Demethylation
I, I-DCA
a (ll, 12) (Hydrolysis)
b p(71) 7%(71) Q b ~Mmg (31, 69, 70, 109~~)
H H
H--'c--c/--H
H / ~'CI
CA a(71)
OH CI
H --- C - - C---C!
H / ~Cl
2,2,2- T ri c h I oro-et hanoi
OH " C!
. C --- C--Ci
~ C l
i
Trichloroacetate
OH H O
H f ~ Cf~ '
H----C ---C - - H H - - - C - -
H ~ ~ H H ~ OH
IN i n lln
Ethanol Acetic Acid
CO 2 + Cells CO 2 + Cells
r
See figure 9
Figure 4. Abiotic and biotic pathways for transformation of 1,1,1-TCA (a=abiotic, b=biotic, p=pure culture, M=mixed culture, mg=methanogens).
89
dehydrogenase 2H (mox) (75, 76)
Ci / Ci C----C--- H
H / N H
H O - ~ ~ H a l o a i k a n e 1, 2-DCA
2 ) 3 dlh:/~en*ase Hydrogenolysis Dehalo-elimination H C I ~ ' ~ (dlha)(75, 76)
C! O H 2HC!
H .--~
HCIC - - - C - - H mg (78) mg (37, 77, 78)
/
q kH %H H C! H H
Chloroethanol
H"-- ~ / ~,C=C /
Alcohol H f C---C--- H H % H
NH
H/C---C
~H
CA Ethylene
2HL]
mg (78)HC!
O = ~ Aldehyde H2 dehydrogenase
2 H ~ T (aid) (75, 76)
cI c o H-"
H /~ - - % H
Chloroacetic acid n
H 2 0 . ~ Haloaikanoic acid dehalogenase HCi (dlhB) (75, 76)
O H ~ 0
H '--~ u---C~o H H / Giycolic acid
CO 2 + Cells
H H
H.._~
C---C-- H/
H / N H
Ethane
Figure 5. Pathways for transformation for I~2-DCA (mgfmethanogens).
90
Methanobacterium thermoautotrophicum [37,77]. Hydrogenotrophic and acetoclastic methanogenic bacteria reductively dechlorinate 1,2-DCA via two reaction mechanisms. Ethylene is formed by dihalo-elimination, and chloroethane (CA) is formed by hydrogenolysis. CA is further transformed to ethane via hydrogenolysis [78] or it may undergo hydrolysis to ethanol. Cells of Methanosarcina barkeri, when grown on H2, converted 1,2-DCA and chloroethane at rates that were higher than acetate- or methanol-fed cells [78].
C a r b o n t e t r a c h l o r i d e (CT) A e r o b i c
Under fully aerobic conditions, CT persists. However, E. coli k12 is capable of transforming CT under low oxygen conditions (~1%) [79].
A n a e r o b i c
As shown in Figure 6, CT is susceptible to transformation by parallel, competing pathways. Transformation of CT to CO 2 occurs in mixed cultures under methanogenic, denitrifying, and sulphate-reducing conditions, but typically, CF is also produced by a parallel pathway [3, 26,30,69,80]. Desulfobacterium autotrophicum [37] and Clostridium sp.
[71] reductively dehalogenated CT to trichloromethane and dichloromethane. Acetobacterium woodii transformed CT to trichloromethane and dichloromethane and to CO 2 by parallel pathways [81]. The CO 2 was subsequently converted to acetate by acetogenesis.
Pseudomonas sp. strain KC rapidly transforms CT to CO2, and to an unidentified nonvolatile fraction under denitrifying conditions [82].
This organism appears to be unique in that it does not produce CF from CT. The transformation is also unusual in that it is regulated by the availability of trace metals, notably iron and cobalt, and it is linked to the mechanism of trace metal scavenging [82,83].
No significant transformation of CT was observed in nitrate-respiring and oxygen-respiring cultures of E. coli k12, except at low oxygen levels (~1%), although fumarate-respiring and fermenting cultures slowly transformed CT to CF and to other products. In these cultures, CF persisted, indicating that other products of CT transformation were generated by parallel pathways and were not derived from CF [79].
Carbon disulphide was an unexpected product of CT transformation in E. coli studies [79]. A recent study of sulphide reactivity [84] demonstrates that CT reacts with HS- to produce CS 2, but it is not known if CT undergoes direct nucleophilic substitution with HS or Sx 2 to form CS 2 or if CT undergoes reduction to form a trichloromethyl radical, which can then react with HS, Sx 2, or $2082" to form CS 2. CS 2 hydrolyzes to CO 2.
About 85% of the CT is ultimately transformed to CO 2 in these systems.
91
i i
CI Ci
C!
% f Ci
2H 20 Carbon tetrachioride- CT
4HC C!
HS" ( ~
R. F
1- ~ 1 C | a(86) b (79) [ I " ~
c I / C . c ! l~i_./ro #~;.8;..,)~ p(79)
~(82,
85)
.C!
C! C! [ ~ C ~ O
/ c_ / L I
Phosgene
L ~ " C d Chloroform-CF
2HCI ~ 2HC!
ic:ol i .coo. !
See Figure 7
2HC! ~ t
c o 2
Figure 6. Pathways of transformation for carbon tetrachloride (affiabiotic, b=biotic, p=pure culture).
The observed product distribution is consistent with the general model of competing parallel pathways presented by Criddle and McCarty [85]
and by Kriegmann-King and Reinhard
[86].92
Chloroform (CF) A e r o b i c
As shown in Figure 7, methanotrophs can transform CF [15,87-89].
The mechanism of MMO-catalyzed oxidations is similar to that of cytochrome P-450-catalyzed oxidations [22,90]. The intermediate product of P-450 transformation is phosgene, and this compound is probably the intermediate produced by MMO reactions as well [15]. Although phosgene is rapidly hydrolyzed to carbon dioxide and chloride, it also binds to
C! \ / CI Cl / c ~H
2H+O2 " ~ / Chl~176176 ~ Mmg (3, 4, 26) y Methane ~ p (37, 71, 81, 92)) . ~ ~ mono-oxygenase N i 20% (71) 30% (69)
H 20 ~ Mmt (15) ~ , 70% (92)
C! / Ci Cl / C ~OH
~
aH C I Mmt (15)
CI
~ c - - - o cI /
C! H
el f c \ n Dichioromethane
See Figure 8
Phosgene H 2 ~ N a (91)
2 H C I ~ CO 2
Figure 7. Pathways of transformation for chloroform (a=abiotic, Mfmixed culture, mgffimethanogens, mtffimethanotrophs).
93 proteins, presumably accounting for its toxicity [91]. Cell-flee MMO extracts also oxidize CF [19]. A recent study demonstrated that formate addition increases CF transformation rate (0.35 da3r 1 for resting cell and 1.5 day -1 for formate-fed cells) and transformation capacity (=
0.0065 mg CF per mg cells for starving cells, and = 0.015 mg of CF per mg cells for formate-fed cells) [66] . These observations indicate that energy substrate alters biomass transformation capacity. However, even in the presence of an energy substrate, biomass transformation capacity had an upper limit, suggesting that toxicity was important.
A n a e r o b i c
Chloroform was dechlorinated by pure cultures of Methanosarcina sp.
strain DCM and Methanosarcina mazei $6. The initial dechlorination product of chloroform was dichloromethane, accounting for about 70 percent of added chloroform. The product of dichloromethane, chloromethane was detected consistently at trace levels but could not be accurately quantified. The production of 14CO2 from [14C]-chloroform and the absence of 14CH4 imply that reaction mechanisms other than reductive dechlorination alone are important [92]. Degradation of dichloromethane by oxidation via phosgene or alcohols was proposed previously, for methanogenic systems [13].
D i c h l o r o m e t h a n e (DCM) A e r o b i c
Pathways for dichloromethane biodegradation are given in Figure 8.
Use of DCM as a growth substrate has been demonstrated in enrichment cultures [93,94], and by pure cultures of Pseudomonas and Hyphomicrobium species [21,95,96]. The ability to grow on DCM requires the biosynthesis of a DCM dehalogenase (100). DCM dehalogenase from a Hyphomicrobium sp. was purified and characterized [97]. DCM degradation by Hyphomicrobium involves nucleophilic substitution by a transferase. The glutathione (GS)-dependent enzyme, glutathione-S- transferase (GS) dechlorinates DCM to S-chloromethyl glutathione.
This compound is nonenzymatically converted to formaldehyde and glutathione [96]. The DCM-degrading methylotrophic bacterium, DCMll, grows more rapidly on DCM than previous isolates- strains DM1, DM2, DM4 and GJ21, and this difference was traced to a difference in enzyme structure [98].
A n a e r o b i c
DCM can serve as a growth substrate under anaerobic conditions [93, 99]. The principal transformation pathways are oxidation to CO 2 and fermentation to acetate. CO2-reducing methanogens use some of the
94
electrons made available from DCM oxidation to form methane - the methyl carbon comes from DCM and the carboxyl carbon from CO 2.
Acetoclastic methanogens produce methane from the acetic acid, formed by fermentation of DCM. DCM degradation is, thus, a disproportionation:
a portion of the DCM is oxidized, making reducing equivalents available for reduction of an equal amount of DCM.
C!
C!