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Spring Trek:  The Next Innovation

By September 1, 2013Uncategorized

Spring: the final frontier. These are the voyages of extremophiles on Earth. Their ongoing missions: to explore extreme new worlds, to seek out new nutrient resources and new metabolisms, and to boldly go any places where no microorganisms have gone before!

Last month I went on a road trip from San Francisco to Chicago with a college friend and the Yellowstone National Park was the highlight of our trip. The best part of Yellowstone was not the beautiful sunsets, the predictable geysers or the free-roaming buffalos, but was the incredibly beautiful hot springs. The Grand Prismatic Spring, the largest hot spring in North America, displayed vivid shades of red, yellow, green against a crystalline blue pool that looks more like a surrealistic Monet painting than reality.

Tourists around us speculated on the origin of the colors. Since the hot springs, as implied in their names, can easily exceed 90ºC, many of them guessed: “it must be some sort of metal” that causes the formation of colors.  This would not be a bad assumption as metals are routinely used to tint windows and fireworks. However, it is actually due to the production of carotenoids and chlorophyll by the microorganisms.  Then the organic pigments, naturally blended in different ratios, create gorgeous color gradients.

Rudy text picture copyMore astonishing than the colors themselves is the fact that the microorganisms form those colors. But how? In the Grand Prismatic Spring, for example, a scientist would be able to find archaea, fungal, algal, protozoa and bacterial communities known as extremophiles that can survive high temperatures in low nutrient conditions. Because of this adverse condition, those microorganisms require unconventional avenues to obtain energy. For instance, while most microorganisms use glycolysis as a catabolic pathway for glucose, extremophiles tend to use the Entner-Doudoroff pathway to oxidize glucose to pyruvate. If alternative glucose metabolism is not rare enough, some extremophiles are even able to use syngas (a mixture of hydrogen and carbon monoxide/dioxide) through the Wood-Ljungdahl pathway in which the final electron acceptor, instead of oxygen, is carbon dioxide. In other words, this latter pathway allows extremophiles to live without air and sugars, an inhospitable environment for most microorganisms.

Although alternative metabolic pathways are interesting, they have also been scrutinized in the industry because petrochemical processes produce copious amounts of syngas and the potential to convert a cheap stream into a specialty chemical can be a profitable endeavor (although it is not commercially viable yet).

Extremophiles not only can survive without air and sugars, but they can also survive at high temperatures. If ordinary microorganisms were subjected to 90ºC, proteins would denature and stop functioning instantaneously (not surprisingly: a common protocol for protein denaturation before running an SDS PAGE requires incubation at high temperatures). However, extremophiles tend to have proteins that are extremely heat stable. At a molecular level, this is achieved by having intramolecular bonds that enable the protein to stay intact at high temperatures. Can you imagine what would the applications of these proteins be? Imagining no more because universities and industries are reaping the benefits of these properties already. Thermostable proteases are being used routinely in detergents to degrade proteinaceous stains as well as to withstand hot wash cycles. Additionally, thermostable amylases are employed in textile processing to desize fabrics but can be subjected to hot process temperatures, and taq polymerase is an enzyme isolated from Thermus Aquaticus in Yellowstone that is used in PCR because it can polymerize DNA while it can withstand the DNA melting temperatures.

Besides aforementioned commercial applications, there are efforts to explore if thermostable enzymes can be used to convert hot gas and methane into biological feedstocks for chemicals and fuels or if these can be used to reduce viscosity of the cellulose-rich mud that is formed in shale-gas drilling sites (commonly known as fracking). Undoubtedly, the ongoing search for thermostable enzymes and their industrial applications will continue for the years to come.

Yet, thermostability is not the only property to be harnessed to create products. Active enzymes at low temperatures are important as well because heating a process in an aqueous environment is energy intensive due to water’s high heat capacity. Thus, having enzymes that work at lower temperature means lower environmental impact. An example of this enzyme would be Optisize® Cool, which I developed the fermentation for in Palo Alto, transferred to one of DuPont’s manufacturing plants and was commercially launched a couple of years ago.

Temperature is not the only condition that extremophiles tolerate. Extremophiles are adept in environments with high osmolarity, which is not trivial. To calculate the osmotic pressure, the Van’t Hoff factor is multiplied by the molarity, the gas constant, and absolute temperature (Osmotic Pressure = iMRT). A 1 molar sodium chloride difference between the inside and outside of the microorganism at 20ºC will exercise 48 atmospheres of gauge pressure on the microorganism, which is roughly equivalent to a 1500 feet pool dive. For the record, extremophiles that can survive up to 5 molar sodium chloride have been reported and that roughly equates to 240 atmospheres or 8000 feet pool dive.

It is known that one of the mechanisms for survival in high osmolarity is through the use of enzymes that are recalcitrant to precipitation or denaturation in high osmolarity conditions. Recalling from undergraduate chemistry/biochemistry courses, those enzymes, like some chemicals, can be salted-out of solution. Precipitation of enzymes inside a cell would be catastrophic as those can form inclusion bodies which stress microorganisms immensely to the point that some will lyse. Thus, extremophiles have developed enzymes that are stable at high osmolarity and that can be useful in applications where enzymes are needed in industrial processes with high solute concentration.

Harsh environments are not limited to high temperatures and salinity or to low nutrient and oxygen, but also include high radiation and pressures or low humidity and temperatures. The quest for finding better enzymes bioprospecting in hot springs and Arctic glaciers will continue in the years to come. The challenge, however, is to find application to enzymes with unique properties.

Although the global market for enzymes is relatively small, at around $3 billion, compared to the biopharmaceutical market, enzymes are present in many consumer products. Products that are already being made with enzymes include clothing, detergents, ethanol, dishwashing soap, paper, animal feed, bread, cheese, denim trousers, wine, juices, yogurt and sugars.  Yet, the list keeps expanding yearly. Who would have thought that something as scientifically inconspicuous as a Yellowstone hot spring held promises for such diverse industries? Who could have imagined that there are more surprises behind the beauty of hot springs? Who would have thought that microorganisms’ enzymes could be the next innovation in the biotech industry?

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