The nature of the EPS components responsible for this process is still not revealed. However, it is quite likely that the apolar regions of proteins provide suitable sites for apolar molecules to bind. Using scanning confocal laser microscopy, Wolfaardt et al. (1995) were the first to demonstrate that herbicides are actually accumulated in the EPS. It is likely that large molecules are trapped in the network of the EPS matrix and bound by weak physicochemical interactions. which multiply by the number of interacting groups. This mechanism will allow exoenzymes to degrade the polymers and result in small oligomers or monomers which can be taken up and metabolized by the cell. CELL WALLS Constituent carboxyl and phosphoryl groups interact passively with available cations and have been shown to be major sites for metal deposition (Ferris and Beveridge, 1984). The localized high concentrations of metals which may develop on bacterial surfaces are subject to mineral formation and diagenesis. which in some cases may be modified by bacterial metabolism (Mclean and Beveridge. 1990). The mechanisms for binding of metals on cell walls are (i) ion exchange reactions, (ii) precipitation, and (iii) complexation (Brierley et al., 1985). The S-Iayer of cyanobacteria has been shown to mediate calcium mineral formation. Mineral formation begins within the large holes of the S-layer array when Ca 2+ binds to negatively charged sites on the S• protein and is joined by sulphate ions, initiating the formation of a mineral aggregate. The S-Iayer exhibits selectivity with respect to the ions bound and subsequently incorporated into carbonate minerals. Celestite and Strontianite, previously thought to be purely evaporitic minerals, can be biologically formed (Schultze• Lam and Beveridge. 1994). Metal binding by Bacillus subtilis walls is considered to be a two stage process. Initially, metal ions are bound to the anionic sites of the peptidoglycan. These bound metals then act as nucleation sites whereby additional metals might be bound (often forming aggregates visible by TEM). Gram-negative bacterial envelopes have a lower overall charge density and are chemically and structurally very different from gram-positive cell walls . The overall contribution of peptidoglycan to metal binding in gram-negative organisms is quite small, due to the limited quantity of this material in the cell envelope and
Sorptionsiresin biofilms
31
its shielding from the environment by the overlying outer membrane. However, by chemical reactions. gram-negative organisms can accumulate substantial amounts of metals at the cell wall. An example: a chemoautotrophic bacterium genus Thiobacillus accumulated silver, forming precipitates of Agz!) on its cell surface (Pooley, 1982). This metal sulfide compound represented up to 25% of the dry weight of the cell. Bacterial heavy metal resistance usually employs active efflux mechanisms, for example resistance to cobalt, cadmium and zinc in Alcaligenes eutrophus strain CH 34. This in tum leads to extracellular precipitation (Collard et al.• 1994). Chafetz and Buczynski (1992) report that the precipitation of calcium carbonate preferably occurred on cyanobacterial filaments in the presence of live bacteria. Dead cyanobacteria were coated with calcium carbonate much more quickly and to a greater extent than live cyanobacteria. The authors conclude that bacteria can playa significant role in producing stromatolites. LIPID MEMBRANES The most probable site of accumulation of lipohilic substances are the lipid membranes. It is surprising that practically no reference could be found in which the accumulation of apolar molecules in lipid membranes was investigated. Assumed that 10% of the dry mass of an average microbial cell consist of lipids and that 20% of a biofilm consist of bacteria, 2% of the overall biofilm dry mass provide a potential capacity for hydrophobic matter. If the biofilm contained 90% water. 0.2% of the wet weight is made up of lipids. In sewage sludge. chlorinated organic compounds were found in concentrations between 15 and 150 mg kg o1 (Friege et al.; 1989). Laschka and Trumpp (1991) report 500 mg kg- 1 AOX in sewer biofilms. These values are far below the lipid content Thus, such amounts may well be taken up by the lipid membranes. This assumption is plausible but not experimentally verified. The above cited work of Wolfaardt et al. (1995) reveal that the EPS seem to be another potential sorbent for apolar molecules. Thus. further research is required in order to obtain deeper insight into the function of lipid membranes as sorption sites for lipophilic molecules. CYTOPLASM The interior of biofilm bacteria is also a potential site for binding and accumulation of dissolved substances (Ford and Mitchell. 1993). Significant intracellular accumulation of metals by bacteria was reported by Charley and Bull (1979). using a mixed culture of bacteria which accumulated as much as 300 mg gol Ag" per dry weight of biomass; however. it seems doubtful that all of this silver was really deposited inside the cell. Cadmium is accumulated internally by a large number of organisms (Ford and Mitchell. 1993). Intracellular formation of a metal sulfide was observed when growing cultures of Klebsiella aerogenes accumulated Cd 2+ ions and inorganic sulfide in a molar ratio close to unity. A subsequent intracellular formation of CdS was noted (Aiking et al., 1984). Uranium, radium and cesium accumulate intracellularly in Pseudomonas aeruginosa (Strandberg et al.• 1981). The ability of certain bacterial cells to accumulate metals intracellularly has been stressed as the most plausible explanation for the removal of metals from the liquid phase. However. it may be doubted that intracellular accumulation is the dominating mechanism in the majority of cases of bio technological sequestering of metals from liquids, in particular as little experimental evidence for that assumption is provided. Furthermore, it is quite likely that the role of the EPS has been underestimated. The intracellular accumulation of apolar organic pollutants such as PCB or DDT seems possible but is not yet verified. REMOBILIZATION Biofilms are not inert chemical structures. They represent a dynamic system in which the various components are synthesized, assembled, modified and finally broken down by autolysins and sloughed off into the environment Thus, they may contribute to the remobilization of the sorbed substances. By nature,
32
H.-C.FLEMMING
the immobilization of metal ions in biomass cannot be irreversible. Fate and transport of the sorbed metal is directly related to the fate of the biofilm. In some cases. lysis of the cell will lead to mineral formation (Ehrlich. 1990) and is responsible for the formation of large ores. However. in other cases the sorbed metal ions will return in their more soluble form and be remobilized. In a study, the remobilization of sorbed metals showed the order Cr 2+<
Sorptionsites in biofilms
33
Hemming, H-C. and Schaule, G. (1989). Biofouling auf Umkehrosmose- und U1U'aftltt:ltionsmembranen. Teil U: Analyse und Enlfernungdes Belages. Vom Wasser 73, 287-301. Hemming, H-C., Schmitt. J. and Marsball, K. C. (1996). Sorption properties of bioftlms. In: Environmental Behaviour 01 Sediments. U. Forsmerand W. CaImano (eds). Springer, Heidelberg,New York., in press. Ford, T. and Mitchell, R. (1993). Microbial transport of toxic metals. In: Environmental Microbiology, R. Mitchell (ed.). John Wiley, New York. pp. 83-101. Friege, H~ Buysch, H. P., Leuchs, W., Hembrock, A. and Kllnig, W. (1989). Belastung von KllItschUlmmen und BlIden mit organiscbenSchadstoffen. Korr. Abw. 36, 601-(j()8. Geesey.G. and Jang, L. (1990). Extracellular polymers for metal binding. In: Microbial MineralRecovery. H. C. Ehrlich and C. L. Brierley (eds). McGraw-Hill,New York, pp. 223-247. Gutekunst, B. (1988). Sielhautuntersucbungen zur Einkreisungscbwermetallhaltiger Einleitungen.Schriftenreihedes Instituts fUr Siedlungswasserwirtschaft, Universilllt Karlsruhe,Band 49. Jang, L. K., Geesey, G. G., Lopez, S. L., Eastman, S. L. and Wichlaez, P. L. (1990). Use of a gel-forming biopolymerdirectly dispensedinto a loop fluidized bed reactor to recoverdissolved copper. Wat. Res. 24, 889-897. Kaplan. D~ Christiaen, D. and Shoshana, A. (1987). Chelating properties of extraeellular polysaccharides from Chiarella spp. Appl EnvirolL Microbiol. 53, 2953-2956. Kennedy. K. J., Le, J. and Mohn, W. W. (1992). Biosorptionof cbloropbenolsto anaerobic granular sludge. WaL Res. 26, 1085. 1092. Konhauser, K. 0 .• Fyfe, W. S.. Ferris. F. G. and Beveridge.T. J. (1993). Metal sorption and mineral precipitation by bacteria in two Amazonian river systems:Rio Solimllesand Rio Negro, Brazil. Geology21. 1103-1106. K1aas. N. and Hemming, H-C. (1996).Sorption of pesticidesto clay minerals. VomWasser, in press. Laschka, D. and Trumpp, M. (1991). Sielhautuntersuchungen zur Loka1isierung von AOX-Eminttenten im Kanalnetz. Korr. Abw. 38. 495-496. Leinfelder, R. R., Nose, M., Schmid, D. U. and Werner. W. (1993). Microbial crusts of the late Jurassic: composition. paleoloecological significanceand importancein reef consttuction. Facies 29. 195-230. McLean, R. J. C. and Beveridge,T. J. (1988). Influenceof metal ion cbarge on their bindingcapacity to bacterial capsules. Abst 88th Ann. Meet Am. Soc. Microbiol. Q 146,p. 307. Ophir, T. and Gutnick. D. L. (1994). A role for exopolysaccbarides in the protectionof microorganismsfrom desiccation. Appl. EnvirolL Microbiol. 60. 740-745. ROberson, E. B. and M. K. Firestone (1992). Relationship between desiccation and exopolysaccbaride production in a soil Pseudomonassp. AppLEnvirolL Microbiol. 58, 1284-1291. Rosenberg, E. and N. Kaplan (1986). Surface-active properties of Acinetobaeter exopolysaccharides. In: Bacterial Outer Membranes as a ModelSyslem, M. Inouye (ed.). Interscience, New York,pp. 311·342. Schmit!,J., Nivens. 0 .. White, D. C. and Flemming, H-C. (1995).Changes of biofilm properties in response to sorbed substances - an Am-ATR study. Wat. Sci. Tech 32(8). this issue. Schultze-Lam, S. and Beveridge. T. J. (1994). Nucleation of suontianite on a cyanobacterialS-Iayer. Appl. EnvirOIL Microbial. 60, 447-453. Strandberg. G. W.. Shumate, S. ~ Parrott, J. R. (1981). Microbioal cells as biosorbents for heavy metals: accumulation of uranium by Saccharomycescerevisiacand Pseudomonas aeruginosa. AppLEnvirolL Microbiol: 41, 237-24S. Sutherland, 1. W. (1984). Microbial exopolysaccbarides - their role in microbial adhesion in aqueous systems. CRC CriL Rev. Microbial. 10, 173-201. Tunlid, A. and Wbite, D. C. (1990). Use of lipid biomarkersin environmentalsamples. In: AnalyticalMicrobiology Methods, A. Fox, S. L. Morgan, L. Larssonand G. Odham (eds), PlenumPress, New York. London, pp. 259-274. Ublinger,D. J. and D. C. White (1983). Relationshipbetween physiological status and formation of extracellular polysaccharide glycocalyx in P. atlantica. AppLEnviron: Microbial. 45,64-70. Wolfaardt,G. M.. Lawrence, J. R.. Headley, J. V., Robarts, R. D. and Caldwell, D. E. (1995). Microbial exopolymers provide a mecbanismfor bioaccumulation of contaminants. Microb. Ecol., in press. Yan,G. and Allen, D. G. (1994). Biosorptionof higb molecularweightorganochlorines in pulp mill effiuent. Wat. Res. 28, 1933• 1941.
Pergamon
WQl. ScL T~ch. Vol. 32, No.8, pp. 27-33, 1995.
PII:S0273-1223(96)OOO04-2
Copyright 01996 IAWQ Printed in Great Britain.AU rights reserved. 0273-1223I9S S9'SO + lHlO
SORPTION SITES IN BIOFILMS Hans-Curt Flemming Lehrstuhl fur WassergUteund Abjallwirtschaft der TU MiUlchen Am Coulombwall;
D-85748 Garching, Germany
ABSTRACT The distribution of pollutants in water is strongly dependent on the processes at the solid-liquid interface, such as sorption. The solid phase is represented by many different materials, mostly minerals but also metals and organics. In technical and natural water systems, biofilms occur to a greater or lesser extent, They will cover the underlying material (substratum) at least partially and represent a gel-type layer, mainly consisting of extracellular polymeric substances (EPS), formed by biofilm organisms, in which the latter are embedded . Thus, the sorption properties of biofilms will significantly determine the overall process. This effect may have been almost completely overseen, e.g., in the assessment of the fate of pollutants in waters, in the process of sediment diagenesis, and in chemical sorption studies. Sorption data can differ considerably between sterile and non-sterile assays; this aspect puts in question many results gained in biologically uncharacterized systems. Sorption in biofllms can be undesired, if pollutants accumulate in biomass such as activated sludge. The same effect, however, can be bioteehnologica1lyexploited, e.g. in biosorption reactors. Biofilms can sorb water, inorganic and organic solutes and particles. As sorption sites can serve: EPS, cell walls, cell membranes and cell cytoplasm. These sites display different sorption preferences, capacities and properties. The situation becomes even more complex as biofilms may respond physiologically to sorbed substances. For example: the uptake of toluene can lead to the formation of uronic acids in the EPS and, thus, to an increased sorption capacity for cations. When decomposing, biofilms will release sorbed substances. This can be of significance if trickling deposition of sewage water on soil is finished , The biomass will decompose, sorbed pollutants are remobilized and can contaminate the ground water if not retained abiotically by other soil components. A substantial research demand is identified regarding the following questions: (i) wbat do biofilms sorb; (ii) what are the binding sites: (iii) what are the sorption mechanisms and capacity, and (iv) what is the remobi1ization potential?
KEYWORDS Bioftlms; extracellularpolymersubstances (EPS);remobilization; sorption. INTRODUCTION The distribution and fate of pollutants in soils, sediment and water is highly influenced by sorption processes. It is well known that "organic matter" plays an important role in both sorption preference and capacity. However, the term is mostly used to describe a black box. Organic matter is composed of humic substances, debris, and microbial biomass. The latter is organized in an immobilized form in biofilms. This is the most dynamic component of organic matter, as it contains living organisms which can respond to enVironmental changes, by the formation of EPS or other metabolites. 27
28
H.-C.FLEMMING
Biofilms are very diverse and one of their major characteristics is heterogeneity. They consist of various microorganisms, develop on various surfaces and under various conditions. They are ubiquitous in natural and most technical water systems. Surfaces will be at least partially covered by biofilms. Dissolved substances will sorb first to biofilms until they may reach the original mineral surface and possibly sorb there. The sorption properties of this "insulating layer" are therefore of importance for the sorption kinetics and mechanisms. For example, the sorption of the pesticide chlortoluron to sediment material was 40% less under sterile than under non-sterile conditions (Klaas and Hemming, in press). The ability of microorganisms to undergo chemical exchanges with their aqueous environment, involving both metal sorption and mineral precipitation, has been largely overseen as an important factor in determining the chemistry of world rivers (Konhauser et al.; 1993). Leinfelder et al. (1993) demonstrated the role of biofilms in the genesis of microbial crusts of the late Jurassic - there are many more examples for the participation of biofilms in mineral genesis. The degree to which the resulting effects can be quantified and predicted depends upon the extent to which the fundamental aspects of sorption are understood, and upon the accuracy with which these phenomena can be characterized and modelled in complex subsurface and water systems. Accounting for biofilm sorption properties is a challenge, but the information will be incomplete and misleading if this one neglects to do so. Information about nature and physico-chemical properties of biofilms is available, but it must be gleaned from very diverse fields. For example, publications on the technical approaches to remove heavy metals and organic pollutants from water streams by immobilized organisms in bioreactors contain a large body of data about biofilms as sorbents. Unfortunately, most of these studies do not distinguish between different sorption sites in the biomass; usually assuming but not verifying that sorption occurs at cell walls. Even in thorough studies such as the one performed on the sorption of organochlorines to biomass by Yan and Allen (1994), there is no indication of a concept of a structured matrix which includes the diverse compartments with different sorption properties. COMPosmON OF BIOFILMS
In general, five regions in a biofilm can be roughly distinguished; they are depicted schematically in Fig. 1: (i) EPS (including capsules) with cationic groups in amino sugars and proteins (e.g.: -NH+-) anionic groups in uronic acids and proteins (e.g.: -COO"; -HP04"- ) apolar groups from proteins (such as in aromatic amino acids) groups with a high hydrogen bonding potential, such as polysaccharides; (ii) Outer membrane and lipopolysaccharides of gram-negative cells with their lipid membrane, and the lipoteichoic acids in gram positive cells; (iii) Cell wall consisting of N-acetylglucosamine and N-acetylmuramic acid, offering cationic and anionic sites; (iv) Cytoplasmic membrane, offering a lipophilic region; (v) Cytoplasm, as a water phase separated from the surrounding water. EXTRACELLULAR POLYMER SUBSTANCES (EPS) EPS consist of polysaccharides, proteins and lipids. In gram-negative cells, the EPS are composed of lipopolysaccharides, capsule polysaccharides, and other excreted polysaccharides and proteins which are less firmly bound to the cell surface. In gram-positive cells, lipoteichoic acids as well as polysaccharides and proteins which are not anchored in the cell wall con tribute to the EPS. Many microorganisms produce EPS, whether grown in suspended cultures or in biofilms. A considerable part of the protein moiety of the EPS may be extracellular enzymes. It seems that the composition of EPS is not constant but influenced by growth conditions and environmental stress (Schmitt et al.. 1995). Many strains form more than one strain-specific type of EPS, and the composition may change during their life cycle (Uhlinger and White, 1983). The EPS molecules provide the forces responsible for cohesion of the biofilm and adhesion to the substratum. This is performed by weak interactions such as van der Waals forces, electrostatic interactions and hydrogen bonds.
29
Sorption sites in biofilms
- - Cy
EPS
Cy
LPS C
Figure I. Different compartments in a biofilm, including a gram-negative (left) and a gram-positive organism (right>. CY =cytoplasm; CM =cytoplasmic membrane; M =murein ; OM =outer membrane; LPS = lipopolysaccbaride; C =capsule; LTA =Iipoteicboic acid.
Water. A common feature of the EPS is that they are highly hydrated; in biofilms a ratio of 1-2% (w/w)
EPS and 98% water is not unusual (Christensen and Characklis, 1990). This affinity to water gives a slimy consistency to biomass and serves as protection against desiccation (Roberson and Firestone, 1992); colanic acid was identified as the dominant water binding component in certain organisms (Ophir and Gutnick, 1994). It is the water bound in biofilms which has to be removed under considerable efforts when sewage sludge is dewatered. In countries with a cold climate, water bound by biofilms in pores and crevices of concrete can cause severe damage if the temperature falls below -10 to -15'C. Then, the water will freeze eventually which leads to frost cracking (Blaschke, 1987). An effective method to weaken the binding force for water could be economically very interesting.
Cations. Because EPS may contain anionic groups such as carboxyl. phosphoryl. and sulphate groups
(Sutherland. 1984). they represent a cation exchange potential. A survey of a wide variety of marine and freshwater bacteria by Kennedy and Sutherland (1987) has shown that bacterial EPS typically contained 20• 50% of their polysaccharides as uronic acids. A wide variety of metal ions is reportedly being bound to EPS (Flemming et al.• in press). Theoretical predictions of metal binding capacities. based on estimated numbers of available carboxyl and hydroxyl groups. suggest a very high capacity, provided in particular by the acidic polysaccharides. Harvey (1981) found a binding capacity for lead of 0.13 IlM Ilg-1 of EPS. He calculated that if EPS represented only a very small proportion of the organic matter in sediments. they could still complex all available Pb2+ in the surface layer sediments of a Palo Alto salt marsh. Adsorption densities as high as 22 ng Ilg-1 have been reported for copper (Kaplan et al., 1987). The stability constants for Ni2+, Cu2+. Pb2+. Cd2+ and Zn2+ complexes with EPS range between 105 and 109 (Kaplan et al., 1987; Geesey et al., 1988). As there exists a competition between H+ and metal ions, the stability constants strongly depend upon the pH value. In studies of freshwater lakes. microbial biofilrns under near-neutral pH scavenged metals up to 12 orders of magnitude higher than biofilms under lower pH (acidic) conditions (Ferris et al., 1989). EPS have been shown to accumulate up to 25% their weight as metal ions (Dugan and Pickrurn, 1972). Alginate has been proposed in biotechnological applications for sorbing dissolved copper from aqueous media (lang et al.• 1990).
H.-C.FLEMMING
30
Anions. In general, there is only very little information available concerning the binding of anions in biofilms. However, sorption of anions must occur, as amino groups in sugars, sugar acids and proteins provide positive charges which can act as anion binding sites. A biofilm which caused irreversible fouling on a reverse osmosis membrane contained accumulated sulphate at levels tenfold higher than the water it was in contact with (Flemming and Schaule, 1989). A biofilm of Pseudomonas diminuta accumulated sulphate, phosphate and nitrate from the nutrient broth (Schaule, unpublished observation). Polar organic molecules. Amino acids are obviously sequestered from aqueous streams by biofilms because they are utilized as substrates (Decho, 1990). The mechanism must involve the EPS as these have to be crossed by a molecule before it can be metabolized in the interior of a cell. Detailed data about the affinity of the EPS for these molecules and the binding capacity could not be found in the literature. Apolar and hydrophobic molecules. Some EPS exhibit surface activity , such as emulsan (Rosenberg and Kaplan, 1986), in particular during growth on hydrophobic nutrients, e.g., oil. grease and fat (Cameron et al.; 1988). In return, surface activity will change the sorption and transport properties of a biofilm in regard to traces of dissolved hydrophobic substances, such as some pesticides. Dohse and Lion (1994) coated sand in a column with EPS of phenanthrene-degrading organisms which caused a significant enhancement of phenanthrene transport. The sorption of apolar substances by EPS is largely unexplored although it must play an important role - not only in trapping these substances in biofilrns but also in the process of adhesion of microorganisms to hydrophobic surfaces, which can be compared to sorption. The flrst cellular components in contact with such a surface are the hydrated, hydrophilic EPS. Still, they provide the adhesion force for the cells. The nature of the EPS components responsible for this process is still not revealed. However, it is quite likely that the apolar regions of proteins provide suitable sites for apolar molecules to bind. Using scanning confocal laser microscopy, Wolfaardt et al. (1995) were the first to demonstrate that herbicides are actually accumulated in the EPS. It is likely that large molecules are trapped in the network of the EPS matrix and bound by weak physicochemical interactions. which multiply by the number of interacting groups. This mechanism will allow exoenzymes to degrade the polymers and result in small oligomers or monomers which can be taken up and metabolized by the cell. CELL WALLS Constituent carboxyl and phosphoryl groups interact passively with available cations and have been shown to be major sites for metal deposition (Ferris and Beveridge, 1984). The localized high concentrations of metals which may develop on bacterial surfaces are subject to mineral formation and diagenesis. which in some cases may be modified by bacterial metabolism (Mclean and Beveridge. 1990). The mechanisms for binding of metals on cell walls are (i) ion exchange reactions, (ii) precipitation, and (iii) complexation (Brierley et al., 1985). The S-Iayer of cyanobacteria has been shown to mediate calcium mineral formation. Mineral formation begins within the large holes of the S-layer array when Ca 2+ binds to negatively charged sites on the S• protein and is joined by sulphate ions, initiating the formation of a mineral aggregate. The S-Iayer exhibits selectivity with respect to the ions bound and subsequently incorporated into carbonate minerals. Celestite and Strontianite, previously thought to be purely evaporitic minerals, can be biologically formed (Schultze• Lam and Beveridge. 1994). Metal binding by Bacillus subtilis walls is considered to be a two stage process. Initially, metal ions are bound to the anionic sites of the peptidoglycan. These bound metals then act as nucleation sites whereby additional metals might be bound (often forming aggregates visible by TEM). Gram-negative bacterial envelopes have a lower overall charge density and are chemically and structurally very different from gram-positive cell walls . The overall contribution of peptidoglycan to metal binding in gram-negative organisms is quite small, due to the limited quantity of this material in the cell envelope and
Sorptionsiresin biofilms
31
its shielding from the environment by the overlying outer membrane. However, by chemical reactions. gram-negative organisms can accumulate substantial amounts of metals at the cell wall. An example: a chemoautotrophic bacterium genus Thiobacillus accumulated silver, forming precipitates of Agz!) on its cell surface (Pooley, 1982). This metal sulfide compound represented up to 25% of the dry weight of the cell. Bacterial heavy metal resistance usually employs active efflux mechanisms, for example resistance to cobalt, cadmium and zinc in Alcaligenes eutrophus strain CH 34. This in tum leads to extracellular precipitation (Collard et al.• 1994). Chafetz and Buczynski (1992) report that the precipitation of calcium carbonate preferably occurred on cyanobacterial filaments in the presence of live bacteria. Dead cyanobacteria were coated with calcium carbonate much more quickly and to a greater extent than live cyanobacteria. The authors conclude that bacteria can playa significant role in producing stromatolites. LIPID MEMBRANES The most probable site of accumulation of lipohilic substances are the lipid membranes. It is surprising that practically no reference could be found in which the accumulation of apolar molecules in lipid membranes was investigated. Assumed that 10% of the dry mass of an average microbial cell consist of lipids and that 20% of a biofilm consist of bacteria, 2% of the overall biofilm dry mass provide a potential capacity for hydrophobic matter. If the biofilm contained 90% water. 0.2% of the wet weight is made up of lipids. In sewage sludge. chlorinated organic compounds were found in concentrations between 15 and 150 mg kg o1 (Friege et al.; 1989). Laschka and Trumpp (1991) report 500 mg kg- 1 AOX in sewer biofilms. These values are far below the lipid content Thus, such amounts may well be taken up by the lipid membranes. This assumption is plausible but not experimentally verified. The above cited work of Wolfaardt et al. (1995) reveal that the EPS seem to be another potential sorbent for apolar molecules. Thus. further research is required in order to obtain deeper insight into the function of lipid membranes as sorption sites for lipophilic molecules. CYTOPLASM The interior of biofilm bacteria is also a potential site for binding and accumulation of dissolved substances (Ford and Mitchell. 1993). Significant intracellular accumulation of metals by bacteria was reported by Charley and Bull (1979). using a mixed culture of bacteria which accumulated as much as 300 mg gol Ag" per dry weight of biomass; however. it seems doubtful that all of this silver was really deposited inside the cell. Cadmium is accumulated internally by a large number of organisms (Ford and Mitchell. 1993). Intracellular formation of a metal sulfide was observed when growing cultures of Klebsiella aerogenes accumulated Cd 2+ ions and inorganic sulfide in a molar ratio close to unity. A subsequent intracellular formation of CdS was noted (Aiking et al., 1984). Uranium, radium and cesium accumulate intracellularly in Pseudomonas aeruginosa (Strandberg et al.• 1981). The ability of certain bacterial cells to accumulate metals intracellularly has been stressed as the most plausible explanation for the removal of metals from the liquid phase. However. it may be doubted that intracellular accumulation is the dominating mechanism in the majority of cases of bio technological sequestering of metals from liquids, in particular as little experimental evidence for that assumption is provided. Furthermore, it is quite likely that the role of the EPS has been underestimated. The intracellular accumulation of apolar organic pollutants such as PCB or DDT seems possible but is not yet verified. REMOBILIZATION Biofilms are not inert chemical structures. They represent a dynamic system in which the various components are synthesized, assembled, modified and finally broken down by autolysins and sloughed off into the environment Thus, they may contribute to the remobilization of the sorbed substances. By nature,
32
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the immobilization of metal ions in biomass cannot be irreversible. Fate and transport of the sorbed metal is directly related to the fate of the biofilm. In some cases. lysis of the cell will lead to mineral formation (Ehrlich. 1990) and is responsible for the formation of large ores. However. in other cases the sorbed metal ions will return in their more soluble form and be remobilized. In a study, the remobilization of sorbed metals showed the order Cr 2+<
Sorptionsites in biofilms
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Hemming, H-C. and Schaule, G. (1989). Biofouling auf Umkehrosmose- und U1U'aftltt:ltionsmembranen. Teil U: Analyse und Enlfernungdes Belages. Vom Wasser 73, 287-301. Hemming, H-C., Schmitt. J. and Marsball, K. C. (1996). Sorption properties of bioftlms. In: Environmental Behaviour 01 Sediments. U. Forsmerand W. CaImano (eds). Springer, Heidelberg,New York., in press. Ford, T. and Mitchell, R. (1993). Microbial transport of toxic metals. In: Environmental Microbiology, R. Mitchell (ed.). John Wiley, New York. pp. 83-101. Friege, H~ Buysch, H. P., Leuchs, W., Hembrock, A. and Kllnig, W. (1989). Belastung von KllItschUlmmen und BlIden mit organiscbenSchadstoffen. Korr. Abw. 36, 601-(j()8. Geesey.G. and Jang, L. (1990). Extracellular polymers for metal binding. In: Microbial MineralRecovery. H. C. Ehrlich and C. L. Brierley (eds). McGraw-Hill,New York, pp. 223-247. Gutekunst, B. (1988). Sielhautuntersucbungen zur Einkreisungscbwermetallhaltiger Einleitungen.Schriftenreihedes Instituts fUr Siedlungswasserwirtschaft, Universilllt Karlsruhe,Band 49. Jang, L. K., Geesey, G. G., Lopez, S. L., Eastman, S. L. and Wichlaez, P. L. (1990). Use of a gel-forming biopolymerdirectly dispensedinto a loop fluidized bed reactor to recoverdissolved copper. Wat. Res. 24, 889-897. Kaplan. D~ Christiaen, D. and Shoshana, A. (1987). Chelating properties of extraeellular polysaccharides from Chiarella spp. Appl EnvirolL Microbiol. 53, 2953-2956. Kennedy. K. J., Le, J. and Mohn, W. W. (1992). Biosorptionof cbloropbenolsto anaerobic granular sludge. WaL Res. 26, 1085. 1092. Konhauser, K. 0 .• Fyfe, W. S.. Ferris. F. G. and Beveridge.T. J. (1993). Metal sorption and mineral precipitation by bacteria in two Amazonian river systems:Rio Solimllesand Rio Negro, Brazil. Geology21. 1103-1106. K1aas. N. and Hemming, H-C. (1996).Sorption of pesticidesto clay minerals. VomWasser, in press. Laschka, D. and Trumpp, M. (1991). Sielhautuntersuchungen zur Loka1isierung von AOX-Eminttenten im Kanalnetz. Korr. Abw. 38. 495-496. Leinfelder, R. R., Nose, M., Schmid, D. U. and Werner. W. (1993). Microbial crusts of the late Jurassic: composition. paleoloecological significanceand importancein reef consttuction. Facies 29. 195-230. McLean, R. J. C. and Beveridge,T. J. (1988). Influenceof metal ion cbarge on their bindingcapacity to bacterial capsules. Abst 88th Ann. Meet Am. Soc. Microbiol. Q 146,p. 307. Ophir, T. and Gutnick. D. L. (1994). A role for exopolysaccbarides in the protectionof microorganismsfrom desiccation. Appl. EnvirolL Microbiol. 60. 740-745. ROberson, E. B. and M. K. Firestone (1992). Relationship between desiccation and exopolysaccbaride production in a soil Pseudomonassp. AppLEnvirolL Microbiol. 58, 1284-1291. Rosenberg, E. and N. Kaplan (1986). Surface-active properties of Acinetobaeter exopolysaccharides. In: Bacterial Outer Membranes as a ModelSyslem, M. Inouye (ed.). Interscience, New York,pp. 311·342. Schmit!,J., Nivens. 0 .. White, D. C. and Flemming, H-C. (1995).Changes of biofilm properties in response to sorbed substances - an Am-ATR study. Wat. Sci. Tech 32(8). this issue. Schultze-Lam, S. and Beveridge. T. J. (1994). Nucleation of suontianite on a cyanobacterialS-Iayer. Appl. EnvirOIL Microbial. 60, 447-453. Strandberg. G. W.. Shumate, S. ~ Parrott, J. R. (1981). Microbioal cells as biosorbents for heavy metals: accumulation of uranium by Saccharomycescerevisiacand Pseudomonas aeruginosa. AppLEnvirolL Microbiol: 41, 237-24S. Sutherland, 1. W. (1984). Microbial exopolysaccbarides - their role in microbial adhesion in aqueous systems. CRC CriL Rev. Microbial. 10, 173-201. Tunlid, A. and Wbite, D. C. (1990). Use of lipid biomarkersin environmentalsamples. In: AnalyticalMicrobiology Methods, A. Fox, S. L. Morgan, L. Larssonand G. Odham (eds), PlenumPress, New York. London, pp. 259-274. Ublinger,D. J. and D. C. White (1983). Relationshipbetween physiological status and formation of extracellular polysaccharide glycocalyx in P. atlantica. AppLEnviron: Microbial. 45,64-70. Wolfaardt,G. M.. Lawrence, J. R.. Headley, J. V., Robarts, R. D. and Caldwell, D. E. (1995). Microbial exopolymers provide a mecbanismfor bioaccumulation of contaminants. Microb. Ecol., in press. Yan,G. and Allen, D. G. (1994). Biosorptionof higb molecularweightorganochlorines in pulp mill effiuent. Wat. Res. 28, 1933• 1941.