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4 Application of CSIA for the Evaluation of the Reductive Dehalogenation Reaction

4 Application of CSIA for the Evaluation of the Reductive Dehalogenation Reaction

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J. Renpenning and I. Nijenhuis



438



Table 18.2  Streitwieser semiclassical limits for KIE during bond cleavage at 25 °C for halogenated hydrocarbons (Cook 1991)

Bond

C–H

C–H

C–C

C–Cl

C–Cl



Frequency (cm−1)

2900



Isotope

12C/13C

1H/2H

12C/13C



1000

750



12C/13C

35Cl/37Cl



KIE

1.021

6.44

1.049

1.057

1.013



Table 18.3  Evaluation of AKIEC values for carbon in established cases of reduction by cleavage

of one C–Cl bond according to Elsner et al. (2005) and Renpenning et al. (2014). In comparison,

carbon kinetic isotope effect (KIE) expected from the Streitwieser Limit for cleavage of a C–Cl

bond ~1.057 (see Table 18.2)

Type of reaction

Reduction of CCl4 by Fe(II)

Reduction of CCl4 by FeS

Reduction of PCE by vitamin B12

Reduction of TCE by vitamin B12



Isotope

12C/13C

12C/13C

12C/13C

12C/13C



AKIEC

1.027–1.033

1.016

1.033–1.053

1.034–1.039



These corrections allow to compare compounds with, e.g., different numbers of

carbon atoms which are subject to the same reaction and can be used to evaluate reaction mechanisms taking place by comparing the values to reactions with

known mechanism (Table 18.2 [KIE] and Table 18.3 [AKIE]). Theoretical isotope

effects can therefore be compared to experimental ones in order to elucidate reaction mechanisms. For example, theoretical KIE and AKIE for the reduction of tetrachloroethene (PCE) by vitamin B12 (Table 18.3) were similar suggesting that the

KIE can be observed for abiotic dehalogenation of PCE in its full magnitude. For

other reactions the AKIE was observed to be lower than the theoretical KIE suggesting that rate-limiting steps prior to the reaction step mask the real magnitude

of the KIE. Additionally, calculation of AKIE will allow to evaluate similarity in

reactions for different substrates, e.g., as done for dichloroethane and dichloropropane dichloroelimination (Fletcher et al. 2009; Schmidt et al. 2014).



18.4.3 Evaluation of Degradation Pathways

Isotope fractionation of compounds subject to degradation often reflects the reaction mechanism involved in its degradation. Therefore, enrichment factors can

be used for identification of degradation pathways, as it was already used for

MTBEs (Kuder et al. 2005; Zwank et al. 2005; Elsner et al. 2007). Halogenated

hydrocarbons can be transformed in situ by different pathways, including abiotic



18  Evaluation of the Microbial Reductive Dehalogenation Reaction …



439



Table 18.4  Experimental carbon isotope enrichment factors (εC) determined for abiotic and

enzymatic catalysis (Dayan et al. 1999; Slater et al. 2003; Nijenhuis et al. 2005; Cichocka et al.

2007, 2008; Elsner et al. 2008; Abe et al. 2009; Schmidt et al. 2010; Clingenpeel et al. 2012;

Cretnik et al. 2013; Renpenning et al. 2014)

Compound



PCE

TCE

cis-DCE

trans-DCE

1,1-DCE

VC

*cometabolic



Abiotic

Fe(0)

−5.7 to −25.3

−7.5 to −13.5

−6.9 to −16.0



−6.9 to −19.3



reaction



Corrinoids (reductive

dehalogenation)

−13.0 to −25.3

−15.0 to −21.3



Biotic

Anaerobic (reductive

dehalogenation)

−0.4 to −16.4

−3.3 to −26.0

−14.9 to −29.7

−20.8 to −30.3

−5.1 to −23.9

−23.2 to −31.1



Aerobic

(degradation)

−11.6 to −14.7*

−0.9 to −9.8



−3.2 to −8.2



and biotic (enzymatic) dehalogenation. Abiotic transformation can be mediated

during reductive dehalogenation by zerovalent iron (ZVI) (Arnold and Roberts

2000; Elsner et al. 2008), mediated by corrinoids (Krone et al. 1989; Glod et al.

1997) or chemical oxidation using, for instance, permanganates or persulfates

(Hrapovic et al. 2005; Tsitonaki et al. 2010). In contrast, biotic transformation

occurs under oxic and anoxic conditions. Aerobic metabolic degradation, however,

was observed only for lower chlorinated compounds, such as vinyl chloride (VC)

and dichloroethene (DCE). Carbon isotope fractionation during aerobic degradation is usually small (Table 18.4) and can be explained by the catalytic reaction

pathways, which do not involve a direct cleavage of the C–Cl bond (Chartrand

et al. 2005; Abe et al. 2009; Mattes et al. 2010; Tiehm and Schmidt 2011; Tiehm

et al. 2008; Clingenpeel et al. 2012). During organohalide respiration C–Cl bonds

are sequentially cleaved leading to formation of lower chlorinated hydrocarbons

(Scholz-Muramatsu et al. 1995; Maymo-Gatell et al. 1997) and carbon isotope

fractionation is generally stronger. The corresponding isotope fractionation is usually larger for VC, cis-DCE (cis-DCE), and trans-DCE, while lower for 1,1-DCE,

trichloroethene (TCE) and especially tetrachloroethene (PCE) and more variable

isotope fractionation was measured (Table 18.4). The cause for the variability of

microbial isotope fractionation will be discussed in detail in Sect. 18.5. The ranges

for carbon isotope enrichment factors do not allow to distinguish abiotic from

biotic, reductive dechlorination, reactions, however, allow distinguishing biodegradation pathways for VC and DCE in situ (Imfeld et al. 2010).

18.4.3.1 Assessment of the Reductive Dehalogenation Reaction

Mechanism

To elucidate the reaction mechanisms of microbial strains capable of reductive

dehalogenation, carbon stable isotope analysis was performed to investigate the



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J. Renpenning and I. Nijenhuis



involved (bio)catalytic step. Isotope fractionation patterns were investigated for

several microorganisms, including members of δ-Proteobacteria, ε-Proteobacteria,

Firmicutes, and Chloroflexi. Despite similarities of the reductive dehalogenase

enzyme (RDase) in all microorganisms, isotope analysis of carbon revealed highly

variable isotope fractionation for different strains during dehalogenation of PCE

and TCE (Table 18.5), making characterization of the reaction mechanism difficult

(Nijenhuis et al. 2005; Cichocka et al. 2008; Lee et al. 2007). Observed variability

in carbon isotope fractionation was thought to be related to the specific corrinoids

incorporated into the RDase enzymes as well as the microbial cell envelope properties or growth conditions (Nijenhuis et al. 2005; Cichocka et al. 2008; Mancini

et al. 2006). Furthermore, effects of substrate properties, i.e., hydrophobicity, were

suggested to be responsible for variability in isotope fractionation (Thullner et al.

2013; Cichocka et al. 2007).

Therefore, the observed variability of isotope fractionation during reductive

dehalogenation was suspected to be a result of isotope masking. Isotope masking in microbial systems is a result of rate-limiting events prior to the actual catalytic reaction, as for instance, extracellular and intracellular mass transfers (Elsner

2010). The effect of extracellular mass transfer was demonstrated to affect observable isotope fractionation in substrate availability studies (Kampara et al. 2009;

Thullner et al. 2013; Aeppli et al. 2009). Similarly, intracellular mass transfer was

demonstrated to affect isotope fractionation, as discussed in Sect. 18.5 (Nijenhuis

et al. 2005; Renpenning et al. 2015b).

18.4.3.2 Role of the Corrinoid Cofactor

The introduction of the dual-element approach offered a major step forward to

overcome the bottlenecks of single-element isotope analysis. Simultaneous analysis of two (or more) elements involved in the bond cleavage potentially elucidates

the real magnitude isotope fractionation, by excluding isotope-masking effects

(Zwank et al. 2005).

Corrinoids are a key cofactor in almost all known RDases (Krone et al. 1989;

Stupperich et al. 1990; Kräutler et al. 2003). Corrinoids, however, were found to

differ in different microorganisms and to affect the reaction rates during reductive dehalogenation significantly (Neumann et al. 2002; Siebert et al. 2002;

Kräutler et al. 2003; Keller et al. 2014). Therefore, corrinoids were suspected to

affect the reaction mechanism. Recent application of dual-element (C/Cl) analysis

however excluded corrinoid cofactors as the responsible reason for the observed

variability in microbial isotope fractionation when carbon was used for single-element analysis only. Different corrinoid cofactors incorporated in RDase enzyme

Sulfurospirillum multivorans (PceA-norpseudo-B12 and PceA-nor-B12) were

observed to result in similar dual-element isotope fractionation for TCE, as well

as PCE. This observation confirmed the minor effect of the corrinoid structure on

isotope fractionation (Renpenning et al. 2014).



18  Evaluation of the Microbial Reductive Dehalogenation Reaction …



441



Table 18.5  Enzymatic and abiotic carbon isotope enrichment factors for dehalogenation of PCE

and TCE catalyzed by corresponding microbial strain or corrinoids

Enzymatic dehalogenation mediated by RDase

Phyla



Organism

Desulfitobacterium

PCE-S



εC–PCE

−5.2 to −8.9



εC–TCE

−10.9 to −12.9



D. restrictus

PER-K23



−4.0 to −6.3



−3.3 to −8.3



G. lovleyi SZ



ns* to −2.3



−8.5 to −12.2



D. michiganensis



−1.7 to −2.6



−3.5 to −7.1



ε-Proteobacteria



S. halorespirans

S. multivorans



−0.5 to −3.2

−0.4 to −2.2



−18.7 to −22.9

−16.2 to −26.0



Chloroflexi



Dhc strain 195



−6.0



−9.6 to −13.7



Dhc strain CBDB1



−1.6



−11.2



Firmicutes



δ-Proteobacteria



Abiotic dehalogenation mediated by corrinoids

Cyanocobalamin

−16.2 to −22.4

Corrinoid type



Norpeudo-B12



−25.3



−18.5



Nor-B12



−23.7



−15.1



Dicyanocobinamide



−25.2



−16.5



Cobaloxime

*ns:



−15.0 to −16.5



−21.5



References

Nijenhuis et al.

(2005), Cichocka

et al. (2007)

Lee et al. (2007),

Renpenning et al.

(2015b)

Cichocka et al.

(2007), Cretnik et al.

(2013), Renpenning

et al. (2015b)

Cichocka et al.

(2007), Renpenning

et al. (2015b)

Cichocka et al. (2007)

Nijenhuis et al.

(2005), Cichocka

et al. (2007), Lee

et al. (2007),

Renpenning et al.

(2014)

Cichocka et al.

(2008), Lee et al.

(2007)

Marco-Urrea et al.

(2011)

Slater et al. (2003),

Nijenhuis et al.

(2005), Cichocka

et al. (2007), Cretnik

et al. (2013),

Renpenning et al.

(2014)

Renpenning et al.

(2014)

Renpenning et al.

(2014)

Renpenning et al.

(2014)

Cretnik et al. (2013)



not significant



Interestingly, variability in the dual-element slopes was still observed for structurally different pure corrinoids. Measured dual-element isotope fractionation

could be distinguished into two groups: Corrinoids containing dimethylbenzamidazole (DMB) as ligand base with a dual-element slope of 4.6–5.0, and nonDMB containing corrinoids with a dual-element slope of 6.9–7.0. The significant



442



J. Renpenning and I. Nijenhuis



differences were attributed to the difference in the lower ligand. Based on the only

available crystal structure of a reductive dehalogenase of S. multivorans, the lower

ligands in enzymes are thought to be bound by the enzyme structure and forced

in a permanent base-off conformation (Bommer et al. 2014). Purified corrinoids,

however, are able to change the conformation according to the redox state of the

cobalt. Therefore, the dissociation of the ligand (base-on/off) during the reaction

may be a rate-limiting step in abiotic dehalogenation reaction, affecting the overall rate of the reaction and masking the isotope fractionation (Renpenning et al.

2014). The effect of mass transfer on isotope fractionation will be discussed in

Sect. 18.5.

18.4.3.3 Proton Transfer During Reductive Dehalogenation

Only few reports are available about proton transfer during dehalogenation and

the corresponding isotope effects of hydrogen. The main reason for that is the

difficulty of hydrogen stable isotope analysis within halogenated compounds

(see Sect. 18.2.3). Thus far only one publication revealed strong isotope effects

for hydrogen during dehalogenation of TCE, cis-DCE, and VC to ethene by a

Dehalococcoides mixed culture (Kuder et al. 2013). The δ2H isotope signature in

the product was enriched in lighter isotopes by ~800 ‰ from +530 ‰ (TCE) to

−270 ‰ (ethene). Each dehalogenation step resulted in an isotopic shift of several

hundred per mil. Although hydrogen isotope fractionation effects during the protonation step are not yet well investigated, they promise a valuable improvement for

characterization of reaction mechanisms by introducing a multidimensional stable

isotope analysis (C, Cl and H).



18.5 Intracellular Mass Transfer and the Effect

on Observed Isotope Fractionation

Variability in microbial isotope fractionation was considered be a result of ratelimiting steps during transport prior to that C–Cl bond cleavage (Nijenhuis et al.

2005; Cichocka et al. 2007; Thullner et al. 2013; Kampara et al. 2008). For

example, mass transfer of the substrate to the enzyme may be affected by extracellular (solubility) and intracellular (membrane barriers, sorption at the membranes or enzymes) rate limitation, resulting in a dilution of the magnitude of

isotope fractionation (Aeppli et al. 2009; Thullner et al. 2013). In microbial systems three main, potentially rate-limiting, barriers can be considered for the substrate: (1) the outer membrane or cell wall, (2) the cytoplasmic membrane in

case of a cytoplasmic location of the enzyme, and (3) the structure and properties of reductive dehalogenase enzyme (Fig. 18.2). Furthermore, the properties of

the substrate, such as solubility and hydrophobicity, may affect the extent of rate



18  Evaluation of the Microbial Reductive Dehalogenation Reaction …



443



Fig. 18.2  A microscale mass transfer at microbial systems, as for instance, dissolution, transport trough the membranes, and enzyme-substrate association potentially affect isotope fractionation. Potentially rate-limiting barriers for the PCE were observed to be the outer membrane and

the cytoplasmic membrane. B Furthermore, in case of a cytoplasmic location of the enzyme, the

structure and the properties of RDase are suspected to have an additional effect on mass transfer

limitation



limitation observed. Indeed, sorption tests to microbial biomass showed in general

significantly higher sorption of PCE compared to TCE for the gram-negative S.

multivorans, as well as a three times higher sorption capacity of PCE for the gramnegative S. multivorans in comparison to the gram-positive Desulfitobacterium

(Renpenning et al. 2015b).

Although mass transfer limitation masks the real magnitude of the reactionspecific isotope enrichment, information can still be used for evaluation and interpretation intracellular microscale mass transfer processes. These masking effects,

however, are only expected to be observed in cases where the catalytic rate at the

enzyme higher is compared to the rate of mass transfer (Sherwood Lollar et al.

2010; Mancini et al. 2006).



18.5.1 Outer Membrane

Microscale mass transfer-induced isotope masking was reported in several bioavailability studies at low substrate concentration and concentration gradients

(Thullner et al. 2008; Kampara et al. 2008). Effect of rate-limiting mass transfer

on isotope fractionation was demonstrated in several studies using high biomass



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