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Systematic and Applied Microbiology 31 (2008) 81–87

www.elsevier.de/syapm

Volatilisation of metals and metalloids: An inherent feature

of methanoarchaea?

Jorg Meyer, Klaus Michalke, Theresa Kouril, Reinhard Hensel�

Department of Microbiology I, University of Duisburg-Essen, Universitaetsstr. 5, D-45117 Essen, Germany

Received 25 September 2007

Abstract

As shown by recent studies, anaerobic members of Archaea and Bacteria are involved in processes that transformionic species of metals and metalloids (arsenic, antimony, bismuth, selenium, tellurium and mercury) into volatile andmostly toxic derivatives (mainly methyl derivatives or hydrides). Since the fact that these transformations proceed inboth environmental settings and in parts of the human body, we have to consider that these processes also interferedirectly with human health. The diversity of the volatile derivatives produced and their emission rates weresignificantly higher in methanoarchaeal than in bacterial strains, which supports the pivotal role of methanoarchaea intransforming metals and metalloids (metal(loid)s) into their volatile derivatives. Compared with methanoarchaea, 14anaerobic bacterial strains showed a significantly restricted spectrum of volatilised derivatives and mostly lowerproduction rates of volatile bismuth and selenium derivatives. Since methanoarchaea isolated from the human gut(Methanosphaera stadtmanae, Methanobrevibacter smithii) showed a higher potential for metal(loid) derivatisationcompared to bacterial gut isolates, we assume that methanoarchaea in the human gut are mainly responsible for theproduction of these volatile derivatives. The observation that trimethylbismuth ((CH3)3Bi), the main volatile derivativeof bismuth produced in human feces, inhibited growing cultures of Bacteroides thetaiotaomicron, a representativemember of the human physiological gut flora, suggests that these volatiles exert their toxic effects on human health notonly by direct interaction with host cells but also by disturbing the physiological gut microflora.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Biomethylation; Gut microbiota; Methanoarchaea; Volatile metal and metalloid derivatives

Introduction

As shown by a series of studies [2,4,10,18–22,29],elements such as arsenic, antimony, bismuth, selenium,tellurium, lead, tin and mercury can be transformed bymethylation and hydridisation into volatile derivatives.The first research into the biosynthesis of these

e front matter r 2008 Elsevier GmbH. All rights reserved.

apm.2008.02.001

ing author. Tel.: +49201 183 3442;

3 3990.

ess: reinhard.hensel@uni-due.de (R. Hensel).

derivatives was performed by Gosio [11], who demon-strated that several Ascomycetes were able to transformspecies of arsenic into a volatile compound, which wassubsequently identified as trimethylarsenic. Generally,such biotransformations seem to be preferred underanaerobic conditions, with members of the metha-noarchaea playing a dominant role [7,8,17,19].

Since most of these volatile products are more toxicthan their inorganic educts [5,12,15,30], these processesdeserve closer public attention, particularly as some ofthese organisms are part of the microbiota from several

ARTICLE IN PRESS

Table

1.

Volatile

metal(loid)derivatives

producedbyvariousmethanoarchaea

andbacteria

Strains

Volatile

metal(loid)derivatives

oftheelem

ents

Sources

References

Arsenic

Selenium

Tellurium

Antimony

Bismuth

Methanoarchaea

Met

han

osp

ha

era

sta

dtm

an

aea

DSM

3091T

(CH

3) 2AsH

,(C

H3) 3As

(CH

3) 2Se,

(CH

3) 2SeS,

(CH

3) 2Se 2,SeX

b(C

H3) 2Te

(CH

3) 3Sb

CH

3BiH

2,(C

H3) 2BiH

,

(CH

3) 3Bi

Humanfeces

Michalkeet

al.,

2007

Met

hanobre

vibact

er

smit

hiiaDSM

2374

CH

3AsH

2,(C

H3) 2AsH

,

(CH

3) 3As

(CH

3) 2Se,

(CH

3) 2SeS,

(CH

3) 2Se 2

,SeX

b(C

H3) 2Te

(CH

3) 3Sb

CH

3BiH

2,(C

H3) 2BiH

,

(CH

3) 3Bi

Humanfeces

Thisstudy

Met

hanoco

ccus

vannie

lii

DSM

1224T

CH

3AsH

2,(C

H3) 2AsH

,

(CH

3) 3As,AsX

b

(CH

3) 2Se,

(CH

3) 2SeS

(CH

3) 2Te

(CH

3) 3Sb

CH

3BiH

2,(C

H3) 3Bi

Marinemud

Michalkeet

al.,

2007

Met

han

oco

ccu

s

ma

ripa

lud

isDSM

2771

LOD

(CH

3) 2Se,

(CH3) 2Se 2

(CH

3) 2Te

(CH

3) 3Sb

(CH

3) 2BiH

,(C

H3) 3Bi

Mud

Michalkeet

al.,

2007

Met

han

ola

cin

iap

ay

nte

ri

DSM

2545T

LOD

(CH

3) 2Se,

(CH

3) 2SeS,

(CH

3) 2Se 2,SeX

b

(CH

3) 2Te

(CH

3) 3Sb

(CH

3) 2BiH

,(C

H3) 3Bi

Marinesedim

ent

Michalkeet

al.,

2007

Met

han

olo

bu

sti

nd

ari

us

DSM

2278T

LOD

(CH

3) 2Se,

SeX

b(C

H3) 2Te

CH

3SbH

2,(C

H3) 3Sb

(CH

3) 3Bi

Sedim

ent

Michalkeet

al.,

2007

Met

han

op

lan

us

lim

ico

la

DSM

2279T

(CH

3) 3As

(CH

3) 2Se,

(CH

3) 2SeS,

(CH

3) 2Se 2,SeX

b

(CH

3) 2Te,

TeX

b(C

H3) 3Sb

(CH

3) 3Bi

Mudofadrilling

swamp

Michalkeet

al.,

2007

Met

han

osa

rcin

ab

ark

eric

DSM

800T

AsH

3,AsX

b(C

H3) 2Se,

(CH

3) 2Se 2

LOD

(CH

3) 3Sb

(CH

3) 3Bi

Anaerobic

sewage

digester

Michalkeet

al.,

2000

Met

han

osa

rcin

am

aze

i

DSM

3647

(CH

3) 3As

(CH

3) 2Se,

(CH

3) 2Se 2

(CH

3) 2Te

(CH

3) 3Sb

(CH

3) 3Bi

Anaerobic

sewage

digester

F.Thomas,

unpublished

Met

han

ob

act

eriu

m

form

icic

um

DSM

1535T

AsH

3,CH

3AsH

2,

(CH

3) 2AsH

,(C

H3) 3As,

AsX

b

(CH

3) 2Se,

(CH

3) 2Se 2

(CH

3) 2Te

SbH

3,CH

3SbH

2,

(CH

3) 2SbH,(C

H3) 3Sb

BiH

3,CH

3BiH

2,

(CH

3) 2BiH

,(C

H3) 3Bi

Anaerobic

sewage

digester

Michalkeet

al.,

2007

Met

han

oth

erm

ob

act

er

ther

ma

uto

trop

hic

usDSM

1053T

AsH

3LOD

LOD

(CH

3) 3Sb

LOD

Sew

agesludge

Michalkeet

al.,

2000

J. Meyer et al. / Systematic and Applied Microbiology 31 (2008) 81–8782

ARTICLE IN PRESSBacteria

Baci

llus

alc

alo

phil

usa

DSM

485T

LOD

LOD

LOD

LOD

LOD

Humanfeces

Thisstudy

Ba

cter

oid

esco

pro

cola

a

DSM

17136T

LOD

(CH

3) 2Se 2

(CH

3) 2Te

LOD

LOD

Humanfeces

Thisstudy

Ba

cter

oid

es

thet

aio

tao

mic

ronaDSM

2079T

LOD

LOD

LOD

LOD

LOD

Humanfeces

Thisstudy

Ba

cter

oid

esvu

lga

tusa

DSM

1447T

LOD

(CH

3) 2Se

(CH

3) 2Te

(CH

3) 3Sb

LOD

Humanfeces

Thisstudy

Bifi

do

ba

cter

ium

bifi

du

ma

DSM

20082

LOD

(CH

3) 2Se 2

LOD

LOD

LOD

Humanfeces

Thisstudy

Bu

tyri

vib

rio

cro

sso

tusa

DSM

2876T

LOD

LOD

LOD

LOD

LOD

Humanfeces

Thisstudy

Clo

stri

diu

mace

ticu

ma

DSM

1496T

LOD

(CH

3) 2Se

(CH

3) 2Te

LOD

LOD

Humanfeces

Thisstudy

Clo

stri

diu

mle

ptu

maDSM

753T

LOD

(CH

3) 2Se,

(CH

3) 2Se 2

LOD

LOD

LOD

Humanfeces

Thisstudy

Coll

inse

lla

inte

stin

ali

sa

DSM

13280T

LOD

(CH

3) 2Se 2

LOD

LOD

LOD

Humanfeces

Thisstudy

Des

ulf

ovi

bri

op

iger

a

DSM

749

LOD

LOD

LOD

LOD

(CH

3) 3Bi

Humanfeces

Thisstudy

Eu

ba

cter

ium

bif

orm

ea

DSM

3989T

LOD

(CH

3) 2Se

(CH

3) 2Te

LOD

LOD

Humanfeces

Thisstudy

Eu

ba

cter

ium

elig

ensa

DSM

3376T

LOD

(CH

3) 2Se 2

(CH

3) 2Te

LOD

(CH

3) 3Bi

Humanfeces

Thisstudy

Lact

obaci

llus

aci

dophil

usa

DSM

20079T

LOD

(CH

3) 2Se

(CH

3) 2Te

LOD

(CH

3) 3Bi

Humanfeces

Thisstudy

Ru

min

oco

ccu

sh

an

sen

iia

DSM

20583T

LOD

(CH

3) 2Se 2

LOD

LOD

LOD

Humanfeces

Thisstudy

LOD,limitofdetection.

aRegarded

asmem

bersofthehumangutflora.

bUnidentified

volatile

metal(loid)s.

cMediatedbyoctamethylcyclotetrasiloxaneortheionophoreslasalocideandmonensin[21].

J. Meyer et al. / Systematic and Applied Microbiology 31 (2008) 81–87 83

ARTICLE IN PRESSJ. Meyer et al. / Systematic and Applied Microbiology 31 (2008) 81–8784

compartments of the human body, such as the oralcavity, the vagina or the gut [1,6,9,16,23,24], suggestingthat their transformation activities could affect humanhealth directly. The gut especially offers sufficient spacefor approximately 1014 anaerobic and facultativelyanaerobic microbes, including methanoarchaea. There-fore, it provides a favourable location for considerableproduction of these volatile derivatives because of itsstrictly anaerobic milieu at virtually constant neutral pHand mesophilic temperature, as well as continuoussubstrate supply. Despite previous assumptions of afacultative presence of methanoarchaea in the humangut, with an estimated proportion of methane producingindividuals of approximately 30–80% in the humanpopulation [13,14,26,27], recent research suggests anobligate presence of methanoarchaea in the humanintestine [6]. According to this, our recent studies ofhuman feces microbiota from 14 volunteers showedsignificant methane production by all feces samples ofthe cohort [K. Michalke et al., unpublished]. Unfortu-nately, our knowledge concerning the abundance anddiversity of the methanoarchaeal population in thehuman intestine and the identity of their members islimited. In fact, to date, only Methanosphaera stadtma-

nae and Methanobrevibacter smithii have been describedas typical members of the human gut microbiota [23,24].

In order to check whether the capability of metha-noarchaea to transform metal(loid)s into volatile deri-vatives represents a specific property of these organisms,we checked growing cultures of methanoarchaea iso-lated from various ecological niches, including themethanoarchaeal human gut inhabitants M. stadtmanae

and M. smithii, for production of volatile derivatives.Comparison with bacterial strains isolated from humanfeces (members of Proteobacteria, Firmicutes, Actino-bacteria and Bacteroidetes), which are considered as themain bacterial phyla represented in the human gutmicrobiota [6], should also provide information con-cerning the preferred role of methanoarchaea inintestinal metal(loid) derivatisation.

Materials and methods

Bacterial strains and culture media

All cultures were grown under strictly anaerobicconditions in butyl rubber-stoppered 120ml serumbottles containing 50ml of the recommended DSMZ-medium [www.dsmz.de] or in butyl rubber-stoppered17ml screw-top test tubes. The media were reduced bythe addition of L-cysteine (0.3–0.5 g l�1), pressurisedwith CO2/H2, N2 or N2/CO2, as recommended, andincubated in the dark in a rotary shaker (150 rpm) at37 1C. To avoid bacterial contamination, ampicillin

(100 mgml�1) was added to the methanoarchaea cul-tures. All strains used in this study and their source ofisolation are listed in Table 1.

Assays for production of volatile metal(loid)

derivatives in cultures of methanoarchaea and

bacteria

The cultures were spiked in the early exponentialgrowth phase (about 106 cellsml�1) with the saltsKH2AsO4, SbCl3, HgCl2, Bi(NO3)3, TeO2 and SeO2

(final concentration: 1 mM each) or with a singlemetal(loid) salt (final concentration: 1 mM). TeO2 andSbCl3 were solved in ethanol. Bi(NO3)3 was solved inpropandiol with 50mM EDTA and subsequent adjust-ment to pH 7.0 with NaOH. The production of volatilemetal(loid) derivatives was followed for up to 14 daysdepending on the growth rate of the respective organ-isms. (CH3)3Bi was synthesised with the aid of a crudeextract of Methanosarcina mazei (B. Huber et al.,unpublished). The volatile metal(loid) derivatives inthe headspace of the cultures were analysed by amodified purge-and-trap gas chromatographic systemcoupled to an inductively coupled plasma mass spectro-meter (ICP-MS; Fisons VG, PlasmaQuad II) [19].Identification was undertaken by parallel ICP-MS andelectron ionisation mass spectrometry (EI-MS) detec-tion after GC separation [17]. Growth of bacterial andmethanoarchaeal strains was followed by measuring theprotein content of the cultures using the protein assay ofBradford [3]. All experiments were performed at least intriplicate.

Results and discussion

Table 1 shows the diversity of the volatile metal(loid)derivatives produced by the various methanoarchaealand anaerobic bacterial strains following addition of amixture of inorganic salts of arsenic, antimony, bismuth,selenium, tellurium and mercury. No strain tested wasable to produce volatile methyl or hydride derivatives ofmercury. The volatile elemental mercury observed in theheadspace seemed to be of abiotic origin, as shown bysterile control samples, which contained comparableamounts of elemental mercury.

As demonstrated in Table 1, members of themethanoarchaea were in general more versatile intransforming metal(loid)s to volatile derivatives ascompared with the bacterial strains. The vast majority,including the human gut inhabitants M. stadtmanae andM. smithii, transformed the five elements arsenic,antimony, bismuth, selenium and tellurium into volatilederivatives. A somewhat restricted capability of transforma-tion was shown by the methanoarchaea Methanococcus

ARTICLE IN PRESS

Table 2. Maximum production rates of methanoarchaea and

bacteria for the formation of volatile bismuth and selenium

derivatives

Microorganisms Maximum production rates

(fmol h�1mg�1 protein)

Bismuth derivatives Selenium

derivatives

Methanoarchaea

M. stadtmanaea (CH3)3Bi: 8.675.7 (CH3)2Se:

206.2725.1

(CH3)2SeS:

2.370.9

(CH3)2Se2:

4.071.2

SeX:b 0.370.1

M. smithiia (CH3)3Bi: 18.673.5 (CH3)2Se:

39.3712.0

(CH3)2SeS:

0.170.1

(CH3)2Se2:

0.170.1

SeX:b 0.170.1

M. vannielii (CH3)3Bi: 24.1673.2 (CH3)2Se:

45.779.0

(CH3)2SeS:

0.170.0

M. maripaludis (CH3)3Bi: 30.171.6 (CH3)2Se:

65.2738.0

(CH3)2Se2:

0.670.2

M. paynteri (CH3)3Bi: 6.973.6 (CH3)2Se:

77.1718.1

(CH3)2SeS:

0.170.1

(CH3)2Se2:

1.670.3

SeX:b 0.270.2

M. tindarius (CH3)3Bi: 16.676.1 (CH3)2Se:

61.9716.3

SeX:b 0.170.0

M. limicola (CH3)3Bi: 18.574.5 (CH3)2Se:

53.7718.5

(CH3)2SeS:

2.170.8

(CH3)2Se2:

0.670.2

SeX:b 0.270.1

M. barkeric (CH3)3Bi: 54.0719.6 LOD

M. mazei (CH3)3Bi:

1698.771628.6

LOD

M. formicicum (CH3)3Bi:

145.4770.5

LOD

M.

thermautotrophicus

LOD LOD

J. Meyer et al. / Systematic and Applied Microbiology 31 (2008) 81–87 85

maripaludis, Methanolacinia paynteri and Methanolobus

tindarius, which were not able to produce volatile arsenicderivatives. The exceptionally low diversity of volatilederivatisation products in the case of Methanothermobacter

thermautotrophicus could be explained by the elevatedgrowth temperature of this organism (Topt: 65 1C), whichmay lead to rapid heat destruction of the volatiles and thusto a seemingly low derivatisation potential [22].

As compared with the methanoarchaeal strains, thebacterial members assigned to the human gut micro-biota produced a significantly smaller spectrum ofvolatile metal(loid) derivatives, both for the number ofmetal(loid)s transformed and the number of derivativesproduced per element. In the cultures of Butyrivibrio

crossotus, Bacillus alcalophilus and Bacteroides thetaio-

taomicron, no volatile derivatives could be observed.The majority of the selected bacterial strains (10 out of14) were able to transform selenium into volatilederivatives and tellurium was transformed in additionto selenium by 6 strains, whereas only 4 bacterial strains(Lactobacillus acidophilus, Eubacterium eligens, Bacter-

oides vulgatus, Desulfovibrio piger) were able to volatiliseadditional elements from bismuth and antimony,respectively.

The higher capability of methanoarchaeal strains totransform metal(loid)s into volatile derivatives, com-pared to the bacterial strains, was also reflected in thederivatisation reaction rates. For this comparison, wechose the derivatisation of selenium, which is atransformation shared by all methanoarchaea tested(with the apparent exception of M. thermautotrophicus)and by most of the bacterial strains. Additionally, weincluded the derivatisation of bismuth to (CH3)3Bi in thecomparison, since this transformation is a healthconcern due to the increasing application of bismuthin cosmetics and pharmaceuticals, as well as the hightoxicity of its permethylated product [28]. As shown inTable 2, the formation rates of the various volatilederivatives of both elements were higher mostly inmethanoarchaea compared to bacteria.

Since methanoarchaea isolated from different envir-onmental locations are characterised by a remarkablyversatile and excessive transformation of metal(loid)s,this ability seems to represent an inherent methanoarch-aeal feature. It seems reasonable to assume thereforethat this high methylation activity on metal(loid)s is due,at least partially, to the multiplicity of their methyl-transfer reactions.

In order to assess the effect of volatile metal(loid)son co-inhabitants of the human gut microbiota, weexposed growing cultures of B. thetaiotaomicron, arepresentative of the so-called physiological microbiotaof the human gut, to (CH3)3Bi and followed its growthresponse. As shown in Fig. 1, an inhibitory effect on thegrowth of B. thetaiotaomicron was apparent at (CH3)3Biconcentrations of 5.7 nM and above. This indicates that

ARTICLE IN PRESS

Table 2. (continued )

Microorganisms Maximum production rates

(fmol h�1mg�1 protein)

Bismuth derivatives Selenium

derivatives

Bacteria

B. coprocolaa LOD (CH3)2Se2:

0.370.1

B. vulgatusa LOD (CH3)2Se:

0.170.1

B. bifiduma LOD (CH3)2Se2:

0.870.2

B. crossotusa LOD LOD

B. alcalophilusa LOD LOD

B.

thetaiotaomicronaLOD LOD

C. aceticuma LOD (CH3)2Se:

0.370.3

C. leptuma LOD (CH3)2Se:

1.870.6

(CH3)2Se2:

0.170.1

C. intestinalisa LOD (CH3)2Se2:

0.370.1

D. pigera (CH3)3Bi: 0.3670.26 LOD

E. bioformea LOD (CH3)2Se:

0.370.0

E. eligensa (CH3)3Bi: 0.0770.01 (CH3)2Se2:

0.270.0

L. acidophilusa (CH3)3Bi: 0.3670.28 (CH3)2Se2:

0.470.1

R. hanseniia LOD (CH3)2Se2:

0.370.1

LOD, limit of detection.aRegarded as members of the human gut flora.bUnidentified selenium derivative.cMediated by octamethylcyclotetrasiloxane or the ionophores

lasalocide and monensin [21].

0.0

0.2

0.4

0.6

0.8

1.0

0 2 4 6 8 10 12 14Incubation time (h)

Opt

ical

den

sity

(600

nm

) Control2.9 nM(CH3)3Bi5.7 nM(CH3)3Bi17 nM(CH3)3Bi

Fig. 1. Influence of (CH3)3Bi on the growth of Bacteroides

thetaiotaomicron. (CH3)3Bi was added before inoculation with

a 1% stock culture of B. thetaiotaomicron.

J. Meyer et al. / Systematic and Applied Microbiology 31 (2008) 81–8786

the volatile (CH3)3Bi exerts toxic effects on bacteriabelonging to the beneficial intestinal microbiota. How-ever, since we do not know the in vivo intestinalconcentrations it seems premature to deduce from thisthat there is a general risk of damaging the intestinalmicrobiota. Nevertheless, an examination of fecessamples taken from volunteers who ingested bismuthsubcitrate (Michalke et al., submitted for publication)showed that the production rates of (CH3)3Bi in thefeces were in the same order of magnitude as thosefound in batch cultures of methanoarchaea. Fecessamples examined produced this compound at rates of2.3–28,000 fmol h�1 g�1 wet weight (corresponding to aproduction of approximately 1012 total cells or approxi-mately 1010 to 1011 methanoarchaeal cells, respectively[6,25]). This is comparable with the productivity of

exponential cultures of M. smithii with rates of3.5 fmol h�1ml�1 at cell counts of 107ml�1. Since thereis a lack of in vivo data for the intestinum, estimates ofthe intestinal (CH3)3Bi concentration have to be basedon data from pure culture experiments with variousmethanoarchaea (Meyer et al., unpublished), and thesehave shown that (CH3)3Bi is accumulated up toapproximately 0.5 nM. From this, the inhibition ofthe gut microbiota by (CH3)3Bi under in vivo conditionsseems to be, at first glance, rather improbable. However,considering that the high cell density in the gut couldfacilitate the transfer of the volatile derivative fromproducer to target cells by direct cell contacts, (CH3)3Bicould nevertheless inhibit the growth of gut co-inhabitants at lower concentrations. Consequently,respective interactions will be investigated in gnotobioticmice.

Acknowledgements

The work was supported by grants from the DeutscheForschungsgemeinschaft (DFG). We would like tothank the working group of A.V. Hirner, Dept. ofEnvironmental and Analytical Chemistry, University ofDuisburg-Essen for analytical assistance.

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