Calcium dynamics in microbialite-forming exopolymer-richmats on the atoll of Kiritimati, Republic of Kiribati, CentralPacificD. IONESCU,1 , 2 S . SPITZER,3 A. REIMER,3 D. SCHNEIDER,4 R. DANIEL,4 J . REITNER,3

D. DE BEER2 AND G. ARP3

1Leibniz Institute for Freshwater Ecology and Inland Fisheries, Experimental Limnology, Neuglobsow, Germany2The Max Planck Institute for Marine Microbiology, Bremen, Germany3Geosciences Center, Georg-August University of G€ottingen, G€ottingen, Germany4Department of Genomic and Applied Microbiology and G€ottingen Genomics Laboratory, Institute of Microbiology and

Genetics, Georg-August University of G€ottingen, G€ottingen, Germany

ABSTRACT

Microbialite-forming microbial mats in a hypersaline lake on the atoll of Kiritimati were investigated with

respect to microgradients, bulk water chemistry, and microbial community composition. O2, H2S, and pH

microgradients show patterns as commonly observed for phototrophic mats with cyanobacteria-dominated

primary production in upper layers, an intermediate purple layer with sulfide oxidation, and anaerobic bot-

tom layers with sulfate reduction. Ca2+ profiles, however, measured in daylight showed an increase of Ca2+

with depth in the oxic zone, followed by a sharp decline and low concentrations in anaerobic mat layers.

In contrast, dark measurements show a constant Ca2+ concentration throughout the entire measured

depth. This is explained by an oxygen-dependent heterotrophic decomposition of Ca2+-binding exopoly-

mers. Strikingly, the daylight maximum in Ca2+ and subsequent drop coincides with a major zone of arago-

nite and gypsum precipitation at the transition from the cyanobacterial layer to the purple sulfur bacterial

layer. Therefore, we suggest that Ca2+ binding exopolymers function as Ca2+ shuttle by their passive

downward transport through compression, triggering aragonite precipitation in the mats upon their aerobic

microbial decomposition and secondary Ca2+ release. This precipitation is mediated by phototrophic sulfide

oxidizers whose action additionally leads to the precipitation of part of the available Ca2+ as gypsum.

Received 21 July 2014; accepted 3 November 2014

Corresponding author: D. Ionescu. Tel.: +49 33082 69969; fax: +49 33082 69917; e-mail:

ionescu@igb-berlin.de

INTRODUCTION

Microbialites (Burne & Moore, 1987) form excellent

archives of past environmental conditions due to the

dependency of many microbial processes on external fac-

tors such as pCO2, temperature, salinity, light intensities

and redox conditions. Therefore, the specific characteristics

of microbialites in marine sedimentary successions poten-

tially provide important information on palaeoenvironmen-

tal parameters in Precambrian to Phanerozoic oceans (e.g.,

Kempe & Kazmierczak, 1990; Riding, 2000).

Mineral formation is influenced by a broad suite of dif-

ferent metabolic processes that can lead to changes in ion

activities and mineral saturation states (see, e.g., Arp et al.,

1999b; Dupraz et al., 2009 for review), dependent on the

bulk hydrochemistry of the environmental site.

While some principal relationships are recognizable (e.g.,

the buffering effect of the dissolved inorganic carbon pool;

Arp et al., 2001), and some mechanisms have been known

for many decades (e.g., effect of photosynthesis on carbon-

ate equilibrium; Cohn, 1864; von Pia, 1934), the effective-

ness is less clear for specific cases. For example, the effect

of sulfate reduction and other heterotrophic processes is

still debated (see Arp et al., 2003; Aloisi, 2008; Meister,

2013, 2014 and Gallagher et al., 2014 for present discus-

sion). Additionally, while oxygenic photosynthesis leads to

© 2014 John Wiley & Sons Ltd 1

Geobiology (2014) DOI: 10.1111/gbi.12120

carbonate precipitation under current atmospheric condi-

tions, it probably played an insignificant role on ancient

Earth given that the first stromatolites are dated much

older than the onset of atmospheric oxygen increase (Sree-

nivas and Murakami, 2005; Sessions et al., 2009). Thus,

chemolitho(auto)trophy may have had a greater impor-

tance on early Earth.

Furthermore, the sometime stimulating, but, more com-

monly, inhibiting effect of foreign ions and extracellular

polymeric substances (hereafter exopolymers) on mineral

nucleation is only partly understood. Indeed, it is postu-

lated that exopolymers not only play a role in the nucle-

ation process, but also affect mineral saturation states in

the microenvironment due to their Ca2+-binding capacities

(e.g., Braissant et al., 2007).

The binding capabilities of exopolymers depend on the

presence of active moieties (e.g., –COOH, –NH3, –SH, –

OH) which can bind divalent cations (Pentecost, 1985;

Decho, 1990; Kawaguchi & Decho, 2002; Braissant et al.,

2007). In turn, the ability of such moieties to scavenge

cations from the matrix depends largely on pH, as binding

increases at elevated pH (Ferris et al., 1989). Thus, exo-

polymers are prone to inhibit calcification in most alkaline

environments or phototrophic zones. In contrast, microbial

degradation of exopolymers is believed to release Ca2+ into

the mat porewater and thus to create a local zone of car-

bonate supersaturation leading to mineral precipitation

(Trichet & D�efarge, 1995; Arp et al., 1999b; Dupraz

et al., 2004).

The investigation of present-day mineralizing benthic

microbial communities (Burne & Moore, 1987), their exo-

polymers and microenvironmental conditions is therefore

crucial to understand past microbialites and their palaeoen-

vironmental significance (Monty, 1977; Kempe & Kazmi-

erczak, 1990; Laval et al., 2002).

Microbial mats on Kiritimati (Trichet et al., 2001) are

well suited for the study of microbial processes involved in

mineral precipitation because of their high mat thickness,

clear lamination, and present-day microbialite formation.

In a previous study, we inferred from water chemistry, mat

thin sections and phylogenetic data that heterotrophic deg-

radation of exopolymers and the associated decrease in

inhibition of nucleation could be crucial for microbialite

formation (Arp et al., 2012). This because oxygenic photo-

synthesis substantially increases aragonite supersaturation in

top parts of the mats, but precipitation is nonetheless lar-

gely located near the basis of the cyanobacterial layer,

where the exopolymer fabric changes due to degradation

(Arp et al., 2012). However, detailed physicochemical data

from within the mat were not available to prove this inter-

pretation.

In March 2011, we conducted a new 2-week expedition

to the island of Kiritimati, to study potential changes in

the hypersaline lake mats since the first field trip in

August/September 2002 and to measure calcium dynamics

and degradation processes with high spatial resolution.

METHODS

Sampling

Sampling was carried out in March 2011 on the atoll of

Kiritimati during a 2-week expedition. Samples were col-

lected from Lake 21 (1°57043.88″ N 157°20000.54″ W).

The sampling locations are shown in Fig. 1. Cores and

mat sections were collected from areas 1 and 2 of Lake 21.

Samples for microprofiling were transferred to a field labo-

ratory immediately upon collection where they were placed

in a deeper vessel and covered with freshly collected water

from the respective area to minimize the formation of

anoxic conditions in the overlaying waters. Mat sections

were used for up to 48 h after collection.

Water chemistry

Water samples for determination of major anions and

cations were collected in pre-cleaned PE-bottles. Samples

for cation analysis were filtered in the field through 0.8-lmmembrane filters (Millipore, Billerica, MA, USA) and fixed

by acidification. Samples were stored cool and dark until

laboratory measurements. Temperature, electrical conduc-

tivity, pH, and redox potential of water samples were

recorded in situ using a portable conductivity meter

(WTW GmbH, Weilheim, Germany) and a portable pH

Fig. 1 The Atoll Kiritimati (central panel) and the sampled sites. Lake 21,

Area 1 and 2 (upper panel). Area 3 (constantly inundated) refers to the

entire ‘pelagic’ part of the lake and is therefore not marked in the map.

The maps were generated using the GOOGLE EARTH software (Google,

Mountain View, CA, USA).

© 2014 John Wiley & Sons Ltd

2 D. IONESCU et al.

meter (WTW GmbH) equipped with a Schott pH-elec-

trode calibrated against standard buffers (pH 7.010 and

10.010; HANNA instruments, Woonsocket, RI, USA).

Major cations (Ca2+, Mg2+, Na+, and K+) and anions (Cl�,F�, Br�, and SO4

2�) were analyzed by ion chromatogra-

phy with non-suppressed and suppressed conductivity

detection (Metrohm, Herisau, Switzerland), respectively.

ICP-OES (Perkin Elmer, Waltham, MA, USA) was used to

determine B, Sr, Ba, Fe, and Mn. Dissolved silica concen-

trations and nutrients (NO2, NH4, PO4) were measured

by spectrophotometric methods (Unicam, Leeds, UK).

Microprofiles

O2, pH, and Ca2+ profiles were measured with Clark-type

and liquid ion exchange microsensors (Revsbech, 1989;

Santegoeds et al., 1998). To minimize the risk of break-

ing the sensor on occasional contact with hard mineral,

all sensors were thick walled with an external tip diameter

of 200–300 lm; however, with a sensing diameter

<50 lm. The microsensors were mounted on a 3-axis

micromanipulator (MM 33; M€arzh€auser, Wetzlar, Ger-

many). The vertical axis was motorized for l-positioning(VT-80 linear stage; Micos, Germany, equipped with a

3564-K-024-BC motor; Faulhaber Group, Sch€onaich,

Germany), and measurements were controlled by

l-PROFILER software (www.microsen-wiki.net). Measure-

ments of all three parameters were conducted simulta-

neously with the sensors <1 cm apart from each other. The

measurements were conducted at 100 lm steps with three

sequential measurements being recorded at each step. Sev-

eral locations in the mat were measured, each until steady

state was obtained. Light was supplied via a Schott KL 1500

LCD lamp (Schott AG, Meinz, Germany) at an intensity of

~2000 lE to mimic the strong environmental light. The pH

and Ca sensors were calibrated against known buffers. Both

Ca and pH microsensors are known to function for short

period of times, thus new sensors were freshly prepared each

day. The O2 sensor was calibrated against air saturated sea

water and brines to account for salinity effect on the sensor.

Light profiles were measured using a flat-end fiberglass

encased in a needle, connected to a USB-4000 spectropho-

tometer (Ocean Optics, Dunedin, Fl, USA; Al-Najjar

et al., 2012) and mounted on a similar microprofiling sys-

tem as above.

RESULTS

Site description and water chemistry

Lake 21 has three main areas: (1) the nearshore area – a

narrow strip of water periodically separated from the main

lake by a 1–3 m line of encrusted microbial mats, (2) the

shallow part of the main lake area, and (3) the deep lake.

Waters of Lake 21 are formed from seawater by evapora-

tion, with Ca2+ reduced due to aragonite and gypsum pre-

cipitation (Table 1). Therefore, alkalinity is lower than

expected by evaporation. Concentrations of silica are

higher than in standard seawater due to the scarcity of dia-

toms that normally take it up for their frustules.

Area 1 is influenced by rain water from the lake sur-

roundings, reducing the water salinity (36 &) and the con-

centrations of Ca2+ (10 mmol L�1) and other ions. Area 2

lies immediately beyond the line of encrusted mats. It is

constantly inundated, has a high salinity (about 140 &),

Ca2+ concentrations of about 28 mmol L�1, and is

exposed to high light intensities (>2000 lE m2 s�1; mea-

sured on 11.03.2011). Area 3 is constantly inundated (up

to 2.5 m), hypersaline (about 172 &), with Ca2+ concen-

trations of about 30 mmol L�1. This area is exposed to

less light and is too deep for wind-driven water movement.

Numerous microbial, chimney-like structures, through

which water of reduced salinities (94 &) and Ca2+ concen-

trations enters the lake, were found in this area of Lake 21

(Fig. 2). All chemical analyses of the water columns above

investigated sites in Lake 21 as well as a seawater sample

from Kiritimati are given in Table 1.

Mat structure

The microbial mats investigated by microsensors (area 1

and 2) show a clear color zonation according to their

microbial community structure. Top sections consist of

orange cyanobacteria-rich layers (L 1 and 2). These are

followed by a green cyanobacteria-rich layer (L 3), a pur-

ple bacterial layer (L 4), and finally reddish to brown

basal layers (L 5 to 9). Note that a gypsum crystal rich

layer and change in mat consistency between L 5 and L

6 marks a former subaerial exposure and discontinuity to

an older, now overgrown microbial mat (i.e., the mat

investigated in 2002: Arp et al., 2012). In total, the

entire microbial mat complex attains thicknesses up to

17 cm (Fig. S1).

The 16S rRNA gene community profile of the microbial

mat of Lake 21 was presented in Schneider et al. (2013)

and was reconstructed here based on the original sequence

data using the SILVA NGS pipeline (Ionescu et al., 2012)

(Fig. 3). The sequenced microbial mat was obtained from

a shallow site at Area 2 of Lake 21 and shows oxygenic

mat parts (L1–3), a macroscopically less obvious purple

layer (L 4), but thick reddish to brownish anoxic basal lay-

ers (L 5) including an older, overgrown mat generation (L

6 to L 9).

In the upper layers of these mats Euhalothece-like cyano-

bacteria (Fig. S2) are the dominant bacterial phototroph

(24% of sequences). Additional light-utilizing bacteria

found in the upper layers of the mat include Salinibacter

sp. (<19%) and Salisaeta sp. (<10%).

© 2014 John Wiley & Sons Ltd

Calcification in EPS-rich microbial mats 3

Tab

le1

Hyd

rochem

ical

properties

ofthewater

from

thedifferentsamplinglocationin

Lake

21an

damarinereference

site

Sample

location

Gen

eral

water

properties

andnutrients

Date

Dep

thSalinity

Den

sity

EC25°C

pH

TpH

Ehpe

TA

O2

NO

2NH4

PO

4

pCO

2ppCO

2

m&

20°C

mScm

�1

°CmV

meq

L�1

lmolL�

1latm

logatm

Seaw

ater

19/03/2011

0.10

34.7

1.0246

53.0

8.360

28.4

––

2.31

197.9

0.14

–0.44

269

�3.57

SESh

ore

Station1(Area2)

13/03/2011

0.05

140.5

1.1051

175.2

7.486

39.9

�30

�0.48

4.79

111.6

0.25

0.78

0.23

3802

�2.42

SESh

ore

Station2(Area3)

13/03/2011

0.10

171.8

1.1290

189.8

7.948

31.7

91

1.51

4.84

78.6

0.05

0.01

0.02

871

�3.06

ECorner

Shore

Station(Area1)

13/03/2011

0.05

35.6

1.0253

53.2

8.705

36.7

133

2.17

3.05

–0.65

2.11

0.42

110

�3.96

Lake

center–Su

rface

14/03/2011

0.10

171.7

1.1289

192.2

8.077

26.5

––

4.80

–0.04

0.00

0.06

575

�3.24

Lake

center–Bottom

14/03/2011

1.20

171.7

1.1288

192.3

8.085

26.3

––

4.80

–0.12

0.25

0.17

562

�3.25

Lake

center–Chim

ney

15/03/2011

1.00

94.4

1.0700

124.8

8.033

28.7

––

3.44

––

––

676

�3.17

Sample

location

Majorions

Ca

Mg

Na

KSr

Ba

FeMn

SiB

FCl

Br

SO4

mmolL�

1

Seaw

ater

10.4

53.6

474

10.3

0.093

0.000048

0.00110

0.00015

0.0036

0.41

0.053

552

0.85

28.6

SESh

ore

Station1(Area2

)27.5

243.6

2090

45.4

0.342

0.000091

0.00322

0.00049

0.1067

2.03

0.296

2425

3.69

122.6

SESh

ore

Station2(Area3)

29.3

298.5

2633

57.1

0.426

0.000163

0.00135

0.00043

0.0546

2.55

0.331

3054

4.68

143.3

ECorner

Shore

Station(Area1)

10.3

55.1

486

10.5

0.108

0.000048

0.00183

0.00009

0.0135

0.53

0.108

564

0.86

30.6

Lake

center–Su

rface

29.4

298.3

2633

57.0

0.426

0.000168

0.00569

0.00061

0.0545

2.54

0.331

3051

4.68

143.1

Lake

center–Bottom

29.9

298.2

2632

56.9

0.425

0.000180

0.00033

0.00035

0.0549

2.55

0.329

3050

4.67

143.1

Lake

center–Chim

ney

19.1

154.6

1363

29.7

0.232

0.000091

0.00023

0.00000

–1.25

0.175

1589

2.41

74.7

Sample

location

Saturationindices

Isotopic

composition

d13C

d18O

d2H

Calcite

Aragonite

Gyp

sum

Halite

Calcite

Aragonite

Gyp

sum

Halite

VPDB

VSM

OW

VSM

OW

logIAP/K

TlogIAP/K

TlogIAP/K

TlogIAP/K

TIAP/K

TIAP/K

TIAP/K

TIAP/K

T&

&&

Seaw

ater

0.86

0.72

�0.66

�2.52

7.24

5.25

0.22

0.003

2.49

0.24

2.12

SESh

ore

Station1(Area2

)0.89

0.76

0.00

�1.18

7.76

5.75

1.00

0.066

�0.58

1.26

0.54

SESh

ore

Station2(Area3)

1.26

1.12

0.13

�0.89

18.20

13.18

1.35

0.129

4.07

2.38

5.23

ECorner

Shore

Station(Area1)

1.20

1.07

�0.67

�2.53

15.85

11.75

0.21

0.003

�2.91

�2.08

�14.46

Lake

center–Su

rface

1.31

1.17

0.16

�0.88

20.42

14.79

1.45

0.132

3.68

2.33

5.53

Lake

center–Bottom

1.32

1.18

0.16

�0.88

20.89

15.14

1.45

0.132

––

–

Lake

center–Chim

ney

0.92

0.78

�0.26

�1.59

8.32

6.03

0.55

0.026

––

–

© 2014 John Wiley & Sons Ltd

4 D. IONESCU et al.

The purple layer is not always unequivocally distinguish-

able by color from deeper layers 5–7 that also have red-

dish–brownish colors, but its upper boundary is indicated

by the onset of phototrophic sulfide oxidizers (Chromati-

ales). This group, however, is most abundant in layer 5,

which is characterized by mm-sized gypsum crystals

(Fig. 3).

Additional bacterial sequences found in significant abun-

dance in and around layers 4 and 5 are associated with

uncultured Anaerolinaceae (<20%), Dichotomicrobium sp.

(<11%), uncultured Desulfobacteraceae (<21%), and Spiro-

chaeta sp. (<28%). Sulfide oxidizing members of the latter

group were recently identified (Dubinina et al., 2011).

Deeper mat layers are characterized by common Desulf-

oarculaceae and Desulfobacteraceae, increasingly abundant

Spirochaeta, various other eubacterial fermenters, as well as

uncharacterized members of the OPB95 phylum and mem-

bers of the Planctomycetes.

Microprofiles

Microprofiles of O2, Ca2+, pH, H2S, and redox potential

were measured in situ in the upper 4.5 cm of the microbial

mats in Area 1 of Lake 21 (Fig. 4). In all cases, the stan-

dard error of replicate point measurements at each depth

within a single profile was <0.01% of the average read,

showing a stable signal.

Due to the weight of the microsensor setup and the

softness of the mat material, in situ measurements were

A

B C

Fig. 2 An underwater chimney-like structure found at groundwater inlets

in Lake 21 (A). The chimney wall consist of a stratified mat lined with a

white colored biofilm (B).The interior wall of the chimney is lined with car-

bonate precipitates of various sizes (C).

Fig. 3 Most frequent genera found in the top layers of Lake 21 as obtained from 454 pyrosequencing data. The color of each symbol represents the

sequence frequency. The size of the symbols is a measure of the inner diversity of each genera based on number of operational taxonomic units obtained

from clustering at 98% similarity. The shape of the symbol represents a rough scale to the sample size for each taxonomic path.

© 2014 John Wiley & Sons Ltd

Calcification in EPS-rich microbial mats 5

limited to a small enclosed area near the shore of the lake

and only one set of such profiles could be obtained. The

water was supersaturated with O2 from the upper photo-

synthetic layer with a second O2 peak evident 3 cm into

the microbial mat. Oxygen was depleted 3.5 cm into the

mat and sulfide coming from the deeper parts penetrated

0.5 cm into the oxic zone. The pH at Area 1 was constant

around 8.7 in the water column and the upper part of the

mat, decreasing from below the second O2 peak to a value

of 6.5 at the lowest point of measurement (4.5 cm).

Redox potential showed a similar trend as the pH profile.

Ca2+ showed a slight increase in the upper parts of the mat

with an ongoing decrease in concentration starting at

about 2.5 cm in the mat, where sulfide becomes totally

depleted.

To obtain reproducible measurements, microprofiles of

O2 and Ca2+ were measured under light and dark condi-

tions in the laboratory in the upper 4.5 cm of cores

retrieved from the microbial mats in Area 1 of Lake 21

(Fig. 5). During illumination an O2 peak evolved at a

depth between 0.5 and 1.5 cm coinciding with a brown

(orange) and green layer in the mat (Fig. 5). Interestingly,

during the light phase, a continuous increase in Ca2+ con-

centrations was measured in the same layers. This increase

was followed by a sharp decrease in Ca2+ at the oxic–

anoxic transition zone at a depth of 2.7 cm. While O2

reached steady state after 30 min, the Ca2+ profile stabi-

lized after 70 min. Under dark conditions, the O2 was rap-

idly consumed alongside with the flattening of the Ca2+

profile. The O2 concentration did not change during dark-

ness; however, the Ca2+ concentration increased continu-

ously down to a depth of at least 4.5 cm reaching steady

state after 70 min. Upon re-illumination, the O2 peak

evolved in the same strata as before and similarly to the

first light period, the Ca concentration in the oxic zone

increased followed by a sharp drop at a depth of 2.7 cm.

Sulfide was not measured in these samples on site due to

technical complications.

Microprofiles of Ca2+, O2, pH, and H2S were measured

again 6–7 months later in cores that were retrieved from

the sampling campaign (Fig. 6). Although the oxygenic

phototrophs were severely impaired by the long storage

time and the organic matter in the core seemed to be

degraded as indicated by the strong smell, similar trends to

those observed on site could be still detected. Ca2+ con-

centration showed an increase during the thin layer of oxy-

genic photosynthesis with no evident decrease in

concentration in the upper 1.5 cm. A decrease in Ca2+

concentration was detected in the purple layer upon O2

depletion and H2S detection, at the oxic–anoxic transition

zone, similar to the observations made in fresh samples.

Similar to previous observations (Arp et al., 2012),

almost no visual evidence of carbonate precipitation except

for scattered aragonite spherulites was observed in the

upper 2.5 cm (the zone of oxygenic photosynthesis). Nev-

ertheless, crystal aggregates were found at the contact of

the green layer to the purple layer at a depth between 1.8

and 2.8 cm. Mat structures were opened by cutting and

found to harbor a continuous layer of carbonate minerals

at the same position (Fig. 7).

A depth profile of the light spectrum was measured in

the microbial mats (Fig. 8), which shows that most of the

visible light is absorbed in the upper 1.5 cm (Fig. 8A).

Analysis of specific wavelengths showed that indeed light

suitable for Chl a and phycobilins (~450–700 nm) is

Fig. 4 O2, Ca2+, pH, H2S, and redox potential microprofiles measured dur-

ing day time in situ in area 1 of Lake 21. H2S and redox measurements

could not be calibrated in the field; hence, scaled raw signals are showed.

Fig. 5 O2 and Ca2+ profiles measured in the light and in the dark in fresh

cores collected from Lake 21. The profiles are aligned with a photograph of

the mat section. Parallel pH and H2S profiles are not available.

© 2014 John Wiley & Sons Ltd

6 D. IONESCU et al.

absorbed early while light suitable for BChl a or BChl c

(>700 nm) penetrates down to 3 cm (Fig. 8B).

DISCUSSION

With respect to calcification mechanisms, microbial mats in

hypersaline lakes with marine-like ion ratios represent sys-

tems between microbial mats of soda-lake (high DIC, low

Ca2+) and microbial mats of karstic freshwater (low DIC,

high Ca2+) (Arp et al., 2001, 2010). While in soda-lake

mats and biofilms, the impact of oxygenic photosynthesis

on the carbonate equilibrium is low and secondary Ca2+

release from degrading exopolymers is a likely calcification

trigger (Arp et al., 1998, 1999b; Arp et al., 2001), cyano-

bacterial biofilms of karstic lakes and streams show a clear

photosynthesis-controlled calcification (Bissett et al.,

2008a,b; Shiraishi et al., 2008).

Hypersaline lakes on Kiritimati (Trichet et al., 2001;

Saenger et al., 2006; B€uhring et al., 2009) intermediate

between soda lake and karstic end-members with respect

to DIC concentrations. The CaCO3 precipitation in mats

from these lakes is located in the deepest parts of the oxy-

genic photosynthetic layer and top parts of the purple layer

(Fig. 8B). The highly photosynthesizing oxygenic top parts

of the mat remain almost free of CaCO3 precipitates,

except for scarce spherulites. A possibly similar situation

has been described from hypersaline lakes on Eleuthera,

where micrite layer forms in deeper parts of the oxygenic

cyanobacterial mat (Baumgartner et al., 2009; Glunk et al.,

2010). Oxygenic photosynthesis, while certainly mostly

increasing CaCO3 mineral saturation states, is therefore

not the direct trigger of CaCO3 precipitation and micro-

bialite formation on Kiritimati, but rather a pre-condition

for that.

Taking into account (i) that exopolymers bind Ca2+ via

their functional groups and kinetically inhibits nucleation

and (ii) mat thin sections demonstrate a degradation-

related change in the exopolymer fabric in lower mat parts

associated with aragonite precipitates, the following

hypothesis has been developed (Arp et al., 2012): (i)

CaCO3 mineral supersaturation is increased substantially

via oxygenic photosynthesis, but immediate precipitation

kinetically inhibited by the produced pristine exopolymers.

(ii) Then, in lower parts of the photic zone, exopolymers

are degraded by heterotrophic bacteria, releasing Ca2+ pre-

viously bound to the exopolymers and decreasing the

kinetic inhibition (Arp et al., 1999a; Braissant et al., 2009;

Glunk et al., 2010). The latter processes finally trigger

CaCO3 mineral precipitation. Alternative explanations of

CaCO3 precipitation in similar microbial mats suggest a

crucial role of sulfate reduction to increase alkalinity and

carbonate mineral supersaturation in the microenvironment

(e.g., Visscher et al., 2000; Baumgartner et al., 2006).

This view, however, has been discussed controversially

(Meister, 2013, 2014; Gallagher et al., 2014).

In the present case of the Kiritimati Lake 21 mats, most

CaCO3 precipitates are found, together with gypsum

(Fig. 8B), at the transition of the lowermost green,

degrading, oxygenic mat layer to the anoxygenic purple

bacterial layer, that is, a zone of fluctuating redox bound-

aries, so that various microbial processes interact.

Thus far, measurements of microscale ionic and physico-

chemical gradients, especially Ca2+ gradients, were not

available, hence, binding and/or secondary release of Ca2+

within the mats remained completely speculative. From

similar lithifying and non-lithifying mats in hypersaline

lakes on Eleuthera, only two daylight Ca2+ profiles (upper-

most 2 mm) are available from, 9-mm thick microbial

mats, respectively, of hypersaline lakes (Baumgartner et al.,

2009: p. 867; see also Dupraz & Visscher, 2005: p. 430).

From Kiritimati, Ca2+ has previously only been measured

Fig. 6 O2, H2S, pH, and Ca2+ profiles measured in the light in cores trans-

ported from Area 3 Lake 21. Measurements were conducted several

months after the original campaign. The layer of phototrophs was highly

degraded by the time of measurement as could be easily seen by visual

inspection.

A B

Fig. 7 An underwater carbonate structure intact (A) and sectioned (B)

showing the carbonate horizon within the purple layer of the microbial

mat.

© 2014 John Wiley & Sons Ltd

Calcification in EPS-rich microbial mats 7

from above (water column) and immediately below the

mats (microbialite porewater), both showing similar con-

centrations (Arp et al., 2012). The present, new microsen-

sor measurements now suggest more complex Ca2+

dynamics than previously assumed (Figs 5, 6 and 9).

During illumination, an increase in Ca2+ concentration is

observed in the upper layers of the mats. We propose that

this is the result of exopolymers degradation by aerobic

heterotrophic bacteria and ensuing release of bound Ca2+.

This increase cannot be the result of carbonate dissolution

because (i) there are nearly no carbonate minerals in these

layers with the exception of small spherulites (Arp et al.,

2012); (ii) carbonate dissolution is a result of acidification,

whereas, during daylight in the upper photic zone, photo-

synthesis leads to a significant increase in pH, as evident

from the measured pH profiles and thus to a supersatura-

tion in carbonates (aragonite).

An increase in pH, such as occurs during daytime in the

photic layer, would lead to deprotonation of the exopoly-

mers and an increase in its Ca2+ binding capacity (Phoenix

et al., 2002; Braissant et al., 2007). Therefore, we con-

clude that during the light period, when O2 is amply sup-

plied by photosynthesis, it is not a pH dependent release

of Ca2+ from exopolymers but rather heterotrophic degra-

dation of the exopolymers leads to release of Ca2+ into the

porewater of the mat as was previously suggested (Arp

et al., 1999a; Dupraz & Visscher, 2005).

The significance of aerobic heterotrophy is further appar-

ent from the Ca2+ profiles obtained under dark conditions.

Upon O2 consumption and cease of aerobic degradation,

Ca2+ rebinds to all available exopolymeric sites. An appar-

ently less efficient, anaerobic degradation of the exopoly-

meric material is then responsible for the continuous

increase in Ca2+ concentration with depth. Repletion of O2

by photosynthesis upon re-illumination regenerates the

same strong increase in Ca2+ concentration as observed

during the first light period. This suggests that the exo-

polymers found in these mats may be easier to degrade

under aerobic conditions, giving the exopolymer composi-

tion a great significance in its role in Ca2+ precipitation.

Fig. 9 A conceptual overview of the Ca2+ precipitation mechanism in the

microbial mats of Lake 21. Idealized Ca2+, O2, pH, and H2S microprofiles

(A) as we would expect them during the light period show an increase in

Ca2+ concentration in the oxic part of the photic zone and a sharp decrease

in the oxic–anoxic transition zone. The EPS in the upper photic zone pre-

vents Ca2+ precipitation and allows only for small spherulites to form (B).

The proposed increase with depth in EPS density leads to an increase in

available Ca2+ binding site and accordingly more Ca2+ is released during

EPS degradation leading to the peak in Ca2+ at the oxic–anoxic transition

zone, where the EPS properties probably change. At this point, Ca2+ precip-

itates as a mixture of aragonite and gypsum. The EPS serves as a passive

Ca2+ shuttle from the water column to the precipitation site. The continu-

ous secretion of EPS by the oxygenic phototrophs (C, D) together with the

heterotrophic degradation of the EPS and its increasing density with depth

due to the weight of new biomass and new EPS, deliver the Ca2+ ions to

the green/purple layer transition where they are precipitated (D).

A B

Fig. 8 Light profiles in the visible and near-infrared range measured in the core from the shore of Lake 21 in which the profiles in Fig. 4 were measured.

Absorbance with depth of the whole spectra (A) was used to generate depth profiles of pigment specific wavelength (B). The line color represents the specific

light spectra. It is evident that infrared light penetrates to depths of up to 3 cm in the mat.

© 2014 John Wiley & Sons Ltd

8 D. IONESCU et al.

The exopolymer properties which inhibit Ca2+ precipita-

tion are maintained over time despite degradation pro-

cesses (Braissant et al., 2009). This is visible in our study

in the measurements obtained from a core which has been

stored for 6–7 months prior to analysis (Fig. 6). Despite

the degradation of the exopolymers and the decay of a

large portion of the oxygenic phototroph community (as

can be seen by the low O2 production), no Ca2+ precipita-

tion is observed below the O2 peak. This results in a wider

Ca2+ maximum. Eventually, the Ca2+ concentration

decreases, as in the fresh samples, at the oxic–anoxic inter-

phase. This shows that the Ca2+ depositing mechanism in

these mats is strongly established.

Decho et al. (2005) have established the significant role

of low molecular weight dissolved organic matter (low-

MW-DOC) in the carbon pool of microbial mats. While

we cannot entirely exclude the role of these compounds in

Ca2+ binding, the study from Decho et al. (2005) shows a

strong decrease in low-MW-DOC after 6 pm, presumably

representing the onset of low-light condition therefore

changing the balance between production and consump-

tion of low-MW-DOC. If a large part of the Ca2+ would

be bound to low-MW-DOC, we believe that we would

observe first an increase in Ca2+ concentration with the

change of light conditions (i.e., turning off the light) at

least as long as O2 was still available.

Precipitates at the green/purple layer transition in Kiriti-

mati Lake 21 microbial mats contain both, aragonite and

gypsum (Fig. S3). Indeed, formation of both precipitates

could be affected by anoxygenic photosynthesis of purple

sulfur bacteria, which have been detected in significant

numbers in layer 4/5 by 16S rRNA gene pyrosequencing.

Only infrared light penetrates to this depth at wavelengths

suitable for Bchl a containing organisms. Gypsum is proba-

bly a result of sulfate originating from sulfide oxidation,

precipitating with Ca2+ ions released from the exopolymers

above. With respect to CaCO3 precipitation, the effect is

less clear. Visscher & Stolz (2005) calculated 0.5 mol of

CaCO3 is precipitated per mole of CO2 fixed by anoxygen-

ic photosynthesis. However, given the decreasing pH val-

ues in the purple layer (Fig. 5), this effect is likely to be of

secondary importance compared to Ca2+ release by aerobic

heterotrophic exopolymer degradation at still high CaCO3

mineral supersaturation inherited from top oxygenic mat

layers.

CONCLUSIONS

1 Thick microbial mats in hypersaline lakes on Kiritmati

investigated in 2011 show active CaCO3 and gypsum

precipitation at the transition of the oxygenic green

layer to anoxygenic purple bacterial layer.

2 Ca2+ microprofiles demonstrate a light dependent, with

depth increasing Ca2+ liberation from degrading exo-

polymers within the oxygenic mat parts. This Ca2+

release during daylight is explained by the aerobic het-

erotrophic degradation of Ca2+-binding exopolymers.

At nighttime, this Ca2+ release is not observed, due to

lack of O2 and slow anaerobic exopolymer degradation.

3 The released Ca2+ is precipitated as aragonite and gyp-

sum at the transition green to purple mat layers due to

a combination of a high aragonite supersaturation,

decrease of kinetic inhibition by exopolymer degrada-

tion, and possibly anoxygenic photosynthesis and sulfide

oxidation.

4 Exopolymers have a significant role in the passive trans-

port of Ca2+ ions from the overlaying waters to the

deep parts of the mat.

ACKNOWLEDGMENTS

This study is part of the Research Unit ‘Geobiology of

Organo-and Biofilms’ funded by the German research

Foundation (DFG-For 571 publication #64). We would

also like to thank the staff of the local Nature Conservation

office on the Island of Kiritimati for their support.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the

online version of this article:

Fig. S1 An intact core sample from Lake 21 showing clear pigmentation to

depths of below 10 cm.

Fig. S2 Transmitted light photomicrographs from the different layers of the

microbial mat in Lake 21.

Fig. S3 Polarized light micrographs of a thin section from a microbialite

from the purple layer of Lake 21.

© 2014 John Wiley & Sons Ltd

Calcification in EPS-rich microbial mats 11