Heat, Hydrogen, and Togas
by Elio
Extremophilic microbes, especially the thermophilic ones, are exceedingly interesting for many industrial processes. And why not, being that high temperatures prompt faster enzymatic reactions. Present-day uses for such organisms include the production of polysaccharide‑, lipid‑, and protein-degrading enzymes among many. It is worth remembering that this pursuit was initiated by Thomas Brock's trailblazing discovery of the heat-stable DNA Taq polymerase from a heat-loving bacterium found in a hot pool of Yellowstone Park. It made the PCR reaction practicable.
Figure 1. Thermotoga maritima EM thin section showing the toga and the large bipolar periplasm. Bar: 1 µm. Source
People in the business have had their eyes on bacteria as producers of hydrogen, a most desirable clean fuel (no CO2 when it burns!). Hydrogen packs more energy per mole than any other fuel, can be used in fuel cells (as in hydrogen-powered cars) to be converted into water and electric current, and can be produced in a variety of ways, including biological. It seems that the industrial production of hydrogen by these means is not yet practical, but given the attention this topic has received, one can expect that progress will be forthcoming. In this spirit, let me introduce Thermotoga, a bacterium that is a prime candidate for the industrial production of hydrogen. I confess, I chose this topic so I could talk about this fascinating organism.
Thermotoga maritima is the type species of its genus and it's the best-known member. It belongs to the phylum Thermotogae, which comprises about a dozen genera. They get their name because they are hyperthermophilic ('Thermo'), growing at temperatures up to about 90°C, the highest known for bacteria and because they are surrounded by a rather unique thick sheath, a toga. A singular feature of these organisms is having large, empty-looking balloons at both ends, which means that they have a huge periplasm. In this space are contained hydrolyzing enzymes such as xylanases (a point that will become important soon). Thermotogae are strict anaerobes and gram negative. Their peptidoglycan is unique in having as much D‑lysine as the usual L‑lysine. And, no LPS (or, at least, no LPS-making enzymes), despite being gram-negative.
Figure 2. The island of Vulcano north of Sicily, showing the "Great Crater," thought by the Romans to be the chimney of Vulcan's forge. Source
The phylum Thermotogae is one the deepest branches in the 16S RNA bacterial tree. Remarkably, members of the phylum carry an unusually large number of Archaea-like genes. These genes comprise about 24% of the ca. 1900 ORFs, which is the highest number of such genes for any bacterium so far. They retain the same gene order as the archaea, suggesting that they were acquired by horizontal gene transfer. The majority of the T. maritima genes related to the archaea are not uniformly distributed. Thus, 49% of transporters (92 genes), 60% of electron transport proteins (28 genes) and 42% of conserved hypothetical proteins (173 genes) are most similar to archaeal genes. Note that they share a love of high temperatures with hyperthermophilic archaea. T. maritima was isolated in 1986 from a geothermal sediment near the (aptly named) small Italian island of Vulcano by Stetter et al. (for a lively vimeo that features Karl Stetter, click here). In the same year and from nearby sites, this group also isolated the hyperthermophilic archaeon Pyrococcus furiosus. Make something of this.
Now for hydrogen production by microbes. The idea is attractive because in theory, fermentation of 1 mol of glucose should yield 4 mol of H2.. In fact, microbial fermentations with various species of Thermotogae have yielded from 1.5 to 3.85 mol of hydrogen per mol of hexoses from carbohydrate-rich wastes such as molasses or cheese whey. In these experiments, these authors used both suspended and attached cells, with about equal results. The values of hydrogen produced were 62 to 74% of the theoretical maximum.
Figure 3. Schematic representation of the diversity of H2 producing biocatalysts. Source
Particularly appealing for the purpose of making hydrogen using microbes is that the same organisms that produce it can also degrade vegetable wastes into fermentable substrates. T. maritima, for example, possesses an array of thermostable hydrolases, including cellulases, invertases, and xylanases. No wonder the Thermotogae have attracted attention of experimenters and modelers alike. These organisms can be fed agricultural waste products that could be hard to dispose of otherwise. Big among them are unused fruit and vegetable material, which can amount to a high proportion of the solid municipal waste (68% in the case of Tunis). Municipal solid wastes consist in good part of discarded fruit and vegetable material that eventually ends up in landfills, thus its biodegradation is a most hoped-for outcome. So, organisms like Thermotoga may rid us of unwanted wastes and make useful products in the process. What is especially nice here is that the Thermotogae, being loaded with hydrolytic enzymes, make it unnecessary to separately turn the waste into fermentable substrates. The organism can degrade both simple sugars and polysaccharides, from pentoses and hexoses to starch and xylans, the main products of fermentation being hydrogen, acetate, and CO2. I can imagine that under some conditions, the bacteria themselves may be a source of, say, valuable proteins. That would do justice to the term used in efficient slaughterhouses, making use of "everything but the squeal." Fruit and vegetable wastes vary of course with the location, time of year, and agricultural practices. In a study carried out in Tunis, the reducing sugars alone comprised about 80 g per liter.
Figure 4. A graphical abstract of the paper discussed. Source
In a recent study, researchers from two Tunisian universities (U. of Carthage and U. of Tunis El Manar), and a French one (U. de Toulon), asked a simple question: can seawater substitute for the expensive and complex salt mixtures used previously in hydrogen fermentation by Thermotoga ? After all, isn't that what the organism thrives in naturally ? The results were most encouraging. Using their medium (seawater supplemented with a source of nitrogen and sulfur), the maximal hydrogen production was about 120 mmol per liter, the equivalent of 3.8 mol per mol of total sugar, which is close to the theoretical yield of 4 mol per mole of glucose. And this in about 6 hours!
For all this promising talk of hydrogen as a common fuel, the reality is that its large-scale use is not yet upon us. The reasons are multiple and beyond the scope of this post. However, there is a race for the best way to generate hydrogen via microbes, including the choice of strains, substrates, fermentation conditions, and other variables. For now, the Thermotogae seem to have the upper hand among the bacteria under consideration. May the scientists who are researching this field continue to be successful, and may their efforts culminate in the production of abundant, clean, and cheap fuel.
