long-term manipulations of intact microbial mat communities in a greenhouse collaboratory:...

ASTROBIOLOGYVolume 2 Number 4 2002copy Mary Ann Liebert Inc

Research Paper

Long-Term Manipulations of Intact Microbial MatCommunities in a Greenhouse Collaboratory Simulating

Earthrsquos Present and Past Field Environments

BRAD M BEBOUT1 STEVEN P CARPENTER1 DAVID J DES MARAIS1

MYKELL DISCIPULO2 TSEGEREDA EMBAYE2 FERRAN GARCIA-PICHEL3

TORI M HOEHLER1 MARY HOGAN4 LINDA L JAHNKE1 RICHARD M KELLER5

SCOTT R MILLER6 LESLIE E PRUFERT-BEBOUT1 CHRIS RALEIGH7

MICHAEL ROTHROCK3 and KENDRA TURK4

ABSTRACT

Photosynthetic microbial mat communities were obtained from marine hypersaline salternponds maintained in a greenhouse facility and examined for the effects of salinity varia-tions Because these microbial mats are considered to be useful analogs of ancient marinecommunities they offer insights about evolutionary events during the 3 billion year timeinterval wherein mats co-evolved with Earthrsquos lithosphere and atmosphere Although photo-synthetic mats can be highly dynamic and exhibit extremely high activity the mats in the pre-sent study have been maintained for 1 year with relatively minor changes The major groupsof microorganisms as assayed using microscopic genetic and biomarker methodologies areessentially the same as those in the original field samples Field and greenhouse mats weresimilar with respect to rates of exchange of oxygen and dissolved inorganic carbon across thematndashwater interface both during the day and at night Field and greenhouse mats exhibitedsimilar rates of efflux of methane and hydrogen Manipulations of salinity in the water over-lying the mats produced changes in the community that strongly resemble those observed inthe field A collaboratory testbed and an array of automated features are being developed tosupport remote scientific experimentation with the assistance of intelligent software agentsThis facility will permit teams of investigators the opportunity to explore ancient environ-mental conditions that are rare or absent today but that might have influenced the early evo-lution of these photosynthetic ecosystems Key Words Microbial matmdashBiogeochemistrymdashBiomarkers Astrobiology 2 383ndash402

383

1Exobiology Branch and 5Computational Sciences Division NASA Ames Research Center Moffett Field Califor-nia

2SETI Institute Mountain View California3Department of Microbiology Arizona State University Tempe Arizona4Institute of Marine Sciences University of California Santa Cruz California6Department of Genetics North Carolina State University Raleigh North Carolina7Montana State University Bozeman Montana

INTRODUCTION

MODERN MICROBIAL MATS are thought to be ex-tant representatives of Earthrsquos most ancient

ecosystems (Walter 1976) Geochemical evidenceof the existence of photosynthetic microbial matsand their mineralized counterparts stromatoliteshas been identified in rocks as old as 30 Ga(Beukes and Lowe 1989) As a living repositoryof genetic physiological isotopic and biogeo-chemical information on the co-evolution of aplanet and the only known biosphere modernmicrobial mats are invaluable objects of studyModern microbial mat studies have provided im-portant insights on rates of biological activity(Revsbech et al 1983 Canfield and Des Marais1993 Des Marais 1995) genetic diversity (Wardet al 1990 Garcia-Pichel et al 1998 Nuumlbel et al2001) stable isotopic fractionation (Schidlowski1988 Des Marais and Canfield 1994) and organic(Boon 1984 Ward et al 1985) and atmospheric(Visscher and Van Gemerden 1991 Visscher andKiene 1994 Hoehler et al 2001) biomarkers aswell as minerals (Reid et al 2000) that have beenused to interpret the fossil record of these com-munities over geologic time

Photosynthetic microbial mats are self-sustain-ing complete ecosystems in which light energyabsorbed over a diel (24-h) cycle drives the syn-thesis of spatially organized diverse biomass Mi-croorganisms with tightly coupled metabolismsin the mat catalyze transformations of carbon ni-trogen sulfur and metals Radiant energy fromthe sun sustains oxygenic and anoxygenic pho-tosynthesis which in turn provides chemical en-ergy (as organic photosynthates and oxygen) tothe rest of the community When oxygenic pho-tosynthesis ceases at night the upper layers of themat become highly reduced and sulfidic (Joslashr-gensen et al 1979) Counteracting gradients ofoxygen and sulfide shape the chemical environ-ment and provide daily-contrasting microenvi-ronments separated on a scale of a few millime-ters (Revsbech et al 1983 Revsbech andJoslashrgensen 1986) While photosynthetic bacteriadominate the biomass and productivity of themat many aspects of the ecosystemrsquos emergentbehavior may ultimately depend on the associ-ated nonphotosynthetic microbial communitiesincluding the anaerobes Additionally transfor-mation of photosynthetic productivity by the mi-crobial community may contribute diagnosticldquobiosignaturerdquo gases that could represent search

targets for remote spectroscopic life detection ef-forts [eg Terrestrial Planet Finder (Des Maraiset al 2002)] To understand the overall structureand function of mat communities it is thus criti-cal to determine the nature and extent of the in-teractions between phototrophic and nonphoto-synthetic microorganisms including anaerobicmicroorganisms

Compared with their historical distributionand abundance microbial mats are today con-fined to a relatively restricted number of habitatsWell-developed modern photosynthetic mats oc-cur in both high-salinity and high-temperatureenvironments This restricted distribution isthought to be due primarily to the higher rates ofgrazing in more moderate modern environments(Garret 1970) although alternative hypotheseshave been forwarded In any event the limitedgeographical distribution of mats on the present-day Earth also serves to limit their usefulness asanalogs of similar ecosystems growing in morediverse ancient environments Environments thatare not well represented on the modern Earth butthat have been extremely important over evolu-tionary time include marine environments hav-ing low concentrations of sulfate andor free oxy-gen (Holland 1984 Canfield and Teske 1996)

The effects on microbial processes of variousenvironmental parameters important in Earthrsquospast (such as sulfate and oxygen concentrations)may be studied in culture However the emer-gent properties of complex microbial ecosystemsare not necessarily predictable or understand-able on the basis of these types of experiments(Wilson and Botkin 1990) Either some microbialprocesses do not occur in culture [eg anaerobicmethane oxidation (Reeburgh 1980) and sulfatereduction under aerobic conditions (Canfield andDes Marais 1991)] or they occur at rates vastlydifferent than rates observed in nature In addi-tion relatively few (1) of the total number ofmicrobes present in nature are available in cul-ture (Ward et al 1990 Amann et al 1995) A mi-crobial mat is a highly complex assemblage of organisms possessing many different modes ofmetabolism all of which are interacting with eachother at some level in beneficial andor compet-itive ways The end products of the metabolismof one group of organisms frequently constitutethe substrates of another These considerationsargue for an alternative approach in which theentire microbial ecosystem is subjected to exper-imental manipulation

BEBOUT ET AL384

To attain several key objectives in astrobiologywe must understand better the mechanisms andrates at which trace gases are produced and con-sumed by microbial ecosystems As dominantcomponents of our biosphere for at least 2 billionyears of its 35 billion year history microbialmats played a pivotal role in shaping the com-position of Earthrsquos early atmosphere including itseventual oxygenation NASArsquos present searchstrategy for the detection of life on extrasolarplanets includes the observation and interpreta-tion of spectral features of biogenic gases in re-mote atmospheres (Des Marais et al 2002) How-ever the current strategy relies principally uponthe detection of O2 and thus might preclude thedetection of life that might resemble our own pre-oxygenated biosphere for as much as 2 billionyears of its early existence Clearly the rates ofproduction and consumption of reduced gases bymicrobial communities over geologic time mustbe better constrained to enhance our search forlife elsewhere The use of simulation facilitiessuch as the greenhouse described here will aug-ment that endeavor

We report here our attempts to maintain intactmodern photosynthetic microbial mats under ap-proximate in situ conditions and to monitor theresponses of these mats to experimental manipu-lations We have constructed a facility in whichphotosynthetic microbial mats can be maintainedover long periods of time using natural illumi-nation and realistic water flow and other envi-ronmental conditions We describe here the re-sults of our first experiment in which we usedthis facility to manipulate the salinity of wateroverlying the mats As variations in salinity alsooccur in the natural environment where thesemats were collected we are able to assess the de-gree to which our experimental facility can re-produce naturally occurring phenomena Futureuses of this experimental system in which con-ditions not presently found in the modern envi-ronment will be simulated are discussed

MATERIALS AND METHODS

Field site

Microbial mats were collected in salterns man-aged by the salt-producing company Exporta-dora de Sal SA de CV located on the PacificOcean side of the Peninsula of Baja California Sur

Mexico This field site was previously describedin detail (Des Marais 1995) Briefly lagoon wa-ter having a salinity of 40permil (parts per thou-sand) is pumped through a series of concentrat-ing ponds and then into crystallizing ponds Thesalinity of the water is gradually raised until itbecomes saturated with respect to sodium chlo-ride at which point the brine is pumped out ofthe crystallizing pond and the salt is harvestedExtensive and well-developed microbial mats oc-cur in concentrating areas in which the salinity isbetween 65permil and 130permil

Microbial mats were collected on May 27 2000from two localities Area 4 near the dike sepa-rating Area 4 from Area 5 (27deg4134509N113deg5502709W) and Area 5 near the dike sepa-rating Area 5 from Area 6 (27deg4441409N113deg5479509W) At the time of the collection thesalinities of Area 4 and Area 5 were 90permil and120permil respectively Microbial mats in both ofthese locations are 5 cm thick and well lami-nated The dominant cyanobacterium of the matcommunity is Microcoleus chthonoplastes Sectionsof mat 20 3 25 cm were cut and removed fromthe bottom of the concentrating pond by diversusing metal spatulas and were immediatelyplaced into tight-fitting black acrylic trays In thisway exposure of the deeper anaerobic layers ofthe mats to air and light was minimized Matswere covered with relatively high-salinity water(180permil) overnight in an effort to slow overallmetabolic rates for transport The trays contain-ing the mats were then transported by vans backto our laboratory in larger plastic trays coveredby tight-fitting plastic film In this way the matswere kept moist but not covered with water andwere exposed to some natural light over the 48h required for the relocation Although six matsfrom Area 5 and 12 mats from Area 4 were main-tained in the greenhouse throughout the entireexperiment results from only the Area 4 matswill be presented here

Greenhouse facility

Upon arrival at Ames Research Center themats were transferred to a greenhouse modifiedfor these experiments by replacing the originalglass with UV-transparent OP-4 acrylic (trans-mission in the UV-B UV-A and visible 90 inthe greenhouse) Mats were placed onto a spe-cially built table (Fig 1) having six clear acrylicflow boxes (150 3 22 cm) each flow box holding

A MICROBIAL MAT GREENHOUSE COLLABORATORY 385

BEBOUT ET AL386

FIG 1

Microbial mats pos

itioned

on the table used in the latter half of the ex

perim

entThr

ee m

ats are incu

bated in

each of the

six acrylic flow box

es T

he po-

sition

s of the

reservo

irs an

d tem

perature con

trol equ

ipmen

t can

be seen

( B) as w

ell a

s the instrumen

t pa

ckag

e ( A

) presen

t on the remotely op

erab

le xyz

table Pho

tocred

it D

an A

ndrews

three trays of mat The depth of the water in theflow boxes can be adjusted with standpipes Forthis experiment the depth of the water overlyingthe mats was 3 cm therefore the total volumeof the water in each flow box at any one time was10 L

Brine used for the experiment was collectedfrom one of the higher-salinity (130permil) concen-trating areas that had retained its original com-plement of seawater solutes and diluted withdeionized water to the appropriate salinity Matswere arbitrarily assigned to one of two salinitytreatments on the table Three of the flow boxesrecirculated brine collected from the field sitewhich was diluted to the in situ salinity of 90permil(hereafter referred to as the NORMAL salinitytreatment) The other three flow boxes recircu-lated brine having an elevated salinity of 120permil(hereafter referred to as the HIGH salinity treat-ment) Water was recirculated from a single reser-voir holding 60 L of brine through the three in-terconnected flow boxes that constituted each ofthe two salinity treatments

Environmental control

The flow boxes assigned to each of the salinitytreatments were interspersed on the table so thatthe treatments alternated (eg NORMAL HIGHNORMAL etc) Salinity was maintained at theselevels over the course of the experiment with ad-ditions of deionized water to replace losses dueto evaporation Temperature control of the air inthe greenhouse was achieved with a combinationof an evaporative cooler and propane heater Precise temperature control of the water being re-circulated over the mats was achieved by circu-lating temperature-controlled water through tita-nium heat-exchanging coils submerged in thereservoirs Normal in situ daily water columntemperature variations were simulated in thegreenhouse by (1) controlling the temperature in-crease (attributable to solar heating) during thedaytime to stay below the maximum temperatureobserved in situ and (2) turning off temperaturecontrol at night to allow the water temperaturein the flow boxes to decrease slowly with the de-crease in greenhouse air temperature This simu-lation of diel temperature variations while not always completely faithful to the precise near-si-nusoidal pattern of temperature change observedin situ (owing to the simplicity of the apparatus)reproduced the normal range of temperatures ob-

served in situ and the approximate duration oftime at each temperature The similarity betweengreenhouse and in situ temperatures was docu-mented by an extensive set of measurements per-formed the year after this experiment was runHowever since no changes had been made to theapparatus since the time of the experiments re-ported here this comparison should be valid forthe period of time discussed here Using sub-mersible data loggers (StowAway TidbiT OnsetComputer Corp Bourne MA) recording at 10-min intervals from June 2001 through September2001 the average temperature in the salterns was228 6 199degC (mean 6 SD for n 5 16914)whereas water temperatures in the greenhouseover the same interval of time were 203 6 262degC(n 5 16191) Water column salinity in the flowboxes was determined using a refractometer(Delta model Bellingham 1 Stanley TunbridgeWells UK) which was corrected for temperatureeffects at each reading Light [as photosyntheti-cally available radiation (PAR)] was monitoredand averaged at 10-min intervals using a datalogger coupled to a terrestrial quantum sensor(LI-1000 and LI-192SA LI-COR Inc LincolnNE) An underwater light sensor (LI 192SA LI-COR Inc) was used to measure irradiance at themat surface in situ on several field trips The irradiance at the mat surface expressed as a per-centage of the irradiance incident upon the sur-face of the pond is comparable with measure-ments of irradiance in the greenhouse

Greenhouse oxygen microelectrode measurements

Oxygen concentrations within the microbialmats were monitored at various times through-out the course of the experiment using oxygenmicroelectrodes We used Clark-type microelec-trodes incorporating guard cathodes (model 737-GC Diamond General Development Corp AnnArbor MI) In the initial phase of the experimentmicroelectrodes were positioned using motorizedmicromaniupulators mounted to bases placedacross the flow boxes Halfway through the ex-periment the microsensors were transitioned toa positioning system using a robotic xyz posi-tioning system installed over the flow boxes (Fig1) In all cases the positioning was controlled byand data were acquired with custom softwarewritten in the LabVIEW programming environ-ment (National Instruments Corp Austin TX)

In general the electrodes were advanced


within the mat at 100-mm steps Oxygen micro-electrode signal output was calibrated to oxy-gen concentrations using a two-point calibra-tion Because the water circulating through theflow boxes was constantly aerated through theaction of the pumps and through contact withthe atmosphere across a large surface area ofwater the electrode current at any point in thewater overlying the mats was taken to be equalto the current produced in air-saturated waterat that particular temperature and salinity Theexact value of this oxygen concentration canthen be identified using published values (Sher-wood et al 1991) The second point in the cali-bration the output of the electrode at an oxy-gen concentration of 0 was provided by theasymptotic minimum of electrode currentwithin the permanently anoxic lower parts ofthe mat

In situ microelectrode measurements

Measurements of in situ concentrations of oxy-gen within the microbial mat were made usinga diver-operated microprofiler (UnisenseAringrhus Denmark) at 100-mm intervals Calibra-tions of the oxygen electrode were performed asdescribed for greenhouse microelectrodes(above) As the ponds are well mixed duringwindy periods of the day electrode readings inthe well-mixed portions of the water columnabove the mat surface were taken to representair-saturated values at a given temperature andsalinity In the morning bottom water can below in oxygen and therefore was not used forelectrode calibrations rather readings closer tothe airndashwater interface were used to calibrate theelectrodes In all cases measurements of the as-ymptotic minimum in electrode current withinthe permanently anoxic lower parts of the matwere used as the zero oxygen concentration cal-ibration point

Community composition analyses

Light microscopy Microbial community compo-sition was followed throughout the course of theexperiment using a variety of techniques First-order observations about the community weremade using light microscopy (Nikon MicrophotFXA Nikon USA) We did not employ quanti-tative microscopy techniques and so these ob-servations will be referred to as ldquononquantita-tiverdquo microscope observations

Cyanobacterial 16S rRNA gene fingerprints bypolymerase chain reaction (PCR)denaturing gradientgel electrophoresis (DGGE) Cores (10 mm in di-ameter) were taken from the microbial mats onSeptember 15 2000 and the photic zone (top 3mm) was aseptically removed The samples werehomogenized in a Tenbroeck tissue grinder(VWR Scientific Products Brisbane CA) Cell ly-sis and DNA extraction were performed as pre-viously described (Nuumlbel et al 1997) In short thehomogenized samples were frozen and thawedrepeatedly followed by incubation with 1sodium dodecyl sulfate and proteinase K DNAextraction was performed using hexadecyl-methylammonium bromide in conjunction withphenolchloroformisoamyl alcohol (25241 byvolume) followed by isopropyl alcohol precipi-tation Cyanobacterialplastid-specific PCR am-plification was carried out using the oligonu-cleotide primers CYA359F (with a 40-nucleotideGC-rich sequence at the 59 end) and CYA805Rwhich were used for specific amplification of 16SrRNA genes from cyanobacteria and plastids(Nuumlbel et al 1997) Each 50-mL PCR mixture con-tained 5 mL of 103 Takara Ex Taqtrade PCR buffer(PanVera Corp Madison WI) 4 mL of Takara de-oxynucleotide triphosphate mixture (25 mMeach) 50 pmol of each primer 200 mg of bovineserum albumin 10 mL of 53 Eppendorf TaqMas-tertrade PCR-enhancer (Brinkmann InstrumentsInc Westbury NY) and 15 ng of DNA extractThermocycling was performed using a Bio-RadiCyclertrade thermal cycler (Bio-Rad LaboratoriesHercules CA) After an initial denaturation at94degC for 5 min (hot start) 25 units of Takara ExTaq DNA polymerase was added to the reactionat 80degC Thirty-five cycles of 1 min each at 94degC(denaturation) 60degC (annealing) and 72degC (ex-tension) were performed and the reaction fin-ished with a final extension at 72degC for 9 minGels were imaged using a Bio-Rad Fluor-Strade Mul-tiImager system For DGGE band sequencingeach band was excised using a sterile scalpel andDNA was allowed to diffuse out for $3 days at4degC in 50 mL of 10 mM Tris buffer One micro-liter of the solution was PCR-amplified using thesame primers reaction mixture and thermocy-cling conditions A kit was used to purify PCRproduct (Qiagen Inc Valencia CA) and 150 ngwas commercially sequenced in two separate re-actions (59 to 39 and 39 to 59) Complementary se-quences were matched aligned and edited usingSequence Navigator (Applied Biosystems Foster

BEBOUT ET AL388

City CA) and submitted to the BLAST search en-gine (National Center for Biotechnology Infor-mation wwwncbinlmnihgov) for phylogeneticmatching

Lipid biomarker analysis Gas chromatographicprofiles of fatty acids (FA) as fatty acid methylesters (FAME) were used to monitor changes ingroup-specific biomarker compounds in the sur-face layers of the greenhouse and field mat com-munities A section (25 cm2 and 1 cm in depth)was removed from a mat in each of the six flowboxes after 4 months dissected frozen andfreeze-dried A similar-sized section was re-moved from freshly collected mats at the field sitein Guerrero Negro in both December 1998 andJune 2001 The sections from the field-collectedmats were dissected shortly after collectionfrozen and returned to NASA Ames ResearchCenter where they were freeze-dried andprocessed

Field and greenhouse mat communities weredissected and processed as follows The floccu-lent orange material on the surface of the mat wasremoved by scraping the surface with a spatulaAn exposed leathery layer of the mat containingextremely high densities of the filamentouscyanobacterium M chthonoplastes (hereaftercalled the Microcoleus layer) 1ndash15 mm thickwas then separated using a razor blade from thedarker-colored gelatinous material that composesthe undermat The freeze-dried mat layers wereground using a glass mortar and pestle Totallipid extracts were prepared by a Bligh and Dyersingle-phase modification (Jahnke et al 1992)Multiple extractions of the cell material recoveredby centrifugation were done until no further pig-ment coloration was apparent (normally four orfive times) A polar fraction (phospholipids andsome glycolipids) was prepared by precipitationin cold acetone The remaining acetone-solublelipids were separated by thin-layer chromatogra-phy using a methylene chloride solvent system(Jahnke et al 1992) The remaining glycolipidspresent in the origin zone (0ndash1 cm) of the thin-layer chromatography plate and the sterols(25ndash35 cm) were recovered from the silica gelby Bligh and Dyer extraction The polar lipid frac-tions were pooled and FAME were prepared bya mild alkaline methanolysis procedure (Jahnkeet al 2001) Gas chromatographyndashmass spec-trometry analyses of FAME or sterols as tri-methylsilyl esters were performed using an HP

5890 gas chromatograph equipped with a JampWDB-5ms (30 m 3 025 mm 025-mm film) capillarycolumn and an HP 5971 mass-selective detectorIdentification of individual compounds was by acombination of standard compounds publishedspectra and relative retention times FAME werequantified using an Agilent 6890 gas chromato-graph equipped with a flame ionization detectorand HP-5 (30 m 3 032 mm 025-mm film) capil-lary column Methyltricosanoate (FAME) wasused as an internal standard Chromatographswere programmed to operate for FAME from60degC to 120degC at 10degCmin then at 2degCmin from120degC to 280degC Total organic carbon (TOC) andstable isotope composition (d13C and d15N) forbiomass was determined using a Carlo Erba CHNEA1108 elemental analyzer interfaced to a Finni-gan Delta Plus XL isotope ratio mass spectrome-ter (Jahnke et al 2001)

Fluxes of oxygen estimated by microsensor measurements

Net fluxes of oxygen across the mat surface (J)were calculated according to Fickrsquos first law ofdiffusion (Berner 1980)

J 5 2fDs (shyCshyz)

where shyCshyz is the change in oxygen concentra-tion with depth near the matndashwater interface in(mMmm) Ds is the sediment (mat in this case)diffusion coefficient (cm22 s21) and f is theporosity (volume of watertotal volume of waterand sediment) fDs within the mat was estimatedas follows For each salinity treatment a high-res-olution (50-mm step) vertical profile through thediffusive boundary layer (DBL) and the mat wasmade D0 (the free solution diffusion coefficient)in the DBL was calculated based on salinity andtemperature of the water column at the time ofthe profile using the equations of Li and Gregory(1974) Since f in water is 1 by definition fD0 inthe DBL is known fDs in the mat was then esti-mated as (shyCshyz) fD0 (in DBL)(shyCshyz) (in mat)Mat fDs estimated in this way was similar topublished values (Glud et al 1995 Wieland et al2001) 1025

Fluxes of oxygen dissolved inorganic carbon(DIC) methane and hydrogen

Fluxes of oxygen (O2) and DIC between themats and the overlying water were measured us-


ing glass benthic flux chambers that allowedunimpeded illumination of the surface with noappreciable (1degC) heating of the chamber Thechamber design and operation follow those ofCanfield and Des Marais (1993) Briefly eachglass chamber (covering an area of mat 0019 or0012 m2) was fitted with a central stirring pad-dle and two sampling ports with septa The glasspaddle rotated at a constant rate of 45 rpmChamber incubations were conducted for full 24-h daily cycles taking samples by syringe every 6h When deployed at sunrise each chamber wasinjected with 100 mL of pure nitrogen (N2) gasto create a headspace to sample at noon and be-fore sunset for acquisition of gas samples (O2CH4 H2 etc) At sunset each chamber was re-deployed and injected with 70 mL of N2 and 30mL of O2 to accommodate O2 demand during thenight Samples were taken at midnight and be-fore sunrise Dissolved and headspace O2 valueswere measured using a Clark-style electrode withan internal reference mounted inside a 21-gaugesyringe needle (model 768 Diamond General De-velopment Corp) and a gas chromatograph re-spectively The gas chromatograph (ShimadzuGC-14A) was fitted with a thermal conductivitydetector and a CTRI column (Alltech AssociatesDeerfield IL) held at 25degC Helium at a flow rateof 30 mLmin was the carrier gas DIC samples(5 mL) were filtered and analyzed in triplicate us-ing a ldquoflow injection analyzerrdquo (Hall and Aller1992) where a sample was injected into an acid-ified aqueous solution that subsequently flowspast a gas-permeable membrane Carbon dioxidefrom the acidified DIC sample traverses thismembrane and enters an alkaline solution thatsubsequently flows through a conductivity de-tector

Hydrogen partial pressures

While actively photosynthesizing mats gener-ate small (1-mL) bubbles that are retained at themat surface with residence times of tens of min-utes These bubbles were collected by means of a3-mL plastic syringe with a plastic pipette tiplodged in the luer fitting (flaring outward to forma collecting funnel) When filled with water andsubmerged this apparatus allows individualbubbles to be picked from the mat surface with-out atmospheric contamination and pooled to avolume of 10ndash25 mL The pooled bubble gas is immediately subsampled with a gas-tight volu-

metric syringe and quantified by gas chromatog-raphy with HgO-reduction detection (modelRGA-3 Trace Analytical Sparks MD) The timefrom sample collection to analysis was generally30 s Partial pressures in bubble samples weredetermined by comparison with standards pre-pared by serial dilution of pure H2 into N2(Hoehler et al 1998) Precision and accuracy aretypically about 65 for sample volumes in therange of 10ndash25 mL

RESULTS

The greenhouse facility as a simulation of the natural environment

Microbial mats kept in the greenhouse facilityretained an overall appearance remarkably simi-lar to that of freshly collected mats In particularno evidence of the mat ldquogreeningrdquo in whichmotile cyanobacteria migrate to the surface of themat (Bebout and Garcia-Pichel 1995) was ap-parent During the first few weeks of greenhouseincubation there was a notable increase in theabundance of loosely attached microbial ldquoflocrdquo atthe surface of the mats as well as the develop-ment of small dark green spots containing largenumbers of cyanobacterial filaments in somemats However after the first 2 months the loosefloc disappeared and the mat surface was smoothand homogeneous in appearance once again Ex-tensive but nonquantitative microscopic obser-vations revealed no major changes in communitycomposition More specifically the major popu-lations of cyanobacteria did not seem to changeand M chthonoplastes remained the dominantphototroph in all of the sections of mat charac-terized microscopically

Molecular analyses based on 16S rRNA genesthrough DGGE fingerprinting were consistentwith the absence of significant shifts in the com-munity The banding pattern of DNA extractedfrom the greenhouse mats was essentially identi-cal to that of freshly collected mats (Fig 2) Twomajor bands were present in all of the mat sam-ples (Band b and Band c) In the greenhouse treat-ments (Fig 2A) Band c represented 84 of the ob-served cyanobacterial diversity gauged as apercentage of total PCR amplificate while Band brepresented 10 These results are consistent withthe structure of field samples (Fig 2B) where Bandc represented 79 of the observed diversity with

BEBOUT ET AL390

Band b accounting for 18 Two other minorbands were found but not in all treatments Banda was present in three of the six greenhouse sam-ples and in the field samples accounting for 3and 2 of the observed diversity respectivelyBand d was found in all six greenhouse samplesat much lower levels (08) and accounted for12 of the observed diversity in the field samplesBLAST similarity searches for the sequencedbands (380 bp in length) were performed Bandb had no close matches to any cultured cyanobac-terium (only 89 similar to Oscillatoria amphigran-ulata strain 23-2 a typical distance between bacte-rial genera) but did match an unidentifieduncultured cyanobacterium from a microbial matin Solar Lake Sinai Peninsula Egypt (Abed andGarcia-Pichel 2001) with 94 similarity TheBLAST results for the remaining two bandsyielded very high similarity (99) to known cul-tured cyanobacteria Band c matched the Mchthonoplastes cluster (Garcia-Pichel et al 1996) andBand dmatched Euhalothece sp strain MPI 95AH13(Garcia-Pichel et al 1998) Though we were un-able to sequence it Band a most likely representsa diatom plastid (according to previous researchand band placement in the denaturant gradient)

The compositions of the total esterified FA(TEFA) in natural and greenhouse mats were inmost respects similar for both the surface and un-derlying Microcoleus layer (Table 1) The amountsof TEFA recovered for the individual mat sam-ples were reasonably consistent The majority ofthis TEFA consisted of n-160 n-161 and n-181which together accounted for 60ndash77 of all FASmaller amounts of other straight-chain saturatedand unsaturated FA (ie n-140 n-170 n-180 n-171 n-201 n-202) together with various termi-nally branched FA (i-140 i-150 ai-150 i-160 i-171 i-170 ai-170) were present in relativelyuniform amounts in all samples However sub-tle differences for TEFA compositions in the sur-face layer and underlying Microcoleus layer of nat-ural mats which were also apparent in thegreenhouse mats even after 4 months of experi-mental treatment (Fig 3) were noted The sur-face layer tended to have higher levels of iso-151and iso-161 than the Microcoleus layer In naturalmat these two iso-FA were composed primarily(90) of D4 positional isomers previously iden-tified in field-collected Nostoc spp (Potts et al1987) All surface layers (in both natural andgreenhouse mats) also contained small amounts


FIG 2 DGGE fingerprints of PCR-amplified cyanobacterial 16S rRNA genes of the six flume treatments (A) andfield samples (B) Arrows indicate bands used for further analysis of the fingerprints Bands b c and d were excisedre-amplified and sequenced

BEBOUT ET AL392

TABLE1

COM

POSITIO

NOFTW

ON

ATURALM

ATS

FROM

THEA

REA4 M

ATD

ISSE

CTED

ATG

UERRERON

EGRO

AND

OFM

ATSC

OLLECTED

FROM

THESA

MEA

REAA

FTERM

AIN

TENANCE

INTHEFL

UM

ESY

STEM

FOR

$4 M

ONTHS

1298 P4n51

90permil flume

120permil

flume

601 P4n51

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Ester-linke

d FA (mg g2

1TOC)

17760

0000

31800

0000

25670

0000

023

380

0000

26520

0000

027

850

000

30870

0000

022

090

000

CN R

atio

980

0000

ND

940

0000

0920

0000

12100

0000

0960

000

ND

ND

d13C (permil)

210

72

6 004

210

23

6 028

2901

6 005

210

59

6 013

2970

6 017

210

50

6 01

2802

6 007

210

56

065

d15N (permil

)2013

6 04

ND

2127

6 006

2092

6 038

2023

6 035

2026

6 018

ND

ND

Error estim

ates sho

wn indicate instrumen

tal precision Sam

ples labe

led N

D w

ere too sm

all to permit a reliable an

alys

is

1 Collected

in D

ecem

ber 19

98 and

June

200

1 from

the

field site Pon

d 4 nea

r Area 5

FIG 3 Relative distribution of various FA characteristic of the surface layer (A) andor the underlying Microcoleus layer(B) for two natural mats collected from Pond 4 near Area 5 in June 2001 (black bars) and December 1998 (gray bars) andfor similar mats maintained for $4 months in flumes at salinities of 90permil (hatched bars) or of 120permil (dotted bars)

BEBOUT ET AL394

of anteiso-171 which was not detected in the Mi-crocoleus layer The most striking difference in thesurface layer however was the presence of rela-tively large amounts of several polyunsaturatedFA (PFA) (n-162 n-182 n-183 n-184 n-204and n-205) The more highly unsaturated C20 FAare generally considered diatom biomarkers (Co-belas and Lechado 1989) The lower Microcoleuslayer was also considerably enriched in severalFA Most pronounced were a 10-methyl-160 as-sociated with some sulfate-reducing bacteria(Dowling et al 1986 Kohring et al 1994) and acy-190 that has been used as a biomarker forThiobacillus spp (Kerger et al 1986) In additionmicrobial mats maintained in the greenhouse re-tained the carbon and nitrogen stable isotopiccomposition of recently collected mats (Table 1)

Measurements of rates of biogeochemical cy-cling in greenhouse mats were similar to ratesmeasured on freshly collected mats (Table 2) Pro-files of oxygen concentration within the green-house mats resembled those obtained in situ inboth absolute concentration and distribution withdepth (data not shown) Oxygen fluxes to the wa-ter column calculated from the concentrationgradients of these profiles agreed with oxygenfluxes calculated from in situ profiles over a widerange of light intensities (Fig 4) Similarly netrates of O2 and DIC production and consumptionas determined using flux measurements weresimilar to those measured in freshly collectedmats (Table 2) after allowances were made for therelationship between rates of photosynthesis andseasonally-dependent levels of illumination inthe greenhouse Fluxes of methane from thegreenhouse mats were reduced to 60 of thevalue measured in freshly collected mats andwere essentially equal day and night (Table 2) Ingas bubbles collected at the mat surface H2 par-tial pressures in the low-salinity control mats were very similar to those observed in the field(Table 2)

The greenhouse as a facility for experimental manipulations

Over the course of the experiment dramaticdifferences in the appearance of mats maintainedat in situ (NORMAL 5 90permil) and elevated(HIGH 5 120permil) salinities became apparentMats held at high salinities assumed a much moreorange color than those incubated at lower salin-ities A change in color can result from physio-

logical (photopigment) changes in an unchangedcommunity or population shifts It is known thatincreased salinity promotes increasingly oxida-tive conditions (Garcia-Pichel et al 1999) and thatM chthonoplastes can respond to increased salin-ity by increasing the cellular levels of carotenoid(Loacutepez-Corteacutes 1990) It is important to note how-ever that in natural field mats yellow-orangemats are typically associated with the dominanceof cyanobacteria from the Halothece cluster

Nonquantitative microscopic observations re-vealed an increase in the abundance of unicellu-lar cyanobacteria that resembled the Halothecetype at the surface of the HIGH salinity mats rel-ative to those maintained at NORMAL salinityInitial DGGE analyses supported this observa-tion with a faint novel band attributable toHalothece which appeared in samples collectedfrom all three HIGH salinity greenhouse matsamples (data not shown) However these initialDGGE samples had been compromised by an ac-cidental thawing The second set of samplestaken at the same time but not thawed beforeDNA extraction did not show this band (Fig 2)Rather this second set of samples showed simi-lar fingerprints for all greenhouse samples re-gardless of the treatment Upon further investi-gation we found that thawing and refreezing ofmat samples before DNA extraction caused thepreferential breakage of large-celled M chthono-plastes and the subsequent degradation of itsDNA Because M chthonoplastes provides the ma-jority of template for PCR amplification DGGEanalyses of samples that had been thawed yieldeda high-resolution fingerprint of the communitymembers present at low numbers It was only insuch analyses that we could detect communityshifts in the form of new bands appearing in allHIGH salinity greenhouses (data not shown) Se-quencing and phylogentic analyses of these novelbands revealed that the new community mem-bers belonged to the Halothece cluster of ex-tremely halotolerant unicellular cyanobacteria(Garcia-Pichel et al 1998) Thus we may havebeen witnessing an incipient community shiftbut not a full replacement of the principalcyanobacterial populations

Some differences between the NORMAL andHIGH salinity mats and between greenhouseand freshly collected mats were apparent in theFA composition An increase in the diatom pop-ulation in the HIGH salinity mats was readilydocumented by increased amounts of two diatom


TABLE2

MEASU

REM

ENTS

OFBIO

GEOCHEM

ICALPROCESS

ES

INFR

ESH

LYC

OLLECTEDM

ATS

AND

MATSM

AIN

TAIN

ED

INTHEG

REENHOUSE

FACIL

ITY

ATNORMAL A

NDHIG

H S

ALIN

ITIE

S

Greenhouse-maintained mats

Freshly collected mat

May 19ndash20 2000

NORMAL

HIGH

NORMAL

HIGH

NORMAL

HIGH

Tem

perature (degC

)Nighttime

16

19

19

16

16

16

16

Day

time

16

21

21

19

19

19

19

DIC

flux (m

mol m

22 h

21 )

Nighttime

260

(002

)486

(036

)mdash

36 (N

A)

307

(005

)38 (N

A)

33 (N

A)

Day

time

2329

(014

)2686

(015

)mdash

256 (N

A)

2374

(008

)221 (N

A)

206 (N

A)

Oxy

gen flux

(mmol m

22 h

21 )

Nighttime

2352

(001

)2445

(002

)mdash

224 (N

A)

2241

(001

)2400

(025

)2334

(001

)Day

time

349

(062

)720

(031

)mdash

56 (N

A)

398

(004

)466

(037

)443

(002

)Metha

ne flux (nmol m

22 h

21 )

Nighttime

381 (547)

179 (236)

mdash18

0 (433)

170 (258)

152 (314)

524 (321)

Day

time

328 (276)

214 (635)

mdash17

6 (683)

174 (116)

200 (738)

653 (211)

[H2] (pp

m)

Stea

dy-state

107 (33)

054

(016

)mdash

067

(018

)028

(008

)mdash

mdashBubb

le46 (29)

43 (20)

29 (10)

40 (0007

6)11 (012)

mdashmdash

n5

26n

58

n5

6n

52

n5

2

Values in

paren

theses rep

resent the

SD of mea

n va

lues F

or flux mea

suremen

ts n

52 The

num

ber of rep

licates for hyd

roge

n bu

bble m

easu

remen

ts is

indicated

An SD

value

of NA in

dicates a flux measu

remen

t of a single sample ( n

51) T

emperatures at w

hich

the

mea

suremen

ts w

ere mad

e are also ind

icated

June 14ndash15 2000

July 6ndash7 2000

November 8ndash9 2000

biomarkers the PFA 204 and 205 and the sterolcomposition Together 204 and 205 accountedfor 438 mg g21 TOC in the HIGH salinity matsand only 316 mg g21 TOC in the NORMAL salin-ity mats Both of these values are considerablyhigher than the natural mat samples (06 mgg21) Sterols a direct indication of the presenceof microeukaryotes in these mat samples werealso more abundant in the HIGH salinity treat-ment with 120 mg g21 versus 074 mg g21 forNORMAL salinity mats The two sterols thatserve as diatom biomarkers 24-methylcholesta-5-en-3b-ol and 24-methylcholesta-522-dien-3b-ol(Volkman 1986) accounted for 80 of the to-tal in both greenhouse samples

There were no differences observed in oxygenprofiles in the NORMAL and HIGH salinity mats(Fig 5) Fluxes of O2 were measured at the out-set (June 2000) of the parallel treatment of themats at two salinities and 5 months later (No-vember 2000) In both cases no differences in O2

fluxes were apparent between the two salinitytreatments with regard to the rate of export of O2to the overlying water column during the day orthe uptake of O2 at night (Table 2) In contrastrates of methane production from the NORMALand HIGH salinity mats which showed no dif-ference for the majority of time the experimentwas running differed at the last sampling (No-vember 8ndash9 2000) with higher rates recorded inthe normal-salinity treatment H2 partial pressurein photosynthetic surface bubbles from the HIGHsalinity mats was about half that in the NORMALsalinity mats after running the experiment for 5months

DISCUSSION

For a period of at least 1 year microbial matsmaintained in the greenhouse facility resembledfield-collected microbial mats with respect to

BEBOUT ET AL396

FIG 4 Microelectrodes were used to monitor oxygen fluxes across the Area 4 mat surfacendashwater interface dur-ing greenhouse [NORMAL (squares) and HIGH (circles) salinity] and in situ (triangles) experimentsPositive fluxesindicate net flux of photosynthetically-generated oxygen out of the mat while negative fluxes indicate net oxygenconsumption Error bars are SE values Mats maintained in the greenhouse retain the general diel pattern observedin situ including a morning peak in photosynthesis Note shading events caused by greenhouse window crossbarsresulting in temporary light limitation of photosynthesis in small areas of the greenhouse mats (eg at 1330 on Day1 and at 1030 on Day 2)

their overall appearance cyanobacterial commu-nity composition and rates of biogeochemical cy-cling High rates of activity of microbial popula-tions in mats contributed to the adaptability ofthese ecosystems However this attribute alsomakes the preservation of mat systems in theiroriginal natural states especially challengingThe phenomenon known as ldquogreeningrdquo in whichmotile cyanobacteria migrate to the surface of themats and remain there in response to lowered ir-radiance is common in mats maintained underartificial illumination (Bebout and Garcia-Pichel1995) For example when removed from theirnatural environment and maintained in large out-door ponds microbial mats collected from hy-persaline Solar Lake Sinai Egypt became ldquoover-grownrdquo with populations of cyanobacteria thatare not necessarily well-represented in situ (Abedand Garcia-Pichel 2001)

Oxygen microprofiles measured using micro-electrodes and oxygen and carbon fluxes mea-

sured using flux chambers were found to be com-parable in greenhouse and freshly collected nat-ural mats While it is reassuring to observe thissimilarity in mat net community metabolism itis likely that these parameters are among the leastsensitive to change since measurements of netcarbon and oxygen flux integrate the rates ofmetabolic activity of most of the microorganismsin the community Increases in the activity of onegroup of microorganisms may be compensatedfor by decreases in the activity of others We alsomeasured a close similarity between greenhouseand in situ bubble H2 partial pressures H2 par-tial pressures in bubbles are extremely sensitiveto changes in the environment of photosynthesisin the uppermost mat layer For example matsremoved from their native pond and placed un-der conditions of high solar irradiance and lowor no water flow frequently exhibited an increasein bubble H2 partial pressures of up to 2 ordersof magnitude The combination of light and flow


FIG 5 Oxygen microelectrode pro-files within microbial mat in the day-time (1000h open symbols) and night-time (2200h solid symbols) Littledifference can be seen in profiles takenin mats incubated at NORMAL (squares)and HIGH (circles) salinity

regimens appears to exert strong and instanta-neous control on bubble H2 partial pressures bysetting the balance between diffusive DIC supplyand light intensity (Lambert and Smith 1980Houchins 1984) The fact that little or no differ-ence in bubble H2 partial pressures was apparentbetween greenhouse and natural mats suggeststhat the basic flow and light regimens of the nat-ural environment are recreated to a very high de-gree in the greenhouse setting

We believe that the greenhouse approach doc-umented here is among the most successful of theefforts to date to produce an environment ap-propriate for the long-term study of these im-portant microbial communities When comparedwith previous efforts to maintain microbial matsour results indicate that two factorsmdashwater flowand the light regimemdashare likely to be more im-portant than others in simulating the field envi-ronment We discuss in more detail the impor-tance of these two factors in our greenhousefacility

The importance of flow in microbial mat me-tabolism has been recognized for at least a decade(Joslashrgensen 1994 Boudreau and Joslashrgensen 2001)The DBL a thin (05 mm) layer of stagnant wa-ter is situated at the interface between any mi-crobial mat community and the water columneven under conditions of high water flow Thetransport of all solutes between the water columnand the mat occurs exclusively by molecular dif-fusion through the DBL Therefore the thicknessof this layer sets the rates at which this exchangemay take place Rates of activity including ratesof primary production in microbial mat commu-nities may be controlled by the rate at which car-bon dioxide and oxygen diffuse across the DBL(Garcia-Pichel et al 1999)

In the greenhouse facility water was con-stantly circulated over the mats The flow veloc-ity was set to 5 cm s21 by adjusting the outputof our water pumps with valves and measuredand maintained by periodic observations of thetransit of small particles or bubbles (through ameasured distance in the flow box over a givenperiod of time) We chose a value of 5 cm s21 forthe greenhouse experiments as (1) it was a rea-sonable approximation of average in situ valuesand (2) that flow velocity is used in a large num-ber of published microelectrode measurements inmicrobial mats (Revsbech and Joslashrgensen 1986)which facilitated the ability to compare our re-sults with those of others

Previous work with these mats revealed thatcompared with the dramatic change in DBL thick-ness that occurs as flow velocities increase from0 (stagnant) to 1ndash3 cm s21 the DBL thickness isrelatively constant once flow velocities exceed1ndash3 cm s21 (Joslashrgensen and Des Marais 1990)Therefore flow variations around the 5 cm s21

value will minimally affect the fluxes in and outof the mat For the flux chamber measurementsreported here motor speeds were adjusted todrive the paddles to produce similar (5 cm s21)flow velocities which were also estimated by tim-ing the transit of small particles within the fluxchambers across the surface of the mats Actualin situ flow velocities also measured by observa-tions of naturally occurring particles underwaterby divers range from 0 (in the morning beforethe wind comes up) to 20 cm s21 under windyafternoon conditions though these values shouldbe regarded as rough estimates owing to the tur-bulent nature of the flow and the difficulty in-herent in making the observations No attemptwas made in the greenhouse to simulate thesedaily fluctuations in flow velocities However thesimilarity of microelectrode-based measurementsof DBL thickness (not shown) and microelectrodeprofile-based oxygen fluxes to those measured insitu (Fig 4) suggests that the greenhouse flumesystem closely reproduces the environmentalflow field and DBL control on matndashwater soluteexchange

In photosynthetic microbial mats all of the en-ergy necessary for growth and maintenance of thecommunity is ultimately derived from the sunOxygen production and consumption by thesephototrophic organisms as well as their sensitiv-ity to UV radiation determine the physical struc-ture of the entire mat community Furthermoremany mat microorganisms are motile utilizinglight andor UV radiation as a cue to adjust theirposition in the mats vertically (Castenholz 1994Bebout and Garcia-Pichel 1995) Therefore thesemats are highly sensitive to changes in PAR (lightwithin the wavelength range from 400 to 700 nm)The use of artificial illumination to drive photo-synthetic activity in microbial mats over longerperiods of time is not attractive from a logisticalperspective owing to the large amounts of heatthat is generated In addition sources of artificiallight rarely reproduce the high irradiance and(even more rarely) the spectral composition ofnatural sunlight For both of these reasons the useof natural sunlight is preferred for the mainte-

BEBOUT ET AL398

nance of these microbial communities Use of agreenhouse facility for microbial mat maintenanceis also the most cost-effective way to deliver therequired visible and UV radiation to microbialmat communities particularly when large areasof mat are required for destructive samplingandor large numbers of replicate measurements

Mats maintained in the greenhouse facilityhave access to natural solar radiation filtered onlythrough OP-4 acrylic and a few centimeters ofwater Therefore both the total PAR irradianceand the exact spectral composition of that PARare essentially unchanged from that of sunlightThe difference in latitude between our green-house and the field site as well as the absence ofa 1-m water column in the greenhouse con-tributed to making the irradiance at the surfaceof the greenhouse mats similar to that in situ Forexample the irradiance measured at the field site(on cloud-free days at high noon) 5 1845 6 1969mE m22 s21 (average reading from four field tripsin May 2000 June 2001 October 2001 and Sep-tember 2002) Attenuation of light by the watercolumn resulted in values measured at the matsurface (on those same cloud-free days) of1130 6 3019 mE m22 s21 In comparison the av-erage reading at the mat surface in the green-house was 1005 6 3962 mE m22 s21 (mean 6 SDn 5 548 values recorded at 10-min intervals from1200h to 1300h from June 2001 through Septem-ber 2001) The greenhouse irradiance record in-cludes cloudy days but these were relatively fewin number owing to the relatively clear summerskies at NASA Ames Research Center

Microbial mats in situ are likely to experiencesome changes in the spectral composition of lightincident upon their surface (relative to sunlight)due to particulates present in the water columnThese changes in spectral composition of the in-coming PAR (relative to sunlight) vary (eg withtime of year due to changes in the communitycomposition of phytoplankton in the salterns andwith time of day due to wind-driven resuspen-sion of detrital material) No attempt was madeto simulate these changes in spectral compositionin the greenhouse Owing to the high diversity ofphototrophic organisms in mats and the wide va-riety of pigments used by those organisms to cap-ture light for photosynthesis the total quantity ofphotons incident upon a mat community is of fargreater importance for community metabolismthan the exact spectral composition of the incom-ing radiation (Prufert-Bebout et al 1998) Addi-

tionally cyanobacterial motility and photosyn-thetic state transitions provide for efficient use ofthe rapidly changing spectral compositions withinmicrobial mat communities over the 1000-mmdepth range within which visible irradiance is ex-tinguished (Ploug et al 1998 Prufert-Bebout et al1998)

Future modifications in greenhouse flow andlight regimens may further improve our ability tosimulate the field environment The one poten-tially significant difference in community com-position observed between greenhouse and nat-ural mats namely the apparent increase in theabundance of diatoms in surface layers as sug-gested by the FAME biomarker data may in factbe explained by differences in the flow regimenexperienced by mats incubated in the greenhouserelative to in situ conditions In the greenhousethe flow of water over the mats while reproduc-ing natural flow velocities and DBL thickness oc-curs 24 h a day In the field environment waterflow over mats is primarily wind-driven and assuch is variable over the course of the day Strongwinds are characteristic of the afternoon as ris-ing warm air is replaced by strong onshorebreezes At night and into the morning there isvery little wind and therefore a much lower flowof water over the mats Recently we have docu-mented a density stratification of the ponds atnight This density stratification in combinationwith high rates of oxygen consumption by themat resulted in a layer of anoxic bottom waterseveral centimeters thick which persisted untillate morning (SR Miller unpublished data) Hy-drogen sulfide produced in the mat by sulfate re-duction reached levels in excess of 185 mM in thislayer (B Thamdrup personal communication)Additionally we recently documented that tem-peratures at the mat surface are often several de-grees higher than those in the middle of the water column and those that we have been sim-ulating in the greenhouse This is presumably be-cause of greater heat retention by the sedimentsunderlying the microbial mats in their natural en-vironment Constant flow over the greenhousemats would tend to diffuse heat from the surfacelayers of the mat resulting in temperatures lowerthan those recorded in situ

The lack of exposure to anoxic sulfidic waterat night as well as greenhouse water tempera-tures lower than those experienced by the matsin situ would tend to increase the abundance ofdiatoms relative to unicellular cyanobacteria in


the surface layers of the greenhouse mats Thephotosynthetic performance of the diatoms maybe inhibited in situ as a result of the sulfide sen-sitivity of oxygenic photosynthesis (Oren et al1979) and unicellular cyanobacteria from micro-bial mats are known to grow optimally at rela-tively high temperatures (Dor and Paz 1989 Garcia-Pichel et al 1998) Future work in thegreenhouse will investigate the extent to whichperiodic andor nighttime periods of low flowas well as periodic higher temperature excur-sions might reduce the tendency of the diatomsto dominate surface layers of the mat

The microbial mats in our greenhouse simula-tion facility represent a resource from which wewould like to obtain diverse observations and asmuch data as necessary to address key questionsin ecology To increase the accessibility of thegreenhouse to members of our research group thefacility is being transformed into a ldquocollaboratoryrdquoThe collaboratory will enable a geographically dis-persed group of scientists to plan experiments op-erate scientific equipment take experimental mea-surements share results and collaborate in realtime with remote colleagues Within the collabo-ratory intelligent software agents will assist in theexperimentation process controlling the hardwaretroubleshooting re-cording results and reportingback to collaborating experimenters The xyz posi-tioning table over the mats is capable of automat-ically positioning sophisticated instruments at anylocation over and within the mats The instrumentpackage currently includes microelectrodes a lightsensor chlorophyll fluorometer a surface detec-tion device and a fiber optic spectrometer The po-sitioning system and instrumentation package areviewable over the Internet (httpgreencamarcnasagov) via a webcam connected to a com-puter located in the greenhouse The advanced au-tomation capabilities in the greenhouse will enablelonger-term experimentation 24 h a day withoutthe costs associated with extensive human super-vision The remote collaboration capabilities willextend this experimental resource to colleaguesnot physically present in the greenhouse

In summary in addition to reproducing ratesof in situ biogeochemical processes greenhousemicrobial mats were responsive to manipulationsof environmental conditions Increasing the salin-ity of water in the greenhouse facility reproducedsome of the community composition changes ob-served in natural mats growing at higher salini-ties The differences observed in the salinity re-

sponses of natural and greenhouse mats may beconsistent with expected differences betweenfield and greenhouse environmental conditions(eg differences in temperature and nighttimeoxygen and hydrogen sulfide concentrations)Therefore we are optimistic that this facility maybe used not only to maintain mats with naturallevels of activity and species composition butalso to explore environmental conditions that arenot presently found in the natural environment(eg low sulfate and low oxygen environments)

ACKNOWLEDGMENTS

This study was supported by grants from theNASA Astrobiology Institute the NASA Exobi-ology Program and the NASA Ames DirectorrsquosDiscretionary Fund The greenhouse xyz tablewas designed and constructed by Dan Andrewsand Brian Koss Code FE NASA Ames ResearchCenter and funded by the NASA Intelligent Sys-tems Program Comments by two anonymous re-viewers greatly improved the manuscript

ABBREVIATIONS

DBL diffusive boundary layer DGGE dena-turing gradient gel electrophoresis DIC dissolvedinorganic carbon FA fatty acids FAME fatty acidmethyl esters PAR photosynthetically availableradiation PCR polymerase chain reaction PFApolyunsaturated fatty acids TEFA total esterifiedfatty acids TOC total organic carbon

REFERENCES

Abed AMM and Garcia-Pichel F (2001) Long-termcompositional changes after transplant in a microbialmat cyanobacterial community revealed using apolyphasic approach Environ Microbiol 3 53ndash62

Amann RI Ludwig W and Schleifer KH (1995) Phy-logenetic identification and in situ detection of indi-vidual microbial cells without cultivation MicrobiolRev 59 143ndash169

Bebout BM and Garcia-Pichel F (1995) UVB-inducedvertical migrations of cyanobacteria in a microbial matAppl Environ Microbiol 61 4215ndash4222

Berner RA (1980) Early Diagenesis A Theoretical ApproachPrinceton University Press Princeton NJ

Beukes NJ and Lowe DR (1989) Environmental con-trol on diverse stromatolite morphologies in the 3000

BEBOUT ET AL400

Myr Pongola Supergroup South Africa Sedimentology36 383ndash397

Boon JJ (1984) Tracing the origin of chemical fossils inmicrobial mats biogeochemical investigations of solarlake cyanobacterial mats using analytical pyrolysismethods In Microbial Mats Stromatolites edited by YCohen RW Castenholz and HO Halvorson Alan RLiss New York pp 313ndash342

Boudreau BP and Joslashrgensen BB eds (2001) The Ben-thic Boundary Layer Transport Processes and Biogeochem-istry Oxford University Press New York

Canfield DE and Des Marais DJ (1991) Aerobic sulfatereduction in microbial mats Science 251 1471ndash1473

Canfield DE and Des Marais DJ (1993) Biogeochemi-cal cycles of carbon sulfur and free oxygen in a mi-crobial mat Geochim Cosmochim Acta 57 3971ndash3984

Canfield DE and Teske A (1996) Late Proterozoic risein atmospheric oxygen from phylogenetic and stableisotope studies Nature 382 127ndash132

Castenholz RW (1994) Microbial mat research the re-cent past and new perspectives In Microbial Mats Struc-ture Development and Environmental Significance editedby LJ Stal and P Caumette Springer-Verlag NewYork pp 265ndash271

Cobelas MA and Lechado JZ (1989) Lipids in mi-croalgae A review I Biochemistry Grasas Aceites 40118ndash145

Des Marais DJ (1995) The biogeochemistry of hyper-saline microbial mats Adv Microb Ecol 14 251ndash274

Des Marais DJ and Canfield DE (1994) The carbon iso-tope biogeochemistry of microbial mats In MicrobialMats Structure Development and Environmental Signifi-cance edited by LJ Stal and P Caumette Springer-Ver-lag New York pp 289ndash298

Des Marais DJ Harwit MO Jucks KW Kasting JFLin DNC Lunine JI Schneider J Seager S TraubWA and Woolf NJ (2002) Remote sensing of plane-tary properties and biosignatures on extrasolar terres-trial planets Astrobiology 2 153ndash181

Dor I and Paz N (1989) Temporal and spatial distribu-tion of mat microalgae in the experimental solar pondsDead Sea area Israel In Microbial Mats The Physiologi-cal Ecology of Benthic Microbial Communities edited byY Cohen and E Rosenberg American Society for Mi-crobiology Washington DC pp 114ndash122

Dowling NJE Widdel F and White DC (1986) Phos-pholipid ester-linked fatty acid biomarkers of acetate-oxidizing sulphate-reducers and other sulphide-form-ing bacteria J Gen Microbiol 132 1815ndash1825

Garcia-Pichel F Prufert-Bebout L and Muyzer G(1996) Phenotypic and phylogenetic analyses show Mi-crocoleus chthonoplastes to be a cosmopolitan cyanobac-terium Appl Environ Microbiol 62 3284ndash3291

Garcia-Pichel F Nuumlbel U and Muyzer G (1998) Thephylogeny of unicellular extremely halotolerant cyano-bacteria Arch Microbiol 169 469ndash482

Garcia-Pichel F Kuumlhl M Nuumlbel U and Muyzer G(1999) Salinity-dependent limitation of photosynthesisand oxygen exchange in microbial mats J Phycol 35227ndash238

Garret P (1970) Phanerozoic stromatolites noncompeti-tive ecological restriction by grazing and burrowing an-imals Science 169 171ndash173

Glud RN Jensen K and Revsbech NP (1995) Diffu-sivity in surficial sediments and benthic mats deter-mined by use of a combined N2O-O2 microsensorGeochim Cosmochim Acta 59 231ndash237

Hall POJ and Aller RC (1992) Rapid small-volumeflow injection analysis for SCO2 and NH4

1 in marineand freshwaters Limnol Oceanogr 37 1113ndash1119

Hoehler TM Alperin MJ Albert DB and MartensCS (1998) Thermodynamic control on H2 concentra-tions in an anoxic marine sediment Geochim Cos-mochim Acta 62 1745ndash1756

Hoehler TM Bebout BM and Des Marais DJ (2001)The role of microbial mats in the production of reducedgases on the early Earth Nature 412 324ndash327

Holland HD (1984) The Chemical Evolution of the Atmos-phere and Oceans Princeton University Press PrincetonNJ

Houchins JP (1984) The physiology and biochemistry ofhydrogen metabolism in cyanobacteria Biochim Bio-phys Acta 768 227ndash255

Jahnke LL Stan-Lotter H Kato K and Hochstein LI(1992) Presence of methyl sterol and bacterio-hopanepolyol in an outer-membrane preparation fromMethylococcus capsulatus (Bath) J Gen Microbiol 1381759ndash1766

Jahnke LL Eder W Huber R Hope JM HinrichsK-U Hayes JM Des Marais DJ Cady SL andSummons RE (2001) Signature lipids and stable car-bon isotope analyses of Octopus Spring hyperther-mophilic communities compared with those of Aquifi-cales representatives Appl Environ Microbiol 67 5179ndash5189

Joslashrgensen BB (1994) Diffusion processes and boundarylayers in microbial mats In Microbial Mats StructureDevelopment and Environmental Significance edited byLJ Stal and P Caumette Springer-Verlag New Yorkpp 243ndash253

Joslashrgensen BB and Des Marais DJ (1990) The diffusiveboundary layer of sediments oxygen microgradientsover a microbial mat Limnol Oceanogr 35 1343ndash1355

Joslashrgensen BB Revsbech NP Blackburn TH and Co-hen Y (1979) Diurnal cycle of oxygen and sulfide mi-crogradients and microbial photosynthesis in acyanobacterial mat sediment Appl Environ Microbiol38 46ndash58

Kerger BD Nichols PD Antworth CP Sand WBock E Cox JC Langworthy TA and White DC(1986) Signature fatty acids in the polar lipids of acid-producing Thiobacillus spp methoxy cyclopropyl al-pha-hydroxy-cyclopropyl and branched and normalmonoenoic fatty acids FEMS Microbiol Ecol 38 67ndash77

Kohring LL Ringelberg DB Devereux R Stahl DAMittelman MW and White DC (1994) Comparisonof phylogenetic relationships based on phospholipidfatty acid profiles and ribosomal RNA sequence simi-larities among dissimilatory sulfate-reducing bacteriaFEMS Microbiol Lett 119 303ndash308


Lambert GR and Smith GD (1980) The hydrogen me-tabolism of cyanobacteria (blue-green algae) Biol RevCambridge Philos Soc 56 589ndash660

Li Y and Gregory S (1974) Diffusion of ions in sea wa-ter and in deep-sea sediments Geochim CosmochimActa 38 703ndash714

Loacutepez-Corteacutes A (1990) Microbial mats in tidal channelsat San Carlos Baja California Sur Mexico Geomicro-biol J 8 69ndash85

Nuumlbel U Garcia-Pichel F and Muyzer G (1997) PCRprimers to amplify 16S rRNA genes from cyanobacte-ria Appl Environ Microbiol 63 3327ndash3332

Nuumlbel U Bateson MM Madigan MT Kuumlhl M andWard DM (2001) Diversity and distribution in hy-persaline mats of Bacteria related to Chloroflexus sppAppl Environ Microbiol 67 4365ndash4371

Oren A Padan E and Malkin S (1979) Sulfide inhibi-tion of photosystem II in cyanobacteria (blue green al-gae) and tobacco chloroplasts Biochim Biophys Acta546 270ndash279

Ploug H Prufert-Bebout L and Joslashrgensen BB (1998)Photosynthetic adaptations to spectral scalar irradianceamong cyanobacteria within a stratified microbial matcommunity In Environmental Controls on CyanobacterialDistribution The Role of Irradiance [PhD Dissertation]University of Aringrhus Aringrhus Denmark

Potts M Olie JJ Nickels JS Parsons J and WhiteDC (1987) Variation in phospholipid ester-linked fattyacids and carotenoids of dessicated Nostoc commune(Cyanobacteria) from different geographic locationsAppl Environ Microbiol 53 4ndash9

Prufert-Bebout L Ploug H Garcia-Pichel F and KuumlhlM (1998) Adaptations of Microcoleus chthonoplastes tobenthic light fields as studied in situ by microsensorsIn Environmental Controls on Cyanobacterial DistributionThe Role of Irradiance [PhD Dissertation] University ofAringrhus Aringrhus Denmark

Reeburgh WS (1980) Anaerobic methane oxidation ratedepth distributions in Skan Bay sediments Earth PlanetSci Lett 47 345ndash352

Reid RP Visscher PT Decho AW Stolz JF BeboutBM Dupraz C Macintyre IG Pinckney J PaerlHW Prufert-Bebout L Steppe TF and Des MaraisDJ (2000) The role of microbes in accretion laminationand lithification in modern marine stromatolites Na-ture 406 989ndash992

Revsbech NP and Joslashrgensen BB (1986) Microelec-trodes their use in microbial ecology Adv Microb Ecol9 293ndash352

Revsbech NP Joslashrgensen BB Blackburn TH and Co-hen Y (1983) Microelectrode studies of the photosyn-thesis and O2 H2S and pH profiles of a microbial matLimnol Oceanogr 28 1062ndash1074

Schidlowski M (1988) A 3800-year-old isotopic recordof life from carbon in sedimentary rocks Nature 333313ndash318

Sherwood JE Stagnitti F Kokkinn MJ and WilliamsWD (1991) Dissolved oxygen concentrations in hy-persaline waters Limnol Oceanogr 36 235ndash250

Visscher PT and Kiene RP (1994) Production and con-sumption of volatile organosulfur compounds in mi-crobial mats In Microbial Mats Structure Developmentand Environmental Significance edited by LJ Stal and PCaumette Springer-Verlag New York pp 279ndash284

Visscher PT and Van Gemerden H (1991) Productionand consumption of dimethylsulfonioproprionate inmarine microbial mats Appl Environ Microbiol 573237ndash3242

Volkman JK (1986) A review of sterol markers for ma-rine and terrigenous organic matter Org Geochem 983ndash99

Walter MR (1976) Stromatolites Elsevier Scientific Am-sterdam

Ward DM Brassell SC and Eglinton G (1985) Ar-chaebacterial lipids in hot-spring microbial mats Na-ture 318 656ndash659

Ward DM Weller R and Bateson MM (1990) 16SrRNA sequences reveal numerous uncultured mi-croorganisms in a natural community Nature 345 63ndash65

Wieland A de Beer D Damgaard LR Khl M vanDusschoten D and Van As H (2001) Fine-scale mea-surement of diffusivity in a microbial mat with nuclearmagnetic resonance imaging Limnol Oceanogr 46 248ndash259

Wilson MV and Botkin DB (1990) Models of simplemicrocosms emergent properties and the effect of com-plexity on stability Am Nat 135 414ndash434

Address reprint requests toDr Brad M Bebout

NASA Ames Research CenterMail Stop 239-4

Moffett Field CA 94035-1000

E-mail BradMBeboutnasagov

BEBOUT ET AL402

INTRODUCTION

MODERN MICROBIAL MATS are thought to be ex-tant representatives of Earthrsquos most ancient

ecosystems (Walter 1976) Geochemical evidenceof the existence of photosynthetic microbial matsand their mineralized counterparts stromatoliteshas been identified in rocks as old as 30 Ga(Beukes and Lowe 1989) As a living repositoryof genetic physiological isotopic and biogeo-chemical information on the co-evolution of aplanet and the only known biosphere modernmicrobial mats are invaluable objects of studyModern microbial mat studies have provided im-portant insights on rates of biological activity(Revsbech et al 1983 Canfield and Des Marais1993 Des Marais 1995) genetic diversity (Wardet al 1990 Garcia-Pichel et al 1998 Nuumlbel et al2001) stable isotopic fractionation (Schidlowski1988 Des Marais and Canfield 1994) and organic(Boon 1984 Ward et al 1985) and atmospheric(Visscher and Van Gemerden 1991 Visscher andKiene 1994 Hoehler et al 2001) biomarkers aswell as minerals (Reid et al 2000) that have beenused to interpret the fossil record of these com-munities over geologic time

Photosynthetic microbial mats are self-sustain-ing complete ecosystems in which light energyabsorbed over a diel (24-h) cycle drives the syn-thesis of spatially organized diverse biomass Mi-croorganisms with tightly coupled metabolismsin the mat catalyze transformations of carbon ni-trogen sulfur and metals Radiant energy fromthe sun sustains oxygenic and anoxygenic pho-tosynthesis which in turn provides chemical en-ergy (as organic photosynthates and oxygen) tothe rest of the community When oxygenic pho-tosynthesis ceases at night the upper layers of themat become highly reduced and sulfidic (Joslashr-gensen et al 1979) Counteracting gradients ofoxygen and sulfide shape the chemical environ-ment and provide daily-contrasting microenvi-ronments separated on a scale of a few millime-ters (Revsbech et al 1983 Revsbech andJoslashrgensen 1986) While photosynthetic bacteriadominate the biomass and productivity of themat many aspects of the ecosystemrsquos emergentbehavior may ultimately depend on the associ-ated nonphotosynthetic microbial communitiesincluding the anaerobes Additionally transfor-mation of photosynthetic productivity by the mi-crobial community may contribute diagnosticldquobiosignaturerdquo gases that could represent search

targets for remote spectroscopic life detection ef-forts [eg Terrestrial Planet Finder (Des Maraiset al 2002)] To understand the overall structureand function of mat communities it is thus criti-cal to determine the nature and extent of the in-teractions between phototrophic and nonphoto-synthetic microorganisms including anaerobicmicroorganisms

Compared with their historical distributionand abundance microbial mats are today con-fined to a relatively restricted number of habitatsWell-developed modern photosynthetic mats oc-cur in both high-salinity and high-temperatureenvironments This restricted distribution isthought to be due primarily to the higher rates ofgrazing in more moderate modern environments(Garret 1970) although alternative hypotheseshave been forwarded In any event the limitedgeographical distribution of mats on the present-day Earth also serves to limit their usefulness asanalogs of similar ecosystems growing in morediverse ancient environments Environments thatare not well represented on the modern Earth butthat have been extremely important over evolu-tionary time include marine environments hav-ing low concentrations of sulfate andor free oxy-gen (Holland 1984 Canfield and Teske 1996)

The effects on microbial processes of variousenvironmental parameters important in Earthrsquospast (such as sulfate and oxygen concentrations)may be studied in culture However the emer-gent properties of complex microbial ecosystemsare not necessarily predictable or understand-able on the basis of these types of experiments(Wilson and Botkin 1990) Either some microbialprocesses do not occur in culture [eg anaerobicmethane oxidation (Reeburgh 1980) and sulfatereduction under aerobic conditions (Canfield andDes Marais 1991)] or they occur at rates vastlydifferent than rates observed in nature In addi-tion relatively few (1) of the total number ofmicrobes present in nature are available in cul-ture (Ward et al 1990 Amann et al 1995) A mi-crobial mat is a highly complex assemblage of organisms possessing many different modes ofmetabolism all of which are interacting with eachother at some level in beneficial andor compet-itive ways The end products of the metabolismof one group of organisms frequently constitutethe substrates of another These considerationsargue for an alternative approach in which theentire microbial ecosystem is subjected to exper-imental manipulation

BEBOUT ET AL384




Field site




Greenhouse facility



BEBOUT ET AL386

FIG 1

Microbial mats pos

itioned


perim

entThr

ee m

ats are incu

bated in

each of the


es T

he po-

sition

s of the

reservo

irs an

d tem

perature con

trol equ

ipmen

t can

be seen

( B) as w

ell a

s the instrumen

t pa

ckag

e ( A

) presen


erab

le xyz

table Pho

tocred

it D

an A

ndrews
















BEBOUT ET AL388







J 5 2fDs (shyCshyz)









RESULTS




BEBOUT ET AL390





BEBOUT ET AL392

TABLE1

COM

POSITIO

NOFTW

ON

ATURALM

ATS

FROM

THEA

REA4 M

ATD

ISSE

CTED

ATG

UERRERON

EGRO

AND

OFM

ATSC

OLLECTED

FROM

THESA

MEA

REAA

FTERM

AIN

TENANCE

INTHEFL

UM

ESY

STEM

FOR

$4 M

ONTHS

1298 P4n51

90permil flume

120permil

flume

601 P4n51

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Ester-linke

d FA (mg g2

1TOC)

17760

0000

31800

0000

25670

0000

023

380

0000

26520

0000

027

850

000

30870

0000

022

090

000

CN R

atio

980

0000

ND

940

0000

0920

0000

12100

0000

0960

000

ND

ND

d13C (permil)

210

72

6 004

210

23

6 028

2901

6 005

210

59

6 013

2970

6 017

210

50

6 01

2802

6 007

210

56

065

d15N (permil

)2013

6 04

ND

2127

6 006

2092

6 038

2023

6 035

2026

6 018

ND

ND

Error estim

ates sho


tal precision Sam

ples labe

led N

D w

ere too sm


alys

is

1 Collected

in D

ecem

ber 19

98 and

June

200

1 from

the

field site Pon

d 4 nea

r Area 5


BEBOUT ET AL394









TABLE2

MEASU

REM

ENTS

OFBIO

GEOCHEM

ICALPROCESS

ES

INFR

ESH

LYC

OLLECTEDM

ATS

AND

MATSM

AIN

TAIN

ED

INTHEG

REENHOUSE

FACIL

ITY

ATNORMAL A

NDHIG

H S

ALIN

ITIE

S



May 19ndash20 2000

NORMAL

HIGH

NORMAL

HIGH

NORMAL

HIGH

Tem

perature (degC

)Nighttime

16

19

19

16

16

16

16

Day

time

16

21

21

19

19

19

19

DIC

flux (m

mol m

22 h

21 )

Nighttime

260

(002

)486

(036

)mdash

36 (N

A)

307

(005

)38 (N

A)

33 (N

A)

Day

time

2329

(014

)2686

(015

)mdash

256 (N

A)

2374

(008

)221 (N

A)

206 (N

A)

Oxy

gen flux

(mmol m

22 h

21 )

Nighttime

2352

(001

)2445

(002

)mdash

224 (N

A)

2241

(001

)2400

(025

)2334

(001

)Day

time

349

(062

)720

(031

)mdash

56 (N

A)

398

(004

)466

(037

)443

(002

)Metha

ne flux (nmol m

22 h

21 )

Nighttime

381 (547)

179 (236)

mdash18

0 (433)

170 (258)

152 (314)

524 (321)

Day

time

328 (276)

214 (635)

mdash17

6 (683)

174 (116)

200 (738)

653 (211)

[H2] (pp

m)

Stea

dy-state

107 (33)

054

(016

)mdash

067

(018

)028

(008

)mdash

mdashBubb

le46 (29)

43 (20)

29 (10)

40 (0007

6)11 (012)

mdashmdash

n5

26n

58

n5

6n

52

n5

2

Values in

paren

theses rep

resent the

SD of mea

n va

lues F

or flux mea

suremen

ts n

52 The

num

ber of rep

licates for hyd

roge

n bu

bble m

easu

remen

ts is

indicated

An SD

value

of NA in


remen


51) T

emperatures at w

hich

the

mea

suremen

ts w

ere mad

e are also ind

icated

June 14ndash15 2000

July 6ndash7 2000





DISCUSSION


BEBOUT ET AL396














BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402




Field site




Greenhouse facility



BEBOUT ET AL386

FIG 1

Microbial mats pos

itioned


perim

entThr

ee m

ats are incu

bated in

each of the


es T

he po-

sition

s of the

reservo

irs an

d tem

perature con

trol equ

ipmen

t can

be seen

( B) as w

ell a

s the instrumen

t pa

ckag

e ( A

) presen


erab

le xyz

table Pho

tocred

it D

an A

ndrews
















BEBOUT ET AL388







J 5 2fDs (shyCshyz)









RESULTS




BEBOUT ET AL390





BEBOUT ET AL392

TABLE1

COM

POSITIO

NOFTW

ON

ATURALM

ATS

FROM

THEA

REA4 M

ATD

ISSE

CTED

ATG

UERRERON

EGRO

AND

OFM

ATSC

OLLECTED

FROM

THESA

MEA

REAA

FTERM

AIN

TENANCE

INTHEFL

UM

ESY

STEM

FOR

$4 M

ONTHS

1298 P4n51

90permil flume

120permil

flume

601 P4n51

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Ester-linke

d FA (mg g2

1TOC)

17760

0000

31800

0000

25670

0000

023

380

0000

26520

0000

027

850

000

30870

0000

022

090

000

CN R

atio

980

0000

ND

940

0000

0920

0000

12100

0000

0960

000

ND

ND

d13C (permil)

210

72

6 004

210

23

6 028

2901

6 005

210

59

6 013

2970

6 017

210

50

6 01

2802

6 007

210

56

065

d15N (permil

)2013

6 04

ND

2127

6 006

2092

6 038

2023

6 035

2026

6 018

ND

ND

Error estim

ates sho


tal precision Sam

ples labe

led N

D w

ere too sm


alys

is

1 Collected

in D

ecem

ber 19

98 and

June

200

1 from

the

field site Pon

d 4 nea

r Area 5


BEBOUT ET AL394









TABLE2

MEASU

REM

ENTS

OFBIO

GEOCHEM

ICALPROCESS

ES

INFR

ESH

LYC

OLLECTEDM

ATS

AND

MATSM

AIN

TAIN

ED

INTHEG

REENHOUSE

FACIL

ITY

ATNORMAL A

NDHIG

H S

ALIN

ITIE

S



May 19ndash20 2000

NORMAL

HIGH

NORMAL

HIGH

NORMAL

HIGH

Tem

perature (degC

)Nighttime

16

19

19

16

16

16

16

Day

time

16

21

21

19

19

19

19

DIC

flux (m

mol m

22 h

21 )

Nighttime

260

(002

)486

(036

)mdash

36 (N

A)

307

(005

)38 (N

A)

33 (N

A)

Day

time

2329

(014

)2686

(015

)mdash

256 (N

A)

2374

(008

)221 (N

A)

206 (N

A)

Oxy

gen flux

(mmol m

22 h

21 )

Nighttime

2352

(001

)2445

(002

)mdash

224 (N

A)

2241

(001

)2400

(025

)2334

(001

)Day

time

349

(062

)720

(031

)mdash

56 (N

A)

398

(004

)466

(037

)443

(002

)Metha

ne flux (nmol m

22 h

21 )

Nighttime

381 (547)

179 (236)

mdash18

0 (433)

170 (258)

152 (314)

524 (321)

Day

time

328 (276)

214 (635)

mdash17

6 (683)

174 (116)

200 (738)

653 (211)

[H2] (pp

m)

Stea

dy-state

107 (33)

054

(016

)mdash

067

(018

)028

(008

)mdash

mdashBubb

le46 (29)

43 (20)

29 (10)

40 (0007

6)11 (012)

mdashmdash

n5

26n

58

n5

6n

52

n5

2

Values in

paren

theses rep

resent the

SD of mea

n va

lues F

or flux mea

suremen

ts n

52 The

num

ber of rep

licates for hyd

roge

n bu

bble m

easu

remen

ts is

indicated

An SD

value

of NA in


remen


51) T

emperatures at w

hich

the

mea

suremen

ts w

ere mad

e are also ind

icated

June 14ndash15 2000

July 6ndash7 2000





DISCUSSION


BEBOUT ET AL396














BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402

BEBOUT ET AL386

FIG 1

Microbial mats pos

itioned


perim

entThr

ee m

ats are incu

bated in

each of the


es T

he po-

sition

s of the

reservo

irs an

d tem

perature con

trol equ

ipmen

t can

be seen

( B) as w

ell a

s the instrumen

t pa

ckag

e ( A

) presen


erab

le xyz

table Pho

tocred

it D

an A

ndrews
















BEBOUT ET AL388







J 5 2fDs (shyCshyz)









RESULTS




BEBOUT ET AL390





BEBOUT ET AL392

TABLE1

COM

POSITIO

NOFTW

ON

ATURALM

ATS

FROM

THEA

REA4 M

ATD

ISSE

CTED

ATG

UERRERON

EGRO

AND

OFM

ATSC

OLLECTED

FROM

THESA

MEA

REAA

FTERM

AIN

TENANCE

INTHEFL

UM

ESY

STEM

FOR

$4 M

ONTHS

1298 P4n51

90permil flume

120permil

flume

601 P4n51

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Ester-linke

d FA (mg g2

1TOC)

17760

0000

31800

0000

25670

0000

023

380

0000

26520

0000

027

850

000

30870

0000

022

090

000

CN R

atio

980

0000

ND

940

0000

0920

0000

12100

0000

0960

000

ND

ND

d13C (permil)

210

72

6 004

210

23

6 028

2901

6 005

210

59

6 013

2970

6 017

210

50

6 01

2802

6 007

210

56

065

d15N (permil

)2013

6 04

ND

2127

6 006

2092

6 038

2023

6 035

2026

6 018

ND

ND

Error estim

ates sho


tal precision Sam

ples labe

led N

D w

ere too sm


alys

is

1 Collected

in D

ecem

ber 19

98 and

June

200

1 from

the

field site Pon

d 4 nea

r Area 5


BEBOUT ET AL394









TABLE2

MEASU

REM

ENTS

OFBIO

GEOCHEM

ICALPROCESS

ES

INFR

ESH

LYC

OLLECTEDM

ATS

AND

MATSM

AIN

TAIN

ED

INTHEG

REENHOUSE

FACIL

ITY

ATNORMAL A

NDHIG

H S

ALIN

ITIE

S



May 19ndash20 2000

NORMAL

HIGH

NORMAL

HIGH

NORMAL

HIGH

Tem

perature (degC

)Nighttime

16

19

19

16

16

16

16

Day

time

16

21

21

19

19

19

19

DIC

flux (m

mol m

22 h

21 )

Nighttime

260

(002

)486

(036

)mdash

36 (N

A)

307

(005

)38 (N

A)

33 (N

A)

Day

time

2329

(014

)2686

(015

)mdash

256 (N

A)

2374

(008

)221 (N

A)

206 (N

A)

Oxy

gen flux

(mmol m

22 h

21 )

Nighttime

2352

(001

)2445

(002

)mdash

224 (N

A)

2241

(001

)2400

(025

)2334

(001

)Day

time

349

(062

)720

(031

)mdash

56 (N

A)

398

(004

)466

(037

)443

(002

)Metha

ne flux (nmol m

22 h

21 )

Nighttime

381 (547)

179 (236)

mdash18

0 (433)

170 (258)

152 (314)

524 (321)

Day

time

328 (276)

214 (635)

mdash17

6 (683)

174 (116)

200 (738)

653 (211)

[H2] (pp

m)

Stea

dy-state

107 (33)

054

(016

)mdash

067

(018

)028

(008

)mdash

mdashBubb

le46 (29)

43 (20)

29 (10)

40 (0007

6)11 (012)

mdashmdash

n5

26n

58

n5

6n

52

n5

2

Values in

paren

theses rep

resent the

SD of mea

n va

lues F

or flux mea

suremen

ts n

52 The

num

ber of rep

licates for hyd

roge

n bu

bble m

easu

remen

ts is

indicated

An SD

value

of NA in


remen


51) T

emperatures at w

hich

the

mea

suremen

ts w

ere mad

e are also ind

icated

June 14ndash15 2000

July 6ndash7 2000





DISCUSSION


BEBOUT ET AL396














BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402
















BEBOUT ET AL388







J 5 2fDs (shyCshyz)









RESULTS




BEBOUT ET AL390





BEBOUT ET AL392

TABLE1

COM

POSITIO

NOFTW

ON

ATURALM

ATS

FROM

THEA

REA4 M

ATD

ISSE

CTED

ATG

UERRERON

EGRO

AND

OFM

ATSC

OLLECTED

FROM

THESA

MEA

REAA

FTERM

AIN

TENANCE

INTHEFL

UM

ESY

STEM

FOR

$4 M

ONTHS

1298 P4n51

90permil flume

120permil

flume

601 P4n51

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Ester-linke

d FA (mg g2

1TOC)

17760

0000

31800

0000

25670

0000

023

380

0000

26520

0000

027

850

000

30870

0000

022

090

000

CN R

atio

980

0000

ND

940

0000

0920

0000

12100

0000

0960

000

ND

ND

d13C (permil)

210

72

6 004

210

23

6 028

2901

6 005

210

59

6 013

2970

6 017

210

50

6 01

2802

6 007

210

56

065

d15N (permil

)2013

6 04

ND

2127

6 006

2092

6 038

2023

6 035

2026

6 018

ND

ND

Error estim

ates sho


tal precision Sam

ples labe

led N

D w

ere too sm


alys

is

1 Collected

in D

ecem

ber 19

98 and

June

200

1 from

the

field site Pon

d 4 nea

r Area 5


BEBOUT ET AL394









TABLE2

MEASU

REM

ENTS

OFBIO

GEOCHEM

ICALPROCESS

ES

INFR

ESH

LYC

OLLECTEDM

ATS

AND

MATSM

AIN

TAIN

ED

INTHEG

REENHOUSE

FACIL

ITY

ATNORMAL A

NDHIG

H S

ALIN

ITIE

S



May 19ndash20 2000

NORMAL

HIGH

NORMAL

HIGH

NORMAL

HIGH

Tem

perature (degC

)Nighttime

16

19

19

16

16

16

16

Day

time

16

21

21

19

19

19

19

DIC

flux (m

mol m

22 h

21 )

Nighttime

260

(002

)486

(036

)mdash

36 (N

A)

307

(005

)38 (N

A)

33 (N

A)

Day

time

2329

(014

)2686

(015

)mdash

256 (N

A)

2374

(008

)221 (N

A)

206 (N

A)

Oxy

gen flux

(mmol m

22 h

21 )

Nighttime

2352

(001

)2445

(002

)mdash

224 (N

A)

2241

(001

)2400

(025

)2334

(001

)Day

time

349

(062

)720

(031

)mdash

56 (N

A)

398

(004

)466

(037

)443

(002

)Metha

ne flux (nmol m

22 h

21 )

Nighttime

381 (547)

179 (236)

mdash18

0 (433)

170 (258)

152 (314)

524 (321)

Day

time

328 (276)

214 (635)

mdash17

6 (683)

174 (116)

200 (738)

653 (211)

[H2] (pp

m)

Stea

dy-state

107 (33)

054

(016

)mdash

067

(018

)028

(008

)mdash

mdashBubb

le46 (29)

43 (20)

29 (10)

40 (0007

6)11 (012)

mdashmdash

n5

26n

58

n5

6n

52

n5

2

Values in

paren

theses rep

resent the

SD of mea

n va

lues F

or flux mea

suremen

ts n

52 The

num

ber of rep

licates for hyd

roge

n bu

bble m

easu

remen

ts is

indicated

An SD

value

of NA in


remen


51) T

emperatures at w

hich

the

mea

suremen

ts w

ere mad

e are also ind

icated

June 14ndash15 2000

July 6ndash7 2000





DISCUSSION


BEBOUT ET AL396














BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402







J 5 2fDs (shyCshyz)









RESULTS




BEBOUT ET AL390





BEBOUT ET AL392

TABLE1

COM

POSITIO

NOFTW

ON

ATURALM

ATS

FROM

THEA

REA4 M

ATD

ISSE

CTED

ATG

UERRERON

EGRO

AND

OFM

ATSC

OLLECTED

FROM

THESA

MEA

REAA

FTERM

AIN

TENANCE

INTHEFL

UM

ESY

STEM

FOR

$4 M

ONTHS

1298 P4n51

90permil flume

120permil

flume

601 P4n51

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Ester-linke

d FA (mg g2

1TOC)

17760

0000

31800

0000

25670

0000

023

380

0000

26520

0000

027

850

000

30870

0000

022

090

000

CN R

atio

980

0000

ND

940

0000

0920

0000

12100

0000

0960

000

ND

ND

d13C (permil)

210

72

6 004

210

23

6 028

2901

6 005

210

59

6 013

2970

6 017

210

50

6 01

2802

6 007

210

56

065

d15N (permil

)2013

6 04

ND

2127

6 006

2092

6 038

2023

6 035

2026

6 018

ND

ND

Error estim

ates sho


tal precision Sam

ples labe

led N

D w

ere too sm


alys

is

1 Collected

in D

ecem

ber 19

98 and

June

200

1 from

the

field site Pon

d 4 nea

r Area 5


BEBOUT ET AL394









TABLE2

MEASU

REM

ENTS

OFBIO

GEOCHEM

ICALPROCESS

ES

INFR

ESH

LYC

OLLECTEDM

ATS

AND

MATSM

AIN

TAIN

ED

INTHEG

REENHOUSE

FACIL

ITY

ATNORMAL A

NDHIG

H S

ALIN

ITIE

S



May 19ndash20 2000

NORMAL

HIGH

NORMAL

HIGH

NORMAL

HIGH

Tem

perature (degC

)Nighttime

16

19

19

16

16

16

16

Day

time

16

21

21

19

19

19

19

DIC

flux (m

mol m

22 h

21 )

Nighttime

260

(002

)486

(036

)mdash

36 (N

A)

307

(005

)38 (N

A)

33 (N

A)

Day

time

2329

(014

)2686

(015

)mdash

256 (N

A)

2374

(008

)221 (N

A)

206 (N

A)

Oxy

gen flux

(mmol m

22 h

21 )

Nighttime

2352

(001

)2445

(002

)mdash

224 (N

A)

2241

(001

)2400

(025

)2334

(001

)Day

time

349

(062

)720

(031

)mdash

56 (N

A)

398

(004

)466

(037

)443

(002

)Metha

ne flux (nmol m

22 h

21 )

Nighttime

381 (547)

179 (236)

mdash18

0 (433)

170 (258)

152 (314)

524 (321)

Day

time

328 (276)

214 (635)

mdash17

6 (683)

174 (116)

200 (738)

653 (211)

[H2] (pp

m)

Stea

dy-state

107 (33)

054

(016

)mdash

067

(018

)028

(008

)mdash

mdashBubb

le46 (29)

43 (20)

29 (10)

40 (0007

6)11 (012)

mdashmdash

n5

26n

58

n5

6n

52

n5

2

Values in

paren

theses rep

resent the

SD of mea

n va

lues F

or flux mea

suremen

ts n

52 The

num

ber of rep

licates for hyd

roge

n bu

bble m

easu

remen

ts is

indicated

An SD

value

of NA in


remen


51) T

emperatures at w

hich

the

mea

suremen

ts w

ere mad

e are also ind

icated

June 14ndash15 2000

July 6ndash7 2000





DISCUSSION


BEBOUT ET AL396














BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402





RESULTS




BEBOUT ET AL390





BEBOUT ET AL392

TABLE1

COM

POSITIO

NOFTW

ON

ATURALM

ATS

FROM

THEA

REA4 M

ATD

ISSE

CTED

ATG

UERRERON

EGRO

AND

OFM

ATSC

OLLECTED

FROM

THESA

MEA

REAA

FTERM

AIN

TENANCE

INTHEFL

UM

ESY

STEM

FOR

$4 M

ONTHS

1298 P4n51

90permil flume

120permil

flume

601 P4n51

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Ester-linke

d FA (mg g2

1TOC)

17760

0000

31800

0000

25670

0000

023

380

0000

26520

0000

027

850

000

30870

0000

022

090

000

CN R

atio

980

0000

ND

940

0000

0920

0000

12100

0000

0960

000

ND

ND

d13C (permil)

210

72

6 004

210

23

6 028

2901

6 005

210

59

6 013

2970

6 017

210

50

6 01

2802

6 007

210

56

065

d15N (permil

)2013

6 04

ND

2127

6 006

2092

6 038

2023

6 035

2026

6 018

ND

ND

Error estim

ates sho


tal precision Sam

ples labe

led N

D w

ere too sm


alys

is

1 Collected

in D

ecem

ber 19

98 and

June

200

1 from

the

field site Pon

d 4 nea

r Area 5


BEBOUT ET AL394









TABLE2

MEASU

REM

ENTS

OFBIO

GEOCHEM

ICALPROCESS

ES

INFR

ESH

LYC

OLLECTEDM

ATS

AND

MATSM

AIN

TAIN

ED

INTHEG

REENHOUSE

FACIL

ITY

ATNORMAL A

NDHIG

H S

ALIN

ITIE

S



May 19ndash20 2000

NORMAL

HIGH

NORMAL

HIGH

NORMAL

HIGH

Tem

perature (degC

)Nighttime

16

19

19

16

16

16

16

Day

time

16

21

21

19

19

19

19

DIC

flux (m

mol m

22 h

21 )

Nighttime

260

(002

)486

(036

)mdash

36 (N

A)

307

(005

)38 (N

A)

33 (N

A)

Day

time

2329

(014

)2686

(015

)mdash

256 (N

A)

2374

(008

)221 (N

A)

206 (N

A)

Oxy

gen flux

(mmol m

22 h

21 )

Nighttime

2352

(001

)2445

(002

)mdash

224 (N

A)

2241

(001

)2400

(025

)2334

(001

)Day

time

349

(062

)720

(031

)mdash

56 (N

A)

398

(004

)466

(037

)443

(002

)Metha

ne flux (nmol m

22 h

21 )

Nighttime

381 (547)

179 (236)

mdash18

0 (433)

170 (258)

152 (314)

524 (321)

Day

time

328 (276)

214 (635)

mdash17

6 (683)

174 (116)

200 (738)

653 (211)

[H2] (pp

m)

Stea

dy-state

107 (33)

054

(016

)mdash

067

(018

)028

(008

)mdash

mdashBubb

le46 (29)

43 (20)

29 (10)

40 (0007

6)11 (012)

mdashmdash

n5

26n

58

n5

6n

52

n5

2

Values in

paren

theses rep

resent the

SD of mea

n va

lues F

or flux mea

suremen

ts n

52 The

num

ber of rep

licates for hyd

roge

n bu

bble m

easu

remen

ts is

indicated

An SD

value

of NA in


remen


51) T

emperatures at w

hich

the

mea

suremen

ts w

ere mad

e are also ind

icated

June 14ndash15 2000

July 6ndash7 2000





DISCUSSION


BEBOUT ET AL396














BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402





BEBOUT ET AL392

TABLE1

COM

POSITIO

NOFTW

ON

ATURALM

ATS

FROM

THEA

REA4 M

ATD

ISSE

CTED

ATG

UERRERON

EGRO

AND

OFM

ATSC

OLLECTED

FROM

THESA

MEA

REAA

FTERM

AIN

TENANCE

INTHEFL

UM

ESY

STEM

FOR

$4 M

ONTHS

1298 P4n51

90permil flume

120permil

flume

601 P4n51

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Surface

Microco

leus

Ester-linke

d FA (mg g2

1TOC)

17760

0000

31800

0000

25670

0000

023

380

0000

26520

0000

027

850

000

30870

0000

022

090

000

CN R

atio

980

0000

ND

940

0000

0920

0000

12100

0000

0960

000

ND

ND

d13C (permil)

210

72

6 004

210

23

6 028

2901

6 005

210

59

6 013

2970

6 017

210

50

6 01

2802

6 007

210

56

065

d15N (permil

)2013

6 04

ND

2127

6 006

2092

6 038

2023

6 035

2026

6 018

ND

ND

Error estim

ates sho


tal precision Sam

ples labe

led N

D w

ere too sm


alys

is

1 Collected

in D

ecem

ber 19

98 and

June

200

1 from

the

field site Pon

d 4 nea

r Area 5


BEBOUT ET AL394









TABLE2

MEASU

REM

ENTS

OFBIO

GEOCHEM

ICALPROCESS

ES

INFR

ESH

LYC

OLLECTEDM

ATS

AND

MATSM

AIN

TAIN

ED

INTHEG

REENHOUSE

FACIL

ITY

ATNORMAL A

NDHIG

H S

ALIN

ITIE

S



May 19ndash20 2000

NORMAL

HIGH

NORMAL

HIGH

NORMAL

HIGH

Tem

perature (degC

)Nighttime

16

19

19

16

16

16

16

Day

time

16

21

21

19

19

19

19

DIC

flux (m

mol m

22 h

21 )

Nighttime

260

(002

)486

(036

)mdash

36 (N

A)

307

(005

)38 (N

A)

33 (N

A)

Day

time

2329

(014

)2686

(015

)mdash

256 (N

A)

2374

(008

)221 (N

A)

206 (N

A)

Oxy

gen flux

(mmol m

22 h

21 )

Nighttime

2352

(001

)2445

(002

)mdash

224 (N

A)

2241

(001

)2400

(025

)2334

(001

)Day

time

349

(062

)720

(031

)mdash

56 (N

A)

398

(004

)466

(037

)443

(002

)Metha

ne flux (nmol m

22 h

21 )

Nighttime

381 (547)

179 (236)

mdash18

0 (433)

170 (258)

152 (314)

524 (321)

Day

time

328 (276)

214 (635)

mdash17

6 (683)

174 (116)

200 (738)

653 (211)

[H2] (pp

m)

Stea

dy-state

107 (33)

054

(016

)mdash

067

(018

)028

(008

)mdash

mdashBubb

le46 (29)

43 (20)

29 (10)

40 (0007

6)11 (012)

mdashmdash

n5

26n

58

n5

6n

52

n5

2

Values in

paren

theses rep

resent the

SD of mea

n va

lues F

or flux mea

suremen

ts n

52 The

num

ber of rep

licates for hyd

roge

n bu

bble m

easu

remen

ts is

indicated

An SD

value

of NA in


remen


51) T

emperatures at w

hich

the

mea

suremen

ts w

ere mad

e are also ind

icated

June 14ndash15 2000

July 6ndash7 2000





DISCUSSION


BEBOUT ET AL396














BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402


BEBOUT ET AL394









TABLE2

MEASU

REM

ENTS

OFBIO

GEOCHEM

ICALPROCESS

ES

INFR

ESH

LYC

OLLECTEDM

ATS

AND

MATSM

AIN

TAIN

ED

INTHEG

REENHOUSE

FACIL

ITY

ATNORMAL A

NDHIG

H S

ALIN

ITIE

S



May 19ndash20 2000

NORMAL

HIGH

NORMAL

HIGH

NORMAL

HIGH

Tem

perature (degC

)Nighttime

16

19

19

16

16

16

16

Day

time

16

21

21

19

19

19

19

DIC

flux (m

mol m

22 h

21 )

Nighttime

260

(002

)486

(036

)mdash

36 (N

A)

307

(005

)38 (N

A)

33 (N

A)

Day

time

2329

(014

)2686

(015

)mdash

256 (N

A)

2374

(008

)221 (N

A)

206 (N

A)

Oxy

gen flux

(mmol m

22 h

21 )

Nighttime

2352

(001

)2445

(002

)mdash

224 (N

A)

2241

(001

)2400

(025

)2334

(001

)Day

time

349

(062

)720

(031

)mdash

56 (N

A)

398

(004

)466

(037

)443

(002

)Metha

ne flux (nmol m

22 h

21 )

Nighttime

381 (547)

179 (236)

mdash18

0 (433)

170 (258)

152 (314)

524 (321)

Day

time

328 (276)

214 (635)

mdash17

6 (683)

174 (116)

200 (738)

653 (211)

[H2] (pp

m)

Stea

dy-state

107 (33)

054

(016

)mdash

067

(018

)028

(008

)mdash

mdashBubb

le46 (29)

43 (20)

29 (10)

40 (0007

6)11 (012)

mdashmdash

n5

26n

58

n5

6n

52

n5

2

Values in

paren

theses rep

resent the

SD of mea

n va

lues F

or flux mea

suremen

ts n

52 The

num

ber of rep

licates for hyd

roge

n bu

bble m

easu

remen

ts is

indicated

An SD

value

of NA in


remen


51) T

emperatures at w

hich

the

mea

suremen

ts w

ere mad

e are also ind

icated

June 14ndash15 2000

July 6ndash7 2000





DISCUSSION


BEBOUT ET AL396














BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402


TABLE2

MEASU

REM

ENTS

OFBIO

GEOCHEM

ICALPROCESS

ES

INFR

ESH

LYC

OLLECTEDM

ATS

AND

MATSM

AIN

TAIN

ED

INTHEG

REENHOUSE

FACIL

ITY

ATNORMAL A

NDHIG

H S

ALIN

ITIE

S



May 19ndash20 2000

NORMAL

HIGH

NORMAL

HIGH

NORMAL

HIGH

Tem

perature (degC

)Nighttime

16

19

19

16

16

16

16

Day

time

16

21

21

19

19

19

19

DIC

flux (m

mol m

22 h

21 )

Nighttime

260

(002

)486

(036

)mdash

36 (N

A)

307

(005

)38 (N

A)

33 (N

A)

Day

time

2329

(014

)2686

(015

)mdash

256 (N

A)

2374

(008

)221 (N

A)

206 (N

A)

Oxy

gen flux

(mmol m

22 h

21 )

Nighttime

2352

(001

)2445

(002

)mdash

224 (N

A)

2241

(001

)2400

(025

)2334

(001

)Day

time

349

(062

)720

(031

)mdash

56 (N

A)

398

(004

)466

(037

)443

(002

)Metha

ne flux (nmol m

22 h

21 )

Nighttime

381 (547)

179 (236)

mdash18

0 (433)

170 (258)

152 (314)

524 (321)

Day

time

328 (276)

214 (635)

mdash17

6 (683)

174 (116)

200 (738)

653 (211)

[H2] (pp

m)

Stea

dy-state

107 (33)

054

(016

)mdash

067

(018

)028

(008

)mdash

mdashBubb

le46 (29)

43 (20)

29 (10)

40 (0007

6)11 (012)

mdashmdash

n5

26n

58

n5

6n

52

n5

2

Values in

paren

theses rep

resent the

SD of mea

n va

lues F

or flux mea

suremen

ts n

52 The

num

ber of rep

licates for hyd

roge

n bu

bble m

easu

remen

ts is

indicated

An SD

value

of NA in


remen


51) T

emperatures at w

hich

the

mea

suremen

ts w

ere mad

e are also ind

icated

June 14ndash15 2000

July 6ndash7 2000





DISCUSSION


BEBOUT ET AL396














BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402




DISCUSSION


BEBOUT ET AL396














BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402













BEBOUT ET AL398












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402












ACKNOWLEDGMENTS


ABBREVIATIONS


REFERENCES






BEBOUT ET AL400




























































BEBOUT ET AL402




























































BEBOUT ET AL402

long-term manipulations of intact microbial mat communities in a greenhouse collaboratory:...

Documents