www.elsevier.com/locate/hydromet

Hydrometallurgy 71 (2003) 301–309

Geomicrobiology of the Tinto River, a model of interest for

biohydrometallurgy

E. Gonzalez-Torila,*, F. Gomezb, N. Rodrıguezb, D. Fernandez-Remolarb,J. Zuluagac, I. Marına, R. Amilsa,b

aCentro de Biologıa Molecular, UAM-CSIC, Cantoblanco, Madrid 28049, SpainbCentro de Astrobiologıa, INTA-CSIC, Torrejon de Ardoz 28850, Spain

cDepartamento de Quımica Fısica Aplicada, UAM, Cantoblanco, Madrid 28049, Spain

Abstract

The Tinto River (Huelva, southwestern Spain) is an extreme environment with a constant acidic pH (mean 2.3), a high

concentration of heavy metals and a remarkable level of microbial diversity (bacteria, archaea, photosynthetic and heterotrophic

protists, yeast and filamentous fungi). The extreme conditions found in the river are the direct consequence of the active

metabolism of chemolithotrophic microorganisms thriving in the rich polymetallic sulfides present in high concentrations in the

Iberian Pyritic Belt. Primary production in the river is driven mainly by oxygenic photosynthesis (protists and cyanobacteria),

although an important part is also due to the activity of chemolithotrophic prokaryotes. Conventional and molecular ecology

techniques were used to study the microbial ecology of the Tinto system. The results of both methods agreed. Although sulfur

metabolism plays an important role in the system, iron seems to be the key element in this habitat. Iron is not only an important

substrate for the rich population of iron oxidizing prokaryotes, but also an electron acceptor for anaerobic respiration in the

anoxic parts of the river. It is also responsible for the maintenance of a constant acidic pH (probably critical for biodiversity) and

for radiation protection. Laminar iron stromatolitic formations can be found along the river. These structures are related to

massive laminated bioinduced iron formations found at different elevations above the current river. The isotopic dating of these

formations leads to the conclusion that the Tinto River corresponds to a natural system and not to an industrial, contaminated

site. A geomicrobiological model of this habitat encompassing most of the geological, physical, chemical and biological

variables is presented and its biohydrometallurgical implications discussed.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Geomicrobiology; Chemolithotrophy; Tinto River; Iron; Acidic pH

1. Introduction

Extremophilic microorganisms capable of develop-

ing in extreme environments have recently attracted

considerable attention because of the challenge that

their discovery has posed to our current notions of the

0304-386X/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0304-386X(03)00169-5

* Corresponding author.

limits of life and its possible origin, but, most impor-

tantly, because of their biotechnological potential. Our

research group has characterized the microbial life of

an acidic habitat, the Tinto River, a 100-km long

acidic river containing a high concentration of heavy

metals in solution, which originates at Pena de Hierro,

at the core of the Rıo Tinto mining district, and flows

into the Atlantic Ocean at Huelva. The river gives its

name to an important mining site, which has been in

E. Gonzalez-Toril et al. / Hydrometallurgy 71 (2003) 301–309302

operation for more than 5000 years. In the past, the

extreme conditions of the river were considered the

consequence of intense mining activity in the area.

Conventional microbial ecology studies showed

that both sulfur and iron oxidizing bacteria are present

at rather high concentrations along the river (Gonza-

lez-Toril et al., 1999; Lopez-Archilla et al., 1993,

2001). In spite of the low pH values and the

corresponding high concentration of heavy metals,

an unexpected level of eukaryotic diversity has been

described in the system (Lopez-Archilla et al., 2001).

Classical microbial analysis has a strong bias as a

result of the absolute requirement of isolation of

microorganisms in selective media prior to their

characterization. The introduction of molecular biol-

ogy techniques, mainly in situ hybridization using

fluorescent probes (FISH) and denaturating gradient

gel electrophoresis (DGGE) enable the resolution of

PCR amplified rDNA using primers with different

phylogenetic specificity, has produced an authentic

revolution in microbial ecology, in general, and in the

study of extreme environments, in particular. The

combination of classical isolation with molecular

ecology techniques has advanced a geomicrobiolog-

ical model of the Tinto system, which has important

implications in biohydrometallurgy.

2. Materials and methods

2.1. Sampling and analysis of physico-chemical

parameters

Samples were collected in triplicate from different

sampling stations along the river. Total content of

metals was measured by atomic absorption spectro-

photometry and X-ray fluorescence reflection. Sulfate

concentrations were determined by a turbidimetric

method (Keith, 1996a) and ferrous iron by a colori-

metric method (Keith, 1996b). Conductivity, pH,

oxygen and redox potential values were measured in

situ using specific electrodes.

2.2. Isolation and phenotypic characterization of

microorganisms

The isolation and characterization of chemolitho-

trophic and heterotrophic bacteria, archaea, photosyn-

thetic and heterotrophic protists, yeast and filamentous

fungi were performed as previously described (Lopez-

Archilla et al., 2001).

2.3. Fluorescence in situ hybridization

The basic protocol described by Amann (1995)

with some modifications required for its adaptation to

acidic environments has been used throughout the

work. One milliliter of each sample was denaturated

with 0.1 ml of formaldehyde solution (37%) and kept

for 4 h at 4 jC. Samples were filtered through GTTP

Millipore filter (0.22 Am) and washed with 10 ml of

minimal Mackintosh medium (Mackintosh, 1978) to

eliminate excess of formaldehyde and heavy metals,

followed by a 10-ml wash with PBS buffer (130 mM

NaCl, 10 mM sodium phosphate pH 7.2) and air

dried. The following fluorescent probes were used:

EUB338 (GCTGCCTCCCGTAGGAGT) for bacteria,

Non338 (ACTCCTACGGGAGGCAGC) as a nega-

tive control, GAM42a (GCCTTCCCACATCGTTT)

for g-proteobacteria, Bet42a (GCCTTCCCACTTCG-

TTT) for h-proteobacteria, Thio1 (GCGCTTTCTGG

GGTCTGC) for Acidithiobacil lus , ALF968

(GGTAAGGTTCTGCGCGTT) for a-proteobacteria,

NTR712 (CGCCTTCG CCACCGGCCTTCC) for

members of the Nitrospira group and FER656

(CGTTTA ACCTCAC CCGATC) for Ferroplasma.

2.4. Denaturating gradient gel electrophoresis

One liter of water from selected sampling stations

was filtered through a 0.22-Am Millipore filter and

washed with Mackintosh minimal media at pH 1.5.

Filters were kept at 4 jC until used. A piece of filter

membrane was treated with 2 ml of SET buffer (25%

saccharose, 50 mM Tris pH 8, 2 mM EDTA) over-

night at � 20 jC. After thawing, the sample 120 Al ofSDS (25%) and 200 Al of pronase (2 mg/ml) were

added and the mixture incubated at 4 jC for 30 min.

Each sample was extracted with 6 ml of PCI (phenol/

chloroform/isoamyl alcohol, 24:24:1) and centrifuged

at low speed to separate the phases. A volume of 0.5

ml of sodium acetate 2 M, pH 5.2 was added to the

water phase and the extraction with 6 ml of PCI

repeated until complete elimination of the interphase.

The nucleic acids were precipitated by addition of 2.5

volumes of ice-cold ethanol (96%) at � 20 jC for 3 h.

E. Gonzalez-Toril et al. / Hydrometallurgy 71 (2003) 301–309 303

The pellet was washed with 1 ml of ice-cold ethanol

(70%), centrifuged at low speed, vacuum-dried and

resuspended in water (MQ). To facilitate a comparison

with the results obtained with FISH, RNA rather than

DNA amplification is convenient. To do so, 50 Al ofeach sample was treated with RNAse-free DNAseI

prior to its amplification with M-ML V reverse tran-

scriptase. The protocols for rDNA amplification and

DGGE were those described by Muyzer et al. (1996).

Each band was amplified and sequenced. Sequences

were analyzed with the BLAST program using the

NCBI data bank and the ARB program using the

Technical University of Munich data bank. The fol-

lowing primers were used for specific amplification:

GM5-GC (CCTAC GGGAGGCAGCAG) and

907RM (CCGTCAATTCMTTTGAGTTT) for

bacterial rDNA, ARC344F-GC (ACGGGGYGCA-

GGCGCGA) and ARC907R (GTGCTCCCGC

GCCAATTCCT) for archaeal rDNA and CYA781R

(GACTACTGGGGTATCTAATCCCWATT) and

CYA781R (GACTACTGGGGTATCTAAYCCCW-

TT) for cyanobacterial and chloroplast rDNA.

3. Results

3.1. Microbial ecology of the Tinto River

The basin of the Tinto River covers an area of 1676

km2. The gentle slope of the river (0.6%) facilitates

the settlement of a dense microbial community on the

riverbed. The river is subject to a Mediterranean type

regime with an extremely variable flow depending on

the season. The highest flow values are reached

during winter (8.1 m3/s) and the lowest during the

summer (0.07 m3/s). Representative values of some of

Table 1

Quantitative ecology of prokaryotes at different sampling sites using FISH

Sampling site Bacteria a-prot h-prot g-p

Origin I 75 – 41 –

Origin II 63 3 – 54

Intersection 67 4 1 9

Anabel’s Garden 70 17 3 15

3.2 69 51 – 1

Berrocal 88 3 – 64

La Palma 43 26 – –

Abbreviations: a-prot =a-proteobacteria, h-prot =h-proteobacteria, g-prot

the physico-chemical parameters measured in differ-

ent sampling stations are shown in Table 1. Comple-

mentary information has been published recently

(Lopez-Archilla et al., 2001). The pH remains low

and rather constant year-round (mean 2.3), regardless

of the temperature or the river flow. This is the

consequence of the buffer effect produced by the

presence of high concentrations of ferric iron along

the river:

Fe3þ þ H2OZFeðOHÞ2þ þ Hþ

FeðOHÞ2þ þ H2OZFeðOHÞþ2 þ Hþ

FeðOHÞþ2 þ H2OZFeðOHÞ3 þ Hþ

When the river is diluted by tributaries or rain,

ferric hydroxide is formed and protons are released,

thus maintaining the acidic pH of the system. During

the summer, when extremely high temperatures are

reached, intensive evaporation is produced, but the pH

is maintained constant because the reverse reaction

operates: ferric hydroxide precipitates dissolve, con-

suming protons.

The Tinto River ecosystem is unique in that its

biological community is exclusively microbial. Mul-

ticellular eukaryotes have not been detected in any of

the sampling stations. In order to elucidate the degree

of biodiversity conventional microbiological methods

were used for the isolation and characterization of

the microorganisms present in the habitat. To date,

we have identified and characterized chemolithotro-

(in %)

rot Nitros Archaea pH Total Fe (g/l)

– – 2.9 0.8

31 1 1.8 19.8

27 – 2.7 9.6

21 5 1.9 2.3

27 – 1.9 1.8

2 – 2.4 3.1

14 1 2.2 0.9

= g-proteobacteria, Nitros =Nitrospira.

E. Gonzalez-Toril et al. / Hydrometallurgy 71 (2003) 301–309304

phic bacteria and archaea (responsible for the ex-

treme conditions of the system), heterotrophic bacte-

ria, heterotrophic and photosynthetic protists, yeasts

and filamentous fungi. A summary of the isolated

prokaryotic microorganisms is presented in Table 2.

The different microbial populations found in the

Tinto River can be grouped according to their

ecological role as primary producers, decomposers

and consumers.

3.1.1. Primary producers

Chemolithotrophic prokaryotes, mainly iron and

sulfur oxidizing bacteria, together with sulfur oxidiz-

ing archaea have been isolated from the Tinto eco-

system (Lopez-Archilla et al., 2001). The iron

oxidizing bacteria have been identified as different

strains of Acidithiobacillus ferrooxidans (formerly

Thiobacillus ferrooxidans) and Leptospirillum fer-

rooxidans based on their morphology, metabolism

and low-frequency restriction fragment analysis

(LFRFA) profiles (Gonzalez-Toril et al., 1999). In

addition to the members of the Acidithiobacillus

genus, other sulfur oxidizing bacteria unable to oxi-

dize iron have been characterized. Chemolithoauto-

trophic archaea related to the Sulfolobales have been

also isolated in the Tinto. Algae accounted for the

Table 2

Comparative prokaryotic ecology of the Tinto River using different

techniques

Isolation DGGE FISH

Acidithiobacillus

ferrooxidans

(different LFRFAsa)

Acidithiobacillus

ferrooxidans

(different sequences)

+ + +

Acidithiobacillus

thiooxidans

Acidithiobacillus

thiooxidans

?

Leptospirillum

ferrooxidans

Leptospirillum

ferrooxidans

+ + +

? Ferroplasma

acidiphilum

+

Acidiphilium sp. Acidiphilim

cryptum

++

Different

Bacillus species

– ?

? Acidimicrobium

ferrooxidans

?

sulfate reducing

? ? related bacteria

? cyanobacteria ?

a LFRFAs: low-frequency restriction fragment analysis.

greatest proportion of biomass (over 65%) in the

system. Together with the chemolithoautotrophic bac-

teria, algae constitute the primary producers of the

system. Members of the Chlorophyta (Klebsormi-

dium, Zignema, Chlamydomonas), Euglenophyta (Eu-

uglena), Bacillariophyta (Diatoms) and Rhodophita

(Galdieria) phyla have been observed and some of

them isolated (Lopez-Archilla et al., 2001).

3.1.2. Decomposers

A high number of heterotrophic bacteria were

isolated from enrichment cultures (Lopez-Archilla et

al., 2001), but many of them did not grow after the

second or third transfer, probably because some com-

ponents of the original inoculum were diluted out,

affecting their growth. Some of the isolates corre-

sponded to the genus Acidiphilium. Members of this

genus have been shown to be frequently associated

with chemolithoautotrophic bacteria, especially iron

oxidizers. Many bacterial isolates were Gram-positive

bacilli, aerobic spore formers of the genus Bacillus.

Within the decomposers, fungi were very abundant

and exhibited great diversity, including yeasts and

filamentous forms. A high percentage of the hypho-

mycetes isolates were able to grow in the Tinto River

conditions. Some of the yeast species isolated can also

be found in other less extreme aquatic environments.

But the isolated Dematiaceae seem to be specific to

the extreme conditions of the habitat, since they are

rarely present in neutral fresh waters (Lopez-Archilla

et al., 2001).

3.1.3. Consumers

Among the eukaryotes, heterotrophic protists con-

stitute the major consumer group in the Tinto ecosys-

tem. Different flagellates (phylum Zoomastigina),

amoebae of the class Lobosea (phylum Rhizopoda),

some representatives of the class Heliozoa (phylum

Actinopoda) and ciliates (Phylum Ciliophora) have

been observed mainly associated to the biofilms

(Lopez-Archilla et al., 2001).

3.2. Bioinduced iron formations in the Tinto ecosystem

Most of the biomass is located on the riverbed in

the form of dense, compact biofilms, mainly com-

posed of filamentous algae and fungi in which bacte-

ria are trapped. Also, heterotrophic protists can be

Fig. 1. Fluorescent in situ hybridization of a sample from the origin

of the Tinto River using (a) DAPI, universal stain, and (b) a specific

probe for Nitrospira (NTR712).

E. Gonzalez-Toril et al. / Hydrometallurgy 71 (2003) 301–309 305

found associated to the biofilms. Significant mineral

precipitation is observed on the surface of the bio-

films, forming iron stromatolites, which grow season-

ally following the exposure of the biofilms to the

water. As mentioned, the system has been altered by

mining activities during the last 5000 years, but

evidence of its antiquity, older than 0.3 My, has been

found in massive laminated iron beds in three iron

rock levels occupying different elevations above the

present river. One of them corresponds to an extensive

formation, more than 35 m above the river and over

10 m thick, organized into two different lithostrati-

graphic units, one with paraconglomerate and massive

facies and the other with laminar-stromatolitic, mas-

sive microbiolitic, as well as some plant fossils. If the

morphology of the outcrops, the strata thickness and

continuity are taken into account, this formation could

be interpreted as an acid lake generated by the

metabolic activity of chemolithotrophic microorgan-

isms. The observation of the actual river iron laminae

allows us to understand the origin of these facies and

their environmental significance.

3.3. Molecular ecology of the Tinto River

As mentioned in the introduction, the use of con-

ventional microbiological techniques has important

limitations for a complete enumeration of the microbial

diversity present in any given system and, most impor-

tant, for the quantification of their populations. The

introduction of PCR based technologies prompted the

accumulation of rDNA sequences from many different

microorganisms, thus allowing the development of

complementary molecular ecology techniques like

FISH and DGGE. One of the goals of this work was

to use in situ hybridization techniques complemented

with DGGE for identification and quantification of

microorganisms present in different sampling stations

along the river and to compare the results with those

obtained using conventional methodologies.

Fig. 1 shows an in situ hybridization performed

with a fluorescent probe specific for members of the

Nitrospira group (in which members of the genus

Leptospirillum are included) compared with DAPI

(universal) stain. The use of probes with different

specificity to hybridize samples collected along the

river generated data on the population of prokaryotic

microorganisms present in the Tinto system. Table 1

shows quantitative FISH results obtained with differ-

ent probes at different sampling sites along the river.

Complementary information was obtained from the

sequence of the different rDNAs amplified by PCR

using selected primers with different specificity and

separated by DGGE. Fig. 2 shows the rDNA bands

resolved after amplification using bacterial and ar-

chaeal universal primers of RNA samples obtained

from different sampling stations.

The diverse bands can be excised, amplified,

sequenced and compared using different programs

(BLAST, ARB) with the rDNA sequence data banks.

Any RNA donor whose homology is greater than 96%

was considered a member of the same species as the

reference sequence. Both techniques, FISH and

DGGE, are complementary in the sense that DGGE

allows possible candidates to be identified and spe-

cific fluorescent probes for in situ hybridization to be

designed. Table 2 shows a summary of comparative

results obtained using conventional and molecular

ecology techniques. It should be noted that there is

substantial agreement among all of them.

Fig. 2. DGGE of amplified rDNA fromRNA extracted from different

river locations : (1) origin (II), (2) origin (I), (3) intersection and (4)

berrocal, using primers with different specificity: Archaea and

Bacteria.

E. Gonzalez-Toril et al. / Hydrometallurgy 71 (2003) 301–309306

Interestingly enough, using DGGE we were able to

amplify rDNAs which according to their sequence

clustered together with sulfate reducing bacteria. Also,

using specific primers for cyanobacteria and chloro-

plast, we amplified several bands whose sequence

clustered with cyanobacteria, strongly suggesting their

presence in the Tinto ecosystem. We are currently

trying to isolate both types of bacteria from enrich-

ment cultures.

4. Discussion

The objective of this work was to compare the

results obtained using conventional microbial techni-

ques with those derived from molecular ecology

techniques, and to generate a functional model of

the Tinto River system. The particular geology and

climatology of the region favor the creation of the

Tinto River’s special environment, which provide the

base on which the biological communities establish

themselves and proliferate. The river rises in the

middle of the Iberian Pyritic Belt, one of the world’s

richest complex polymetallic sulfide deposits. The

abundance of sulfide minerals, mainly pyrite and

chalcopyrite, provides the necessary substrate for the

development of chemolithotrophic bacteria, responsi-

ble for the extreme conditions of the habitat. The high

water table, which has been a serious hindrance to the

exploitation of the mines in the past, maintains the

river flow during the extremely hot summers, in the

absence of rain and with a high rate of evaporation.

The Tinto River originates at Pena de Hierro from

very close tributaries, which have quite different

microbial content, probably related to the character-

istics of the mineral substrates that feed them. ‘‘Origin

I’’ is less acidic (mean 2.9) and has a rather low

content in iron (mean 0.8 g/l) when compared to

‘‘Origin II’’, which has a higher concentration of

oxidized iron (mean 19.8 g/l) and is more acidic

(mean pH 1.8). In the first case, there is a high

concentration of unidentified bacteria belonging to

the h-proteobacteria, which might have a strong sulfur

oxidizing capability, because of the high content of

sulfate in the water and its pH. ‘‘Origin II’’ holds a

high concentration of different strains of At. ferroox-

idans and L. ferrooxidans. This site seems to be the

output of a large underground reactor, which may be

in contact with an acidic lake formed in an abandoned

open mine pit (Pena de Hierro). This acidic lake has a

diameter of about 500 m and is 50 m deep. Prelim-

inary results indicate that this lake is anoxic. Because

of its difficult accessibility, a minisubmarine has been

designed to explore the characteristics of this interest-

ing opaque habitat in collaboration with the Centro de

Astrobiologıa. This lake is a good indicator of the

water table level in the area, allowing the problems

that the water table caused to past and present mining

activities to be explained, and raises serious questions

about the flooding of open pits as a safe environmen-

tal practice in sulfidic mine closing projects.

The ‘‘Intersection’’ sampling site refers to the area

in which both streams (Origin I and II) join with an

almost neutral tributary (pH between 3.9 and 4.5). The

microbial content of the ‘‘Intersection’’ site and its

physico-chemical conditions correspond to the differ-

ent contributions of these streams to the river. Another

interesting observation can be made at the sampling

station known as ‘‘Anabel’s Garden’’. This site was

considered in the beggining of the work as the origin

of the Tinto system, although careful Global Position-

ing System (GPS) measurements have proven that it is

located at a lower altitude than the ‘‘Origin’’. ‘‘Ana-

bel’s Garden’’ is the origin of a tributary that crosses

the modern Rıo Tinto mine operation and was used by

the British company as a source of acidic waters for

E. Gonzalez-Toril et al. / Hydrometallurgy 71 (2003) 301–309 307

direct copper extraction from the river using cemen-

tation procedures. This tributary has a broader spec-

trum of microbial diversity, not only at the prokaryotic

level (Table 2), but also at the eukaryotic one, exhib-

iting important blooms of algae, mainly Diatoms and

Euglenas, especially during spring and summer.

This stream gives rise to the ‘‘3.2’’ site, which

corresponds to a reservoir of acidic waters used for

cementation. This reservoir was originally 12 m deep.

Today, it is half full of sediment. Interestingly enough,

the microbial composition of this site is rather differ-

ent from the ‘‘Anabel’s Garden’’ site which feeds it.

There is an important increase in cell density, and in

relation to prokaryotes the number of bacteria that

give a positive signal with the a-proteobacteria probe

is rather high, whereas the concentration of At. fer-

rooxidans seems rather low. An interesting feature of

this reservoir is that from several centimeters below

the surface to its bottom it is completely anoxic,

which might explain the difference in bacterial pop-

ulation observed in its waters.

The ‘‘Berrocal’’ site is representative of the middle

course of the river. This site gives reproducible results

in which the proportion of At. ferrooxidans is rather

high when compared to other sampling sites along the

river. This is the part of the river in which important

fields of iron stromatolites can be found. Also, the

flow of the river in this section is rather slow,

facilitating the formation of biofilms and their

corresponding mineral precipitation, and produces in

many places (especially in the meanders) deep basins,

which are basically anoxic. Finally, the sampling

station of ‘‘La Palma’’, near the mouth of the river,

shows that more than fifty per cent of the biota at this

site is eukaryotic. Large numbers of filamentous algae

thrive on the surface of the rocks where dark ferric

iron waters do not impede sunlight penetration. There

are some important and reproducible differences be-

tween ‘‘La Palma’’ and ‘‘Berrocal’’ in terms of their

prokaryotic populations, which are probably related to

the physico-chemical differences between both sites,

mainly a lower content of heavy metals, especially

iron, in the lowest part of the river.

DGGE techniques amplified two sets of signatures

from RNA samples from several sites which clustered

with bacteria with interesting properties. One of these

corresponds unequivocally to cyanobacterial sequen-

ces. This observation is interesting because up to now

no one has been able to isolate a cyanobacteria that

can grow at acidic pH. So far, we have been unable to

identify the possible candidates and to isolate them.

One of the bands has a sequence 93% homologous

with Stanieria cyanosphaera, others cluster with un-

identified cyanobacteria. The other signature clusters

with sulfate reducing bacteria. For years, our group

has been interested in the isolation of acidic sulfate

reducing bacteria to explain the recycling of reduced

sulfur substrates for use by strict chemolithotrophic

bacteria along the main basin of the Tinto River.

Knowledge of the area in which the signal has been

collected and the sequence of its rDNA should help in

the design of tools to isolate these bacteria which have

important biotechnological applications (Sen and

Johnson, 1999).

As mentioned, both sulfur and iron have a funda-

mental role in this fluvial ecosystem. Of these, much

emphasis has been given in the literature to the sulfur

cycle bacteria, probably because from a biochemical

point of view much more energy can be obtained from

its reduced forms. Interestingly enough, iron bacteria

have recently acquired a more predominant role in

environmental microbiology since the discovery of

the importance of iron in bioleaching operations

(Brierley, 2001), its use as reducing power for anaer-

obic photosynthesis (Ehrenreich and Widdel, 1994)

and its use as substrate for anaerobic respiration

(Straub et al., 1996). We believe that the Tinto River

is a system controlled by iron (Fig. 3). Iron is not only

a source of energy for iron oxidizing prokaryotes (At.

ferrooxidans, L. ferrooxidans and Ferroplasma acid-

iphilum), but also can be used as an electron acceptor,

both in aerobic (At. thiooxidans and Acidiphilium sp.)

and anaerobic conditions (At. ferroxidans). In addi-

tion, ferric iron is responsible for the maintenance of a

constant pH in the ecosystem, a property that must be

important to the maintenance of a high level of

eukaryotic diversity found in the system. Finally, acid

ferric iron solutions provide good UV irradiation

protection.

We know that iron is an important limiting factor

for the growth of phytoplankton in the oceans

(Martin, 1990). We know that most cells, both

prokaryotes, but especially eukaryotes devote an

important amount of genetic information and energy

to scavenging iron. A possible advantage to the

photosynthetic protists that thrive in the extreme

Fig. 3. Geomicrobiological scheme of the Tinto River.

E. Gonzalez-Toril et al. / Hydrometallurgy 71 (2003) 301–309308

conditions of the Tinto is an unlimited supply of

iron. Physiological studies of acidophilic photosyn-

thetic protists isolated from the Tinto River done in

collaboration with M. Sogin’s group at the Marine

Biology Laboratories of Woods Hole show that

adaptation of protists and probably other eukaryotes

to acidic environments is much easier than had been

thought.

We are living in an iron-limited world. But there

was a time when iron was abundant in the oceans

(Martin, 1990). Most of the iron was sequestered in

massive iron deposits, probably as a result of the

evolution of life (Trendall and Morris, 1993). Today,

at Rio Tinto, we have a rather unique extreme acidic

environment which is the product of chemolithotro-

phic metabolism controlled by iron. It produces iron

stromatolites that prove that the system operated

long before the mining activities started in the area,

and that therefore the system is natural and not the

product of industrial contamination. Probably, the

characteristics of the river enabled the ancient min-

ers to discover the Iberian Pyritic Belt. The chemo-

lithotrophs involved in the production and

maintenance of this environment are responsible

for two critical cycles of nature: the iron and the

sulfur cycles. Some of these microorganisms are

important in several processes of biohydrometallur-

gical interest: acid mine drainage, biomining and

bioremediation.

Obviously, there are many variables in this

complex model that have not been considered in

this preliminary model: i.e. the role of the high

level of eukaryotic diversity in the system, which is

currently being analyzed by molecular techniques,

or the function of metal accumulating plants, etc.

Understanding the model is vital not only to

increase our knowledge of a peculiar life style,

but also to improve the performance of biotechno-

logical processes and to search for new applications

for the microorganisms that thrive in the red waters

of the Tinto River.

Acknowledgements

Thanks are given to an anonymous reviewer for

his/her helpful comments. This work was supported

by grants BIO99-0184 and BX2000-1385 from the

Ministerio de Educacion y Cultura and 07M/0023/199

from the Comunidad Autonoma de Madrid and an

institutional grant from the Fundacion Areces to the

CBM.

E. Gonzalez-Toril et al. / Hydrometallurgy 71 (2003) 301–309 309

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