The Nucleus.

38(2): 131-135 (2001)

MICROBIAL BIOFILMS FOR ENVIRONMENTAL WASTE MANAGEMENT: AN OVERVIEW M. Ashraf, Mazhar H. Naqvi, M. Mohsin Iqbal Soil Biology Division, Nuclear Institute for Agriculture and Biology (NIAB), P.O. Box #128, Jhang Road, Faisalabad. Key words: biofilms, organic contaminants, bioreactors, exo-polysaccharides, microbial consortia, decontamination, biotransformation, bioremediation, toxic heavy metals

Micro-organisms attach to the environmental surfaces by means of a tangled network of exo-polysaccharides, in the assemblages termed as Biofilm. Because of the unique physical, biological and chemical properties, the microbial biofilms serve both deleterious and beneficial purposes in the environment and in modulated wastewater treatment plants. Sewage treatment, denitrification of the drinking water, hazardous xenobiotic detoxification, heavy metal removal from the contaminated waters and the soils as well as environmental monitoring biosensor fabrication are some beneficial industrial applications of the microbial biofilm for cleanliness of the natural environment. An overview of bio-chemical aspects of the microbial biofilms for understanding their impact and importance in environmental/atmospheric cleanliness has been presented.

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1. Introduction Microbes adhere to the environmental surfaces ranging from human tooth and intestine to the metal surfaces of the condenser tubes exposed to turbulent flow of water, for their physiological functioning, support and protection [2,6,8]. Their association with these surfaces involves the synthesis of extra cellular homo- or hetero polymers of sugars called exo-polysaccharides (EPS). These extracellular polymers are fabricated and extended from the cells to the surfaces in a very organized manner forming a tangled and channelized network of the polymeric fibres [2]. The whole assemblage including the resident microbes and the channelized network of the EPS is termed as Biofilm. Because of its polyelectrolyte nature the microbial biofilms are highly absorptive and can collect significant quantities of silt, clay heavy metals and other detritus from their immediate environment [8]. Thus their unique physical, biological and chemical properties make them a very useful tool for environment cleanliness. Since the microbial biofilms are implicated in the natural and modulated environmental cleanliness systems being used for purification of the drinking water, detoxification of the oil spills, removal of heavy metals and biodegradation of the hazardous xenobiotics in the contaminated waters and the soils, as well as environmental monitoring biosensor fabrications [3,5], an overview of the physico-chemical and biological aspects of the microbial biofilms is being presented here.

2. Process of the Biofilm Formation The attachment of the microbes with the surfaces and the formation of biofilm is the consequence of the following processes (Fig. 1).

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2.1.Conditioning of the surface The conditioning of surfaces involves the adsorption of organic substrates to the physical surface so that it would be suitable for the adsorption of the microbes. The factors like the nature of the fabricating materials, surface tension, electrophoretic mobility, roughness and wettability of the surfaces control the conditioning process [8]. The adsorption of organic substrates brings a change in the physico-chemical properties of the surface, which facilitate the invasion of the microbes of the surface. The analysis of the organic substrates adsorbed to the surfaces indicated that the organic substance is the mixture of polysaccharides, proteins and glycoproteins. 2.2. Adsorption of the cells to the conditioned surface The course of conditioning follows the adhesion of surface by free floating or planktonic microbes (bacteria, algae, fungi). Initially the attachment of the microbes to the surface is reversible i.e., the microbes adsorbed to the surface at this stage are in Brownian motion and a mild rinsing could readily remove them from the surface. The attachment of the microbes with the conditioned surfaces at this stage is the result of some week forces like surface charge of the microbes, and the Van der Waals and electrostatic forces of the interacting molecules [8]. 2.3. Formation of a tangled and channelled network of EPS A prolong microbial adhesion results an irreversible attachment of the microbes to the surface. This firm attachment of the microbes with surface requires severe mechanical or chemical treatment for its removal. Essentially, the irreversible or permanent attachment of the microbes is the consequence of the production of extra cellular cementing substances called exo-polysaccharides (EPS). The network of EPS fibres anchors the microbes to the surface and

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connects their cells like strands of spaghetti [2]. With the passage of time, a diversified microbial consortium occupies the sticky web of the tangled polysaccharide fibres. The network of EPS provides cohesive forces within the biofilm, draws nutrients from the medium, protects microbes from sudden environmental shocks, and adsorbs heavy metals and other organic detritus from the surroundings. Aside from serving as means of intercellular communication within the biofilm, the network of EPS enhances the intercellular transfer of genetic material and acts as short-term energy storage house. The physical properties like diffusiability, thermal conductivity, and rheology of the microbial biofilms are also influenced by the EPS [8]. Chemical analysis showed that the EPS are mostly comprised of neutral and acidic sugars (e.g., glucose, galactose, mannose, rhamnose, ribose, fucose, uronic and gluconic acids, etc.).

3. Nuisance effects of the Microbial Biofilms Microbial biofilms are considered to be nuisance because these badly affect every thing from human health to important industrial products [2,5,8,9]. Reduction of sulphur to SO2 by anaerobic, and oxidation of metals by aerobic microbial population of the biofilms, respectively, results in plugging and corrosion of the water supply pipes (Fig. 2) [2]. The polyelectrolyte nature of the EPS part of the biofilms makes them an electron sink at the cathode and serves as conductor for interrupting the electronic signals of the computer chips, a deleterious effect causing the malfunctioning of the computers [2]. The formation of the microbial biofilms on wetted surfaces of the water supply pipes and tubes increases the fluid frictional resistance, reduces the cross sectional area for flow, enhances the roughness of the surface and viscoelasticity of the fluids [8].

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Development of biofilms on heat transfer tubes impedes the flow of heat across the interfaces hence a loss of energy occurs. An accumulation of the microbial biofilms on the surfaces of the sensors, submarine periscopes and sight glasses reduces the effectiveness of these instruments. A release of detached biofilms in water distribution system and from cooling towers not only lowers the quality of the drinking water but also increases the incidence of pathogen dissemination in the atmosphere [8]. Dental plaques and various other animal and human diseases of lungs, intestinal and urinary tracts are the result of the development of the microbial biofilms within the tissues of the organs. Moreover, the development of the microbial biofilms reduces the effectiveness of the antibiotics against the pathogens [2].

4. Benefits of the Microbial Biofilms Because of the physico-chemical and biological properties, the biofilms are highly beneficial for removing organic and inorganic contaminants from the natural environments and in the modulated systems [5,8]. Microbial biofilm formation with the plant roots protects the plants against soil borne diseases and improves crop productivity by regulating nutrient and water flow across the roots, as well as by improving the physico-chemical characteristics of the soils [9]. The microbial biofilms associated with the soil particles help degrade and detoxify the hazardous organic and inorganic agrochemical contaminants. A renegade of microbes entrapped in the biofilms is implicated in detoxification and removal of the heavy metals and other organic contaminants from drinking and sewerage water supplies (Fig. 2) [1,3,4].

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Detoxification of the industrial and agricultural organic and inorganic wastes for atmospheric cleanliness is achieved, either through oxidation of carbon compounds, chelation of heavy metal contaminants and their bio transformation into non-toxic or less toxic organic compounds, nitrification and denitrification of the nitrogenous compounds, or biodegradation of xenobiotics, toxic fuels and the oil spills.

5. Removal of Heavy Metals Detoxification and removal of the heavy metals from industrial and domestic wastes waters and from terrestrial ecosystems are the function of combined or the individual, microbial and EPS components of the biofilms. EPS because of the presence of negatively charged functional groups like pyruvate, phosphate, hydroxyl, succinyl and uronic acid binds and transforms the toxic heavy metals into non-toxic insoluble organic or inorganic salts [4,7,10]. These immobilized metals are then subsequently carried to the microbial cell walls where these are either taken inside of the cells and assimilated into non-toxic organic compounds or transformed from insoluble toxicant to soluble intoxicant form [7].

6. Bioremediation and Detoxification of Hazardous Organic and Inorganic Materials A wide variety of organic and inorganic contaminants including long-lived petroleum derived aliphatic or aromatic hydrocarbons; chlorinated pesticides, preservatives, and insulators are disseminated in the environment (waste water, soil, drinking water, rivers, etc.) through anthropogenic, agricultural and industrial

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activities. A substantial portion of the pollutant flux is comprised of the labile substitutes for persistent herbicides and pesticides, including organophosphates, carbamates and triazines. For detoxification of the pollutants, microbial transformations change the form, phase or redox state of the contaminant [3]. A complete degradation of the organic contaminants into CO2 or H2O represents a direct transformation of the pollutant while a sequestration or chelation of the resulting inorganic products with the microbial metabolites is an example of an indirect transformation. Conversely, the organic and inorganic wastewater pollutants are either electron donors or acceptors [1]. The detoxification of these contaminants is therefore, achieved by oxidation-reduction process that allows a specific group of bacteria to grow while inhibiting the growth of the others. For example, NH4+ a common inorganic wastewater pollutant is converted to NO3 when oxygen is supplied in the system, here, the NH4+ acts as electron donor. The presence of oxygen in the system triggers the growth of a selected group of nitrifying bacteria that converts the reduced form of N to oxidized NO3. In the presence of an appropriate electron donor e.g., organic matter, reduced sulphur or H2 and under anoxic conditions, the denitrification of NO3 results in the formation and evolution of N2 gas. NO3 in this case acts as electron acceptor. Similarly, degradation of particulate organic matter is carried out by a group of aerobic heterotrophs when oxygen is supplied as electron acceptor, organic matter in this case would be the electron donor. However, under anoxic conditions consortia of fermenting microbes including methane-producing archaebacteria would develop and convert the biochemical oxygen demand (BOD) of the system into CH4, which then will be evolved from the liquid as gas. Because of the importance and usefulness of the microbial biofilms in detoxification of organic and inorganic

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contaminants in the terrestrial and water ecosystems, rotating biological contractors, fluidised-bed, and packed-bed bioreactors as well as other biofilm and activated sludge systems are fabricated for anaerobic wastewater treatment, bioremediation of the toxic industrial wastes and biological treatment of the drinking water [1,5]. Aside from these modulated bioreactors, development of biofilms of a variety of microbial consortia capable of degradation and detoxification of the contaminants in the contaminated soils and the ground water is being accomplished by controlled supply and injection of the nutrients and the substrates (required to develop a specific microbial conglomeration) in the water aquifers.

The work was supported in part jointly by IFS, Sweden and COMSTECH under research grant NO. C/3015-1. The authors are grateful for acknowledging the assistance of Mr. Ansar Mahmood for preparation of the manuscript and the Centre for Biofilm Engineering (CBE), Montana State University, USA, for permitting the use of demonstration figures.

References [1] B.E. Rittmann, Environmental Microbiology, Innovations in Biological Processes for Pollution Control, ed. R. Mitchell (Wiley-Liss, Inc. USA, 1992) p.265-296. [2] [3] C. Potera, Science. 273: 1795-1797 (1996). D.G. Capone and J.M. Bauer, Environmental Microbiology, Microbial Processes in Coastal Pollution, ed. R. Mitchell (Wiley-Liss, Inc. USA, 1992) p.191-237.

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[4] [5] [6]

J. L. Geddie and Sutherland, J. Appl. Bacteriol. 74: 467-472 (1993). J.D. Brayers, J Appl Bacteriol. 74: 98s-109s (1993). N. Bhosle, P.A. Suci, A.M. Baty, R.M. Weiner, and G.G. Geesey, J. Colloid and Interf. Sci. 205: 89-96 (1998).

[7]

T. Ford and R. Mitchell, Environmental Microbiology, Microbial Transport of Toxic Metals, ed. R. Mitchell (Wiley-Liss, Inc. USA, 1992) p.83-101.

[8]

W.G. Characklis and K.E. Cooksey, Advances Appl. Microbiol. 29: 93138 (1983).

[9]

Y. Alami, W. Achouak, C. Moral and T. Heulin, Appl. Environ. Microbiol. 66: 3393-3398 (2000)

[10]

Z. Lewandowski, Corrosion 98. 296: 1-15 (1998).

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Figure 1. The process of microbial biofilm formation and its consequent

interaction with the substrate (Adopted from ASM collection of CBE, at University of Montana, USA)

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Figure 2. Illustration of development of microbial biofilms in water supply pipes

and their role in purification and decontamination of the drinking water and corrosion of the water supply pipes (Adopted from ASM collection of CBE at University of Montana, USA).

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