Some like it hot

In the hot springs of Yellowstone Park, US; Iceland; New Zealand; and Russia, microbes thrive in temperatures up to 100°C. The volcanically heated waters can also be very acidic or alkaline, and yet despite these harsh conditions, there are single-celled bacteria, and the single-cell archaea, that thrive. Deep underwater at the bottom of the ocean, geothermal vents spew out gases and water up to 400°C, and similar microbes survive there too.

Normally, cells would be expected to die at these temperatures. Heat denatures the proteins that enzymes are made of, stopping them from working; it also breaks the chemical bonds that hold the lipids in cell membranes together, and destroys the DNA double helix.

Yet thermophilic enzymes remain intact and functional, offering several industries the potential to boost reaction rates and improve productivity. Their stability makes them ideal for use in the food, brewing, pulp and paper and feed processing industries. Many of these industries already use enzymes from mesophilic bacteria, which grow at moderate temperatures of between 20 and 45°C, but thermophiles have the added advantage of being resistant to harsh conditions such as chemical denaturing agents, wide pH ranges, and non-aqueous solvents.

Frank Robb, a researcher at the University of Maryland’s School of Medicine in Baltimore, US, has spent many years investigating how thermophiles maintain stable proteins in the face of intense heat. He has identified ‘chaperone’ or heat shock proteins that the thermophilic bacteria produce to allow them to fold the proteins necessary for life. These chaperone proteins continue functioning at high temperatures by refolding proteins that have denatured, stopping them from aggregating and sticking together.

Chaperones work on disordered proteins by binding specifically to them, and not to native proteins, and maintaining the solubility of their client proteins by obscuring their hydrophobic surfaces. Some classes of chaperones, like HSP60 or HSP70, are powered by ATP hydrolysis to actively fold proteins,’ explains Robb.

As well as molecular chaperones, Robb explains that thermophilic enzymes are also more compact and densely packed together than other enzymes, which minimises internal cavities in the hydrophobic core of the protein. Having smaller cavities, he says, makes the protein less susceptible to thermal denaturation because the core ‘resists conformational writhing and wriggling’.

Thermophilic enzymes also contain more ionic pair interactions between the amino acids. ‘Having extra ion pairs helps to make proteins more stable because the ion pair is effective over a much longer distance (over 3Ä) than van der Waals forces,’ says Robb. They also contain more acidic and basic amino acids and have more charged ions on their internal surface. As long as the charges are organised so that they exert attractive rather than repulsive forces on each other, this helps to hold the protein together.

Additionally, lipid cell membranes in thermophilic bacteria are protected from breaking apart because they are held together by ether bonds instead of the weaker ester bonds.

The super stability and heat resistance of thermophilic enzymes makes them a superior choice over mesophiles for most applications. Indeed, the polymerase enzyme from Thermus aquaticus, an aerobic thermophile discovered in the hot springs of Yellowstone National Park, US, is now widely used in the polymerase chain reaction, which is used by forensic scientists to amplify isolated DNA. Another thermophilic enzyme currently on the market is Novozyme’s Lipozyme, which originates from Thermomyces lanuginosus. The enzyme is an effective catalyst for ester hydrolysis and is used in the commercial manufacture of Pregabalin, an anticonvulsant drug.

Despite these examples, however, thermophilic enzymes have yet to make a big impact on industry. Over the past 15 years researchers have tried to expand the use of enzymes from thermophiles into other areas. Theoretically, all scientists need to do to widen the use of thermophiles is harvest the super enzymes and put them to use. However, this hasn’t proved easy.

One of the main challenges for scientists studying thermophiles is the difficulty involved in culturing them in the lab. Typically only 1–10% of the total microbial population from a hot spring or geothermal vent will grow in the lab or under industry conditions.¹ So many scientists have chosen to skip the culturing step, instead taking the relevant gene from the microorganism and inserting it into Escherichia coli. In this way, they have been able to show the potential, at least on the lab scale, for these enzymes.

One area where thermophiles could be particularly useful is in the production of butyrate, a precursor of butyric acid, which is produced on an industrial scale and used as a feedstock for the synthesis of plastics, plasticisers and surfactants. Various derivatives of butyric acid, such as esters, are used in the food and perfume industries, as well as in the chemical and pharmaceutical industries. A recent study led by Lonnie Ingram, director of the Florida Center for Renewables at the University of Florida, US, showed that the thermophile Clostridium thermobutyricum could ferment sugars into butyric acid at a yield that is among the highest reported for batch fermentations using mesophilic bacteria.²

The microorganism was able to convert 85% of the total sugars from juice and bagasse into a yield of 0.42g of butyrate per gram of sugar fermented. This equates to approximately 90kg of butyric acid per tonne of fresh sweet sorghum stalk, which is equal to the most productive microorganisms such as Clostridium butyricum and Clostridium acetobutylicum. However, the reactions were carried out at 50°C rather than 37°C, leading to higher reaction rates and productivity and less contamination and cooling costs than are currently possible with the mesophilic bacteria.

Hot fuels

Meanwhile, other researchers are investigating the use of thermophiles in the production of biofuels.

Liquid biofuels such as bioethanol, biodiesel, biobutanol and biokerosene are formed from the fermentation of starch and lignocellulose from plant dry matter. However, because cellulose is so resistant to being broken down by enzymes, industries must first pretreat plant materials using heat and strong acids and bases to break the chemical bonds, sometimes adding other enzymes, and then cooling the products – a costly process.

Yet many thermophiles produce enzymes that are able to break down carbohydrate polymers, such as starch, cellulose and hemicellulose, at high temperatures.

Lee Rybeck Lynd, professor of engineering and of biological sciences at Dartmouth University, US, studies the production of energy from plant biomass. He currently leads the biomass deconstruction and conversion research for the US department of energy-funded Bioenergy Science Center (BESC), for which thermophiles are a major focus. Lynd believes that thermophiles could solve this industrial problem. His research group is investigating the possibility of ‘consolidated bioprocessing’ (CBP), ie converting cellulose from plants into ethanol in a single step without the costly pretreatment steps. Although his group is focusing on the production of ethanol, the method could be used to make a broad range of fuels and chemicals.

Recently, Lynd’s team investigated the ability of six thermophilic microorganisms to breakdown switchgrass into soluble products,³ which could then be fermented and ultimately used as fuel. The researchers showed that Clostridium thermocellum was twice as effective at breaking down switchgrass as the fungal enzymes currently used by industry. They also showed that the thermophiles were able breakdown switchgrass that hadn’t been pretreated, representing a reduction in the costs and a potential boost to the efficiency of biofuel production. ‘In light of rapid recent advances, there are indications that thermophiles are knocking on the door with respect to industrial applications for the production of biofuels,’ says Lynd.

According to Lynd, ‘It is becoming clear that the thermophilic, cellulolytic bacterium Clostridium thermocellum and some other thermophiles are able to achieve several-fold higher solubilisation of grassy cellulosic feedstocks with no pretreatment other than autoclaving. The challenge is to combine this distinctive ability to access cellulosic biomass with production of fuel molecules at industrially-viable yield and titer. Great strides are being made using genetic techniques that have only recently become available.’

Another group led by Rajesh Sani, assistant professor of biology at the South Dakota School of Mines and Technology in Rapid City, US, is also investigating how thermophiles could improve biofuel production. Over the past decade, Sani has been looking for thermophiles that can naturally degrade and ferment cellulose and xylan, a polysaccharide found in plant cell walls and some algae.

So far, his group has identified more than 9,000 microorganisms living in the deep biosphere of Sanford Underground Research Facility at Homestake Gold Mine in South Dakota, US. Here, 300 to 5,000 ft deep underground, extreme conditions exist – low nutrients, extreme temperatures, pressure and pH, and low oxygen concentrations, no light, and the possibility of toxic metals. His group has also isolated thermophilic microbes from a compost facility in Rapid City, South Dakota, and from the Hot Springs State Park in Wyoming. Many of the thermophiles found, include those belonging to the genera Brevibacillus, Bacillus, Clostridium, Geobacillus, Paenibacillus, Thermus and Thermoanaerobacterium.

Sani believes that these microbes can circumvent the multiple steps of fuel production, including pre-treatment, saccharification, fermentation, and separation of the product. Similar to Lynd’s work, this is a one-step process. In a recent study,⁴ Sani and his team showed that at 70°C, thermostable hydrolytic enzymes from a Geobacillus sp. converted untreated prairie cord grass and corn stover into fermentable sugars much more effectively than commercial enzymes in use. For example, the Geobacillus xylanase was able to convert 69% of the feedstock to sugars, compared with two commercial enzymes, Cellic-HTec2, and AccelleraseXY, which converted 49%, and 29%, respectively.

Cost-intensive physical, chemical, biological pretreatment operations and slow enzymatic hydrolysis carried out at less than 50°C make the overall process of lignocellulosic conversion into biofuels less economical than available fossil fuels, says Sani. ‘However the unique characteristics of the lignocellulose-deconstructing enzymes produced by mine and compost thermophiles include optimum temperatures of above 70°C, wide pH ranges (4–8) and greater thermostability – for example, at 70ºC, the half-life of the Geobacillus xylanase was found to be 12 days,’ explains Sani. ‘Our thermophiles also produce biohydrogen or bioethanol in a single step using inexpensive regional untreated biomass such as prairie cordgrass and corn stover, as well as mixed food and human wastes.’

However, although thermophiles are making some progress in the biofuel sector, extending their use into other industrial sectors remains a challenge. Kian Mau Goh, a researcher on thermophiles and a lecturer at the Universiti Teknologi, Malaysia, explains: ’Easily isolated thermophiles have long been used in biotechnology. The challenge is how to find new or novel strains, especially those so-called “unculturables” from heated areas such as hot springs, volcanic area and deep sea vents.’

References

1 M. Bouzas et al, Protein and peptide letters, 2006, 13(7)645.

2 K.T. Shanmugam et al, Bioresource Technol., 2015, doi: 10.1016/j.biortech.2015.09.062.

3 L.R. Lynd et al, Biotechnol. Biofuels, 2016, doi: 10.1186/s13068-015-0412-y.

4 R.K Sani et al, Front Bioeng Biotechnol., 2015, doi:10.3389/fbioe.2015.00084