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 H_2.. 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.
 

(Click to enlarge )
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.