Jet-Suspended, Calcite-Ballasted Cyanobacterial Waterwarts in a Desert Spring

9 Pages·2002·3 MB·English

by  Pichel-Garcia, Ferran

(2002)
JET-SUSPENDED, CALCITE-BALLASTED CYANOBACTERIAL.WATERWARTSINA
DESERT
BmanD.
tmentof Arizona State ,Tempe, USA
and
Jack
DepartmentofGeological Sciences, Arizona State ,Tempe, 85287,USA
Wedescribe a populationof colonial cyanobacteria (“pozas”),which are famousfor their beauty and the
(waterwarts)that develops as the dominant primary biological diversity they harbor (Grall 1995).
producer in a bottom-fed, warm springin bacteriaareoftendominantprimaryproducersin cal-
the Cuatro Cienegas karstic region of the Mexican careous freshwater springs (Pentecost and
Chihuahuan Desert. The centimeter-sizedwaterwarts 2000).In most cases they are sessile,benthic, or epi-
weresuspendedwithina central, conicallyshaped,6-m phytic are also typically associated with the pre-
deepwellby upwelling waters. Waterwarts were built cipitation of microcrystalline calcite (algal micrite),
byan unicellular cyanobacterium and whichinsomecasesresultsinthe formation ofmacro-
supported a communityofepiphyticfilamentouscyano- scopic stromatolitic structures (“living”stromatolites).
bacteria and diatoms but were free of heterotrophic Thealgalflorain the Cuatro Basinfreshwa-
bacteriainside. Sequenceanalysisof genes ters is no exception, particularly with regard to the
revealed that this cyanobacteriumis onlydistantly re- widespread formation of laminated calcified
lated to severalstrainsof other unicellular gal communities (Winsborough and Golubic 1987,
teria Cyanothece, Waterwarts Winsborough etal.1994).
contained orderly arrangements of mineral During a surveyof Cuatro cyanobacterial
madeupofmicrocrystallinelow-magnesiumcalcitewith communities, we noted Escobedo’s warm spring, a
high levels of strontium and sulfur. were sheltered, small,fast flowingspringwhere planktonic
95.9% glycan, 2.8% cells, and 1.3%mineral populations of marble-sized colonies of blue-green al-
grainsandhad a buoyant density of 1.034 An gae developed. Although buoyant planktonic cyano-
analysis of the hydrologicalproperties of the spring bacterialpopulationsareknown hard-waterlakes
welland the waterwartsdemonstrated that both large (Konopka 1989) and small karstic sinkholes
colony size and the presence of controlled amounts et al. the nature of the spring itself made
of mineralballastare required to prevent the popula- the presence oflarge-sizedcyanobacterialassemblages
tion from being washed out of the well. The unique anapparentparadox.First,planktonic populations do
hydrological characteristicsof the spring have likely not usually develop in swift streams and spring-fed
selectedforboth traits. Themechanismsbywhichcon- small lakes in the area. Second, planktonic popula-
trolled nucleation of extracellularcalcite is achieved tionsof large colonial cyanobacteria, such as
remaintobeexplored. or arepositivelybuoy-
antbyvirtue of their intracellular gas vesicles
Keyindexwords: buoyancy; calciteprecipitation;col-
1994)and develop in open waterswhere wind-forcing
onyformation;cyanobacteria;deserts;warmsprings
circulates them in the mixed Under
Abbreviations: BLAST, basic local alignment search tered conditions, suchasthose reigningin Escobedo’s
tool; DAPI, DGGE, spring, positively buoyant cyanobacteria should float
denaturing gradient gel electrophoresis; ICDD, In- to thesurfaceandsubsequently be washedout.
ternational Center forDiffractionData Mostcyanobacteria-dominated communitiesfound
in waters of the Cuatro Basin undergo ex-
tensive calcification. This is also the case along the
TheCuatro Basin (Coahuila,Mexico)isa shoreledgesofEscobedo’sspring, where calcifying
complex karstic systeminwhichthe underlyingCreta- anobacteria and diatoms form stromatolitic mats.
ceous limestone, dolomite, and gypsum formations However, such calcification would force the
are actively dissolved by an aquifer of distant origin. wartsto sinkto the spring’sbottom. Here we present
This results in the formation of innumerable springs, a addressing various aspects of this most un-
surfaceand underwater streams, and sinkholes usual cyanobacterial habitat.
MATERIAL\ ANDMETHODS
Received26September Accepted14 2002. and warm spring (N
forcorrespondence:e-mail 54’229,W102”04’590)surfacesthrough alarge (ca.
CALCITEBALLASTED CYANOBACTERIAL WATERWARTS 421
wide) travertine mound that isarelic of alonghistory of car- weight-to-volume ratios. The groupswere pooled anddried at
bonate spring deposition. It haslongbeenusedasalocalrecre- C overnight to obtain a measure of dry weight and then
ational area. Wevisitedthe springduringDecember 2000,car- ashedat650"Cfor6 h toobtain theashcontent.
ryingoutobservations and taking samples of the biota,andin waterwartswere
April 2001, conducting morphometric, chemical, and biologi- embedded in aliquid,50"C,1.5%aqueousagar solution. After
calmeasurements andsampling.Abathymetricmap (Fig.1)of agar solidification, thick sections (1mm) were cut from agar
the present springwas done by triangulation from the shore blockscontainingwateiwartsusing a tabletop microtome. Sec-
and manual depth sounding. Flow measurements were con- tions were placed on Petri dishes to prevent desiccation
ducted thewestern inletandthemain outlet byspatialinte- andobserved within 1-3h.Some sections werestainedwith
grationofpoint offlowvelocitytakenonacross- diamidino-2-phenylindole (DAPI,Sigma)byimmersion in a
sectional plane, ca. 30 cm apart.Watervelocitywas measured solution for 25min andwashed for min in distilled
withan probe (GlobalWater Instrumentation,Inc., water. Cyanobacterial cells were adequately stained, demon-
Gold River, USA).Dissolvedoxygenandtemperature pro- strating complete penetration of DAPI into the sections. For
files were measuredwith oxygenmeter (YellowSprings In- overall morphology, sections were photographed nnder a
struments,YellowSprings, USA)attached to a submersible lion dissectingphotomicroscope (Nikon, Tokyo,Japan)
sensor. Light intensity (downwelling irradiance)depthprofiles with basal illumination. For determination of the distribution
weremeasured with aLiCormeter (LiCor, Lincoln, NE,USA) ofheterotrophic bacteria,DAPI-stained sections were
attached to a submersible cosine-corrected PAR sensor and photographedunder oil immersion with aNikon Eclipse
Cor).Samples ofwaterwarts were collected by diving,using a dual-interference photomicroscope
small fish net. Samples for molecular analyses were placed in (Nikon)under excitation (excitation, 340-380nm;emission,
sterile microcentrifuge tubes. immediately frozen in liquid 435-485nm).Blue excitation (excitation.4.50-490; emission
on site, transported, and kept frozen (-80"Cor lower) until wasused todetect gand red
DNAisolation. Samples forvolumetric,gravimetric, andminer- cence. Initial mineralogical analyses were carried usinga
alogical analyses were stored in local water in a plastic kon Eclipse E600 polarizing photomicroscope (Nikon) under
and transported to the laboratoryforanalyses. Samples bright-field (plane-polarized)andcrossed-polarized illumination.
for analyses were immediately fixed with 3% partitioning Watenvarts were collected,
formaldehyde. Unfixed samples remained apparently un- strained, blotted, andtheirvolumemeasuredasexplained above.
changed forseveralmonthsifkept at room temperature under The suspensionwas blended to uniformity twice for 1min in a
gentle shaking and moderate light intensities (50-100 blender and dispensed into conical-end plastic
PAR).Storageat4"C caused apparentdisin- centrifuge tubes (Dynamic Corp, New Hartford, CT, USA).
tegrationofthecellular fraction within3-4weelts. Tubeswere for40minat andthe clear super-
For size distribution natant decanted. Fifty milliliters of distilled water was added,
deterinination, 100 were randomly selected, depos- and the mixture blended, centrifuged, and decanted again.
ited on a Petri dish containing 1%solidified agar, and their Pellets were collected and resuspended in 200 of'distilled
maximum diameter measured to the nearest millimeterwith a water to which 0.5 of a detergent solution (RBS
ruler.For the determination ofbuoyant density,fivegroupsof ScientificProducts, Brisbane, CA,USA)was added. The
waterwarts were collected, strained, blotted on paper towels, mixturewasblended again 10timesfor seach,allowing1-2
and weighed to yield around g each. Theywere min in between for the to settle. After two additional
pended in 50 ofdistilled water a cylinder,and hour-longsettlingtimesat C,remainingfoamwas
the newvolumewasrecorded.Waterwart volume wasobtained Atthispoint,cells,minerals,andextracellularglycancouldbe
by subtraction. Buoyant densities were calculated from the separated differential centrifugation 20 The
N
1. Escobedo's warm spring: general view(left)andbathymetricmap (right). Asamplingandreference line abovethe spring
transectingthe central wellcanbeseenin the photograph, whichisdrawn (AB) inthemap reference.
422 FERRAN GARCIA-PICIHELETAL.
supernatantswere clear, indicating that no lossof phycobilins of1 TakaraEx PCRbuffer Corporation,
had taken place, and thus cell integrity was ensured. Micro- Madison, USA), 8 of Takara mixture (2.5
scopicinspectionofthecellularfractionrevealedintactcellsaf- each),50pmol ofeachprimer,200 ofBSA,20 of5
terthisprocedure.For purification, thelastcentrifugation step PCR-enhancer (Brinkmann Instruments,
wasrepeated three times,withgentlemixing,eachtimedecant- Inc., Westbury, USA),and15 ofisolated Afteranini-
ingthepolysaccharide-rich supernatantandsubstituting it with tialdenaturation at 94"Cfor5 (hotstart),2.5 units ofTakara
distilled water. Thecellfractionandmineral phase wereseparated Ex DNApolymerasewas added to the reaction at C.
bycareful pipetting andcollectedincustom-graduated cyclesof'1min each at94"C(denaturation),60"C(an-
tubes,which were used todetermine the packedvol- nealing),and C(extension)wereperformedandthereaction
umeofeach fraction. Afterseveralwasheswithdistilledwater,the finishedwithafinalextension at72"C for 9min. Quantification
mineral fraction wasusedformineralogicalanalyses. of PCR productwasdoneasaboveforisolatedDNA.
Confirmation (DGGE),
ofmineralogy wasdoneusingx-raypowder diffraction in com- DGGEwasused toseparateandcharac-
binationwithelectron microprobe analysisofpolishedthinsec- terize genefragments.DGGE a gradient of
tions.Fine-grained mineral separates wereobtained from colo- icaldenaturants (urea andformamide)in a gel
nies by centrifuging (see above). Grains were suspended in toseparate fragmentsofequal length butdifferentmelting
ethanolandthen dispersedontoa sample holder ("zero behaviors sequence). Four hundred nanograms of PCR
background"thin plate) to form athinlayercoveringan product was electrophoresed throngh a denaturing
area about 1 X-raydiffraction analysiswas carriedout us- gradient according to etal. for4h at200Vin a
ing a Siemens automated x-ray fitted with a Bio-Rad universalmutation detection systemandana-
anode x-raytube (Cu K = 1.5406Angstroms). The in- lyzed asdescribed abovefor agarose gels. For DGGEband se-
strumentwasoperatedina summation step-scanmode at 40 quencing, each band was excised using a sterile scalpel and
(30 anddatawere obtained overa 2-thetarange of5-90 DNAallowedtodiffuseoutfor at least3claysat Cin of
degreesusinga 7-degreedetector.Mineralogicalidentifications 10 Tris OnemicroliterofthesolutionwasPCRam-
were made using an on-line powder diffraction database pro- plified using the primers, reaction mixture,
vided by the International Center for Diffraction Data (ICDD, clingconditions,andproduct quantificationasabove.Akitwas
Elemental analysesof mineral separates were used to PCRproduct (Qiagen, Inc.,Valencia,
accruedout on a Jeol JXA-8600 electron probe and ngwas commercially sequenced in twoseparate reac-
USA, MA, on polished thin sections of tions (5' to and 3' to 5'). Complementary sequences were
mineralgrain mounts. matched, aligned, and edited using Sequence Navigator (Ap-
composition. To the composi- plied Foster City, USA) and submitted to the
tionofeukaryoticandcyanobacterial formingorin- Basic Local Search Tool (BLAST,National Center
habiting the community,we microscopy,cultiva- for Information, for
tion,andfingerprintinganalyses of genes.Enrichment logenetic matching. Sequences obtained from DGGE bands
cnlturesweresetuponbothagar-solidifiedandliquid a-f have been deposited in with accession numbers
medium.Wholewateiwarts.pieces,andslurries thereof were respectively.
usedasinoculumandincubated at room temperatnre at light in-
tensitiesbetween50and100pmol Thinsections
RESULTS
(asabove)andslurries were used formicroscopy.Molecularmeth-
odsaredetailedin the followingsections. andphysical-chemicalchar-
DNA methods, designed for samples rich in Most of the spring basin is shallow m
mucopolysaccharides,were used to isolate DNA water- deep, Fig. 1).A central, 6-m deep, conical well, dug
warts.Inthefirstmethod,a single washomogenized
several decades ago by the townspeople, contains two
in 1 ofextraction buffer (see below)using a tis-
suegrinder International,WestChester,PA,USA).After thirds ofEscobedo's water volume,which amounts to
adding5 ofextraction buffer tothehomogenate,itwasex- 440 The bottom sedimentis softcarbonate sand.
truded through a chilled C)French press (American Surface water and groundwater feed the spring. A
Company,Inc.,SilverSpring,MD,USA)at800psitolyse
smallstream, originating in a nearby springhead,pro-
cells.DNAwasisolatedaccordingto etal.(1997).Briefly,
vides 0.063 of well-oxygenated, relatively cold
the sample was repeatedly frozen and thawed in extraction
buffer containing the mucopolysaccharide-precipitatingcom- water through an inlet situated at the western shore.
pound CTAB,followedbyincubationwith SDSandproteinase Water temperatures atthis inletarevariable andclose
K. Phenol, chloroform, andisoamylalcoholweresubsequently to ambient air temperature. Most of the water, how-
used to isolate DNA,which was then precipitated in isopropyl
ever,jets into thespring from severalcavernousopen-
alcohol.Inthesecondmethod,a single washomoge-
nizedasbeforebutwasnotextrudedthrough the French press. ings through the travertine at the bottom of the cen-
The homogenate was to freeze-thaw cycles and its tral well. Water exits the spring through a single
DNAisolated with acommercial plant DNAisolation kit (Mo outlet channel at the shore.The total discharge
BioLaboratories, Inc., Solana Beach, CA,USA).Forcultivated
measured through the outlet was 0.658 Mass
isolates, cell lysis was accomplished through homogenization
balance then requires that the deep source account
andfreeze-thaw cyclesas described above, but in Tris
buffer instead ofextraction buffer. In addition. cells were incu- For at least 90% of water inputs. According to the
bated in a gentlyboilingwaterbath for 10 Quantification above values, the average residence time of water in
of isolated DNA was done with standard agarose gel electro- the spring is 669 (11.1min). The water from the
phoresisfollowedbyethidium bromide staining.Gelswereana-
deep source is warm C) and severelydepleted
lyzedin aBio-Rad system (Bio-Rad Lab-
oratories, Hercules, USA) using a Bio-Rad EZ in dissolved oxygen (around 4% air saturation) but
precision molecularmassruler. does not bear appreciable hydrogen sulfide. Colder
PCR of Primers (denser) water from the western spring inlet flows
and et al. specific cyanobacteria
into Escobedo without mixing completely and sinks
and plastids, were used for amplification of ca. 600base pair
down into the mainwellwhere turbulent mixingwith
long genefragments in a Bio-Rad
cycler.Each PCRreaction contained the following: 10 deep, warmer water occurs. A lane of dark mineral
CALCITEBALLASTED 423
precipitate5 (probablyMn-bearing) on thespringsed- colonies suspended by the upward flow within the
iment marks its course.Mixing cold andwarm waters boundsofthemainwellconstituted themostconspic-
then flowupward and in a direction toward the uous component of primary producers. Because of
outlet. Because of the large contribution of the low their morphology, we refer to them as waterwarts
warm source, and the short residence time, the (Fig. 3).We estimate that the population in the well
entire spring basin is poor and warm even in the may have been in tlie tens of thousands of individual
shallowsand close to the surface.Vertical profiles of watenvarts. A small portion of this population was ly-
temperature, dissolved oxygen,and light penetration ingon the steeplyslopedsedimentof thewell,partic-
for the mainwellarepresentedinFigure2.Thewater ularly on southern side. Interestingly, this large
was clear with spectrally averaged transmittance population was not alwaysvisible during our observa-
around 65% in thePARregion. tional period. During episodesoflowwater flowfrom
Unicellular planktonic primary pro- the bottom springs, waterwarts sank to the well bot-
ducers were not observed in water sample5 nor were tom and rolled into its cavernous openings, com-
conspicuousbenthicbloomspresentonthesediment, pletely disappearing from view until flow resumed
even though they were all within the euphotic zone again. The episodes of waterwart disappearance we
(Fig. 2). Heterogenous stromatolitic assemblages of were able to witness lasted on the order of hours to
calcifyingcyanobacteria and diatoms,typicalof many days.
springsin theregion (Winsboroughetal. were composition. Waterwarts were
present on the shore ledges only. A population of composed of roughly spheroidal to elongated colonies
brown, centimeter-sized, gelatinous cyanobacterial of bacilloid unicellular cyanobacteria embedded in a
large amount gel-like glycan (Fig.3).According to
botanical taxonomy (Komarek andAnagnostidis
they would be assigned to the genus Ac-
Temperature cordingto bacteriological taxonomy (Castenholz
they would be assigned to the genus How-
32 33 34 35 ever, presently recognized genera of unicellular
I bacteria do not represent phylogenetically coherent
units at the molecular or biochemical level, including
Oxygen (% air saturation) and (Garcia-Pichelet al. 1998).
We thus refrain from using taxonomic epithets that
mightinduce unfounded ecologicalorphysiologicalas-
sociations.Thecyanobacteria were distributed
out the colony, although higher population densities
occurredattheperiphery.Theglycanwascolorless,and
theoverallbrownappearanceofthecolonieswasdueto
the cell’s phycobilin complement (abundant
One to several of these colonies, together
with smaller budding new colonies,formed a cohesive
waterwart. mean waterwart size (max. diameter)
was0.995cm (n andthe rangewas cm,
but the sizedistribution wasskewedtowardlargersizes.
Themodalsizewas0.8 Microscopic observations of
thick sections (1mm)revealed thein-
terior ofthecolonieswascompletely ofbacteria
other than cyanobacteria. The waterwarts supported a
diverse assemblage of epiphytic microalgae, which in-
cluded cyanobacteria and diatoms
(mostly .This assemblage was alsofound
intheinternal parts ofwaterwarts,but onlyonthecon-
tact surface between colonies and not within theglycan
ofeachcolony. Mineral werealwaysobserved
within watenvarts. Crystals were white to orange and
ranged in length from 30 to The crystallites
were most abundant on the contact surface between
103 104 colonies,but somemostlylarger were always
present within the extracellular glycan of colonyinteri-
Irradiance (PAR, ors (Fig. 3).The surface of the crystalliteswas free of
bacteria, asjudged by staining. Differential cen-
trifugation yielded the following volumetric partition-
2. Vertical profiles oflight oxygen
andtemperature (0)inEscobedo’s well. ing:glycan cells andmineral grains
/ -
424 F E WGARCIA-PICHELETAL.
FIG.3. AspectsofEscobedo'swaterwartpopulation andtheirstructure. (A)Underwater viewofEscobedo's deepsource (from
above)withcloudsofwaterwartsbeingpushed upwardbythejettingwaters. (B)Underwaterviewofthepopulationofwaterwartsat
shallowdepthafterdisturbancebyaswimmer (B.D.W.).(C)Photomicrograph ofasectionedwaterwart,madeupofloboseglycan
colonies. Dark areascloseto the surfaceareaccumulations ofbrown-colored unicellular cyanobacteria.Stringsofcrystallites(dark
dots) can he seen at the contactsurface ofadjacent coloniesand deepwithin a colony,inside the glycan matrix. (D)Photomicro-
graphoftheexternalwaterwartappearance. (E)Close-upofacontactregionbetween individualcoloniesshowingthepopulationsof
unicellularcyanobacteriaintheglycanmatrixandagroupofcrystallites.Noticetheextracellularsheathsandtrichomesofepiphytic
cyanobacteria. (F)Glycan,cells,andmineralgrainsfrombulkwaterwartslurryafterdifferentialcentrifugation.
of the total waterwart volume. With respect to their cyanobacterial line of descent (cyanobacteria and
weightcomposition,waterwarts were 96.2%water (blot- plastids) using DGGE separation demonstrated the
ted wet weight ratio) and 3.78% presence of at least six well-differentiated
solids. The warts had an average buoyant density of genealleles(Fig.4).Oneofthese alleles (field sample
1.034 0.014 significantlylighterthan the aver- bandb in Fig.4)wasbyfarthemostcommonproduct
age buoyant density of typical bacterial cells. Experi- in thePCRamplification, regardless oftheDNAisola-
ments conducted in the laboratorydetermined that an tion method used, and likelycorresponds to the uni-
averageminimal upward flowvelocityof0.76 cellular cyanobacteria forming the colonies. BLAST
neededtokeepwaterwartssuspended. similarity analyses of DNA sequences (ca. 600 base
Identityof waterwart component. Microscopic pair long), obtained after excision, reamplification,
observation indicated that the bulk of waterwart bio- andsequencingofDGGEfieldbands,clearly associate
mass, responsible for its formation, corresponded to field sample band a to the diatom plastids line of de-
colonial unicellular cyanobacteria. However,attempts scent with 97% similarity to Amphora, and
to obtain them in culture using various combinations Odontellaplastids. BLASTanalysis of the main band b
of culture conditions failed repeatedly. Enrichment did not find very close matches, yielding a group of
cultures yielded typically Pseudanabaena, unicellular cyanobacteria glauca,
and filamentous cyanobacteria, as well as othecePCC 7424,and CyanotheceATCC 51142) as
diatoms .Allwere likelyenriched from the closestmatches, but onlywith 89%sequence similar-
epiphytic community. Culture-independent finger- ity.Additionally, this sequencewas93% similar tovar-
print analysis of the waterwart community based on ious isolates, albeit only 400basepaircould
PCRamplification of genesspecificfor the be compared in this case. No clear matches were
CALCITEBALLASTED CYANOBACTERIAL WATERWARTS 425
a
a,
TI. FIG 4 Denaturing gradient gel
6) electrophoreticseparation (DGGE
gerprint)of gene fiagments
0 PCR amplified specifically from
phototrophs and
Amplified fragments
equallength,buteachbandrepresents
a genealleleofuniquemelt-
ing characteristics (sequence) Isolated
DNAfromfield samples of
or isolated from unialgal, clonal
from enrichment cultures of
were used as template for
separate PCR amplification and the
products loaded in each lane (as la-
beled). In the case of field samples,
two different DNA icolation methods
used (A and B), and each
loadedintwoseparate lanes foreaseof
Specific arelabeled
fordiscussion (see text)
found for the remaining field bands (c,d, e, and the primary diffraction peak from an expected
Band c had sequence similarity to various of 3.030 Angstroms (purecalcite) to an observed
strains of and Se- of 3.028 Angstroms (Fig. 5).This suggestiveof some
quences from bands c, d, and f were very similar typeoflimited cation substitution.Electronmicroprobe
among themselves and all gave a strain of analysisofindividualmineral grains (Table1)revealed
as the closest cultivated match, thatthe oflowmagnesiumcalcite,with
but againwith only 90%similarity. With respect tothe an averageMgcontent of 1.76%byweightandwith no
identity of the isolates enriched from waterwarts, sev- measurable iron. Strontium averaged 1.74%by weight.
eral observations are interesting to note. Isolates 1 Surprisingly, averaged 2.03% byweight.
and6, morphologically a thin 1.5 widegreen
had indistinguishable DGGE band positions DISCUSSION
between them, but this band position was not repre- Natural selection Escobedo’s
sented in thefield sample fingerprints, indicating that water residence time (11min) is significantlyshorter
they were not among the dominant epiphytes. The than the fastest doubling times measured in any mi-
same holds true for isolate 2,a 2-3 wide croorganism. This is the likely reason that no typical
Isolate morphologically, an 8-10 wide planktonic populations exist there.Tomaintain a sta-
matched the position offield band but itis ble population, attachment to resist flow, or move-
yetunclear if thisimpliesalsosequencesimilarity.Iso- ment to oppose it, is necessary. The lack of benthic
lates 3 and 5 were brown, phycoerythrin-containing, populations may be due to efficient grazing on the
3-5 wide filamentous forms, morphologically as- soft sediment by local fish populations. Sinking by
signableto TheDGGEfingerprintsofiso- gravity may provide the necessary opposing force to
lates 3 and5arequite intriguing because theyseem to theupward flow of waterwithin themainwell.Averti-
contain at leastfour divergent gene alleles cal profile of space-averaged upward flow velocity
andcoincidewithfieldbands c-f. within the central well was calculated from total
Identity mineral The mineral precipitate spring discharge and well cross-sectional area as a
found within the colonies was determined to be cal- function ofdepth (Fig.6).According tothese calcula-
cite using a combination of optical and tions,minimalsinkingordownward swimming speeds
x-ray diffraction analyses. Optically, the crystallites between 1.2and0.5 areneeded tokeepa pop-
showed a bladed internal fabric, suggesting that they ulation deeper than 1m, thus avoiding lateral flow
are in fact crystal aggregates rather than single crys- and Maximal swimming speedsforunicellu-
tals. X-ray diffraction results revealed that although lar prokaryotes andeukaryotes (ca. 0.6 and 1
the mineral fraction belonged to the calcite family, respectively, Garcia-Pichel are too slow by one
there was a consistent displacement of d-spacing of order ofmagnitude to counteractsuch flowrate.The
FERRAN ETAL.
5. X-ray formin-
eral separates obtained by
of Ma-
3 jor and minor peak positions are
indicativeofcalcitewith butcon-
sistent shift from expected values for
calcite. peak position
2000 forcalcite based onICDDda-
tabase (entry 240027) corresponds to
a 2-theta angle of 29.453 (d-spacing
0 I I I I I 1
55 1100 2200 3300 4400 5500 6600 7700 8800 9900
2-Theta
sinking of roughly spherical particles in fluids is well millimeter- to aggregates. Subtract-
described by Stokes Law, provided that the particles ing the experimental value of average upward flow
sink in an environment of low Reynolds Num- to levitate waterwarts (0.76 from the
ber. The Reynolds Number gauges the impor- flowvelocityprofile in Figure 6,one can estimate the
tance of versus inertial forces that oppose actual average net speed and direction waterwarts
movementwithin afluid. StokesLawstates that parti- atdifferentdepthswithin thewell (indicatedas veloc-
cles will sink with a constant, terminal velocity, V, ity vectors in Fig. 6).According to this model,
equalto 2g (p (9 where isthedensity
oftheparticle andgistheaccelerationofgravity.Ter-
minal sinking velocities, with 1,of0.22 Upwelling water velocity
(or2.17 canbecalculatedfortypical 1
wide bacterial cells (1.08 Guerrero et al. 0.5 2 2.5
1985).Because this velocity is much smaller the
average upward fluxvelocity0.5-1mdeepinthewell,
typical bacteria would be easily swept away. Solving
Stokes equation to match the flow velocity at 1 m
depth (0.56 andusing 0.007 and
0.997 typicalforfreshwaterat we
determine that to maintain single microbial cells
within thewell,theyshouldbeatleast3mmindiame-
n
ter. In fact, this situation is no longer appropriately
described by Stokes Law,because the corresponding
is larger than one and inertial forces become im-
portant. It is clear that the hydrological regime
present in Escobedo during active flow periods will
tend towash outplanktersof small sizeand selectfor
TABLE 1. Elemental composition for mineral grains extracted
from Escobedo's accordingto electron microprobe
analysis.
cont t Standard ot
Element j
1.76 0.69 28
Fe b.d. 28
2.84 0.84 25"
1.74 2.45 (0-12.7) 28
FIG. 6. Vertical profile of space-average upwelling wa-
b.d. belowdetectionlimit. Minimum detection for tervelocityin Escobedo's mainwell.Flowshavebeencalculated
Fewas0.053,for-Mg forS andforSr0.026. well cross-sectional area and spring outflow. Drawn at
Three analyses were run before the addition of a depth isthe calculatedaverage vertical velocityvector
standard. asafunctionoflocalflowandsinkingspeed.
CALCITEBALLASTED WATERWARTS 427
warts are pushed upward rapidly at depth, slowing wart’s glycan could be such that initial steps of calcite
down as they reach the average depth of zerovelocity nucleation are prevented at sites and yet pro-
(approx. 1.35 m). Above that depth they sink moted or allowed at particular sites, so that enough
into the well. This simple model explains the contin- ballast is attained. The preferred distribution of cal-
ued presence of thewaterwart population in thewell, cite in the interfaces between individual colonies and
with thesingleimposed parametric valueof appropri- the orderly arrangement of microcrystalline aggre-
atelevitationvelocity.Thefactors affecting suchveloc- gatesimplies such acontrolled mechanism, but direct
ity, under obvious biological control,are discussed in evidenceforthis hypothesis must be sought in chemi-
thefollowingsection. cal analyses purified glycan. Interestingly, the fact
Given that much of the calcite isfound at the interfaces be-
the measured buoyant density (1.034 of tweencolonies,incloseproximityto theepiphytic
terwartsandtheirvolumetric partitioning intoglycan, croalgae, suggests the epiphytes may control ballast
cellular,and mineral fractions, some relevant calcula- precipitation. If this were corroborated,itwould indi-
tions are possible. First, it can be calculated that with cate that the coordinated action of the community as
typical bacterial buoyant cell densities of 1.08 a whole, not the unicellular cyanobacteria form-
a typical density for calcite of 2.75 cells ing the colonies themselves, is needed to achieve hy-
contribute 0.030 to the buoyant density of drodynamically stable
terwarts,whereas calcite concretionscontribute 0.035 Strontium incalcite. Furtheranalysesof the
The rest, 0.968 is contributed by the calcite ballast is warranted given the consistent dis-
hydrated glycan. The buoyant density of the glycan placement of d-spacing of the primary x-ray diffrac-
fraction must be ca. 1.010 very close to the tion peak and the percent averages by weight of Sr
density at C.Thus,striving to increase size and in the calcite. Cyanobacteria have been in-
byaccumulatinglargeamountsofextracellular glycan volved with the nucleation of strontium calcite else-
will be at the expense of a loss in buoyant density of where (Ferrisetal. 1995).TheeffectofSrsubstitution
thecolonyandpartlydefeatthe purpose ofincreasing onlatticestructureandcrystalformandthecoordina-
sinkingvelocity. Without the mineral fraction (main- tion siteforsulfurarestillunderinvestigation. Ofspe-
taining cellsand glycan in the same volumetric ratios cialinterest in this regard isthe precise
as determined) the waterwart’s density should be cal process that controls carbonate precipitation, the
1.012 Thus, the mineral fraction, although role theglycanmatrix incrystalnucleation,andthe
small, makes a significant contribution to the water- potential for biosignatures specific to
wart’s ballast andallowsit to sinkataspeedsufficient thisenvironment.
toremainwithin theboundsofEscobedo’smainwell.
ballast. Whereas most CONCLUSIONS
algae in the region are calcifying and stromatolitic The particular hydrological conditions created in
(Winsboroughand Golubic 198’7,Winsborough et al. Escobedo’sspring after digging the central well dur-
including other algal communities in Esco- ingthe 1960slikelysetthe stagefor the selection ofa
bedo’s spring, waterwarts apparently control this pro- unique form ofjet-suspended, calcite-ballasted, colo-
cess. Under a purely thermodynamical view, the nial community simplebut appar-
terwart’s main constraint in controlling extracellular ently sufficient adaptations to the local environment.
mineral ballast isnotsomuch howtopromote itsfor- In the case of Escobedo’s spring “buoyostat,”the se-
mation but rather how to avoid its generalized occur- lectiverules aresimple:too heavyand sink
rence. In macroscopic assemblages of photosynthetic and are eventually buried in the soft turbated sedi-
organisms (such as benthic biofilms or large colo- ment, or too light and they be washed out down-
nies),diffusion limitation of masstransportpromotes stream. Only within the physical bounds allowed
localized photosynthesis-driven increases in environ- the hydrodynamic regime, their fitness coefficient is
Thisin turn raisesthe carbonate ion con- 1;otherwise, it is 0. In our view, the combination of
centration,further increasing the calcite macroscopic colony formation and controlled calcite
factors in the immediate vicinityof the cells (de precipitation represent a solution to the prob-
Vrind-de and deVrind 1997).To avoidgeneral- lem, because both traits are common
ized calcite precipitation in the waterwarts, the initial bacteria. We are not aware of the presence of sus-
nucleation process must be prevented. It is known pended waterwarts in other springs in the area or
thatpolymeric substancescan act to modifythenucle- elsewhere.Morphologically similar (macroscopic
ation energy for calcite (Manoliet al. 199’7,Hosoda colony-forming cyanobacteria areknown
and Kato 2001). Both promotion and prevention ef- from benthic environments, but they only rarely and
fectscan be found in the chemically diverse temporarily become planktonic (Komarek and
polymers of cyanobacteria. Pentecost and Bauld 1999). It isinteresting to speculate thatEsco-
(1988)demonstrated that extracellular glycansheaths bedo’s waterwarts may have originated from benthic
of some and promote nucleation, colonial forms already adapted to life soft spring
but not those of and We sediment,similar to those described for
suggest that the chemical composition of the water- (DoddsandCastenholz 1987).