A bright red river meanders through the countryside of southwestern Spain, its water acidic enough to eat through metal. Such an image brings to mind the worst excesses of industrial pollution, and scientists long assumed that a local copper mine had contaminated the Tinto River.

Mining activity at the Tinto River dates back at least 5,000 years, and while it has altered the river it is not solely responsible for the river’s conditions. Acid rock drainage is a natural process that occurs when water, oxygen, and bacteria interact with sulfide minerals, producing highly acidic solutions. The Tinto River runs through the Iberian Pyritic Belt, one of the biggest complex sulfide formations in the world (pyrite, or iron sulfide (FeS2), is also known as ‘fool’s gold’).

Ricardo Amils is the director of the laboratory of applied microbiology at the Center for Molecular Biology at the Autonomous University in Madrid, and is associated with the Spanish Centro de Astrobiologia. Amils has been studying the Tinto River’s ecosystem for over 10 years. He says the water’s red color and average pH of 2 is due to this natural abundance of sulfide. Amils believes that bacteria living in the river turn this sulfide into sulfuric acid, giving the river its low pH. Other bacteria oxidize the iron, giving the river its signature red color. Although both sulfur and iron naturally oxidize when exposed to air, the bacteria act as catalysts, speeding up the reactions considerably.

“The water table and the high temperatures all year around favor the growth of chemolithotrophic bacteria, which exist in high amounts in the river and probably in underground waters,” says Amils. “The most important characteristics of the system – sulfuric acid and high concentrations of ferric iron – are products of chemolithotrophic activity using pyrite and other sulfidic minerals.”

Chemolithotrophy is a metabolic process used by microorganisms to obtain energy from inorganic molecules. In the case of the Rio Tinto, “rock eating” bacteria like Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans get energy by oxidizing the ferrous iron (Fe2+) in the pyrite, turning it into ferric iron (Fe3+) (The Acidithiobacillus also get energy by oxidizing the sulfide). Because very little energy is generated in the oxidation of ferrous to ferric iron, these bacteria must oxidize large amounts of iron in order to grow. As a result, relatively little bacterial growth results in massive amounts of ferric iron precipitation.

It is not precisely known how bacteria oxidize the ferrous iron. Scientists believe the process relies on both chemical and biological forces working together.

The combination of sulfuric acid and ferric iron in the water produces conditions that promote the oxidation of other metals such as arsenic, copper, cadmium and nickel. Many metals become much more soluble when oxidized, so this increases the concentration of metals in the river.

The chemistry of the Tinto River is not completely unique – the Iron Mountain mine in California, for instance, can reach negative pH levels due to chemolithotrophic activity – but the length of the Tinto River makes it an ideal place to study life based on sulfide and iron.

“Microbes and substrates are available in many places around the world,” says Amils. “What makes the Tinto different is the proportion: a 90 kilometer-long acidic river. This probably facilitated the adaptation of many different systems.”

Indeed, bacteria are not the only life forms found in the river. Amils’s team has collected about 1,300 different organisms, including archaea, yeast, fungi, and protists. The most abundant biomass in the river seems to be algae. Blooms of algae often coat the surface of the water, turning the red water green and producing bubbles of oxygen. Amils thinks it is strange how eukaryotic organisms like algae are able perform in such harsh conditions of acidity and heavy metal concentrations (Eukaryotes are organisms that have a DNA-holding nuclear membrane in their cells).

But Ken Nealson, an astrobiologist with NASA’s Jet Propulsion Laboratory, says keeping acid out of a cell is not that difficult for eukaryotes. Simple eukaryotes can often survive in low or high pH environments, as well as in extremes of salinity and dryness.

“Studies of any extreme environment always extend our appreciation of evolution and adaptation,” says Nealson. “One thing that is often forgotten is that very seldom is only one thing extreme, and adaptation to one variable almost always is connected with adaptations to others.”

Figuring out the biology of the Tinto River could help Amils and others understand the development of early life on Earth. For instance, the Tinto River may be a real-life model for Proterozoic life (2.5 billion to 544 million years ago), since aerobic algae first appeared during the Proterozoic. But Amils says the biology of the Tinto River may have a connection even further back in the past, with the Archaean era (3.8 to 2.5 billion years ago).

“Most of the biological microbes that you can find now days in Rio Tinto probably existed in the Archaean,” says Amils. He cites recent molecular evidence for the presence of algae 2.7 billion years ago in Pilbara, Australia. There is also evidence for the complete operation of the sulfur cycle in Pilbara, with oxidation and reduction (or ‘redox reactions’) dating back 3.5 billion years.

“Iron was massively precipitated in Pilbara, and all these ingredients are operation in this very moment in Rio Tinto,” says Amils. “The only difference is that the Australian Archaean system was in a shallow marine environment, while Rio Tinto corresponds to a terrestrial aquatic system.”

Research into Tinto river biology also could determine whether biological iron fractionation could be a useful biomarker in the search for life on Earth and on other planets. In other words, could we look at the division of iron isotopes in a rock as evidence that iron-based life once existed there?

“Research in the Rio Tinto will almost certainly help us to determine whether iron fractionation occurs in such acidic environments,” says Nealson. “Whether it is a good biomarker will depend on the results. Even if not, the river is not excluded from being useful for learning about iron metabolism in other environments.”

By studying the Tinto River’s biology, says Amils, we can better understand how a biological system based on iron functions.

“Until now biohydrometallurgists have concentrated on sulfur because they thought that life based on iron could not be efficient,” says Amils. “Rio Tinto and other systems show that this is not true. But we still need to determine what are the products of this metabolism, how can we recognize them, and what are the suitable instruments to be used for its detection.”

Perhaps organisms similar to those found in the Tinto River could have developed on Mars. The Martian soil appears reddish because of iron in the soil, and some scientists believe that Mars once had rivers and other surface water. Mars is also a very sulfur-rich planet, so microorganisms on Mars also could survive by oxidizing sulfide.

“We need to learn more about Mars before we know if this is a good analogue,” says Nealson. “However, given that sulfuric acid production requires oxygen, and there may not be easy ways to generate this acid without it, knowing the oxidative history of Mars will be very important. Either way, the Rio Tinto is a great place to understand adaptation and evolution of life and constrain our thinking about life elsewhere.”

Researchers from the Centro de Astrobiologia have suggested that the Tinto River also makes a good Europa analogy. Jupiter’s moon Europa is thought to have an acidic, salty ocean under its outer layer of ice. Thus, the Tinto River could represent a unique biological setting to investigate the possibility of sulfur-based life on Europa.
What’s Next

Amils is collaborating with different groups of the NASA Astrobiology Institute to characterize the iron formations of the Tinto River and the eukaryotic diversity of the system. He is also using conventional and molecular techniques to characterize the prokaryotic diversity of the river in order to understand how the system works.

“We believe that chemolithotrophy is an ancient energy system, and we need to know more about it,” says Amils. “We have to learn what happened with iron in the Archaean, for instance. There are many questions that we can start to address using a living system. This is the advantage of the Tinto.”