Biofiltration – the treatment of fluids by microorganisms immobilized into the filter bedding material: a review

Bioresource Technology 77 (2001) 257±274

Review paper

Bio®ltration ± the treatment of ¯uids by microorganisms immobilized into the ®lter bedding material: a review Yariv Cohen * Department of Soil Sciences, Swedish University of Agricultural Sciences, Box 7014, 750 07 Uppsala, Sweden Accepted 12 May 2000

Abstract Bio®ltration is distinguished from other biological waste treatments by the fact that there is a separation between the microorganisms and the treated waste. In bio®ltration systems the microorganisms are immobilized to the bedding material, while the treated ¯uid ¯ows through it. In recent decades, a vast amount of literature has been written on single experiments involving the treatment of ¯uids by immobilized microorganisms. Several arti®cial immobilization methods have been examined and impressive results have been achieved in the treatment of ¯uids with one of the arti®cial immobilization methods ± the entrapment of microorganisms within polymer beads. This method, even though it needs to be improved, seems to have a future potential in commercial bio®ltration systems. The methods of arti®cial immobilization of microorganisms within bio®ltration systems have several advantages, but also su€er from several disadvantages in comparison to the treatment of ¯uids by naturally attached microorganisms. Understanding the mechanisms and forces responsible for the attachment of microbes to the bedding material, in attempt to improve this attachment, is of the utmost importance. Further improvement of the arti®cial entrapment of microorganisms within polymers will allow the exploitation of the advantages of this method in the treatment of ¯uids. The aim of this review essay is to introduce the main principles of two immobilization processes ± the self-attachment of microorganisms to the bedding material and the arti®cial entrapment of microorganisms within polymer beads. Both treatments of liquids and gases with each immobilization process are discussed. The advantages and disadvantages of each immobilization process are pointed out and di€erent aspects of the ¯uid treatment with the two immobilization processes are compared. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bio®ltration; Immobilization; Entrapped; Bio®lm; Bio®lter

1. Introduction To control and reduce ¯uid pollution is a topic of great importance to society. Wastewater discharged into natural aquatic ecosystem causes problems of eutrophication and toxic e€ects. Wastewater, which penetrates into the ground contaminates groundwater and reduces the quality of drinking water supplies. Ammonia and hydrogen sulphide emissions, as well as other volatile compounds from wastewater treatment plants and composting facilities contribute to the greenhouse e€ect and produce acid rain. Bio®ltration seems to be an interesting alternative in controlling these liquid and gaseous emissions. Biological treatment of wastes is considered to be an ecient treatment with a relatively

*

Tel.: +46-18-671-495; fax: +46-18-672-795. E-mail address: [email protected] (Y. Cohen).

low cost compared to other physical or chemical treatment methods. In common biological treatments, microorganisms are mixed with the waste material. The microorganisms decompose the waste material and convert it to microbial biomass and energy. There is no separation between the microorganisms and the treated waste. One such treatment system is the activated sludge in sewage waste treatment, in which the microorganisms are suspended within the treated liquid. A second step of treatment is needed in this system to separate the microbial biomass from the treated ¯uid. Bio®ltration is distinguished from other biological waste treatments by the fact that there is a separation between the microorganisms and the treated waste. In bio®ltration the microbial biomass is static ± immobilized to the bedding material, while the treated ¯uid is mobile ± it ¯ows through the ®lter. This construction creates a separation between the microbial biomass and

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the treated ¯uid (although this separation is not complete and biomass to a certain extent leaches into the treated ¯uid). The immobilization of microorganisms to the bedding material can be divided into two main immobilization processes: (1) the self-attachment of microorganisms to the ®lter bedding material, which is de®ned as `attached growth', (2) the arti®cial immobilization of microorganisms to the bedding material. There are several methods for arti®cial immobilization of microorganisms to a support material (®ve of them will be reported here). One of them, which seems to have a potential future application, is the arti®cial entrapment of microorganisms within polymer beads. Apart from the immobilization process, bio®ltration systems can be divided into two di€erent treatment systems according to the phase of the treated ¯uid, i.e., systems treating gas and those treating liquids. There is a considerable di€erence in the operation of systems treating di€erent phases of ¯uid, even though based upon the same bedding material. In bio®ltration systems the pollutants may be removed from the ¯uid in several ways. They can be adsorbed to the microbial ®lm or to the bedding material. In bio®lters treating gas, the pollutants might be adsorbed to the water that clings to the bedding material. The main way of pollutant removal in bio®ltration systems, however, is the biological degradation of the waste. In this way the contaminants are incorporated into the microbial biomass or used as energy sources (electron donors or electron acceptors).

The aim of this review essay is to introduce the main principles of two immobilization processes, namely the self-attachment of microorganisms to the bedding material and the arti®cial entrapment of microorganisms within polymer beads. Treatment of both liquids and gases will be discussed and di€erent aspects of the two immobilization processes will be compared (see Fig. 1). 2. Attached growth 2.1. The advantages of attached microbial ®lm compared to suspended microorganisms in the degradation of ¯uid pollutants 2.1.1. Higher biomass concentrations In natural and arti®cial aquatic ecosystems, the concentration of microorganisms upon submerged surfaces is generally higher than in the free water. For instance, attached bacteria were found to exceed the suspended population by 3±4 orders of magnitude in oligotrophic alpine streams and 200-fold in sewage e‚uent (McLean et al., 1994). Since the concentration of the attached microorganisms is generally higher than that of the suspended microorganisms, a higher microbial concentration could be maintained within systems treating ¯uids with attached microorganisms. Senthilnathan and Ganczarczyk (1990) reported that the suspended biomass concentration within activated sludge is maintained in the range of 700±2500 mg/l (expressed in terms of mixed liquor volatile suspended solids (MLVSS)),

Fig. 1. Schematic illustration of the immobilization processes and ¯uids treatment as will be presented in this review.

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while in trickling ®lters, for example, the attached microbial biomass ranges from 2000 to 100 000 mg MLVSS per litre of ®lter volume. The higher concentration of biomass within attached growth systems is believed to be the main reason for the advantages of these systems, leading to an ecient treatment with a more compact treatment system. 2.1.2. Higher metabolic activity In many cases, a higher metabolic activity was measured within attached growth treatment systems compared to that of the suspended growth systems. It is widely agreed that attached growth anaerobic digesters can handle higher loading rates with shorter hydraulic retention times than their suspended counterparts (Polprasert, 1989), suggesting that the attached bio®lm is more ecient in waste decomposition than the suspended microorganisms. One explanation for this increased biodegradation activity might originate in the higher amount of active biomass concentration within the attached growth systems. This higher activity has also been attributed to the high concentration of nutrients around attached bio®lms (Madigan et al., 1997). Due to its slimy nature, the bio®lm traps particulate matter from the treated ¯uid, so the nutrient concentration around bio®lms is usually higher than that of the free ¯uid. This high nutrient concentration may increase the microbial growth rate and enhance the degradation activity. Another explanation for this increased biodegradation activity is a physiological di€erence between attached and suspended microorganisms (Senthilnathan and Ganczarczyk, 1990; Lazarova and Manem, 1995). It has been suggested that with the attachment, the `switching on' of di€erent genes occur, which leads to a physiological di€erence between the attached and the suspended microorganisms. This di€erence ®nds expression in a faster growth rate, increased metabolic activity and greater resistance to toxicity. 2.1.3. Greater resistance to toxicity A greater resistance to toxicity is usually seen within attached bio®lms compared to suspended microorganisms. For instance, attached bacteria in a hospital environment were able to resist antiseptic concentrations of up to 20 000 mg chlorhexidine per litre for a period of 27 months, whereas the suspended organisms of the same species were killed at a much lower concentration of 500 mg/l (Marrie and Costerton, 1981). Some attribute this phenomenon to the physiological di€erence between attached and suspended microorganisms (Senthilnathan and Ganczarczyk, 1990; Lazarova and Manem, 1995). Others explain this phenomenon by suggesting that the increased concentration of nutrients around bio®lms help the microorganisms to survive in

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the higher concentration of toxic compounds (Madigan et al., 1997). Another explanation for this increased resistance to toxicity within attached bio®lms is the protective e€ect of the extracellular matrix. The di€usion barrier, together with the ion exchanger role of this matrix, reduces the concentration of toxic compounds within the bio®lm. This is considered to be one of the main reasons for the higher resistance towards toxic compounds within attached bio®lms (Lazarova and Manem, 1995). 2.1.4. Better sludge properties There is a considerable di€erence in the properties of the sludge which is produced by attached growth treatment or suspended growth treatment. Usually suspended growth treatment produces a sparse sludge with a high volume per unit biomass. Filamentous microorganisms are usually dominating (Wanner, 1994), which may cause problems of bulking and foaming in wastewater treatment plants. On the other hand the nature of the sludge produced by attached growth treatment, which consists of sloughed bio®lm, is usually more dense and in many cases reduces settling problems and precludes bulking and foaming problems (Droste, 1997). Even though attached bio®lms possess a variety of advantages compared to suspended microorganisms in the treatment of ¯uids, treating ¯uids with suspended microorganisms will, in some cases, be superior to the treatment of ¯uids with an attached bio®lm. Free microorganisms being immersed in a ¯uid have a relatively small di€usion barrier around themselves compared to a bio®lm, which is surrounded by a di€usion resistant extracellular matrix. Due to that fact, at low concentration of nutrients, suspended microorganisms can show a faster growth rate than attached bio®lms. The di€usion restriction of the bio®lm might reduce oxygen penetration into a thick bio®lm and limit the amount of aerobic decomposition. In many cases the growth of the bio®lm cannot be controlled e€ectively. This can lead to variable reactor performance and uncontrolled sloughing. To bene®t from the advantages of attached growth treatments, these treatment systems must be designed correctly and match the properties of the treated ¯uid. 2.2. The structure and properties of bio®lms 2.2.1. The bio®lm thickness The bio®lm thickness is in¯uenced by several factors. These include the rate of ¯ow through the bio®lter, the bedding material construction and the di€erent treatment system designs. Generally, a rapid ¯ow through the bio®lter will limit the growth of bacterial ®lms to small thicknesses. Microorganisms form thinner layers

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upon smooth surfaces in comparison to microbial ®lms upon porous material (Kosaric and Blaszczyk, 1990) and each treatment system has a typical bio®lm thickness (Senthilnathan and Ganczarczyk, 1990). The bio®lm thickness usually varies from tens of micrometres to more than 1 cm, although an average of 1 mm or less is usually observed (Wanner and Gujer, 1984). However, the whole bio®lm is not active. The activity increases with the thickness of the bio®lm up to a level termed the `active thickness'. Above this level, the di€usion of nutrients becomes a limiting factor, thus di€erentiating an `active' bio®lm from an `inactive' bio®lm (Lazarova and Manem, 1995). 2.2.2. The extracellular matrix Within bio®lms the total cell volume is only a small part of the total volume. The extracellular matrix, a polymer matrix in which cells and colonies are embedded, accounts for the majority of the bio®lm volume (Lazarova and Manem, 1995). The extracellular matrix structure and composition varies between bio®lms, but generally polysaccharides predominate the extracellular matrix and represent up to 65% of this material, while proteins usually comprise up to 10±15% of its total biomass (Lazarova and Manem, 1995). Most of these polymers are anionic as evidenced by their staining with cationic ruthenium red and their ability to bind metals (McLean et al., 1994). 2.2.3. The heterogeneous structure of bio®lms Even though bio®lms seem to consist of a homogeneous layer, there is a considerable non-uniformity within bio®lms. It has been shown that the dissolved oxygen di€usivity is not constant through the bio®lm, but decreases with depth (Wanner and Gujer, 1984). This suggests that there is a density gradient within bio®lms (Masuda et al., 1991). One explanation for this density gradient may be the di€erent growth rates of the microorganisms, which are located at di€erent depths within the bio®lm (Bishop et al., 1995). The traditional perception of the ¯ow pattern around bio®lms describes two separated zones: the bio®lm zone and the ¯uid zone. The bio®lm zone contacts the ¯uid zone with its outer plane and the ¯uid does not penetrate into the bio®lm, but ¯ows upon it. This perception may not be accurate. Lewandowski et al. (1995) showed by nuclear magnetic resonance imaging (NMRI) experiments that bio®lms could have a complex heterogeneous structure consisting of cell clusters separated by interstitial voids. Water can ¯ow through the entire bio®lm creating two ¯ow ®elds; one above the bio®lm and another within the bio®lm. These ¯ow ®elds interact with each other in a complex manner. It is assumed that these ¯ow patterns enhance the substrate ¯ux from the bulk liquid to the bio®lm.

2.3. The mechanisms and forces responsible for the attachment of microorganisms to a surface 2.3.1. Microbial structures Microorganisms possess a variety of structures which are used for attachment. These include ®mbria (pili), capsules (glycocalyx), various holdfast structures, stalks, cell wall components and slimes (McLean et al., 1994). It is believed that the main microbial structure which is involved in attachment is the glycocalyx. The glycocalyx consists of extracellular polysaccharides, which were found to have no other special properties beside their participation in the attachment process (Jones et al., 1969). Besides the glycocalyx, any extension from the bacterial cell often comes into contact with surfaces and is involved in attachment. 2.3.2. Forces governing microbial attachment Several forces are involved in microbial attachment to a surface. Usually, none of the forces could be considered as the dominant force. The strength of the attachment and the composition of the forces, which govern it change with di€erent environmental conditions, di€erent microbial species, di€erent surface properties and with di€erent ¯uid properties. (a) Electrostatic interactions. The electrostatic interactions involved in microbial attachment are mostly of ionic and hydrogen bonding which, although being individually weak when compared to covalent bonds, are nevertheless capable of producing relatively ®rm binding, if the number of bonds is suciently large (Tampion and Tampion, 1987). The electrostatic forces are highly signi®cant in the initial stages of adsorption. Since the surface charge of almost all microorganisms is negative (Kolot, 1988) and in many cases the bedding material is also negatively charged, an electrostatic repulsive force can prevent attachment. Usually other stronger forces overcome this repulsive barrier and negatively charged cells do attach to negatively charged material, such as glass and ceramics. In other cases, where the attachment surface is positively charged, the electrostatic forces can dominate the attachment process (Crope, 1970). (b) Covalent bond formation. The outer surfaces of microbial cells and cell walls contain large quantities of a variety of reactive groups. Covalent bonds can form between those ligands on the cell surface and speci®c groups, which are located on the bedding material surface. Usually covalent bonds are formed between organic groups on the bedding material and the microbial ligands (Cochet et al., 1990). (c) Hydrophobic interactions. Since the attachment of microorganisms to the bedding material takes place in a liquid environment, hydrophobic interactions between the microorganisms and the bedding material are very important. Hydrophobic groups on the microbial

Y. Cohen / Bioresource Technology 77 (2001) 257±274

surface can interact with hydrophobic groups on the bedding material, while removing the water molecules which separate the microorganisms from the surface. It has been reported that hydrophobic surfaces are more prone to colonization by dental and soil bacteria than are their hydrophilic counterparts (McLean et al., 1994). (d) Partial covalent bond formation between microorganisms and hydroxyl groups on surfaces. Since the microbial immobilization is performed in solution, hydroxides are formed instead of di€erent metal oxides incorporated in the bedding material (Kolot, 1988). Suitable amino or carboxyl groups on the cell surface can replace those hydroxyl groups. As a result, partial covalent bonds form between the microbial cells and the bedding material. 2.4. The bedding material construction and its in¯uence upon the forces governing microbial attachment The selection of a bedding material will depend upon many factors including the resistance to microbial degradation, mechanical strength, type of ¯uid, surface characteristics and the cost of the material. 2.4.1. Organic bedding material It is widely accepted that organic material has a higher adsorbtivity compared to inorganic material. For instance, the microbial adsorption is 248 and 2 mg/g on wood chips and inorganic silica, respectively (Gemeiner et al., 1994). The reason for the higher adsorption of organic material compared to inorganic material is due to the larger variety of reactive groups, such as carboxyl, amino, hydroxyl etc., which are located on the organic material surface. Organic bedding materials usually also contain a certain amount of nutrients, which help the microorganisms to attach and grow. As organic bedding materials are often biodegradable, they need to be replaced at higher frequency than other non-biodegradable bedding materials. 2.4.2. Inorganic bedding material Inorganic bedding material is usually considered to be resistant to microbial attack, to exhibit high thermostability and to have good ¯ow properties (Gemeiner et al., 1994). The inorganic bedding material can be subdivided into grafted and ungrafted material. In grafted material, organic groups are attached to the inorganic material surface by various coupling agents. Since organic material contains a large variety of reactive groups, this application usually improves the adsorbance performance of the inorganic material. Inorganic material usually consists of a variety of metal oxides. Since the attachment of microorganisms to the bedding material occurs in aqueous conditions,

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metal hydroxides are formed on surfaces of inorganic material instead of metal oxides (Kolot, 1988). As a result, partial covalent bonds form between the microorganisms and these hydroxides. Many of the inorganic materials, such as glass or ceramics, are negatively charged. The high biomass accumulation of negatively charged microorganisms on negatively charged inorganic material suggests that other forces overcome the electrostatic repulsion between the microorganisms and the bedding material. One explanation for this might be the partial covalent bonds formed between inorganic metal hydroxides and the microbial surface. There is a potential to increase the microbial retention capacity of the bedding material by incorporating speci®c metals into the bedding material. Incorporation of Fe3‡ within a bedding material increased the binding of yeast cells by almost 50% (Kolot, 1988). 2.4.3. Charged bedding material Since the surface charge of microorganisms is negative in general, it follows that microorganisms will attach readily to the surface of positively charged material. Many bedding materials, especially inorganic materials, are negatively charged. Rouxhet and Mozes (1990) state that the microbial adhesion to those materials might be promoted by treatments applied to the bedding materials, in order to modify their surface properties and, thereby, decrease electrical repulsion or achieve attraction. In their paper, they present an overview of such treatments. The use of charged bedding material could become a problem if substrate, product, and/or residual contaminants are charged and interact with the bedding material. This can lead to di€usion limitations and reaction kinetic problems (Bickersta€, 1997). 2.4.4. Porous bedding material The degree of porosity of the bedding material is an important factor in the adsorption of microorganisms to the bedding material. A bedding material, which has a high void space inside allows the microorganisms to attach under low shear conditions, while the ¯uid outside the bedding material can be moving at a high speed. It has been shown (Van Den Berg and Kennedy, 1981) that despite the fact that polyvinyl chloride (PVC) is frequently used as a packing material in wastewater treatment, it took a considerably longer time to reach a maximum loading rate with PVC than with porous red draintile clay and grey potters' clay. According to Tampion and Tampion (1987), the maximum accumulation of biomass occurred when pores sizes were one to ®ve times the bacterial size. In synthetic support material the degree of porosity can, to a certain limit, be controlled. Gemeiner et al.

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(1994) found that porous glass could be prepared at any  desired pore diameter over the range of 20±2000 A. 2.5. The in¯uence of ¯uid properties on microbial attachment 2.5.1. pH Marcipar et al. (1978), studied the adsorption of microorganisms to an inorganic ceramic bedding material as a function of pH 4 and 6. They found that the rate of adsorption was rather speci®c for each microbial strain, but at the lower pH, the percentage of microorganisms adsorbed was higher. A possible explanation for this higher attachment of microorganisms at lower pH arises from the in¯uence of pH on the microbial surface charge. Most microbes have a dipolar character, which can function as a cation or anion depending on the pH of the solution. For instance, raising the pH from 3 to 7.5 resulted in an increase of cell charge from )10 to )30 mV and the opposite tendency, the decrease in the cell charge, has been achieved by decreasing the pH (Kolot, 1988). It is reasonable that at low pH values, the negative charge of the microbial surface is reduced, which leads to a reduction in the electrostatic repulsion between the microorganisms and negatively charged bedding material. This reduction in the electrostatic repulsion might be the cause of the greater microbial attachment at low pH values. 2.5.2. Salt content Meadows (1971) has reported that he was able to detach microbial cells from sand by washing with distilled water, but when salt was present in the water the microbial leaching decreased considerably. The explanation for this high attachment of microorganisms in the presence of salts is also the in¯uence of these salts upon the microbial surface charge. Kolot (1988) showed that an increase in Ca-ion concentration from 0.01% to 1% resulted in an eight-fold decrease of the charge on the microbial cell surface. Placed in distilled water, the negative charge of the microbial cell surface increased ®ve-fold ()7.9 to )39 mV). In cases, where the microorganisms and the bedding material are negatively charged, the divalent positively charged ions reduce the electrostatic repulsion between the microorganisms and the bedding material and may even act as bridges between those two negatively charged surfaces (Tampion and Tampion, 1987). According to DiCosmo et al. (1994), in cells of C. roseus the maximum level adhesion occurred in the presence of 0.1 M AlCl3 . Adhesion decreased, however, when the concentration was either greater or less than 0.1 M AlCl3 . The explanation for the maximum attachment at a speci®c salt content of 0.1 M AlCl3 is probably the achievement of an optimal surface charge

for the attachment. In 0.1 M AlCl3 solution, the electrophoretic mobility of the microbial cells was close to 0. At that point electrostatic repulsion between the microbial cells and the bedding material was relatively low, resulting in increased adhesion. With increasing concentrations of AlCl3 above 0.1 M, adsorption decreased because the adsorption of the ions to the microorganisms and the bedding material resulted in charge reversal from a negative to a positive sign. This charge reversal probably resulted in an electrostatic repulsion between the positively charged microorganisms and the positive bedding material. It is assumed that for each microbial species and sort of bedding material, there is an optimal salt content of the treated ¯uid, which can enhance the microbial attachment. 2.6. The di€erences in the treatment of a di€erent ¯uid phase (liquid or gaseous) by attached growth bio®ltration systems 2.6.1. The di€erences between the liquid and the gaseous phase Liquids and gases di€er from each other in many aspects. They have a di€erent density, di€erent viscosity, di€erent thermal properties and they di€er in some of their ¯uid dynamics. There is a considerable di€erence in the di€usion of substrates through a gaseous or an aqueous phase. For instance, the di€usion coecients of substrates such as oxygen and carbon dioxide are about 10 000 times smaller in water than they are in air (Cussler, 1997). Liquids and gases also di€er in the thickness of the boundary layer that surrounds surfaces which are immersed in those ¯uids. A ¯uid in contact with a solid surface does not move relative to that solid surface. As distance increases from the solid surface, the velocity of the ¯uid rises asymptotically to match that of the free stream. A boundary layer is de®ned as the distance from the solid surface to the point where the velocity of the ¯uid equals a certain percentage of the main stream. In that layer substrates usually cannot be delivered by convection to the solid surface, instead di€usion is the dominant way of transporting substrates through that layer to the solid surface. The boundary layer, which surrounds a solid surface in a gaseous medium is thicker than that in an aqueous medium, given the same ¯ow conditions. Generally, including the di€erences in di€usivity and the boundary layer thickness, the transport of a substrate to a consumption site in a gas medium is 300 times that in aquatic conditions (Denny, 1993). Due to the di€erent properties of liquids and gases, the systems treating these di€erent ¯uids di€er in many aspects. (a) Di€erent construction of the bio®ltering system. The main di€erence in the construction of systems

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treating liquids and those of gas treatment originates in the low water content of the treated gases. Water is one of the essential compounds for a living bio®lm. To compensate for the low water content of gases, water must be added arti®cially in such systems. There are two main ways of adding water to systems treating gas: (1) humidifying the gas prior to its entrance to the bio®lter, (2) addition of water by spraying it directly onto the bedding material inside the bio®lter. Even in bio®lters treating humidi®ed gas, water is usually added in the form of spraying upon the bedding material in an attempt to maintain the optimal water content. The two di€erent constructions of bio®lters treating gas are presented in Fig. 2. The basic construction of bio®lters which treat liquids di€ers slightly from the bio®lters that treat gas. In bio-

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®lters treating liquids, a considerable amount of microbial biomass sloughs o€ into the treated ¯uid. This sloughed microbial biomass needs to be further separated from the treated liquid. The basic construction of a trickling bio®lter, which treats liquids, is presented in Fig. 3. (b) Di€erent route of substrate transport from the ¯uid to the bio®lm. In systems treating liquids, substrates are conveyed with the ¯uid currents to the border of the boundary layer surrounding the bio®lm. From this border of the boundary layer, substrates di€use through the boundary layer towards their consumption sites within the bio®lm. In gas-treating systems, an aqueous layer surrounds a notable portion of the bio®lm. The substrates are conveyed with the gas ¯ow currents and di€use through the

Fig. 2. The two di€erent constructions of bio®lters treating gas: (a) Bio®lters treating humidi®ed gas. In these systems a small amount of water is sprayed upon the bedding material to maintain optimal water content. (b) Trickling bio®lters ± in these bio®lters the gas is not humidi®ed. The water trickles freely through the bedding material and is recycled to a certain extent.

Fig. 3. The construction of a trickling bio®lter treating wastewater. Usually the wastewater that ¯ows into the bio®lter is settled in a primary settling tank. The wastewater is dosed over the bedding material and trickles through it. The e‚uent is settled again in a second settling tank to separate the sloughed microbial biomass from the treated water.

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gaseous boundary layer, which contacts the water surrounding the bio®lm. To reach the bio®lm the substrates must dissolve in the aqueous medium ®rst and then di€use through the liquid towards the consumption sites within the bio®lm. (c) Di€erent bio®lm structure. There is a considerable di€erence in the shear stress exerted upon the bio®lm in systems treating liquids and those of gas treatment. In systems treating liquids, the movement of the liquid through the bedding material exerts a considerable hydrodynamic shear stress. This hydrodynamic shear stress forces the bio®lm to be constructed in a quite condensed and slimy form to withstand it. In bio®lters treating gas, water clings to the bedding material by surface tension and does not ¯ow rapidly. So in those bio®lters, more elevated structures of microorganisms can be seen, such as elevated fungal hyphae (Devinny et al., 1999). Bio®lters treating gas may include a ®lm of standing water outside the bio®lm. If the thickness of this ®lm is enough, free swimming microbial communities can be maintained within the ®lm (Devinny et al., 1999). Free swimming microbial communities cannot be maintained in liquid-treating systems. In such systems, free swimming microorganisms will be swept away with the treated ¯uid. (d) Di€erences in acclimatisation. There is a notable di€erence in the acclimatisation process that occurs within bio®lters treating gas and those of liquid treatment. Wastewater always carries high densities of a large variety of microorganisms and only a short time is needed to establish best-adapted species in the bio®lter. In the gaseous phase, on the other hand, the density of

the microorganisms is much lower and therefore much more time is needed. A gas-treating bio®lter used to degrade methyl tertiarybutyl ether, for example, showed no activity for a year and then suddenly became very e€ective (Devinny et al., 1999). 2.7. Treatment of liquids by attached growth systems 2.7.1. Di€erent designs of attached growth systems for treating liquids The systems which treat liquids with attached bio®lms can be divided, generally, into four main groups according to the main mode of operation. In each group, a variety of di€erent designs is usually seen. Usually, a certain range of biomass concentration and bio®lm thickness characterizes each group of treatment systems. The description and biomass accumulation of the di€erent groups of systems, which treat liquids with attached bio®lms, are presented in Table 1. 2.8. Treatment of gases by attached growth systems 2.8.1. The advantages of the bio®ltration process in comparison to the traditional technologies of gas treatment The traditional technologies of gas treatment are founded on physical, chemical and thermal mechanisms (adsorption to activated carbon, adsorption to a liquid phase, condensation, thermal incineration, etc.). Usually, those technologies su€er from high cost and secondary pollution, which include a contaminated solid adsorbent phase, which requires regeneration or

Table 1 The di€erent designs and biomass accumulations of the main systems which treat liquids with attached bio®lms. The data were obtained from a comparison made by Senthilnathan and Ganczarczyk (1990) Treatment system

Description

Biomass concentration (expressed in terms of MLVSS)

Bio®lm thickness

Bio®lm active thickness

Trickling ®lters

The wastewater is uniformly dosed over the bedding material and trickles downward through it. Forced aeration is usually not designed Circular discs made of a lightweight material are half submerged in the wastewater and rotate at low speed. Oxygen is transferred from the open air to the exposed bio®lm growing upon the discs The bedding material is completely submerged in the treatment basin. Usually, air di€users located at the bottom of the bedding material aerate the basin The bio®lm is grown on particles of a medium such as sand. The wastewater is pumped through the reactor at a high rate, thereby suspending the medium particles

2000±100 000 mg/l

20±12 000 lm (average 3000 lm)

50±70 lm

10 000±20 000 mg/l

500±4000 lm

Similar to that of trickling ®lters

No information available

No information available

No information available

10 000±50 000 mg/l (average 16 000 mg/l)

Less than 100 lm (average 40 lm)

Usually all the bio®lm thickness is active

Rotating biological contactors Submerged bio®lters Fluidized bed reactors

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disposal, and/or a contaminated liquid stream that requires further treatment. On the other hand, the treatment of gases by bio®ltration systems is considered to achieve, in some environmental conditions, results at a relatively low cost and without secondary pollution (Corsi and Seed, 1995). It is common that bio®lters treating gas are used in the treatment of dilute, high-¯ow gas streams. According to Devinny et al. (1999), bio®lters treating gas are most cost-e€ective for waste gas ¯ows of 1000 to 50 000 m3 hÿ1 and pollutant concentrations up to 1 gmÿ3 . 2.8.2. The role of water in the treatment of gases by attached growth bio®lters The adsorption of the gaseous pollutants to the aqueous phase. In bio®lters, gases are adsorbed to an aqueous phase ®rst and within that aqueous phase the microbial degradation occurs. The adsorption of contaminants, present in gases, from the dilute gaseous phase to the aqueous phase, concentrates the pollutants. For any volume within a bio®lter, more contaminant is likely to be in the water phase than in the gas phase (Devinny et al., 1999). The gas usually remains within the bio®lter for a short period of time. During this time, pollutants are dissolved and concentrated in the aqueous phase. A typical vapour residence time for commercial and industrial applications ranges from 25 s for low concentrations to over a minute for high concentrations of gaseous pollutants (Leson and Winer, 1991). If the contaminants were not retained for a much longer time in the aqueous phase, biodegradation could not occur. Other aspects of the importance of the aqueous phase. In many cases, missing nutrients are added to the water sprayed in the bio®lter. Gibbons and Loehr (1998) showed that nitrogen availability could limit the performance of bio®lters treating gas. A free water phase, which contacts the bio®lm in bio®lters treating gas, can remove possible toxic degradation by-products from the bio®lm and aid in the diffusion of hydrophilic pollutants into it (Devinny et al., 1999). The temperature of the treated gas can rise as it passes through the bio®lter, due to the oxidation of the organic compounds. This heat can be controlled by the addition of water to the bio®lter. Usually, the heat produced within gas-treating bio®lters leads to evaporation, due to the higher content of water in the warmer air, so water must be added in an attempt to keep the optimal moisture content within bio®lters treating gas. The optimal moisture content within bio®lters treating gas usually ranges between 30% and 60% on a weight basis, depending upon the media used (Williams and Miller, 1992; Corsi and Seed, 1995; Li et al., 1996).

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3. Arti®cial immobilization of the microorganisms to the ®lter bedding material 3.1. Methods of arti®cial immobilization of microorganisms 3.1.1. Microencapsulation The microencapsulation method consists of wrapping droplets containing microorganisms with a thin membrane. The microorganisms can freely move within their own capsule, consuming substrates that penetrate through the membrane cover. Many di€erent materials have been used to construct microcapsules, nylon and cellulose nitrate have proven popular. Usually, the diameter of these microcapsules varies from 10 to 100 lm (Bickersta€, 1997). The main advantage of this technology is the low di€usion restriction of the thin membrane. However, several practical problems have limited the use of this technology. First, the toxicity of the membranes in many cases causes the loss of catalytic activity within the immobilized microorganisms. Secondly, the growth, the cell division or the gas produced may cause the mechanical rupture of the encapsulation membrane (Tampion and Tampion, 1987). 3.1.2. Membrane separation The main principle of this method is to separate the microorganisms from the bulk ¯uid by the use of sheets of membrane. The membranes will allow the substrates to penetrate to the microorganism's zone, while preventing the microorganisms from mixing with the ¯uid to be treated (Iorio and Calabro, 1995; Sutton and Mishra, 1996). The membranes used in this method are usually porous ultra®ltration membranes (separates in the range of 0.002±0.1 lm), but non-porous membranes can also be applied (Gaeta, 1995). In some cases, special membranes which are selective to di€erent compounds are used, such as membranes which separate CO2 and H2 S from methane (Drioli, 1995). Whether the membrane is porous or non-porous, the main problem associated with this technology is the fouling (Gaeta, 1995). The pores in a porous membrane may clog and a non-porous membrane can be covered by a bio®lm that alters its performance. The conventional method of membrane cleaning, back ¯ushing against the normal direction of ¯ow, may damage the microbial cells, and other chemical and/or physical methods cannot be applied directly in bio®ltration systems, due to the harmful e€ect they cause to the immobilized microorganisms. 3.1.3. Covalent bonding and covalent crosslinking The outer surfaces of microbial cells contain large quantities of a variety of reactive groups. The covalent

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bonding method includes the creation of covalent bonds between those reactive groups and di€erent ligands on the bedding material. Usually di€erent coupling agents are used in order to activate the ligands on the microbial cells and the bedding material. The most commonly used coupling agent is probably glutaraldyde although carbodiimine, isocyanate and amino silane have also been frequently used (Tampion and Tampion, 1987). The main problem associated with this technology is that the microorganisms are exposed to potent reactive groups, which exert toxic e€ects. Beside the toxicity problems, where viable microorganisms are covalently bound, any cell division is likely to result in cell leakage from the bedding material, so low cell loading is usually achieved compared to other immobilization methods (Tampion and Tampion, 1987). The covalent crosslinking method is support free and involves the joining of the microorganisms to each other to form a large, three-dimensional complex structure. This structure is used as a bedding material. The methods of crosslinking normally involve covalent bond formation between the microorganisms. Therefore, this immobilization method usually su€ers from the toxicity problem associated with the covalent bonding method. 3.1.4. Entrapment within polymers This method consists of trapping microorganisms within a three-dimensional polymer matrix. The pores in the matrix are smaller than the microbial cells, keeping them trapped within the material, but the pores still allow the penetration of substrates through the polymer matrix towards the trapped microorganisms. The main disadvantage associated with this method is the higher di€usion restriction which some polymer materials possess compared to the other immobilization methods. However, compared to the other immobilization methods, this method bene®ts from some advantages. These include the achievement of a high viable

biomass concentration, higher resistance to toxic compounds within the treated ¯uid, the possibility of immobilizing together di€erent species of microorganisms separated physically from each other, greater plasmid stability within genetic engineered microorganisms, which are immobilized in this method, etc. A comparison between the di€erent arti®cial immobilization methods is presented in Table 2. 3.2. The entrapment of microorganisms within polymer beads 3.2.1. The procedure of entrapping microorganisms within polymer beads The general procedure of entrapping microorganisms within polymer beads consists of suspending the microorganisms within a liquid solution which contains macromolecule monomers. The next step is to gelate the solution so the microorganisms are entrapped within the matrix. The gelation of the solution is created by the linkage of the macromolecules to each other and by this forming a polymer three-dimensional network. The gelation of the macromolecules can be done by a variety of physical or chemical methods, depending on the nature of the monomers used. These methods include lowering or raising the temperature, the ionotropic gelation of macromolecules with di- and multi-valent cations and other di€erent chemical or photochemical reactions. In attempts to form spherical beads containing entrapped microorganisms, the gelation of the monomers can be done by adding the preparation dropwise into an ice-cold bu€er or a di€erent chemical solution (Woodward, 1988). After the production of the polymer beads, they can be utilized as is, or placed in a nutrient solution to encourage additional cell growth inside the beads prior to use. Alternatively, the beads can be dried after either of these processes and stored until use. Dried

Table 2 A comparison between the di€erent methods for the arti®cial immobilization of microorganisms to the bedding material Di€usion restriction

Biomass concentration

Toxicity problems

Mechanical stability

Complexity of the application

Micro encapsulation

Relatively low

High

Fouling can cause severe di€usion restrictions No

High

Low ± the capsules tend to rupture High

Complex

Membrane separation Covalent bonding

Su€er from severe toxicity problems No

Complex

Low

High

Simple

Covalent crosslinking

No

Moderate

High

Simple

Entrapment within polymers

Relatively high ± varies with the polymer material kind and construction

High

Su€er from severe toxicity problems Su€er from severe toxicity problems Moderate toxicity problems

Varies greatly with the kind of polymer

Simple

Y. Cohen / Bioresource Technology 77 (2001) 257±274

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beads have been successfully stored for up to 3 years at 4°C (Cassidy et al., 1996). 3.2.2. Polymers used in the entrapment of microorganisms Many di€erent polymers have been used for the entrapment of microorganisms. Generally, they can be divided into natural and synthetic materials. In both of these cases, the material must be hydrophilic, so that the substrates can di€use into the beads (Sumino et al., 1992). Natural polymers, such as alginate and carrageenan, are mainly isolated from algae. Natural monomers are usually brought to gelation by either cooling and/or contact with a solution that contains di€erent ions. Synthetic polymers, on the other hand, can be brought to gelation by a wide range of chemical or photochemical reactions, so a great variety of materials have been used for entrapment, such as polyacrylamide, polyvinyl alcohol (PVA) polypropylene glycol, etc. Generally, natural polymers are considered less physically stable than synthetic material. It has been shown that natural polymers dissolve in domestic wastewater, while synthetic polymers remain stable in the same conditions (Leenen et al., 1996). Furthermore, it is believed that natural polymers are more vulnerable to biodegradation than synthetic material. On the other hand, di€usivity is considered to be higher in natural polymers and the procedure of preparing the polymer beads is usually less hostile than that in synthetic polymers (Leenen et al., 1996). A comparison between some natural and synthetic polymers used for microbial entrapment, with the objective of selecting a bedding material which has the desired characteristics for application in domestic wastewater, has been made by Leenen et al. (1996) and is presented in Table 3. 3.2.3. The three-dimensional structure of the polymers The three-dimensional structure of the polymers used for the entrapment of microorganisms varies greatly with the di€erent kinds of material used. In carrageenan polymers, which may be brought to gelation by cooling, the polymer exists in solution as a random coil. When cooling, a three-dimensional polymer network builds

Fig. 4. Acrylamide polymer construction. Modi®ed from Gemeiner et al. (1994).

up, with double helical structures, which are connected in several junction zones. Further cooling leads to aggregation of these junction zones and a complex threedimensional structure is formed (Gemeiner et al., 1994). The structure of the synthetic acrylamide polymer can be seen in Fig. 4. Generally, the mechanical strength of the polymer matrix increases with the increase in the concentration of the monomers used and the cross-linking agent. At the same time, however, the pore size of the polymer matrix decreases. 3.2.4. The di€usional properties of the polymer matrix The actual size, form and density of the polymer matrix determines the di€usional properties of the matrix. Renneberg et al. (1988) found out that it was mainly the pore size and the water content which in¯uenced the oxygen di€usivity within di€erent polymers. An increase in the di€usivity was correlated to an

Table 3 Comparison between natural polymers (carrageenan and alginate) and synthetic polymers (PVA, polyethylene glycol (PEG) and polycarbamoyl sulphonate (PCS)) used for microbial entrapment, applied in the treatment of domestic wastewatera

a

Characteristics

Natural polymers Carrageenan

Ca-alginate

PVA

PCS

PEG

Solubility Biodegradability Stability Di€usivity Growth Immobilization procedure

High Possible Low Very good Good Simple

High Possible Low Very good Good Simple

Low/not Low High Good Moderate Laborious

Low/not Low High Moderate Moderate Laborious

Low/not Low Medium Not determined Good Laborious

Modi®ed from Leenen et al., (1996).

Synthetic polymers

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increase in the pore sizes of the polymers and polymers with higher water content showed also the highest oxygen di€usivity. Hu et al. (1993) suggested that the wide porosity range of polyurethane, a synthetic polymer, potentially eliminate di€usional barriers. The actual size of the polymer beads can also greatly e€ect the di€usion of substrates into the beads. Generally, in polymer beads with a large diameter, an anoxic region is formed in the centre of the bead (Zhou and Bishop, 1997). In order to avoid the loss of activity due to di€usional limitation, 0.2±1.2 mm in diameter is most likely the most e€ective bead size (Ogbonna et al., 1991). Beside the properties of the polymers, the microbial loading within the polymer beads greatly a€ects the di€usion of substrates into the beads. Usually with high microbial densities, oxygen is consumed faster than it can di€use into the beads. For instance, with high initial microbial densities the microbial growth was limited to the outer 50±150 lm of the beads (Cassidy et al., 1996). Even though the main strategies of reducing the diffusional limitations within polymers are the control of the pore size of the matrix and the bead size, other methods such as the oxygenation of the bulk ¯uid and the incorporation of microbial oxygen generators within the polymer beads have been suggested (Ogbonna et al., 1991).

that the number of living bacteria entrapped within synthetic acrylamide beads was maximal at a certain acrylamide concentration of 15±18% (Sumino et al., 1993). The number of living bacteria decreased in the region of 10% or less and 20% or more acrylamide. The explanation for this maximal microbial concentration at a certain polymer concentration might be that bacteria escape easily from the loose lattice structure at low polymer concentration and the permeability of the substrates is lowered as a result of a reduction in the di€usion coecient at high polymer concentration (Sumino et al., 1993).

3.2.5. Improvement of the entrapping material by incorporation of di€erent compounds Several materials have been added to the preparation solution in order to increase the resistance to disruption of the polymer beads. For instance, derivatives of alginic acid (e.g., propylene glycol ester), ethyleneimine or colloidal silica have been used as hardening materials for alginate polymer beads (Gemeiner et al., 1994). The incorporation of nutrients within the polymer beads has also been shown to improve the microbial microenvironment and enhance microbial activity. The addition of skim milk to the bead formulation increased activity and growth of bacteria entrapped within alginate beads (Cassidy et al., 1996). Freeze-drying of natural polymer beads has been shown to be an e€ective method for improving their structural and mechanical properties (Nussinovitch et al., 1996; Tal et al., 1997).

3.2.7. Changes in microbial properties after entrapment Several changes in microbial properties have been seen after entrapment. Kolot (1988) reported that a higher activity and shorter generation time have been seen in entrapped microorganisms compared to free microorganisms. She further reported that a change in the macromolecular components occurred ± more DNA, glycogen and glucan but less trehalose were observed. A shift in the optimum pH (0.5±1 units) and a temperature optimum shift of about 10°C were observed. Cassidy et al. (1996) reviewed several researches, which showed a higher resistance to toxicity of microorganisms entrapped within polymers in comparison to suspended microorganisms. Sumino et al. (1993) compared the sludge production between a mixed culture of microorganisms entrapped within acrylamide beads and that of conventional methods of wastewater biological treatment. The production of sludge by the entrapped microorganisms was about one-third to one-®fth of the sludge produced by conventional activated sludge. It has been shown that it took 22 h for microorganisms entrapped within carrageenan polymer beads to reach a steady-state oxygen consumption rate (Zhou and Bishop, 1997). This indicates that the microorganisms need time to acclimatise to the polymer environment and maybe even carry out some physiological changes. In summary, despite the fact that some evidence of physiological changes is available in the literature, the precise physiological change within the microorganisms due to the entrapment process is not known.

3.2.6. Microbial biomass concentration within polymer beads A relatively high biomass concentration can be achieved by entrapping microorganisms within polymer beads. According to Sumino et al. (1993), microorganisms can be concentrated at a value of up to 100 g/l in a polymer matrix. Generally, a maximum microbial loading is usually correlated to a certain concentration of monomers and cross-linking agents. For instance, it has been shown

3.2.8. The high plasmid stability within microorganisms entrapped in polymer beads Plasmids are extrachromosomal genetic material, which consists of circular double-stranded DNA molecules that are capable of self-duplication and can be transferred between the microbial cells (Bryers and Huang, 1995). There are several ways of plasmid transmission between the microorganisms. These include the active transfer of plasmids from one microbial cell to another,

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the passive microbial adsorption of plasmids from the surroundings and the passive plasmid transfer from one bacterium to another by phage particles (a virus that attacks bacteria). Genetic engineering of microorganisms usually involves the insertion of plasmids that bear arti®cially inserted genes. The plasmid stability ± the transfer of the plasmids between the growing microorganisms within a bio®lter ± is a very important factor in bio®lters that use genetically engineered microorganisms. Generally, it has been shown that plasmid-free cells exhibit higher turnover rates than their plasmid-bearing counterpart (Bryers and Huang, 1995), so after several generations plasmid-free cells usually dominate plasmidbearing cells within a bio®lter. Surprisingly, very high plasmid stability has been found in microorganisms which are entrapped within polymer beads. For instance, the plasmid stability of plasmid-bearing E. coli which were entrapped within carrageenan beads was higher in comparison to a suspended culture of bacteria (de Taxis du poet et al., 1986). Bacteria extracted from those beads and re-suspended were shown to have the same plasmid loss frequency as suspension cultured cells. This higher plasmid stability within entrapped microorganisms has been con®rmed by other authors (Sayadi et al., 1989). Nasri et al. (1987) reported that when plasmid-bearing and plasmid-free cells were entrapped together within polymer beads, plasmid free cells did not overrun the culture. Even though the plasmid stability cannot be explained by considering one single factor, the main explanation for this higher plasmid stability within entrapped microorganisms seems to be the absence of competition between the plasmid-bearing and plasmidfree microorganisms within the polymer beads. 3.2.9. Large scale production of polymer beads For the commercial use of microorganisms entrapped within polymer beads, simple and ecient methods for large-scale production of polymer beads must be applied. Generally, it has been concluded that the high cost of the production of polymer beads can sometimes be compensated for by the reduced costs for the construction of a smaller treatment system (Leenen et al., 1996). Several di€erent systems for large-scale polymer bead production are reported in the literature. The most promising method seems to be the large-scale production of polymer beads by breaking up a capillary jet with a sinusoidal signal of a vibration exciter (Hunik and Tramper, 1993). In this system, the aqueous gel solution is pressed at such a high ¯ow rate through a small ori®ce that a jet is formed. With a membrane, a sinusoidal vibration of certain frequency is transferred to the liquid.

269

This signal causes the break-up of the jet in uniform droplets. It is assumed that a 100-fold increase in ¯ow rate can be achieved by this method compared to the dripping method (Hunik and Tramper, 1993). Cassidy et al. (1996) summarized the di€erent production rates of several large-scale bead production systems. A resonance nozzle technique allowed production of uniform alginate beads in the rate of 24 l/h. A production rate of 1.05 l/h of alginate micro-beads (500 lm diameter) has been achieved using a rotating disk atomizer which disperses the aqueous alginate solution in air. A maximum production rate of 27.6 l/h of uniform (2 mm diameter) j-carrageenan beads was obtained by breaking up a jet of j-carrageenan solution with sinusoidal vibrations, as described before. 3.3. Treatment of liquids through microorganisms entrapped within polymer beads 3.3.1. Removal of nitrogen by photosynthetic microorganisms Generally, there are two main ways for the biological removal of nitrogen from waste liquids. They include: (1) The uptake of nitrogen in the assimilative way ± the incorporation of nitrogen within the microbial biomass. (2) The oxidation of the reduced nitrogen (ammonium) to nitrite/nitrate followed by the reduction of these compounds to N2 , which is released to the atmosphere. The incorporation of nitrogen within microbial biomass is assumed to be very ecient by using photosynthetic microorganisms. These microorganisms have the ability to utilize inorganic nitrogen forms, such as nitrite, nitrate or ammonium as the sole nitrogen source for growth. Several non-N2 ®xing cyanobacteria and micro-algae have been investigated in attempts to produce a system which removes nitrogen. Chevalier and de la Noue (1985), showed that cells of Scenedesmus immobilized in carrageenan beads are as ecient as free cells in taking up ammonium from secondary urban e‚uents. Vilchez and Vega (1991) established the optimal conditions for nitrite uptake by Chlamydomonas reihardtii cells immobilized in calcium alginate beads. In another publication (Vilchez and Vega 1995) they reported that a maximum nitrite uptake rate of 90 lmol/h was achieved, but a minimum nitrite concentration of 0.6 mM in the culture media was required. Garbisu et al. (1991) investigated the removal of nitrate from water by Phormidium laminosum entrapped within, or adsorbed on to, polyurethane foam. The main disadvantage of the systems which use photosynthetic microorganisms is that ecient illumination must be provided. This energy investment can be omitted by removing the nitrogen the other way, by nitri®cation±denitri®cation processes.

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3.3.2. Biodegradation of pollutants by combining oxidative and reductive processes Oxygen, owing to limitation of its uptake and di€usion, seldom penetrates more than a few hundred micrometres into polymer beads (dos Santos et al., 1996a). This creates two di€erent zones within the polymer beads: an aerobic outer zone and an anaerobic inner zone. When a mixture of aerobic and anaerobic bacteria are entrapped within polymer beads, the two di€erent zones within the beads will cause the dominance of anaerobic bacteria within the core of the beads and aerobic bacteria at the periphery of the beads. Since the anaerobic and the aerobic microorganisms are immobilized close to each other, di€erent pollutants can be degraded by a sequencing aerobic±anaerobic or anaerobic±aerobic degradation. 3.3.2.1. Degradation of xenobiotics by combining reductive and oxidative processes. Several xenobiotics can be degraded into harmless compounds only by combining reductive and oxidative processes. Beunink and Rehm (1988) established a synchronous anaerobic and aerobic degradation of DDT by co-immobilization of Alcaligenes sp. and Enterobacter cloacae entrapped within Caalginate. In another publication (Beunink and Rehm, 1990) they used the restriction of oxygen transfer in Caalginate beads for a coupled reductive and oxidative degradation of the xenobiotic 4-chloro-2-nitrophenol by using the same microorganisms. In these systems, the substrate for the anaerobic reduction process passes through the outer aerobic layer (it cannot be degraded by the aerobic microorganisms) and is reduced within the core of the bead, where anaerobic conditions exist. The reduced product di€uses out from the anaerobic core of the bead, and in the outer aerobic layer this reduced product is oxidized by the aerobic microorganisms immobilized at the periphery of the bead. Beunink and Rehm (1990) concluded that the diameter of the alginate beads is a main factor determining the properties of this mixed culture system. 3.3.2.2. Nitrogen removal by combining oxidative and reductive processes. The other main way of biological nitrogen removal from waste liquids, apart from its incorporation within microbial biomass, involves the removal of nitrogen by nitri®cation±denitri®cation processes. dos Santos et al. (1996b) investigated a system that uses the oxidative and reductive environments within the polymer beads to remove nitrogen via nitri®cation±denitri®cation processes. In this system, the nitri®er Nitrosomonas europaea and either of the denitri®ers Pseudomonas denitri®cans or Paracoccus denitri®cans were co-immobilized by entrapment within double layer polymer beads. The di€erent microorganisms were separated physically by entrapment within two di€erent

layers that constructed the beads. The inner core of the bead was made of alginate polymer in which either P. denitri®cans or P. denitri®cans were entrapped, while the outer layer of the beads consisted of j-carrageenan in which N. europaea was entrapped. According to dos Santos et al. (1996a), if both microorganisms are simply mixed and directly entrapped within a polymer bead, the fast-growing denitrifying cells will overgrow the slow-growing nitrifying cells once in the presence of an organic carbon source. Physical separation of the two microbial populations gives an advantage to the nitri®ers and avoids the possibility that these cells will be out-competed by the denitrifying cells. High nitrogen removal rates (up to 5.1 mmol Nmÿ3 polymer sÿ1 ) were achieved in continuous ¯ow under aerobic conditions. Chemical analyses showed that ammonia was primarily converted into molecular nitrogen. No nitrous oxide was formed. The intermediary product, nitrite, was not detected, so it was concluded that the nitrite formed by the oxidation of ammonia was immediately decomposed via dissimilatory reduction by the denitrifying microorganisms. In conventional nitri®cation/denitri®cation systems, nitrite is normally ®rst converted into nitrate at the expense of oxygen. The nitrate is then reduced again to nitrite and further reduced until molecular nitrogen is formed. dos Santos et al. (1996a) stated that these two steps (the oxidation of nitrite to nitrate and then the reduction of nitrate back to nitrite) could be circumvented by the above-mentioned system. In addition, the acid produced in the nitri®cation step could partially be neutralized by the base resulting from the denitri®cation. To summarize, it was concluded that the ammonia removal rates obtained were higher than those reported for nitri®cation with entrapped microorganisms, and the ammonia removal rates in activated sludge processes, although somewhat lower than those for ¯uidized-bed and other attached growth systems. 3.4. Treatment of gases through microorganisms entrapped within polymer beads 3.4.1. Achievements in ammonia and hydrogen sulphide removal Huang et al. (1996) investigated the removal of hydrogen sulphide by autotrophic and heterotrophic microorganisms entrapped within Ca-alginate polymer beads, in a laboratory-scale experimental bio®lter. The autotrophic microorganisms employed in those bioreactors included Thiobacillus thioparus, Thiobacillus concretivorus, Thiobacillus denitri®cans and Thiobacillus intermedius. The heterotrophic microorganisms included Xanthomonas sp, Hyphomicrobium sp and Pseudomonas putida. The bio®lters maintained a high removal performance (>95%), when ¯ow rates were between 18 and

Y. Cohen / Bioresource Technology 77 (2001) 257±274

93 l/h. The autotrophic bio®lter showed a high anity for H2 S, but failed to reliably remove low concentrations of H2 S over a long-term period. However, the heterotrophic bio®lter exhibited the opposite tendency. Chung et al. (1996) showed a high H2 S removal eciency, greater than 98%, with T. thioparus entrapped within Ca-alginate beads. The pH drop was insigni®cant in this bio®lter during 3 months of operation. The bio®lter had excellent adaptability to upset conditions even at a 12-fold shock loading. The major metabolic products were identi®ed as elemental sulphur or sulphate. The activated beads exhibited excellent mechanical strength in the continuous experiments. The optimal inlet S-loading was noted to be 25 gmÿ3 hÿ1 . The authors report that this new technology bio®lter system reduces the required working volume and enhances H2 S removal eciency under low-concentration conditions compared to conventional gas bio®ltration. Chung et al. (1997) immobilized Arthrobacter oxydans by entrapment within Ca-alginate beads, in a packed column bio®lter treating ammonia. The bio®lter showed high eciency (>97%) in the removal of NH3 , at a ¯ow of 36 l/h with a pH control. A high maximum removal rate (1.22 g of N/day. kg bead) was achieved. The ability for removing NH3 at high inlet concentration and temperature suggests that this immobilized A. oxydans CH8 bio®lter has potential in processing NH3 gas. Chung et al. (1997) states that the great disadvantage of the conventional attached growth bio®ltration systems, which is the environmental risk due to bioaerosols (fungi and bacteria) which can be released from the bio®lter into the ambient air, can be overcome by the entrapment of microorganisms within polymers.

4. Comparison between systems ± attached growth and microbial entrapment within polymer beads 4.1. The advantages of attached growth systems Generally, it is much easier to provide only the bedding material and to let the microorganisms, which are present in the waste ¯uid, attach to this bedding material naturally than to produce a bedding material that consists of entrapped microorganisms within polymer beads. Usually the cost of producing the polymer beads is higher than many of the bedding materials used for attached growth. Besides the higher cost of producing polymer beads, several of the synthetic polymers used su€er from toxic e€ects toward the entrapped microorganisms. These synthetic polymers are considered as resistant polymers that can remain stable under the harsh conditions of several waste ¯uids and seem to be the most suitable polymers for wastewater treatment.

271

Another advantage of the attached microorganisms is the relatively low di€usion restriction that the bio®lm possesses compared to several polymer materials. The di€usion restriction is considered to be the main problem of the entrapment technology. The treatment of a ¯uid containing many di€erent contaminants is usually more e€ective by the great variety of microorganisms present within attached bio®lms in comparison to the single or few microbial species which are entrapped within polymer beads. The changing population within attached bio®lms can, furthermore, allow those treatment systems to adapt to changes in the ¯uid content. The entrapped microbial population cannot change due to changes in the ¯uid content, so those systems cannot treat waste sources, which change with time. 4.2. The advantages of systems based upon microorganisms entrapped within polymer beads The main advantage of the entrapment technology is the control of the dominant microbial species within the bio®lter. In attached bio®lms there is almost no control of the dominant species of bacteria within the bio®lter, while in the entrapment technology a desired microbial species can be maintained within the bio®lter with no competition with other microorganisms present in the waste ¯uid. The potential to immobilize di€erent species of microorganisms together but physically separated (so no competition between the di€erent microorganisms will occur) can allow the bio®lter to accomplish complex degradation procedures which involve several di€erent sequencing steps. Both reductive and oxidative processes can be carried out in an aerobic treatment reactor, due to the oxygen gradient within the polymer beads. The high plasmid stability within microorganisms which are entrapped in polymer beads allows for the possibility of using genetically engineered microorganisms. The experience of introducing genetically engineered microorganisms in open environmental systems, such as attached growth bio®lters, has produced poor results. The engineered microorganisms usually lose out in the competition with the native microbial population. The reduced production of sludge by the entrapped microorganisms and the high biomass concentration within the polymer beads can produce compact and ef®cient treatment systems in comparison to di€erent bio®lm based treatment systems. The bioaerosols released from gas-treating bio®lters, based upon attached bio®lms, are considered to be an environmental risk due to the pathogenic e€ect of several bioaerosols on humans. The treatment of gases by entrapped microorganisms reduces the amount of bioaerosols released from such bio®lters.

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The nature of the polymer bead bedding material makes it easy to store and shift. The change of a portion of the bedding material that contains, for instance, old and less e€ective microorganisms is possible with this technology. The di€erences between attached growth systems and systems based upon entrapment technology in some aspects of the ¯uid treatment are presented in Table 4. 5. Conclusions The comparison between attached growth systems and systems based upon entrapment technology shows that each of the treatment systems is considered to be most e€ective at di€erent ¯uid characteristics. For instance, attached growth is the optimal immobilization process for bio®lters which treat a ¯uid containing many di€erent contaminants and/or a ¯uid which changes its content with time. The entrapment technology, on the other hand, can be the optimal immobilization process for bio®lters which treat a ¯uid containing few contaminants, which need to be degraded by a complex sequencing degradation process. Neither of the immobilization processes can be considered as superior to or replace the other immobilization process.

The incorporation of microorganisms, entrapped within polymer beads, into commercial bio®ltration systems is in its very preliminary stages. Despite the problems related to this immobilization method, such as di€usion restrictions and the toxicity of some polymer production processes, this technology seems to have many advantages. These advantages include the use of a selected microbial species (this selected microbial species might even be genetically engineered), the potential to carry out complex degradation processes, the ability to carry out oxidative±reductive processes, etc. It seems that with the improvement of the polymer beads, di€usional properties and the development of mild entrapment procedures, this technology has a future potential in commercial bio®ltration systems. It is very complicated to interfere and change the microbial surface characteristics in attached growth systems. In these open environmental systems the use of genetically engineered microorganisms usually ends in poor results. In attempts to enhance and improve the attachment of microorganisms in such systems, the most possible option will be to alter properties of the bedding material. Understanding the forces that are responsible for microbial attachment to the bedding material will help in the selection of a suitable bedding material for a particular application.

Table 4 Some di€erences between attached growth systems and entrapment technology for ¯uid treatment Principal aspects

Immobilization process Attached microorganisms

Entrapped microorganisms

Production of bedding material

Simple, low cost

Adaptation to changes in the ¯uid content Di€usion restriction

The microbial population can adapt to changes in the ¯uid content Lower di€usion restriction

The eciency of the treatment of a ¯uid that contain many di€erent contaminants

The great variety of microorganisms can e€ectively treat a ¯uid that contains many di€erent contaminants Limited control Not possible

Complex, high cost, some resistant synthetic polymers su€er from toxicity problems The microbial population cannot adapt to changes in the ¯uids content Several polymer materials su€er from relatively high di€usional restrictions The small number of microbial species cannot e€ectively treat a ¯uid that contains many di€erent contaminants The desired dominant microbial species within a bio®lter can be controlled Possible

Not possible

Possible

Low

High

Relatively high

Low

Higher amount of sludge produced Lower resistance

Lower amount of sludge produced Higher resistance

Very complex

Simple

Depends on the di€erent system design ± generally, very complex

Simple

The control of the dominant species of microorganisms within the bio®lter The possibility of carrying out a sequencing degradation process The possibility of carrying out an oxidative±reductive process in an aerobic ¯uid environment Plasmid stability within the microorganisms Bioaerosols production from gas treating bio®lter Sludge production Resistance to high concentrations of toxic compounds within the treated ¯uid The possibility of storing bedding material that contains microorganisms The possibility of changing a portion of the microorganisms within the bio®lter

Y. Cohen / Bioresource Technology 77 (2001) 257±274

The treatment of ¯uids by bio®ltration systems, based upon attached growth and entrapped microorganisms, seems to be, in some environmental conditions, more ecient than the treatment of ¯uids by suspended microorganisms. This allows the bio®ltration treatment systems to be more compact than the treatment systems based upon suspended microorganisms. However, the existing bio®ltration systems su€er from many di€erent problems, such as uncontrolled microbial growth, aeration problems, etc. The improvement of the existing bio®ltration systems is still a necessity. A good bio®lter will achieve a high eciency of pollutant removal by providing the best conditions for the microorganisms it contains. Understanding the microbial needs, the ¯uid physics, mass transfer calculations, degradation pathways and ways of microbial immobilization will help to utilize the enormous power of the microorganisms in the control of ¯uid pollution. Acknowledgements With deep appreciation I want to thank Dr. Holger Kirchmann, Dr. Anna M artensson and Dr. Michael Robinson for their continued support and encouragement when writing this review, for spending their time reading the manuscript and for giving useful advice. References Beunink, J., Rehm, H.-J., 1988. Synchronous anaerobic and aerobic degradation of DDT by an immobilized mixed culture system. Appl. Microbiol. Biotechnol. 29, 72±80. Beunink, J., Rehm, H.-J., 1990. Coupled reductive and oxidative degradation of 4-chloro-2-nitrophenol by a co-immobilized mixed culture system. Appl. Microbiol. Biotechnol. 34, 108±115. Bickersta€, G.F., 1997. Immobilization of enzymes and cells. In: Bickersta€, G.F. (Ed.), Immobilization of Enzymes and Cells. Humana Press, Clifton, UK. Bishop, P.L., Zhang, T.C., Fu, Y.-C., 1995. E€ects of bio®lm structure, microbial distribution and mass transport on biodegradation processes. Water Sci. Technol. 31 (1), 143±152. Bryers, J.D., Huang, C.-T., 1995. Recombinant plasmid retention and expression in bacterial bio®lm cultures. Water Sci. Technol. 31, 105. Cassidy, M.B., Lee, H., Trevors, J.T., 1996. Environmental application of immobilized cells: a review. J. Ind. Microbiol. 16, 79±100. Chevalier, P., de la Noue, J., 1985. Wastewater nutrient removal with micro-algae immobilized in carrageenan. Enzyme Microb. Technol. 7, 395±400. Chung, Y.-C., Huang, C., Tseng, C.-P., 1996. Operation optimization of Thiobacillus thioparus CH11 bio®lter for hydrogen sul®de removal. J. Biotechnol. 52, 31±38. Chung, Y.-C., Huang, C., Tseng, C.-P., 1997. Biotreatment of ammonia from air by an immobilized Arthrobacter oxydans CH8 bio®lter. Biotechnol. Prog. 13, 794±798. Cochet, N., Lebeault, J.M., Vijayalakshmi, A., 1990. Physicochemical aspects of cell adsorption. In: Tyagi, R.D, Vembo, K. (Eds.), Wastewater Treatment by Immobilized Cells.

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