Arno Gasteiger

Some like it hot

Boiling water and the stench of sulphur hardly seem a combination conducive to life, yet some bacteria thrive in such hostile environments. Biologists are beginning to think that the first life forms on Earth may have been similar to these hardiest of living things.

Written by       Photographed by Arno Gasteiger

Descending to the seventh circle of hell, Dante and his guide the poet Virgil encountered “an open stretch of flatland/whose soil refused the roots of any plant” surrounded by a “boiling crimson river.”

Substitute orange-tinted pools of bubbling water and mud for the crimson river and you have an accurate description of the Whakarewarewa thermal reserve in Rotorua. Demonstrating that one man’s inferno can indeed be another’s tourist heaven, this reserve is frequented not by the tor­tured souls of the violent damned but by hundreds of thousands of delighted camera-toting visitors each year.

Not everyone who comes to the reserve, however, sports a Handycam and a Hawaiian shirt. Others stalk the banks of the steaming pools, sampling the waters and scraping the surrounding rocks with serious purpose. They are microbiologists and biochemists, molecular biologists and protein engineers, all bound together by a common enthusiasm for the indigenous denizens of this singular environment: extreme thermophilic bacteria.

I saw Whakarewarewa up close for the first time in 1990. I had been working with thermophilic bacteria only in the laboratory, growing cultures of them in a heated two-litre flask to extract their proteins for further study.

Largely because of their extremely unusual living requirements, I no longer thought of them as naturally occurring organisms, ex­isting freely in an open environment. It was an eye-opening experience when I first visited them at “their place,” rather than having them over to mine Pohutu, the geyser, was erupting with casual vio­lence, and steam and sulphurous stench was everywhere. Not the sort of place you would expect to find much living. I tested the water temperature with a small elec­tronic thermometer it read a scalding 80°C at the edge of one large pool. And yet, submerged in this enormous, open-air kettle, there they were: reddish-brown mats composed of many species of thermophiles (literally “heat-lovers”) growing on rocks. Their indi­vidual names are just as dramatic and descriptive as the places they are found: Haloferax vokanii, Thermoplasma acidophilum, Sulfolobus acidocaldarius to name but a few.

Thermophilic bacteria can be encountered in many locales around the world: along the Pacific Ring of Fire (the active volcanic zone girdling the Pacific Oceanof which New Zealand is part), in Yellowstone National Park and on volcanic islands such as Iceland.

All but a handful of extreme thermophiles belong to a group of organisms known as the archaebacteria. This group of single-celled organisms have been a puzzle for scientists since their discovery in the 1970s because they differ from other bacteria in several ways. With few exceptions, they live in most peculiar habitats, the sulphurous pools of Whakarewarewa being but one example.

The thermophile Thermoplasma acidophilum, for instance, was first obtained from a burning refuse pile at a coal mine in Indiana. Other species have been recovered from a smouldering uranium mine and reservoirs of hot crude oil kilometres beneath the seafloor.

Most archaebacteria share unique features which have prompted evolution­ary biologists to give them a branch of their own in the tree of life (see sidebar, page 84). This status alone would render them worthy of study, but their unusual properties have also piqued the interest of biotechnologists, who see opportunities to exploit them for human benefit. Most intriguingly of all, the archaebacteria may hold the keys to discovering how life first developed on this planet.

Archae bacteria are divided into three main groups. One includes the heat-loving extreme thermophiles, such as the evocatively named Pyrococcus furiosus. Found in such environments as thermal pools and submarine laval vents (“black smokers”), and respiring elemental sulphur rather than oxygen, they are exqui­sitely adapted to their extreme habitats.

A second group consists of the methanogens. Adapted to living in swampy regions and in the intestines of cows and other ruminants, these organisms are named for their ability to convert carbon dioxide into energy-rich methane. Whilst this property is considered a boon by biotechnologists, many a sharemilker has doubtless wrinkled his nose at their capacity to enhance bovine flatulence.

The third group contains the halophiles, organisms which are capable of colonising some of the least hospitable habitats in the world: searing salt flats such as those dotted around Utah. These microbes are typically reddish-brown in colour, and are responsible for the deterioration of salted foods the red patina which forms on salted fish that has been stored for a while in humid conditions is due to the growth of some halobacterial species.

Totally in keeping with their seemingly quixotic range of habitats, the archaebacteria prove to be just as unusual in their internal make-up. At first glance, they look very prokaryotic simple and single-celled, just like most other bacteria. Prokaryotes are so named because they lack a cell nucleus, the com­partment which in other cells holds the genetic machinery (katyon means “kernel” in Greek). Under the microscope they appear as small spheres or rods about 1 micron (one thousandth of a millimetre) in diameter, and have no obvious internal structures.

Eukaryotic cells (eu being from the Greek for “true”)the type of cells of which you and I are made are about 10 to 30 times larger in diameter, and are u fled with membranous bags of material called “organelles,” specialised struc­tures about the size of prokaryotic cells which perform specific functions such as energy generation and, in plants, photosynthesis

The archaebacteria share some biochemical processes with prokaryotes, but in o r respects they are more closely related to eukaryotic organisms. For example many archaebacterial proteins are more similar in structure to their eukaryotic equivalents is than they are to the proteins of regular bacteria. And the archaebacteria share patterns of sensitivity to certain common antibiotics with higher organisms, including humans. Of course, they also possess unusual features which enable them to live in their extreme habitats.

The upshot is that archaebacteria are now considered to be in a class of their oNx it, as different from normal bacteria as they are from blueberry bushes.

T ese biological curiosities have also proved to be journeyman labourers, able i do a number of useful jobs, thanks, in part, to their peculiar habits. The salt- ving halophiles were an early focus of interest, owing to their role in the rioration of salted foodstuffs. In Thailand, a widely used condiment known as Nam Pla (fish sauce) is the product of the action of halophilic archaebacteria on salted fish, and actually contains the bacteria themselves. Nam Pla is one of the few examples of humans purposefully, indeed gleefully, eating bacterial slurries (yoghurt being another).

More recently, archaebacteria have been playing a central role in the bur­geoning biotechnology industry, worth SUS10 billion dollars per annum and counting. Because of their vulnerability to the same types of antibiotics as humans, a number of research groups are exploring the possibility of using halophiles to screen new anticancer treatments.

On a different front, halophilic archaebacteria have been shown to contain light-sensitive “purple membrane” (PM) proteins, which change colour from purple to yellow when exposed to a source of light. This property has excited the designers of computer memories, as such light-sensitive digital changes (1 = purple, 0 = yellow, for example) may enable the development of reversible holographic memory stores.

PM proteins are capable of storing the interference patterns which consti­tute holographic recordings as purple and yellow areas, just as silver halide photographic media store patterns as black or white dots. PM proteins have two major advantages over photographic techniques: first, they are capable of stor­ing the holographic information for much longer, and, second, they can be genetically engineered to enhance and/or alter this property.

An allied potential application of PM proteins is as the next generation of computer microprocessors. Silicon-based microchips are speedily approaching a limit in miniaturisation and, therefore, processing speed. The two alternative states of PM proteins’ colour cycle (purple and yellow) could be used as the basis of an optical switch analogous to the switch formed by the conducting/ non-conducting states of a semiconductor. Who knows: tomorrow’s computers might contain “biochips,” and Silicon Valley be renamed Bacterial Alley.

In a more conventional application, archaebacterially-produced methane (by the methanogens) can be used as an energy source, and some countries are already processing agricultural and domestic wastes for methane (natural gas) production. Some methanogens also produce the related gas ethane, when fed with ethanol (the most common type of alcohol). As well as having a higher energy content than methane, ethane can also be used to generate ethylene, the basic component of polymer plastics.

On an equally large scale, the unusual fatty compounds (or lipids) produced by archaebacteria also harbour industrial potential. Enhanced Oil Recovery (FOR) is a procedure by which residual crude oil in a deposit can be forcibly extracted by the high pressure injection of water. Water displaces the oil, which is then driven to the surface through an oil well. If the viscosity of the injected water could be increased and its surface tension lowered, the process would be much more efficient. Archae bacterial lipids have properties which cause exactly these changes when they are added to water, and the high temperature of most oil deposits (60°C) would not adversely affect thermophiles or their heat resistant components.

In the field of biotechnology, extreme thermophiles have not been that lucrative

to date. David Saul of the University of Auckland’s thermophile re­search group explains that while many industrial chemical reactions work well at high temperatures, the temperatures at which enzymes (protein catalysts) from extreme thermophiles work best is often too high “unless you want to make caramel from starch!”

The major commercial application of thermophilic products has been in genetic research. The enzyme DNA polymerase can be used in a reaction known as the Polymerase Chain Reaction (PCR), which can make millions of copies of an infinitesimal amount of DNA a process known as amplification. It is commonly used to make the genetic equivalent of a single straw buried within a haystack into a great pile of absolutely identical straws. Prior to the advent of PCR, analysis of rare DNA (such as a single gene amidst all the DNA in a cell) was impossible.

“PCR has revolutionised genetic engineering, molecular diagnostics, micro­biology, ecology you name it and is now one of the most widely used genetic engineering techniques,” Saul says.

The enzymes used in the reaction must be capable of repeatedly surviving very high temperatures (up to 95°C) for short periods of time. Suitable enzymes have been sourced from a number of thermophilic organisms, the most com­mon being Thermus aquaticus.

One field in which PCR has had particular impact is the study of microor­ganisms themselves. Until a decade ago, about 5000 species of bacteria of all types had been described, and few more were expected to be discovered. Even under the best light microscope, bacteria are just specks, so study was usually based on the properties of bulk cultures.

Then someone carried out PCR on an uncultured field sample containing bacteria, and found evidence of myriad new varieties. It turns out that labora­tory culture methods are very selective, and perhaps as many as 99 per cent of bacterial species do not grow under present culture regimes. Furthermore, many of the new varieties have unusual and potentially interesting features, so PCR has opened up a vast and exciting new world of microbes.

Microbiologists are now claiming that perhaps 95 per cent of all species on Earth are microbes, and that most of the world’s biomass is microbes too.

Other thermophilic enzymes are gradually coming into use in less specialised industrial processes. Amylase breaks down starch naturally for the baking and brewing industries, and protease, an enzyme which digests other proteins, is used in food processing and in the development of “gets-your-whites-whiter” detergents.

The capacity of some archaebacterial extreme thermophiles to respire sul­phurous compounds rather than oxygen is also exploitable: their unique me­tabolisms (known by the tongue-twisting term “chemolithoautotrophic”) can process mineral sulphides, enabling them to leach metals from their ores. Copper and gold are two of the metals which are being released in this fashion in bioreactors on an industrial scale.

Given these successes, David Saul is highly optimistic for the future. “There is immense potential in these hot pool inhabitants,” he says. “New Zealand companies and the government need to realise that there is a valuable and underexploited resource living in our own backyard.”

[Chapter Break]

At about  the same time that I was meeting my research subjects au naturel in Whakarewarewa, a debate was raging in the microbiological community. The question being asked was a deceptively simple one: what is the maximum temperature at which life survive. The subject is not as esoteric as it seems. When life was first forming on this planet three-and­a-half billion years ago, Earth was much hotter than it is today perhaps 80-90°C, or even higher.

The high ambient temperature of the young Earth was due to several factors. The thin atmosphere at that time did little to prevent bombardment by meteor­ites. These impacts generated heat, perhaps enough to boil the surfaces of the newly-forming oceans. Also, higher amounts of carbon dioxide and other “greenhouse” gases in the atmosphere would have trapped more of the Sun’s radiant heat a scenario humans are now trying to avoid recreating.

If life arose in such conditions, then the earliest lifeforms must have been thermophilic in nature. Asking questions about the lifeforms which now dwell in the hottest contemporary locations on Earth and attempting to understand the basis of their thermophily might, therefore, provide insights into what those most primitive of organisms looked like, and how they might have evolved.

Until the beginning of this decade, it was thought that the upper tempera­ture limit for life must be below 100°C, the boiling point of water (since water is easily the dominant constituent of all living cells). No organism wants to be broiled.

Nevertheless, this seemingly obvious view was challenged on a number of fronts, the most dramatic being a claim that life could be maintained up to 2 50°C if the ambient pressure were high enough to prevent water from boiling, and hold it in its liquid form the kind of conditions which could be encoun­tered in a “black smoker” thousands of metres below the ocean surface.

Sure enough, a creature has been found which lives at a startling 113°C. It is an archaebacterium called Pyrococcus, a submarine thermal vent-dweller. So far, it holds the record for the upper temperature limit for a living organism.

But Professor Roy Daniel’s research group in Hamilton has isolated an enzyme from a Fijian bacterium that works at 130°C, fuelling speculation that creatures might be able to tolerate even higher temperatures.

Rotorua's waters and rocks, such as these at Waimangu Thermal Valley, are tinted both by minerals and by mats of bacteria and algae.
Rotorua’s waters and rocks, such as these at Waimangu Thermal Valley, are tinted both by minerals and by mats of bacteria and algae.

Heat has the same effect on organisms as it does on a breakfast egg. Eggs are containers of proteins in solution, chiefly egg albumen. At “normal” tempera­tures, say 30-37°C, such proteins remain in solution, their flexible structures vibrating in a comfortable molecular dance. Raise the temperature to boiling, and this comparatively leisurely waltz becomes a break-dancing frenzy of death. The protein structure becomes hyperenergised, and individual molecules col­lide and begin to fuse.

This process, known as denaturation, leads to the aggregation of proteins, solidifying them and bringing them out of solution. Proteins in this hard-boiled state are now functionally useless, and, even if the ambient temperature is returned to normal, they will stay denatured until destroyed by your stomach enzymes along with your toast and coffee.

The cells of all organisms on Earth are, in a similar fashion, bags of protein with some DNA and lipids thrown in. Their fate at boiling temperatures is the same as that importunate egg.

Except for thermophilic cells. These cells not only thrive in high tempera­tures, they have an absolute requirement for them. Look at a thermophilic bacterium at 30°C and you will see a collapsed, dormant thing, much like a human cell would look at -50°C. This extraordinary situation is made possible by the adaptations of these cells to their singular habitat. For example, they have thermostable proteins, which are stiffer than regular proteins. Being more rigid, they are able to hold together when hyperenergised by heating. The corollary is that at lower temperatures the molecules are too rigid to perform at all. At 30°C such a protein is, in effect, frozen solid. DNA in the cells of thermophiles is coiled differently (either relaxed or positively supercoiled) from the configuration it has in all other cells, and membranes may be single-layered and hence less likely to fall apart when heated.

[Chapter break]

How could life have originated and evolved in an environment as inhospitable as that of infant Earth? Could it be that the ancestor of all life on Earth was akin to a thermophilic archae bacterium?

The eclectic astronomer Fred Hoyle has suggested that life was de­posited here via one of those previously mentioned meteorite impacts. Whilst it is unlikely that a fully functioning organism could materialise by that fashion, it is well known that a certain type of meteorite, called a carbonaceous chondrite, is packed with the amino acids from which proteins are made and the hydrocarbons and alcohols which form cell membranes. The tails of some comets, including Halley’s, are also rich in organic material which could be showered on to the planet during a close fly-by. By a variety of chemical processes, which are reproducible in a laboratory, such constituents can form larger molecules, given an input of energy such as that from a bolt of lightning.

However, the latest research suggest a different scenario. Where hot, mineral-rich waters associated with volcanic activity contact cooler seawater, thermophilic organisms can actually gain energy by coupling the synthesis of large organic molecules from carbon dioxide to the oxidation of hydrogen. Improbable as it sounds, thermophiles around deep-sea vents today can gain metabolic energy by synthesising the constituents of their own cells, and it seems likely that simple metabolism could have originated with thermophilic archae bacteria in similar settings three or four billion years ago.

Whether life was seeded from the cosmos or bubbled up from the depths, a critical step in the process was almost certainly the production of a molecule such as RNA. This sister to the genetic material DNA is not only capable of acting as a surrogate genetic source (for example, in retroviruses, such as HIV virus), but can catalyse certain biochemical reactions in an enzyme-like fashion.

The waters of the Champagne Pool at Waiotapu are gently simmering, and contain a variety of extremely thermophilic archaebacteria. Such natural cauldrons, with their invisible fauna of biological curiosities, may one day be treasured as a biochemical resource, not just a tourist mecca.
The waters of the Champagne Pool at Waiotapu are gently simmering, and contain a variety of extremely thermophilic archaebacteria. Such natural cauldrons, with their invisible fauna of biological curiosities, may one day be treasured as a biochemical resource, not just a tourist mecca.

In a pre-life scenario, these twin attributes would give RNA the role of a staging post on the road to fully-functioning DNA and protein, and place it high on the list of candidates for the first self-replicating molecule on the planet. The further evolution of RNA is not, and may never be, clear especially since RNA is notoriously unstable at high temperatures but some­how a fully integrated system for both reproducing genetic information and translating it into protein-mediated action was developed.

Given that all this molecular activity was probably happening in a hot environment, it is not too much of a stretch to suppose that the first functioning organism resembled a thermophilic archae bacterium. A great many examples of thermophilic “true” bacteria also exist (although they tend to live in cooler habitats than the archae bacterial thermophiles), and it is possible that two separate prokaryotic thermophilic lineages arose at around the same time.

The more complex eukaryotic lineage is thought to have formed some time later by the physical and genetic intermixing of examples of both of the primi­tive prokaryotic families. Such a relationship would have started out as symbio­sis, but then become something more intimate. There are plenty of examples of symbiotic microbes today (e.g. gut microbes in ruminants), and there is good evidence that genes are exchanged between different groups of symbionts in the one host, and even between them and the host.

This theory of the origin of eukaryotes explains why some of the membra­nous structures in eukaryotic cells contain vestiges of their own independent genetic material, and why some DNA and protein structures of the archae bacteria seem to be so closely related to those found in higher organisms.

[Chapter Break]

If life really did evolve in this way, then the tourist paradise of Whakarewarewa may represent a modern glimpse into the very oldest popu­lated environment on Earth, and the organisms that nestle in its limpid, steaming pools may not be antediluvian so much as ante-pretty- much­ every thing.

Ignoring the tourist trappings of the place, and trying to blinker out the paved walkways between the pools, it is possible to acquire a sense of the ancient power that lies at the heart of the area: the power of genesis itself, the stench hanging in the air being that of life’s first ferment.

It is this power and beauty which draws people by the thousands to such places each year, whether they are there to be entertained or to study.

If Dante was correct, and such places exist in a Hell somewhere, one can be sure that at least some of the Earth’s denizens the thermophilic bacteria and, especially, the archaebacteria unlike those “souls concealed within these moving fires/each one swathed in his burning punishment” would be happy to call that Hell home.

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