Download Microbial metabolism of short-chain unsaturated hydrocarbons

FEMS Microbiology Reviews 63 (1989) 235-264
Published by Elsevier
235
FEMSRE 00124
Microbial metabolism of short-chain unsaturated hydrocarbons
S. Hartmans, J.A.M. de Bont and W. Harder 1
Division of Industrial Mierobiology, Department of Food Seience, Agricultural University, Wageningen,
and 1 Department of Microbiology, State University of Groningen, Haren, The Netherlands
Received 10 February 1989
Accepted 17 April 1989
Key words: Ethene; Acetylene; Alkenes; Epoxides
1. INTRODUCTION
The microbial degradation of hydrocarbons has
been studied extensively during the last decades.
In general, reviews of such studies grouped the
hydrocarbons in classes of compounds for collective discussion. Microbial degradation of aromatic
hydrocarbons has been reviewed several times [1,2]
which is in keeping not only with the often detailed physiological and genetic studies undertaken but also with the importance of the mineralization of these compounds in the carbon cycle
in nature. Aliphatic long chain hydrocarbons have
mostly been dealt with as a distinct group of
hydrocarbonous compounds that enter the environment through unintentional and undesirable
spillage from the petrochemical industries [3]. They
have also been considered as cheap substrates in
biotechnological processes [4]. Methane utilization
is generally treated as a specific topic, mainly
because the methane-utilizing bacteria comprise a
distinct group of organisms with rather specific
biochemical characteristics [5]. Terpenes have also
been reviewed as a separate class of compounds.
Correspondence to: S. Hartmans,Division of Industrial Microbiology, Departmentof Food Science,AgriculturalUniversity,
P.O. Box 8129, 6700 EV Wageningen,The Netherlands.
These natural products have practical applications
especially in the flavour and fragrance industries
and their potential as substrates in biotechnological processes employing micro-organisms as biocatalysts has been reviewed [6].
Until recently, the microbial metabolism of unsaturated short chain hydrocarbons received considerably less attention. Nonetheless, this group
comprises the important naturally occurring plant
hormone ethene (ethylene). Furthermore, many of
the gaseous alkenes and alkynes are used extensively in the petrochemical industry. The recent
interest in organisms degrading gaseous alkenes
and alkynes stems mainly from applied aspects.
The organisms involved may be employed either
in the treatment of waste gases or in the biotechnological production of epoxides. However, the
ecological implications are also important. The
fate of gaseous hydrocarbons is studied with increased interest because of their contribution to
the production of carbon monoxide and their involvement in the production and degradation of
ozone [7].
The microbial degradation of the heterogeneous group of short chain unsaturated hydro-.
carbons and its implications has received only
limited attention in reviews on other classes of
hydrocarbons [8,4]. The intention of the present
article is to focus on the short chain unsaturated
0168-6445/89/$03.50 © 1989 Federationof European MicrobiologicalSocieties
236
h y d r o c a r b o n s . It will c o n c e n t r a t e on the b i o d e g r a d a t i v e routes e m p l o y e d b y the various microo r g a n i s m s involved a n d b o t h a p p l i e d a n d ecological aspects will b e considered,
2. P R O P E R T I E S , S O U R C E S A N D
SHORT-CHAIN OLEFINS
SINKS OF
Some relevant p r o p e r t i e s of the h y d r o c a r b o n s
u n d e r review have b e e n c o m p i l e d in T a b l e 1.
Several c h l o r i n a t e d alkenes are also i n c l u d e d because of their extensive use as i n d u s t r i a l solvents.
F r o m the table it is obvious t h a t all c o m p o u n d s
are gaseous or volatile at a m b i e n t c o n d i t i o n s with
the exception of some of the c h l o r i n a t e d c o m p o u n d s . G a s e o u s s u b s t r a t e s which are p o o r l y solu-
Table 1
Physical properties of selected olefins
Compound
Ethyne
(acetylene)
Ethene
(ethylene)
Propene
(propylene)
1-Butene
cis-2-Butene
trans-2-Butene
Butadiene
Isoprene
(2-methylbutadiene)
Styrene
(phenylethene)
Chloroethene
(vinyl chloride)
1,1-Dichloroethene
(vinylidene chloride)
Boiling
point
( o C at
1 Atm)
Explosion
limits
in air
(V//V%)b
- 84.0
1.15 c
2.4-83
- 103.7
9.5 c
2.7-34
- 47.6
-6.3
- 138.9
- 105.5
- 4.4
8.9 c
10 c
4.5 c
cis-l,2-Dichloroethene
trans- l,2-Dichloro-
ethene
Trichloroethene
(trichloroethylene)
Tetrachlorethene
(perchloroethylene)
a mol m
Partioning
coefficient
(air/water
at 30 ° C) a
3 gas phase (mol
34.1
2.0-11.1
1.6-10
1.4-16.3
-
1.0- 9.7
145.2
0.2 d
1.1- 8.8
- 13.4
1.26 e
3.6-31
37
60.3
1.25 e
0.19 e
6.5-15.5
_
47.5
0.46 e
_
87
0.49 e
121.1
0.91 e
m -3
-
water p h a s e ) - 1 ; b [12]; c [13];
d determined experimentally; e [14].
b l e in w a t e r offer c e r t a i n a d v a n t a g e s w h e n studying their m i c r o b i a l m e t a b o l i s m . A t low s u b s t r a t e
c o n c e n t r a t i o n s a n d at low cell densities gas chrom a t o g r a p h i c h e a d - s p a c e analysis a f f o r d s very sensitive, r a p i d a n d reliable m o n i t o r i n g of biological
processes in c o m p a r i s o n w i t h o t h e r s u b s t r a t e s that
dissolve well in water. H o w e v e r , the d i s a d v a n t a g e s
of using gaseous r a t h e r t h a n solid or liquid substrates have b e e n m o r e o b v i o u s to microbiologists.
A s a consequence, research on the m e t a b o l i s m of
gaseous s u b s t r a t e s has b e e n p e r f o r m e d in only a
few research groups. W o r k in the field of gaseous
c o m p o u n d s therefore lags b e h i n d that involving
the m o r e usual substrates. A m i c r o b i o l o g i s t is
g e n e r a l l y m o r e f a m i l i a r with h a n d l i n g glucose t h a n
with h a n d l i n g a s u b s t r a t e such as ethene. A d i s a d v a n t a g e of a p o o r l y w a t e r - s o l u b l e gaseous substrate is the special a t t e n t i o n t h a t needs to be
given to the kinetics of gas transfer to the a q u e o u s
phase. T h e i n c u b a t i o n systems used o b v i o u s l y need
special c o n s i d e r a t i o n s , not o n l y to keep the substrate a v a i l a b l e to the o r g a n i s m s b u t s o m e t i m e s
also to allow r e m o v a l of gaseous r e a c t i o n p r o d u c t s
such as c a r b o n dioxide. A n o t h e r i m p o r t a n t aspect
is that certain m i x t u r e s o f gas a n d air m a y explode. E t h e n e in air for i n s t a n c e forms explosive
m i x t u r e s in c o n c e n t r a t i o n s b e t w e e n 2.7% a n d 34%.
Nevertheless, m i c r o - o r g a n i s m s have not o n l y been
grown on gaseous s u b s t r a t e s in sealed b a t c h cultures b u t also in o p e n c h e m o s t a t cultures. W o r k
with c h e m o s t a t cultures using gaseous substrates
has m a i n l y b e e n p e r f o r m e d with m e t h a n e - u t i l i z i n g
b a c t e r i a [9,10] a n d m o r e recently also with ethenea n d p r o p e n e - u t i l i z i n g b a c t e r i a [11].
T h e c o m p o u n d s listed in T a b l e 1 are p r e d o m i n a n t l y of a n t h r o p o g e n i c origin with the n o t a b l e
e x c e p t i o n of ethene a n d isoprene. E t h e n e is a
special a n d i n t e r e s t i n g case. It is a simple, rather
reactive m o l e c u l e t h a t can u n d e r g o various reactions. F o r this r e a s o n it is p r o d u c e d b y m a n in
massive q u a n t i t i e s f r o m fossil reserves a n d is used
as a b u i l d i n g b l o c k for synthesis in the p e t r o c h e m ical industry. E t h e n e also p l a y s an u n e x p e c t e d l y
i m p o r t a n t role in p l a n t physiology. It is a gaseous
h o r m o n e t h a t acts o n all p l a n t s a n d is not only
p r o d u c e d b y p l a n t s b u t also b y b a c t e r i a a n d fungi.
T h e v a r i o u s effects o f ethene o n p l a n t s have b e e n
s t u d i e d a n d d e s c r i b e d in detail [15,16] b u t the
237
molecular basis for the observed phenomena is
still unresolved [17]. It remains to be shown how
this simple unsaturated hydrocarbon exerts its
drastic effects on plants at concentrations well
below 1 ppm.
The relative contributions to ethene emissions
into the atmosphere from biogenic and anthropogenic sources have been estimated by Sawada and
Totsuka [7]. Biogenic sources taken into account
were both plants and micro-organisms from terrestrial as well as aquatic ecosystems. The conclusions of Sawada and Totsuka are that most ethene
released is of biogenic origin (74%), while the
anthropogenic ethene (26%) originates mainly from
burning biomass and to a lesser extent from the
combustion of coal and fuel oil [7]. The total
amount of ethene released into the atmosphere by
both biogenic and anthropogenic sources was
estimated at 35 x 1 0 6 tonnes per year which is
approximately half the quantity produced and
processed annually by the petrochemical industry.
The volatile compound isoprene is also excreted by many plants [18]. Emission rates are
dependent on temperature and light intensity and
can account for 0.1 to 2% of the carbon fixed
during photosynthesis by oak plants [19]. Isoprene
emissions from natural vegetations have also been
quantified and were shown to be significantly
higher than the contribution from forest fires [20].
In contrast to the situation for isoprene and
ethene, the other compounds in Table 1 are predominantly of anthropogenic origins. The sources
of such compounds sometimes are directly linked
to a specific human activity. For instance chlorinated hydrocarbons are used on a large scale as
solvents. Many of these toxic compounds, including chlorinated ethenes, are persistent in the environment and consequently are widely distributed in ground water. In industrial areas concentrations up to 100 ~tg per 1 have been detected
[21,22]. Recently however, there have been several
reports on the biotransformation of chlorinated
ethenes which will be discussed in more detail in
sections 4.7 and 6.1.2.
The gaseous, non-chlorinated compounds may
also be attributed to specific sources. For example,
more than 90% of the acetylene generated originates from automobile exhaust, making it a useful
tracer gas in air pollution studies [23]. The atmospheric concentrations of the non-methane hydrocarbons ( N M H C ) have been monitored several
times at different locations. In urban areas the
N M H C - c o n c e n t r a t i o n was in the range of
250-1000 ppbC while concentrations in samples
from rural locations were 10-100 fold lower. At
both types of locations parafins are the major
class of compounds detected while olefins were
usually present in the range of 6-12% [24,25]. In
several rural air samples ethene and isoprene were
the major olefins detected [24] although in other
cases isoprene was not present in sufficient quantities to guarantee correct identification [25].
Short-chain unsaturated hydrocarbons have a
short atmospheric lifetime. Ethene is predominantly destructed in the troposphere by reactions
with O H radical or with ozone, while only a small
percentage diffuses into the stratosphere [7]. It is
unlikely that the non-methane hydrocarbons from
anthropogenic sources that are released into the
atmosphere will affect or will be affected by micro-organisms in either terrestrial or aquatic ecosystems. Nevertheless, as will be discussed in section 4, several bacterial species have the ability to
completely metabolize these compounds. In section 5 some considerations will be presented which
could explain these metabolic capabilities. An important part of the reasoning will be that traces of
the so called anthropogenic volatile hydrocarbonS
are also formed by microbial activities.
3. B I O T R A N S F O R M A T I O N OF UNSATURATED
HYDROCARBONS:
GENERAL
ASPECTS
Unsaturated hydrocarbons m a y be metabolized
by an initial attack on the unsaturated moiety of
the molecule (Fig. 1) or by an attack elsewhere on
the molecule. The following reactions may be observed at the unsaturated moiety of the molecule:
(i) oxidation with hydrogen peroxide and halide
ions by haloperoxidases, (ii) oxidation by mono-.
oxygenases using molecular oxygen as oxidant,
(iii) addition of water to the unsaturated bond and
(iv) reduction of the alkene or alkyne. T h e addition of ammonia to an unsaturated carbon-
238
0
/\
R ---HC--CH--R'
R--HC~CH--R'~
OH H
I
I
R--HC--CH--R'
R-- H2C--CH2--R'
Fig. 1. Biotransformations of the alkenic bond.
carbon bond, e.g. the formation of aspartate from
fumarate and ammonia by aspartate ammonialyase, will not be discussed.
3.1. Oxidation of unsaturated carbon-carbon bonds
3.1.1. Oxidation by haloperoxidases
Haloperoxidases c a t a l y z e the formation of
a,fl-halohydrins from alkenes [26] and a-halogenated ketones from alkynes [27] in the presence of
halide ion and hydrogen peroxide. In the absence
of halide ions, the action of a chloroperoxidase
may also result in the formation of epoxides [28].
At higher concentrations of halide ions, vicinal
dihalogenated products are also formed from alkenes [29]. The role of these enzymes in nature is
not always clear [30] and they presumably are not
involved in the metabolism of short chain unsaturated hydrocarbons.
3.1.2. Oxidation by monooxygenases
Alkane metabolism has been studied for many
years and several times alkenes have been proposed as intermediates in the degradation ot
long-chain alkanes. This assumption was based on
the detection of trace amounts of 1-alkenes after
growth with the corresponding alkanes (see [31]
for references) and the formation of 1-decene from
decane by cell-free extracts of Gandida rugosa
[32]. Reduction of N A D + in the presence of alkane by crude extracts of Candida tropicalis was
later shown to be caused by impurities in the
alkanes used [31]. Arguments against the involvement of 1-alkenes in the degradation pathway of
alkanes to primary alcohols and subsequently
carboxylic acids are however, abundant [33] and it
is now generally accepted that degradation of
aliphatic hydrocarbons in general proceeds via
initial oxidation by a monooxygenase yielding
primary or secondary alcohols [8].
The first evidence for oxidation of the double
bond of an alkene was the isolation of 1,2-dihydroxyhexadecane from cultures of Candida lipolytica growing on 1-hexadecene [34]. Later it was
shown that a large amount of the oxygen in the
diol was derived from molecular oxygen [35]. Using
heptane-grown Pseudomonas cells van der Linden
[36] demonstrated the formation of 1,2-epoxyheptane from 1,heptene. Subsequently many more
bacteria were found that possessed monooxygenases capable of epoxidating alkenes (see [37] for a
review). Although alkane- and alkene-grown cells
can generally epoxidate alkenes, reports concerning the metabolism of 1-alkenes with a chain-length
longer than C 5 show that the epoxide is not a
major intermediate in the degradative pathways,
which generally proceed via oxidation of the
saturated terminal methyl group. The first reports
of an epoxidation reaction actually participating
in the complete metabolism of an alkene were on
ethene metabolism in Mycobacterium E20 [38,39].
3.2. Hydratation of unsaturated carbon-carbon bonds
Hydratation of fumarate to malic acid in the
citric acid cycle is probably the best known biochemical hydratation of a double carbon-carbon
bond. A similar hydratation reaction was reported
by Wallen et al., who demonstrated formation of
10-hydroxystearic acid from oleic acid by a Pseudomonas sp. [40]. Further work by Schoepfer [41]
revealed that the 10-hydroxystearic acid produced
was optically active and had the R-configuration.
In a chloroacrylic acid-degrading bacterium, we
have recently detected two chloroacrylic acid hydratases, acting on either the cis- or the trans-isomer of this acid (unpublished results).
Hydratases acting on unsubstituted alkenes are
less well studied. Iida and Iizuka reported the
enzymatic conversion of 1-decene to 1-decanol in
crude extracts of a decane-grown Candida rugosa,
although it is not clear if this reaction participates
in decane catabolism [42]. To our knowledge the
only report on degradation of an aliphatic alkene
involving a hydratase is the degradation of 1hexadecene by a methanogenic enrichment culture
239
[43]. It is implicated that anaerobic 1-hexadecene
metabolism proceeds via an initial hydratation to
1-hexadecanol.
Degradation of acetylenic bonds is associated
with hydratation of the triple bond. These hydratation reactions are discussed in more detail in
section 4.1 of this review.
3.3. Reduction of unsaturated carbon-carbon bonds
Apart from the reduction of the 2,3-trans double bond in fatty acid synthesis, examples of reductases acting on double bonds are scarce. One
example is the hydrogenation of unsaturated fatty
acids by anaerobic rumen bacteria [44]. Until now
there is no evidence that such a reduction of
unsaturated carbon-carbon bonds is involved in
the mineralization of unsaturated hydrocarbons.
Although reduction of chlorinated ethenes under
methanogenic conditions results in sequential dechlorination, it is not clear if the double bond
itself is ultimately reduced [45].
The enzymatic reduction of acetylene and
several other unsaturated compounds by the action of a nitrogenase is a special case involving a
special enzyme.
4. SPECIFIC
WAYS
BIODEGRADATION
PATH-
4.1. Ethyne and acetylenic compounds
4.1.1. Ethyne (acetylene)
The first report on the biodegradation of ethyne
(acetylene) by aerobic bacteria was published in
1932 by Birch-Hirschfeld [46] who described
growth of Myobacterium lacticola with ethyne as
sole carbon and energy source. Almost fifty years
later a Nocardia rhodochrous, which requires the
pyrimidine moiety of thiamine for growth [47],
and a Rhodococcus A1 [48] were isolated with
ethyne as carbon source. The Rhodococcus A1 also
utilized propyne as growth substrate. The description of the strain designated M. lacticola could fit
both the Nocardia and the Rhodococcus A1. The
Mycobacterium was grown in the presence of soil
extract, which could fulfill the growth factor
requirement of the Nocardia rhodochrous. None of
the above strains were capable of growth with
ethene. Other aerobic acetylene-utilizing bacteria
have been isolated [49,50], but these organisms
were not studied in detail.
The observation of Birch-Hirschfeld that
acetaldehyde accumulated when ethyne-grown
cells were incubated in the presence of ethyne was
confirmed by the Bont and Peck [48]. Kanner and
Bartha [51] also identified acetaldehyde as an intermediate in ethyne metabolism. They also
showed that growth of the N. rhodochrous with
ethyne and ethanol resulted in similar activities of
acetaldehyde dehydrogenase, acetothiokinase and
isocitrate lyase. However, ethyne hydratase, the
enzymic activity that would explain acetaldehyde
formation from ethyne, could not be detected in
cell-free extracts of ethyene grown N. rhodochrous
[51].
In cell-free extracts of ethyne grown Rhodococcus A1 ethyne consumption and acetaldehyde formation were detected when the extract was
incubated under an atmosphere of nitrogen [48].
Aerobic incubation of the extract resulted in irreversible inactivation of ethyne hydratase activity.
Using dialyzed cell-free extracts of Rhodococcus
A1 a stoichi0metric conversion of ethyne to
acetaldehyde was observed.
In crude extracts the K m of the hydratase for
ethyne was calculated to be 0.6 m M [48]. The
concentration of ethyne in the gas phase which
corresponds with this value is 1.5% [v/v]. In order
to grow at ethyne concentrations far below the K m
of the hydratase, Rhodococcus A1 probably
synthesizes large amounts of the enzyme to ensure
a sufficient carbon flux. When these cells subsequently are exposed to high concentrations of
e t h y n e an u n b a l a n c e d o v e r p r o d u c t i o n of
acetaldehyde results. It should however be borne
in mind that the K m was determined with crude
extracts and not with whole cells. The K m for
these two situations need not necessarily be the
same.
Anaerobic degradation of ethyne has also been
observed [52]. With sulphate as an electron acceptor an enrichment culture which grew anaerobically at the expense of ethyne was obtained.
Acetate was identified as an intermediate in the
degradation of ethyne to carbon dioxide [53].
Schink recently reported the isolation of strictly
240
anaerobic acetylene-utilizing bacteria [54]. These
isolates were assigned to a new species in the
genus Pelobacter, namely P. acetylenicus. Acetylene was fermented by disproportionation to acetate and ethanol. Acetylene hydratase was not
detected in cell-free extracts but the enzymes necessary for acetaldehyde metabolism were present
in high activities.
4.1.2. Other acetylenic compounds
To study enzymatic transformations of the
acetylenic bond, Yamada and Jakoby [55] enriched for organisms capable of utilizing the nongaseous and water-soluble acetylenedicarboxylic
acid. From a Pseudomonas isolated on acetylene
dicarboxylic acid a more than 100-fold purified
enzyme preparation was obtained which transformed acetylenedicarboxylic acid to pyruvate and
carbon dioxide in a cofactor-independent reaction
(EC 4.2.1.72). The mechanism by which this reaction proceeds was not elucidated. Neither of the
potential intermediates, oxaloacetate (which would
result from a hydratation) or acetylene monocarboxylic acid (which would result from an initial
decarboxylation of the acetylene dicarboxylic
acid), resulted in pyruvate formation with the
partially purified protein preparation.
To provide further information on the mechanism of this reaction a Pseudomonas was isolated
with acetylene monocarboxylic (propynoic) acid
as the sole carbon source [56]. From extracts of
this strain an enzyme was partially purified (93fold) which catalyzed the hydratation of propynoic acid to malonic semialdehyde (3-oxo-propionic acid) without the addition of cofactors under an atmosphere of helium (EC 4.2.1.71). Acetylenedicarboxylic acid and propynol did not serve
as substrates.
Also using propynoic acid, de Bont et al. isolated a Gram-negative and a Gram-positive
bacterium [57]. Interestingly, when propynol was
used as a carbon-source in enrichment cultures
only fungi were isolated. Using 3-butyn-l-ol as
carbon source both fungi and Gram-negative
bacteria were isolated [57]. From these isolates the
facultative methylotroph Pseudomonas BB1 was
chosen for further study of 3-butyn-l-ol metabolism [58]. Cell-free extracts of 3-butyn-l-ol grown
Pseudomonas BB1 contained a phenazine methosulphate-dependent alcohol dehydrogenase and
3-butynoic acid hydratase activity yielding succinic semialdehyde (4-oxo-butyric acid). With propynoic acid as a substrate malonic semialdehyde
was formed in cell-free extracts. Although Yamada
and Jakoby [56] did not test 3-butynoic acid as a
substrate for their enzyme preparation, it probably
resembles the hydratase activity of 3-butyn-l-ol
grown Pseudomonas BB1.
The metabolism of acetylenic compounds thus
seems to be catalyzed by hydratases, and until
now at least three different enzymes capable of
hydratating the acetylenic bond in different molecules have been described: (i) an oxygen sensitive
ethyne hydratase [48], (ii) an acetylene dicarboxylate hydratase (EC 4.2.1.72) yielding pyruvate and
CO 2 [55] and (iii) an acetylenecarboxylate hydratase (EC 4.2.1.71) yielding malonic semialdehyde [58,56].
4.2. Ethene
Ethene (ethylene), the simplest olefin, can be
oxidized to epoxyethane by several types of microorganisms, including methanotrophs [59], alkaneutilizers [60] and Nitrosomonas europaea [61].
These organisms, however, are not able to grow
with ethene, but other bacteria have been isolated
which utilize ethene as the sole source of carbon
and energy.
Ishikura and Foster [35] isolated an orange-yellow pigmented 'ethylene bacterium' from soil. It
was a Gram-positive, motile, non-sporulating rod
that was also capable of growth with ethanediol.
In 1976 both Heyer [62] and de Bont [63] reported
the isolation of several Mycobacteria capable of
growth with ethene as sole carbon and energy
source. Subsequent enrichment cultures with
ethene have always resulted in the isolation of
Mycobacteria, although very recently non-mycobacterial ethene utilizers have been isolated by
Mahmoudian and Leak [64]. Using propene or
1-butene as the carbon source in enrichment cultures, van Ginkel et al. [65] in most cases isolated
Xanthobacters. These Xanthobacter strains, along
with Nocardia H8 which was isolated with 1-
241
hexene as a carbon source, were all capable of
growth with ethene. However, the growth rates of
these strains with ethene as substrate were lower
than those of the ethene-utilizing Mycobacteria.
This could explain why Mycobacteria are almost
always isolated when ethene is used as carbon
source in batch-type enrichment cultures.
Growth of the 'ethylene bacterium' in the presence of labelled oxygen on ethene resulted in
incorporation of significant amounts of 180 in cell
material as compared to growth with acetate under the same conditions [35]. This would implicate
the involvement of a monooxygenase type of reaction in the assimilation of ethene into cell material.
More specific evidence for the involvement of
such an enzyme was obtained in studies with
Mycobacterium E20. In this ethene-utilizing
Mycobacterium it is possible to accumulate
epoxyethane from ethene when whole cells are
incubated in the presence of fluoroacetate [38],
implicating that ethene is metabolized via
epoxyethane in a reaction catalyzed by a monooxygenase.
When similarly inhibited cells were incubated
with either 1SO2 o r H2180 it was established that
the oxygen atom in epoxyethane was indeed derived from molecular oxygen [39]. Monooxygenase
activity with ethene was confirmed using cell-free
extracts of ethene-grown Mycobacterium E20. The
reaction was shown to be O 2- and N A D H - d e p e n dent. N A D H could be replaced by N A D P H , although this resulted in a lower specific activity.
Whole cells of ethene-grown Mycobacterium
E20 also oxidized ethane to some extent but this
capacity was not present in crude extracts of
ethene-grown cells. Ethane-grown ceils oxidized
ethane and ethene, but it was not possible to
detect any monooxygenase activity in crude extracts of these cells. Apparently Mycobacterium
E20 is capable of synthesizing two different monooxygenases: a soluble alkene monooxygenase after
growth with ethene and an unstable alkane monooxygenase after growth with ethane. This dependency of the monooxygenase induction on the
growth substrate was further confirmed when the
enantiomeric composition of the epoxypropane
produced by ethene- and ethane-grown Mycobacterium E20 was compared (see 6.2.3).
Although it has been known for some time that
microorganisms can oxidize alkenes to the corresponding epoxyalkanes [36], very little is known
about the further metabolism of these compounds.
Microbial metabolism of epoxides has recently
been reviewed by Weijers et al. [66].
The metabolism of epoxyethane, the most simple epoxide, was studied in Mycobacterium E20.
When ethane-grown Mycobacterium E20 was incubated with ethane, in the presence of fluoroacetate, acetate accumulated analogous to epoxyethane accumulation from ethene. This could implicate that epoxyethane, analogous to acetate in
ethane metabolism, is metabolized in a CoA-dependent reaction. Cell-free extracts of ethenegrown cells of Mycobacteriurn E20 were able to
catalyze the oxidation of epoxyethane. The reaction was completely dependent upon the presence
of N A D + and CoA, and the epoxyethane degradation rate was approximately doubled by adding
F A D to the reaction mixture [38]. Besides N A D +,
CoA and FAD, a fourth unknown dissociable
cofactor was involved in the enzymic conversion
of epoxyethane. This dialysable, heat stable cofactor was present in ethene-grown cells, but not in
ethanol-grown cells. The nature of the unknown
cofactor was not elucidated. Evidence that the
product of the epoxyethane dehydrogenase reaction was acetyl-CoA was sought in experiments
using [14C]epoxyethane. Incubation of cell-free extract with the radioactive epoxide and the required
cofactors along with citrate synthase, oxaloacetate
and fluorocitrate resulted in radioactivity in
ether-extracts which cochromatographed with
citrate. Omission of citrate synthase or oxaloacetate from the complete reaction mixture resulted
in almost no radioactivity in the citrate spot. It is
not clear whether one single enzyme or an enzyme
complex is responsible for the oxidation of
epoxyethane and much remains to be elucidated
concerning this novel enzymic activity.
Anaerobic degradation of ethene has until now
not been reported. The strict oxygen-dependency
of ethene-degradation strongly suggests that
ethanol formation due to hydration of the
double-bond does not occur. The activation of the
double-bond apparently is only brought about by
a monooxygenase.
242
4.3. Propene and 1-butene
Analogous to the situation with ethene, many
bacteria are capable of oxidizing propene to
1,2-epoxypropane [59,60]. In contrast to ethene,
however, propene is an asymmetric molecule with
an, unsaturated and a saturated carbon-carbon
bond, allowing more than one possibility for initial enzymic attack.
With propane-grown cells of Mycobacterium
convolutum acrylic acid was identified as oxidation
product of propene, indicating the initial formation of 3-hydroxy-l-propene [67]. But with other
bacteria grown on propane, epoxypropane was
reported as an oxidation product of propene with
only trace amounts of 3-hydroxy-l-propene being
detected [60]. With Pseudomonas fluorescens
N N R L B-1244 the propene consumption rate was
20% higher than the 1,2-epoxypropane formation
rate. The 1,2-epoxypropane degradation rate was
not reported so that only a minimal ratio of
epoxidation versus hydroxylation of 6 can be
calculated. Although the presence of two different
enzymes could not be ruled out, both hydroxylation and epoxidation of propene to 3-hydroxy-1propene and 1,2-epoxypropane, respectively, is
probably effected by the same enzyme system in
both Pseudomonas fluorescens N R R L B-1244 and
Brevibacterium sp. strain CRL56 [60]. This situation has also been encountered in Nitrosomonas
europaea [68].
The utilization of propene as a carbon and
energy source is less frequently described. The
first report of an organism capable of growth with
this gaseous compound was a 'methanbakterium'
isolated in 1930 [69]. Cerniglia et al. [67] also
isolated a propene-utilizing organims. They proposed, on the basis of isocitrate lyase activities
and the fatty acid composition of their strain PL-1
after growth with different substrates, that propene was metabolized via initial attack at the
double bond resulting in a C 2 + C 1 cleavage. Experiments with Mycobacterium Pyl [70,71] and
Xanthobacter Py2 [72], which were both isolated
with propene as carbon source, and Nocardia By1
and Xanthobacter By2 [65] which were enriched
with 1-butene, revealed that in these strains 1-alkenes were epoxidized to the corresponding 1,2-
epoxyalkanes. In the strains Pyl and Py2,
NADH-dependent propene-monooxygenase activity was detected in crude ceil-free extracts. 1,2epoxypropane, the product of the monooxygenase
reaction, was also utilized as growth substrate.
Both strains can also utilize 1-butene as a growth
substrate. In contrast to strain Pyl, strain Py2 also
grows on ethene although the growth rate is low.
In Mycobacterium Pyl isocitrate lyase activity
was induced after growth on propene, 1,2-propanediol and acetate, indicating that these substrates
are metabolized via acetyl-CoA. G r o w t h on
1-butene and propionic acid did not result in
isocitrate lyase induction. These results correspond to the results obtained by Cerniglia et al.
[67], but further indications as to how 1,2epoxypropane is metabolized are still lacking. An
analogous reaction as was proposed in ethenemetabolism [38] in which epoxyethane is oxidized
to acetyl-CoA seems very unlikely in view of the
increased isocitrate lyase activities after growth
with propene.
Another possibility for the further metabolism
of 1,2-epoxypropane in Mycobacterium Pyl via
1,2-propanediol and propionaldehyde as was
shown in Nocardia A60 [73] was ruled out by
carrying out simultaneous adaptation experiments
with propene- and 1,2-propanediol-growth cells
(Hartmans, unpublished results). 1,2-Propanediol
metabolism in Mycobacterium Pyl proceeds via
acetol which is subsequently cleaved into acetate
and formaldehyde by acetol monooxygenase [74].
Although this explains the induction of isocitrate
lyase after growth with 1,2-propanediol in Mycobacterium Pyl, the metabolic pathway of propene
degradation via 1,2-epoxypropane to acetyl-CoA
still remains to be elucidated.
4.4. 2-Butene
In contrast with ethene, propene and 1-butene,
2-butene is an internal alkene thus possessing only
saturated terminal carbon atoms. This difference
in chemical structure is reflected in the ability of
organisms to degrade the compound. It is not
utilized as a growth substrate by at least six different 1-alkene-utilizers tested, including ethene-,
propene- or 1-butene-utilizers [65]. Fujii et al.
243
however, have described two Mycobacteria which
were isolated with propene and 1-butene, respectively, that were also able to grow on the C z to Ca
saturated hydrocarbons and both isomers of 2butene [75]. Both strains grew very poorly with
ethene and did not grow with 1,3-butadiene as a
carbon source.
Enrichment cultures with trans-2 butene as the
carbon-source resulted in three bacterial isolates,
two strains of the genus Nocardia and one Mycobacterium [65]. One of the Nocardia strains, a
red-pigmented bacterium designated as Nocardia
TB1 was chosen to study trans-butene degradation
[76]. Strain TB1 was also capable of growing on
the C 3 to C 6 alkanes but did not grow with
methane, ethane, 1,3-butadiene or the C 2 to C 6
1-alkenes. Growth with cis-butene was extremely
slow and doubling times on butane and transbutene were 6 and 30 hours, respectively.
Using arsenite as inhibitor, butyric acid and
crotonic acid accumulated when butane- or transbutene-grown cells were incubated with their respective growth substrates.
Surprisingly, trans-butene- and butane-grown
cells oxidized trans-butene at a higher rate than
butane, the substrate which supports the faster
growth, cis-butene was degraded at the same rate
as trans-butene.
Based on enzymic activities of trans-butene,
butane and succinate grown cells a degradative
pathway as shown in Fig. 2 was proposed [70].
Although 2,3-epoxybutane was degraded by
trans-butene-grown cells of strain TB1, it was not
considered an intermediate in trans-butene
metabolism as its degradation did not result in
increased CO2-formation by washed cells, whereas
the oxidation of crotonic alcohol and trans-butene
itself did. Degradation of 2,3-epoxybutane resuited in the excretion of an unidentified product,
probably originating from a hydroxylation reaction by the monooxygenase.
Further evidence that Nocardia TB1 contains
an alkane-type monooxygenase was obtained when
the enantiomeric composition of epoxides formed
by both butane- and trans-butene grown cells was
analyzed (Table 4).
4.5. Butadiene and isoprene
H3C-CH2~CHT CH3
02~
NADH
02tNADH
H3C-CH : CH-CH3
%
H3C-CH2-CH2-CH2
I x~.-NADH
°°
"H
I NAD
NADH
L
H3C- Cl4=CH -(H 2
['~NADH
°°
NADH
~'-~NADH
H3E-CH2-IEH2-C~0
H3C-CH ~CH-C ~0
ATP+ ~oA
ATP~-- CoA
~OH
\OH
!
H~E-CH2-CH2-C~ ~
"CoA
H3C-CH =CH-C ~0
"CoA
13-oxidation
Fig. 2. Proposed degradative pathway of butane and transbutene in Nocardia TB1 [76].
Isoprene (2-methylbutadiene) is a naturally occurring compound, whereas its non-methylated
analogue butadiene, to our knowledge, is not
formed biologically [77]. Butadiene can be compared to ethene, with respect to the unsaturated
character of its carbon atoms, with the two double
bonds probably behaving as a conjugated system.
Microbial utilization of butadiene has been reported by Watkinson and Somerville who isolated
a Nocardia species from enrichments with butadiene as the sole carbon and energy source [78].
Respiration rates of butadiene-grown cells with
butadiene and 1,2-epoxy-3-butene as substrate
were similar. This oxidative capacity was absent
from acetate-grown cells. Based on isocitrate lyase
activities butadiene metabolism in Nocardia sp.
249 was thought to proceed via acetate. The degradation p a t h w a y that was p r o p o s e d for
butadiene metabolism was very speculative, and
not based on measurement of enzymic activities or
identification of possible intermediates. It was
suggested that butadiene is epoxidized to 1,2-
244
epoxy-3-butene, which subsequently would be hydrolyzed to the corresponding diol and oxidized to
2-oxo-3-butenoic acid. Oxidative decarboxylation
to acrylic acid followed by hydratation to lactate
and oxidation to pyruvate would after decarboxylation, eventually result in acetate formation. Interestingly, oxidation of racemic 1,2-epoxy-3butene was not complete, and it was shown that
the remaining epoxide material was optically active, thus implicating that the epoxide degrading
enzymic activity was stereoselective. Based on this
observation it was concluded that the epoxidation
of butadiene by the Nocardia species is stereospecific [78].
Enrichments using different soil samples and
butadiene or isoprene as a carbon source in all
cases resulted in the isolation of pink-pigmented
bacteria belonging to the genus Nocardia [65,77].
All isolates were capable of growth on both substrates, possibly suggesting a connection between
the degradation pathway of the two alkadienes. In
cell-free extracts of alkadiene-grown Nocardia IP1
oxidation of these compounds was N A D H - and
oxygen-dependent, indicating that these compounds are degraded by a monooxygenase [77].
Incubation of washed cell suspensions of alkadiene grown Nocardia TB1 with the respective
growth substrates in the presence of 1,2-epoxyalkanes as competitive inhibitors of epoxide degradation, all the possible mono- and diepoxides
of butadiene and isoprene could be detected. Although it was proposed that the initial step in the
metabolism of both compounds Nocardia TB1
probably was the formation of an epoxide, the
degradation pathway of these alkadienes remains
to be elucidated.
4.6. Styrene
Although styrene contains an aromatic nucleus,
it can also be classified as a substituted alkene.
Styrene can be oxidized by Methylosinus trichosporium OB3b [79] and Nocardia corallina B-276
[80]. In both cases the alkenic moiety of the molecule is attacked, resulting in the formation of
styrene oxide (7,8-epoxyethylbenzene or phenyloxirane).
Omori et al. [81] were, to our knowledge, the
first to attempt the isolation of styrene-utilizers.
They tested 101 soil samples without success,
probably because the concentration of styrene that
was used in the enrichment cultures was too high
(2% w / v ) . Sielicki et al. [82] using a concentration
of 1% (w/v), which is still much more than the
solubility of styrene in water, obtained a mixed
culture utilizing styrene. In ether extracts from
styrene cultures phenylacetate and 2-phenylethanol were identified. Using a pure culture of a
styrene-utilizing Pseudomonas Shirai and Hisatsuka [83] also demonstrated accumulation of 2phenylethanol from both styrene and styrene
oxide. Based on these results it was proposed that
styrene oxide is an intermediate in the transformation of styrene to 2-phenylethanol. Baggi et al. [84]
isolated phenylacetic acid and 2-hydroxyphenylacetic acid from styrene-grown cultures of a Pseudomonas fluorescens, once more indicating that
styrene metabolism involves initial attack of the
ethylenic bond.
Using low concentrations of styrene it was recently shown (van der Werf and Hartmans, unpublished results) that styrene utilizers are very
abundant. F r o m all soil samples tested, bacterial
strains were isolated that could utilize styrene as a
sole carbon and energy source. In several of these
strains an oxygen- and N A D H - d e p e n d e n t styrenedegrading enzymic activity was present in cell-free
extracts after growth with styrene. The further
metabolism of the probable oxidation product
styrene oxide, was in some cases via phenylacetaldehyde which was formed by a styrene oxide
isomerase activity. Reduction or oxidation of the
phenylacetaldehyde thus formed would then have
resulted in either the formation of 2-phenylethanol
or phenylacetic acid, the intermediates previously
isolated by other groups [83,84].
2-phenylethanol formation from styrene could
also result from a hydratation reaction. Indeed
very recently anaerobic isolates with styrene as
sole carbon and energy source were shown to
produce 2-phenylethanolas intermediate in styrene
degradation [85].
F r o m the above we can conclude that there are
at least two different modes of initial attack of
styrene, both involving the alkenic bond.
245
4. 7. Chlorinated ethylenic compounds
Vinyl chloride (chloroethene), the simplest
chlorinated ethylenic compound is aerobically
utilized as a sole carbon and energy source by
Mycobacterium L1 [86]. Ethene, but not propene,
also supports growth of this strain. Growth on
vinyl chloride and ethene both result in alkene
monooxygenase induction. Oxidation of vinyl
chloride by vinyl chloride- and ethene-grown cells
can be competitively inhibited by ethene or propene, indicating that in vivo vinyl chloride is
degraded by the alkene monooxygenase. The
product of the monooxygenase reaction with vinyl
chloride has not been identified, but probably is
chloroepoxyethane (chlorooxirane). The metabolism of chlorooxirane, which is a very unstable
compound that rearranges to chloroacetaldehyde,
has not been studied. However, using washed
cell-suspensions it was possible to detect chlorideion formation after incubation with vinyl chloride,
but not with chloroacetaldehyde, indicating that
this compound is not an intermediate in vinyl
chloride metabolism. Although vinyl chloride
grown Mycobacterium L1 cells were capable of
oxidizing dichloroethene isomers, strain L1 did
not grow with chloroethenes other than vinyl chloride.
Until now, no other bacterial strains are known
that can grow with chlorinated ethenes. Biotransformation of ethenes with more than one
chloride substituent, especially trichloroethene and
tetrachloroethene, however, has been extensively
studied the last decade. Both oxidative and reductive transformations have been reported (see [87]
for a recent review).
Oxidative transformation of di- and trichloroethenes by mixed- and pure-cultures of methane
utilizers has been reported by several authors
[21,88,89]. It is generally accepted that the initial
oxidation step is brought about by the a-specific
methane monooxygenase resulting in the formation of the relatively unstable epoxides. Any further transformations that take place are still obscure, although, using labeled trichloroethene, it
was possible to detect the formation of labeled
carbon dioxide in a pure culture of a methaneoxidizing bacterium [89]. Tetrachloroethene was
not degraded by the methane-utilizing cultures.
Nelson et al. [90] recently reported that toluene
stimulates the trichloroethene-degradative ability
of the natural microflora of environmental water
samples. Several pure cultures capable of utilizing
toluene also degrade trichloroethene. These
authors also presented evidence that a toluene
dioxygenase is involved in trichloroethene degradation by one of the pure cultures. Studies with
toluene induced cells of Pseudomonas putida F1
[91] revealed that trichloroethene degradation by
these cells was at a significantly greater initial rate
than by Methylosinus trichosporium OB3b. Mutants
of Pseudomonas putida F1 defective in the gene
encoding for the oxygenase component of toluene
dioxygenase failed to degrade trichloroethene,
whereas a spontaneous revertant simultaneously
regained the ability to oxidize toluene and to
degrade trichloroethene, thus confirming the role
of the oxygenase component of toluene dioxygenase in trichloroethene degradation. The
three isomeric dichloroethenes were also degraded
by Pseudomonas putida F1, but tetrachloroethene,
chloroethene (vinyl chloride) and ethene were not.
Until now, tetrachloroethene degradation under aerobic conditions has not been reported. Under methanogenic conditions, however, a sequential transformation of tetrachloroethene via trichloroethene and dichloroethenes to chloroethene
(vinyl chloride) was observed in anaerobic laboratory columns fed with an acetate-containing
medium [45]. The transformation of tetrachloroethene to trichloroethene is also accomplished by
pure cultures of several Methanosarcina spp., although the transformation rate differs significantly between strains [92].
5. E C O L O G I C A L ASPECTS
The interesting question arises as to how the
organisms described above acquired their metaboric potential to degrade the various gaseous and
volatile olefins. Several of these compounds are
assumed to have entered the environment during
this century due to human activities. It would be
interesting to know if the microbes capable of
metabolizing these compounds have also only re-
246
cently acquired this capacity; or have these degradative capacities evolved over a much longer
period of time as a result of the presence of these
supposedly anthropogenic compounds arising from
natural sources?
Before speculating on these aspects, it seems
worthwhile to first examine the situation for the
natural compound ethene. The other short chain
unsaturated hydrocarbons, with the exception of
isoprene, are generally considered to be anthropogenic and will be discussed subsequently. Another
interesting aspect of the short chain unsaturated
hydrocarbons is their inhibitory effect on certain
metabolic processes. These effects will be discussed in section 5.3 and attention will be given to
the use of acetylene as a tool to determine in situ
metabolic activities.
5.1. Ethene." formation and degradation in nature
Ethene presents a special ecological case because the gas has such pronounced effects on
plants and fruits at very low concentrations. In
soil it may affect plant life when accumulating due
to the production by microorganisms or to a lesser
extent by plants. It also accumulates during storage of harvested fruits, vegetables and flowers
through the endogenous production by the plant
material.
Several relevant aspects of ethene in nature
were discussed 10 years ago by Primrose [93] and
his review presently still offers a solid basis for
our understanding of the role of microbes in the
production and utilization of ethene. New insights, however, have emerged during the last decade. Especially on the biosynthesis of the gas,
substantial progress has been made. It has now
been clearly established that at least three different routes for the biosynthesis of ethene exist. In
plants, methionine serves as the precursor and
yields ethene through S-adenosyl-methionine and
1-aminocyclopropane-l-carboxylic acid [94,17]. In
bacteria ethene is also formed from methionine
but in a different fashion. In E. coli [94] and other
microorganisms [96] 2-keto-4-methylthiobutyric
acid is an intermediate which is subsequently converted into ethene and methanethiol [97]. In the
fungus Penicillium digitatum ethene is formed from
H~N-CH-COOH
CH2
EhH2
$1
rl2N-CH-EOOH
i
EH 2
CH2
*~-Adenosyt
Plants
EH]
~
H~N
• ,, / COOH
sCx
~ H2C=CH~
H2C-- [H2
O2-dependent
CH~
H2N- CH -COOH
i
CH2
5H 2
~
S
[0OH
O=C
Ecoli
!H 2
CH?
~
H2C:CH 2
Oz-dependenf
i
[H~
EOOH
O=C
Pemolhum
[H2
0 3-
~H~
~lgltafum
depend enf
~
H2[zCH 2
EOOH
Fig. 3. Biosynthetic pathways of ethene formation.
a-ketoglutarate as was shown using a combination
of partially purified enzyme preparations [98]. The
reaction also required Fe2+-ions and L-arginine
but the carbon atoms of ethene were derived from
a-ketoghitarate as shown with 14C-labelled substrates. In a later communication it was stated
that oxygen is also essential for ethene formation
[99].
The three biosynthetic pathways leading to
ethene formation are shown in Fig. 3. It is interesting to note that in all three routes enzymic
reactions are implicated that require molecular
oxygen. Since ethene formation has also been observed under strict anaerobic conditions, it is
therefore obvious that other pathways leading to
ethene formation remain to be discovered.
The rate at which ethene is produced by either
plants or microorganisms varies considerably. For
plants this rate varies from species to species and
organ to organ and it also depends on the stage of
development of the plant [15]. For microorganisms the rate of ethene production depends
very much on the organism tested, and also on the
culture conditions applied. Ethene-producing microorganisms have been compiled from the literature by Primrose [93] and his conclusions were
that such organisms are ubiquitous in soil and
water. This concept was supported by Fujii et al.
[75] who screened 296 microorganisms of which 51
produced ethene. The positive strains included
bacteria as well as fungi and yeasts. The best
producing organism in terms of quantities formed,
247
was a Penicillium digitatum strain. By mutant
selection it was eventually possible to obtain a
growth-suppressed mutant that was able to convert 2.1% of the carbon present in glucose into
ethene-carbon [99].
In soil the situation is clearly different and here
only very minute quantities are produced. It is
very difficult to determine the absolute quantity of
ethene produced in soil since under aerobic conditions the gas is also oxidized by ethene-utilizers
[100,101,63] resulting in a considerably lower net
observed production, or in no apparent production at all. This point has not always been kept in
mind by many researchers studying the production of ethene in soil and consequently has resuited in some in interesting theories [102-108].
Another favoured approach in the area of ethene
formation in soil has been to amend soil with
different substrates that are potentially able to
generate ethene [102,106-108]. The substrates were
generally added in relatively high concentrations
(mM-range). Obviously, when such excessive
amounts, of for instance methionine, are added to
aerobic soil, the many different organisms able to
form ethene from this compound in only a few
enzymic steps will do so immediately, resulting in
unusually high ethene evolution rates. The
ethene-utilizers present clearly cannot immediately oxidize this excess of ethene formed, resulting
in a transient accumulation of ethene. Such experiments have little bearing on the acutal situation in soil; they can only give indications as to
potential precursors for ethene formation. Realistic rates for ethene formation can only be obtained under anaerobic conditions. Such rates,
when recalculated on a basis of ng ethene (g
soil) -a day 1 vary from 0.01-10 [109] and 0.06-2.5
[107] for various soils to 600 for pine-needle litter
[104] and to a difficult to explain high rate of 7000
in a silt loam soil [105].
Estimation of ethene formation rates in soil is
interesting to know, but more important for the
ecosystem are the in situ concentrations of the gas.
Several measurements in field situations have been
reported by K.A. Smith and colleagues [110,111].
From their results it can be generalized that under
anaerobic conditions ethene may accumulate to 10
ppm (determined in the gas phase) or sometimes
even higher, whereas under aerobic conditions the
gas is almost never present at concentrations of
more than 0.1 ppm. This concentration incidently
is approximately the threshold concentration for
the action of ethene as a plant hormone. The
ethene-utilizing bacteria, in keeping the concentrations below this level in aerobic soil, thus hold a
key position in maintaining the soil environment
suitable for plant roots. When assessing the effects
of ethene production in soil on plant growth [112],
the significance of these bacteria should be given
explicit attention.
The implicit premise made on the functioning
of the ethene-utilizers in soil is that they are able
to degrade ethene at the extremely low concentrations prevailing in soil. A concentration of 0.1
ppm in the gas-phase corresponds to a concentration of 0.45 nM in the water-phase. The Michaelis-Menten constant of the ethene-utilizing Mycobacterium E3 has been determined and was estimated to be 100 ppm [113] ethene in the gas-phase
corresponding to 0.45 ~M in the water-phase. For
comparison the K m for methane of the purified
methane monooxygenase from Methylococcus
capsulatus (Bath) is 3 /~M [114]. This observation
does not make it likely that such organisms are
actively engaged in the oxidation of ethene at the
concentrations present in aerobic soils. Nevertheless, van Ginkel et al. [115], using a compost filter
with its indigenous population in a packed bed
through which 2 ppm ethene in air was passed
continuously, demonstrated ethene degradation at
this low concentration. They also demonstrated
that the organisms responsible for ethene degradation were actually able to grow under these conditions, as evidenced by an increase in ethene-utilization rate in time. It would seem that this remarkable capacity of the ethene-utilizers to
metabolize ethene at these low concentrations,
and to obviously benefit from this, is only possible
when they grow mixotrophically. Components
from the compost almost certainly will have functioned as supplementary carbon and energy
sources for the bacteria.
Plants not only produce ethene, they also
metabolize ethene and during the last decade considerable progress has been made concerning the
metabolism of ethene in plants. Until recently it
248
was believed that higher plants were not able to
metabolize the hormone, but by using 14C-labeled
ethene it has now been firmly established that
different products are formed in plants [116,117].
The rates at which ethene is metabolized are,
however, extremely low and in the order of 0.5
nmol (kg dry weight)-1 hr-1. This process therefore will not be significant as an ethene sink in
nature. Interestingly, in plants ethene is attacked
in the same way as in bacteria. In both cases the
initial step in ethene metabolism is the oxidation
to epoxyethane. The values reported for the K m of
ethene oxidation by the plant monooxygenases
are, depending on the plant being studied, in the
range of 1 nM to 1 /*M [117].
5.2. Supposedly anthropogenic short chain unsaturated hydrocarbons
5.2.1. Biogenic formation
The generally accepted view on atmospheric
hydrocarbons with respect to their origin is that a
division can be made in biogenic and anthropogenic hydrocarbons [7,24,25]. The biogenic hydrocarbons are methane, ethene, isoprene and the
terpenes, but these hydrocarbons may of course
also be found in anthropogenic emissions. The
anthropogenic hydrocarbons supposedly enter the
biosphe, re only as a result of human activities and
thus should be 'new' compounds for microorganisms. This division on the basis of the source
of the hydrocarbons may be useful to determine to
what extent hydrocarbons in air can be attributed
to certain emissions. But the division on biogenic
versus anthropogenic is misleading in understanding the metabolic potential of micro-organisms.
Indeed several reports are available which demonstrate the biological formation of most compounds
considered in this section.
The production of ethane, propane, butanes,
ethene, propene and 1-butene from soil have been
reported [118,119]. The gases were observed under
anaerobic conditions, which would indicate the
involvement of anaerobic bacteria. However, a
more pronounced formation of the gases was observed when soil initially kept anaerobic was subsequently made aerobic. Also, amending soil with
various substrates enhanced the formation of the
gases. These observations led Goodlass and Smith
[119] to the hypothesis that substrates first mobilized under anaerobic conditions stimulate the formation of gases under aerobic conditions.
The involvement of aerobic organisms as anticipated by Goodlass and Smith [119] has found
strong support by interesting work published by a
group of Japanese researchers [120-123]. They
screened m a n y bacteria, yeasts and fungi under
aerobic conditions and m a n y strains were found
that produced ethane, ethene, acetylene, propane,
propene, butanes, butenes and pentanes. Even
aerobic methane formation was detected. Recently
it was shown that acetylene, contrary to the general belief, is not exclusively connected with human activities. It is apparently also produced by
natural processes in seawater [124].
The quantities that are formed by the various
processes described above are minute and consequently concentrations of these gases are always
low in nature. But from time to time, depending
on the local conditions in for instance soil, the
compounds will be present in appreciable quantities with concentrations in the gas-phase reaching
the ppm-range [119]. The presence of these compounds in these concentrations implies that microorganisms have been exposed to these substrates
in the course of evolution and it therefore is quite
understandable that so many organisms have
acquired the capability to grow on short chain
unsaturated hydrocarbons.
5.2.2. Enzymes with relaxed substrate specificity
The capability of various organisms to grow on
substrates not abundantly present in nature could
also be the result of the action of certain enzymes
with relaxed substrate specificity. As will be discussed in more detail in the following section on
applied aspects, enzymes involved in the initial
biodegradation of hydrocarbons generally oxidize
a broad range of substrates. An organism able to
grow on a natural substrate may thus also be able
to grow on a xenobiotic compound with similar
chemical or structural properties. Such an example
has been described by van Ginkel et al. [77] who
isolated bacteria on the biogenic isoprene (2methyl-l,3-butadiene) and on the anthropogenic
1,3-butadiene. Strains isolated on isoprene or on
249
butadiene strongly resembled each other and
moreover, all strains isolated were able to grow on
both substrates. It was thus anticipated that a
connection existed between the pathways for isoprene and butadiene metabolism and that the
ability to degrade the anthropogenic 1,3-butadiene
was a mere reflection of the ability to degrade the
biogenic isoprene. A similar mechanism was also
envisaged by Schink [54] when explaining the ecological significance of acetylene fermentation in
anaerobic environments. He speculated thai the
observed hydratation of acetylene to acetaldehyde
in Pelobacter acetylenicus was due to an unspecific
hydratase that in natural environments would be
involved in the detoxification of not only acetylenic
compounds, but also nitriles and cyanides.
5.2.3. Recently evolved enzymes and biodegradative
routes
A prerequisite for growth with novel compounds is that the necessary degradative enzymic
activities are present. In some cases effective enzymic activities may already be present in the
microorganism thus only requiring an appropriate
regulation of enzyme synthesis to allow degradation of the novel compound. Very often a mutation resulting in constitutive synthesis of the required enzyme is sufficient. If sufficiently effective
enzymes are not at hand, microorganisms can
acquire new metabolic activities as a result of
mutations affecting the rates of enzyme synthesis
or the structure of enzyme proteins [125,126].
These studies share the fact that microorganisms
were challenged to develop new pathways by altering their own genetic information due to mutations in structural a n d / o r regulatory genes, or
perhaps by recruitment of single 'silent genes'.
In nature however, much more genetic information is present than in a single microorganism.
Another strategy therefore is, that the degradation
capabilities of an organism can be expanded by
recruitment of genetic information from the environment. An example is the in vivo construction
of an organism with the novel capacity to degrade
4-chlorobenzoate. In Pseudomonas sp. B13 the
first step in 3-chlorobenzoate degradation is the
oxidation of 3-chlorobenzoate by a very specific
1,2-dioxygenase. This dioxygenase does not oxidize
4-chlorobenzoate and hence strain B13 cannot
grow with 4-chlorobenzoate as substrate. By direct
transconjugant mating between the TOL plasmidcontaining Pseudomonas putida PaW1 as donor of
the non-specific toluate 1,2-dioxygenase and strain
B13 as recipient it was possible to select 4-chlorobenzoate-degrading derivatives. The enzymatic,
regulatory and genetic aspects concerning this and
other genetically manipulated strains degrading
chlorinated aromatic compounds have been reviewed by Reineke [127] and others [128]. Interestingly, 4-chlorobenzoate-degrading organisms isolated directly from nature do not degrade 4-chlorobenzoate via 4-chlorocatechol but via a hydrolytic reaction to 4-hydroxybenzoate (see [127] for
references).
As has already been pointed out in the previous
sections the unsaturated short chain hydrocarbons, with the exception of the chlorinated
analogues, cannot generally be considered as true
xenobiotics, thus making it difficult to judge
whether the degradative pathways involved have
evolved very recently or have existed in nature for
a long time. Chloroethene (vinyl chloride) can be
considered as a true xenobiotic which has only
recently entered the environment, directly, and
indirectly via the anaerobic transformation of other
chlorinated ethenes (see section 4.7), from anthropogenic sources. It is the only chlorinated ethene
which, until now has been shown to support
aerobic growth, but the metabolic pathways still
needs elucidating. It is therefore difficult to
speculate on the evolutionary events that took
place resulting in the capacity to degrade vinyl
chloride.
With other chlorinated hydrocarbons, which
also have just recently entered the environment,
more progress has been made. The aerobic degradative pathways of 1,2-dichloroethane and dichloromethane metabolism have been described
[129,130] and the enzymes performing the initial
dehalogenation step purified [131,132]. The dichloromethane dehalogenases (group A) from
several dichloromethane utilizers isolated from
different environments have been characterized
and were shown to have the same immunological
properties and identical N-terminal amino acid
250
sequences [133]. This could be an indication that
this enzyme has evolved only very recently.
A novel dichloromethane dehalogenase (group
B) has recently been characterized [134] which had
only one of the 15 amino acids of the N-terminus
in the same position as in the group A enzyme.
The group B enzyme has a higher specific activity
than the group A enzyme but represents a lower
fraction of total protein when compared with
organisms having the group A enzyme. Nevertheless, the new isolate grew at a higher rate (0.22
h -~) with dichloromethane than the strains with
the group A enzyme (0.07-0.10 h - l ) . The K m of
the group B enzyme was however, also somewhat
higher so that it will be very interesting to see
which enzyme type will predominate under different ecological conditions and in what way any
further evolution will take place.
5. 3. Inhibitory effects on metabolic functions
Ethene and its multiple functions as a hormone
in plant physiology has of course attracted the
attention of researchers in areas other than plant
physiology. It has been attempted to assess a role
for ethene in the production of cultivated
mushrooms [135] and the production of secondary
metabolites by Aspergilli and Streptomycetes
[136,137] but no direct evidence concerning an
effect of ethene was given. Arguments indicating a
role for ethene as a critical regulator in soil [103]
and a causative agent in fungistatis [138] were
refuted in later publications [109,139]. An effect
on methanogenic bacteria has only been noted at
elevated levels. Oremland and Taylor [140] showed
that in anaerobic sediments methanogenesis was
reversibly inhibited by 5% ethene in the gas-phase.
Schink [141] later confirmed that inhibition of
methanogenesis is due to a direct inhibition of the
methanogens, with 0.5% ethene in the gas-phase
already resulting in more than 98% inhibition of
methane formation with several different substrates. In conclusion, however, it may be stated
that the biological effects of ethene are confined
to plants and that microorganisms are affected by
the gas in isolated cases only.
Interestingly this situation is reversed in the
case of acetylene. This gas has only limited effects
on the physiology of whole plants but it is known
that m a n y metabolic processes in bacteria are
blocked by acetylene. The effects of acetylene on
enzymes has very recently been reviewed by Hyman and Arp [142]. They have collected detailed
information on the effects of acetylene on the
enzymes nitrogenase, hydrogenase, ammonia monooxygenase, methane monooxygenase, nitrate reductase and nitrous oxide reductase. Therefore,
only limited attention has to be given here to the
effects of the gas. In the present context, only the
use of acetylene in ecological studies will be briefly
considered. Three activities can be monitored in
situ in ecosystems by employing acetylene as a
selective inhibitor i.e. nitrogen fixation, denitrification and methane oxidation. The reduction of
acetylene to ethene has been used extensively in
ecological studies to assess nitrogen fixation rates.
The standard assay employs 10% acetylene in the
gas-phase. The merits of the technique, as well as
the precautions to be taken when applying it in
ecosystems, have been discussed in detail in the
literature. A recent, very interesting observation is
that under certain conditions acetylene is not only
reduced to ethene but also to traces of ethane
[1431.
Acetylene is also used for in situ measurements
of denitrification rates, generally resulting in a
complete inhibition of N20-reduction activity at
acetylene concentrations of 0.1%. The merits of
the acetylene method for assessing denitrification
rates have been summarized by Knowles [144].
Much less attention has been given to acetylene
as a useful tool in the determination of methaneoxidizing bacteria in situ. Methane formation and
methane consumption are interrelated in nature at
interfaces where oxygen meets anaerobic zones. In
the anaerobic zone methane is produced and at
the interface methane is oxidized. Such interfaces
occur for instance in stratified lakes, in the upper
mud-layer of ditches or at the root-soil interface
of plants growing in submerged soils. Using
acetylene it is possible to determine methane
oxidation rates in such complex systems since the
inhibition of methane oxidation is complete at 10
p p m acetylene, whereas methane formation is only
inhibited at acetylene concentrations of approximately 100 p p m as determined for pure cultures
251
[145] or higher as determined for marine sediments [146]. The difference in net methane accumulation in the presence and absence of acetylene
in the lower ppm-range therefore is equal to the
amount of methane oxidized in the system.
Methane oxidation rates using acetylene as an
inhibitor have been determined in a marine environment [147] and in rice paddy fields [148]
although it has not always been realized that
acetylene has a dual function as inhibitor of both
methane formation and methane oxidation [149].
As a general rule, it should be kept in mind
that acetylene has m a n y different effects on microorganisms present in an ecosystem under study,
and that therefore artifacts may be detected rather
than quantifying the metabolic rates of the microbial p o p u l a t i o n present in the ecosystem
[150,49,145].
6. A P P L I E D ASPECTS
6.1. Biodecontamination of Unsaturated compounds
In several instances the presence of alkenic
compounds is a problem in air or water environments. Low concentrations of ethene in the atmosphere of fruit storage facilities can have devastating effects. The presence of the carcinogen vinyl
chloride or the odorous styrene can be a serious
problem in the atmosphere of the polymer industry. The less volatile chlorinated ethenes like
the suspected carcinogens tetrachloroethene and
trichloroethene are frequently found as contaminants in drinking-water aquifers [21].
6.1.1. Removal of gaseous contaminants
Gaseous contaminants may be effectively removed through biofiltration using undefined support materials for the active biomass. Such systems are operated successfully when very dilute
and undefined mixtures of odorous gaseous compounds are to be eliminated. The microbial population in such system is very heterogeneous and is
not only thriving on the gaseous substrates to be
removed but also on components of the support as
for instance compost [151]. More defined systems
however, may also be of use whenever a defined
gas mixture is to be treated. In such cases pure
cultures, immobilized on a defined support, may
be applied. Several of such systems have been
studied recently, including the removal of aldehydes and ketones [152] and ethene [113].
To prevent the accumulation of ethene in storage places for fruit and flowers ventilation of the
atmosphere is often applied. However, ventilation
is impracticable when the temperature a n d / o r the
gas composition in the warehouse differ from the
open air. As an alternative for chemical or physical methods to remove ethene from the storage
atmosphere the possibility of applying a bioreactor has been investigated [113]. It was shown that
immobilization of ethene-utilizing cells of Mycobacterium E3 on lava or perlite did not result in a
decrease in the maximal specific oxidation rate or
in an increase in the apparent K m in comparison
with free cells. However, the cell densities employed were quite low, thus requiring residence
times of 15 to 30 s to decrease the ethene concentration from 3.2 p p m to 1 ppm. The Vma~ of
Mycobacterium E3 is 50 nmol ethene rain -1 (rag
protein) -~ and the K m for ethene is 100 p p m in
the gas phase [113]. Although the results seem
promising it would be advantageous to have a
biocatalyst with a significantly higher affinity towards ethene.
Application of Mycobacterium L1 to remove
vinyl chloride from waste gases has also been
proposed recently [86]. The stability of vinyl chloride degradation by washed cells was, however,
not very high and depended strongly on the biomass concentration. It was shown later (Hartmans, unpublished results) that this inactivation is
caused by the excretion of a toxic product, probably chloroepoxyethane. Using growing cultures
in a chemostat it was possible to degrade vinyl
chloride quite efficiently, but also under these
conditions autoinactivation of the vinyl chloridedegrading population sometimes occurred when
there were irregularities in the vinyl chloride
supply. Due to the involvement of the highly toxic
chloroepoxyethane in the degradative pathway of
vinyl chloride, Mycobacterium L1 does not seem
to be a suitable biocatalyst for the removal of
vinyl chloride from waste gases.
252
Styrene removal from waste gases has also been
reported [153] using a biofilter which was inoculated with a Nocardia sp. The styrene in styrene
containing air (1.3 g m 3) was completely degraded by passing the air through a 2.5 meter
biofilter at a superficial gas flow of 100 m h -1.
However, no comments were made on the stability
of the system. Before becoming applicable in a
practical situation the stability of this type of
reactor with a monoculture immobilized on a defined cartier will have to be enhanced.
It is reasonable to expect that further research
in this field will lead to successful bioreactors for
the effective removal of polluting gaseous compounds.
6.1.2. Removal of chlorinated ethenes with
methane-utilizers
The increasing frequency with which chlorinated ethenes are detected in ground water has
stimulated research concerning the degradation
and removal of these compounds. Currently, treatment of ground water containing chlorinated hydrocarbons consists of pumping the water to the
surface and stripping out the components in aeration towers or removing the pollutants with a
sorbent [21]. Recently it was shown that exposure
of a soil column to natural gas resulted in mineralization of trichloroethene [22]. Since then there
have been several reports concerning the degradation of chlorinated ethenes by mixed and pure
methane-utilizing cultures [21,89,88]. The degradation of trichloroethene is proposed to proceed via
the epoxide which spontaneously breaks down to
other products [89]. Using trans-l,2-dichloroethene it was shown that both mixed and pure
cultures of methane-utilizers transformed this
compound to the relatively stable epoxide [89].
Application of methane-utilizing cultures to
treat ground water does not seem very realistic as
one of the most important pollutants, tetrachloroethene, is not degraded under these conditions.
6.2. Biotechnological production of epoxides from
alkenes
Epoxides are reactive molecules and there are
many chemical syntheses in which epoxides can
function as intermediates. In the past ten years the
possibility of using bacteria to transform alkenes
to the corresponding epoxides has been suggested
by several authors. This is also reflected by the
patient literature (see [4,154]). The capacity to
epoxidate alkenes is not limited to alkene-utilizing
bacteria. In theory any microorganism containing
a monooxygenase with activity towards alkenes
could be exploited. Furthermore, a process has
been considered utilizing isolated enzymes (haloperoxidase, halohydrin epoxidase and pyranose-2oxidase) in which D-fructose is a co-product in the
transformation of an alkene to an epoxyalkane
[1551.
6.2.1. Bacteria that can epo,,idate alkenes
Microbial epoxidation of 1-alkenes was first
demonstrated by van der Linden [36] who detected the formation of 1,2-epoxyoctane from loctene by heptane-grown Pseudomonas aeruginosa. The capacity to epoxidate 1-octene was
only present in alkane-grown cells and it was
suggested that the epoxidation of the 1-alkene was
effected by the alkane-hydroxylating activity. 2octene was not epoxidated under the same conditions.
In 1963 Coon and co-workers demonstrated
octanol-formation in crude extracts of an octanegrown Pseudomonas. It was shown that N A D H
and molecular oxygen were required for activity.
Further work by the same group revealed that the
hydroxylation system of Pseudomonas oleovorans
consisted of three proteins (see review of May
[37]). Subsequently M a y and Abbott [156] showed
that the enzyme system purified from Pseudomonas oleovorans also catalyzes the epoxidation of
1-octene to 1,2-epoxyoctane. It was thus confirmed that the hydroxylation and epoxidation
reactions are accomplished by the same monooxygenase system. Further work [157] revealed
that with 1-decene as substrate, 1,2-epoxydecane
and 9-decene-l-ol were formed as oxidation products. The epoxide formation however, predominated. Using alkadienes it was shown that
with decreasing chain length the epoxidation rate
decreases rapidly. In contrast, the hydroxylation
rate of the methyl-group of an alkane is less
affected by the chain length. As a consequence,
253
propene and 1-butene are only hydroxylated by
the enzyme system from Pseudomonas oleovorans
[1571.
The methane monooxygenase from Methylococcus capsulatus (Bath), which is also a multicomponent enzyme and which has been very thoroughly
studied by the group of Dalton (see [158] for a
recent publication), exhibits a different substrate
specificity. Unlike the terminal alkane hydroxylase from Pseudomonas oleovorans the methane
monooxygenase oxidizes n-alkanes to mixtures of
the corresponding 1- and 2-alcohols [159].
Using soluble extracts of Methylococcus
capsulatus (Bath) Dalton and co-workers also
showed that the short-chain 1- and 2-alkenes were
epoxidated, in contrast to the hydroxylation reactions which were observed with extracts from
Pseudomonas oleovorans [157]. Of the 31 different
compounds oxidized by cell free extract from
Methylococcus capsulatus (Bath) [159] only 12 were
oxidized by whole cells [59]. With the exception of
chloromethane and bromomethane the oxidation
of these compounds, including the alkenes, was
dependent on, or significantly stimulated by, formaldehyde. It was suggested that the oxidation of
formaldehyde supplied the reducing power necessary for the in vivo oxidation of the various compounds by methane monooxygenase. With chloromethane and bromomethane the probable reaction
product of monooxygenase activity is formaldehyde, thus explaining the inability of exogenously
supplied formaldehyde to stimulate the oxidation
of these halomethanes. The restricted range of
compounds oxidized by whole cells, compared
with cell-free extracts of Methylococcus capsulatus
(Bath), was not further discussed [59]. With Methylosinus trichosporium OB3b Higgins et al. found
that with whole cells of this methanotroph a much
larger range of compounds was oxidized [79].
However, no oxidation rates were presented. The
apparent differences in the range of compounds
oxidized by whole cells of Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b
may, in part, be a result of the different incubation conditions used. Hou et al. compared three
different methanotrophs with respect to their
capacity to epoxidate various alkenes, and also
reported that methane metabolites stimulated
epoxide formation rates [160]. There were no significant differences between the tested methanotrops.
Besides methanotrophs, various bacteria isolated with short-chain alkanes have also been
screened for their capacity to epoxidate short-chain
alkenes [60]. In contrast with octane-grown Pseudomonas eleovorans, enrichments with propane as
carbon-source resulted in organisms which formed
1,2-epoxypropane from propene. Although traces
a 3-hydroxy-l-propene were detected after incubation of propane-grown cells with propene, 1,2epoxypropane was the major oxidation product.
Hyman and Wood [61] demonstrated that
Nitrosomonas europaea cells containing ammonia
monooxygenase activity were capable of forming
epoxyethane from ethene. From inhibition and
competition experiments they concluded that the
epoxidation of ethene is probably accomplished
by ammonia monooxygenase. The highest rate of
epoxyethane formation was obtained by adding
hydrazine as donor of reducing power. Ammonia
monooxygenase also oxidized many other alkenes
and alkanes [68].
Although epoxides are intermediates in the alkene-metabolism of short-chain alkene-utilizing
bacteria, and therefore also substrates for these
bacteria, it is possible to produce epoxyalkanes
with alkene-utilizing bacteria by exploiting the
substrate specificity of the epoxide-degrading enzymic activities of these bacteria [161]. In this way
for example, it is possible to form 1,2-epoxypropane with ethene-grown Mycobacteria and epoxyethane with propene-grown cells [162]. Furthermore, Furuhashi et al. [163] reported the accumulation of 1,2-epoxypropane during growth of
Nocardia corallina B-276 on propene.
A general conclusion concerning the specificity
of the monooxygenases from the different types of
organisms can be that alkane- and methane-grown
bacteria can perform epoxidation as well as hydroxylation reactions, whereas alkene-grown
bacteria can only perform the epoxidation of alkenes (Fig. 4).
6.2.2. Biotechnological production of short-chain
epoxides
The biotechnological production of epoxides
254
Atkene-grown bocterio
H]C-( CH2)n- CH= CH2
~
/0\
HI C-(CHz)n- CH- C H2
0H
i
H2C ~ (ell2) n- CH = EH2
H~C-(CHz)-CH: CH2 / / , F , /0\
_
n
- - ~ ~ . H3C-(CH2)n CH-CHz
AlkcLne- g r own bacferlo
Fig. 4. General modes of oxidative attack of hydrocarbons by
alkene- and alkane-grown bacteria.
can be envisaged in several ways. Application of
enzyme preparations seems very unrealistic due to
the low stability of the monooxygenases in vitro.
Employment of whole cells containing m o n o oxygenase activity seems a much more attractive
alternative. Two possible modes of operation, using
whole cells containing monooxygenase activity towards alkenes, have been described. Furuhashi et
al. [163] described the production of 1,2-epoxypropane using Nocardia corallina B-276 growing on
propene. Apparently in this organism the oxidation of propene proceeds at a higher rate than the
consumption of the product 1,2-epoxypropane.
After 5 days of incubation of Nocardia corallina
B-276 growing on propene a 1,2-epoxypropane
formation of 0.6 grams per litre was reported
[163]. The specific rate of 1,2-epoxypropane formation was not reported, but can be estimated to
have been approximately 1 nmol rain i (mg dry
weight)-1. Alternatively, epoxides can also be produced with non-growing cells in which a monooxygenase activity is present.
6.2.2.1. Epoxidation by non-growing cells. By
using non-growing cells for the production of
epoxides much higher yields, based on the alkene
substrate, can be realized. Indeed most of the
literature concerning the biotechnological production of epoxides has focussed on systems using
non-growing cells. In Table 2 some typical initial
1,2-epoxyalkane formation rates by resting cellsuspensions of different bacteria are shown.
To realize the biotechnological production of
short-chain epoxides several novel bioreactor configurations have been proposed. The aim of these
gas-solid and multiphase bioreactors is to facilitate the supply of the relatively low water-soluble
gaseous substrates (oxygen and alkene) and to
efficiently remove the inhibitory product [71,164,
165]. Theoretically the gas-solid bioreactor, with a
minimal amount of water surrounding the immobilized biocatalyst, would seem ideal to facilitate mass-transfer of gaseous substrates and product. However, this minimizing of the water-phase
can become critical, as for several alkene-utilizers
it was shown that lowering the water-activity to
99% resulted in a 70% decrease in the alkene
oxidation rate [166].
The multiphase bioreactor proposed for the
production of 1,2-epoxypropane [164] has the disadvantage that the biocatalyst has to be immobilized to facilitate separation of the biocatalyst and
the organic phase, although for the production of
1,2-epoxyoctane the classical stirred tank reactor
with an organic phase has also been studied [167].
The most essential component of a biotechnological process however, is not the bioreactor but
the biocatalyst. If non-growing cells are to be used
as biocalyst for the biotechnological process of
epoxide production two major problems, besides
the stability and initial activity of the monooxygenase, are cofactor regeneration and product
inhibition.
6.2.2.2. Cofactor regeneration and co-substrate.
With Methylococcus capsulatus strains it was shown
Table 2
Epoxide formation rates by resting cell-suspensions of various
bacteria
Organism
Growth
substrate
Formation rate nmol min
m g protein ]
1,2epoxyethane
1,2epoxypropane
1,2epoxybutane
methane
33
33
11
methane
16
30
4
propane
16
40
5
0
16
14
46
0
0
Methylococcus
capsulatus
C R L M1 [160]
Methylosinus
trichosporium
OB 3b [160]
Brevibacterium
CRL56 [60]
Mycobacterium
E3 [161]
ethene
Xanthobacter
Py2 [162]
propene
255
that a co-substrate could enhance initial epoxide
formation rates [160,59]. The variations in the
magnitude of the reported enhancement can possibly be ascribed to the different cultivation conditions used resulting in different endogenous respiration rates.
Although, due to its extremely low growth rate,
Nitrosomonas europaea does not seem a suitable
biocatalyst for the production of epoxides it is
interesting to note the effect of an external source
of reducing power on epoxyethane production by
this organism. Hyman and Wood [61] reported a
10 to 60-fold increase in the epoxyethane formation rate by whole cells of Nitrosomonas europaea
by adding hydrazine as reductant. The highest rate
of epoxyethane formation that was reported was
250 nmol min-1 (mg protein) -1. In this case the
transformation rate of ethene to epoxyethane was
apparently completely determined by the supply
of reducing power.
In addition to increasing the initial epoxide
formation rate a co-substrate can also result in an
increased stability of the epoxide formation. Using
a small gas-solid bioreactor with Mycobacterium
Pyl immobilized on sand it was shown that the
addition of propionaldehyde as co-substrate resuited in an increased epoxyethane production
over a period of several days [71].
Working with several different ethene-utilizing
Mycobacteria immobilized on lava it was found
that the different strains did not all react in the
same manner with respect to the addition of
ethanol or ethylacetate as co-substrate during the
oxidation of propene to 1,2-epoxypropane [168].
With strains 2W and Eul the rate of 1,2-epoxypropane formation increased significantly when
ethanol or ethylacetate were simultaneously supplied with the substrate propene. Furthermore,
with ethylacetate as co-substrate biocatalysis was
possible for a prolonged period of time with these
two strains. With strain E3 co-substrates had no
effect on 1,2-epoxypropane formation. The most
obvious co-substrate, the growth substrate ethene,
cannot be applied during the 1,2-epoxypropane
production for several reasons. Ethene and propene are transformed by the same monooxygenase, so that the presence of ethene results
in a lower propene turnover and furthermore the
presence of 1,2-epoxypropane competitively inhibits the degradation of epoxyethane [161] and
hence the production of the desired reducing
equivalents. An alternative is to operate the
gas-solid bioreactor in a mode in which ethene
and propene are supplied in alternating cycles of
15 h. In this manner 1,2-epoxypropane production, although at low rates, was maintained for 20
days [168]. Besides replenishment of reducing
equivalents during the ethene cycle, presumably,
de novo synthesis of the alkene monooxygenase
also takes place.
6.2.2.3. Product toxicity. The lower epoxides are
reactive molecules with the capacity to alkylate
biological macromolecules (see [169] for references). Epoxyethane for example, due to its gaseous nature at room temperature, is often used to
sterilize heat sensitive materials. Besides high conversion rates the economics of a biotechnological
process for the production of epoxides to a large
extent also depends on the product concentration
that can be achieved. Higher product concentrations result in lower purification costs. For this
reason, when comparing potential biocatalysts for
the production of epoxides it is relevant to know
the epoxide tolerance. Habets-Criitzen and de Bont
[169] have compared the short-term effect of 1,2epoxypropane on the propene oxidation rate of an
ethene-utilizer, an ethane-utilizer and a methanotroph (Table 3).
The 1,2-epoxypropane concentrations resulting
in approximately 50% inhibition of the propene
oxidation rate by whole cells were 20 mM, 1 mM
and 5 mM for the ethene-, ethane- and methaneutilizer respectively. When Mycobacterium E20 was
grown on ethene instead of ethane, an inhibition
pattern comparable to that of the ethene-grown
Mycobacterium E3 was obtained, indicating that
Mycobacterium E20 possesses two distinct monooxygenase activities, as previously assumed [39].
By performing respiration experiments with other
readily oxidizable substrates at the same 1,2epoxypropane concentrations, it was shown that
the inhibition was specific for propene oxidation.
Furthermore, using strain E3 it was shown that
the inhibition of propene oxidation was irreversible. Apparently the epoxide reacts preferentially
with the monooxygenase. Surprisingly, the inhibi-
256
Table 3
Inactivation by 1,2-epoxypropane of propene oxidation by
whole cell suspensions and cell-free extracts of different bacteria
[169]
Organism
Mycobacterium E3
(ethene grown)
Mycobacterium E20
(ethane grown)
1,2-epoxypropane
concentration
(mM)
Propene oxidation
rate
(relative activities)
Whole
Cell-free
cells
extract
0
5
10
50
100
500
0
0.03
0.09
0.4
100
75
63
38
15
1
1130
84
49
29
1
(methane grown)
tion
of p r o p e n e
-
15
-
7
2.5
Methylosinus
trichosporium OB3b
100
100
98
97
91
33
-
0
1O0
1O0
0.5
1
5
10
25
90
78
39
22
5
100
95
72
56
27
oxidation by
whole
cells of
Mycobacterium E3 was m u c h s t r o n g e r t h a n the
i n h i b i t i o n o f cell-free extract. H o w e v e r , the results
o b t a i n e d with cell-free extracts m a y b e m i s l e a d i n g
b e c a u s e u p o n s o n i c a t i o n m o r e t h a n 90% of the in
vivo activity is lost. A l k e n e m o n o o x y g e n a s e is a
m u l t i c o m p o n e n t e n z y m e [169], so thot it c o u l d b e
p o s s i b l e t h a t the 1 , 2 - e p o x y p r o p a n e reacts with the
c o m p o n e n t w h i c h is n o t rate l i m i t i n g in vitro.
Based o n the relative e p o x i d e t o l e r a n c e of the
alkene-utilizing M y c o b a c t e r i a , o n e c o u l d c o n c l u d e
that these b a c t e r i a are m o r e s u i t a b l e for the p r o d u c t i o n of e p o x i d e s t h a n m e t h a n e - a n d ethaneutilizing bacteria.
A l t h o u g h several p a t e n t s have b e e n registered
for the b i o t e c h n o l o g i c a l p r o d u c t i o n of epoxides,
e c o n o m i c factors still f a v o u r the use of c h e m i c a l
processes, as extensively d o c u m e n t e d b y D r o s z d
[171]. H e h o w e v e r also c o n c l u d e d , t h a t the biot e c h n o l o g i c a l p r o d u c t i o n of e p o x i d e s e n r i c h e d in
a n o p t i c a l i s o m e r m i g h t be interesting. E p o x i d e s
o f three or m o r e c a r b o n a t o m s c o n t a i n at least one
a s y m m e t r i c c e n t r e (Fig. 5). If it is p o s s i b l e to
p r o d u c e these e p o x i d e s stereospecifically such a
p r o d u c t w o u l d have a higher value t h a n the
racemic e p o x i d e a n d c o u l d f o r m the s t a r t i n g p o i n t
of s o m e interesting s t e r e o c h e m i c a l syntheses.
6.2.3. Stereospecific production of epoxides
M a y a n d c o - w o r k e r s were the first to r e p o r t on
the e n a n t i o m e r i c c o m p o s i t i o n of b i o l o g i c a l l y p r o d u c e d epoxides. T h e 1 , 2 - e p o x y o c t a n e p r o d u c e d
f r o m 1-octene b y Pseudomonas oleovorans was 92%
in the R - f o r m a n d 8% in the S - f o r m (see [172]).
W i t h several o t h e r b a c t e r i a 1,2-epoxyoctane, 1,2e p o x y d e c a n e , 1 , 2 - e p o x y t e t r a d e c a n e a n d 1,2-epoxyh e x a d e c a n e were also p r e d o m i n a n t l y p r o d u c e d in
the R - f o r m (see [172]). T h e s e strains were n o t
active with s h o r t - c h a i n alkenes. T h e 1,2-epoxytet r a d e c a n e p r o d u c e d with Nocardia corallina B-276
was m o r e t h a n 86% in the R - f o r m [163]. A l t h o u g h
they d i d n o t d e t e r m i n e the e n a n t i o m e r i c c o m p o s i tion F u r u h a s h i et al. a s s u m e d t h a t the 1,2-epoxyp r o p a n e p r o d u c e d b y the s a m e o r g a n i s m was also
p r e d o m i n a n t l y of the R - f o r m . H a b e t s - C r i i t z e n et
al. [172] a n a l y z e d the e n a n t i o m e r i c c o m p o s i t i o n of
several e p o x i d e s f o r m e d b y a l k e n e - u t i l i z i n g
bacteria. W h e n g r o w n on alkenes all these strains
also p r e d o m i n a n t l y f o r m e d the R - f o r m o f 1,2e p o x y p r o p a n e a n d 1 , 2 - e p o x y b u t a n e . O n e strain,
Mycobacterium E20, w h e n g r o w n o n ethane, p r o d u c e d r a c e m i c 1 , 2 - e p o x y p r o p a n e [172]. This conf i r m e d the a s s u m p t i o n that Mycobacterium E20
c o n t a i n s different m o n o o x y g e n a s e s d e p e n d i n g on
the g r o w t h s u b s t r a t e used. This o b s e r v a t i o n with
H
R
0
HI 1 ~ 1 1
H
0
R
H
R-1,2-epoxyaqkane
HI 1 ~ 1 1
H
H
R
.S' 1 , 2 - e p o x y a l k a n e
Fig. 5. Epoxidation of 1-alkenes can result in two stereoisomers
of the 1,2-epoxyalkanes.
257
Table 4
Enantiomeric composition of epoxyalkanes produced by various bacteria a
Bacterial strain
Methylococcus capsulatus (Texas)
Methylosinus trichosporium OB3b
Pseudomonas sp. P9y
Nocardia sp. TB1
Nocardia sp. TB1
Mycobacterium parafortuTtum E3
Mycobacterium aurum L1
Mycobacterium aurum L1
Xanthobacter sp. Py2
Nocardia sp. By1
Nocardia sp. IP1
Nocardia sp. IP1
Nocardia sp. IP1
Nocardia corallina B-276
Nocardia corallina B-276
Nitrosomonas europaea ATCC 19178
growth substrate
methane
methane
propane
butane
trans-butene
ethene
ethene
vinyl chloride
propene
1-butene
butane
1,3-butadiene
isoprene
ethene
propane
CO2/NH 4
1,2epoxypropane
R
S
1-chloro-2,3epoxypropane
R
S
1,2epoxybutane
R
S
56
54
45
68
60
91
99
99
91
90
91
86
87
32
52
48
nd
nd
45
69
63
84
90
87
44
46
55
32
40
9
1
1
9
10
9
14
13
68
48
52
-
-
59
60
99
98
97
41
40
1
2
3
1
99
1
99
99
99
99
83
89
14
1
1
1
17
11
86
trans-2,3epoxybutane
R
S
nd
nd
55
31
37
16
10
13
-
-
87
95
99
84
84
29
13
5
1
16
16
71
51
50
49
50
-
-
85
87
82
89
78
81
81
80
80
73
-
15
13
18
11
22
19
19
20
20
27
-
a [173] relative values in %, +2%.
Mycobacterium E20 prompted further investigations concerning the enantiomeric composition of
short-chain epoxyalkanes produced by different
bacteria [173]. The epoxides formed by methane
utilizers tested were racemic (Table 4). Propaneand butane-grown strains tested also produced
racemic epoxides. In contrast all strains grown on
unsaturated hydrocarbons formed optically active
epoxyalkanes. Two Nocardia species, strains IP1
and B-276 differed in that they also produced
predominantly the R-form of the optically active
unsubstituted epoxides, after growth on alkanes.
Interestingly, the ammonia monooxygenase from
Nitrosomonas europaea exhibited an opposite stereospecificity compared with all the other strains
that produced optically active epoxides.
The S-form of 1-chloro-2,3-epoxypropane has
the same steric conformation as the R-form of
1,2-epoxypropane. Of the above epoxides, 1chloro-2,3-epoxypropane is the most interesting
chiral starting material for the synthesis of optically active compounds due to the presence of two
different reactive groups. Unfortunately, 1-chloro2,3-epoxypropane is also the most inhibitory of
the above epoxides, so that even though the optical purity can be very high, it will prove difficult
to produce it economically in a biotechnological
process.
Recently several other interesting epoxides for
the synthesis of chiral products, nave been produced using alkene-utilizers [174], but unfortunately the optical purity was not as high as with
1-chloro-2,3-epoxypropane. Stereospecific epoxidation of substituted alkenes has also been reported [175]. Using bacteria belonging to the genera Rhodococcus, Mycobacterium, Nocardia and
Pseudomonas it was possible to epoxidate 4-(2methoxyethyl)phenylallylether stereospecifically
yielding optically pure epoxides in the S-config0
/
Hu ~ l l
CH3
CH3
X0
H3C t l ~ u
H
H
H
CH3
(2S.3S)
(2R,3R)
Degradation
products
Fig. 6. Stereoselective degradation of 2S,3S-epoxybutane by
Xanthobacter Py2 resulting in optically pure 2R,3R-epoxybutane [176].
258
uration. These epoxides were subsequently chemically transformed to the r - b l o c k e r S ( - ) Metoprolol in optical purities ranging from 95 to
99%.
An alternative approach leading to optically
active epoxides is stereoselective degradation of
one of the isomers. It was recently shown that
using the propene-grown Xanthobacter Py2 it was
possible to produce enantiomerically pure 2r,3repoxybutane from a racemic mixture of trans-2,3epoxybutane due to the stereoselective degradation of only the 2s,3s-isomer (Fig. 6) [176].
Although very high enantiospecificities have
been observed we know of only one strain which
is used commercially to produce an optically active 1,2-epoxide. Serva (Heidelberg, F.R.G.) supplies 1,2-epoxytetradecane consisting of at least
97% R-isomer which has been produced using
Nocardia corallina B-276 according to the procedure described by Furuhashi and Takagi [177]. It
is not quite clear how this value of 97% relates to
the value reported earlier [163] of 1,2-epoxytetradecane produced by the same strain containing
more than 86% of the R-isomer.
Summarizing one can conclude that the choice
of organism is crucial when considering the biotechnological production of optically active
epoxides. General rules are difficult to make. One
could be that methane-utilizers form epoxides
racemically whereas alkene-utilizers generally produce optically active epoxides. Alkane-utilizers
form a less homogenous group with respect to the
stereospecificity of performed epoxidation reactions. When considering the production of novel
epoxides it is generally not possible to predict
which bacterial strain will produce the epoxide in
the highest optical purity [174]. Therefore a
screening programme is necessary to select the
best organism for a specific epoxidation reaction.
7. C O N C L U D I N G R E M A R K S
Summarizing the previous sections one can
conclude that a surprising amount of work concerning the microbial degradation and formation
of short-chain unsaturated hydrocarbons has been
published during the last few decades. The earlier
work mainly concerned the role of ethene in soil
ecosystems in relation to its plant-hormone activity. Subsequent work focused on the degradation
of the various short-chain olefins and questioned
the previously assumed anthropogenic nature of
many of the compounds under review. The capacity to degrade the short-chain olefins is apparently
limited to a few genera, e.g. Mycobacterium,
Nocardia, Rhodococcus and Xanthobacter. It is
noteworthy that Pseudomonads, which are generally considered to be metabolically very versatile,
were only isolated in a few cases e.g. with olefins
of intermediate chain length (1-hexene) or substituted olefins (styrene and butynol).
The possibility of producing (optically active)
epoxides with (alkene-utilizing) microorganisms
has inspired several research groups during the
last decade. Other recent publications concerning
applied aspects bacterial alkene metabolism focus
on the potential of using microorganisms to produce olefins from renewable sources and the possibilities of biologically degrading environmental
contaminants like chlorinated ethenes.
In spite of the significant research efforts described above, many questions remain to be
answered. A detailed understanding of many degradative pathways is still lacking and the significance of the olefin-degrading organisms in the
natural ecosystem still needs quantitation. Future
work could thus lead to the discovery of novel
enzymatic activities and should result in a better
understanding as to how these olefin degrading
microorganisms have evolved and of their function in natural ecosystems.
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