Millimeter-scale resolution of trace metal distributions in microbial mats from a hypersaline...

Millimeter-scale resolution of trace metal distributions inmicrobial mats from a hypersaline environment in BajaCalifornia, MexicoM. A. HUERTA-DIAZ,1 F . DELGADILLO-HINOJOSA,1 A. SIQUEIROS-VALENCIA,1

J . VALDIVIESO-OJEDA,2 J . J . REIMER2 ,* AND J . A . SEGOVIA-ZAVALA1

1Instituto de Investigaciones Oceanologicas, Universidad Autonoma de Baja California, Ensenada, Baja California, Mexico2Posgrado en Oceanografıa Costera, Instituto de Investigaciones Oceanologicas/Facultad de Ciencias Marinas, Universidad

Autonoma de Baja California, Ensenada, Baja California, Mexico

ABSTRACT

Microbial mats from two ponds with different salinities from the saltern of Guerrero Negro (Mexico) points

toward millimeter-scale coherent variations in trace metal (Me) concentrations (Cd, Co, Cu, Fe, Mn, Ni, Pb,

Zn). Total, HCl-leachable and pyrite-associated Me showed a trend of increasing concentrations with

increasing depth suggesting gradual addition of reactive Me probably as a result of metal sulfide precipita-

tion at depth. The trends in Me profiles can be ascribed to the establishment and maintenance of microz-

ones that promote geochemical processes, bacterial population distributions, and differential mass transport

within the mats. Degrees of trace metal pyritization (1 ± 1% for Zn to 24 ± 7% for Cd) as well as metals

associated with the pyrite fraction (<1.4–36 ± 18 nmol g�1 for Zn and Mn, respectively) were low, as

expected from a reactive Fe-limited system like Guerrero Negro. Calculated enrichment factors showed that

Ni (2.6 ± 2.1), Co (5.5 ± 4.0), Pb (9.4 ± 7.4), and Cd (57 ± 39) were, on average, enriched in the micro-

bial mats of Guerrero Negro. Natural enrichments of Cd, Pb, and Co in sediments along the coast of Baja

California and metabolical requirements of Co and Ni by the predominant cyanobacteria in the Guerrero

Negro mats may explain these enrichments. Metal characteristics in microbial mats could be advanta-

geously used as biosignatures to identify their presence in the geological record or in other planetary

systems.

Received 2 November 2011; accepted 22 August 2012

Corresponding author. M. A. Huerta-Diaz. Tel.: +52-646-174-4601 Ext. 167; fax: +52-646-174-5303;

e-mail: [email protected]

*Present address: Post-doctoral Fellow, Programa Mexicano del Carbono, Centro de Investigacion Cientifica

y de Educacion Superior de Ensenada, Carretera Ensenada-Tijuana No. 3918, Ensenada, Baja California,

22860, Mexico

INTRODUCTION

Microbial mats are multilayered sheet accumulations of het-

erotrophic and autotrophic micro-organisms arranged in

coherent, laminated structures (Bender et al., 1995; Des

Marais, 1995). These biological structures are embedded in

a polysaccharide matrix excreted by cyanobacteria and other

bacteria (Des Marais, 1995). Microbial mats represent lami-

nated layers (some as fine as 1–400 lm) of phylogenetically

and metabolically diverse microbial communities, whose

arrangement is based upon the flow of energy and organic

carbon substrates (Fike et al., 2008). Modern microbial

mats are remarkably cosmopolitan, with some of their most

important representatives found practically in every country

of the world (Esteve et al., 1992).

Microbial mat ecosystems support most of the major

biogeochemical cycles within a vertical dimension of only a

few millimeters (Paerl & Pinckney, 1996). The presence of

vertical zonations of light, O2, H2S, pH, and sulfur bacte-

ria contribute to the development of steep physical–chemi-

cal gradients within the microbial mat (Jørgensen & Des

Marais, 1986; Fike et al., 2008), which in turn may cause

© 2012 Blackwell Publishing Ltd 1

Geobiology (2012) DOI: 10.1111/gbi.12008

distinctive vertical trace metal distributions. Furthermore,

intense bacterial production of sulfur compounds (Des

Marais, 2003), as well as high sulfate reduction rates

(Canfield & Des Marais, 1991) supported by aerotolerant

sulfate reducers (Teske et al., 1998) suggest that these

processes may play an important role in the precipitation,

dissolution, fixation, and alteration of iron sulfide (e.g.,

iron monosulfides, pyrite) and Fe oxihydroxide minerals

(Visscher et al., 1998; Castanier et al., 2000; Visscher &

Stolz, 2005), and associated trace elements. These pro-

cesses and the presence of the laminations in the mats

underscore the importance of studying the fine (millimeter

resolution) vertical distribution of trace metals in microbial

mats.

Studies performed on modern microbial mats and stro-

matolites, their lithified counterparts (Walter, 1976; Can-

field & Des Marais, 1993; Reid et al., 2000), have

provided information needed to interpret biosignatures

and geochemical patterns left in the siliciclastic rock record

over geologic time by fossil stromatolites and other soft

microbial mat features (e.g., Grotzinger & Knoll, 1999;

Schieber et al., 2007). In applying the use of metal biosig-

natures, one question that may arise is how similar the ver-

tical distributions of trace metals are among microbial mats

from different environments. As a first approximation, we

compare the vertical distribution of trace metals (Cd, Co,

Cu, Fe, Mn, Ni, Pb, Zn) in three different geochemical

fractions (total, HCl, and pyrite) at fine resolution (mm

scale) in three mats from a hypersaline environment located

within the saltern of Guerrero Negro, Baja California Sur,

Mexico. Their behavior is then analyzed in terms of enrich-

ment factors and degrees of trace metal pyritization which

can be explained by a combination of physical, chemical,

and biological processes. We postulate that the presence of

laminations appears to be an important factor affecting the

enrichment and fine scale vertical distributions of trace

metals within the microbial mat owing to the establish-

ment and maintenance of geochemical and biological

micro zones.

DESCRIPTION OF THE STUDY AREA

The city of Guerrero Negro has a population of approxi-

mately 30 000, and it is located in the northern limit of a

salt evaporation complex, or saltern (Fig. 1). The climate

of the region is arid (annual precipitation ranges from 15

to 120 mm per year; Des Marais et al., 1992), with sus-

tained winds during the day that average approximately

18 km per h. The saltern is composed of a series of evapo-

ration and crystallization ponds (~250 km2) on the site of

a natural hypersaline environment located within the Vizca-

ino Biosphere National Reserve. It was diked around 1971

for the production of sea salt (Jørgensen & Des Marais,

1986) and concessioned to the Exportadora de Sal S.A.

Company (ESSA). The saltern system consists of a series of

13 evaporations ponds, 1 to 1.5-m deep (concentration

area; Figure 1) constructed approximately 2 m above sea

level that spans a salinity gradient from 40 PSU (Ojo de

Liebre Lagoon) up to saturation with sodium chloride

(crystallization area; Fig. 1). There are no permanent rivers

in the region, and runoff input to the ponds is minimal,

except during the occasional periods of heavy rain or hurri-

canes. Water from Ojo de Liebre Lagoon is introduced

into the saltern system by a series of pumps into evapora-

tion pond 1 (Fig. 1) and then flows through the ponds in

increasing numerical order via meter-wide openings in the

dikes.

Permanently submerged and well-developed (3 to 8-cm

thick) benthic microbial mats dominated by the cya-

nobactyeria Microcoleus were found in evaporation ponds 4–

8 (Des Marais, 1995) at the time of sampling; however, the

most coherent cyanobacterial mats were located in ponds 4

and 5. Rubbery, cohesive mats in these ponds have a

smooth, olive-tan surface with dark green to orange–brown

internal laminations that can be 1-lm to 3-mm thick (Des

Marais, 1995; Fike et al., 2008). The dominant component

of the microbial mats is the cosmopolitan cyanobacteria

Microcoleus chthonoplastes (Canfield & Des Marais, 1993),

although other filamentous (Oscillatoria spp., Spirulina

labyrynthiformis) and unicellular (Synechococcus sp.) cyano-

bacteria have been observed in the upper 300-lm photic

zone (D’Amelio et al., 1989). Pond number 5 has been

described as iron limited by Huerta-Diaz et al. (2011)

implying that reactive Fe may severely limit the formation of

pyrite in this pond and, probably, other ponds as well.

METHODOLOGY

All laboratory materials were washed with phosphate-free

soap, rinsed three times with distilled water, and left for

24 h in a 5% HCl solution. The material was then rinsed

three times with deionized water (Milli-Q grade) and left

semiclosed to dry in a hood under laminar flow. All

reagents used were of reagent grade or better.

Sample collection

Microbial mats from evaporation ponds 4 (cores 4A and

4B; S = 91) and 5 (core 5; S = 119) were collected by a

diver using a 7.6 cm i.d. PVC core liner on June 17 (cores

4A and 5) and 19 (core 4B), 1999 at a water depth of

approximately 1 m. Collected cores consisted of mats and

underlying sediments, but only the mats were sampled.

Thickness of the mats was 19, 21, and 11 mm for cores

4A, 4B, and 5, respectively. The mats were immediately

sliced in the field every 2 mm using a nylon string, each

sample placed in 50-mL plastic centrifuge tubes, preserved

in ice and later frozen at �10 °C in the field laboratory.

© 2012 Blackwell Publishing Ltd

2 M. A. HUERTA-DIAZ et al.

Salinity was measured with an Atago 2442-W10 handheld

refractometer. Percentage by weight of water and solid for

each sample was calculated by drying approximately 1–2 g

of wet sample in an oven at ~80 °C, until a constant

weight was obtained.

Sequential chemical extraction

Trace metals (Cd, Co, Cu, Fe, Mn, Ni, Pb, Zn) in mats

were separated into the operationally defined fractions HCl

and pyrite using the sequential extraction method devel-

oped by Huerta-Diaz & Morse (1990). The method

involves the treatment of a wet sample equivalent to 2.5 g

of dry mat (estimated from the previously calculated per-

centage by weight of water and solid). The first fraction is

obtained from the digestion of the sample for 16 h with

20 mL 1 N HCl. This so-called HCl fraction includes car-

bonates, Fe and Mn oxides, and acid volatile sulfide or

AVS. The silicate fraction, composed mainly of clays, was

extracted with two successive digestions (1 and 16 h,

respectively) using 30 mL of 10 M HF. This fraction was

not measured because it is not important in the processes

of early diagenesis. Finally, the residue from the previous

fraction was digested for 1 h with 10 mL of concentrated

HNO3 to obtain the so-called pyrite fraction (and the trace

metals associated with this mineral phase). Hence, the

extraction procedure of Huerta-Diaz & Morse (1990)

selectively separate aluminosilicate (non-reactive) from dia-

genetic (reactive fraction = HCl + pyrite fractions) metals.

The reproducibility of the method, as reported by Hu-

erta-Diaz & Morse (1990), is generally around ±10% for

the metals measured in this study, except for Cd (~24% in

the pyrite fraction), in which case closeness to its detection

limit may have accounted for its high variability (Huerta-

Diaz & Morse, 1990). It is important to mention that

trace metal results obtained with sequential extraction

procedures are, by definition, operationally defined by the

method of extraction. Generally, there is no suitable

Fig. 1 Location of sampling sites in the salt works of Guerrero Negro. Microbial mats 4A and 4B were collected in Pond number 4, whereas mat 5 was

collected in Pond 5.


Millimeter-scale distributions of trace metals in microbial mats 3

Certified Reference Material (CRM) available to confirm

the validity of the extractions, except for a few exceptions

that are related to very specific extraction schemes; how-

ever, for the particular extraction procedure used in this

work, the reader is referred to the study by Huerta-Diaz &

Morse (1990), who performed an extensive validation of

the method: (i) by analyzing samples before and after the

HNO3 leaching to verify the integrity/dissolution of pyrite

before/after this acid attack step; (ii) by correlating pyrite

extractions carried out by our method and by an indepen-

dent method that demonstrated quantitative extraction and

(iii) by summing the Fe concentrations of the three frac-

tions (HCl + silicate + pyrite) and then comparing them

to total extractions.

Results from the HCl and pyrite fractions for Fe (FeHCl

and Fepyr, respectively) and other trace metals (MeHCl and

Mepyr, respectively) were combined to calculate the degree

of pyritization (DOP for Fe) and the degree of trace metal

pyritization (DTMP for the other metals) of the samples.

The DOP and DTMP terms, introduced by Berner (1970)

and Huerta-Diaz & Morse (1990), respectively, were used

to evaluate the amount of Fe present as (or Me associated

with) pyrite relative to the so-called ‘reactive fraction’

(FeRF = Fepyr + FeHCl and MeRF = Mepyr + MeHCl). The

DOP and DTMP values were calculated using the follow-

ing equations:

DOPð%Þ ¼ FepyrFepyr þ FeHCl

� 100 and

DTMPð%Þ ¼ MepyrMepyr þMeHCl

� 100

ð1Þ

Total trace metal extraction

Total trace metals (Cd, Co, Cu, Fe, Mn, Ni, Pb, Zn, Al)

were extracted following the method of Carignan & Tes-

sier (1988). In this method, 0.5 g (dry weight) of sedi-

ment is placed in a 150-mL Teflon beaker covered by a

glass watch made of Teflon. The sample is digested during

30 min with 15 mL of concentrated HNO3 at 150 °Cunder reflux, and then, evaporated to dryness at 200 °C.Next, 4 mL of concentrated HClO4 is added to the sample

and heated under reflux during 30 min. The sample is

allowed to cool down during 30 min followed by addition

of 10 mL of concentrated HF and evaporation to dryness

at 200 °C (approximately 12–19 h). Finally, the sample is

allowed to cool down, the beakers are rinsed with 5% HCl

and the solution is transferred to 50-mL volumetric flasks.

The final solution was always transparent and without any

visible residue. A Varian SpectraAA 220 Fast Sequential

flame atomic absorption spectrophotometer was used to

measure metal (Me) concentrations. Included in each batch

of samples were blanks whose measured values were always

below their respective detection limit. Throughout this

work and independently of their concentration levels, all

metals associated with mats will be designated as trace met-

als. Samples from this study were analyzed concurrently

with those described by the study by Huerta-Diaz et al.

(2011) for a core collected in Pond 5 in December 1998

and, therefore, their detection limits (DL) and CRM

results are the same. The DL (in nmol g–1) for (i) Cd, (ii)

Co, (iii) Cu, (iv) Fe, (v) Mn, (vi) Ni, (vii) Pb, (viii) Zn,

and (ix) Al were (for HCl, pyrite, and total fractions,

respectively) (i) 1.2, 1.1, 14; (ii) 3.1, 9.6, 120; (iii) 3.6,

3.3, 43; (iv) 14, 3.6, 800; (v) 5.5, 7.5, 220; (vi) 9.4, 9.4,

46; (vii) 5.1, 6.0, 75; (viii) 1.2, 2.1, 31; (ix) 1.9 lmol g-1.

The recovery percentages of the CRM BCSS-1 (Beaufort

Chemistry Standard Sediment; National Research Council

of Canada), which were used to ascertain the accuracy and

precision of the total extraction (for Al, Cd, Co, Cu, Fe,

Mn, Ni, Pb, Zn), averaged 99 ± 5% and ranged from 92%

for Cd to 105% for Ni. The BCSS-1 CRM was used mainly

because no certified standard for microbial mats (or analo-

gous matrix) is commercially available. Our experience

indicated that mats have matrix characteristics similar to

those found in sediments with high organic matter content

(>10% organic C dry weight; e.g., Carignan & Tessier,

1988), in which cases our total extraction scheme was suc-

cessfully applied.

Geochemical normalization with Al and enrichment factor

calculation

Geochemical normalization of metal concentrations with a

conservative element is based on the assumption that there

are certain elements that represent proxies for the clay min-

eral concentration (Kersten & Smedes, 2002). This

approach has the main advantage of compensating for the

mineralogical and natural grain size variability of trace ele-

ment concentrations in sediment (Loring, 1991; Birch,

2003). One of the best normalizing agents for evaluating

trace metal concentrations in estuarine and coastal sedi-

ments is Al (e.g., Goldberg et al., 1979; Schropp et al.,

1990; Summers et al., 1996; Weisberg et al., 2004)

because of its minimal anthropogenic contamination and

high natural concentration (second most abundant metal

in the Earth’s crust). Additionally, the metal to Al propor-

tions in the crust is relatively constant, and it is a structural

element of clays (Schropp et al., 1990; Summers et al.,

1996). Use of Al as a normalizing element can be used to

calculate enrichment factors of trace elements (EFMe) using

the following equation:

EFMe ¼ðMe=AlÞsample

ðMe=AlÞbackground

!ð2Þ

where, (Me/Al)sample represents the Al-normalized concen-

tration of the metal (Me) in the sediment sample on a



molar ratio basis. It is generally accepted that, for the cal-

culation of metal enrichments, it is preferable to use regio-

nal background concentrations, represented by (Me/

Al)background in equation (2). Nevertheless, because this

information has not been reported for the study area, the

values of Turekian & Wedepohl (1961) and Li & Schoon-

maker (2005) for the average composition of sedimentary

shale rocks were used instead.

RESULTS AND DISCUSSION

Total trace metals

Average total trace metal concentrations (Table 1) in our

three mats ranged from 19 ± 5 nmol g�1 (Cd) to 101 ±60 lmol g�1 (Fe) and 627 ± 480 (Al), comparable with the

range of 25 ± 19 nmol g�1 (Cd) to 154 ± 124 lmol g�1

Table 1 Average concentration values (± one standard deviation) for total, HCl, pyrite, and reactive (HCl + pyrite) fractions and for enrichment factors for

mats 4A, 4B, and 5

Element Mat 4A (n = 10) Mat 4B (n = 11)

Mats 4A and 4B combined

(n = 21) Mat 5 (n = 6)

All mats combined

(n = 27)

Total fraction (nmol g�1)

Al (2.2 ± 1.3) 9 102 (6.6 ± 3.2) 9 102 (4.5 ± 3.3) 9 102 (12.5 ± 4.1) 9 102 (6.3 ± 4.8) 9 102

Cd 16 ± 4 22 ± 4 19 ± 5 18 ± 4 19 ± 5

Co 183 ± 40 246 ± 54 216 ± 57 228 ± 63 219 ± 57

Cu 77 ± 31 126 ± 33 102 ± 40 84 ± 20 98 ± 37

Fe (55 ± 30) 9 103 (109 ± 52) 9 103 (83 ± 50) 9 103 (162 ± 53) 9 103 (101 ± 60) 9 103

Mn (0.90 ± 0.48) 9 103 (2.0 ± 0.8) 9 103 (1.5 ± 0.9) 9 103 (4.4 ± 2.0) 9 103 (2.1 ± 1.7) 9 103

Ni 342 ± 59 385 ± 75 364 ± 69 376 ± 27 367 ± 62

Pb 104 ± 26 114 ± 38 109 ± 33 81 ± 37 103 ± 35

Zn 185 ± 54 386 ± 141 290 ± 148 339 ± 57 301 ± 133

HCl fraction (nmol g�1)

Cd 5.6 ± 1.2 6.3 ± 1.3 5.9 ± 1.3 5.3 ± 1.6 5.8 ± 1.3

Co 54 ± 14 55 ± 14 55 ± 13 54 ± 14 54 ± 13

Cu 19 ± 5 24 ± 10 22 ± 8 21 ± 6 22 ± 8

Fe (9.1 ± 3.0) 9 103 (14 ± 7) 9 103 (12 ± 6) 9 103 (14 ± 5) 9 103 (12 ± 6) 9 103

Mn (0.21 ± 0.19) 9 103 (0.31 ± 0.21) 9 103 (0.26 ± 0.20) 9 103 (0.24 ± 0.06) 9 103 (0.26 ± 0.18) 9 103

Ni 47 ± 13 65 ± 17 56 ± 17 55 ± 13 56 ± 16

Pb 30 ± 8 22 ± 7 26 ± 8 21 ± 8 25 ± 8

Zn 36 ± 5 45 ± 8 41 ± 8 46 ± 7 42 ± 8

Pyrite fraction (nmol g�1)

Cd 1.7 ± 0.3 1.8 ± 0.7 1.7 ± 0.5 2.1 ± 0.3 1.8 ± 0.5

Co 12 ± 2 16 ± 2 14 ± 3 14 ± 2 14 ± 3

Cu 4.8 ± 4.0 6.6 ± 2.4 5.7 ± 3.3 4.1 ± 1.6 5.4 ± 3.0

Fe (1.8 ± 0.7) 9 103 (2.2 ± 1.1) 9 103 (2.0 ± 1.0) 9 103 (2.1 ± 1.1) 9 103 (2.0 ± 1.0) 9 103

Mn 19 ± 6 26 ± 12 23 ± 10 36 ± 18 26 ± 13

Ni 14 ± 4 11 ± 6 12 ± 6 8.0 ± 3.9 11 ± 5

Pb 11 ± 2 5.4 ± 2.5 8 ± 4 <4.9 7.3 ± 3.9

Zn <1.4 <1.4 <1.4 <1.4 <1.4

Reactive fraction (nmol g�1)

Cd 7 ± 1 8 ± 1 7.7 ± 1.3 7 ± 2 7.6 ± 1.3 [42 ± 7]

Co 66 ± 16 71 ± 15 69 ± 15 68 ± 14 69 ± 15 [32 ± 7]

Cu 24 ± 9 31 ± 11 28 ± 11 25 ± 6 27 ± 10 [29 ± 7]

Fe (11 ± 3) 9 103 (17 ± 8) 9 103 (14 ± 7) 9 103 (16 ± 4) 9 103 (14 ± 7) 9 103 [18 ± 9]

Mn (0.23 ± 0.19) 9 103 (0.33 ± 0.22) 9 103 (0.28 ± 0.21) 9 103 (0.28 ± 0.06) 9 103 (0.28 ± 0.18) 9 103 [19 ± 10]

Ni 60 ± 16 75 ± 22 68 ± 20 63 ± 15 67 ± 19 [18 ± 4]

Pb 41 ± 8 28 ± 7 34 ± 10 26 ± 9 32 ± 10 [33 ± 10]

Zn 36 ± 5 46 ± 8 41 ± 9 46 ± 7 42 ± 9 [16 ± 5]

Enrichment factors (molar ratio)

Cd 94 ± 34 45 ± 22 68 ± 37 17 ± 3 57 ± 39

Co 9.3 ± 4.0 4.1 ± 1.6 6.6 ± 4.0 1.7 ± 0.3 5.5 ± 4.0

Cu 1.6 ± 0.5 0.92 ± 0.31 1.3 ± 0.5 0.32 ± 0.17 1.0 ± 0.6

Fe 0.90 ± 0.04 0.58 ± 0.03 0.73 ± 0.17 0.46 ± 0.04 0.67 ± 0.19

Mn 0.81 ± 0.05 0.58 ± 0.06 0.69 ± 0.13 0.66 ± 0.11 0.68 ± 0.12

Ni 4.8 ± 1.9 1.8 ± 0.7 3.1 ± 2.1 0.84 ± 0.24 2.6 ± 2.1

Pb 17 ± 6 6.4 ± 3.0 11 ± 7 2.0 ± 0.5 9.4 ± 7.4

Zn 2.0 ± 0.6 1.3 ± 0.4 1.6 ± 0.6 0.58 ± 0.11 1.4 ± 0.7

Values are also shown for mats 4A and 4B combined and for all mats (4A, 4B, and 5) combined. Numbers in brackets represent the percentage of reactive

metals relative to the total concentrations.



(Fe) and 609 ± 311 (Al) reported for a microbial mat tran-

sect from pond number 5 (Yevenes-Burgos, 2004). Total

trace metals also showed a general trend of increasing con-

centrations with increasing depth in the mat (Figure 2).

This trend may imply that either eolian input has been

decreasing with time due to changes in intensity and direc-

tion of the prevailing winds, or that aluminosilicates are

being affected by mat microbial activity. Salt production in

the saltern depends on the presence of constant winds that

efficiently mix the shallow water column of the ponds.

Considering that salt production has not been affected

because the salt company started operations in the area,

changes in wind intensity and/or direction does not appear

to be an important factor. However, it has been reported

that pH in mats can become alkaline (e.g., D’Amelio et al.,

1989; Ley et al., 2006), reaching values as high as 11 dur-

ing daytime (Decho et al., 2010), at least in the top 0.5–

2.0 mm of the mat. Partial dissolution of aluminosilicates

can easily be achieved in the presence of this high pH

which, in turn, could reduce the concentrations of trace

metals associated to clays, at least in the top layer of the

mat (Figure 2). This hypothesis is further supported by the

reported presence of diatoms on the surface of the Guerre-

ro Negro mats (e.g., Nubel et al., 1999; Omoregie et al.,

2004), probably stimulated by Si fluxes originated from

the dissolution of aluminosilicates in the upper portion of

the mats. Interestingly, the minima observed between 5

and 7 mm in Fig. 2 could be the result of dilution of clay

minerals by heavy rain. If an accretion rate of 0.45 cm per

year is used for our microbial mats (average of the 0.4–

0.5 cm per year range reported by Des Marais et al.,

1992), then the depth of 5–7 mm roughly corresponds to

1997, the year that Hurricane Nora passed over Guerrero

Negro (25 September 1997). The low values observed

below 15 mm could have been produced by a less intense,

but similar process, like a ‘wet’ period produced by the

negative ENSO condition present from 1990 to 1995.

When total trace metals were normalized with Al

(Fig. 3), however, Me/Al profiles were completely differ-

ent from those obtained for total metals (Fig. 2), an

indication that mineral clays are not controlling the con-

centrations of trace metals. Me/Al profiles showed high

values at mid-depths (e.g., Zn/Al), or remained relatively

constant (e.g., Fe/Al and Mn/Al) with depth. The con-

stancy in the Fe/Al and Mn/Al profiles suggests that Fe

and Mn are mainly associated with clay minerals with little

contribution from diagenetic components, as expected

from an environment limited by reactive Fe. The high val-

ues present at mid-depths for the other metals (Figure 3)

suggest that diagenetic processes, rather than clay minerals,

are affecting the concentrations of trace metals at these

depths, and that Hurricane Nora and the negative ENSO

Cdtot (nmol g–1)

Dep

th (

mm

)

0

5

10

15

20

Cotot (nmol g–1) Cutot (nmol g–1) Fetot (mol g–1)

Mntot (mol g–1)

Dep

th (

mm

)

0

5

10

15

20

Nitot (nmol g–1) Pbtot (nmol g–1) Zntot (nmol g–1)

A B DC

F G IH

Altot (mol g–1)

0 10 20 30 0 100 200 300 400 0 50 100 150 200 0 100 200 300

0 2 4 6 8 0 200 400 600 0 50 100 150 200 0 200 400 600 800

0 1000 2000

E

Mat 4A Mat 4B Mat 5

Fig. 2 Mat profiles of trace metals associated with the total fraction (Metot) for mats 4A, 4B, and 5.



condition period probably contributed with additional

loads of reactive trace elements (at 5–7 and below 15 mm,

respectively). Hence, changes in deposition processes

appear to be unimportant in controlling the distribution of

total metals, and their increasing concentrations with depth

in the mat can be a consequence of the gradual addition of

reactive trace metals as a result of authigenic mineral for-

mation at depth and addition by rainfall of reactive metals

from the surroundings. As we will discuss later, bacterial

extracellular polymeric substances (EPS) probably play an

important role in trace element distributions by quelating

metals and entrapping particulate material.

One question that may arise is whether it is appropriate

to use Al in evaporation ponds where microbial mats thrive

and in which inputs of clay minerals are mainly eolian and

irregular. To try to answer this question, we compared the

total metal concentrations (Metot) and Me/Al ratios of our

mats with those reported for sediments from the Mexican

Pacific Ocean (Nava-Lopez, 2002), an area relatively close

to our study area. Additionally, total concentrations of mat

and sediment from Solar Lake and Port Said, Egypt (Taher

et al., 1994) were further considered. The results of this

exercise indicate that although significant differences were

found between mats and sediments for some of the values,

the differences are neither so large nor consistent to conclu-

sively separate the mats from the sediments (see Fig. 4A,B).

Cd/Al (molar ratio)

Dep

th (

mm

)0

5

10

15

20

Co/Al (molar ratio) Cu/Al (molar ratio) Fe/Al (molar ratio)

Mn/Al (molar ratio)

Dep

th (

mm

)

0

5

10

15

20

Ni/Al (molar ratio) Pb/Al (molar ratio) Zn/Al (molar ratio)

0.00000 0.00008 0.00016 0.000 0.001 0.002 0.0000 0.0004 0.0008 0.0 0.1 0.2 0.3

0.000 0.003 0.006 0.0000 0.0025 0.0050 0.0000 0.0004 0.0008 0.0012 0.000 0.001 0.002

A B DC

E F HG

Mat 4A Mat 4B Mat 5

Fig. 3 Mat profiles of total trace metals (Me) normalized with aluminum (Al) for mats 4A, 4B, and 5.

Element

Me/

Al (

mo

lar

rati

o)

10–5

10–4

10–3

10–2

10–1

100

To

tal c

on

cen

trat

ion

(nm

ol g

–1)

100

101

102

103

104

105

106

107

Cd Cu Pb Co Zn Ni Mn Fe

Cd Cu Pb Co Zn Ni Mn Fe Al

GNMPOPS-MEPS-MDSL

B

A

Fig. 4 Average (± one standard deviation) (A) total trace metal concentra-

tions and (B) metal to aluminum ratios (Me/Al) of Guerrero Negro (GN)

mats from evaporation ponds 4 and 5 combined. Values reported for sedi-

ments and microbial mats from other hypersaline locations and for sedi-

ments close to the study area (Mexican Pacific Ocean) are included as

reference: Mexican Pacific Ocean (MPO; Nava-Lopez, 2002), Solar Lake

(SL), Egypt (Taher et al., 1994), and Port Said, Egypt that includes

mat-enriched (PS-ME) and mat-deficient (PS-MD) sediments (Taher et al.,

1994).



The similarity of mats with sediments is probably due to

the intrusion of sediment particles into the mucilaginous

layer of the mat (e.g., Dupraz & Visscher, 2005) known as

EPS. Bacterial EPS are made mostly of heteropolymeric,

high molecular weight, polysaccharides (10–30 kDa) rich in

hexoses, proteins, and uronic acid (Bhaskar & Bhosle, 2005;

Omoike & Chorover, 2006). The presence of this acid gives

an overall negative charge to the polymer and, therefore, the

capacity to adsorb a wide range of metals (e.g., Pb, Co, Sr,

Cr, Th, Cd; Bhaskar & Bhosle, 2005; Mikutta et al., 2012).

Metal sorption occurs mainly through the formation of

unidentate, bidentate, and multidentate complexes with the

EPS molecules (Decho, 2000). Additionally, the EPS is able

to trap and retain the finest sediment fraction, a fraction that

because of its relatively high surface area, the biotic and min-

eral fractions in the mat cannot be effectively separated

(Webster-Brown & Webster, 2007).

Enrichment factors

Enrichment factors (molar ratios) for all mats combined

ranged from 0.67 ± 0.19 for Fe to 57 ± 39 for Cd

(Table 1). Metal enrichment or impoverishment in this

study was arbitrarily defined as EFMe � 2 and

EFMe � 1/2 = 0.5, respectively, with a zone of ‘normal-

ity’ represented by the EFMe values in between. According

to this definition, none of the metals are impoverished and

Fe (0.67 ± 0.19), Mn (0.68 ± 0.12), Cu (1.0 ± 0.6), and

Zn (1.4 ± 0.7) are neither impoverished nor enriched (i.e.,

they are within the normality zone), whereas Ni

(2.6 ± 2.1), Co (5.5 ± 4.0), Pb (9.4 ± 7.4), and Cd

(57 ± 39) are generally enriched. Our average EF values

compare favorably with those previously reported by

Huerta-Diaz et al. (2011) and Yevenes-Burgos (2004) for

mats of Pond 5 (Ni: 2.1 ± 1.0 and 3.4 ± 3.2, Co:

2.2 ± 0.4 and 8.3 ± 7.7, Pb: 2.8 ± 1.6 and 23 ± 43, Cd:

35 ± 10 and 94 ± 133). The high enrichment values can-

not be ascribed to contamination inputs from the city of

Guerrero Negro, which is too small (~30 000 inhabitants)

and too far (~28 km) from ponds 4 and 5 to represent a

significant source of anthropogenic emissions to the study

sites. The high EF values of Ni, Co, Pb, and Cd could be

partially caused by evapoconcentration processes, which

could in turn produce saturation of the overlying water fol-

lowed by mineral precipitation (Fig. 5). Another possibility

would be that dissolved metals, enriched in the water col-

umn by the evapoconcentration processes, were eventually

transferred to the mats via scavenging by biogenic particles

and dissolved organic matter produced in the overlying

water (DOC = 1.7–2.7 m in Pond 5; Des Marais et al.,

1989). Our hypothesis regarding the combination of phys-

ical, chemical, and biological processes as the mechanisms

Fig. 5 Conceptual model for the different processes involving trace metals in ponds 4 and 5. Note that the figure was not drawn to scale.



responsible for the trace metal enrichment and distribution

in the microbial mats from Guerrero Negro has substantial

support and could be especially true for Cd, whose biogeo-

chemical cycle is closely linked to that of nutrients (Bru-

land, 1983) and organic matter. Hence, Cd enrichment

factors could be further enhanced in microbial mats, which

have been determined to be the most productive aquatic

ecosystems on Earth (Canfield & Des Marais, 1994). Fur-

thermore, Cd has been reported as significantly enriched in

plankton off Baja California (Martin & Broenkow, 1975)

and in sediments from the continental shelf of Baja Califor-

nia (EFCd = 23 ± 10; Nava-Lopez, 2002) and Sebastian

Vizcaıno Bay (adjacent to Ojo de Liebre Lagoon;

EFCd = 11 ± 8; Daessle et al., 2000). Similar processes

may also explain Pb and Co enrichments which are also

enriched in the shelf sediments of Baja California

(EFPb = 3.7 and EFCo = 2.1, respectively; Nava-Lopez,

2002). Whether these enrichments are occurring only in

evaporation ponds 5 and 6, in all the evaporation ponds,

or in the whole region of Guerrero Negro is a question

that we hope to address once trace metal analyses of soils

surrounding the ponds and adjacent sediments from Ojo

de Liebre Lagoon are completed. This work is part of

future research proposed for this particular region.

Once in contact with the microbial mat surface, metals

could become associated with Mn or Fe oxihydroxides,

bound to EPS, or incorporated into living organic matter

through the process of photosynthesis or other metabolic

processes. For example, Synechococcus (a conspicuous

cyanobacteria of Guerrero Negro mats; D’Amelio et al.,

1989) requires 0.08–1.43 lmol mol�1 Co:C to thrive.

(Saito et al., 2002), with similar metabolic requirements

probably applying to Ni (e.g., Price & Morel, 1991).

Later, the metals can be released at depth by interactions

with EPS or when the organic matter is oxidized by het-

erotrophic processes (e.g., oxic respiration, denitrification,

sulfate reduction) and, eventually, precipitated as sulfides

in the presence of dissolved sulfides (e.g., Heggie & Lewis,

1984; Gendron et al., 1986). The liberated metals could

subsequently coprecipitate with Fe sulfides (mainly pyrite

and AVS), minerals that readily precipitate given the pres-

ence of substantial amounts of dissolved sulfide (e.g., Des

Marais, 1995), especially in the deeper parts of the mats

(Fig. 5). This process may explain the accumulation of

trace metals at depth, especially in the reactive relative to

the pyrite fraction, because formation of iron sulfides is

hindered by lack of reactive Fe. Interestingly, Gehrke et al.

(1998) found that EPS are involved in metal sulfide (e.g.,

pyrite) dissolution through the attachment of lipopolysac-

charide-containing EPS to the sulfide substrate. The pro-

cess is mediated by exopolymer-complexed Fe(III) ions

that allow the establishment of an electrochemical inter-

action with the negatively charged pyrite surface. Some

minerals that are bound to EPS can change the extent and

rate of trace metal sorption by inducing mineral-specific

alterations of the pore-size distributions or by providing

additional sites for complexation (Omoike & Chorover,

2006; Mikutta et al., 2012). Other mechanisms involved

in metal sorption are cation exchange with Ca or Mg,

microprecipitation of metals, and global electric field

surrounding the ligand (Guibaud et al., 2006). Given the

ability of bacterial EPS to initiate the development of fine-

grained minerals by quelating dilute metals on their surface

(Beveridge et al., 1997), it is reasonable to assume that

EPS can be responsible, to a significant extent, of the

enrichment of some of the total metal concentrations mea-

sured in our mats. Geochemical studies of trace metals (Fe,

Cu, Pb, Zn, Ni, Ag, Cd) in microbial mats from the Lake

Vanda region in Antarctica showed that an apparent trace

metal enrichment in the mats, relative to the sediment

beneath, was due to the incorporation of fine (submicron)

sediment particles in the mucilaginous matrix of the mat

(Webster-Brown & Webster, 2007). According to these

authors, although loosely adhering sediment was removed

during ultrasonic cleaning of the sample, it was clear from

microscopic examination that fine sediments remained

strongly bound within the biological matrix.

HCl and pyrite-associated trace metals

Metals associated with the pyrite fraction were generally

low, as expected from reactive Fe-limited systems, with

values ranging from <1.4 to 23 ± 10 nmol g�1 for Zn and

Mn, respectively, for mats in Pond 4. For the mat in Pond

5, the range was from <1.4 to 36 ± 18 nmol g-1 for Zn

and Mn, respectively (Table 1). Metals associated with the

HCl fraction (Table 1) in the mat from Pond 5 ranged

from 5.3 ± 1.6 to 239 ± 63 nmol g�1 for Cd and Mn,

respectively, quite similar to the range found in mats from

Pond 4 (5.9 ± 1.3 to 261 ± 196 for Cd and Mn, respec-

tively). Similarly to the total profiles, HCl- and pyrite-asso-

ciated trace metals (Cd, Co, Cu, Fe, Mn, Ni, Pb, Zn) also

showed a general trend of increasing concentration with

increasing depth in the mat (Figs 6 and 7, respectively),

probably a consequence of the gradual addition of reactive

trace metals as a result of organic-matter mineralization. In

a number of them, the concentration increment was almost

linear with depth, as shown by the significant correlations

obtained from regression analyses of HCl or pyrite frac-

tions vs. depth in the mat (Table 2). In particular, Ni in

both fractions showed significant correlations (P � 0.05

or better) with depth in all mats (cores 4A, 4B, 5), with

the rest of the trace metals showing significant correlations

in some cores and in some of the fractions. Only Zn

showed no significant correlations with depth in the mat

along the different cores and fractions, possibly due to its

very low concentrations. Overall, of the 48 calculated con-

centrations vs. depth linear regressions, slightly more than



half (25 equivalent to 52%) were significantly correlated

(P � 0.05 or better; Table 2).

The reasons for the observed coherent trends in trace

metal profiles can be ascribed to geochemical processes,

bacterial population distributions, and differential mass

transport within the mats. In modern marine stromatolites,

biogeochemical and microbial processes (cyanobacterial

photosynthesis, sulfate reduction, and anaerobic sulfide oxi-

dation) are responsible for micrometer-scale CaCO3 precip-

itation, whereas aerobic respiration and aerobic sulfide

oxidation cause CaCO3 dissolution (Visscher et al., 2000;

Dupraz & Visscher, 2005; Dupraz et al., 2009; Puckett

et al., 2011). First-order increase trends in sulfide concen-

tration with depth in the mat have been reported (e.g.,

Jørgensen et al., 1979; Revsbech et al., 1983; Canfield &

Des Marais, 1993) arising from localized bacterial sulfate

reduction and/or other sulfur metabolisms (Fike et al.,

2008). Additionally, the laminar structure of the mats pro-

motes the establishment and maintenance of microzones,

which in turn will affect the molecular diffusion of sulfides

(Paerl & Pinckney, 1996) and other dissolved components.

Hence, increasing sulfide concentrations may imply increas-

ing levels of iron sulfides like pyrite and acid volatile sulfide

(AVS) and associated trace elements with depth. Because

these metals are represented in the HCl (AVS is extracted

in this fraction) and pyrite fractions (i.e., the reactive frac-

tion = MeHCl + Mepyr), it is reasonable to assume that they

will be present primarily in these two fractions and mini-

mally in the total fraction (see Table 1 for reactive fraction

percentages as a function of their total concentrations), as

they generally represent small enrichments over the back-

ground alumnosilicate values. Indeed, this behavior is what

we observed in the millimeter-scale profiles of HCl, pyrite,

and, to a certain extent, total fractions (Figs 6, 7 and 2,

respectively); they were near absent, however, in the

Al-normalized profiles (Fig. 3).

The operationally defined reactive fraction (equivalent to

the sum of MeHCl + Mepyr), as its name implies, represents

the pool of a given metal that is available to be transformed

into other mineral phases, or to be released into the inter-

stitial water for further interactions with the biota. By con-

trast, the silicate fraction is essentially non-reactive toward

the formation of new minerals and does not play an active

role in terms of early diagenetic transformations. For exam-

ple, whereas the oxyhydroxide mineral ferrihydrite has a

half-life of 4 h in a 1 mM sulfide solution, sheet silicate iron

has a half-life of 100 000 years (Canfield et al., 1992).

Another characteristic of silicate minerals is that they exhibit

high concentrations of certain trace metals (e.g., Fe, Mn,

Zn), while others like Cd tend to accumulate in the reactive

CdHCl (nmol g–1)

Dep

th (

mm

)0

5

10

15

20

CoHCl (nmol g–1) CuHCl (nmol g–1) FeHCl (mol g–1)

MnHCl (mol g–1)

Dep

th (

mm

)

0

5

10

15

20

NiHCl (nmol g–1) PbHCl (nmol g–1) ZnHCl (nmol g–1)

0 2 4 6 8 10 0 20 40 60 80 100 0 10 20 30 40 50 0 10 20 30

0.0 0.2 0.4 0.6 0.8 0 20 40 60 80 100 0 20 40 60 0 20 40 60 80

A B DC

E F HG

Mat 4A Mat 4B Mat 5

Fig. 6 Mat profiles of trace metals associated with the HCl fraction (MeHCl) for mats 4A, 4B, and 5.



Cdpyr (nmol g–1)

Dep

th (

mm

)0

5

10

15

20

Copyr (nmol g–1) Cupyr (nmol g–1) Fepyr (mol g–1)

Mnpyr (mol g–1)

Dep

th (

mm

)

0

5

10

15

20

Nipyr (nmol g–1) Pbpyr (nmol g–1) Znpyr (nmol g–1)

0 1 2 3 4 0 5 10 15 20 25 0 4 8 12 16 0 1 2 3 4 5

0.00 0.04 0.08 0 5 10 15 20 25 0 4 8 12 16 0 1 2 3 4

A B C D

E F G H

Mat 4A Mat 4B Mat 5

Fig. 7 Mat profiles of trace metals associated with the pyrite fraction (Mepyr) for mats 4A, 4B, and 5.

Table 2 Slopes (± standard error) and correlation coefficients (r) obtained from regression analyses of HCl or pyrite fraction vs. depth in mat

Element Core

HCl fraction slope

(nmol g�1 mm�1) r

Pyrite fraction slope

(nmol g�1 mm�1) r

Cd 4A 0.133 ± 0.050 0.683* 0.034 ± 0.014 0.640*

Cd 4B 0.163 ± 0.039 0.810** �0.075 ± 0.024 �0.717*

Cd 5 0.28 ± 0.16 ns 0.036 ± 0.029 ns

Co 4A 1.57 ± 0.58 0.692* 0.286 ± 0.090 0.749*

Co 4B 1.97 ± 0.25 0.934*** 0.195 ± 0.051 0.788**

Co 5 2.4 ± 1.4 ns 0.25 ± 0.30 ns

Cu 4A 0.52 ± 0.22 0.651* 0.55 ± 0.13 0.832**

Cu 4B 1.15 ± 0.31 0.780** 0.326 ± 0.047 0.918***

Cu 5 1.10 ± 0.49 ns 0.14 ± 0.21 ns

Fe 4A (2.6 ± 1.5) 9 102 ns �7 ± 41 ns

Fe 4B (8.4 ± 2.4) 9 102 0.756** 83 ± 49 ns

Fe 5 (1.12 ± 0.35) 9 103 0.851* (�2.3 ± 0.8) 9 102 �0.829*

Mn 4A 17.3 ± 8.9 ns 0.16 ± 0.36 ns

Mn 4B 21.6 ± 7.4 0.699* 0.69 ± 0.57 ns

Mn 5 11.2 ± 6.3 ns �4.35 ± 0.92 �0.922**

Ni 4A 1.72 ± 0.49 0.779** 0.49 ± 0.19 0.676*

Ni 4B 2.22 ± 0.38 0.888*** 0.77 ± 0.18 0.822**

Ni 5 2.77 ± 0.98 0.818* 0.96 ± 0.21 0.914*

Pb 4A 1.03 ± 0.32 0.752* 0.064 ± 0.097 ns

Pb 4B 0.82 ± 0.23 0.762** 0.04 ± 0.13 ns

Pb 5 1.37 ± 0.80 ns �0.25 ± 0.44 ns

Zn 4A �0.05 ± 0.30 ns �0.022 ± 0.012 ns

Zn 4B 0.69 ± 0.32 ns 0.035 ± 0.045 ns

Zn 5 1.24 ± 0.70 ns – –

***P � 0.001; **P � 0.01; *P � 0.05; ns, not significant.



fraction. In our samples from Guerrero Negro, reactive

concentrations were represented from 16 ± 5 to 42 ± 7%

for Zn and Cd, respectively, relative to their respective total

concentrations (Table 1). Hence, more than 50% of the

trace element concentrations in our samples were distrib-

uted in the silicate fraction, as is normally expected consid-

ering that sediments are mostly composed of clay minerals

(15–50%; Griggs & Hein, 1980; Schulz & Zabel, 2000).

Degree of pyritization and degree of trace metal

pyritization

Evidence that iron limitation prevents pyrite formation in

mats and underlying anoxic-sulfidic sediments from Pond

5 in the saltern of Guerrero Negro was recently reported

by Huerta-Diaz et al. (2011). They measured very low

concentrations of FeRF relative to other sedimentary envi-

ronments, a slight impoverishment of total Fe, intermedi-

ate DOP (12–50%) values, and high degrees of

sulfidization (DOS: up to 100%). Our Fe data set for

microbial mats from Pond 4 (4A and 4B combined) may

suggest that the same condition may exist in this pond as

well, because FeRF values ranged from 6.3 to 33 lmol g�1

(average: 14 ± 7 lmol g�1, n = 21; Table 1), very similar

to the 10–33 lmol g�1 (average: 20 ± 12 lmol g�1,

n = 5) range reported by Huerta-Diaz et al. (2011) for a

mat collected previously to this study in Pond 5.

In general, average degrees of pyritization (DOP) and

degrees of trace metal pyritization (DTMP) within all mats

studied here, were relatively low (1 ± 1% for Zn to

24 ± 7% for Cd), an expected result for a sedimentary envi-

ronment in which the production of pyrite and AVS are

limited by the availability of reactive Fe (Huerta-Diaz

et al., 2011). AVS is comprised mainly of mackinawite and

greigite, minerals potentially capable of incorporating

important amounts of trace elements. Hence, it should be

reasonable to assume that an unknown part of the metal

sulfide pool could be transferred to the HCl fraction,

decreasing the DTMP levels in the process. Unfortunately,

no direct method has been developed for determining

AVS-associated trace metals (however, for an indirect eval-

uation method, see the study by Huerta-Diaz et al.,

1998). Our results simply show that low concentrations of

pyrite lead to low concentrations of pyrite-associated met-

als and, therefore, to low DTMP values, as shown in

Zn Mn Fe Ni Cu Co Pb Cd

DO

P o

r D

TM

P (

%)

0

10

20

30

40

Element

Mat 4A Mat 4B Mat 5

Fig. 8 Box plots showing degrees of pyritization (DOP for Fe) and degrees of trace metal pyritization (DTMP for all other metals) for mats 4A, 4B, and 5.

Elements are arranged in increased order of overall (all mats combined) degree of pyritization (DOP) values. Open symbols represent average values.

Table 3 Correlation coefficients (r) and their significance (P) obtained by

comparing the concentrations of mat 4A with mat 4B for the given fraction

(total, HCl, pyrite, and reactive)

Element Total fraction HCl fraction Pyrite fraction Reactive fraction

Cd ns ns ns ns

Co ns ns ns ns

Cu ns 0.901*** 0.744* 0.901***

Fe ns 0.708* 0.682* 0.752*

Mn ns ns 0.692* ns

Ni ns 0.758* 0.835** 0.807**

Pb ns ns ns ns

Zn ns ns ns ns

***P � 0.001; **P � 0.01; *P � 0.05; ns, not significant.



Fig. 8. These findings are consistent with those of Huerta-

Diaz & Morse (1992), who showed that low DOP values

generally corresponded to low DTMP values in sediments

from a wide variety of marine environments. As can be

seen in Fig. 8, differences in DOP and DTMP between

the different mats generally were not statistically significant,

except in the cases of Ni and Pb for mats 4A and 4B.

With the exception of DTMP-Zn, all DTMP values were

rather similar to the DOP values. Complete pyritization

(close to 100%) of reactive Fe is to be expected in Fe-lim-

ited sedimentary environments with abundant dissolved

sulfide present, characteristics that appear to be present in

ponds 4 and 5 (Des Marais, 1995; Huerta-Diaz et al.,

2011). The reason for the low to medium DOP values

obtained in these two ponds lies in the different iron

sulfide pools (pyrite and AVS) normally present in marine

sediments, combined with the low concentrations of Fe

available for pyrite formation (Huerta-Diaz et al., 2011)

and, possibly, the presence of ‘amorphous’ Fe silicates

(Canfield, 1989).

Reactive trace metalconcentrations in mats4A and 4B (nmol g–1)

Rea

ctiv

e tr

ace

met

alco

nce

ntr

atio

ns

in m

at 5

(n

mo

l g–1

)

0.0

0.2

0.4

20.0

40.0

60.0

80.0

100.0

Total trace metalconcentrations in mats4A and 4B (nmol g–1)

To

tal t

race

met

alco

nce

ntr

atio

ns

in m

at 5

(n

mo

l g–1

)

0

10

20

200

400

600

Pyrite trace metalconcentrations in mats4A and 4B (nmol g–1)

Pyr

ite

trac

e m

etal

con

cen

trat

ion

s in

mat

5 (

nm

ol g

–1)

0.00

0.03

0.06

5.00

10.00

15.00

20.00

1:1 L

ine

4A vs. 5:Y = (36–15) + (1.19–0.10)Xr2 = 0.75P = < 0.001n = 48

4B vs. 5:Y = (30–13) + (0.74–0.05)Xr2 = 0.80P = < 0.001n = 48

4A vs. 5:Y = (0.6–1.8) + (1.17–0.06)Xr2 = 0.88p = < 0.001n = 48

4B vs. 5:Y = (1.5–1.4) + (1.04–0.05)Xr2 = 0.92p = < 0.001n = 48

4A vs. 5:Y = (0.93–0.68) + (0.67–0.09)Xr2 = 0.53P = < 0.001n = 48

4B vs. 5:Y = (0.37–0.48) + (0.86–0.07)Xr2 = 0.76P = < 0.001n = 48

4A vs. 5:Y = (1.1–2.2) + (1.09–0.06)Xr2 = 0.87P = < 0.001n = 48

4B vs. 5:Y = (1.0–1.5) + (1.05–0.04)Xr2 = 0.93P = < 0.001n = 48

1:1 L

ine

1:1 L

ine

1:1 L

ine

HCl trace metalconcentrations in mats4A and 4B (nmol g–1)

0.0 0.2 0.4 20.0 40.0 60.0 80.0 100.0

0 10 20 200 400 600

0.00 0.03 0.06 5.00 10.00 15.00 20.00

0.0 0.2 20.0 40.0 60.0 80.0

HC

l tra

ce m

etal

con

cen

trat

ion

s in

mat

5 (

nm

ol g

–1)

0.0

0.2

20.0

40.0

60.0

80.0

Cd, Co, Mn, Ni, Pb, ZnCu,

Fe in mol g–1

Mn in mol g–1

Red symbols: Mat 4A vs. Mat 5; Yellow symbols: Mat 4B vs Mat 5

Fig. 9 Similarities between microbial mats 5 and 4A or 4B, where metal concentrations for total, HCl, pyrite, and reactive fractions from mat 5 are compared

with those of mat 4A (gray symbols) or 4B (white symbols). Because the differences in concentrations for a given fraction encompassed in some cases

are close to eight orders of magnitude (e.g., pyrite), different concentration units were used for constructing this figure. All concentrations are expressed

in nmol g�1, except when noted. Dashed lines represent the ideal 1:1 relationship. Note the breaks in the X and Y axes.



Trace metal associations

An important characteristic of the different trace metal pro-

files is their shape similarity, despite the fact that the mats

were collected in different evaporation ponds with different

salinities (mats 4A and 5, S = 91 and 119, respectively)

and at slightly different dates (mats 4A and 4B, June 17

and 19, respectively). Furthermore, given the complex and

biologically diverse composition of the microbial mats from

Guerrero Negro (Ley et al., 2006), it is surprising that

such similarity exists, which is quite noticeable in the case

of the HCl and reactive fractions and, to a certain extent,

pyrite and total (Figs 2, 6, and 7). The degrees of similar-

ity between mats 4A and 4B were simply established by

calculating the correlation coefficient (r) obtained by com-

paring the concentration values between these two cores

for a given depth and fraction (total, HCl, pyrite, and reac-

tive). The results of this calculation are shown in Table 3,

where the HCl, pyrite, and reactive fractions showed sig-

nificant correlations (P � 0.05 or better) for Cu, Fe, Mn

(only for pyrite), and Ni. Based on these results, we could

postulate that Cu and Ni could be used as a pair of ele-

ments suitable as biosignatures to test the past presence of

soft mats in the geological record. The high r2 values for

the reactive (HCl + pyrite) fraction suggest that minerals

produced by the metabolic activities of mat micro-organ-

isms play a significant role in the generation of the profiles.

In contrast, no significant correlations were found for the

total fraction, probably due to variable eolian inputs from

the surrounding desert and occasional rains which intro-

duce small-scale variations in the profiles due to the incor-

poration of aluminosilicate particles to the mats.

Metal concentrations for total, HCl, pyrite, and reactive

fractions from mat 5 were compared with those of mat 4A

or 4B to find similarities between the first six mat sections

(1–11 mm), the only depths common to all three mats.

Results from this exercise (Fig. 9) show that similarities

between mat 5 and 4A or 4B are more evident for the

reactive and HCl fractions, followed by the total and pyrite

fractions. Considering that the trace metals associated with

the pyrite fraction are not important due to the reactive Fe

limitation in pyrite formation, then the geochemical rele-

vance of the reactive and HCl fractions is further enhanced

in the mat ecosystem (Table 1). These results suggest that

the geochemistry of trace metals in the mats is not con-

trolled by iron sulfide minerals, as is the case in most mar-

ine systems (e.g., Huerta-Diaz & Morse, 1992), but rather

by the as yet unknown mineral phases that are part of the

dominant HCl fraction. Linear regression analyses of trace

metal concentrations in mat 4A or 4B vs. those in mat 5

gave significant relationships (P � 0.001) in all cases

(total, HCl, pyrite, and reactive fractions, Fig. 9). How-

ever, coefficients of determination (r2) for the regression

analyses of mat 4A vs. mat 5 and mat 4B vs. mat 5 were

higher for the reactive (0.87 and 0.93, respectively) and

HCl (0.88 and 0.92, respectively) than for the pyrite (0.53

and 0.76, respectively) and total (0.75 and 0.80, respec-

tively) fractions (Figure 9). Furthermore, the slopes of the

regression lines were closer to unity for the reactive

(1.09 ± 0.06 and 1.05 ± 0.04, respectively) and HCl

(1.17 ± 0.06 and 1.04 ± 0.05, respectively) than for the

pyrite (0.67 ± 0.09 and 0.86 ± 0.07, respectively) and

total (1.19 ± 0.10 and 0.074 ± 0.05, respectively) fractions

(Fig. 9). The similarities between mats 4A and 4B or

between these two mats and core 5 are most probably

related to the internal laminations of the mats. These lami-

nations reflect the relative contributions over time by the

bacterial community (Des Marais, 1995; Fike et al., 2008),

whose distribution is apparently the result of strong spatial

gradients of light intensity and redox conditions (Fike

et al., 2008). These localized metabolic activities taking

place at submillimeter scales is present not only in the

uppermost few millimeters of the mat (close to the photo-

synthetically active surficial layer), but also at the depth

(e.g., Bebout et al., 1994, 2004; Visscher et al., 1998,

2000; Fike et al., 2009). Thus, our results indicate that

the vertical distributions of reactive trace metals within the

mat are probably associated with these gradients, and that

they are reflected in the relatively constant rate of metal

concentration increments with depth. Our results indicate

that the metal gradients are more evident for the reactive

rather than for the total trace elements, a consequence of

remobilization produced by the remineralization of organic

matter and/or EPS influence on reactive minerals.

CONCLUSIONS

Our results indicate that trace metals in soft, non-lithified

microbial mats present several characteristics that could be

advantageously used as biosignatures to identify their pres-

ence (and those of stromatolites, their mineralized counter-

parts) in the geological record or in other planetary

systems (e.g., Mars). The main characteristics of these

microbial systems are (i) they are enriched with Ni, Co,

Pb, and Cd; (ii) exhibit relatively similar profiles of reactive

metals, which generally show increasing concentrations

with increasing depth in the mat; (iii) impoverished con-

centrations of total and reactive Fe, which in turn produce

low pyrite concentrations and, as a consequence produce

(iv) low to moderate DOP and DTMP values. Therefore,

the presence of laminations that promote the establishment

and maintenance of geochemical and biological microz-

ones, appear to be an important factor affecting the vertical

and horizontal distributions of trace metals within the

microbial mat. Future research should focus on increas-

ingly finer vertical resolution (lm scale) using mats and

stromatolites from different extreme environments (e.g.,

temperature, salinity) and in fossil microbial systems.



ACKNOWLEDGMENTS

We thank the management and technical staff of the Com-

panıa Exportadora de Sal S.A. (ESSA) for their assistance

and support. To Brad Bebout and David Des Marais of

NASA-Ames for their advice and for allowing us to accom-

pany them on their permits. The comments of two anony-

mous reviewers improved this work significantly and is

greatly acknowledged. This work was funded by the Cons-

ejo Nacional de Ciencias y Tecnologia (CONACYT) pro-

ject number CB-2005-1-025341. Support for J.V.-O. and

J.J.R. in the form of Consejo Nacional de Ciencia y Tec-

nologıa (CONACYT) Research Assistantships is greatly

acknowledged. The authors declare that they have no con-

flict of interest to declare.

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