368

bioremediation

Jeff Cole

Controlling environmental nitrogen through microbial

metabolism

The major sources of environmental nitrogen are the result of process

intensification by human activities such as the chemical synthesis of nitrogenous

fertilizers, internal-combustion engines, intensive farming and the use of xenobiotic

chemicals in industrial processes. Recent advances in the biochemistry, genetics

and ecology of nitrifying and denitrifying bacteria can now be exploited in the control

of environmental pollution from these sources. However, frequently, biotreatment

must be combined with physical and chemical processes to achieve satisfactory

remediation.

There are four major sources of environmental nitrogen: (1) chemical reactions resulting from natural phenomena such as the synthesis of nitrogen oxides by the action of lightning in the atmosphere; (2) bacterial N 2 fixation; (3) animal excrement and the biodegra- dation (or 'putrefaction') of dead plant and animal ma- terials; (4) human activities such as the chemical syn- thesis of nitrogenous fertilizers, industrial processes involving nitrogen compounds, the use of internal- combustion engines, which generate nitrogen oxides, intensive farming in enclosed spaces and the use of recalcitrant xenobiotic chemicals. The first three sources of environmental nitrogen are usually counter- balanced without the need for human intervention by microbial activity. The need - and hence the oppor- tunities - for biotechnological intervention arise, to a large extent, from process-intensification by human activities in the fourth category.

Microorganisms are essential for both the formation and the subsequent interconversions of nitrogen com- pounds in the environment. Nitrogen assimilated by plants in the form of ammonia and nitrate is eventu- ally released either as ammonium ions, or as organic nitrogen compounds which are readily metabolized to ammonia. The nitrogen excreted by mammals as urea is also readily converted to ammonia; microorganisms in soil and water are a prolific source ofurease which rapidly releases the nitrogen atoms of urea as am- monia. The nitrogen atoms of the majority of recal- citrant, xenobiotic chemicals can also be metabolized to ammonia at a slow rate, provided that their con- centrations are low enough to permit the active microbial species to survive. The major problem is to accelerate this metabolism sufficiently to cope with the

j . Cole is at the School of Biochemistry, University of Birmingham, Birmingham, UK B15 2TT.

rate at which the contaminant is delivered to the en- vironment.

Although ammonia is extremely toxic to mammals, it is rapidly oxidized in any well-aerated environment by nitrifying bacteria, which convert it first to nitrite, and ultimately to nitrate. In poorly aerated soils and sediments, nitrate is then reduced by denitrifying bac- teria to N2, intermediates in this process being nitrite, nitric oxide and nitrous oxide. Consequently, micro- bial metabolism of three molecular species: ammonia; nitrate; and N2, dominates the biological nitrogen cycle (Fig. 1). The processes of putrefaction, nitrifi- cation and denitrification have also provided the basis of by far the largest (if not the most glamorous) of biotechnologies, the safe disposal of sewage and the supply of potable water.

Nitrification: the oxidation o f a m m o n i a to nitrate The presence of ammonia in agricultural soils is a

valuable resource that the farmer seeks to conserve; its absence necessitates the application of expensive nitrogenous fertilizer. In contrast, ammonia in fresh water represents a problem because, as well as its offens- ive smell, it is a primary cause of eutrophication and is toxic to animals. In any well-aerated soil or water, nitrifying bacteria rapidly oxidize ammonia to nitrate, a process that is beneficial in waste-water treatment plants. Although nitrate is also a perfectly acceptable source of nitrogen for plant growth, it is less accept- able to the farmer than ammonia for at least two reasons. First, before the nitrogen can be assimilated into the pool of amino acids, plants must provide reducing equivalents (which otherwise would be used for CO 2 fixation) to reduce nitrate back to ammonia. Hence, biomass production under these conditions is less than when ammonia is used as the nitrogen source. Second, in contrast to ammonia, which is retained in

TIBTECH AUGUST 1993 (VOL 11) © 1993, Elsevier Science Publishers Ltd (UK)

369

bioremediation

the topsoil by binding to anionic soil particles, nitrate is readily leached by rainwater into the unproductive anaerobic layers of soil, from where it is either lost from the biosphere by denitrification, or transferred into the underground aquifers.

Most nitrification is catalysed by a specialized group ofchemolitbotrophic bacteria-bacteria that conserve, for growth, energy released from chemical oxi- dation-reduction reactions in which the electron donor is an inorganic ion or molecule 1,2. Three chemi- cal intermediates in nitrification are recognized; hydroxylamine, nitroxyl (a very reactive chemical species that remains enzyme-bound), and nitrite (Fig. 2).

Although the most intensively studied nitrifying bacteria are obligate autotrophs, various heterotrophs also oxidize organic nitrogen compounds to nitrite or nitrate 2 4. One of the many uncertainties about nitri- fication is whether the alternative processes contribute 0.1%, 1% or even ]0% to the global oxidation of ammonia 1,2. Enzymes responsible for heterotrophic nitrification have not been characterized adequately, so there remains doubt as to whether ammonia is first released and then oxidized by the same mechanisms as those used by the conventional nitrifying bacteria. Alternatively, it has been suggested that organic derivatives (based on an unspecified group, R), such as R. .NHOH, R . N O o r P,~.NO2, might be generated as intermediates from which the corresponding in- organic nitrogen compounds, NH2OH , ( N O H ) and N O 2 - , are released.

Loss of nitrogen from the biosphere by denitrification

There are essentially three, physiologically distinct types of microbial nitrate reduction: denitrification; nitrate assimilation; and the dissimilatory reduction of nitrate to ammonia. The latter two processes essen- tially reverse nitrification and therefore compete with denitrification to decrease nitrogen in the biosphere. The various subdivisions of each of these processes have been summarized according to the types of enzyme involved, their genetic regulation, whether energy is conserved as a proton-motive force, and the end products s 7.

Even among the conventional denitrifying bacteria, there are wide variations in the end products of nitrate- and nitrite-reduction. First, in some but not all bacteria, nitrate inhibits, represses or competes for electrons with nitrite-, nitric oxide- and nitrous oxide reductases, so nitrite accumulates in the immediate environment 8. Second, some bacteria do not possess genes for one or more of the denitrification enzymes 9. For example, some bacteria are unable to reduce N2 0 , which is therefore the end product of denitrification. Third, the four reductases differ in pH optima, sensi- tivity to oxygen inactivation or repression, and to vari- ous other inhibitors, so environmental factors play a critical role in determining whether denitrification is partial or complete. Genes for some or all of the de- nitrification enzymes may exist on unstable plasmids; the products of nitrate reduction therefore depend on

Sewage Bird/animal sludge excreta

Industrial ~ ~ Cyanide wastes ~ ~ ¢ ~ / / - Nitriles

Herbicides, ~ ~ Detritus pesticides Orgo.nic N

Fish -.~ ,~"

J NH20H

NITRIFIEATION I

[NOHI

Aerobic process

~ . Anaerobic process

' FIXATION

N2

t N20

J NO NO ~

NO~ Nitrate from

soil water

Biodegrodation End Products

OENITRIFICATION

Figure 1 The biological nitrogen cycle. Virtually all organic nitrogen com- pounds entering the environment are degraded to ammonia, which is then oxidized by the aerobic nitrifying bacteria to nitrate (open arrows on the left). In anaerobic soils and sediments, nitrate is reduced to nitrite which can then either be lost from the biosphere as gaseous NO, N20 or N2, or be converted back to ammonia by fermentative bacteria. Thus, ammonia (as NH4+), nitrate and N 2 dom- inate the biological nitrogen cycle.

the proportion of plasmid-deficient bacteria in the population ~0. Finally, even under optimal growth con- ditions, some bacteria produce far more of one enzyme than another, resulting in the accumulation of the intermediate that is the substrate for the least active e n z y m e .

Waste-water treatment A major environmental problem is the removal of

nitrogen from waste water. Many water authorities use an anaerobic denitrification step as the first, or even the only, process for water purification. This simul- taneously removes much of the organic carbon and all of the nitrate without incurring the expense of adding substrates such as methanol, ethanol or acetate. If the concentration of ammonia in the resulting water exceeds the legal limit, it and any remaining organic carbon is removed in a subsequent nitrification stage. This results in potable water with little organic carbon, but retains the low but measurable concentration of nitrate generated from ammonia. If the nitrate con- centration is still too high, the water can be recycled until this problem is solved, but this only generates another problem - how to dispose of sewage sludge.

Activated sludge Activated-sludge plants generate vast quantities of

virtually useless sludge which, in the past, has been digested anaerobically and then dumped, often in landfill sites. The problem is therefore particularly acute in countries such as Japan and The Netherlands where population densities and land prices are

TIBTECH AUGUST 1993 (VOL 11)

370

bioremediation

Pk60 redx~ N H 3 ~ P/+60 o

HI

Nitrosomonas electron-transfer

chain

½ 02 H20

NO2

Nitrobacter electron-transfer

chain

~a02 H20

Figure 2 Electron-transfer processes in the aerobic nitrifying bacteria, show- ing the cyclical role of cytochrome P460 in the oxidation of ammonia to nitrite by Nitrosomonas spp.

especially high. Yokohama, for example, is Japan's second largest city with 89% of its 3.2 million people connected to the sewage system. Even after anaerobic digestion and de-watering, this generates a formidable 77 000 tonnes of dry solids each year. Most countries either dump the solids at sea - a practice now strongly discouraged - or incinerate it at considerable cost. Each year since 1977, 600 tonnes of the dried solids from Yokohama have been sold as a fertilizer, effec- tively replacing at least half of the normal requirement for chemical fertilizers. Because of their microbial ori- gin, the sewage solids are a rich source of nitrogen, calcium and phosphorus, although they are slightly deficient in potassium. Trials have established that they support equivalent yields of crops such as cabbage, chrysanthemums, spinach, potatoes, turnips, green soybeans and radishes; French beans, which give up to 80% better yields on sewage solids than on organic or conventional fertilizers, are the star pefformeff 1.

So why is this technology not used more widely? Most sewage sludge is enriched for toxic, heavy metals and is therefore unusable on food crops. Although this is apparently not a problem with sludge from Yokohama C i t y 11 , elsewhere in Japan alternative technologies have been developed. Over 200 in- cinerators are used to dispose of the 1.4 million tonnes of sludge generated annually. Some of them are fed by domestic combustible waste to minimize costs. For ex- ample, the Kyoto sewage works is designed to incin- erate 30 tonnes day -1 of biologically generated sludge with 400 tonnes day 1 of normal refuse 12. The heat generated can then be used for power generation. In Osaka, coke is added to the incinerator to generate 25 tonnes day 1 of slag which is sold for road building.

Recent developments in the control of environmental nitrogen

The design of domestic waste-water-treatment plants is dictated more by economics and the standards set by regulatory authorities for nitrate levels in water

than by biochemical logic. Full-scale denitrification plants have been in operation for many years in France and the USA, with the addition of reduced car- bon compounds such as methanol as a source of elec- trons. More than 50 plants in the USA use the D E N I T E ® deep-bed denitrification technology (developed by Tektron Technologies Inc., Houston, TX, USA), with methanol as the added substrate. In contrast, only one D E N I T E ® plant, and that only at the pilot-plant scale, is operative in the U K 13. This has been shown to remove 85% of the typical 30 mg nitro- gen per litre in settled sewage from the activated- sludge process 13. Although methanol is cheap, it is also toxic, and therefore more-expensive electron donors such as acetate and ethanol are also used.

In the Blankaart region of Belgium, intense farming activity results in very high concentrations of nitrate in the reservoirs during winter, but not during sum- mer. In contrast to the limit of 25 mg nitrogen per litre recommended by the EC Drinking Water Directive 80/778 (P,e£ 14), throughout the 1980s, special dis- pensation has been granted on a yearly basis for the 50mgl q statutory limit to be exceeded. When legis- lation was passed for the introduction of more-strin- gent limits, both the economics and scientific feasi- bility of adding a methanol-driven denitrification stage to the water purification process were investigated is. The scientific data from pilot-plant experiments are summarized in Table 1. Both the stability of the pro- cess and the need to achieve virtually the complete removal of nitrate dictate that the process should oper- ate under conditions of excess methanol; conse- quently, a further stage to remove methanol, together with on-line continuous monitoring is essential to ensure that the methanol concentration in drinking water is never above 0.25 mg1-1. The implementation of the EC Urban Waste-water Treatment Directive 91/271 (Ref. 16) has imposed even higher standards, requiring treatment plants serving populations of greater than 100000 to achieve less than 10mg nitro- gen per litre in their effluend 3.

Anaerobic nitrification and aerobic denitriflcation Broda 17 has suggested that it should be possible for

bacteria with the appropriate enzyme complement to use ammonia as the electron donor for denitrification in the absence of oxygen, the overall reaction being thermodynamically favourable:

5 NH4 + + 3 N O 3 -9 4 N 2 + 9 H 2 0 + 2 H + (G o' = -1483.5kJ per reaction)

Although it is not yet certain that chemolithotrophic bacteria are involved, such bacteria have recently been discovered and used in the patented Anamox T M (Gist- Brocades) process 18. The significance of this develop- ment is that, as 02 is clearly not essential for nitrifi- cation, it should be possible to operate a totally anaerobic waste-water treatment process that is capable of achieving nitrification and denitrification simultaneously. I f the ratio of nitrate to ammonia is unfavourably low, presumably the balance could be

TIBTECH AUGUST 1993 (VOL 11)

371

bioremediation

restored by limited aeration to convert some of the ammonia to nitrate by aerobic nitrification.

Some nitrifying bacteria also carry genes for de- nitrification that are regulated by the availability o f O 2 (Refs 19, 20). Although denitrification is normally repressed by excess 02, in some bacteria, denitrifi- cation continues even when the dissolved 0 2 concen- tration is sufficient to saturate cytochrome oxidase 21. As ammonia mono-oxygenase is also inhibited or inactivated by excess 02, nitrification, like denitrifi- cation, is more rapid under conditions of 0 2 limi- tation. Oxygen concentrations as low as 401XM are sufficient for ammonia oxidation to nitrite, though higher concentrations are required for nitrite oxi- dation 1,22. Consequently, some nitrifying bacteria can simultaneously denitrify either nitrate or nitrite to N20 or N2, but only when the supply of 0 2 is care- fully regulated. There are clearly considerable prob- lems in operating a treatment plant that relies solely on this fine balancing act to achieve one-step removal of ammonia as N2; too frequently the process is only partially successful, resulting in the production of the 'greenhouse gases', N O and N 2 0 , which are major contributing factors in the production of acid rain.

Recalcitrant and toxic nitrogenous wastes The introduction of xenobiotic chemicals into the

environment to control one problem often generates another, an example being the use of N-containing heterocyclic pesticides and herbicides such as linuron or metamitron (Fig. 3). Considerable effort is currently being made to develop techniques for detecting such compounds in the parts per billion concentration range, and to isolate microorganisms that are able to degrade them. Bacteria able to degrade linuron were isolated by sequential-enrichment culture in a mini- mal salts medium. The result was a stable mixed culture from which pure microbial cultures could be isolated, none of which alone was able to metabolize the pesticide 23. Attempts to isolate a metarnitron- degrading organism were more successful, resulting in the purification of a Rhodococcus sp. that totally degraded the herbicide in less than 24h in minimal salts medium supplemented with ammonia (N. R. Parekh, S. J. Welch, S. J. Roberts and A. Walker, unpublished). In neither of these examples, however, were either the biochemical pathway or the final degradation products identified. Although further studies in this important research area are clearly required, traces of many xenobiotics are effectively removed from water supplies in a tertiary treatment process involving large filters of granular activated carbon. This again highlights the good sense behind what is already common practice in the water indus- try - the combination of physical, chemical and bio- logical treatment processes to control the various forms of environmental nitrogen.

Sometimes it is necessary to use a specific mixture of organisms or to add an additional nutrient to degrade a recalcitrant nitrogen compound. For example, although a mixture of an Arthrobacter sp. and

Table 1. Effect of different methanol concentrations on the efficiency of nitrate-removal from a water supply a

Conc. in feed {mg 1-1) Conc. in effluent (mg 1-1) Limiting substrate Nitrate Methanol Nitrate Nitrite Methanol

Methanol 66.0 29.3 30.0 4.9 0.0

Nitrate 65.5 53.0 1.0 0.0 2.5

Neither 66.5 50.1 2.0 1.8 0.1

aData from Ref. 14.

a Corynebacterium sp., isolated from an esturine sedi- ment, rapidly oxidized acetaldoxime to nitrate, neither bacterium alone catalysed this reaction at a significant rate 24. One of the priority pollutants on the US Environment Protection Agency (EPA) list is the organic nitrogen compound, 2,4-dinitrophenol, which can be degraded readily in the presence of low concentrations of easily degradable carbon compounds (glucose in the range 50-500mgl q appears to be par- ticularly effective) 2s.

Cyanide and nitriles are both important toxic wastes 26. Cyanide is produced in large quantities for use in metal (especially gold)-extraction, electro- plating, polymer, steel, organic-chemical and related industries. It is commonly found in industrial effluents from mining, coal liquefaction and gasification, steel manufacture and organic-chemical factories. Aerobic sewage systems can adapt to degrade effluents con- taining up to 200mgV of cyanide, and a full-scale commercial process for biodegrading cyanide in mine washings at the rate of 20000m 3 per day has been developed at the Homestake Mine, SD, USA 26.

Large quantities of nitriles such as acrylonitrile, methacrylonitrile, adiponitrile, acetonitrile, and acetone cyanohydrin are used to synthesize complex organic chemicals, or as monomers or monomer pre- cursors to synthesize polymers. For example, the Nitto Chemical Company, Japan, was one of the first to use the bioconversion of acrylonitrile to produce 6000 tonnes year -1 of acrylamide. Such processes generate high concentrations of highly toxic wastes which must be degraded without release into the environment. Despite this toxicity, the halogenated aromatic nitriles, bromoxynil, ioxynil and dichlorobenil, are amongst the most widely used pesticides and herbicides.

Over 70 different types ofcyanogenic glycosides are produced by more than 2000 species of plants, includ- ing cassava, sorghum, lima beans, alfalfa, flax and bam- boo (reviewed in Ref. 26). Such cyanogenic plants can cause acute and chronic cyanide poisoning in humans and domestic animals. Cyanide is released from the plants by the action of[3-glycosidases and ot-hydroxy- nitrile lyase:

cyanogenic nitrile --+ sugars + o~-hydroxynitrile ---> H C N + carbonyl compound

Unlike most of the other inorganic nitrogen com- pounds, cyanide is both produced and metabolized by eukaryotic as well as prokaryotic microorganisms

TIBTECH AUGUST 1993 (VOL 11)

372

bioremediation

(reviewed in 1Ke£ 27). The most prolific fungal sources of cyanide are probably the cyanogenic glycosides pro- duced by the plants that host phytopathogenic basidio- mycetes, ascomycetes and zygomycetes 28. The pathogenic fungi synthesize [3-glucosidases which release cyanide from c(-hydroxynitriles produced as intermediates in glycoside degradation 28. Other fungi generate cyanide from their own metabolic pre- cursors 29.

Small quantities of cyanide are also generated by algae; e.g. synthesis of cyanide from amino acids by Chlorella vulgaris is catalysed by both D- and z-amino acid oxidoreductases in the presence o f O 2 and either appropriate divalent metal ions or peroxidase 3°.

The best-studied cyanogenic bacteria are Chromo- bacterium violaceum and various pseudomonads, the major difference between them being that C. violaceum also degrades cyanide to [3-cyanoalanine 27. The source of cyanide is glycine, although optimal conditions vary considerably. Cyanogenesis occurs late in the growth cycle, typical of secondary metabolite production.

The availability of microbes that can degrade cyanide rapidly to nontoxic end products would therefore be of considerable significance in environ- mental biotechnology. Some of the many reports of such cultures have been reviewed 21. Three enzymes able to degrade cyanide have been identified in C. violaceum. The most significant appears to be the soluble, cytoplasmic [3-cyanoalanine synthase, which catalyses the reaction31:

CN + cysteine (or O-ace@ serine) -+ /3-cyanoalanine + H2S (or acetate)

In non-cyanogenic phytopathogenic fungi, cyanide is hydrolysed to formamide which can then be further metabolized to NH4 + and formate.

A cautionary note was sounded when, during a search for anaerobes capable of degrading cyanide, a Klebsiella planticola culture that could use cyanide anaerobically as a sole nitrogen source was isolated 32. The conversion of H C N to NH4 + was shown con- vincingly to be due to an abiotic, chemical reaction with the reducing sugar added as a carbon source for microbial growth. This raises the question as to whether some of the earlier, less meticulous work on cyanide metabolism might have been based on chemi- cal rather than enzyme catalysed-reactions.

Future prospects The best way to control environmental nitrogen is

clearly to prevent its accumulation in the first place. The generation of domestic sewage and agricultural wastes are obvious examples of why this is sometimes impossible, so the biochemical reactions of the bio- logical nitrogen cycle will continue to be exploited to contain environmental pollution by converting am- monia and organic nitrogen compounds to atmospheric N 2. This requires the combined activities of a wide variety of putrefying microbes, followed by bacterial nitrification and denitrification. We have seen how recent developments in the biodegradation of toxic,

xenobiotic compounds and in our knowledge of the biochemical and genetic regulation of nitrification and denitrification have provided new ways for control- ling environmental nitrogen. Nevertheless, current processes are inadequate to deal with many recalcitrant chemicals, so further intense research will clearly be profitable. Bioremediation alone is unlikely to succeed unless it is combined judiciously with physical and chemical treatment processes, a practice that for years has been routine in the waste-water industry.

References 1 Prosser, J. I. (1986) in Nitrification (Vol. 20, Spedal Publications Soc.

Gen. Microbiol.) (Prosser, J. I., ed.), pp. 63-78, IRL Press 2 Prosser, J. I. (1989) Adv. Microb. Physiol. 30, 125 181 3 Focht, D. D. and Verstraete, W. (1977) Adv. Microbiol. Ecol. 1,

135~14 4 Killham, K. (1986) in Nitrification (VoI. 20, Special Publications Soc. Gen.

Microbiol 0 (Prosser, J. I., ed.), pp. 95-115, ItKL Press 5 Cole, J. A. (1988) in The Nitrogen and Sulphur Cydes (Cole, J. A. and

Ferguson, S. J., eds), Syrup. Soc. Gen. Microbiol. 40, pp. 281-330, Cambridge University Press

6 Cole, J. A. (1989) in Molecular and Genetic Aspects of Nitrate Assimi- lation (Wray, J. L. and Kinghorn, J. R., eds), pp. 229-243, Oxford University Press

7 Page, L., Griffiths, L. and Cole, J. A. (1990) Arch. Microbiol. 154, 349454

8 Can', G.J., Page, D. and Fergnson, S.J. (1989) Eur.J. Biockem. 179, 683-692

9 Payne, W.J. (1973) Baaeriol. Rev. 37, 409 452 10 Romerrnann, D. and Friedrich, B. (1985)J. Baaeriol. 160, 852 854 11 Noguchi, H. and lto, H. (1992)3'. Inst. Water Environ. Manage. 6,

576-582 12 Tebbutt, T. H. Y. (1992)_/. Inst. Water Environ. Manage. 6, 628-632 13 Upton,J, Fergusson, A. and Savage, S. (1993)_/. Inst. Water Env. Man-

age. 7, 1-12 14 Council of European Communities OffidalJouma1229, 30 August 1980 15 Germonpre, R., Liessens, J. and Verstraete, W. (1991) in Environ-

mental Biotechnology 1991 (Verachtert, H. and Verstraete, W., eds), pp. 4%52, Koninklijke Vlaamse Ingemeursvereniging

16 Coundl of European Communities OfftciaI-/ourna113 5 /140, 30 May 1991 17 Broda, E. (1977) Z. Allg. Microbiol. 17, 491-493 18 van der Graaf, A., Mulder, A., Slijkhuis, H., Robertson, L. A. and

Kuenen, J. G. (1990) in Proceedings of the 5th European Congress of Biotechnology (Christiansen, C., Munck, L. and Villadsen, J., eds), pp.388-391, Munksgaard International

19 Poth, M. (1986) Appl. Environ. Microbiol. 52, 957 959 20 Pdtchie, G. A. F. and Nicholas, D. J. D. (1972) Biochem. J. 126,

1181 1191 21 Robertson, L. A. and Kuenen, J. G. (1984) Arch. Microbiol. 139,

351-354 22 Helder, W. and de Vries, R. T. P. (1983) Neth.d. Sea Res. 17, 1-18 23 Roberts, S.J., Walker, A., Waddington, M.J. and Welch, s.J. (1991)

in Pesticides in Soils and Water, BCPC Monograph 47, pp. 51-58 24 Rho,J. (1986) Can.J. Microbiol. 32, 243-247 25 Hess, T. F., Silverstein, J. and Schmidt, S. K. (1993) Water Environ.

Res. 65, 73-81 26 Knowles, C.J. and Wyatt, J. W. (1992) Syrup. Soc. Gen. MicrobioI. 48,

113-128 27 Knowles, C. J. and Bunch, A. W. (1986) Adv. Micro& Physiol. 27,

73-111 28 Colotelo, N. and Ward, E. W. B. (1961) Nature 189, 242 243 29 Ward, E. W. B. and Thorn, G. D. (i961) Can@ Bot. 44, 95 104 30 Pistorius, E. K., Gewitz, H. S., Voss, H. and Vennesland, B. (1977)

Planta 128, 73-80 31 Macadam, A. M. and Knowles, C.J. (1984) Biochim. Biophys. Aaa

786, 123-132 32 Hope, K. M. and Knowles, C.J . (1991) FEMS Microb. Lett. 80,

217-220

TIBTECH AUGUST 1993 (VOL 11)