Monodeuterated Methane, an Isotopic Tool To Assess Biological Methane Metabolism Rates

19 Pages·2017·1.75 MB·English

RESEARCH ARTICLE
AppliedandEnvironmentalScience
crossm
Monodeuterated Methane, an Isotopic
Tool To Assess Biological Methane
Metabolism Rates
D
o
JeffreyJ.Marlow,a*JoshuaA.Steele,a*WiebkeZiebis,bSilvanScheller,a* w
n
DavidCase,aLindaM.Reynard,cVictoriaJ.Orphana
lo
DivisionofGeologicalandPlanetarySciences,CaliforniaInstituteofTechnology,Pasadena,California,USAa; a
d
DepartmentofBiologicalScience,UniversityofSouthernCalifornia,LosAngeles,California,USAb;Department e
ofHumanEvolutionaryBiology,HarvardUniversity,Cambridge,Massachusetts,USAc d

f
r
o
ABSTRACT Biological methane oxidation is a globally relevant process that medi- m
ates the flux of an important greenhouse gas through both aerobic and anaerobic Received18July2017 Accepted31July
h
metabolic pathways. However, measuring these metabolic rates presents many ob- 2017 Published23August2017 tt
CitationMarlowJJ,SteeleJA,ZiebisW, p
stacles, from logistical barriers to regulatory hurdles and poor precision. Here we SchellerS,CaseD,ReynardLM,OrphanVJ. ://
present a new approach for investigating microbial methane metabolism based on 2017.Monodeuteratedmethane,anisotopic m
tooltoassessbiologicalmethanemetabolism s
hydrogen atom dynamics, which is complementary to carbon-focused assessments p
rates.mSphere2:e00309-17.https://doi.org/10 h
of methanotrophy. The method uses monodeuterated methane (CH3D) as a meta- .1128/mSphereDirect.00309-17. e
r
bolic substrate, quantifying the aqueous D/H ratio over time using off-axis inte- EditorSusannahGreenTringe,DOEJoint e
.
grated cavity output spectroscopy. This approach represents a nontoxic, compara- GenomeInstitute a
s
tively rapid, and straightforward approach that supplements existing radiotopic and Copyright©2017Marlowetal.Thisisan m
open-accessarticledistributedundertheterms .
stable carbon isotopic methods; by probing hydrogen atoms, it offers an additional oftheCreativeCommonsAttribution4.0 o
r
dimension for examining rates and pathways of methane metabolism. We provide Internationallicense. g
/
direct comparisons between the CH D procedure and the well-established 14CH ra- AddresscorrespondencetoJeffreyJ.Marlow, o
3 4 [email protected]. n
diotracer method for several methanotrophic systems, including type I and II aerobic *Presentaddress:JeffreyJ.Marlow, N
methanotroph cultures and methane-seep sediment slurries and carbonate rocks un- DepartmentofOrganismicandEvolutionary o
v
der anoxic and oxic incubation conditions. In all applications tested, methane con- Biology,HarvardUniversity,Cambridge, e
Massachusetts,USA;JoshuaA.Steele,Southern m
sumption values calculated via the CH D method were directly and consistently pro-
3 CaliforniaCoastalWaterResearchProject, b
portional to 14C radiolabel-derived methane oxidation rates. We also employed this CostaMesa,California,USA;SilvanScheller, e
r
method in a nontraditional experimental setup, using flexible, gas-impermeable bags DepartmentofBioproductsandBiosystems, 1
AaltoUniversity,Espoo,Finland. 9
to investigate the role of pressure on seep sediment methane oxidation rates. Re- ,
Solicitedexternalreviewers:PeerTimmers, 2
sults revealed an 80% increase over atmospheric pressure in methanotrophic rates Wetsus,EuropeanCentreofExcellencefor 0
the equivalent of ~900-m water depth, highlighting the importance of this parame- SustainableWaterTechnology;TinaTreude, 1
8
ter on methane metabolism and exhibiting the flexibility of the newly described UniversityofCalifornia,LosAngeles. b
Thispaperwassubmittedviathe y
method. mSphereDirect™pathway. g
u
IMPORTANCE Microbial methane consumption is a critical component of the global e
carbon cycle, with wide-ranging implications for climate regulation and hydrocarbon s
t
exploitation. Nonetheless, quantifying methane metabolism typically involves logisti-
cally challenging methods and/or specialized equipment; these impediments have
limited our understanding of methane fluxes and reservoirs in natural systems, mak-
ing effective management difficult. Here, we offer an easily implementable, precise
method using monodeuterated methane (CH D) that advances three specific aims.
3
First, it allows users to directly compare methane consumption rates between differ-
ent experimental treatments of the same inoculum. Second, by empirically linking
the CH D procedure with the well-established 14C radiocarbon approach, we deter-
3
mine absolute scaling factors that facilitate rate measurements for several aerobic and
anaerobicsystemsofinterest.Third,CH Drepresentsahelpfultoolinevaluatingthere-
3
lationship between methane activation and full oxidation in methanotrophic metabo-
lisms. The procedural advantages, consistency, and novel research questions enabled
July/August2017 Volume2 Issue4 e00309-17 msphere.asm.org 1
Marlowetal.
by the CH D method should prove useful in a wide range of culture-based and envi-
3
ronmentalmicrobialsystemstofurtherelucidatemethanemetabolismdynamics.
KEYWORDS environmentalmicrobiology,metabolicratemeasurement,methane,
stableisotopeprobing
Methane-consuming microbial processes represent an important component of
biogeochemicalcyclesinnaturalfreshwaterandmarineenvironments,aswellas
in human-impacted systems. In terrestrial soils, methane production in rice fields,
anoxicwetlands,andthawingpermafrostsupportsmethanotrophiccommunities(1–4).
Inmarinesettings,anestimated85Tgofmethaneperyear,derivedfrombiogenicand
D
thermogenicsources,entersthesubseafloor,thevastmajorityofwhichisanaerobically o
w
consumedinanoxicsediments(5).Muchofwhatremainsistakenupinmicrooxicor
n
oxic zones of the sediment or water column by aerobic methanotrophic microorgan- lo
a
isms (6). Methanotrophy is also of interest in a range of human-impacted contexts, d
e
includinggroundwater(7,8),wastewatertreatmentplants(9),landfills(10),shalegas
d
(11),coalbedharvesting(12),andoilspills(13). f
r
o
Inadditiontoitsclimaticandeconomicimplications,thebiochemicaldetailsof
m
the methanotrophic process have stimulated many investigations. The sulfate-linked
h
anaerobic oxidation of methane (AOM; reaction 1) has proven particularly enigmatic; tt
p
this process typically involves a mutualistic relationship between anaerobic metha- :
/
/
notrophic(ANME)archaeaandsulfate-reducingbacteria(SRB)(14–16),althoughnitrate m
s
(17, 18) and, potentially, metals such as iron and manganese (19–21) can serve as p
alternativeelectronacceptorsforsomeANMElineages.Methaneisoxidizedaerobically h
e
(reaction2)bymembersoftheclassesGammaproteobacteria(e.g.,typeIandtypeX) re
andAlphaproteobacteria(typeII);verrucomicrobialrepresentativescanperformaerobic .a
s
methanotrophy under extremely acidic conditions (22, 23). Methane is converted to m
methanol,whichisfurtheroxidizedtoformaldehyde;assimilatorypathwaysbranching .o
at this point can incorporate carbon into central metabolism through the ribulose rg
/
monophosphate (RuMP) cycle (type I and type X methanotrophs) or the serine cycle o
(typeII). n

N
CH (cid:2)SO2(cid:3) →HCO(cid:3)(cid:2)HS(cid:3)(cid:2)HO (reaction1) o
2 4 3 2 v
e
CH (cid:2)2O →HCO(cid:3)(cid:2)HO(cid:2)H(cid:2) (reaction2) m
4 2 3 2
b
Methanotrophy is both a biogeochemically relevant activity that modulates the e
r
globalclimateandapoorlyunderstoodbiochemicalprocess;giventhisdualrole,there
1
is substantial interest in measuring its rate and in understanding elemental flows 9
,

throughmetabolicpathways.Theoxidationofmethaneinenvironmentalsampleshas 2
0
traditionally been studied using a few techniques. Numerical models incorporating 1
8
environmentalsedimentprofilesofsulfateandmethaneconcentrationscanbeusedto
b
back-calculate methane consumption rates (24). 13CH can be used to probe rates y
4
under controlled conditions (25–28), but the presence of natural 13C in marine dis- g
u
solved inorganic carbon (DIC) pools requires long incubations as well as accurate e
s
measurements of isotopically resolved concentrations of reactants and products (29). t
Gas chromatography (GC) quantification of dissolved (30–32) or headspace (33, 34)
methane concentrations has also been demonstrated as a rate measurement tool,
though low concentrations can hamper reproducibility and exacerbate background
contaminationissues,particularlyinfield-basedsettings(35).Perhapsthemostsensi-
tive approach uses radiolabeled 14CH to track the oxidation of methane-associated
4
carbontoinorganiccarbonspecies(36,37).Tritiatedmethanewasintroducedforwater
columnaerobicmethaneoxidationmeasurementsduetoitshigheractivityperradio-
nuclide (6, 38). Logistical challenges and health and safety regulations led Pack et al.
(29)todevelopanacceleratormassspectrometrydetectionmethodthatrequires103
to 105 less radiolabel than previous 14C and 3H approaches, though the analytical
procedureremainslabor-intensive.
Despite the range of methods available, measurement of microbial methane utili-
July/August2017 Volume2 Issue4 e00309-17 msphere.asm.org 2
MonodeuteratedMethane
zation rates remains cumbersome, and a precise, safe, and easily enacted approach
would be a welcome contribution for a diverse array of researchers. Nearly all of the
aforementionedapproachesarecarbonbased;ahydrogen-basedtraceroffersacom-
plementary approach to investigations of methane biochemical dynamics. Here we
introduceanovelmethodforbiologicallymediatedmethanotrophyratemeasurement
that utilizes monodeuterated methane (CH D) as a substrate and measures the D/H
3
ratiooftheaqueoussolution.Thisapproachoffersseveraladvantagesforprospective
users: it does not require the logistical, safety, and administrative hurdles associated
withradiotracerssuchas14CH and[3H]CH ,itcomparesfavorablyintermsofequip-
4 4
mentcostandportability,anditprovidesanadditionalanalyticaloptionthatenables
hydrogen stable isotope-based measurement of methane activation that is comple- D
mentarytocarbon-basedstableisotope(13C)orradiocarbon(14C)methods.Asaproof o
w
ofconcept,weapplythemonodeuterated-methaneapproachtopressurizedmethane n
seep sediment incubations in order to test the role of an important environmental lo
a
variableonmethanotrophicratesundernontraditionalempiricalconditions. d
e
d

RESULTSANDDISCUSSION fr
o
Comparison of CH D and 14CH rates in aerobic methanotroph cultures. D/H m
3 4
ratios were acquired and corresponding values of methane consumption were calcu- h
lated at eight points during the Methylosinus trichosporium growth curve and seven tt
p
points of the Methyloprofundus sedimenti growth curve. Three measurements of 14C :/
/
m
distributionswereacquiredforeachstrain,targetingexponentialandstationaryphases
s
(Fig. 1). The type II alphaproteobacterial methanotroph M. trichosporium exhibited p
h
methane consumption rates more than an order of magnitude greater than those of e
r
M. sedimenti (gammaproteobacterial type I methanotroph), yet the scaling factor e
.
relating the CH D- and 14CH -derived rates was remarkably consistent in both cases. a
3 4 s
Scalingfactorswerecalculatedforbothexponentialgrowthandstationaryphase,using m
data points from both CH D and 14CH experiments. The M. trichosporium rate value .o
3 4 r
calculated from the CH D experimental treatment point (47.5 h, 4.16 (cid:2) 104 nmol of g
3 /
methane consumed) was compared with the rate determined from the 14CH experi- o
mental treatment point (47.5 h, 2.78 (cid:2) 104 nmol of methane consumed), yi4elding a n
N
scalingfactorof1.5forexponential-phasegrowth.Similarly,datafromtheexperimen- o
taltreatmentpointat140h(5.27(cid:2)104nmolofmethane,CH3D)and166.5h(4.24(cid:2) ve
104nmolofmethane,14CH )wereusedforM.trichosporium’sstationary-phasescaling m
4 b
factor. Equivalent values were determined for M. sedimenti using the following data e
points: 7.07 (cid:2) 103 nmol of methane after 140 h with CH D and 3.35 (cid:2) 103 nmol of r
3 1
methaneafter102hwith14CH fortheexponentialgrowthphase,and7.53(cid:2)103nmol 9
4 ,
ofmethaneafter476hwithCH Dand4.30(cid:2)103nmolofmethaneafter432hwith 2
3 0
14CH forthestationaryphase(Fig.1).Itshouldbenotedthatsimultaneoussampling 1
4
8
of CH3D and 14CH4 experiments was not always possible, as they were conducted at b
differentinstitutions.Nonetheless,theopticaldensityat600nm(OD )andrate-based y
600
growthcurvesindicatethatallsamplingoccurredwithinthedesignatedgrowthphase g
u
(Fig.1andseeFig.S1inthesupplementalmaterial). e
s
Inthisway,theratiosofmethaneconsumptionratesderivedfromtheCH Dmethod t
3
(using equations 1 to 7 [see Materials and Methods]) and the 14CH method (using
4
equation8)canbecompared.ThisvalueishereinreferredtoastheD/14Ctracerratio.
This ratio can be used to evaluate the consistency of the monodeuterated-methane
method compared with the well-established 14CH approach and as a potent investi-
4
gatory tool to probe the relationship between partial and complete metabolism of
methane.
D/14C tracer ratio values for aerobic methanotroph cultures tested in this study
are shown in Table 1; their consistency is a promising indicator of the utility of the
monodeuterated-methaneapproach.Bydividingthemethaneactivationratesderived
fromD/Hvalues(R [see“RatemeasurementsderivedfromCH Daddition”below])
CH3D 3
by 1.5, an estimate of full-oxidation methanotrophy—that is, the complete biological
oxidationofmethanetocarbondioxide—canbeattained.
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Marlowetal.
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FIG1 AmountofmethaneconsumedovertimeforculturesofthetypeIImethanotrophM.trichospo- o
rium(a)andthetypeImethanotrophM.sedimenti(b)usingC (valueswerederivedfromtheCHD n
corr 3
method[circles])andthe14CH method(diamonds),calculatedasdiscussedinthetext.14CH-derived N
4 4
data convey values of methane consumption and full oxidation, while CHD-derived data provide a o
3 v
measureofmethaneactivation.Errorbarsshowstandarderrorsforthreebiologicalreplicates,exceptfor e
the14CH killedcontrol(n(cid:5)1).Obscureddatapointsexhibitedvaluesbetween(cid:4)60and110nmolfor m
4
theresultsinpanelaandbetween0and60nmolfortheresultsinpanelb. b
e
r

1
ComparisonofCH Dand14CH ratemeasurementsinenvironmentalmethane 9
3 4 ,
seep samples. Methane consumption rates under oxic (Fig. 2a) and anoxic (Fig. 2b) 2
0
microcosmincubationconditions,derivedfrombothCH Dand14CH measurements, 1
3 4
8
are provided for five different sample types from marine methane seeps (active

b
sediment, low-activity sediment, active porous carbonate, active massive carbonate, y

and low-activity massive carbonate) and were calculated from data collected after g
u
4days(oxic)or8days(anoxic)ofincubation. e
s
TheD/14Ctracerratiowas1.66(cid:3)0.02standarderror(SE)fortheoxicand1.99(cid:3)0.04 t
SEfortheanoxicincubations(Table1).Theserelativelyconsistentvaluesacrossphysical
substratetype(sedimentandcarbonatesofvariouslithologies)andcollectionsiteactivity
level(activeandlowactivity)suggestanunderlyingmetabolicbasisoftheD/14Ctracerratio
thatisunperturbedbyphysicochemicalfactorsorrelativeactivitylevels.
UnderstandingtheD/14Ctracerratio.TheCH Dand14CH approachesquantify
3 4
distinct aspects of methanotrophy: methane activation and complete conversion to
CO ,respectively.The14CH techniquequantifiestheamountof14C(initiallysupplied
2 4
asmethane)thatisfullyoxidizedandpersistsassolublespecies(HCO (cid:4))oracid-labile
3
precipitation products (CaCO ). The CH D protocol, on the other hand, reports the
3 3
extent to which methane-derived hydrogen atoms are detected in water. Abiotic
exchange between methane- and water-associated hydrogen atoms is not expected.
Indeed,D/Hratiosinkilled-controlexperimentsremainedstable(e.g.,exhibitingavalue
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MonodeuteratedMethane
TABLE1 D/14Ctracerratiosfortheexperimentaltreatmentsaddressedinthisstudya
Ratioin:
Exponential Stationary Oxic Anoxic
Sampletested phase phase incubations incubations
Aerobicmethanotrophcultures
M.trichosporium 1.5 1.48
M.sedimenti 1.54 1.59
Methaneseepsedimentsandcarbonates
A.Sed-5128 1.62 2.05
L.Sed-5043 1.71 2.01
A.Carb-5305 1.65 1.96
D
A.Carb-5152 1.63 2.08 o
L.Carb-5028 1.69 1.86 w
n
aCulturesoftheaerobicmethanotrophsM.trichosporiumandM.sedimentiweretestedalongside lo
environmentalsamples,sediments(Sed)andcarbonaterocks(Carb),fromHydrateRidgemethaneseeps. a
“A”referstositesofactiveseepage,while“L”indicateslocationsoflowseepageactivity,whereclearsigns d
ofcontemporarymethaneseepagewereabsent.(Seethetextforadditionalsamplingdetails.) e
d

f
r
o
m
of1.40(cid:2)10(cid:4)4(cid:3)3.1(cid:2)10(cid:4)8SEat time zero [T0] and 1.40 (cid:2) 10(cid:4)4(cid:3) 2.9 (cid:2) 10(cid:4)8SE h
at 140 days [T ] during experimentation withM. trichosporium[data are incorpo- tt
140 p
rated into Fig. 1a]). The activation of methane thereby indicates enzymatic func- :/
/
m
tionalization, but the ultimate fate of each hydrogen atom during methane oxida-
s
tion is not known. p
h
The flow of methane-derived hydrogen atoms through anaerobic and aerobic e
r
methanotrophicmetabolismswasexaminedinanattempttopredictivelyevaluate e
.
the consequence of monodeuterated-methane reactions. Previously published re- a
s
ports were used to compile Fig. 3 (39–41) and Fig. 4 (42), which trace anaerobic and m
aerobicmethanemetabolisms,respectively,withaspecificfocusonhydrogenatoms. .o
r
Inthiscontext,ourobservationsofrelativelyconsistentbutdistinctD/14Ctracerratios g
/
foranaerobicandaerobicmethanotrophy(Table1)likelyreflectdifferentaspectsofthe o
n
twometabolicpathways.InAOM,metaboliteback-flux(43)mayincreasetheD/Hratio;
N
in aerobic methanotrophy, biomass growth represents a substantial carbon and hy- o
drogenshunt. v
e
TheD/14Ctracerratioinanaerobicmethanotrophy.AOMisdepictedinFig.3via m
b
the reverse-methanogenesis pathway, which is believed to be enacted by anaerobic e
r
methanotrophicarchaeabasedongenetic(41,44,45)andproteomic(46,47)data.In
1
this metabolic process, methyl-coenzyme M reductase (Mcr) activates methane and 9
,

generates methyl-coenzyme M (methyl-CoM). A tetrahydromethanopterin molecule 2
0
supplants CoM, and subsequent carbon oxidation steps release hydrogen atoms into 1
8
the medium. Ultimately, the number of methane-derived hydrogen atoms that enter
b
water-exchangeable products determines the physiological interpretation of aqueous y

D/Hratios.Forexample,ifjustonemethane-derivedhydrogenentersanintermediate g
u
andisfreelyexchangeablewithwater,thenobservedwater-baseddeuteriummustbe e
s
multiplied by 4 (to account for methane’s hydrogen-carbon stoichiometry [see equa- t
tion 5 in Materials and Methods]) and the appropriate primary isotope effect (not
evaluatedhere)toarriveattheactualquantityofactivatedmethanemolecules.Inthis
context,theexperimentalD/14Ctracerratiovaluesmayprovideusefulinsight.AD/14C
tracer ratio of 2 for the reverse-methanogenesis pathway suggests that for every
methane molecule that is fully oxidized to CO , two hydrogen atoms enter water-
2
exchangeableintermediates.
However,theback-reactionofenzymaticprocesses(48)mayleadtoheightenedD/H
ratios in the absence of full carbon oxidation. For example, upon the activation of
methanebyMcr,HS-coenzymeB(HS-CoB)andCH -S-CoMform,withthethiolhydro-
3
genexchangingwithwater-boundhydrogen.IftheinitiallyformedS-boundhydrogen
isdeuterium,thisatomthenexchangeswith1Hfromwater.UponMcrback-reaction,
CH isformedandtheaqueousdeuteriumcausesaheightenedD/Hratiodespitealack
4
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FIG2 Methanotrophyinoxic(a)andanoxic(b)incubationsofactiveandlow-activityseepsedimentand N
carbonaterocks(n(cid:5)3inallcases).Valuescompareratesofmethaneconsumptionandfulloxidation o
v
derived from 14CH4 measurements (blue) and rates of methane activation derived from the CH3D e
approach(green,R values).Valuesarereflectiveofrockandinitialsedimentvolumes(notincluding m
addedwater).RatesCHd3eDrivedfromtriplicateA.Sed-5128killed-controlincubationsweresubtractedfrom b
allsamples.Standarderrorbarsareprovided. e
r

1
9
,

ofcompletemethaneoxidation(Fig.3).Weanalyzedtheremainingheadspaceofseep 2
0
sediment incubations for the formation of CH from CH D via 1H-nuclear magnetic 1
4 3
8
resonance(NMR)spectroscopy.Overthecourseof58daysintriplicateactive-sediment
b
5128(A.Sed-5128)incubationspreparedwithexclusivelyCH Dheadspace,CH inthe y
3 4
headspaceincreasedfrom0.33%(cid:3)0.02%SEto4.48%(cid:3)0.27%SE.Ifthisdemonstrated g
u
reversibilityreflectsonlytheback-reactionofMcr,thentheCH increase(4.15%)must e
4 s
be multiplied by 4 ((cid:5)16.6%) to reflect the actual percentage of headspace methane t
that was re-formed by Mcr; if the reversibility reflects back-reaction of the entire
pathway,thennoscalingfactorisneeded.Fullmethaneoxidationratesmeasuredvia
14CH indifferentreplicatesofthesameinoculum(Fig.2b)revealedthat4.1%ofthe
4
availablemethanewasobservedinthefullyoxidizedstate(i.e.,as14C-labeleddissolved
inorganiccarbon[DIC])duringthe58-dayincubationandthusdidnotparticipateinthe
back-reaction.Anestimated95.9%oftheinitialmethaneremainedatthetimeofNMR
measurement, meaning that the amount of initial CH D that may have re-formed as
3
CH through a partial or complete back-reaction is between 3.98 and 15.92%. For
4
clarity, these calculations neglect isotope effects and activity by methanogens, the
latter of which was highly endergonic given the lack of added hydrogen or acetate.
These factors can be explored through further experimentation. Reversibility can be
evaluatedinfuturestableisotopeworkby(i)includinga[13C]DICsourceinthewater
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MonodeuteratedMethane
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,

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0
1
FIG3 Schematicdiagramdemonstratingthepotentialfateofmethane-associatedhydrogenatomsin 8

the reverse-methanogenesis pathway. Hydrogen atoms are distinguished by color and superscript b
y
number, and potential exchanges with inter- and intracellular water are shown; asterisks represent
g
location-specific ambiguity. Potentially detectable methane-derived hydrogen atoms (four, occurring
u
throughouttheoxidationpathway)andcarbonatoms(one,requiringfulloxidation)arehighlightedin e
orangeandpurpleboxes,respectively.Shorterback-fluxarrowsreflecttheobservationthatallenzymes s
t
(85)andtheentirepathway(43)havebeenshowntobereversible.Forfiguresimplicity,notallcofactors
orisotopicallydistinctback-fluxproductsareshown.Enzymeabbreviationsareinblack-linedboxes,and
the extended dashed line represents the cell membrane. Fd , oxidized ferredoxin; Fd , reduced
ox red
ferredoxin;MF,methanofuran;H4MPT,tetrahydromethanopterin.
and measuring 13CH and/or (ii) utilizing commercially available multiply deuterated
4
methaneastheinitialheadspaceandquantifyingallpossibleisotopologues.Nonethe-
less,eventheupperboundofpartiallyandreversiblyoxidizedCH Dsuggeststhatthe
3
majority of the D/H change is attributable to reactions indicative of net methane
consumption, if not complete oxidation.
The D/14C tracer ratio in aerobic methanotrophy. In aerobic methanotrophic
cultures,aD/14Ctracerratioof~1.5wasobserved,suggestingthatonaverage,2.67of
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FIG4 Schematicdiagramdemonstratingthepotentialfateofmethane-associatedhydrogenatomsin b
the aerobic methanotrophy pathway. Hydrogen atoms are distinguished by color and superscript e
r
number;asterisksrepresentlocation-specificambiguity.Potentiallydetectablemethane-derivedhydro- 1
genatomsandcarbonatomsarehighlightedinorangeandpurpleboxes,respectively.Mmoenzymes 9
arenotbelievedtoperformreversiblereactions.FDH,formatedehydrogenase;CytCox,oxidizedcyto- , 2
chromec;CytCred,reducedcytochromec;MDH,methanoldehydrogenase;pMMO,particulatemethane 0
monooxygenase;sMMO,solublemethanemonooxygenase. 1
8

b
y

the four methane-derived hydrogen atoms likely enter water-exchangeable products g
u
during the course of a full-oxidation pathway. M. trichosporium is a type II metha- e
s
notroph,amemberoftheAlphaproteobacteriathatusestheserinepathwayforcarbon t
assimilation;M.sedimentiisagammaproteobacterialtypeImethanotrophthatusesthe
RuMPcarbonassimilationpathway(49).ThepathwaydatapresentedinFig.4suggest
that all methane-bound hydrogens are water exchangeable during the catabolic
oxidationofmethanetocarbondioxide.Thus,toachieveaD/14Ctracerratiolessthan
4,asubstantialproportionofmethane-derivedformaldehydewouldneedtoproceed
downtheassimilatorypathway,arequirementthatwaslikelymetgiventhecultures’
increaseincelldensity(Fig.S1).Intriguingly,theD/14Ctracerratiosweresimilarforthe
two cultured organisms despite their distinct metabolic pathways; a similar phenom-
enonofconsistentcarbonconversionefficiencywasrecentlyobservedamongdistinct
aerobicmethanotrophcommunitiesinEnglishriverbeds(50).Previousstudiesofaerobic
methanotrophycomparedratesderivedfromradiolabeledcarbon(14C)-andhydrogen
(3H)-basedapproaches,yieldingunpredictableratiosspanningmultipleordersofmag-
July/August2017 Volume2 Issue4 e00309-17 msphere.asm.org 8
MonodeuteratedMethane
nitude(29,51).Thesefindingswereattributedtodiscrepanciesinincubationtemper-
atures and metabolic priming effects between methods, highlighting the need for
consistentexperimentalparameters.
The oxic incubations of methane seep sediment produced a D/14C tracer ratio of
1.66(cid:3)0.02SE.Giventhattheknownmodesofbiologicalmethaneoxidation—typeI
andtypeIIaerobicmethanotrophyandreverse-methanogenesisanaerobicmethanot-
rophy—boundthisobservedvalue,itappearslikelythattheoxicsedimentincubations
supported a mixture of both aerobic and anaerobic methane oxidation processes.
Aerobicmethaneoxidationlikelydominated,basedonthe~7(cid:2)104-Papartialpressure
ofO andtheproximityoftheD/14Ctracerratiotothatoftheaerobicmethanotrophic
2
cultures,butanoxicnicheslikelyremainedordevelopedintheincubationbottles. D
Specializedapplicationofmonodeuteratedmethane:examiningmethaneac- o
w
tivation under pressure. To demonstrate the utility of the CH D rate measurement n
3
approachinnontraditionalempiricalcontexts,wesoughttoevaluatetheinfluenceof lo
a
in situ pressure on methanotrophic rates of Hydrate Ridge seep sediment microbial d
e
communities. Material collected for microbiological studies of AOM is frequently ob- d

tained from marine settings of various depths that are subjected to distinct and fr
o
substantial pressure regimes (52). Pressure is not always rigorously incorporated into m
microcosmexperiments,thoughevidencesuggeststhatitcanbeanimportantdeter- h
minant of methanotrophic rates (53–56). In addition, some procedural aspects of the tt
p
14CH4protocol,includingheadspacesamplingandfull-volumetransfer,arenotestab- ://
m
lished for use with Mylar bags, which lack gas-tight sampling ports, making the
s
monodeuterated-methaneapproachanappealingalternativeinthiscontext. p
h
Parallel seep sediment incubations were subjected to 0.1 MPa (atmospheric pres- e
r
sure)and9.0MPa(equivalenttoan~900-mdepth).Nitrogenintheformofammonium e
.
(500 (cid:4)M NH Cl) or the amino acid glycine (500 (cid:4)M) was added to assess whether a
4 s
distinct nitrogen sources influenced AOM rates. Methane consumption rates derived m
from heightened D/H ratios are shown in Fig. 5. A significant increase in methane .o
r
consumptionwasobservedunderbothliveconditionsathighpressure,corresponding g
/
to sediment incubated with glycine (samples 1a and 1b) and ammonium chloride o
n
(samples 2a and 2b). Neither live controls lacking CH3D (samples 3a and 3b) nor N
autoclaved,killedcontrols(samples4aand4b)showedactivationofCH3D(seeTableS1 o
v
for sample setup details). The simulation of in situ Hydrate Ridge pressures led to a e
79.5%((cid:3)6.5%SE)increaseinrelativemethaneconsumptionrates.Incubationwith500(cid:4)M m
b
glycineratherthanammoniumathighandlowpressuresresultedinsmallbutconsistent e
rate increases of 12% (cid:3) 4.1% SE, potentially reflecting the energetic and biosynthetic r
1
distinctionbetweenexogenousaminoacidsandunprocessedfixednitrogen. 9
,

Previous reports have found a wide range of different pressure-related effects. 2
0
In a sulfate-coupled AOM bioreactor, pressures were varied from 1 to 8 MPa, and 1
8
sulfideproductionapproximatelytripled(55).Comparedwithtreatmentat0.101MPa,
b
a10.1-MPabioreactorwithsedimentfromEckernfördeBaydemonstratedacessation y

of methanogenesis, a 4-fold increase in methane oxidation rates, and high relative g
u
abundances of ANME-2a/b and ANME-2c (56). A continuous incubation system with e
s
Black Sea microbial mats at 16 MPa measured a 10- to 15-fold increase in methane- t
dependent sulfide generation compared with ambient pressure (57). Methane partial
pressures of 1.1 MPa led to a 5-fold increase in sulfate reduction rates relative to
ambientatmosphericpressurewithHydrateRidgesedimentsdemonstratingmethane-
dependentsulfatereduction(31).WithmethaneseepsedimentfromtheJapanTrench,
however, methane-driven sulfate reduction rates did not correlate with changing
pressure(58).Nauhausetal.(54)suggestedthatthepressure-inducedrateincreasesare
due more to heightened methane solubility and bioavailability than to physiological
effects or biomolecular reordering. Bowles et al. (53) presented a very different per-
spective by showing a 6- to 10-fold AOM rate increase at 10 MPa when methane
concentrationswereheldconstant.Deconvolvingthesetwoinfluencesandhowthey
depend on community composition or physicochemical parameters is feasible with
pressure chamber experiments utilizing monodeuterated methane. Intriguingly, in
July/August2017 Volume2 Issue4 e00309-17 msphere.asm.org 9
Marlowetal.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
s
p
h
e
r
e
.
a
s
m
FIG5 PressureexperimentresultsshowingmethaneconsumptionratesderivedfromaqueousD/Hvalues, .o
withstandarderrorbars,ofseepsedimentsamplesfollowing38-dayincubationswithCHDat9.0MPa r
3 g
(graybars,“b”samples)or0.1MPa(greenbars,“a”samples).Additionaldetailsonsampletreatmentscan /

befoundinTableS1.Valuesarereflectiveofrockandinitialsedimentvolumes(notincludingaddedwater). o
n

N
o
combination with a carbon-based isotopic probe, aqueous D/H measurements might v
e
be used to evaluate predictions that AOM at heightened pressure exhibits decreased m
b
back-fluxes (39, 59) and, if so, whether the barrier occurs after complete or partial e
oxidation. More broadly, understanding the relative contributions of environmental and r
1
physiologicaleffectstomethaneoxidationwillhelpconstrainmethanefluxesacrossalarge 9
,
envelopeoftheplanet’smethanotrophicallyactivezones. 2
0
Using monodeuterated methane in experimental investigations. Based on 14CH 1
4
8
groundtruthexperimentswithaerobicmethanotrophiccultures,oxicseepsediment,and
b
anoxicseepsediment,aswellastheproof-of-conceptpressurizedexperiments,webelieve y

thatthemonodeuterated-methaneapproachtomethaneoxidationratemeasurement g
u
isausefuladditiontoabiogeochemist’stoolset(Table2).Comparedwithradiolabel e
s
approaches (14CH , [3H]CH , 35SO 2(cid:4)), the method requires less safety-oriented plan- t
4 4 4
ning and is procedurally simpler, more affordable, and less susceptible to hydrogen-
associatedisotopefractionationeffects(relativeto3H).Ourresultsalsosuggestthatthe
monodeuterated-methanetechniqueappearstobeamoreprecisemethodbasedon
standarderrorcalculations(Fig.1and2;TableS2).Directcomparisonsofenvironmental
incubationsarecomplicatedbythemicroheterogeneityofseepsettings(60,61),aswell
as the fact that different aliquots of the same initial material were used in our
experiments. Analysis of culture-based and seep substrate experiments reveals that
standard errors from CH D-derived values were between 1.56 times lower (M. tricho-
3
sporiumcultures)and4.76timeslower(anoxicseepsubstrateincubations)thanthose
derivedfrom14CH -basedvalues(TableS2).
4
Becausethemonodeuterated-methanemethodfocusesonmethane-boundhydro-
gen atoms, it offers information about methanotrophic systems that is different from
July/August2017 Volume2 Issue4 e00309-17 msphere.asm.org 10