Development of a process for the production of propylene oxide in methane oxidising bacteria

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Propylene O xide in M e th a n e -o x id isin g B a c t e r ia

by

Motoshl Suzuki

T his t h e s is i s p re s e n t e d fo r the d e g re e o f Doctor o f P h ilosop h y

Department o f B i o l o g i c a l Sciences

U n iv e r s ity o f Warwick

ACKNOWLEDGMENTS

SUNNARY

LIST OP FIGURES

LIST OF TABLES

CHAPTtat 1 G e n e r a l In trodu ction

1.1 The concept o f a e th y lo tro ph y

1.2 O ccu ren ce, ecology and h i s t o r y o f

th e i s o la t i o n o f a e t h a n e -o x id is in g b a c t e r i a

1.3

1.4

1 .4 .1

1 .4 .2

1 .4 .3

1 .4 .4

1 .4 .5

1 .4 .6

C l a s s i f i c a t i o n o f a e t h a n e -o x id is in g b a c t e r i a

P h y s io lo g y and b lo c h e a la t r y o f o b li g a t e a e t h a n e -o x id is in g b a c t e r i a

B a s i c growth re q u ir e s e n ts

C arb o n a e t a b o lis a

C^-coapound a s a i a ll a t i o n pathways

R lb u lo s e aonophosphate pathway (RKP)

S e r i n e pathway

R i b u lo s e diphosphate pathway o f C0_ a s s l a i l a t i o n

1.5

1

.

1 .5 .3

1 .6

1.10

1.11

Methane oxid ation

TWo types o f aethane aonooxygenase. s o lu b le - a n d p a rt lc u la te -IM O

S o l u b l e aethane aonooxygenase

P a r t i c u l a t e aethane aonooxygenase

M ethanol o x id a tio n

G row th on aethanol and IMO a c t iv i t y

Secon dary a lc o h o l o x id a t io n

Formaldehyde o x id a tio n

F o rn a te oxid ation

The in d u s t r i a l a p p lic a t i o n s o f a e th y lo tro p h s

PAGE NO.

oxygenated compounds

22

1.13 I s »e th a n o l dehydrogenase a b le to su p ply

e le c t ro n s to the UNO? 27

1.14 The s t a b i l i t y o f b i o c a t a ly s t 28

1.14.1 The s t a b i l i t y o f the s o lu b le MMO 28

1.14.2 The s t a b i l i t y o f the p a r t ic u la t e MMO 31

1.15 In h i b it io n and I n a c t iv a t io n o f b io c a t a ly s t 32

1.15.1 I n h i b it io n o f NMO by c h e la t o rs 32

1.15.2 In h i b it io n o f c e l l growth by n i t r i t e 33

1.16 I n a c t iv a t io n o f NNO by ace tyle n e 34

1.17 I n a c t iv a t io n o f b i o c a t a l y s t by epoxides 36

1.16

1.19 S t a b i l i s a t i o n o f b i o c a t a ly s t 39

CHAPTER 2 M a te r ia ls and Methods

2.1

2.2

2 .3 Maintenance and growth 41

2.4 Dry «»eight e stim atio n s o f c u ltu re s and

c e l l suspensions 44

2 .5 P re p a ra tio n s o f c e l l e x t r a c t s 44

2.6

2.7 Methanol dehydrogenase a ssa y 45

2.8

2.9 Im m obilized c e l l - b l o f l l m re a c to r

(IB P -r e a c t o r ) 46

2.10

o f P0 produ ction and I t s r e a c t iv a t io n 47

2.11

2.12

2.13 Oases 50

CHAPTER 3

3 .1 .1

3 . 1. 2

3 .1 .3

3-2

3.2 .1 3.2 .2

3 .2 .3

3 .2 .*

3 -2 .5

3 .2 .6

3 -4 .1

3 -4 .2

3.*».3

3 .4 .4

3-5

3 -5 .1

The Produ ction o f Oxygen C on tain in g Compounds by M e th a n e -o x id isin g B a c t e ria

Reactions by s o lu b le MHO c e l l s 51

In tro d u c tio n

51

Experimental

52

R esu lts and d is c u s s io n

32

The e v a lu a tio n o f re a c tio n s and compounds

38

In tro d u c tio n

38

The produ ction o f prim ary a lc o h o ls

58

The produ ction o f secondary a lc o h o ls and

aethyIketones 62

The produ ction o f epoxides

63

The produ ction o f hydroxy-cyclic-com pounds

63

The produ ction o f aldehydes from halogensted

compounds and o th e rs 64

Comment 64

S e le c tio n o f t a r g e t compound and d is c u s s io n

66

The energy supply f o r the produ ction

o f PO 69

In tro d u c tio n

69

9

A lc oh ols as e le c t ro n donor

70

Methane as an e le c t ro n donor

73

Hydrogen a s an e le c t ro n d on or

75

D isc u ssio n

77

The c u lt iv a t io n o f h igh a c t i v i t y

MNO c e l l s

78

In tro d u c tio n

78

The vh o la c e l l MK) a c t i v i t y o f

aethanotrophs

8

D isc u ssio n

8

MonooxvKenase in Whole C e l l s

4.1 In t ro d u c tio n 91

<

1.2

o f non-PO production 91

4 .2 .1 Experim ental 92

4 .2 .2 R e s u lt s 92

4 .2 .2 .1 C e ll s 92

4 . 2 . 2

.2

o f p a r t ic u la t e MHO c e l l s . 92

4.3 The e f f e c t o f PO c o n c e n tra tion o f the growth

o f Nethylococcus c a p s u la tu s (Bath) and

M eth y lo cy stis parvus (OBBP) 94

4.4 I n a c t iv a t io n o f c e l l s u n d e r the c o n d itio n s o f

PO p ro du ctio n w ith PO accum ulation

97

4 .4 .1 Experim ental 99

4 .4 .2 R e s u lt s 99

4.5 D isc u ssio n 103

C hapter 5 In a c t iv a t io n o f Whole C e l l s under the

C on ditio n s o f PO P ro d u c tion without PO Accumulation

5-1 In t ro d u c tio n 109

5-2 The o ptim izatio n o f r e a c t i o n c o n d itio n o f

I B F -r e a c t o r H O

5-3 In t e r p r e t a t io n o f k i n e t i c s o f in a c t iv a t io n

on im m obilized c e l l - b l o f l l m r e a c to r 115

5 . * Use o f the IB F -r e a c t o r 116

5 .4 .1 Comparison o f peak p r o d u c t i v it y and whole

c e l l a c t i v i t y

116

5 .H .2 E f f e c t o f pH on the i n a c t i v a t i o n 118

5 .4 .3 E f f e c t o f temperature on th e in a c t iv a t io n 120

5 .4 .4 C assants on r e s u lt s co n ce rn in g the

X B F -reac tor 120

5-5 E f f e c t o f e x t r a c e ll u l a r PO con ce n tration

on the in a c t iv a t io n un der the co n d itio n s

5 - 5 .1 Experimental 123

5 - 5 - 2 R e s u lt s 124

5 - 6 D isc u ssio n 127

CHAPTER

6

been In a c tiv a te d by a c e ty le n e or P ropylen e Oxide

6 .1 In tro d u c tio n 129

6 .2 The in a c t iv a t io n o f MHO u s in g ace tyle n e

and i t s r e a c t iv a t io n i n v iv o

130

6 . 2 . 1 In tro d u c tio n

130

6 . 2 . 2 The e f f e c t o f carbon s o u rc e s in the

r e a c t i v a t i o n process 131

6 . 2 . 2 . 1 Methane 131

6 . 2 . 2 . 2 Methanol 137

6 . 2 . 2 . 3 Formaldehyde 140

6 .2 . 2 . 4 Formate 140

6 .3 The e f f e c t o f oxygen on the re a c tiv a tio n

o f NMO 143

6 .4 The e f f e c t o f n itro g e n so u rc es on

r e a c t iv a t io n ₁₄₃

6 .5 The e f f e c t o f su lph u r sou rc es 146

6 . 6 The e f f e c t o f tem perature on re a c t iv a t io n 146

6 .7 The re a c tiv a tio n o f c e l l s which are

in a c t iv a t e d by e x t r a c e l l u l a r PO 150

6 . 7 . 1 Experimental 152

6 . 7 . 2 R e s u lt s and d is c u s s io n 152

6 .8 Repeated r e a c t iv a t io n o f c e l l s fo llo w in g

in a c t iv a t io n 155

6 .9 P r o t e in s y st h e s is d u rin g the re a c t iv a t io n 155

6 .1 0 D isc u ssio n 158

CHAPTER 7

o f PO Production and i t s R e a c tiv a tion

the co n d itio n s o f PO produ ction 166

7 . 3 E f f e c t s o f pH, te a p e ra t u re and carbon

so u rc es on the r e a c t i v a t i o n o f p ro p y le n e -

in a c t iv a t e d c e l l s 177

7 - 3 . 1 The e f f e c t o f pH on the r e a c t i v a t i o n o f

p ro p y le n e -in a c tiv a te d c e l l s 177

7 - 3 . 2 The e f f e c t o f te a p e ra t u re on the

r e a c t i v a t i o n o f p r o p y le n e -in a c tiv a te d

c e l l s 177

7 . 3 . 3 The e f f e c t o f carbon sou rc es on the

r e a c t iv a t io n o f p r o p y le n e -in a c tiv a te d

c e l l s 177

7 .3 .* » The e f f e c t o f copper on the r e a c t iv a t io n

o f p r o p y le n e -in a c tiv a te d c e l l s 183

7 . 3 . * . l Experim ental 185

7 . 3 - * . 2 R e s u lt s and D isc u ssio n 185

CHAPTER

8

Mechanisms under the C on ditions o f PO Produ ction

8 . 1 In tro d u c tio n 191

8 . 2 PO con ce n tration i n the c e l l under the

c o n d itio n s o f PO produ ction 191

8 . 2 . 1 In tro d u c tio n 192

8 . 2 . 2 Experim ental 192

8 . 2 . 3 R e s u lt s and d is c u s s io n 193

8 . 3 P o s s i b le in a c t iv a t io n mechanisms o f MMO

by I n t r a c e ll u l a r PO 197

8 . 3 . 1 Experim ental 197

8 . 3 . 2 The e f f e c t o f methane on in a c t iv a t io n

o f c e l l s 197

8 . 3 . 3 The e f f e c t o f ammonia on in a c t iv a t io n 199

Î . 3 . * D isc u ssio n 199

8 . * P o s s ib le in a c t i v a t i o n mechanisms o f

co n cealed in a c t iv a t io n 203

8

8

3.2

8

.

8.8

8

8

8.1

8 .5

CHAPTER 9

9

.

9

2.2

9 .2 .2 .1

9

2

2.2

9 .2 .3

9 -8

9

8.1

9

8.2

By-product form ation and in a c t iv a t io n

d u rin g the o x id a tio n o f propylene

205

How does P0 a c t as an in a c t i v a t o r w it h in the c e l l s under c o n d itio n s o f

h igh P0 production? 206

Experimented. 208

R e s u lt s and d is c u s s io n 208

The e f f e c t o f e le c t r o n donors on

in a c t i v a t i o n

212

Formaldehyde 212

Comments 119

The I n h i b it io n o f R e a c tiv a tio n

In tro d u c tio n ???

The e f f e c t o f n i t r i t e on in a c t iv a t io n

and r e a c t iv a t io n

2

The e f f e c t o f n i t r a t e on r e a c t iv a t io n 222

The in h ib i t io n o f r e a c t i v a t i o n by n i t r i t e 223

Experim ental 223

R e s u lt s 223

The accumulation o f n i t r i t e 226

Experim ental 226

N i t r i t e accum ulation fo llo w in g the

in a c t iv a t io n

227

The e f f e c t s o f oxygen and an en e

rgy-supply on the accum ulation o f n i t r i t e

227

The e f f e c t o f n i t r i t e on the produ ction

o f P0 238

The in h ib i t io n o f r e a c t i v a t i o n by

h i s t i d i n e

236

I s r e a c t iv a t io n c o n t ro lle d ?

238

Experim ental 239

The e f f e c t o f methanol on the r e a c t i v a t i o n

o f MMO 241

9 .4 .4 D isc u ssion 243

CHAPTtjt 10 Process E valu atio n and the

Economics o f P0 Produ ction

10.1 In tro d u c tio n 247

10.2 S e le c t io n o f s u i t a b le o rg a n isa s 248

10.2 .1 In tro d u c tio n 248

10 .2 .2 Experim ental 230

1 0 .2 .3 R esu lts 230

1 0 .2 .3 .1 The r e a c t iv a t io n p a t t e r n s o f

th erm otolerant aethanotrophs 230

1 0 .2 .3 -2 The r e a c t i v a t i o n p a t t e r n s o f a e s o p h ilic

aethanotrophs

233

10.3 Which o rg a n is a i s b e t t e r as a b io c a t a ly s t

f o r the produ ction o f P07

236

10.4 The stoic h iom etry o f th e P0 p rodu ction and

methanol consumption

239

10.3 S u bstra te s consumption f o r r e a c t iv a t io n 264

10 .3 .1 In tro d u c tio n 264

10 .3 .2 Experim ental 264

10 .3 .3 The nec essa ry amount o f carbon source

f o r the r e a c t iv a t io n

263

10 .3 .4 The nec essa ry amount o f n itrogen

source f o r the r e a c t i v a t i o n 268

10 .3 .3 The nec essa ry amount o f sulphur source

f o r the r e a c t iv a t io n

271

10 .3 .6 D isc u ssio n 271

10.6 P rocess economics 273

10.6.1 The b i o l o g i c a l c h lo ro h y d rin p ro

cess-Cetus P ro cess

273

10.6 .2 The produ ction o f P0 u s in g the

g r o w i n g - c e lls process

277

10

6

2.2

1 0 .6 .2 .3 P0 produ ction under grow in g c e l l

co n d itio n s u sin g M eth y lo cy stis pai

1 0 .6 .2 .4 Process economics o f g r o w in g -c e ll

process

10.6.3 Flow sheet o f the tw o -st a g e

r e a c t iv a t io n process

10.6.4 The economics o f p ro du cin g P0

u sin g the tw o -stage r e a c t iv a t io n process

278

279

284

287

CHAPTER

11

R eferences

_$

l

I should l i k e to thank s in c e r e ly ey s u p e r v is o r . P r o fe s s o r Howard

D alton f o r h i s encouragement and a d v ic e over the p a s t two y e a r s. I

would a l s o l i k e t o thank D r.S .H . S t a n le y , N r.A .O . R ich ards f o r the

experim ental a d v ic e and M r.J. Carney and M r.a. MacDonald f o r t h e ir

e x c e lle n t te c h n ic a l a s s is ta n c e and a l l o f the o t h e r members o f

M ic ro b io lo g y 1 a t B i o lo g i c a l S c ie n c e s over the l a s t '4 y e a r s .

F i n a l ly , I would l i k e to thank M r.T. Yamamoto, D r .T . Y o r l f u j l ,

M r.T. T etsu ka, M r.N. Murakami, M r.S . Kamata and a l l o f th e members o f

Biochem ical Research L aboratory and a ls o H E members f o r t h e i r support

The a l a o f t h is p r o je c t I s to develop a p ro c e ss f o r the production

o f p ro p y le n e o x id e (P O ), u s in g m eth an e-oxidisin g b a c t e r i a . A t the

b e g in n in g , a d i f f i c u l t p r o b le e needed to be s o lv e d f o r the development o f t h is p ro c e s s l . e . the s h o r t li f e - s p a n o f the b io c a t a ly s t . E x p e ria e n ts showed th at the c e l l s o f the e e t h a n e -o x id is in g bacterium, M ethylococcua capsulatu s (B a th ) l o s t t h e ir c a t a l y t i c a c t i v i t y w ith in 30

minutes un der the con d ition s o f h igh PO p rodu ction . The in a c tiv a tio n o f

the b i o c a t a l y s t was la r g e ly Independent o f exte rn a lly -a c c u m u la te d PO but was t o t a l l y dependent on PO produced in v iv o under c o n d itio n s o f high PO

p ro d u c tio n . The c e l l s l o s t t h e i r a c t iv i t y w ith ou t any e x te rn a l

accum ulation o f PO under those con d ition s where PO was produced. P r io r

to the re se a rc h o f the p re se n t w r i t e r , i t had been concluded that

e x t e rn a l PO in a c tiv a te d the b io c a t a ly s t . A s p e c i f i c PO p ro d u c tiv it y o f

■o re than 700 n ao l PO produced/aln/ag c e l l s was ob ta in e d in the work

re po rted h e r e . However, by in c r e a s in g the PO p r o d u c t iv it y more than 200

mU/mg c e l l s , the c e l l s l o s t t h e i r a c t iv i t y r a p i d ly and t h e ir h a l f - l i f e

la s t e d

7

In o r d e r to overcome the s h o r t l i f e - s p a n o f the b io c a t a ly s t , a

r e a c t i v a t i o n o f the in a c tiv a te d c e l l s had to b e d e v is e d . The

m e th a n e -o x id isin g b a c t e r ia c o n t a in an enzyme, methane monooxygenase

(IM

0

P0. The UNO was ir r e v e r s i b l y in a c tiv a te d by a c e ty le n e o r by P0, however

t h is in a c t i v a t e d MN

0

r e a c t i v a t i o n treatm ent. T h is r e a c t iv a t io n p ro c e ss i s a phenomenon not

p r e v io u s ly known about. I n o r d e r to r e a c t iv a t e the In a c tiv a te d MM0, the

c e l l s re q u ir e d carbon, n itro g e n and sulphur so u rc e s . In ad d ition , a

s u i t a b l e oxygen and temperature regime was re q u ir e d f o r the re a c tiv a tio n

p r o c e s s . The re q u ir emen t o f n u trie n t s fo r r e a c t i v a t i o n and the

in h i b i t i o n o f re a c tiv a tio n by th e ad dition o f chloram phenicol le d to the co n c lu sion t h a t p ro te in s y n th e sis was a sso c ia te d w ith the re a c tiv a tio n

p ro c e s s . Furthermore, i t was found that UNO s y n th e sis was completely

in h ib i t e d by a d e te c ta b le amount o f methanol in the c e l l suspensions. Copper was n o t re qu ired f o r the re a c tiv a tio n o f c e l l s which contained

p a r t i c u la t e NN

0

Two ty p es o f in a c t iv a t io n mechanism were assumed under the

c o n d itio n s o f P0 production. These are the in a c t i v a t i o n o f NN0 and the

i n a c t i v a t i o n o f the b io c a t a ly s t by a means not y e t i d e n t i f i e d . When the

MIO o n ly was In a c tiv a te d , i t was re a c tiv a te d q u ic k ly . However when

th ese c e l l s w ere in a c tiv a te d un der con dition s o f h igh P0 production, they r e q u ir e d th ree times as lo n g a period f o r com plete re a c tiv a tio n

than d id th ose c e l l s which had been in a c tiv a te d by a c e ty le n e . This

d e la y in th e re a c tiv a tio n p ro c e s s was thought to be due to a concealed

i n a c t i v a t i o n (u n id e n t ifie d in a c t i v a t i o n ) fa c t o r . The l a t t e r was thought

to be caused by the accum ulation o f P0 w ith in the c e l l s . The

i n t r a c e l l u l a r P0 concentration was c a lc u la t e d on the b a s is o f the r e t e n t io n tim e o f P0 in the c e l l s , and i t s con c e n tra tion appeared to be

r e la t e d t o P0 p ro d u c tiv it y . The concealed in a c t i v a t i o n was assumed to

be due to a s o lv e n t -lik e e f f e c t o f P0 in the c e l l s and not from an a lk y la t i o n e f f e c t .

I n o r d e r to develop a P0 produ ction p ro cess u s in g the re a c tiv a tio n systam , H e th y lo c y stls parvus (0BBP) was s e le c te d as the b e s t organism

from 25 methanotrophs. The r e a c t iv a t io n system, the g r o w in g -c a ll

p ro c e ss ( s i n g l e sta g e ) and the tw o -sta g e r e a c t i v a t i o n p ro cess were

d esign e d and o pe rated. U sin g th e g r o w in g -c e ll p r o c e s s , continuous P0

p ro du ctio n was achieved a t a r a t e o f

12

2 .9 -1 The schematic diagram o f the IB F -r e a c t o r 1»8

3 -4 .1 The a c t iv e s i t e o f aethanol dehydrogenase 74

3 -5 .1 The e f f e c t o f co p per concentration s on

whole c e l l MMO a c t i v i t y

83

3 - 5 .2 The e f f e c t o f a e th a n o l con ce n tration on

whole c e l l MMO a c t i v i t y

87

3 -5 -3 The e f f e c t o f tem perature on whole c e l l

MMO a c t i v i t y

88

4 .2 .1 The s t a b i l i t y o f C e l l s under c o n d itio n s

o f non-PO p ro du c tio n

93

4 .2 .2 The s t a b i l i t y o f c e l l s under ana erobic

con d ition s

95

PAGE NO.

1 . 5 - 1 T h e o x i d a t i o n o f a e t h a n e b y m e t h a n o tro p h s 10

4 .2 .3 The decay o f whole c e l l a c t i v i t y d u rin g

s to ra g e in phosphate b u f f e r

96

4 .4 .1 In a c tiv a tio n o f c e l l s under the c o n d it io n

4 .4 .2 I n a c t iv a t io n o f c e l l s under the c o n d it io n

o f PO produ ction w ith PO accum ulation

High biom ass-m oderate c on ce n tration o f PO

accumulation

101

4 .4 .3 I n a c t iv a t io n o f c e l l s under the c o n d it io n s

o f PO p ro du ctio n w ith PO accum ulation

High b io m ass-h igh con ce n tration o f PO

accumulation

102

5 -2 .1 The time co u rse o f PO produ ction i n the

IB F -r e a c t o r

111

5 - 2 .2 The e f f e c t o f oxygen con ce n tration on

the peak p r o d u c t i v it y in the IB F -r e a c t o r 112

5 * 2 .3 The e f f e c t o f methanol c on ce n tration on

the peak p r o d u c t i v it y in the I B F -r e a c t o r 114

5 - 3 .1 The i n v i s i b l e I n a c t iv a t io n d u rin g PO

produ ction under lim it in g c o n d it io n s in

the IB F -r e a c t o r H

7

5 .4 .1 The e f f e c t o f pH on the in a c t i v a t i o n o f

5 . * . 2 The e f f e c t o f temperature on the

in a c t iv a t io n o f c e l l s in the I B F -r e a c t o r 121

5.1».3 The r e la t io n s h i p between peak p r o d u c t iv it y

and the time e la p se d u n t il the a c t i v i t y

decreased to h a l f o f the peak a c t i v i t y

122

5 .5 *1 The e f f e c t o f th e e xtern a l PO con c e n tra tion

on the i n a c t i v a t i o n o f c e l l s

125

5 .5 * 2 The e f f e c t o f th e e xtern a l PO con c e n tra tion

on the i n a c t i v a t i o n and on the p r o d u c t iv it y

o f the c e l l s 126

6 .2 .1 The e f f e c t o f methane as a carbon so u rc e

in the r e a c t i v a t i o n process

133

6 .2 .2 The e f f e c t o f methanol as a carbon source

in the r e a c t i v a t i o n process 134

6 .2 .3 The r e a c t i v a t i o n o f Hethylococcus

capsu latu s (B a t h ) c e l l s in which UNO was

com pletely in a c t iv a t e d by a c e ty le n e

135

6 .2 .4 The r e a c t i v a t i o n o f a c e t y le n e -in a c t iv a t e d

c e l l s by the endogenous energy in

6 .2 .5 The e f f e c t o f aethane con ce n tration in a i r

d u rin g the r e a c t i v a t i o n p ro cess in

Methylococcus cap su la tu s (B ath )

138

6 .2 .6 The e f f e c t o f aeth an ol on the r e a c t i v a t i o n

ra t e o f the in a c t iv a t e d c e l l s in

Methylococcus cap su la tu s (B ath ) 139

6 .2 .7 The e f f e c t o f fo ra ald eh y d e on the

r e a c t iv a t io n a s a carbon and energy

source in M ethylococcus capsu latu s (B a t h ) l 4 l

6 .2 .8 The e f f e c t o f f o r a a t e on the r e a c t i v a t i o n

as a carbon and energy source in

Methylococcus cap su latu s (B ath ) 142

6 .3 *1 The e f f e c t o f oxygen on the r e a c t i v a t i o n

o f MMO i n M ethylococcus c ap su latu s (B a th ) 144

6

r e a c t iv a t io n o f MMO in Methylococcus

cap su la tu s (B a t h ) IU

5

6 .4 .2 The e f f e c t o f th e n itro g e n sources on

the r e a c t i v a t i o n o f NNO in M eth y lo cy stis

6 . 5 . 1

6

.

5.2

6

6.1

6 .7 .1

6.7.2

6 .7 .3

6

8.1

6 .9 .1 «

r e a c t iv a t io n o f MMO in Methvlococcus

capsu latu s (B a t h ) 148

The e f f e c t o f c y s te in e as a s u lp h u r so u rc e

on the r e a c t i v a t i o n o f MMO in Methylococcus

capsu latu s (B a t h ) 149

The e f f e c t o f tem perature on the r e a c t i v a t i o n

o f MMO in M ethylococcus c a p su la tu s (B a th ) 150

The r e a c t i v a t i o n o f the P O -in a c tiv a te d c e l l s

and both P 0 - and a c e t y le n e -in a c t iv a t e d c e l l s

o f M ethylococcus capsu latu s (B ath ) 153

The e f f e c t o f e x t e r n a l PO c o n c e n tra tion on

the in a c t i v a t i o n ' and the r e a c t i v a t i o n o f

c e l l s o f M ethylococcua cap su la tu s (B a th ) 154

The r e a c t i v a t i o n o f whole c e l l MMO a c t i v i t y

o f the P O -in a c tiv a te d c e l l s measured u s in g

d i f f e r e n t e le c t r o n donors

156

Repeated r e a c t i v a t i o n o f c e l l s f o llo w in g

in a c t iv a t io n b y ace tyle n e and e x t e r n a lly

added PO 157

P ro te in s y n t h e s is d u rin g the r e a c t i v a t i o n

o f MHO in M ethylococcus capsula t u s (B a th ) The e f f e c t o f t h e s u l p h u r s o u r c e o n t h e

6 . 9 . 1 b

7.2 .1

7 .2 .2

7 .2 .3

7 -2 . *

7 .2 .5

o f MHO i n M eth y lo cy stis parvu s (OBBP) l60

The r e a c t i v a t i o n o f c e l l s In a c tiv a te d

under the c o n d it io n o f PO p ro du ctio n -

low PO p r o d u c t i v it y ( I ) 168

The r e a c t i v a t i o n o f c e l l s In a c t iv a t e d

under the c o n d it io n o f PO p ro du ctio n

-low PO p r o d u c t i v it y ( I I ) 169

The r e a c t i v a t i o n o f c e l l s In a c t iv a t e d

under th e c o n d it io n o f PO p ro du ctio n -

moderate p r o d u c t iv it y

170

The r e a c t i v a t i o n o f c e l l s in a c t iv a t e d

under the c o n d it io n o f PO p ro du ctio n -

high PO p r o d u c t i v it y

171

The r e la t io n s h i p between r e s i d u a l

a c t i v i t y a t th e begin n in g o f the

r e a c t iv a t io n p ro c e ss and the time

re q u ir e d f o r complete r e a c t i v a t i o n

173

r a t e i n the e x p o n e t la l phase and the peak

p r o d u c t iv it y

17

7 * 2 .7 The r e a c t i v a t i o n o f c e l l s in a c t iv a t e d

under the c o n d it io n o f h igh PO p ro du c tio n

176

7 .3 .1 The e f f e c t o f pH on the r e a c t i v a t i o n o f

p ro p y le n e -in a c tiv a te d c e l l s I

78

7 * 3 .2 The e f f e c t o f temperature on the

r e a c t i v a t i o n o f propylene in a c tiv a te d

c e l l s o f Methylococcua cap au latua ( B a t h ) ,

a : r e a c tio n tem perature 45*C, b : r e a c t io n

temperature 40*C 179

7 .3 .3 The e f f e c t o f methanol as the carbon

source on the r e a c t iv a t io n o f p ro p y le n e

-in a c t iv a t e d c e l l s o f Methylococcua

c ap su latu s (B a t h ) 182

7 - 3 - 1» The e f f e c t o f peak p ro d u c tiv it y on the

l a g p e rio d observed d u rin g the i n i t i a l

p e rio d o f r e a c t i v a t i o n in Methylococcua

cap su la tu s (B a t h ) 183

7*3*5 The e f f e c t o f v a r io u s carbon so u rc es on

the I n i t i a l r e a c t i v a t i o n r a t e in

Methylococcua cap su la tu s (B ath ) 184

r e a c t i v a t i o n o f a c e t y le n e -in a c t iv a t e d

c e l l s o f M ethylococcus c a p su la tu s (B a th ) 186

7 - 3 - 7 The e f f e c t o f copper ions on the

r e a c t i v a t i o n o f p ro p y le n e -in a c tiv a te d

c e l l s o f Me th ylococcu s c a p su la tu s (B a th ) 187

7 - 3 - 8 The e f f e c t o f copper ions on the

r e a c t i v a t i o n o f a c e ty le n e -in a c t iv a t e d

c e l l s o f Me th y lo c y s t i s parvus (OBBP) 188

7 - 3 - 9 The e f f e c t o f copper ions on the

r e a c t i v a t i o n o f p ro p y le n e -in a c tiv a te d

c e l l s o f Me th y lo c y s t i s parvus (OBBP)

189

8 .2 .1 E stia a te d i n t r a c e l l u l a r PO con c e n tra tion

d u rin g the b io t r a n s fo r a a t i o n o f p ropylen e

195

8 .2 .2 S e qu e n tia l PO produ ction and th e change o f

e s t i a a t e d i n t r a c e l l u l a r PO c o n c e n tra tion

196

8 .3 - 1 The e f f e c t o f aethane on the i n a c t i v a t i o n

o f c e l l s un der con d ition s o f PO produ ction

i n the IB F -r e a c t o r

7 - 3 - 6 T h e e f f e c t o f c o p p e r i o n s o n t h e

o f c e l l s u n der conditions o f PO produ ction

in the I B F -r e a c t o r

8 - 3 -3 P o s s ib le ro u te s o f in a c t i v a t i o n o f MMO

a c t iv e s i t e by PO

8 .4 .1 In te rm itte n t re a c tio n and the in a c t iv a t io n

o f c e l l s o f Methylococcus c a p su la tu s (B ath )

8 .4 .2 The i n a c t i v a t i o n o f c e l l s f o llo w in g the

o x id a tio n o f ethylene in Methylococcus

c ap su latu s (B ath )

8 .4 .3 The i n a c t i v a t i o n o f enzymes under the

c o n d itio n o f PO production i n M ethylocytia

parvus (OBBP)

8 .4 .4 I n a c t iv a t io n o f enzymes by th e a d d itio n o f

ace ty le n e o r propylene o x id e and t h e ir

r e a c t i v a t i o n in M eth ylocystis parvus (OBBP)

8 .4 .3 The i n a c t i v a t i o n o f enzymes fo llo w in g the

a d d itio n o f h igh PO con ce n tration s and

t h e ir r e a c t i v a t i o n in M eth y lo cy stis parvus

8 . 3 - 2 The e f f e c t o f a u o n l a o n t h e i n a c t i v a t i o n

200

202

204

207

209

210

8 .4 .6 The i n a c t i v a t i o n o f enzymes fo llo w in g the

a d d itio n o f benzene and t h e i r r e a c t i v a t i o n

in M e th y lo c y stis parvus (OBBP) (1 ) 213

8 .4 .7 The In a c t iv a t io n o f enzymes fo llo w in g the

a d d itio n o f benzene and t h e i r r e a c t i v a t i o n

in H e th y lo c y s tis parvus (OBBP) (2 ) 214

8 .4 .8 The e f f e c t o f formaldehyde as an e le c t r o n

donor on th e in a c tiv a tio n o f Methylococcus

c ap su latu a (B ath ) in IB F -r e a c t o r 216

8 .4 .9 The i n a c t i v a t i o n o f c e l l s f o llo w in g the

a d d it io n i s formaldehyde and t h e ir

r e a c t i v a t i o n in Wethylococcus cap su latu a

(B ath ) 217

8 .4 .1 0 The i n a c t i v a t i o n o f enzymes fo llo w in g the

a d d it io n o f formaldehyde and t h e ir

r e a c t i v a t i o n in M eth y lo cy stis parvu s (OBBP) 218

8 .4 .1 1 The e f f e c t o f formate as an e le c t r o n donor

on the i n a c t i v a t i o n o f c e l l s i n the IB F

-r e a c t o -r

220

9 * 2 .1 The e f f e c t o f n i t r a t e con c e n tra tion on

r e a c t i v a t i o n in Hethylococcua cap su latu a

o f n i t r i t e and r e a c t iv a t io n in M eth y lo cy stis

parvus (OBBP) 228

9 .2 .3 a The accum ulation o f n i t r i t e f o llo w in g the

a d d it io n o f PO in M eth y lo cy stis p arvu s

(OBBP) 229

9 .2 .3 b The accum ulation o f n i t r i t e f o llo w i n g the

p ro du ctio n o f PO in M eth y lo cy stis p arvu s

(OBBP) 230

9 .2 .4 The accum ulation o f n i t r i t e f o llo w in g

the a d d i t i o n o f ace tyle n e under the

c o n d it io n o f low d is s o lv e d oxygen

c o n c e n tr a tio n s in M eth y lo cy stis p arvu s

(OBBP) 232

9 .2 .9 The accum ulation o f n i t r i t e fo llo w in g

the treatm en t o f high d is s o lv e d oxygen

c o n c e n tr a tio n con dition i n M e th y lo c y stis

parvus (OBBP) 233

9 .2 .6 The e f f e c t o f n i t r i t e on th e p ro du c tio n o f

PO in M ethylococcus c ap su latu a (B a th ) 235

9 .4 .1 The r e p r e s s io n o f r e a c t i v a t i o n MMO by

methanol i n M eth ylocystis p a r ''. - (OBBP) 240

9 .*.2

9 - * . 3

10.2.1

1 0.2 . 2

1 0 .2 .3

1 0 .2 .4

1 0 .3 -1

10.3 -2

o f MHO i n M ethylocystis p a rv u s (OBBP) 242

A d a p ta tion o f methane-grown c e l l s to the

methanol c u lt u r e by u sin g a c e ty le n e

-i n a c t -i v a t -i o n techn-ique 245

R e a c tiv a tio n o f a c e ty le n e -in a c t iv a t e d

c e l l s o f thermo to le ra n t methanotrophs 251

R e a c tiv a tio n o f p ro py le n e -in a c tiv a te d

c e l l s o f thermo to le ra n t methanotrophs

252

R e a c tiv a tio n o f a c e t y le n e -in a c t iv a t e d

c e l l s o f a e s o p h ilic methanotrophs 254

R e a c t iv a t io n o f p ro p y le n e -in a c tiv a te d

c e l l s o f m esoph ilic methanotrophs 255

The e f f e c t o f re action tem peratures

on the In a c t iv a t io n and r e a c t i v a t i o n

o f c e l l s 257

The e f f e c t o f re action tem peratures on

the in a c t i v a t i o n and r e a c t i v a t i o n o f

c e l l s I n Methylococcus c a p s u la tu s (B ath ) T he e f f e c t o f fo rm a te o n t h e r e a c t i v a t i o n

10 .4 .1

1 0 .5 .1

1 0 .5 .2

1 0 .5 .3

10 .5 .4

1 0 .5 .5

10.5.6

The s t o ic h lo a e t r y o f the b u t a n o l consumption

and P0 p ro du c tio n in M e th y lo c v stis parvus

(0B8P) 2*3

The n e c e s sa ry amount o f methanol f o r the

r e a c t i v a t i o n o f a c e t y le n e -in a c t iv a t e d

c e l l s i n Methylococcus c a p s u la tu s (B ath ) 266

The n e c e s sa ry amount o f methanol f o r the

r e a c t i v a t i o n o f p ro p y le n e -in a c tiv a te d

c e l l s in Methylococcus c a p s u la tu s (B ath ) 267

The n e c e ssa ry amount o f n i t r a t e f o r the

r e a c t i v a t i o n o f a c e t y le n e -in a c t iv a t e d

c e l l s 269

The n e c e s sa ry amount o f n i t r a t e f o r the

r e a c t i v a t i o n o f p r o p y le n e -In a c t iv a t e d

c e l l s 270

The n e c e ssa ry amount o f s u lp h a te f o r the

r e a c t i v a t i o n o f a c e t y le n e -in a c t iv a t e d

c e l l s 272

The n e c e s sa ry amount o f s u lp h a te f o r the

r e a c t i v a t i o n o f p r o p y le n e -in a c tiv a te d

10.6 .1 PO p ro du c tio n under g r o w i n g - c e ll c o n d itio n

u s in g M eth y lo cy stis p arvu s (OBBP) - low

biom ass c o n d itio n 280

1 0 .6 .2 P0 p ro du c tio n under g r o w i n g - c e ll c o n d itio n

u s in g M eth y lo cy stia p arvu s (OBBP) - h igh

biom ass c o n d itio n

281

1 0 .6 .3 The con c e p tu a liz e d p ro c e ss f lo w -s h e e t o f

the g r o w i n g - c e ll process 285

The con c e p tu a liz e d p ro c e s s f lo w -s h e e t o f

the tw o -st a g e r e a c t i v a t i o n p ro c e ss 289

The e f f e c t o f a d d it iv e s on the p reven tion

o f In a c t iv a t io n and the a c c e le r a t io n o f

r e a c t i v a t i o n 301

1 1 .1 .2 The e f f e c t o f sodium c arb o n ate on the P0

p r o d u c t i v it y in v a r io u s m e th a n e -o x id isin g

b a c t e r i a

303

1 1 .1 .3 The e f f e c t o f melamine on the prev en tio n

o f i n a c t i v a t i o n and the a c c e le r a t io n o f

1

3.1

o x i d i s i n g b a c t e ria *

1 .11 .1 S u b s tra te s p e c i f i c i t y o f MMO in v iv o

and i n v i t r o 23

1

17.1

organism s 37

2 .2 .1 Com position o f n i t r a t e m in eral s a l t (NMS)

medium <12

3 .1 .1 O x id a t iv e products produced by whole c e l l s

o f Methylococcua c a p su la tu s (B ath ) which

con tain ed soluble-MNO 53

3 .2 .1 Param eters f o r e v a lu a t in g compounds 59

3 .2 .2 G rou ping o f the r e a c tio n s by MMO 60

3 .2 .3 E v a lu a t io n o f p rodu cts by MHO 65

3 .3 .1 Economie comparison o f produ cts 67

3 .3 .2 D ir e c t e p o x idation p ro c e s s e s o f propylene

3.1». 1 E f f e c t o f a lc o h o ls as e le c t r o n donors 71

3-1».2 Whole c e l l a c t i v i t y »e n su re d u s in g

■ethane as the e le c t ro n donor 76

3.5*1 MMO a c t i v i t y in c e l l e x t r a c t s and whole

c e l l MMO a c t i v i t i e s 80

3 .5 *2 Whole c e l l MMO a c t i v i t y o f v a r io u s

■ethan otrophs 82

3 -5 -3 R e la t io n s h ip between b io a a s s concentration

and w h ole c e l l MMO a c t i v i t y

85

4 .3 •1 E f f e c t o f PO con cen tration in the c u lt u re

on c e l l growth

98

4 .5 .1 I n a c t iv a t io n o f whole c e l l s by e x t e r n a lly

added PO in Nethylococcus c a p su la tu s (B ath ) 105

9 *2 .1 The i n h i b i t i o n o f r e a c t i v a t i o n o f a c e ty le n e

-in a c t i v a t e d c e l l s by the a d d it io n o f n i t r i t e

225

10 .3 .1 The t y p i c a l p ro p e rtie s o f the th ree ■ethane-

o x i d i s i n g b a c t e r i a , M ethylococcus capsu latu s

(B a t h ), M eth ylocystls p arvu s (OBBP) and

10 .4 .1

10.6.1

10.6 .2

10 .6 .3

The s t o ic h io a e t r y o f the »e t h a n o l

consumption and P0 p ro du c tio n 26l

The b a s i c a l parameters f o r th e c a lc u la t io n

o f P0 p ro du c tio n c o st i n t w o -st a g e

r e a c t i v a t i o n process

291

The P0 p ro du c tio n c o st in tw o -st a g e

r e a c t i v a t i o n process

293

The c o s t a n a ly s is o f to w e rs, v e s s e ls and

In 1972, Colby and Zataan proposed and d e fin e d the tern

'm e th y lo tro p h y '. A cc o rd in g to t h e ir d e f i n i t i o n , m ethylotrophs a r e

o rg a n is e s c a p ab le o f o b t a i n in g energy by the o x id a tio n o f C j-g ro w th

s u b s tr a te s and the a s s im i la t io n o f carbon such as formaldehyde o r as a

m ixture o f formaldehyde and carbon d io x id e , b u t always by pathways that

a r e d is t in g u is h a b le from the C a lv in c y c le . T h is group o f organism s can

be fu r t h e r s u b -d iv id e d in t o two g r o u p s :- o b li g a t e and fa c u lt a t iv e

m ethylotrophs, the l a t t e r group h aving th e a d d i t i o n a l c a p a b i li t y to grow

and r e p li c a t e on a v a r i e t y o f oth e r c a r b o n -t o -c a r b o n bond compounds.

M ethylotrophs, as d e fin e d above, a r e organism s which can u t i l i z e

methane, methanol, N-m ethyl compounds, S -m eth yl compounds as t h e i r s o le

sources o f carbon f o r grow th and r e p l i c a t i o n . The e x te n siv e and v a rie d

p h y sio lo gy and b io ch em istry o f a l l the d i f f e r e n t types o f methylotrophs

have been com prehensively w ritt e n about i n a number o f re view s by

Quayle, 1972, Colby e t a l . . 1979; H ig g in s e t a l . . 1981a; Anthony, 1982;

Large and Bamforth, 1988. The re a d e r, t h e r e f o r e , should use th ese

sources o f re fe re n c e f o r more d e t a ile d in fo rm a tio n on m ethylotrophy.

1 .2 Occurrence , E c o lo g y and H isto ry o f th e I s o la t i o n o f Methane-

o x id is in g B a c t e r ia

The e x te n t o f tu rn o v e r o f methane w it h in the bio sp h e re as th e major

carbon source o f m e th an e-o x id isin g b a c t e r i a (methanotroph) i s n o t w idely

a p p re c ia te d . In 1976, E h h alt reported ap pro xim ately 50% o f a l l org a n ic

carbon, decomposed by a n a e ro b ic m ic r o flo r a i s converted in to methane.

The methane amounts to 5 - 3 “ 8.1 mega tonnes p e r y e a r.

B io gen ic methane I s the major so u rc e o f atmospheric methane and i s

methane gen erated by methanogenic b a c t e r i a i s due m ainly to the

a c t i v i t i e s o f m e th an e-o x id isin g b a c t e r i a which a r e w id e ly d is t r ib u t e d in

the environment. In c lu d in g the p o s s i b i l i t y th at some methane may be

o x id is e d a n a e r o b ic a lly (Pan gariban e t a l . . 1979; Reeburgh,

1981

Although m e th a n e -o x id isin g b a c t e r i a a r e now known to b e w id ely

d is t r ib u t e d in n a t u r e , p r i o r to 1970, o n ly th ree s p e c ie s o f

m eth an e-o xid isin g b a c t e r i a had been I s o la t e d and c h a r a c t e r iz e d (P o s te r

and D a v ie s.

1966

96

d e s p ite the f a c t th a t th e f i r s t i s o l a t e had been found e a r l y in th is

century by Sohngen (1 9 0 6 ). The d i f f i c u l t y o f i s o l a t i n g p u re c u lt u r e s ,

p a r t i c u la r ly o f m e th an e-o x id isin g b a c t e r i a was c e r t a i n l y due to the la c k

o f a r e l i a b l e enrichm ent and i s o la t i o n te ch n iqu e. When en rich ed

c u lt u re s were p la t e d on a g a r p la t e s un der an atmosphere o f methane and

a i r , many scaven gers formed c o lo n ie s w h ich , when r e p la t e d on fr e s h aga r

p la t e s , formed f u r t h e r c o lo n ie s even w it h o u t methane.

The o th e r problem encountered was sy m b io s is . I t was sometimes found

th at m e th a n e -o x id isin g b a c t e r i a grew w e l l i n a n ix e d c u lt u r e (Lin ton and

Buckee, 1977- Imai e t m l . . 1986). The enrichm ent and i s o l a t i o n

technique was d r a m a t ic a lly transform ed i n 1969-1970 by W hittenbury and

h is c o lle a g u e s . They d e v is e d sim ple and e f f e c t i v e tech n iqu es f o r the

is o l a t i o n o f m e th a n e -o x id isin g b a c t e r i a (W h itten bu ry , 1969; Whittenbury

e t a l . , 1970b) from which they is o la t e d more than a hundred d i f f e r e n t

s t r a i n s . The succ ess o f t h e i r techniqu es d e riv e d from th e employment o f

a s h o r t e r enrichment p e r i o d . This li m i t e d lo s s e s o f b a c t e r i a

experienced as a r e s u l t o f p red atio n and overgrow th g row in g on

s u b s tr a te s o th e r than methane.

W hittenbury e t a l . (1970a) c l a s s i f i e d t h e ir i s o l a t e s In t o f i v e

grou ps; M ethylococcus. Methylomonaa. Me th y lo b a c t e r . M eth ylosln u s and

1.3 C la s s i f i c a t i o n o f M eth a n e-oxidisin g B a c t e r ia

Confusion r e l a t i n g to the nomenclature and taxonomy o f

■ e th a n e -o x id is in g b a c t e r i a has not y e t been c o n c lu s iv e ly re so lv e d ,

however the b e s t scheme produced to d a te i s W hittenbury e t a l . (1970a)

who found a c o r r e la t i o n between the type o f aeabrane arrangement, and

the «ean s employed f o r carbon a s s im i la t io n . Type 1 organisms a ssim ilate

carbon by the r i b u l o s e monophosphate pathway, whereas organisms

p o sse ssin g Type I I membrane systems used the s e r i n e pathway.

T a b le 1 .3 -1 shows the c l a s s i f i c a t i o n o f o b li g a t e m ethane-oxidising

b a c t e r i a proposed b y the above au th o rs. However, s in c e the adoption o f

t h is scheme o f c l a s s i f i c a t i o n a number o f r e p o rt s have appeared with

su gg est th a t the two groups are not as c l e a r l y d e fin e d as was f i r s t

th ought. I t has been shown th at some o f Type I and I I methanotrophs can

p ossess both h e x u lo se phosphate synthase and hydroxypyruvate reductase

a c t i v i t y and th is i s in d ic a t iv e o f the presen c e o f the r i b u lo s e

monophosphate c y c le and the s e rin e pathway r e s p e c t iv e ly which has le d to

the p ro p o sa l o f th e t h ir d group o f methanotrophs, the Type X group

(W hittenbury and D a lt o n , 1981).

The c r i t e r i a u se d to re so lv e the problem o f c l a s s i f i c a t i o n o f

■ethanotrophs a r e , a t p resen t, u n s a t is fa c t o r y s in c e the n atu re o f the

organisms makes t h i s ta sk d i f f i c u l t . I n 198**. Galchenko and Andreev

d evised a scheme f o r the c l a s s i f i c a t i o n o f methanotrophs which was based

:-C la s s i f i c a t i o n to O b lig a t e M e t h a n e -u t i li s i n g B a c t e ria

C haracter

Membrane arrangement

R estin g s ta g e

Carbon A s sim ila tio n

TCA c y c le

G lucose-6-phosph ate

and 6-ph osphogluconate

dehydrogenase

Examples

Type I

Bundles o f v e s i c u l a r

d is c s

Cysts (A z o t o b a c t e r

-11km)

R ibu lose monophosphate

pathway

Incomplete (la c k s

2-o x 2-o g lu ta ra te

dehydrogenase)

Type I I

P a ire d membranes in

la y e r s around

p eriph e ry

Exospores o r " l i p i d -

c y s t s "

S e rin e pathway

Complete

Hethylococcu» a m a m i

m t t r i — » He thy l o e « t i «

by W hittenbury e t a l . 1970b).

I t i s o f paramount im portance t h a t th e problem r e la t i n g to c o rr e c t

c l a s s i f i c a t i o n o f methanotrophs i s th o r o u g h ly r e so lv e d , not ju s t because

i t would g iv e academ ic s a t i s f a c t i o n , b u t because o f the im p lic a t io n s f o r

in d u s t r i e s . For exam ple, in both Europe and Japan, m ic ro b ia l p aten ts are

iss u e d acc ordin g t o genus and/or s p e c ie s th ese can cover a l l the sp e c ie s

which b elo n g to t h a t genus. Even i f new s p e c ie s are found, these

s p e c ie s a r e r e s t r i c t e d by the p aten t which has been a lre a d y is s u e d . But

the p aten t has no r e s t r i c t i o n on any o t h e r genus. The confu sion o f

c l a s s i f i c a t i o n makes i t easy to pro po se a new genus. In t h is case a

p aten t which has be e n is s u e d , has n o t r e s t r i c t i o n f o r the newly proposed

genus. I n the U n it e d S t a t e s , p a te n ts a r e is s u e d acc ordin g to sp e c ie s

o n ly , so the above problem does not a r i s e .

1.4 P h ysiolo g y and Biochem istry o f O b li g a t e M eth a n e-oxidisin g

B a c t e r ia

1 .4 .1 B a s ic Growth Requirements

M e th a n e -o x id isin g b a c t e r i a a r e s t r i c t l y a e r o b ic , due to t h e ir need

f o r gaseous oxygen i n the i n i t i a l o x id a t i o n o f methane (H ig g in s and

Quayle, 1970). They a r e cap able o f u t i l i s i n g e i t h e r methane o r methanol

a s a s o le source o f carbon and en e rg y . The growth o f m eth an e-oxidisin g

b a c t e r i a i s i n h i b i t e d by the a d d it io n o f o rd in a ry h etero tro ph ic

m e ta b o lite s in norm al c o n ce n tration s (E c c le s t o n and K e lly , 1972, 1973)«

No growth fa c t o r s a r e re q u ir e d f o r th e grow th o f these organisms as they

sou rc e, c a lc iu m , Magnesium, p o t a s s iu e , s u lp h a t e , phosphate and trace

elements (D a lt o n and W hittenbury, 1976). Some amino acids stim u late the

growth o f m eth an e-o xid isin g b a c t e r i a (M u r r e ll , 1981).

1 .4 .2 Carbon Metabolism

M e th a n e -o x id isin g b a c t e r i a a r e c a p a b le o f o x id is in g methane to

carbon d io x id e com pletely. Methane i s f i r s t o f a l l o x id is e d to methanol

by way o f the a c t io n o f a methane monooxygenase (MHO). Methanol i s then

fu r t h e r o x id is e d to formaldehyde by a methanol dehydrogenase.

Formaldehyde can be a ssim ila t e d in to the c e l l to fo ra c e l l m a te ria ls o r

i t can be fu r t h e r o x id ise d by way o f a d i s s l a l l a t o r y rou te v i a formate

and f i n a l l y t o carbon d io x id e , to p ro v id e th e c e l l with energy f o r i t s

a s s i a i la t o r y pathway. Both a s s i a i la t o r y and d i s s i a i la t o r y pathways work

sim u ltan eo u sly i n the c e l l .

1 .4 .3 Cl-compound A s sim ila tio n Pathways

Three d e v ic e s f o r the a s s im ila t io n o f Cj-compounds a r e recognized

to d a t e : - the r lb u i o s e monophosphate pathway f o r formaldehyde

a s s im i la t io n , th e s e rin e pathway and the r l b u l o s e diphosphate pathway

(C a lv in c y c le ) f o r carbon d io x id e a s s im i la t io n . Some organisms may use

more than one mechanise f o r C ^ -a s s im i la t io n e i t h e r when su b je c t e d to

d i f f e r e n t grow th c o n d itio n s; o r indeed when two mechanisms a r e used

sim u lta n e o u sly . However th ere i s no c o n v in c in g evidence a s y et o f two

o r more com plete C .- a s s i m ila t i o n pathways o p e ra t in g sim u ltaneously in

any one m ic ro-organism . N e v e rt h e le s s , I t I s c l e a r that c e r t a in

C j - u t i l i z e r s , w h i le usin g one pathway as th e m ajor source o f fix e d

cartoon a r e c a p a b le o f a s s im i la t in g sm all amounts o f C1-compound by a

R ib u lo s e monophosphate pathway (R M P ), a pathway o f formaldehyde

a s s im ila t io n was i n i t i a l l y proposed by Kemp and Quayle (1967) and

fu r t h e r e la b o r a t e d by Lawrence and Q u a yle (1970a) and Kemp (1 9 7 4 ). The

o v e r a ll e f f e c t o f the c y c le i s to s y n t h e s iz e a C^-compound from th ree

m olecules o f formaldehyde. The c y c le i s conven iently d iv id e d in t o three

s ta g e s ; f i x a t i o n , cleavag e and rearrangem ent.

S tage I . F ix a tio n : by th e a c t io n o f hexulose phosphate synthase,

th ree m o le c u le s o f formaldehyde a r e condensed with th re e molecules o f

r ib u lo se -5 -p h o s p h a t e to y i e l d th ree m o le cu les o f fr u e tose-6 -p h osph ate.

S tage 2 , Cleavage: one m olecule o f h exu lose-6-phosph ate i s

isom erised t o fru c tose -6 -ph o sp h a te and t h i s i s then s p l i t in to two C^-

compounds. T h i s bein g ach ieved e i t h e r by the enzymes o f the g l y c o ly t i c

sequence o r b y the Entn er-D oudoroff pathway enzymes.

Stage 3 . Rearrangement: t h is s t a g e in v o lv e s the re g e n e ra tio n o f the

th ree m ole c u le s o f r ib u lo se -5 -p h o s p h a t e from the two m olecules o f

fr u c t o se -6 -p h o sp h a te and one m olecule o f glycera ld eh yd e-3 -p h osph a te

produced in s t a g e s 1 and 2.

1 .4 .5 S e r in e Pathway

The ca rb o n a s s im ila t io n pathway i n the Type I I m eth an e-oxidisin g

b a c t e r i a w ere f i r s t stu died by Q uayle and co-workers (Lawrence e t a l . .

1970«. 1 9 7 0 b). The o v e r a ll r e s u lt o f th e pathway i s t o in co rp orate two

m olecules o f formaldehyde and one m o le c u le o f carbon d io x id e in t o a

C^-compound. 3~phosphoglycerate. The c y c le i s i n i t i a t e d by two

m olecules o f s e r i n e which a r e f o rmed from two m olecules o f g ly c in e plu s

in to 3 -ph osp h oglyc era te f o r a s s i m i la t i o n in to c e l l carbon o r converted

in to phosphoenol pyruvate (P E P ). PEP c arbo xylase c a t a ly s e s the

c a r b o x y la t io n o f PEP w ith carbon d io x id e to fo r a o x a lo a c e t a t e , which

su b seq u e n tly forms m alyl-CoA. The a a ly l- C o A i s then c leav ed in t o two

C2 u n it s t o a c t as fu r t h e r a c c e p t o rs o f C1 u n its to m aintain the

c y c le o f r e a c t i o n s .

1 .4 .6 R ib u lo s e Diphosphate Pathway o f Carbon D iox id e A s sim ila tio n

M ethylococcus cap su la tu s ( B a t h ) , a Type I methanotroph, appeared to

use on ly th e r i b u lo s e monophosphate pathway f o r a s s im i la t io n (S tr^ a

e t a l . . 197*0 • However, i t h as now been shown to possess

hydroxypyru vate redu ctase, a key enzyme in the s e r i n e pathway, although

a t low l e v e l (Reed, 1976). Furtherm ore the presence o f the key enzymes

o f the C a lv i n c y c le , r i b u lo s e dip h osp h ate c a rb o x y la se and

p h o sp h o ribu lo k in ase were dem onstrated i n c e l l e x t r a c t s o f Hethylococcus

c a p su la tu s (B ath ) (T a y lo r , 1977)* The r a t e o f C02 f i x a t i o n by whole

c e l l was lo w , and c o n trib u te d o n ly a bo u t 2.5% (w t/w t) o f the t o t a l

c e l l c a rb o n . F ix a tio n was o b se rv e d o n ly w ith the presence o f methane,

in d i c a t i n g an energy requirem ent f o r in c o rp o r a tio n (T a y lo r e t a l . ,

1980).

R ib u lo s e diphosphate c a r b o x y la s e from t h is organism r e q u ir e s a

d iv a le n t c a t io n f o r a c t i v i t y . I t h as an a lk a lin e pH optimum; i s

in h ib i t e d b y 6-phosphogluconate and p o ssesses an oxygenase a c t i v i t y

(T a y lo r e t a l . . 1980). T h is s eco n d ary a c t i v i t y o f th e enzyme gen erates

phosphoglucon ate, which may b e s u b se q u e n tly c le a r e d by a s p e c i f i c

phosphogluconate phosphatase. T h is was observed i n e x t r a c t s o f

M ethylococcus capsu latu s (B a t h ) (T a y lo r e t a l . , 19 81 ). The m etabolic