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mulation of plasmid DNA. Proceedings of Ninth European Congress of vaccination. Nature 400, 269–271 Chitin deacetylases: new, versatile tools in
biotechnology
Iason Tsigos, Aggeliki Martinou, Dimitris Kafetzopoulos and Vassilis Bouriotis Chitin deacetylases have been identified in several fungi and insects. They catalyse the hydrolysis of N-acetamido bonds of chitin, converting it to chitosan. Chitosans, which are produced by a harsh thermochemical procedure, have several applications in areas such as biomedicine, food ingredients, cosmetics and pharmaceuticals. The use of chitin deacetylases for the conversion of chitin to chitosan, in contrast to the presently used chemical procedure, offers the possibility of a controlled, non-degradable process, resulting in the production of novel, well-defined chitosan oligomers and polymers.
Chitin, a homopolymer comprising ␤-(1-4)-linked various fractions of acetylated units. It is biodegradable,
N-acetyl-D-glucosamine residues is one of the non-toxic to animals (in mice, the LD was Ͼ16 g kgϪ1), most abundant, easily obtained and renewable soluble in acidic solutions, available in various physical natural polymers, second only to cellulose. It is com- forms and much more tractable than chitin2,3. Thus, monly found in the exoskeletons or cuticles of many chitosan offers properties with great potential for many invertebrates and in the cell walls of most fungi1.
Because of its high crystallinity, chitin is insoluble in Today, several companies are producing chitin and aqueous solutions and organic solvents2.
chitosan products on a commercial scale; the majority Chitosan is a polycationic biopolymer that occurs are located in Japan, where Ͼ100 billion tons of naturally or is obtained by the N-deacetylation of chitosan are manufactured each year from the shells of chitin; its name does not refer to a uniquely defined crabs and shrimps, an amount that accounts for ~90% compound but rather to a family of copolymers with of the global chitosan market (approximately four tril-lion yen). The major areas of application include watertreatment, biomedical applications (including wound I. Tsigos, A. Martinou and D. Kafetzopoulos are at the Institute of dressing and artificial skin) and personal-care products2–13.
Molecular Biology and Biotechnology, Foundation of Research and In addition, oligomers of chitin and chitosan have Technology, Crete, Greece. V. Bouriotis (bouriotis@imbb.forth.gr) isat the Division of Applied Biology and Biotechnology, Department of also attracted considerable attention because they have Biology, University of Crete, Crete, Greece. been reported to exhibit certain interesting physiological 0167-7799/00/$ – see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01462-1 The catalytic action of chitin deacetylases; chitin is deacetylated by the enzyme chitin deacetylase to form chitosan and acetate.
activities, such as antitumour and antimicrobial activ- Chitin deacetylases
ity4,11 and elicitor activity for plants4. Furthermore, Chitin deacetylases have been purified and character- chitin and chitosan oligomers are soluble in aqueous ized from several fungi. The most well-studied enzymes solutions, and can be easily characterized by a variety are those from the fungi Mucor rouxii18–20, Absidia coerulea21, of analytical procedures (e.g. nuclear magnetic resonance Aspergillus nidulans22 and two strains of Colletotrichum spectroscopy14 and mass spectroscopy15).
lindemuthianum23,24. The most important characteristics Presently, chitosan is produced from chitin via a harsh of the enzymes are summarized in Table 1. All the thermochemical procedure. This process shares most of enzymes are glycoproteins and are secreted either into the disadvantages of a severe chemical procedure; it is the periplasmic region or into the culture medium.
environmentally unsafe and not easily controlled, lead- Furthermore, all enzymes exhibit a remarkable thermal ing to a broad and heterogeneous range of products16.
stability at their optimum temperature (50ЊC), and Similarly, chitosan oligomers are prepared by partial exhibit a very stringent specificity for water-soluble acid-hydrolysis of chitosan polymers. A chemo-enzym- ␤-(1,4)-linked N-acetyl-D-glucosamine polymers.
atic method using lysozyme and tri-N-acetylchitotriose Nevertheless, they vary considerably in their molecu- derivatives as substrates has also been reported for the lar weight and carbohydrate content, and display a wide preparation of specific chitosan oligomers17. However, range of pH optima. One interesting property with a the resulting products from both methods are mixtures potential biotechnological application for the enzymes of randomly deacetylated chitosan oligomers with from C. lindemuthianum and A. nidulans is that, apartvarious degrees of polymerization.
from their thermal stability, they are not inhibited by The use of chitin deacetylase for the preparation of acetate, a product of the deacetylation reaction22–24.
chitosan polymers and oligomers offers the possibility The respective genes of chitin deacetylases from the of the development of an enzymatic process that could fungi M. rouxii26, C. lindemuthianum27 and Saccharomyces potentially overcome most of these drawbacks. Chitin cerevisiae28,29 have been cloned, sequenced and charac- deacetylase (CDA; EC 3.5.1.41) catalyses the hydrolysis terized. Furthermore, the expression of chitin deacetyl- of N-acetamido bonds in chitin to produce chitosan ase genes from C. lindemuthianum and M. rouxii in (Fig. 1). The presence of this enzyme activity has been Escherichia coli30 and Pichia pastoris31 respectively, have reported in several fungi18–24 and insect species25. This article reviews the most important characteristics ofchitin deacetylases, and highlights some initial studies Biological role
exploiting their potential use in the deacetylation of Two different biological roles have been suggested chitinous substrates for the production of oligomer and for fungal CDAs, namely their involvement in cell- polymer products with novel characteristics.
wall formation and plant–pathogen interactions. The Table 1. Characteristics of chitin deacetylases from different sources
Mucor Absidia Aspergillus
Colletotrichum
Colletotrichum
coerulea
nidulans
lindemuthianum
lindemuthianum
(ATCC 56676)
(DSM 63144)
Abbreviations: Min. DP, minimum degree of polymerization of a chitin oligomer required for catalysis; NA, not available. involvement of CDA in cell-wall chitosan biosynthesiswas demonstrated for the first time during studies to Glossary
investigate chitin and chitosan biosynthesis in fungi. Inthe case of the fungus M. rouxii, it was revealed that Single chain The enzyme forms an active enzyme–poly-
chitin synthase operates in tandem with chitin deacety- mer complex and catalyses the reaction in a ‘zipper’ lase; chitin synthase synthesizes chitin by the polymeriz- fashion towards one end of the chitin chain; it does notform an active complex with another substrate until it ation of N-acetyl-D-glucosamine residues from uridine 5-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), Multiple chain or random type The enzyme forms an
and chitin deacetylase hydrolyses the N-acetamido active enzyme–polymer complex and catalyses the bonds in the chitin chains, acting more efficiently on hydrolysis of only one acetyl group before it dissociates nascent rather than on microfibrillar chitin32. Similar results were also obtained for the fungus Absidia coerulea, Multiple attack The enzyme forms an enzyme–polymer
where it was also found that CDA was localized near complex and further catalyses the hydrolysis of several the inner face of the cell wall (periplasmic region)21.
acetyl groups before it dissociates and forms a new This spatial arrangement of CDA is in agreement with active complex with another polymer chain previous studies, which reported the presence of chitin Degree of multiple attack The maximum number of
acetyl groups that can be hydrolysed by the enzyme
synthase activity and chitin biosynthesis in the plasma membrane fraction of fungi33. A similar biological rolehas been reported for the two CDAs (Cda1p andCda2p) from S. cerevisiae. It was shown that these high-pressure liquid chromatography (HPLC) and the enzymes are required for correct ascospore wall for- results were further verified using 1H-NMR spec- mation28. Furthermore, it was found that Cda2p was troscopy. It was observed that the length of the oligomer the most active enzyme and performed most of the was important for enzyme action (Fig. 2). The enzyme deacetylation tasks in the ascospore cell wall29.
could not effectively deacetylate chitin oligomers with An alternative biological role, namely the involve- a degree of polymerization lower than three. Tetra-N- ment of the enzyme in plant–pathogen interactions, has acetylchitotetraose and penta-N-acetylchitopentaose been suggested for the CDA from Colletotrichum linde- were fully deacetylated by the enzyme, however, in muthianum, considering that the fungus is a plant tri-N-acetylchitotriose, hexa-N-acetylchitohexaose and pathogen, and the enzyme is extracellular and active hepta-N-acetylchitoheptaose, the reducing end-residue on chitin oligomers. Taking into account that chitin remained intact. Furthermore, the enzyme initially oligomers (tetramer to hexamer) elicit plant-defence removed an acetyl group from the non-reducing end- mechanisms (callose formation, lignification and residue of all chitin oligomers with a degree of poly-synthesis of coumarin derivatives)34,35, whereas their merization greater than two, and further catalysed the deacetylated forms do not36,37, it has been proposed that hydrolysis of the next acetamido groups in a progres- CDA might deacetylate chitin oligomers that arise from sive fashion. This mechanism resembles the mode of the fungus cell wall subsequent to the activity of plant action that the enzyme exhibits on polymeric substrates39.
chitinases, and thereby diminish their elicitor activity23.
Chitin oligosaccharides (DP2–4) have also been used Finally, a role for CDA during the penetration pro- as model substrates to study the mode of action of CDA cess of the fungus hypha (which contain a considerable from C. lindemuthianum41. Using HPLC and fast-atom- amount of chitin) in plant tissues has been proposed.
bombardment mass spectrometry (FAB-MS), it was Because decreasing levels of acetylation result in less- found that in a similar manner to the M. rouxii enzyme, efficient hydrolysis of chitin by plant endochitinases, it CDA from C. lindemuthianum could fully deacetylate was suggested that the penetrated hypha might be tetra-N-acetylchitotetraose. However, it could alsoprotected by enzymatic deacetylation38.
fully deacetylate tri-N-acetylchitotriose and the non-reducing end-residue of di-N-acetylchitobiose. Fur- Mode of action
thermore, it was shown that under specific conditions The mode of action of CDA has been studied in both (in the presence of 3 M sodium acetate), this enzyme chitosan polymers39 and chitin oligomers40,41. The mode could also catalyse the reverse deacetylation reaction42.
of action of CDA from M. rouxii has been investigated Using FAB-MS and NMR it was demonstrated that on an ~32% randomly deacetylated water-soluble chito- CDA from C. lindemuthianum could acetylate chito- san substrate, with an average degree of polymerization biose (GlcNGlcN) and convert it into 2-acetamido-2- of 30. Using 1H- and 13C-NMR spectroscopy, it was deoxy-D-glucopyranosyl-(1-4)-2-amino-2-deoxy-D- found that the enzyme hydrolysed the acetyl groups of the substrate according to a multiple-attack mechanism In summary, enzymatic deacetylation of both chitin (see Glossary) with a degree of multiple attack of three.
oligomers and chitosan polymers is a well-defined reac- This is the maximum number of successive deacetyl- tion, in contrast to chemical deacetylation, in which ations that could be achieved by the enzyme because the hydrolysis of the N-acetamido bonds of N-acetyl- the maximum number of the consecutive N-acetyl-D- D-glucosamine residues is performed in a random fash- glucosamine residues that were found in this substrate ion. Therefore, enzymatic deacetylation involving chitin deacetylases offers a possibility for the preparation of The mode of action of CDA from M. rouxii on chitin specific novel chitosan oligomers and polymers.
oligosaccharides (DP 2–7) has also been studied40. Thesequence of chitin oligomers following enzymatic Genes
deacetylation was identified by the alternative use The CDA genes from M. rouxii, C. lindemuthianum of two specific exoglycosidases in conjunction with and S. cerevisiae have been cloned and characterized26–28.
Mode of action of chitin deacetylase from Mucor rouxii on chitin oligomers. Blue spheres correspond to N -acetyl-D-glucosamine residues;green spheres to their deacetylated counterparts (D-glucosamine). Numbered arrows indicate the order and the site of deacetylation. Theenzyme initially deacetylates the non-reducing end-residue of the oligomers, and further catalyses the hydrolysis of the next acetamidogroups in a progressive fashion. Tetra-N-acetylchitotetraose and penta-N-acetylchitopentaose are fully deacetylated; in tri-N-acetylchitotriose,hexa-N -acetylchitohexaose and hepta-N-acetylchitoheptaose, the reducing end-residue remains intact.
The enzymes are highly homologous and, furthermore, peptidoglycan deacetylases, although this function has there is a universal conserved region that exhibits a significant similarity to the rhizobial nodulation pro- Thus, the similarities between the CDAs highlights teins43,44 (NodB proteins), certain regions in microbial the catalytic domain in the CDA sequences and can acetyl xylan esterases and xylanases45,46, and several direct the design of an enzyme with improved efficiency uncharacterized open reading frames (ORFs) in Bacillus in the deacetylation of chitinous substrates.
sp. (Fig. 3). This conserved region has been assigned asthe ‘nodB homology domain’ because of its similarity Applications of chitosan
to NodB proteins. Apart from this region, no other The following major characteristics of chitosan make homologies were found between chitin deacetylases this polymer advantageous for numerous applications: (1) it has a defined chemical structure; (2) it can be NodB proteins are chitooligosaccharide deacetylases chemically and enzymatically modified; (3) it is physi- that are essential for the biosynthesis of bacterial nodu- cally and biologically functional; (4) it is biodegradable lation signals, termed Nod factors43. Nod factors from and biocompatible with many organs, tissues and cells; different rhizobial species share a common basic struc- and (5) it can be processed into several products includ- ture: they are all N-acetylglucosamine oligomers, with ing flakes, fine powders, beads, membranes, sponges, an N-acyl substitution at the non-reducing end-residue.
cottons, fibres and gels2–4. Furthermore, its main source This N-acyl substitution is required for the biological (crab and shrimp shells) is produced as a byproduct of activity of all Nod factors because N-acetylglucosamine the seafood industry. Consequently, chitosan has found oligomers fail to elicit any nodulation-specific responses considerable application in various industrial areas in host plants. NodB proteins specifically remove the N-acetyl group from the non-reducing terminal residue of As a result of its high molecular weight, cationic char- N-acetylglucosamine oligomers, thus providing the nec- acter and gel-forming ability, chitosan has been exten- essary free amino group for the subsequent N-acylation44.
sively used in industry, foremost as a flocculent in the The nodB homology domain present in Cellulomonas clarification of waste-water and the detoxification of fimi and Clostridium thermocellum xylanases was found to hazardous waste2,4,5. It was found that the adsorption be functional, exhibiting deacetylase activity against capacity of chitosan varies with the amino-group con- xylan, and thus contributing to the efficient hydrolysis tent, and that polymers with ~50% deacetylation were of acetylated xylan by xylanases45,46. Finally, the ORFs the most effective5. The United States Environmental from Bacillus sp., which also exhibit significant similar- Protection Agency has also approved the use of com- ity with CDAs have been suggested to correspond to mercially available chitosan in potable-water purification The NodB homology domain. Chitin deacetylases from Mucor rouxii, Saccharomyces cerevisiae and Colletotrichum lindemuthianum arealigned with NodB from Rhizobium melitoti, Xylanase D from Cellulomonas fimi, and acetyl xylan esterase A from Streptomyces lividans. Theblack segment indicates the NodB homology domain. Abbreviations: CDA, chitin deacetylase; NodB, nodulation protein B; XylD, xylanase D;AxeA, acetyl xylan esterase A; aa, amino acids.
systems4 up to a maximum level of 10 mg lϪ1. Fur- pharmaceutical product (TegasorbTM, a wound-healing thermore, chitosan has been used as a paper-coating product), several other important applications of chito- material, increasing the physical strength of cellulose san as an excipient in pharmaceutical products are cur- paper and improving the printing quality with anionic rently being investigated6. Interestingly, in all cases the degree of polymerization and N-acetylation of the Chitosan has also been used for the clarification of oligomers and polymers proved to be extremely impor- beverages (such as fruit juices and beers) and in the tant, because both these parameters influence not only agricultural sector2,4. Furthermore, chitosans (Ͼ70% their biochemical characteristics but also their bio- deacetylated) have recently been introduced to the compatibility and immunological activity3. For exam- nutritional-supplement market as a weight-loss aid and ple, it was shown that chitin oligomers (DP 4–7) dis- a cholesterol-lowering agent6,47. They are also used as played a strong immuno-enhancing effect, inhibiting a constituent of many food products, particularly in the growth of various tumour mice cells, whereas Japan2, and are presently approved as a food additive in chitosan oligomers (DP 2–6) did not exhibit such an effect4. Furthermore, high molecular weight chitosans Chitosan polymers and oligomers have recently with a high degree of deacetylation were more effec- attracted considerable attention in the pharmaceutical tive in inhibiting bacterial growth than chitosans with and biomedical fields because of their favourable char- a lower molecular weight and degree of deacetylation11.
acteristics, such as biocompatibility, biodegradability, Owing to its cationic character and the presence of antimicrobial action and non-toxicity to animals.
reactive functional groups, chitosan has also been used Although chitosan has only been used in one registered in the development of controlled-release technologies.
Table 2. Applications of chitosan polymers and oligomers
Application
Examples
Recovery of metal ions and pesticides, removal of phenols, proteins, radioisotopes, 2,4,5 PCBs and dyes, recovery of solid materials from food-processing wastes Seed- and fruit-coating, fertilizer and fungicide Clarification and de-acidification of fruits and beverages, colour stabilization, reduction of lipid adsorption, natural flavour extender, texture-controlling agent, food preservative and antioxidant, emulsifying, thickening and stabilizing agent, livestock and fish-feed additive, and preparation of dietary fibres Treating major burns, preparation of artificial skin, surgical sutures, contact lenses, 3,7–13 blood dialysis membranes and artificial blood vessels, as antitumour, blood anticoagulant, antigastritis, haemostatic, hypocholesterolaemic and anti-thrombogenic agents, in drug- and gene-delivery systems, and in dental therapy Immobilization of enzymes, as a matrix in affinity and gel permeation Synthetic fibres, chitosan-coated paper, manufacturing material for fibres, In such systems, the rate of drug administration is con- chitin, has been used as a substrate. The degrees of trolled while prolonging the duration of the therapeu- deacetylation obtained were 0.5% and 9.5%. These tic effects6,8. Chitosans used for these purposes have relatively low degrees of deacetylation indicate that the been fabricated in the form of gels, microspheres and enzyme is not very effective in deacetylating insoluble microcapsules, and it was found that the products were chitin substrates. Similar results were also obtained more stable and the rate of drug release was more sus- using chitin deacetylases isolated from different tained when chitosans with a high molecular weight sources21,23,24. Pretreatment of crystalline chitin sub- and degree of deacetylation were used6.
strates before enzyme addition seems to be necessary in In addition, chitosan polymers have a moisturizing order to improve the accessibility of the acetyl groups effect on the skin, offer protection from mechanical to the enzyme and therefore to enhance the yield and hair damage and exhibit an anti-electrostatic effect on the rate of the deacetylation reaction. Experiments have hair. Their moisturizing effect (which is proportional also been performed using chitin deacetylase from to their molecular weight and degree of deacetylation) M. rouxii and partially deacetylated water-soluble chi- is comparable to that of an aqueous 20% propylene- tosans as substrates39,52. It was shown that the enzyme glycol or hyaluronic acid solution. They also protect was effective in deacetylating polymers, with up to 97% the skin from microbial infections. Consequently, they deacetylation52, although this procedure has not yet have been used widely in the cosmetic industry for skin- been optimized. Moreover, because enzymatic and hair-care products2. Finally, chitosan oligomers and deacetylation is not a random process like chemical polymers, as well as their derivatives have been used deacetylation, new polymers with potentially different extensively as analytical reagents, for example, in various physical and chemical characteristics can be produced.
enzymatic reactions as substrates for chitinases, chito- The advantages of an enzymatic process are more sanases and lysozymes48,49. However, a broader use of evident in the deacetylation of chitin oligomers. These chitosan has been significantly restricted, primarily as a compounds, in contrast to their corresponding pol- result of the current methods of preparing these poly- ymers, are soluble in aqueous solution and are there- mers, which lead to products exhibiting non-uniform fore more accessible for enzyme action. Furthermore, enzymatically deacetylated oligomers, compared withchemically deacetylated oligomers, can easily be pro- Enzymatic vs chemical deacetylation
duced and have a different distribution of N-deacetyl- Presently, chitin and chitosan are produced from crab ated residues. A variety of well-defined chitosan oligomers and shrimp shells by a thermochemical procedure. can be produced from a single enzymatic deacetylationFirst, shells are deproteinized under alkaline conditions, step (Fig. 2). Another example indicating the specificity and are subsequently demineralized to remove CaCO of the enzymatic method and the facile preparation under acidic conditions to produce pure chitin. The of products is the selective N-deacetylation of p-nitro- N-deacetylation of chitin is either performed hetero- phenyl N,NЈ-diacetyl-␤-chitobioside [(GlcNAc) -pNP] geneously16 or homogeneously50. In the heterogeneous by CDA from Colletotrichum lindemuthianum48. The method, chitin is treated with a hot, concentrated solu- enzyme specifically deacetylates the non-reducing end tion of NaOH and chitosan is produced as an insolu- of the glycoside, converting it to p-nitrophenyl-2- ble precipitate. Chitosan prepared by this method is acetamido-4-O-(2-amino-2-deoxy-␤-D-glucopyranosyl) ~85–93% deacetylated. According to the homogeneous -2-deoxy-␤-D-glucopyranoside (GlcNGlcNAc-pNP).
method, chitin is also treated with NaOH, but under This disaccharide derivative can be used to further clas- milder conditions. This method results in a water- sify chitinases degrading [(GlcNAc) -pNP]. This reac- soluble chitosan with an average degree of deacetylation tion can also be performed chemically, but a number of synthetic and purification steps would be required.
However, both methods have three critical disadvan- Finally, the reverse deacetylation reaction catalysed tages: (1) they consume considerable amounts of energy; by CDA from C. lindemuthianum also has several inter- (2) they waste a large amount of concentrated alkaline esting applications. One example is in the synthesis solution, resulting in an increase in the level of envi- of a triacetylated chitosan tetramer [(GlcNAc) GlcN], ronmental pollution; and (3) they lead to products with which can be used to explore the physiological activ- a broad range of molecular weights and a hetero- ity of partially deacetylated chitin oligomers53. The geneous extent of deacetylation. However, a uniform enzymatic reaction exhibits a regioselectivity that is material with specific physical and chemical properties hard to achieve by chemical methods.
is required for many high-value biomedical appli- In conclusion, the development of a controllable cations. Furthermore, the degree and distribution of process using the enzymatic deacetylation of chitinous deacetylation has been found to influence the physical substrates presents an attractive alternative process that and chemical properties, as well as the biological activ- can, in principle, result in the preparation of novel ities of this polymer and subsequently the properties of the pharmaceutical or other industrial formulations thatare based on chitosan. In order to overcome these Conclusions
drawbacks in the preparation of chitosan, an alternative There is an increasing interest in chitin deacetylases enzymatic method exploiting chitin deacetylases has and as new primary structures become available, the application of molecular biological techniques are The effectiveness of chitin deacetylase for the prepa- anticipated to provide the tools to tailor and manipu- ration of chitosan has been tested using an enzyme iso- late chitin deacetylases, resulting in enzymes with novel lated from the fungus M. rouxii51. Chitin, both in its properties that can be used for the preparation of crystalline and its chemically modified form, amorphous chitosan polymers and oligomers. Future developments in both basic research and biotechnological applications 25 Rajulu, S. et al. (1986) Natural deacetylation of chitin to chitosan in
the abdominal cuticle of the physogastric queen of Macrotermetesestherae. In Chitin in Nature and Technology (Muzzarelli, R. et al., eds), Acknowledgments
The authors’ work was funded by the EPET II pro- 26 Kafetzopoulos, D. et al. (1993) The primary structure of a fungal
chitin deacetylase reveals the function for two bacterial gene prod- gram (97EKBAN2-1.2-104) of the European Union, ucts. Proc. Natl. Acad. Sci. U. S. A. 90, 8005–8008 supported by the Hellenic General Secretariat of Research 27 Tokuyasu, K. et al. (1999) Cloning and expression of chitin deacetyl-
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Particle-based biofilm reactor technology
Cristiano Nicolella, Mark C.M. van Loosdrecht and Sef J. Heijnen Particle-based biofilm reactors provide the potential to develop compact and high-rate processes. In these reactors, a large biomass content can be maintained (up to 30 g lϪ1), and the large specific surface area (up to 3000 m2 mϪ3) ensures that the conversions are not strongly limited by the biofilm liquid mass-transfer rate. Engineered design and control of particle- based biofilm reactors are established, and reliable correlations exist for the estimation of the design parameters. As a result, a new generation of high-load, efficient biofilm reactors are operating throughout the world with several full-scale applications for industrial and municipal waste-water treatment. Biofilm reactors are used in situations wherein the operated at high biomass concentrations to treat the
reactor capacity obtained by using freely sus- large volumes of dilute aqueous solutions that are typi- pended organisms is limited by the biomass cal of industrial and municipal waste-waters without concentration and hydraulic residence time. This can the need for separating the biomass and the treated be the case either for slow-growing organisms (e.g.
nitrifiers, methanogens), whose growth in suspension Although the use of biofilms overcomes limitations requires long residence times, or for diluted feed caused by a low reactor-biomass concentration, for streams (often present in waste-water treatment pro- high reactor capacities, a new bottleneck has to be con- cesses), in which only a very low biomass concentration sidered because the delivery of poorly soluble substrates can be achieved without biomass retention. In these (e.g. oxygen) to the biofilm surface might become lim- cases, biofilms are an effective solution to successfully iting. Systems with static biofilms (e.g. trickling filters) retain biomass in the reactors and to improve the vol- have small specific biofilm surface areas (typically less umetric conversion capacity. Biofilm reactors are not than 300 m2 biofilm mϪ3 reactor) available for substrate particularly useful when fast-growing organisms (i.e.
transport and reaction, and thus a limited reactor with a maximum specific growth rate Ͼ0.1 hϪ1) or capacity (the oxygen-transfer rate is typically less than concentrated feed streams are used1 (e.g. in industrial 3 kg mϪ3 dϪ1 for trickling filters). Therefore, static fermentation processes). In these situations, sufficient biofilm reactors can be useful if the biomass retention biomass will be formed to metabolize the substrate with and not the mass transfer is the main requirement, for relatively short residence times without the need for example, when large volumes of liquid with very low any form of retention; it is the oxygen supply to the substrate concentrations have to be treated (e.g. the liquid phase, not the biomass concentration, which is removal of xenobiotics from ground water). For more- often the limiting factor. For this reason, in the major- concentrated streams, the enlargement of the biofilm ity of industrial fermentation processes where high specific surface area can lead to a substantial reductionsubstrate concentrations are used, biofilm formation is in the reactor volume and the area requirements of the either unnecessary or even disadvantageous, and the process. A dramatic increase in biofilm surface area can range of applications of immobilized-cell systems in be obtained by growing biofilms as small particles. The industry is mainly limited to waste-water treatment choice of the optimal particle size is a compromise processes2,3. Biofilms are extensively used in environ- between the conversion rate and the particle sedi- mental biotechnology because biofilm reactors can be mentation rate. If the particles become too small (i.e. their settling velocity is too small), the process mightagain be limited by the biomass concentration that is C. Nicolella (c.nicolella@afnovell.reading.ac.uk) is at the Department achievable in the reactor, as for cell suspensions.
of Food Science and Technology, University of Reading, PO Box 226, Gravity separation can be enhanced by growing the Reading, UK RG6 6AP. M.C.M. van Loosdrecht (mark.vanloosdrecht@stm.tudelft.nl) and S.J. Heijnen are at the Kluyver Institute for Biotech- biomass in the form of dense spherical aggregates.
nology, Delft University of Technology, Julianalaan 67, 2628 BC These aggregates can either form spontaneously as large, dense granules4,5, or attached to suspended carriers6 0167-7799/00/$ – see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01461-X

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