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"opened" compared to the B-DNA duplex and might be favourable for the binding of specific proteins, homologous sequences of nucleic acids and for the catalytic action of enzymes on DNA. Alternatively, syn and anti folding may be involved in the introduction of positive and negative supercoils into relaxed DNA and may represent a transient stage in chromosome con- densation.

Acknowledgement The critical reading of the manuscript by Dr Tony Maxwell, the typing by Mrs Ulla Gervind Richards and the drawing of figures by Mrs Zsuzsa Nigovicz-Pal are gratefully acknowledged.

References I Fieldhouse, J (1981) Biochem Educ 9, 88 2Banfalvi, G (1984) Biochem Educ 12, 155-156 3Banfalvi, G (1986) Biochem Educ 14, 50-59 4johnson, D and Morgan, A R (1978) Proc Natl Acad Sci USA 75,

1637-1641 5Stent, G (1958) Adv Virus Res 5, 95-149

~Zubay, G (1958) Nature 182, 1290-1292 7McGavin, S, Wilson, H R and Barr, G C (1966) J Mol Biol 22,

187-191 SK6hnlein, W and Hutchinson, F (1976) Mol Gen Genet 144, 323-331 ')Evdokimov, Y M, Akimenko, N M, Kadikov, V A, Vengerov, Y Y,

Pyatigorskaya, L, Platonov, A L and Varshavskii, Y M (1976) Mol Biol (Moscow) 10,657

l°Arnott, S, Chandrasekaran, R and Martilla, C (1974) Biochem J 141, 537-543

11Zimmerman, S B, Cohen, G H and Davis, D R (1975) J Mol Bio192, 181-192

J2Scovell, W M and Reaoch, R S (1979) JAmer Chem Soc 101, 174

13McGavin, S (1977) Heredity 39, 15-25 t4Cavalieri, L F, Fiaston, R and Rosenberg, B H (1961) Nature 189,

833-834 15Hall, C E and Cavalieri, L F (1961) J Biophys Biochem 10, 347

16McGavin, S (1971)J Mol Biol 55,293-298 17Holliday, R (1964) Genet Res 5,282-304 18Kikuchi, Y and Nash, H A (1979) Proc Natl Acad Sci USA 76~

3760-3764 1~ West, S C, Cassuto, E and Howars-Flanders, P (1981) Proc Natl Acad

Sci USA 78, 2100-2104 Z°Cavalieri, L F and Rosenberg, B H (1961) Biophys J 1,337-347 21Lim, V I and Mazarov, A L (1978) FEBS Lett 88, 118-123 eZHopkins, R C (1984) Comments Mol Cell Biophys 2, 153-178 23Hopkins, R C (1986) J Theor Biol 120, 215-222

Journals and Books Available A Canadian scientist is willing to give away the following: Canadian Journal of Microbiology (Vol 1 to Vol 33, 1987); Applied and Environmental Microbiology (Vol 45, 1983 to Vol 52, 1986); Microbiological Reviews (Vol 29, 1965 to Vol 50, 1986); as well as about fifty books mostly on microbiological topics including several volumes of Annual Reviews of Micro- biology (between 1973 and 1981).

Any library, department of biochemistry or microbiology or individual interested and prepared to pay the costs of crating and shipping to destination, of part or all of these, should write to Professor F Vella, Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OW0

Anaerobic Digestion: A Case Study

J J BERMIODEZ, M CANOVAS, A HOWELL* and J L IBORRA**

Department of Biochemistry University of Murcia, Spain and * School of Chemical Engineering University of Bath, UK

MANJON, J A

Introduction Anaerobic digestion, ie the degradation of organic matter to methane by microbiological action in the absence of molecular oxygen, occurs in natural environments such as freshwater and marine sediments, the ruminal cavities of animals, and others. It is responsible for such phenomena as 'Will o' the wisp', but its importance extends to different fields, ranging from Ecology to Chemical Engin- eering, with special emphasis on Microbiology and Bio- chemistry.

It represents a typical subject where interdisciplinary approaches are not only desirable but necessary, as well as an interesting case of scientific and technical application of natural processes. Because of these and other reasons, anaerobic digestion has attracted considerable interest in recent years, both theoretical and practical. Several excellent reviews 1-3 are available.

In anaerobic digestion, several groups of bacteria, each including different species, develop a wide range of metabolic activities with the final result of degrading organic matter, lipids, proteins and carbohydrates, to methane and carbon dioxide as the main components of a gaseous mixture, biogas (Fig 1). Among the different bacterial groups, the terminal one, the methanogens,

Hydrotysis

Fermentation

ParticuLate organic material ]

I -~ Carbohydrates [ L ~ d T 1

lamina acids, sugars I I Fatty acids I

Anaerobic oxidation

Intermediary products

Propionate, butyrate.

J Acetate J~ ' ---[ Hydrogen I

Acetotroph ~ H y d r o g e n o t r o p h

Figure 1 Proposed scheme for the anaerobic digestion of domestic wastes. Adapted from Kaspar and Wuhrmann 5

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belong to the Archaebacteria, and show unique physio- logical and metabolic properties. 5 They reduce one- carbon compounds and acetic acid to CH4 using hydrogen produced by other populations: acidogenic and acetogenic bacteria. Therefore, H2-concentration balance (by means of so-called 'interspecies hydrogen transfer') is essential for the maintenance of the system. Some biochemical aspects of methanogenesis are still under investigation: ATP synthesis coupled to CH4 production, cell carbon assimilation, and the mechanism of the reduction reaction (methylreductase system) are the most important.

Methanogens are strict anaerobes and have narrow pH tolerances, eg pH 6.5-8.0, lower values are inhibitory and higher values may be toxic. Many inhibitory effects have been described: metals, H2S, NH3, long-chain fatty acids, phenolic substances and others may inhibit methano- genesis at certain concentrations. Nevertheless, the effect of volatile fatty acids (acetic, propionic and butyric acids) is the most important as they are intermediates in the process and their action may be both on pH, with acidification (souring) and on the bacteria, with toxicity of their unionized forms.

Immobilized Cell Systems From the foregoing it is evident that the industrial application of anaerobic digestion may be troublesome. Another important drawback is the slow growth rate of methanogenic bacteria, with doubling times ranging from several hours to days. For this reason, any system with cell suspension growth, in which the solids retention time (SRT) equals the hydraulic retention time (HRT) is unable to treat the high volume-low strength wastes characteristic of anaerobic digestion process. We can define the HRT as the ratio between the total volume of the reactor and the flow of liquid to be treated, and the SRT as the ratio between the weight of solids present in the system and the weight of solids leaving the system per unit time. Due to the slow growth rates usually associated with methanogenic bacteria, the small flow rates needed to keep HRT less or equal than SRT makes suspension growth systems uneconomic. 6

Fortunately, methanogenic bacteria show the property of adhering to insoluble support materials to produce biofilms or of sticking together to form aggregates. This property has been utilized in high-rate or second gener- ation digesters. Although the mechanisms of adhesion and support colonization are not well understood, 7 they lead to structures (biofilms, flocs, granules) with cellular stabilization and operational advantages.

Immobilization of biomolecules and cells is a bio- technological technique that has received considerable attention in recent years. Different methods are described by different authors, because no classification is absolute. In principle six immobilization methods can be distin- guished: 8 covalent coupling, including cross-linking, adsorption, affinity (biospecific) immobilization, entrap- ment in a three-dimensional polymer network, confine- ment in a liquid-liquid emulsion, and capture behind

semipermeable membranes. In the anaerobic digestion field, adsorption is the most important method, but the existence of ionic bonds between extracellular polymers and charged support surfaces cannot be neglected. Although there are references to other methods, the simplicity and inexpensiveness of adsorption is of fundamental importance for a large-scale process such as waste water treatment. Furthermore, adsorption is a reversible method, and this means that in principle, the support can be recovered and reused.

Many types of digesters, some adapted from classical chemical reactors and others specially designed for anaerobic digestion, have been used in different scales. Considering only high-rate digesters (those allowing SRTs higher than HRTs) and the main configurations are:

- - U p f l o w Anaerobic Sludge Blanket (UASB): developed by Lettinga et al. 9 Based on the ability of cells to form bacterial aggregates without need of support material.

- - Anaerobic Contact Process. A high biomass con- centration is kept by separating the cells from the effluent stream and carrying them back to the digester.

- -S ta t i c systems. Usually known as anaerobic filters. 1° These systems promote the adhesion of cells to the surface of large particles as rocks or stones.

--Expanded/Fluidized Beds. The distinction be- tween the two systems is not always clear, depend- ing on the existence of relative movement between the support particles.

- -Two-phase Digesters. Operated with physical separation between acidogenesis and methano- genesis. 11

Using the principles associated with anaerobic filters, Kennedy and van den Berg 12 developed the downflow stationary fixed film concept (DSFF). This includes recirculation of the reactor liquid from the top to the bottom in order to control the biofilm width by means of the shear forces, and allows the bacterial cells to attach themselves to the surface of a support. Needle-punched polyester has shown to be an adequate support material because of its roughness, weight and porous structure. The authors have used this type of reactor configuration and support material for several long-term experiments conducted with synthetic feed mediums (volatile fatty acids as carbon and energy sources) and have applied the system to the treatment of cheese whey, a waste material whose large scale production and polluting potential makes it interesting to stabilize with associated production of energy (methane). Detailed descriptions of the results obtained are presented elsewhere.13-15

The start-up period of any anaerobic digester being the most unstable situation in the process, a great number of contributions has been made dealing with its reduction. Several theoretical aspects are of interest in this respect. The quality of the inoculum in terms of bacterial cells concentration and its distribution according to the variety

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of groups involved in the process, is essential for a short and stable start-up. The presence of inhibitory or toxic substances will result in poor reactor performance. The promotion of bacterial adhesion in order to increase its concentration in the reactor is also of key importance. Other factors such as optimum temperature and pH conditions, appropriate HRTs and organic loadings should be provided for the achievement of a successful operation. But probably the most important feature of a stepped, loading start-up using synthetic feed mediums is the imposition of a stratified pattern for growth and adhesion, provided that, after an optimum methanogenic- acetogenic populations consortium has been established, the supply of hydrocarbons or other complex nutrients to the system results in a fast development of acidogenic bacteria without process upset.

In this respect successful runs have been accomplished with the above mentioned systems.14 Figures 2 and 3 show scanning electron micrographs of both uncolonized and colonized supports. It can be seen that the bacterial population is surrounded by an organic matrix that keeps it adherent to the support.

Figure 2 Electron micrograph of the uncolonized support (x 1000)

Figure 3 Electron micrograph of biofilm (x 2200)

Conclusion Anaerobic digestion is a natural process with an important global impact on mineralization of organic matter, 2 and involves some very ancient bacteria whose unique features make them most interesting for the microbiologist as well as for the biochemist. Its application as a way of stabilizing

organic pollutants began in the last century but only the energy crisis of the 1970s and increasing ecological concern made it an attractive process for full-scale waste treatment and energy production.

On resolving the drawbacks associated with the com- plex nature of the process, Biotechnology has adopted some of the natural strategies: immobilization and juxta- position of the species. Immobilization of bacteria is not only a procedure to increase the active biomass inside the digesters and, proportionally, its degradative capability, but also provides a mechanism of natural protection, particularly for the sensitive methanogens, against physico-chemical stresses.

Experiments developed in order to optimize the startup period of a fixed-film reactor (the most troublesome stage in reactors operation) demonstrate that the procedure promoted a stratified ecology in the biofilm, with an inner methanogenic layer and an outer acidogenic layer. These results have been confirmed by other authors 16 and are being utilized in the elaboration of mathematical models for biofilm development. We hope that interdisciplinary approaches will help the elucidation of this process which, from our point of view, is an example of the importance of an understanding of nature for the solution of everyday problems.

Acknowledgements J J Bermfidez is fellow of the 'Consejerfa de Industria, Comercio y Turismo', Comunidad Aut6noma de la Regi6n de Murcia, Murcia, Spain. This work has been partially supported by a grant No. 24/82 of the Governments of Spain and the United Kingdom (Acciones Integradas Hispano-Britfinicas).

References 1Hawkes, F R (1984) in 'Biomethane, Production and Uses'. Turret- Wheatland Ltd, London. pp 41-60

2Schoberth, S M (1984) in 'Biomethane, Production and Uses', Turret- Wheatland Ltd, London, pp 61-78

3Harper, S R and Pohland, F G (1986) Biotechnol Bioeng 28,585-602 4Wolfe, R S (1979) Antonie van Leeuwenhoek 45, 353-364

5Kaspar, H F and Wuhrmann, K (1978) Appl Env Microbiol 36. 1-7

6Hall, E R and Melcer. H (1984) Water and Pollution Control, March/April, 1984

7Costerton, J W, Geesey. G G and Cheng, K J (1978) Sci Amer 238, 86-95

SMattiasson, B (1983) qmmobilized Cells and Organelles'. chapter 2, CRC Press, Boca Raton, Florida

9Hulsholf Pol, L M, Dolfing, J, de Zeeuw, W and Lettinga, G (1982) Biotech Lett 4, 392-396

l°Young, J C and McCarty, P L (1969) J Water Pollut Control Fed 41, 160

~Cohen, A, Breure, A M, Van Andel. J G and Van Deursen. A (1982) Water Res 16, 449-455

12Kennedy, K J and van den Berg, L (1984) in 'Comprehensive Biotechnology' (Editor Moo-Young, M) 4, 180-201

13Cfinovas-Dfaz, M and Howell, J A (1986) Biotech Lett 8, 379-384

14Cfinovas-Diaz, M and Howell, J A (1987) Biotechnol Bioeng. 30. 289-296

15Cfinovas-Diaz, M (1985) PhD Thesis, University of Wales

16Oackley, D L, Wase. D A J and Forster, C F (1985) Presentation at "Multi-Stream '85', Subject Groups Symposium. Institution of Chemical Engineers, The City University, London, April 1985

BIOCHEMICAL EDUCATION 16(2) 1988