Microbial metabolism of dimethyl sulphide and related compounds

MÏCROBIAL METABOLISM OF DIMETHYL SULPHIDK

AND RELATED COMPOUNDS

STELLINGEN

1. De door Kiene et al. (1986) gedane uitspraak, dat de door hen

ten gevolge van dimethylsulfide- en

methylmercaptaan-fermen-tatie gemeten C02:CH4 productieverhouding overeenkomt met die

welke door Zinder & Broek (1978) werd gevonden, is onjuist.

- Kiene, R.P., Oremland, R.S., Catena, A., Miller, L.G. &

Capone, D.G. (1986). Appl. Environ. Microbiol. 5_2,

1037-1045.

- Zinder, S.H. & Brock, T.D. (1978). Nature

212_, 226-228.

2. Sand (1987) concludeert ten onrechte dat methylmercaptaan

niet kan bijdragen aan betoncorrosie door thiobacilli.

- Sand, W. (1987). Appl. Environ. Microbiol.

S2_, 1645-1648.

3. De door Kanagawa & Kelly (1986) gemeten relatieve toename in

02-gebruik bij de oxydatie van oplopende concentraties

dime-thylsulfide door Thiobacillus thioparus TK-1 is vermoedelijk

toe te schrijven aan ontkoppeling van de energiegenerering.

- Kanagawa, T. & Kelly, D.P. (1986). FEMS Microbiol. Lett.

_34, 13-19.

- Mhatre, S.S., Chetty, K.G. & Pradhan, D.S. (1983).

Biochem. Biophys. Res. Commun. 110, 325-331.

4. De keuze van een "trickling-filter" voor de behandeling van

toxisch afvalwater is niet gelukkig.

- Dit proefschrift.

5. Het ligt in de lijn der verwachtingen dat het spectrum van

fysiologische typen dimethylsulfide-oxyderende micro-organis­

men even groot is als dat van de sulfide-oxyderende.

- Dit proefschrift.

6. De conclusie van Schoenen & Colbourne (1987) dat hun metho­

den, voor de bepaling van de bacteriegroeibevorderende wer­

king van materialen, vergelijkbare resultaten opleveren, is

ietwat gechargeerd, gezien het ontbreken van enige correlatie

tussen hun meetresultaten.

- Schoenen, D. & Colbourne, J.S. (1987). Zbl. Bakt. Hyg.

B

}Ü^, 505-510.

7. De door Garrity et al. (1980) en Brown et al. (1981) voorge­

stelde opsplitsing van de Legionellaceae in de geslachten

Legionella, Fluoribacter en Tatlockia op basis van

DNA-ver-wantschap tussen de verschillende soorten is prematuur en

ongewenst.

- Garrity, G.M., Brown, A. & Vickers, R.M. (1980). Int.

J. Syst. Bacteriol. 29.' 609-614.

- Brown, A., Garrity, G.M. & Vickers, R.M. (1981). Int.

J. Syst. Bacteriol. _3_1' 111-115.

MICROBIAL METABOLISM OF DIMETHYL SULPHIDE

AND RELATED COMPOUNDS

PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Technische Universiteit Delft, op gezag

van de Rector Magnificus, prof. drs. P.A. Schenck,

in het openbaar te verdedigen ten overstaan van

een commissie aangewezen door het College van Dekanen

op dinsdag 27 september 1988 te 16.00 uur

door

GERTRUDIS MARIA HUBERTINA SUYLEN

DIT PROEFSCHRIFT IS GOEDGEKEURD DOOR DE

PROMOTOR PROFESSOR DOCTOR J.G. KUENEN

This study was carried out at the Department of Microbiology and Enzymology

of the University of Technology Delft, The Netherlands

Voor pap en mam, die niet achter,

VOORWOORD

Met een opgelucht, maar ook wat weemoedig gevoel begin ik aan dit dankwoord. Het boekje is klaar, maar daarmee is tevens een eind gekomen aan de regelmatige bezoeken aan het microbiologisch lab in Delft. De Fransen hebben een mooie uitdrukking voor deze gemoedstoestand: "partir, c'est mourir un peu".

Rest mij nog de plezierige taak om eenieder die aan de totstand­ koming van dit proefschrift heeft bijgedragen te bedanken.

Allereerst wil ik mijn promotor Prof. Dr. J.G. Kuenen danken voor zijn nimmer aflatende belangstelling voor de "beestjes", zijn opbouwende kritiek op de artikels en hoofdstukken, en zijn goede coaching bij de voorbereiding van lezingen. Gijs, jouw opmer­ kingen waren evenzovele zetten in de goede richting.

My (laboratory) roommate Lesley Robertson, thanks for the countless friendly "Denglish" chats, I will never forget that we were taking each other in turn out for a stroll in the garden. Peter Large, jouw hulp bij de enzymisolatie was onmisbaar. Bovendien heb ik genoten van de vele gedachtenwisselingen over Nederlandse en Engelse literatuur.

Hans van Dijken: de weinige gesprekken die ik met je voerde waren altijd een bron van inspiratie.

Mijn doctoraalstudenten en stagiaires: Ron van Poelgeest, Hans Verbeek, Marjolein Reichenfeld, Ed Dakkenhorst, Eric Winkel en Guus Stefess, jullie leverden niet alleen een bijdrage aan het onderzoek maar zorgden mede voor de prettige sfeer op het lab. Cornel Verduijn bedankt voor de "spoed"enzymisolatie op het eind. Maudy Smith, Hans en Elly Bonnet en Eduard van de Bosch, mijn dank voor de vriendschap die ik van jullie heb ondervonden.

Zonder de mensen van de algemene en technische dienst zouden de fermentoren niet zo "gesmeerd" hebben gelopen en zou er waarschijnlijk geen "beestje" wat te eten hebben gehad.

Theo Scheulderman bedankt voor de hulp bij alle computertech­ nische zaken.

Tenslotte wil ik alle overige leden van de "Bacterie-" en "Gistgroep" die hier niet met name zijn genoemd bedanken voor hun collegialiteit.

CONTENTS

VOORWOORD

CHAPTER 1: Introduction

1. Dimethyl sulphide production and the global sulphur cycle

2. Biological and industrial production of dimethyl sulphide

Microbial metabolism of dimethyl sulphide General characteristics of hyphomicrobia Biochemistry of hyphomicrobia

1 Oxidation of C,-compounds 2 Assimilation of C,-compounds 3 Metabolism of C~-compounds

A Basis for restricted me thy lotrophy in hyphomicrobia Physiology of hyphomicrobia

Physiology and ecology of reduced sulphur compound oxidizing bacteria

1 Physiology of the chemolithoheterotrophic sulphur oxidizers

Biochemistry of sulphur compound oxidation Outline of this thesis

A.2. A.2. A.2. A.2. A.3 5.1 5.1. 5.2 6. 12 19 27 29 29 3A 36 37 39 Al A3 AA 50

CHAPTER 2: Reevaluation of the dimethyl sulphide oxidizing

capacity of Thiobacillus MS1 51

Abstract 51 In trod uction 51 Materials and methods 52

Results 55 Discussion 59

CHAPTER 3: Chemostat enrichment and isolation of Hyphomicrobium

EG, a dimethyl sulphide oxidizing methylotroph 61

Abstract 61 Introduction 62 Materials and methods 63

Results 65 Discussion 7 2 C H A P T E R 4 : C h e m o l i t h o t r o p h i c p o t e n t i a l of a H y p h o m i c r o b i u m s p e c i e s , c a p a b l e of g r o w t h on m e t h y l a t e d s u l p h u r c o m p o u n d s 76 A b s t r a c t 76 Introduc tion 77 Materials and methods 78

Results 81 Discussion 90

CHAPTER 5: Methyl mercaptan oxidase, a key enzyme in the metabolism of methylated sulphur compounds by

Hyphomicrobium EG v 92

Abstract 92 Introduction 92 Materials and methods 93

Results 98 Discussion 106

CHAPTER 6: Considerations on toxicity of small (in)organic reduced sulphur compounds for Hyphomicrobium EG

and other microorganisms 109

Abstract • 109

Introduction 109 Materials and methods 110

Results 114 Discussion 121

CHAPTER 7: Summary 125

REFERENCES

ABBREVIATIONS

CURRICULUM VITAE

CHAPTER 1

INTRODUCTION

1. Dimethyl sulphide production and the global sulphur cycle

All models for the yearly circulation of sulphur on a global scale require the release of some volatile or gaseous form of sulphur from terrestrial and/or marine surfaces to the atmosphere in order to balance the cycle (Conway, 1942; Junge, 1960; Eriksson, 1963; Kellogg et al., 1972). Formerly (e.g. Rodhe & Isaksen, 1980) the oceans have been suggested as a source of (biogenic) H~S, emitting the amount of sulphur to the atmosphere theoretically needed to complete the budget, which equals about

-1 12

80 Tg S year (1 Tg= 10 g) and is comparable to the anthropo­ genic sulphur emissions.

Only two processes lead to an appreciable amount of sulphate reduction to H„S in the global sulphur cycle: the chemical

2 +

reduction of seawater sulphate by Fe in basaltic rocks, as a result of its cycling through hydrothermal systems of the submarine ridges (Von Damm, 1983), and the biological dissimila-tory sulphate reduction. In the first process the H-S emitted from the submarine hot springs is immediately utilized by the rich and abundant ecosystems found on the ocean floor near hydro-thermal vents. In these systems sulphide-oxidizing bacteria, free-living as well as symbiotic within certain tissues of bivalve molluscs and tube worms, obtain metabolically useful energy from the aerobic chemolithoautotrophic oxidation of H~S on which these systems thrive (Cavanaugh et al., 1981; Felbeck & Somero, 1982; Cavanaugh, 1983; Jannasch & Taylor, 1984). Therefore this H„S will not contribute to atmospheric emissions. The H-S produced by dissimilatory sulphate reduction, a process commonly observed in aquatic (marine) environments where organic matter and sulphate are abundant and oxygen is depleted (e.g.

sedimentary pore waters, stratified basins), usually does not enter the atmosphere in large quantities, since it will probably be oxidized before it reaches the air/water interface. A notable exception is formed by the tidal mud flats along the sea coasts all over the world where, at least on a local scale, substantial quantities of H~S may be produced (Jeirgensen, 1982). The oxidation of sulphide can either be due to microbial metabolism of sulphide at the oxic/anoxic interface or to the chemical reaction between H~S and the dissolved 0- in these waters. In the past the latter has often been used as an argument to support the assumption that the H„S flux from continental surface waters or the open oceans is negligible (Östlund & Alexander, 1963),

although widely divergent halflife values for H„S in sea water are reported ranging from minutes (Jdrgensen et al., 1979) to a couple of hours (Almgren & Hagström, 1974) to about 50 hours (Chen & Morris, 1972), depending on e.g. temperature and salt concentration of the water and the presence of trace metals.

Finally, study of the stable isotope ratio of sulphur in atmospheric sulphur compounds provides a means of discriminating between their origin. When 6 S values of sulphate in rain samples from industrial and remote areas were compared (Jensen & Nakai, 1961; Rafter, 1965), it appeared that the samples with the lowest sulphate concentration, and therefore probably the least

C 34

polluted, had the highest o S values. This indicated that sea salts ( o3 4S value +20.1 + 0.3, Thode et al., 1961) and fossil

fuels (o3 4S value of coal +11.9 to 23.9 (Jensen & Nakai, 1961)

and of petroleum -9.8 to +16 (Manowitz & Tucker, 1969)) were mainly contributing to the sulphate in the air of these regions,

(34

whereas biogenic H„S with its much lower o S value (-23 to +6; Ault & Kulp, 1959) formed only a minor contribution. These

results are inconsistent with the "older" models for the global sulphur cycle which assume that about 50 percent of the total amount of atmospheric sulphur is contributed by H„S from the biosphere (Eriksson, I960; Junge, 1960; Kellogg et al., 1972; Rodhe & Isaksen, 1980).

Even though the presented arguments that H„S is not responsible for essentially all the flux of reduced sulphur from the oceans to the atmosphere are based on circumstantial evidence, it seems

l i k e l y t h a t a m o r e s t a b l e ( v o l a t i l e ) s u l p h u r c o m p o u n d w i t h a ( 3 4 r e l a t i v e l y h i g h o S v a l u e w o u l d b e r e s p o n s i b l e f o r t h e t r a n s f e r of s u l p h u r i n t o t h e a t m o s p h e r e . F u r t h e r m o r e , i n f u t u r e i t s h o u l d b e a t t e m p t e d t o m e a s u r e t h e c o n c e n t r a t i o n o f H „ S i n s u r f a c e w a t e r s a n d t h e o v e r l y i n g a t m o s p h e r e i n o r d e r t o b e a b l e t o d e t e r m i n e t h e m a g n i t u d e ( a n d d i r e c t i o n ) o f t h e H~S f l u x . L o v e l o c k e t a l . ( 1 9 7 2 ) w e r e t h e f i r s t t o d e t e c t d i m e t h y l s u l p h i d e (DMS) i n s u r f a c e o c e a n w a t e r s a n d t h e y s u g g e s t e d t h a t t h i s c o m p o u n d w a s f i l l i n g t h e r o l e w h i c h w a s p r e v i o u s l y a t t r i b u t e d t o H _ S . S i n c e t h e n v a r i o u s w o r k e r s , m a k i n g u s e o f d i f f e r e n t t r a p p i n g a n d d e t e c t i o n t e c h n i q u e s f o r DMS, h a v e d e t e r m i n e d t h e DMS c o n c e n t r a t i o n i n s e a w a t e r a n d t r i e d t o e s t i m a t e t h e s e a t o a i r f l u x o f DMS ( L i s s & S l a t e r , 1 9 7 4 ; N g u y e n e t a l . , 1 9 7 8 ; B a r n a r d e t a l . , 1 9 8 2 ) . S i n c e t h e i r c a l c u l a t i o n s w e r e , b a s e d o n a l i m i t e d n u m b e r o f m e a s u r e m e n t s d o n e o n s a m p l e s t a k e n a t d i f f e r e n t s i t e s t h e f l u x v a l u e s w e r e r a t h e r d i v e r g e n t . On t h e b a s i s o f a v e r y e x t e n s i v e s e t o f d a t a A n d r e a e a n d

Table 1. Estimates of the emission of sulphur t o the atmosphere as p a r t i c u l a t e s and a s g a s e s . Emission r a t e s of the various sulphur compounds a r e given in Tg S/year (Andreae,

1985).

S02 H2S COS DMS CS2 SO^2 - Other T o t a l

Sea spray 40-300 40-300 Dust 3-30 3-30 Total p a r t i c u l a t e s 40-330 40-330 Volcanoes 8 1 0.01 0.01 (<3) S o i l s and p l a n t s - 3-41 0 . 2 - 0 . 6 0.2-4 0 . 6 - 0 . 8 (8?) (8?) Coastal wetlands - 0.9 0.13 0.6 0.07 Biomas9 burning* 7 ? 0.11 - ? ? Oceans ( g a s e s ) - 0-15 0.4 40 0.4 T o t a l gases 15 5-58 0 . 8 - 1 . 2 40-45 1.1-1.3 (<3) 1 65-125

*: All gasoous emissions (other than COS) assumed t o be S02>

? 1 0.13 9 12 5-47 1.8 7 40-56

Raemdonck (1983) calculated a global weighted-mean DMS concentra­ tion in surface waters of 102.4 ng 1 , and predicted a global

-2 -1

mean emission rate of 290 ug of S (DMS) m day representing a 12 ' -1

flux of 38.5x10 g of sulphur year . This estimate of the biogenic sulphur flux from the oceans appears to be rather precise since inclusion of new data in the above calculation does not alter its outcome (Andreae, 1986). Moreover it agrees with the excess sulphate (that fraction of sulphate in aerosols which is not accounted for by sea spray) deposition rates of 209 and

-2 -1

202 ug m day measured in the Southern Hemisphere (Bonsang et al., 1980; Andreae, 1982), where man-made sulphur emissions are negligible.

A summary of the world's natural sulphur emissions based on current information is given in Table 1, which demonstrates that the transfer of DMS from marine environments into the atmosphere makes up about one-half of these emissions. Combined with the

( 34

aforesaid this would suggest a very positive o S value of DMS. _{( 34} Preliminary data indicated the o S value of DMS to be +17 (Calhoun et al., 1987).

2. Biological and industrial production of dimethyl sulphide

DMS can be produced from several compounds in biological systems (Figure 1 ) , e.g. through the cleavage of sulphonium compounds or by the reduction of dimethyl sulphoxide (DMS0).

A linear relationship was shown to exist between DMS concen­ tration and primary production in ocean surface waters, when the data were grouped according to wide regions of marine produc­ tivity (Andreae & Barnard, 1984). Furthermore, in several cases a close resemblance was found between the vertical distribution of DMS and chlorophyll a_ in marine water columns (Andreae & Barnard,

1984). This suggested that phytoplankton was responsible for the emission of DMS. The seasonal variation in DMS concentration (high in spring and summer, the algal growing seasons; low during autumn and winter) further supported this assumption (Turner & Liss, 1987). Upon lumping a large data set no significant correlation was found between DMS concentration and chlorophyll.

H

3

C

s , °

S*-CH

2

-CH

2

-C^

H

3

C

/ S

0H

dimethyl -G-propiothetin

H

3

C NH

2

O

/

S - C H

2

- C H

2

- C H - C

x

H

3

c' OH

S- methylmethionine

NH

2

'hT ^N

j : - C H - C H

2

- C H

2

- S . - C H

2

0

OH NH

2

CH

S - adenosy l methionine

3

ANJ « / H

OH OH

CH

3

CH3-VCH3

1

cr

trimethylsuiphonium chloride

O

H

CH3-S-CH3

dimethyl sulphoxide

Figure 1. Structure formulae of the various compounds from which DMS can be formed .

However, when the data were grouped according to the dominant algal species in the various samples significant relationships were shown to exist, demonstrating at the same time that the different algal species produce varying levels of DMS (Turner & Liss, 1987).

Challenger and Simpson (1948) showed that dimethyl sulphonium propionate (DMSP), which has since been found to be the major sulphur compound in nearly all algal species (Table 2 ) , was enzymatically cleaved into DMS and acrylate. The function of this cleavage remains as yet unknown. It appears to take place continuously at a low rate, which is enhanced under external stress conditions such as increasing salinity (Vairavaraurthy et al., 1985) and zooplankton grazing on phytoplankton (Dacey & Wakeham, 1986). The latter resulted in DMS production rates which

Table 2. Algal genera containing dimethyl sulphonium propionate (DMSP) or able to produce DMS.

References: 1= Andreae 1980a, 2= Craigie et al. 1967, 3= White 1982, 4=,Challenger et al. 1957, 5= Katayama 1962, 6= Tocher et al. 1966, 7= Ishida 8 Kadota 1968, 8= Dacey S Wakeham 1986, 9= Turner & Liss 1987, 10= Vairavamurthy et al. 1985, 11= Barnard et al. 1984, 12= Ackman et al. 1966, 13= Lovelock et al. 1972, 14= Haas 1935, 15= Challenger & Simpson 1948, 16= Reed 1983, 17= Rasmussen 1974.

Organism Reference

Bacillariophyta (diatoms)

Skeletonema, Thalasslosira 1 Chlorophyta

.Carteria, Chlorococcum, Cladophora, Codium, 2, 3, 4, 5, 6 Enteromorpha, Micromonas, Monostroma,

Oedogonium, Prasinocladus, Spongomorpha, Tetraselmis, Ulothrix, Ulva

Dinophyta (Pyrrhophyta or dlnoflaRellat.es)

Amphidinium, Gymnodinium, Cyrodinium, 7, 8, 9 Prorocentrum

Haptophyta (coccolithophorids)

Emiliania, Hymenomonas, Phaeocystis, 9, 10, II, 12, 6 Syracosphaera

Phaeophyta

Dlctyopteris, Egregia, Endatachne, Halidrys, 3, 4, 13 Laminaria, Macrocystis, Pelvetia

Prasinophyta

Platymonas 1 Rhodophyta

Ceramium, Corallina, Gelidium, Gigartina, 3, 4, 14, 15, 16 Gracilaria, Piocamium, Polysiphonia, Soliera

Cyanobacteria (formerly called Cyanophyta)

Anacystis, Microcoleus, Oscillatoria, 17, 3 Phormidium, Plectonema, Synechococcus

w e r e a p p r o x i m a t e l y 24 t i m e s h i g h e r than those of p h y t o p l a n k t o n a l o n e , g i v i n g rise to DMS c o n c e n t r a t i o n s of about 200 nM (Dacey & W a k e h a m , 1 9 8 6 ) , w h i c h would be s u f f i c i e n t to s u p p o r t the g r o w t h of D M S - u t i l i z i n g b a c t e r i a .

marine algae (Dickson et al., 1980, 1982; Vairavamurthy et al. ,

1985). This was also suggested by experiments indicating DMSP production by some algal species to occur under nitrate limita­ tion, when synthesis of other osmoregulants such as proline and glycine betaine is restricted (Turner & Liss, 1987).

DMS emitted from land surfaces (Banwart & Bremner, 1976a, 1976b; Adams et al., 1981) is likely to originate from S-methyImethionine and S-adenosylmethionine, important plant sulphur compounds. Since the amount of DMS volatilized from decaying leaves is 10-100 times higher than that emitted by living foliage (Lovelock et al., 1972) microorganisms are likely to be involved in this process. It is unknown whether the plants themselves also contain enzymes for the cleavage of the above compounds. Several authors have provided definite evidence that microorganisms produce DMS and other, mainly organic, volatile sulphur compounds from various sources (Kadota & Ishida, 1972; Rasmussen, 1974; Bremner 8 Steele, 1978). One of these organisms, Pseudomonas MS, was shown to be able to grow on trime thyIsulpho-nium chloride. Growth on this compound involved transfer of a

5

methyl group to tetrahydrofolate yielding DMS and N -methy1tetra-hydrofolate. The latter compound was further oxidized to methylene tetrahydrofolate, which was either oxidized or assimi­ lated into cell material. The DMS formed during trime thylsul-phonium chloride breakdown was liberated in the medium and was not metabolized by this organism (Wagner et al., 1966, 1967; Kung & Wagner, 1970).

Reduction of DMSO (which itself is formed through oxidation of DMS) has been demonstrated in both eucaryotic and procaryotic microorganisms (Zinder & Brock, 1978a). Several strains of Saccharomyces cerevisiae and Saccharomyces uvarum have been shown to produce DMS during fermentation from ale wort but also from defined glucose- and DMSO-containing mineral salts media (Anness, 1980; Anness & Bamforth, 1982), the former process being partly responsible for the presence of DMS in beer. A great variety of prokaryotic microorganisms reduced DMSO, including members of as widely divergent families as the Enterobacteriaceae and the Rhodospirillaceae, some of which, e.g. various Rhodopseudomonas spp., Proteus vulgaris and Escherichia coli have been shown to be

able to use this compound as a terminal electron acceptor during growth under anaerobic conditions (Yen & Marrs, 1977; Zinder & Brock, 1978b; McEwan et al., 1983; Bilous & Weiner, 1985). The latter process appears to be dependent on the type of electron donor used: e.g. lactate and formate proved to be inappropriate energy sources for Escherichia coli during anaerobic growth with DMSO, in contrast with glycerol which could serve this purpose under these conditions (Bilous & Weiner, 1985).

Taken together the above biological processes represent a considerable DMS flux (Table 1 ) , which is due to the production of low concentrations of this compound over a vast area (e.g. the oceans cover 71% of the earth's surface). In contrast, several types of industry, e.g. oil refineries, sewage treatment plants,

Table 3. Composition of the "black liquor" (according to Hagglund, 1951) and the average concentration of methylated sulphur compounds in the condensates of the waste gases formed during alkaline pulping (according to Sivela, 1980).

Black liquor (data are expressed in % of the weight of the wood)

Cellulose' Alkali lignin Resin , fats etc.

Lactones and hydroxy (saccharinic , lactic) acids Acetic acid

Formic acid Methanol

Lignin soluble in acid solution

42.8 21.6 2.2 18.2 3.2 1.7 0.4 6.9

Condensates (data given in mg/1)

Hydrogen sulphide Methyl mercaptan Dimethyl sulphide Dimethyl disulphide 39.0 94.0 16.6 21.7

animal farms, and more in particular sulphate kraft mills, can give rise to high local concentrations of DMS which may cause environmental pollution problems.

Sulphate kraft mills isolate cellulose plant fibers for the production of paper and textile such as rayon, silk and cord. They pulp wood by cooking it with aqueous alkali, which is recovered from the so-called "black liquor" (Table 3) and replenished with sodium sulphate to replace the lost alkali. The sulphate is reduced to sulphide when the organic matter is burned in order to recover the alkali. Usage of this alkali (enriched in sulphide) results in a better yield and quality of the pulp from conifers (Hagglund, 1951). A disadvantage of this process is formed by the unpleasant odours which emanate from it, due to the reaction of sulphide with the methoxyl groups of lignin which gives rise to the formation of methyl mercaptan ( M M ) , dimethyl disulphide (DMDS) and DMS (Hynninen, 1971). The latter compounds are to be found in the condensate from the waste gases (Table 3) along with varying amounts of turpentine oil and methanol, which is formed by the reaction of hydroxide with the methoxyl groups of lignin. The amount of volatile sulphur compounds formed in the sulphate process depends on the sulphidity of the cooking liquor, the methoxyl group content of the wood and the cooking-tempera­ ture and -time.

Some characteristics of these sulphides are given in Table 4.

Table 5. Odour threshold concentration of hydrogen sulphide and the methylated sulphur compounds according to Seluyzhitskii (1972) and the Merck Index (1976).

Compound Odour threshold concentration (ppb) liquid phase gas phase

Hydrogen sulphide Methyl mercaptan Dimethyl sulphide Dimethyl disulphide 0.4 2 1 8.5 - 1000 0.9 - 8.5 0.6 - 40 0.1 - 3.6

S i n c e m e t h y l a t e d s u l p h u r c o m p o u n d s a r e , a p a r t f r o m b e i n g m a l o d o r o u s ( T a b l e 5 ) , a l s o r a t h e r t o x i c ( L j u n g g r e h & N o r b e r g , 1 9 4 3 ; M h a t r e e t a l . , 1 9 8 3 ; s e e a l s o T a b l e 4 ) t h e c o n d e n s a t e s n e e d t o b e p u r i f i e d b e f o r e t h e y c a n b e d i s c h a r g e d .

C o n s i d e r i n g t h e l a r g e s c a l e b i o l o g i c a L p r o d u c t i o n o f DMS i t

Table 4 . General c h a r a c t e r i s t i c s of some v o l a t i l e sulphur compounds.

Hydrogen s u l p h i d e ( s u l p h u r e t t e d hydrogen, h y d r o s u l -phuric a c i d ) H2S Methyl mercaptan ( m e t h a n e t h i o l , mercaptomethane, t h i o m e t h y l a l c o h o l , m e t h y l s u l f h y -d r a t e ) CH3SH Dimethyl s u l p h i d e (methyl s u l p h i d e , t h i o b i s m e t h a n e ) CHrtSCHo Dimethyl disulphide (methyl disulphide) CH3SSCH3

colourless gas; offensive odour; sweetish taste; soluble in water and alcohol; mw. 34.08; m.p. -83.8 °C; b.p. -60.2 °C; d 1.1895 referred to air; autoignition temp. 500 F

highly toxic by inhalation; strong irritant to eyes and mucous membranes; highly flammable; dangerous

fire risk; explosive limits in air 4.3 to 46%; tolerance 10 ppm in air

water-white liquid when below boiling point or colourless gas; powerful unpleasant odour; mw. 48.11; m.p. -121 °C; d 0.87; flash point below 0

C; b.p. 5.96 C; slightly soluble in water; soluble in alcohol, ether, petroleum, naplita

flammable; dangerous fire risk; highly toxic; strong irritant; explosive limits in air 3.9 to 21.8%; tolerance 0.5 ppm in air

colourless volatile liquid; disagreeable odour; soluble in alcohol and ether; poorly soluble in water; mw. 62.13; d 0.845; m.p. -83 °C; b.p. 37.5

C; evolves SO» when heated; autoignition tempera­ ture 403 °F; flash point -36 °C

dangerous fire risk; moderate explosion risk; irritates mucous membranes; paralyzes voluntary muscles and finally also respiratory muscles; flammable limits in air 2.2 to 19.7%

mw. 94.20; m.p. -85 °C; b.p. 109 °C; d 1.046; flash point 24 C; flammable liquid; poorly soluble in water; offensive odour; toxicity greater than that of dimethyl sulphide; symptoms are similar, however irritation is stronger and paralysis of external respiration occurs without foregoing paralysis of locomotion

should be expected that this compound is also metabolized by microorganisms. Surprisingly enough, very little is known about microbial DMS metabolism. The current state of affairs regarding this subject is reviewed in the next paragraph. This lack in knowledge combined with the fact that DMS was rather recalcitrant in waste purification making use of biofilters of crushed, graded softwood bark (Sivela & Sundman, 1975) or of an activated sludge process (Imai, 1983), formed the incentive for the present study, which is mainly concerned with the isolation and characterization of organisms able to metabolize DMS. It was conducted to gain a better insight in the metabolic pathway(s) involved in the break­ down of this compound. It was anticipated that this knowledge would lead to an understanding of DMS recalcitrance and even­ tually to an improved purification procedure.

3. Microbial metabolism of dimethyl sulphide

Looking at it from a microbiologist's point of view, DMS is a rather "hybrid" compound. It is to be expected that organisms which can metabolize DMS are either able to use both the methyl groups and the sulphur moiety of this compound, or else live in a consortium where separate organisms are responsible for the breakdown of the different parts of DMS. Obvious candidate DMS-utilizers therefore might be colourless sulphur bacteria (and photosynthetic bacteria) with some methylotrophic character­ istics, or vice versa, and methanogens.

Having established the groups of organisms among which DMS-metabolizers are most likely to be found, by no means gives a clue to the way this compound is metabolized, since these groups not only harbour a number of different genera but also micro­ organisms with a widely divergent type of physiology. To make the reader acquainted with the terminology in the remainder of this section a summary of the most frequently used terms is given. Methylotrophic bacteria are those microorganisms able to grow on reduced C.-compounds (compounds without carbon-carbon bonds, such as methanol, formate, mono-, di- and trimethylamine) . Based on their nutritional characteristics they can be divided into two

Table 6. Different nutritional types of methylotrophic bacteria and their assimilatory pathways, red.: reduced; org.: organic; inorg.: inorganic; comp.: compound.

Nutritional type Carbon source red. other C0„

org. comp. comp.

V

Energy source Assimilatory pathway red. other inorg.

org. comp. comp. comp. Cl " O b l i g a t e methylotroph S e r i n e pathway RuMP pathway F a c u l t a t i v e methylotroph S e r i n e pathway RuMP pathway RuBP pathway g r o u p s , w h i c h c a n b e s u b d i v i d e d d e p e n d i n g o n t h e p a t h w a y by w h i c h t h e o r g a n i s m s b e l o n g i n g t o t h e s e g r o u p s i n c o r p o r a t e c a r b o n i n t o c e l l m a t e r i a l ( T a b l e 6 ) . T h e d i f f e r e n t p h y s i o l o g i c a l t y p e s o f s u l p h u r - o x i d i z i n g b a c t e r i a a r e g i v e n i n T a b l e 7 . A t p r e s e n t o n l y v e r y l i m i t e d d a t a a r e a v a i l a b l e o n t h e m i c r o b i a l m e t a b o l i s m o f DMS. I n 1 9 8 0 S i v e l a d e s c r i b e d t h e i s o l a t i o n o f a n o b l i g a t e l y c h e m o -l i t h o t r o p h i c , D M S - u t i -l i z i n g , T h i o b a c i -l -l u s s p e c i e s f r o m a b i o f i l t e r , w h i c h h a d b e e n u s e d t o p u r i f y w a s t e w a t e r a n d - g a s f r o m a p a p e r m i l l o f m e t h y l a t e d s u l p h u r c o m p o u n d s . T h i s o r g a n i s m ,

Table 7 . D i f f e r e n t p h y s i o l o g i c a l types of reduced sulphur compound o x i d i z i n g b a c t e r i a .

P h y s i o l o g i c a l type Carbon source CO. organic compound Energy source reduced organic sulphur compound compound Obligate chemolithoautotroph Facultative chemolithoautotroph Chemolithoheterotroph Heterotroph

designated Thiobacillus MSI, was able to co-metabolize DMS when grown in a chemostat under thiosulphate limitation. It appeared to gain energy from the oxidation of this compound, as was reflected by the increased cell yield on thiosulphate plus DMS-containing medium as compared to that on thiosulphate only medium. Thiobacillus MSI was reported to possess enzymes of the serine pathway, enabling it to assimilate the methyl groups of DMS into cell carbon, whereas enzymes necessary for methyl group dissimilation, formaldehyde- and formate dehydrogenases , were absent. This suggested that the above mentioned energy gain was due to the oxidation of the sulphur atom of DMS. Since some of the published data were open to question the organism was reevaluated for its DMS-utilizing potential and will be discussed further in chapter 2 of this thesis.

Very recently, Kanagawa and Kelly (1986) described the break­ down of dimethyl sulphide by mixed cultures of Pseudomonas sp. AK-2 and a Thiobacillus thioparus strain named TK-1. These organisms had been isolated from activated sludge acclimatized to 0,0-dimethyl phosphorodi thioate ( (CH-O) „P( S )SH ; Kanagawa et al., 1982). Strain TK-1 utilized dimethyl phosphorodithioate as energy source and oxidized it via 0,0-dimethyl phosphorothioic acid ((CH30)2P(S)0H) to dimethyl phosphoric acid ((CH30)2P(0)OH) and

sulphate, whereas strain AK-2, a facultative methylotroph, was responsible for the breakdown of dimethyl phosphoric acid to inorganic phosphate.

During cultivation on DMS both organisms appeared to persist, suggesting their coordinate action in the breakdown of this compound. However, Thiobacillus thioparus could be grown on its own on DMS. It oxidized this compound with an apparent K of

r m

45 uM. Experiments in which the organism was cultured on mixtures ' 14

of DMS and NaH C0~ showed that after an initial period during which only labelled carbon was incorporated into biomass, the percentage C in total cell carbon gradually decreased, indica­ ting that DMS-carbon was now also assimilated. This suggested that the methyl groups of DMS were first oxidized to C0„, which gradually diluted the labelled bicarbonate and therefore also the label in the biomass, rather than directly assimilated via the serine pathway as suggested by Sivela (1980). These authors found

that suspensions of the Thiobacillus thioparus sp. oxidized 1 mol of DMS with the concomitant utilization of 0.9-2.1 mol 02. It was

proposed that this would be consistent with the following hypothetical reaction:

(CH3)2S + 2 02 + 2 H20 2 CHQ0H + H-SO.

3 2 4 (1) However, methanol was not detected during growth and moreover, since the organism was unable to oxidize methanol, this would not

14

explain the results of the CO„-fixation experiments. Therefore they suggested dissimilation of DMS to occur via a pathway similar to the one described for Hyphomicrobium S (de Bont et al., 1981; Figure 2 ) , followed by assimilation of the formed CO». However, no data were given in support of this theory and unravelling of the metabolic pathway in this organism must await further experiments.

Certainly the observation that Thiobacillus thioparus, so far known as an obligate chemolithoautotroph, appears to grow auto-trophically on DMS is rather interesting. Other thiobacilli belonging to this physiological group have only been shown to be capable of assimilating some amino acids and other organic acids into their biomass up to approximately 20% of their total cell carbon (Smith et al., 1967; Kuenen & Veldkamp, 1973; Matin,

1978). Until now only facultative chemolithotrophs like Thiobacillus versutus (A„), Thiobacillus novellus and Paracoccus denitrificans , which grow chemolithoautotrophically on thiosul-phate (Chandra & Shethna, 1977; Gottschal & Kuenen, 1980a; Friedrich & Mitrenga, 1981), have been shown to be able to grow autotrophically on C^-compounds (Cox & Quayle, 1975; Chandra &

Shethna, 1977; Kelly & Wood, 1.982). A list of organisms able to (co)metabolize both reduced C.- and inorganic sulphur compounds is given in Table 8.

Zinder and Brock (1978c) reported the production of methane and carbon dioxide from MM and DMS by anaerobic lake sediments. Chloroform inhibited this activity, suggesting the involvement of methanogenic bacteria. This was not so surprising since many methanogens have been shown to be able to use reduced C,-com-pounds as growth substrates (Balch et a l . , 1979; Walther et al.,

°2

H

2<>2

C H3S H

^X?*

H2S

? - * - H

2

s q

;

O "20

CH

3

-§-CH

3

■ ^ — ^

NADH*H* NAD*

0

2

H

2

0

CH3-S-CH3 ^ >

2 a t

^

NADHUH* NAD*

assimilation in cell material

HCHO

V ^ ? » HCOOH J L > C0

2

NAD* NADH^H*

NAD» NADH»H«

Table 8. Organisms able to (co)metabolize reduced sulphur com­ pounds and C,-compounds . (Note: DMSO and DMS are "hybrid" com­ pounds belonging to both groups.)

Organism Assimilatory Reference pathway Hyphomicrobium EG Hyphomicrobium S Hyphomicrobium Z-3 Hyphomicrobium Z-116 Paracoccus denitrificans Rhodopseudomonas palustris Thiobacillus MSI Thiobacillus novellus Thiobacillus thioparus TK-1 Thiobacillus versutus (A„) (Thiocystis spp. (Unidentified methanogen This thesis De Bont et al. 1981 Vedenina 1987 Vedenina 1987

Friedrich & Mitrenga 1981 ; Cox & Quayle 1975

Yoch & Lindstrom 1967 RuBP/serine Sivela 1980

RuBP Chandra & Shethna 1977

RuBP Kanagawa & Kelly 1986 RuBP Kelly & Wood 1982 RuBP Zeyer et al. 1987)

Kiene et al. 1986) serine serine serine serine RuBP RuBP

1981; Sowers & Ferry, 1983). Upon addition of micromolar concen­ trations of C-MM to anoxic freshwater sediments labelled methane and carbon dioxide were produced in a ratio of 3:1,

14

whereas with C-DMS this ratio was about 7:1. The former ratio indicated that MM was fermented according to the following equation:

4 CH3SH + 2 H20 -> 3 C H4 + 4 H2S + C 02 (2)

In order to explain the higher ratio found with labelled DMS the authors suggested that MM could also be metabolized as follows:

Indeed, when the sediments were incubated with MM in a hydrogen atmosphere the ratio increased. However, this could very well be due to catalysis of the following reaction which is well established in methanogens:

CO, + 4 H, C H4 + H20 (4)

Although methanogens seemed to be responsible for the DMS and MM utilization in these environments, the authors did not succeed in isolating methanogens capable of DMS and/or MM metabolism from these sediments. Neither could they show the utilization of these compounds by pure cultures of Methanobacterium ruminantium, Methanobacterium thermoautotrophicum and Methanosarcina barker!.

Recently, Kiene et al. (1986) showed that upon addition of 14

micromolar concentrations of C-DMS to a diversity of anoxic 14

sediments, ranging from fresh water to hypersaline, CH, and 14

CO,, were produced in a ratio of- 0.06. When molybdate, an inhibitor of sulphate reduction (Oremland & Taylor, 1978), was added this was increased to 1.8, whereas addition of 2-bromo-ethanesulphonic acid, an inhibitor of methanogenesis (Gunsalus et al., 1978) decreased it to 0, but did not block C0„ production. This very elegant work suggested that sulphate reducers and methanogens were competing for DMS when it was present in low concentrations. In high concentrations DMS appeared to be a substrate for methanogens only. An obligately methylotrophic methanogen was isolated which was capable of growth on DMS. This organism metabolized DMS, with a transient appearance of MM, giving rise to a CH,/C0„ ratio of 2.8 suggesting DMS metabolism according to: (CH3)2S + 2 HT + 2 e' CH. + CH-SH 4 3 (5) CH-SH + H20 -» 0.5 CH. + 0.5 C0„ + H„S + 2 HT + 2 e (6) 4 2 2 Sum: (CH3)2S + H20 1.5 C H4 + 0.5 C 02 + H2S (7)

Again, the increase in CH /C0„ ratio upon addition of molybdate, could have resulted from anaerobic, autotrophic hydrogen oxida­ tion according to equation 4 since sulphate reducers normally

make use of the H„ present. In a hydrogen atmosphere the authors indeed found an increased ratio of 1.4.

The results of these workers are difficult to reconcile with those of Zinder and Brock (1978c) and it seems that work with 14 35

C S-DMS might shed some light on the reason for these differences .

Very recently, Zeyer et al . (1987) reported the oxidation of DMS to DMSO by enrichment cultures of phototrophic purple bacteria, and a Thiocystis species isolated from these cultures. This oxidation appeared to be dependent on light intensity: it did not take place in cultures incubated in the dark and it was slow in cultures exposed to full daylight. High concentrations of sulphide (>1 mM) also reduced this DMS oxidation. The authors proved that DMS supported growth of these cultures and their experiments indicated that the electrons liberated during the oxidation of DMS to DMSO were quantitatively used to reduce C0~ to biomass.

De Bont et al. (1981) isolated a Hyphomicrobium species, Hyphomicrobium S, using ordinary garden soil as an inoculum for batch enrichment cultures on dimethyl sulphoxide (DMSO) as the sole source of carbon, sulphur and energy. The organism proved to be an obligate methylotroph which could only grow very slowly on DMSO (its u was 0.017 h ) and unlike other hyphomicrobia _{/ m a x ' '}

v could not be grown, either aerobically or anaerobically, on any

other C,-compound. The authors showed that Hyphomicrobium S reduced DMSO to DMS with a mainly NADH-dependent DMSO reductase. Since oxidation of DMS in cell-free extracts of the organism only occurred under aerobic conditions in the presence of NADH it seemed likely that a mono-oxygenase was responsible for the oxidation of DMS to formaldehyde and MM. Indeed, accumulation of MM (but not of formaldehyde) was shown to occur in whole cells upon addition of DMS in the presence of ethyl mercaptan, which partly inhibited MM oxidation. The authors did not succeed in following the formation of metabolic products of MM oxidation in cell suspensions of the organism. However, upon addition of ethyl mercaptan to whole cells they found an accumulation of acetal-dehyde, suggesting formaldehyde to be a product of MM breakdown. The sulphur moiety of DMSO was ultimately oxidized to sulphuric

acid and formaldehyde could either be dissimilated to C0~ by way of NAD -dependent formaldehyde and formate dehydrogenases present in the cells or assimilated via the serine pathway. This led de Bont et al. (1981) to propose the metabolic pathway illustrated in Figure 2.

The main questions which remained unanswered in this study were how the organism metabolized MM, whether it gained energy from the oxidation of the sulphur moiety of DMS(O) and how DMS(O) metabolism was regulated.

In 1982 we isolated a Hyphomicrobium species which could grow relatively rapidly on DMSO and DMS, and it was decided to study both its carbon and sulphur metabolism. Therefore some of the general characteristics of this me thylotrophic genus and its metabolic peculiarities are discussed in the next section. As the DMS(0)-metabolizing Hy phomicrobium species is not only a methylo-troph but also a sulphur oxidizer, some of the relevant litera­ ture on the physiology and ecology of the colourless sulphur bacteria is reviewed in section 5.

4.1 General characteristics of hyphomicrobia

The genus Hyphomicrobium belongs to the group of prosthecate bacteria which reproduce by a budding process. Other members of this group include the genera Hyphomonas and Pedomicrobium (Bergey's Manual of Determinative Bacteriology 8th Edition, 1974). Bacteria of the latter genus are morphologically different from those belonging to the other two: they produce hyphae from several sites of the cell surface, whereas in Hyphomicrobium and Hyphomonas these only emanate from one or both poles of the cell (Harder & Attwood, 1978). Hyphomicrobium and Hyphomonas can be

discriminated on the basis of their nutritional requirement. It is now generally acknowledged that hyphomicrobia only grow on reduced C,-compounds, a limited number of C,-compounds and 3-hydroxybutyrate which are metabolized via acetyl-CoA whereas Hyphomonas spp. are unable to grow on C,-compounds (Harder & Attwood, 1978).

they were outcompeted by other microorganisms in enrichment cultures on high concentrations of substrate. In the course of time however, isolation procedures were developed based on the organism's ability to grow oligotrophically (Zavarzin, 1960; Hirsch & Conti, 1964) and/or on its capacity to grow on methanol under anaerobic conditions using nitrate as an electron acceptor (Sperl & Hoare, 1971; Attwood & Harder, 1972). Since the latter

technique rapidly yields pure cultures of hyphomicrobia it is now frequently used in the isolation of these bacteria. The organism's ability to thrive under oligotrophic conditions also was responsible for the false claims of its slow growth on C,-C,-compound s.

Hyphomicrobia can be recognized by their rather distinct life cycle, which was studied in detail by Moore and Hirsch (1973). Reproduction takes place by a budding process followed by an asymmetric division. This process is initiated by polar wall growth resulting in the formation of a filament or hypha at one end of a mature mother cell. At the end of the hypha a swelling is formed which will develop into a bud. This will grow one to three polar or subpolar flagellae, after which a septum is formed in the filament just below the bud. The still immature bud will then break loose and mature further during the so-called swarmer cell stage of the life cycle. Upon maturation the swarmer will shed its flagellae at which point it becomes a mother cell. After a reproductive cycle the hypha of a mother cell will recommence growth and go through another cycle of bud formation. Older cells may have branched hyphae, but bud formation will only occur at the end of one branch (Hirsch, 1974). Under certain environmental conditions (e.g. in the presence of high concentrations of Fe , Mn , NOo ) the lyfe cycle is more complex than described above, and polymorphic forms of hyphomicrobia can be observed, ranging from Y-shaped cells and microcolonies to complex cell aggregates

(Harder & Attwood, 1978).

Hyphomicrobia are found in both oligotrophic and eutrophic environments such as lakes, brooks, seas, acid mine water, drinking water distribution systems, sewage sludge and polluted streams (Harder & Attwood, 1978). Furthermore, they have been found in close association with both fresh water and marine algae

(Geitler, 1965), which are able to produce DMS (see also Table 2 ) .

4.2 Biochemistry of hyphomicrobia

In the past years excellent reviews have been written on C,-metabolism in general (Anthony, 1975, 1982; Quayle, 1972, 1980a, 1980b; Higgins et al., 1981), whereas several other publications focussed more in particular on C,-metabolism in hyphomicrobia (Harder & Attwood, 1978; Meiberg, 1979). Therefore, this section only means to give a short summary of the typical C,-metabolic pathways operating in hyphomicrobia . The routes for the dissimi­ lation and assimilation of C.-compounds will be treated separately in the next two paragraphs.

A.2.1 Oxidation of C,-compounds

The dissimilation of C.-compounds generally proceeds through their oxidation via formaldehyde and formate to C0„ (Figure 3 ) .

The oxidation of methanol is catalyzed by an ammonia or methylamine activated methanol dehydrogenase (MDH) which oxidizes a rather wide range of primary alcohols. In vitro it uses phena-zine methosulphate or Wurster's blue as an electron acceptor and

o

2

V i

H

2

0

■^~- CH

3

0H

CH

Z

NADH.H* NAD*

N-mtthyl compounds P Q Q P Q Q H2 H2O HCHO ^ * -X -X H2 HCOOH

_{7 ^ c o}

2

NAD* NADH»H*

Figure 3. General pathway of C.-compound oxidation.

1= methane monooxygenase; 2= methanol dehydrogenase; 3= formalde­ hyde dehydrogenase; 4 = formate dehydrogenase (Anthony, 1982).

it h a s a p H - o p t i m u m of 9 ( A n t h o n y , 1 9 8 2 ) . S a l i s b u r y et a l . ( 1 9 7 9 ) and Duine and h i s c o l l e a g u e s (for a r e v i e w of their work see D u i n e & F r a n k , 1 9 8 1 ) i n d e p e n d e n t l y c h a r a c t e r i z e d the p r o s t h e t i c g r o u p of t h i s e n z y m e , w h i c h t h e y named p y r r o l o - q u i n o l i n e q u i n o n e ( P Q Q ) , and w h i c h p r o v e d to b e a 2e /2H redox c a r r i e r ( D u i n e et a l . , 1 9 8 1 ) . E v e r y MDH m o l e c u l e a p p e a r e d to c o n t a i n two of t h e s e p r o s t h e t i c g r o u p s ( D u i n e et a l . , 1 9 8 1 ) , w h i c h could o c c u r in e i t h e r of t h r e e f o r m s : an o x i d i z e d ( q u i n o n e ) form P Q Q , a r e d u c e d ( q u i n o l ) f o r m P Q Q H „ and a q u i n o n e free r a d i c a l form ( P Q Q H ' ) . T h e i s o l a t e d M D H is a m i x t u r e o f M D H , and M D H , and c o n t a i n s

red ox 1

P Q Q H ' . Upon a d d i t i o n of m e t h a n o l the a b s o r p t i o n s p e c t r u m and the p r o p e r t i e s and c o n t e n t of P Q Q H ' r e m a i n e d u n c h a n g e d , s u g g e s t i n g that the M D H , form of the e n z y m e is in a half o x i d i z e d s t a t e

ox 1 ' u n a b l e to c o n v e r t s u b s t r a t e i n t o p r o d u c t . T h e c o m p l e t e l y o x i d i z e d form of t h i s e n z y m e (MDH ) i s p r o d u c e d by e l e c t r o n t r a n s f e r to ox r J an e l e c t r o n a c c e p t o r ( e . g . b u r s t e r ' s b l u e ) in the p r e s e n c e of the a c t i v a t o r N H - . M D H c o n t a i n s v i r t u a l l y no free r a d i c a l and _{3 ox} J r e a c t s with s u b s t r a t e ( S ) to form an a d d u c t MDH - S . T h i s r e a c t i o n p r o b a b l y r e q u i r e s a high p H . F i n a l l y the r a t e - d e t e r ­ m i n i n g step of the r e a c t i o n , the c o n v e r s i o n of m e t h a n o l , w h i c h a l s o r e q u i r e s N H „ , t a k e s p l a c e , y i e l d i n g M D H , ( D u i n e et a l . , 1 9 8 6 ) . D u r i n g t h i s c a t a l y t i c c y c l e o - q u i n o n e is r e d u c e d to q u i n o l .

M D H is c o u p l e d to the e l e c t r o n t r a n s p o r t c h a i n at the level of c y t o c h r o m e c. R e c e n t l y it w a s shown that a n a e r o b i c a 1 1 y i s o l a t e d p r e p a r a t i o n s of t h i s e n z y m e c o n t a i n e d a heat s t a b l e , low m o l e c u l a r w e i g h t f a c t o r , w h i c h e n h a n c e d the rate of c y t o c h r o m e c r e d u c t i o n via M D H . T h i s f a c t o r could r e p l a c e the a c t i v a t o r NH_ in the MDH d y e - a s s a y at pH 9. I t s a c t i v i t y w a s d e s t r o y e d by 0~ in the p r e s e n c e of M D H and c y t o c h r o m e c, w h i c h r e n d e r e d the e n z y m e N H o - d e p e n d e n t ( D u i n e et a l . , 1 9 8 6 ) . F o r m a l d e h y d e o x i d a t i o n /in h y p h o m i c r o b i a is m e d i a t e d e i t h e r by a N A D ( P ) - l i n k e d G S H - ( i n ) d e p e n d e n t f o r m a l d e h y d e d e h y d r o g e n a s e , a g e n e r a l a l d e h y d e d e h y d r o g e n a s e , or m e t h a n o l d e h y d r o g e n a s e ( A n t h o n y , 1 9 8 2 ) . T h e r e l a t i v e i m p o r t a n c e of these e n z y m e s in the o x i d a t i o n of f o r m a l d e h y d e w a s s t u d i e d by M a r i s o n and A t t w o o d ( 1 9 8 0 ) . S i n c e a l m o s t a l l of t h e c o m p o u n d s h y p h o m i c r o b i a are a b l e to g r o w on a r e m e t a b o l i z e d v i a the i n t e r m e d i a t e f o r m a t i o n of an

a l d e h y d e , it is d i f f i c u l t to draw any c o n c l u s i o n s about the i n v o l v e m e n t of an a l d e h y d e d e h y d r o g e n a s e in the m e t a b o l i s m of f o r m a l d e h y d e by these m i c r o o r g a n i s m s . T h e a u t h o r s m e n t i o n e d above t h e r e f o r e studied c e l l - f r e e e x t r a c t s of v a r i o u s f a c u l t a t i v e m e t h y l o t r o p h s grown on C . - c o m p o u n d s or m u l t i - c a r b o n s u b s t r a t e s . They showed that these c o n t a i n e d a d y e - l i n k e d f o r m a l d e h y d e d e h y d r o g e n a s e i r r e s p e c t i v e of the g r o w t h c o n d i t i o n s of these b a c t e r i a , that the a c t i v i t y of this e n z y m e w a s low w h e n compared to that of other C . - s p e c i f i c e n z y m e s and that it was not induced during g r o w t h on C , - s u b s t r a t e s . In view of this it seemed u n l i k e l y that the d y e - l i n k e d a l d e h y d e d e h y d r o g e n a s e a c t i v i t y found in c e l l - f r e e e x t r a c t s of h y p h o m i c r o b i a is of any i m p o r t a n c e in C , - m e t a b o l i s m . K o h i e r and S c h w a r t z ( 1 9 8 2 ) h o w e v e r came to an o p p o s i t e c o n c l u s i o n . They s u b j e c t e d c e l l - f r e e e x t r a c t s of s e v e r a l m e t h a n o l - g r o w n H y p h o m i c r o b i u m s t r a i n s to p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s and p e r f o r m e d a c t i v i t y tests on these gels with n i t r o b l u e t e t r a z o l i u m in the p r e s e n c e of f o r m a l d e h y d e or b e n z a l -d e h y -d e as a s u b s t r a t e . E v e r y e x t r a c t was teste-d with both s u b s t r a t e s , each on a s e p a r a t e g e l . C o m p a r i s o n of these gels showed that in some c a s e s t h e i n t e n s i t y of the a c t i v e bands (which were v i s i b l e as black p r e c i p i t a t e s of f o r m a z a n ) was a l m o s t e q u a l , l e a d i n g the above a u t h o r s to c o n c l u d e that dye-linked a l d e h y d e d e h y d r o g e n a s e s were i n v o l v e d in f o r m a l d e h y d e o x i d a t i o n . In gels c o n t a i n i n g c e l l - f r e e e x t r a c t of H y p h o m i c r o b i u m X h o w e v e r , the i n t e n s i t y of the e n z y m e b a n d s a c t i v e w i t h f o r m a l d e h y d e was only a f r a c t i o n of that of b a n d s a c t i v e with b e n z a l d e h y d e . Since the o x i d a t i o n rate of f o r m a l d e h y d e in the tested s t r a i n s was s i m i l a r , it is clear that H y p h o m i c r o b i u m X must c o n t a i n a n o t h e r f o r m a l d e h y d e d e h y d r o g e n a s e .

M a r i s o n and Attwood ( 1 9 8 0 ) s h o w e d that both H y p h o m i c r o b i u m X and H y p h o m i c r o b i u m v u l g a r e 3 c o n t a i n e d a m e t h a n o l d e h y d r o g e n a s e , w h i c h showed a high a c t i v i t y with f o r m a l d e h y d e , w h e r e a s the latter o r g a n i s m a l s o c o n t a i n e d a NAD - d e p e n d e n t f o r m a l d e h y d e d e h y d r o g e n a s e . T h i s s u g g e s t e d that these two e n z y m e s could be r e s p o n s i b l e for f o r m a l d e h y d e o x i d a t i o n in h y p h o m i c r o b i a . Since MDH - m u t a n t s showed u n i m p a i r e d f o r m a l d e h y d e o x i d a t i o n rates and w h o l e c e l l s of H y p h o m i c r o b i u m X in which M D H was blocked by c y c l o p r o p a n o l c o n t i n u e d to c o n v e r t f o r m a l d e h y d e , the role of MDH

NAD(P)H*Ht NAD(P)*-HCHO (CH3)2NH | ^ § ^ NH3 ♦ PQQH2 XH2 " z O l ' P Q Q NAD(P)H.H*. v

5

COOH i NAD(P)*> ^H2Q HCHOl H20 jHCHO CH3NH2 ( C H2)2 NH2CHCOOH C-NHCH3 (CH2)2 NH2CHCOOH N H3 -H₂70 U2 ,13 H20 ^NH-; HCHO COOH ?H3 , 1 0=CCOOH « ( C H2)2 / NH2CHCOOH / ~ * N H3 CH NH2CHCOOH COOH ( C H2)2 CH3NHCHCOOH

v

cooH j yr*

( C H2)2 ^ 9 < ' fHCHÖl X* H2 NHCHCOOH ' ^ NAD(P)H*H* NAD(P)* CH3 CH3NHCHCOOH XH2

Figure 4. Dissimilation of N-methyl compounds to formaldehyde. 1= tetramethylammonium monooxygenase; 2= TMA monooxygenase; 3= TMAO demethylase; 4 = TMA dehydrogenase; 5= DMA monooxygenase; 6 = DMA dehydrogenase; 7 = y-glutamylmethylamide synthetase; 8= N-methyIglutamate synthase; 9= N-N-methyIglutamate dehydrogenase; 10= N-methylalanine synthase; 11= N-methylalanine dehydrogenase; 12= de'amination of alanine.; 13= methylamine oxidase; 14= methylamine dehydrogenase (Meiberg, 1979; Anthony, 1982).

in f o r m a l d e h y d e o x i d a t i o n in vivo is q u e s t i o n a b l e ( D u i n e et a l . , 1 9 8 6 ) . N a t u r a l l y it c a n n o t be excluded that d i f f e r e n t s i t e s of MDH c a t a l y z e m e t h a n o l and f o r m a l d e h y d e d e h y d r o g e n a t i o n , in w h i c h case MDH could still be r e s p o n s i b l e for f o r m a l d e h y d e o x i d a t i o n . R e c e n t l y , E g g e l i n g and Sahm ( 1 9 8 4 , 1985) discovered a NAD - d e p e n ­ dent f o r m a l d e h y d e d e h y d r o g e n a s e in R h o d o c o c c u s e r y t h r o p o l i s , which required an e x t r a compound for a c t i v i t y . This f a c t o r proved to be h e a t - s t a b l e and r e d u c i b l e . F u r t h e r research proved that it was neither G S H , T H F , PQQ nor c o e n z y m e A (Eggeling & S a h m , 1 9 8 5 ; Duine et a l . , 1 9 8 6 ) . A s i m i l a r factor and e n z y m e proved to be present in m e t h y l a m i n e g r o w n T h i o b a c i l l u s v e r s u t u s and m e t h a n o l -grown H y p h o m i c r o b i u m X (Duine et a l . , 1 9 8 6 ) . This m i g h t i n d i c a t e that this e n z y m e is r e s p o n s i b l e for f o r m a l d e h y d e o x i d a t i o n in hyphomic r o b i a .

In most m e t h y l o t r o p h s f o r m a t e is o x i d i z e d by a NAD - d e p e n d e n t formate d e h y d r o g e n a s e . In H y p h o m i c r o b i u m X t h i s e n z y m e is inhibited by NADH and ATP and t h e r e f o r e might play a role in the r e g u l a t i o n of the e x t e n t of a s s i m i l a t i o n and d i s s i m i l a t i o n of f o r m a l d e h y d e ( M a r i s o n , 1 9 8 0 ) .

A s c h e m a t i c r e p r e s e n t a t i o n of the v a r i o u s e n z y m e s involved in the m e t a b o l i s m of some m e t h y l a t e d a m i n e s is given in F i g u r e A. In h y p h o m i c r o b i a t r i m e t h y l a m i n e is oxidized by way of a NAD -i n d e p e n d e n t d e h y d r o g e n a s e ( M e -i b e r g , 1 9 7 9 ) , w h e r e a s d-ime t h y l a m -i n e o x i d a t i o n is m e d i a t e d by a m o n o - o x y g e n a s e u n d e r a e r o b i c and by a d e h y d r o g e n a s e under a n a e r o b i c c o n d i t i o n s (Meiberg et a 1. , 1 9 8 0 ) . The o x i d a t i o n of m e t h y l a m i n e in h y p h o m i c r o b i a p r o c e e d s via the ( i n d i r e c t ) f o r m a t i o n of N - m e t h y l g l u t a m a t e ( M e i b e r g & H a r d e r , 1 9 7 8 ) and its s u b s e q u e n t o x i d a t i v e d e m e t h y l a t i o n by way of a NAD - ( i n ) d e p e n d e n t d e h y d r o g e n a s e ( A n t h o n y , 1 9 8 2 ) .

R e c e n t l y , G h i s a l b a et a l . ( 1 9 8 6 ) isolated 28 H y p h o m i c r o b i u m spp. able to u t i l i z e m e t h y l p h o s p h a t e s and m e t h y l p h o s p h o n a t e s as the sole s o u r c e of c a r b o n and e n e r g y . T h e s e s t r a i n s could not only grow on m e t h y l a t e d p h o s p h o r c o m p o u n d s , but a l s o on m e t h y ­ lated a m i n e s , m e t h a n o l and C ^ - c o m p o u n d s such as e t h a n o l and a c e t a t e , and t h e r e f o r e were restricted f a c u l t a t i v e m e t h y l o t r o p h s . The b r e a k d o w n of the m e t h y l a t e d p h o s p h a t e s w a s very s i m i l a r to that of m o n o m e t h y l s u l p h a t e , which was e a r l i e r shown to be m e t a b o ­ lized by h y p h o m i c r o b i a via m e t h a n o l and s u l p h a t e ( G h i s a l b a &

Kiienzi, 1983). All isolates proved to degrade methy Iphospha tes to phosphate and methanol, which was subsequently utilized as the growth substrate.

The same researchers (Schar et al., 1986) also studied the break­ down of dimethyl formamide. They proved that the Pseudomonas spp. responsible for the degradation of this compound were first hydrolyzing it by way of a novel enzyme, N,N-dimethy1 formamidase, to dimethylamine and formate. The latter compounds were further degraded to ammonia and carbon dioxide, following known metabolic pa thwa ys.

4.2.2 Assimilation of C,-compounds

The main problem methylotrophs are faced with during growth on C.-compounds is the formation of carbon-carbon bonds, in order to synthesize C~-compounds . These "units" in turn can function as building blocks in the metabolic routes leading to the formation of the main cell constituents, which are rather universal in different organisms.

In principle three different pathways can be used in the aerobic bacteria for the synthesis of Co-compounds from one-carbon compounds namely:

- The ribulose bisphosphate cycle of CO- fixation (Bassham et al., 1954), which is the autotrophic mode of growth on C,-compounds and which is confined to a few facultative autotrophs, which can gain energy from light or the oxidation of reduced inorganic substrates, and Pseudomonas oxalaticus (Anthony, 1982).

- The ribulose monophosphate cycle of formaldehyde fixation (Kemp, 1964; Str«Sm et al . , 1974), which has four potential variants and is rather widespread among both obligate and facultative me thy lotrophs and is the only route for one-carbon compound assimilation in obligate methanotrophs (Anthony, 1982).

- The serine pathway in which both formaldehyde and C0„ are fixed (Large et al., 1961; Bellion & Hersh, 1972), and which also functions in a wide range of obligate and facultative

methylo-3- phosphoglycerat* 10 2 2-phosphoglycerate cell material

X

H20 phosphoenol pyruvate

co

2

serine pathway lloacel

f NADH.H* r V N A D * Pi oxalodcetate malate C o A y - ATP ' V V A D P - P I malyl-CoA 15. 2glyoxylate ^ -« J6. - - acetyl-CoA J17 2formaldehyde * - 2N5'K)-methylene THF citrate isocitrate I ^ N A D P * ^1 9 IK succinate T NADPH*H* oxaloacetate N^-methenyl THF 120 F P H2 4

A'

I C L * glyoxylate cycle fumarate X H2 THF ATP A D P . P i formate ; * ' ' * ■f* - N,0-formyt THF malate N A D * F i g u r e 5. S e r i n e pathway of C , - c o m p o u n d a s s i m i l a t i o n . 1= n o n e n z y m a t i c r e a c t i o n ; 2= f o r m a l d e h y d e d e h y d r o g e n a s e ; 3= f o r m y l T H F s y n t h a s e ; 4 = m e t h e n y l T H F c y c l o h y d r o l a s e ; 5= m e t h y l e n e T H F d e h y d r o g e n a s e ; 6 = s e r i n e t r a n s h y d r o x y m e t h y l a s e ; 7 = s e r i n e -g l y o x y l a t e a m i n o t r a n s f e r a s e ; 8= h y d r o x y p y r u v a t e r e d u c t a s e ; 9 = g l y c e r a t e k i n a s e ; 10= p h o s p h o g l y c e r a t e m u t a s e ; 11= e n o l a s e ; 12= P E P c a r b o x y l a s e ; 13= raalate d e h y d r o g e n a s e ; 14= m a l a t e t h i o k i n a s e ; 15= m a l y l - C o A l y a s e ; 16= u n k n o w n r e a c t i o n s ; 17= c i t r a t e s y n t h a s e ; 18= a c o n i t a s e ; 19= i s o c i t r a t e l y a s e ; 20= s u c c i n a t e d e h y d r o g e n a s e ; 21= f u m a r a s e ; ICL= i s o c i t r a t e lyase ( A n t h o n y , 1 9 8 2 ) .

trophs (Anthony, 1982).

A schematic representation of the latter pathway which operates in hyphomicrobia is given in Figure 5. During its functioning two molecules of formaldehyde condense with two molecules of glycine resulting in the formation of two serine molecules. These undergo a series of reactions giving two 2-phosphoglycerate molecules, of which one is assimilated into cell material (via 3-phospho-glycerate) and the other is converted into phosphoenolpyruvate (PEP). Via car boxy la t ion of this compound the third C.-unit (CO,,) is introduced into the cycle yielding oxaloacetate and subse­ quently malate. Malate is activated to malyl-CoA, which is cleaved to acetyl-CoA and glyoxylate which is converted to glycine. To complete the pathway another molecule of glyoxylate should be regenerated from acetyl-CoA. In microorganisms possessing isocitrate lyase this is achieved via the glyoxylate cycle. Organisms lacking this enzyme including hyphomicrobia operate a so-far unknown acetyl-CoA oxidation route yielding glyoxylate.

A.2.3 Metabolism of C~-compounds

During growth on ethanol Hyphomicrobium spp. induce a NAD -dependent ethanol dehydrogenase which converts ethanol into acetaldehyde. This in turn is oxidized either to acetyl-CoA by a CoA-dependent acetaldehyde dehydrogenase or to acetate by the action of a NAD -dependent dehydrogenase. Acetate is subsequently metabolized to acetyl-CoA by acetothiokinase. This reaction was also shown to occur in acetate-grown hyphomicrobia.

Since 3-hydroxybutyrate is converted to two molecules of acetyl-CoA, growth on this C,-compound is in effect similar to that on a C-j-compound (Attwood & Harder, 1973, 1974). Acetyl-CoA enters the tricarboxylic acid cycle, giving rise to the production of energy and reducing power. As mentioned above hyphomicrobia lack isoci­ trate lyase, and thus the route whereby cell constituents are made from acetyl-CoA remains to be elucidated.

4.2.4 Basis for restricted methylotrophy in hyphomicrobia

Hyphomicrobia are so-called restricted facultative methylo-trophs with a rather limited substrate range being C,- and a few C~-compounds and 3-hydroxybutyrate. In some obligate methylo-trophs the inability to utilize other substrates than the one-carbon compounds can be attributed to the lack of 2-ketoglutarate dehydrogenase (e.g. Colby & Zatman, 1972, 1975). This results in an incomplete TCA cycle, preventing these organisms from gaining energy and reducing power from it. The same situation exists in the obligate chemolithotrophs, which also lack or have very low concentrations of this enzyme (e.g. Smith et al., 1967; Kelly, 1971; Kuenen & Veldkamp, 1973; Matin, 1978). However, since some hyphomicrobia are able to grow on C„-compounds which are metabo­ lized via acetyl-CoA, they must possess a complete TCA cycle. Studies on cell-free extracts of Hyphomicrobium X and G showed this indeed to be the case and demonstrated that these organisms lack pyruvate dehydrogenase (Attwood & Harder, 1973, 1974), preventing them to convert C_-C, compounds into acetyl-CoA, needed for the functional operation of the TCA cycle. (Other pyruvate metabolizing enzymes like pyruvate carboxylase, malic enzyme (malate dehydrogenase; decarboxylating, NAD(P) ) and PEP synthase were also absent, see Figure 6.) Evidence for this key role of the pyruvate dehydrogenase complex was obtained in work with mutants of the facultative methylotroph Pseudomonas AMI , which had lost the activity of this enzyme. This privation resulted in a restricted facultatively methylotrophic behaviour of the organism, i.e. these mutants could only grow on C,- and C?-compounds and 3-hydroxybutyrate (Bolbot & Anthony, 1980).

Further proof indicating the lack of this enzyme to form the basis for the restricted facultative methylotrophy in hyphoml-crobia came from experiments in which the cloned pyruvate dehydrogenase genes from Escherichia coli were introduced into Hyphomicrobium X (Dijkhuizen et al., 1984). This conferred the ability to grow on pyruvate and succinate upon the organism. Since no other novel growth substrates were identified, it seems likely that the organism has additional metabolic lesions, as was suggested by the work of Harder et al. (1975). They showed that

Cj-compounds C_/C,-compound» 3 O C 3 -compounds formaldehyde pyruvate Cj-compounds 3-tiydroxybutyrate energy

Figure 6. Basis for restricted facultative methylotrophy in hyphomicrobia (Harder & Attwood, 1978).

1= PEP carboxylase; 2= malate dehydrogenase; 3= pyruvate kinase; 4= PEP synthase; 5= pyruvate carboxylase; 6= malie enzyme= malate dehydrogenase (decarboxylating; NAD(P)); 7= pyruvate dehydro­ genase .

during growth of Hyphomicrobiuro X on methanol in the presence of 14

various C-labelled compounds, several of these (e.g. pyruvate and succinate) accumulated inside the cells, whereas others (e.g. citrate and glucose) did not, indicating the absence of transport systems for the latter compounds.

Upon addition of C„-C, compounds to m or ethanol-limited chemostat cultures of hyphomicrobia only a small (0-5%) increase in cell yield was observed (Meiberg, 1979), whereas the increase could amount to about 30% when these compounds were added to cultures of obligate chemolithotrophs (Smith et al., 1967; Kelly, 1971; Kuenen & Veldkamp, 1973; Matin, 1 9 7 8 ) . Harder and Attwood (1978) suggested that growth of hyphomicrobia on methanol or methylated amines was energy-limited, rather than carbon-limited. This hypothesis was based on the rapid non-enzymic condensation reaction of tetrahydrofolate (THF) with formaldehyde, which is known to take place in these organisms. They postulated that formaldehyde would only become available for the dissimilatory enzymes (and yield energy) when all THF would be in the methylene-THF form. Since ATP is needed for the operation of glycerate kinase and 3-phosphoglycerate kinase in the serine pathway this cycle would be blocked until all THF in the cell would be bound. Only then would ATP be generated from formaldehyde, activating the kinases resulting in carbon assimi­ lation. Although this theory explains the lack of cell yield increase of C.-compound grown cultures of Hyphomicrobium spp. supplied with additional carbon sources, it gives no clue why addition of these compounds to e.g. ethanol-grown hyphomicrobium cultures (which gain energy through the operation of the TCA cycle) had no influence on their yield either. Therefore eluci­ dation of this matter must await further experimental evidence.

A.3 Physiology of hyphomicrobia

As mentioned above hyphomicrobia are unable to use multi-carbon compounds as additional multi-carbon source. Furthermore, their affinity constant for methanol e.g. is not significantly lower than that of other methylotrophs (Meiberg, 1979), which poses