110 Prokaryotic Diversity

Microbial Mats

Microbial mats or large biofilms may represent the earliest forms of prokaryotic life on Earth; there is fossil evidence of their presence starting about 3.5 billion years ago. It is remarkable that cellular life appeared on Earth only a billion years after the Earth itself formed, suggesting that pre-cellular “life” that could replicate itself had evolved much earlier. A microbial mat is a multi-layered sheet of prokaryotes ((Figure)) that includes mostly bacteria, but also archaeans. Microbial mats are only a few centimeters thick, and they typically grow where different types of materials interface, mostly on moist surfaces. The various types of prokaryotes that comprise them carry out different metabolic pathways, and that is the reason for their various colors. Prokaryotes in a microbial mat are held together by a glue-like sticky substance that they secrete called extracellular matrix.

The first microbial mats likely obtained their energy from chemicals found near hydrothermal vents. A hydrothermal vent is a breakage or fissure in the Earth’s surface that releases geothermally heated water. With the evolution of photosynthesis about three billion years ago, some prokaryotes in microbial mats came to use a more widely available energy source—sunlight—whereas others were still dependent on chemicals from hydrothermal vents for energy and food.

Stromatolites

Fossilized microbial mats represent the earliest record of life on Earth. A stromatolite is a sedimentary structure formed when minerals are precipitated out of water by prokaryotes in a microbial mat ((Figure)). Stromatolites form layered rocks made of carbonate or silicate. Although most stromatolites are artifacts from the past, there are places on Earth where stromatolites are still forming. For example, growing stromatolites have been found in the Anza-Borrego Desert State Park in San Diego County, California.

The Ancient Atmosphere

Evidence indicates that during the first two billion years of Earth’s existence, the atmosphere was anoxic, meaning that there was no molecular oxygen. Therefore, only those organisms that can grow without oxygen—anaerobic organisms—were able to live. Autotrophic organisms that convert solar energy into chemical energy are called phototrophs, and they appeared within one billion years of the formation of Earth. Then, cyanobacteria, also known as “blue-green algae,” evolved from these simple phototrophs at least one billion years later. It was the ancestral cyanobacteria ((Figure)) that began the “oxygenation” of the atmosphere: Increased atmospheric oxygen allowed the evolution of more efficient O₂-utilizing catabolic pathways. It also opened up the land to increased colonization, because some O₂ is converted into O₃ (ozone) and ozone effectively absorbs the ultraviolet light that could have otherwise caused lethal mutations in DNA. The current evidence suggests that the increase in O₂ concentrations allowed the evolution of other life forms.

Prokaryotes in the Dead Sea

One example of a very harsh environment is the Dead Sea, a hypersaline basin that is located between Jordan and Israel. Hypersaline environments are essentially concentrated seawater. In the Dead Sea, the sodium concentration is 10 times higher than that of seawater, and the water contains high levels of magnesium (about 40 times higher than in seawater) that would be toxic to most living things. Iron, calcium, and magnesium, elements that form divalent ions (Fe²⁺, Ca²⁺, and Mg²⁺), produce what is commonly referred to as “hard” water. Taken together, the high concentration of divalent cations, the acidic pH (6.0), and the intense solar radiation flux make the Dead Sea a unique, and uniquely hostile, ecosystem¹ ((Figure)).

What sort of prokaryotes do we find in the Dead Sea? The extremely salt-tolerant bacterial mats include Halobacterium, Haloferax volcanii (which is found in other locations, not only the Dead Sea), Halorubrum sodomense, and Halobaculum gomorrense, and the archaean Haloarcula marismortui, among others.

Unculturable Prokaryotes and the Viable-but-Non-Culturable State

The process of culturing bacteria is complex and is one of the greatest discoveries of modern science. German physician Robert Koch is credited with discovering the techniques for pure culture, including staining and using growth media. Microbiologists typically grow prokaryotes in the laboratory using an appropriate culture medium containing all the nutrients needed by the target organism. The medium can be liquid, broth, or solid. After an incubation time at the right temperature, there should be evidence of microbial growth ((Figure)). Koch’s assistant Julius Petri invented the Petri dish, whose use persists in today’s laboratories. Koch worked primarily with the Mycobacterium tuberculosis bacterium that causes tuberculosis and developed guidelines, called Koch’s postulates, to identify the organisms responsible for specific diseases. Koch’s postulates continue to be widely used in the medical community. Koch’s postulates include that an organism can be identified as the cause of disease when it is present in all infected samples and absent in all healthy samples, and it is able to reproduce the infection after being cultured multiple times. Today, cultures remain a primary diagnostic tool in medicine and other areas of molecular biology.

Koch’s postulates can be fully applied only to organisms that can be isolated and cultured. Some prokaryotes, however, cannot grow in a laboratory setting. In fact, over 99 percent of bacteria and archaea are unculturable. For the most part, this is due to a lack of knowledge as to what to feed these organisms and how to grow them; they may have special requirements for growth that remain unknown to scientists, such as needing specific micronutrients, pH, temperature, pressure, co-factors, or co-metabolites. Some bacteria cannot be cultured because they are obligate intracellular parasites and cannot be grown outside a host cell.

In other cases, culturable organisms become unculturable under stressful conditions, even though the same organism could be cultured previously. Those organisms that cannot be cultured but are not dead are in a viable-but-non-culturable (VBNC) state. The VBNC state occurs when prokaryotes respond to environmental stressors by entering a dormant state that allows their survival. The criteria for entering into the VBNC state are not completely understood. In a process called resuscitation, the prokaryote can go back to “normal” life when environmental conditions improve.

Is the VBNC state an unusual way of living for prokaryotes? In fact, most of the prokaryotes living in the soil or in oceanic waters are non-culturable. It has been said that only a small fraction, perhaps one percent, of prokaryotes can be cultured under laboratory conditions. If these organisms are non-culturable, then how is it known whether they are present and alive? Microbiologists use molecular techniques, such as the polymerase chain reaction (PCR), to amplify selected portions of DNA of prokaryotes, e.g., 16S rRNA genes, demonstrating their existence. (Recall that PCR can make billions of copies of a DNA segment in a process called amplification.)