Feel de­hy­drated? Dig for some wa­ter then!

by Christoph

Cyanobac­te­ria of the genus Chroococ­cid­iop­sis are known to sur­vive harsh en­vi­ron­men­tal con­di­tions, high and low tem­per­a­tures, ion­iz­ing ra­di­a­tion, and high salin­ity. Add re­sis­tance to de­hy­dra­tion or, in the ex­treme form, desic­ca­tion and you have a se­ri­ous chal­lenger of Deinococ­cus to the ti­tle "cham­pion of re­silience." Like the lat­ter, Chro­o­coccidiopsis is eas­ily found among other cyanobac­te­ria in the At­a­cama desert in Chile, one of the dri­est places on the planet with un­for­giv­ing sun­light. No won­der that they are pet mi­crobes of exo­bi­ol­o­gists pon­der­ing terra­formation on Mars; they were al­ready re­cruited to the ISS as as­tro­nauts, in­clud­ing for space­walks.
 

Fig­ure 1. Chroococ­cid­iop­sis iso­lated from li­me­stone (CaCO₃) from Hawaii with baeo­cyte for­ma­tion, that is, cells formed by mul­ti­ple divi­si­ons with­out in­ter­me­di­ate growth (ar­rows). Pho­to: Burkhard Büdel, Col­lec­tion of ter­restrial cy­anobacteria and cyanobionts, TU Kai­sers­lau­t­ern, Ger­many. Source. Fron­tispiece: Cyano­bacteria in a soil gyp­sic crust sam­ple from the At­a­cama Desert Source

Chroococ­cid­iop­sis is not en­demic to the At­a­cama, it's al­so found in the Mo­jave desert (USA), and the desert-like Al‑Jafr basin (Jor­dan.) But, im­por­tantly, it's not a true ex­tremophile, that is, an or­gan­ism with op­ti­mal growth in ex­treme en­vi­ron­men­tal con­di­tions, as it is read­ily found in tem­per­ate cli­mates world­wide and eas­ily cul­ti­vated in the lab. Chroococ­cid­iop­sis is also known as "blau­grüne Fel­skugel" (blue-green rock globe) to phy­col­o­gists, the branch of bo­tany that deals with al­gae, in­clud­ing the cyanobac­te­ria as 'blue‑green al­gae' for tra­di­tion, and the Ger­man name "Fel­skugel" in­di­cates a pen­chant of these pig­mented (pho­to­syn­thetic), coc­coid bac­teria for rocks (Fig­ure 1). In­deed, they are mostly found on and within min­eral stones, which are, at least in thin lay­ers, par­tially translu­cent like quartz (cal­cium car­bon­ate) and gyp­sum (cal­cium sul­fate, see Fron­tispiece.)

Fig­ure 2. SEM mi­cro­graphs of frac­tured soil gyp­sum spec­i­mens from At­a­cama site AT326b ©, re­vealing uni­cellular cyano­bacteria and cell divi­sions (ar­rows). Scale bar: 5 mm. Source

It is gen­er­ally as­sumed that Chroococ­cid­iop­sis pro­tects it­self from ex­ces­sive sun­light by grow­ing within the rock and get­ting there through mi­cro-cracks or crevices. In­deed, es­pe­cially gyp­sum has a rather brit­tle tex­ture (Fi­gure 2). The At­a­cama is so dry be­cause very lit­tle mois­ture is trans­ported in­land from the Pa­cific Ocean. So, it was thought that the bac­te­ria ben­e­fit from oc­ca­sional dew when grow­ing on the un­der­side of rocks, as this de­sert has a hefty daily tem­per­a­ture gra­di­ent.

This com­fort­ing ex­pla­na­tion − be­ing con­sis­tent − got fis­sures when Wei Huang and cowork­ers from the Ki­sailus lab at the Dept. of Ma­te­ri­als Sci­ence and En­gi­neer­ing, UCI, Irvine, CA, took a closer look at cyanobac­te­r­ial in­clu­sions in gyp­sum rock sam­ples from the At­a­cama (their sam­pling point). More pre­cisely, they looked at thin sec­tions of sam­ples by Fourier-trans­form in­frared spec­troscopy (FTIR) and found in re­gions en­riched in cyanobac­te­ria sig­nals for gyp­sum (CaSO₄·2H₂O) sur­rounded by an­hy­drite (CaSO₄). They hy­poth­e­sized that the cyanobacte­ria gained ac­cess to the H₂O bound as "wa­ters of crys­tal­liza­tion" in the gyp­sum to meet their own de­mands (see here for the dif­fer­ent crys­tal struc­tures of CaSO₄·2H₂O and CaSO₄). Af­ter all, gyp­sum (CaSO₄·2H₂O) con­tains 20% (weight) H₂O.

Fig­ure 3. Cyanobac­te­ria cul­ture on gyp­sum sam­ples. X‑ray dif­frac­tion (XRD). Gyp­sum rock con­trol, not ex­posed to mi­crobes (black curve), sam­ples cul­tured in low con­cen­tra­tion (LC) and high con­cen­tra­tion (HC) of cyanobac­te­ria in both dry and liq­uid medium envi­ronments. Dif­fraction peaks la­beled with black squares repre­sent the an­hy­drite phase, while those lab­eled with stars are from gyp­sum. XRD was with Cu-Kα ra­di­a­tion, a gen­er­a­tor volt­age of 45 kV, and tube cur­rent of 40 mA. The scan range (2θ) was from 10° to 70°; In­ten­sity is given in ar­bi­trary units (a.u.). Source

For the ex­per­i­ment, they in­oc­u­lated au­to­claved gyp­sum rock "coupons" (0.5×0.8 ×0.5 mm, made with a di­a­mond saw) with a Chroococ­cid­iop­sis iso­late from the At­a­cama and in­cu­bated them at two dif­fer­ent cell den­si­ties in 96-well plates un­der two dif­fer­ent cul­ture con­di­tions: 1. "dry con­di­tion" with no fur­ther liq­uid than the ini­tial in­ocu­lum, and 2. "wet con­di­tions" by adding cul­ture medium dur­ing the in­cu­ba­tion pe­riod. Af­ter 30 days, cells on and within the gyp­sum coupons had a bright green color, in­di­cat­ing the pres­ence of pho­to­syn­thetic pig­ments. Elec­tron micro­scopy re­vealed the pres­ence of cells and biofilm at and close to the gyp­sum crys­tals with an­hy­drite crys­tals near­by. In­ter­est­ingly, Chroococ­cid­iop­sis cells (and biofilm) grew pref­er­en­tially on the "nar­row side" of the gyp­sum crys­tals and not on the "broad side" (see here, and Fig­ure 4, H). When they an­a­lyzed the ground coupons by X‑ray dif­frac­tion (XRD), they found sig­nals for an­hy­drite only for the "dry con­di­tion" sam­ples, and stronger sig­nals for the sam­ples with higher cell num­bers in the in­ocu­lum (Fi­gure 3). The "wet con­di­tion" sam­ples gave sig­nals for gyp­sum only, as did the con­trol (a mi­nor flaw: the au­thors do not in­di­cate whether the con­trol "with­out bac­te­ria" was in­cu­bated un­der "dry" or "wet" con­di­tions.) This is a clear in­di­ca­tion that Chroococ­cid­iop­sis can ac­cess the crys­tal wa­ter in CaSO₄·2H₂O dur­ing growth. But ap­par­ently it does so only if no other source of H₂O is avail­able. But how does Chroococ­cid­iop­sis "nib­ble" on gyp­sum? FTIR analy­sis and Ra­man spec­troscopy of gyp­sum coupons cul­tured with high cell con­cen­tra­tions showed sig­nif­i­cantly more in­tense ab­sorp­tion bands for C=O and O−H than for those with the small­er in­oc­ula, point­ing to the pres­ence of acid moi­eties, for ex­am­ple, or­ganic acids in the biofilm sur­round­ing the cyanobac­te­ria. This was as close as they could get − mass spec­trom­e­try might even­tu­ally tell more − to pin­point­ing which se­creted metabolite(s) sol­u­bi­lize the CaSO₄·2H₂O crys­tals to ac­cess the H₂O.

Fig­ure 4. Four stages of gyp­sum−an­hydrite phase trans­for­ma­tion. Stage I: micro­organisms at­tach on the gyp­sum crys­tals and form a biofilm. A TEM im­age high­light­ing the gyp­sum crys­tals. Stage II: gyp­sum dissolu­tion and wa­ter ex­tract­ion with sub­sequent an­hydrite nano­crystal precip­ita­tion. C A porous struc­ture is obs­erved at the peri­phery of the gyp­sum par­ticles, in­dicat­ing their dis­solu­tion. Stage III: An­hydrite crys­tal growth. E SEM im­age shows large, faceted, an­hydrite par­ticles on the sur­face of gyp­sum. Stage IV: Com­ple­tion of the gyp­­sum-an­hy­drite phase trans­form­ation. G SEM im­age of an­hydrite par­ticles, high­light­ing sur­face facet­ing. H Sum­mary and schem­atic of micro­organ­ism in­duced gyp­sum–an­hydrite pha­se trans­form­ation (see text for de­tails). Source

Based on their ex­per­i­men­tal re­sults, Huang et al. estab­lish a "time­line" for the gyp­sum-dig­ging by Chroococci­diopsis: cells at­tach and form biofilms on the {011} pla­nes of gyp­sum crys­tals. Gyp­sum dis­solves, and wa­ter ex­trac­tion oc­curs. Based on the crys­tal struc­ture of gyp­sum, the wa­ter of crys­tal­liza­tion layer is ex­posed to the {011} planes, but not to the {010} planes (see here). As the sin­gle crys­talline gyp­sum dis­solves and loses wa­ter of crys­tal­liza­tion, it trans­forms via pre­cip­i­ta­tion of nanocrys­talline an­hy­drite. These an­hy­drite nanocrys­tals pre­cip­i­tate on the sur­faces of gyp­sum crys­tals. Short-­range align­ment of the an­hy­drite nanocrys­tals is ob­ser­ved. Large mi­crom­e­ter-sized an­hy­drite crys­tals are for­med via par­ti­cle at­tach­ment and align­ment (Fig­ure 4).

Three fi­nal digs

1  Dig­ging for wa­ter of crys­tal­liza­tion in gyp­sum-rich rocks or soil is an amaz­ing skill but prob­a­bly not unique to Chroococ­cid­iop­sis. Sara Pala­cio and col­leagues from the In­sti­tuto Pire­naico de Ecología (IPE-CSIC) in Jaca, Spain, could show in an ear­lier study that the crystalliza­tion wa­ter of gyp­sum rocks is a rel­e­vant wa­ter source for the gypsum‑specialist He­lianthe­mum squa­ma­tum, a plant from the Cis­taceae fam­ily of ~200 species, known to pre­fer dry and sunny habi­tats, and grow­ing well with its shal­low roots on poor soils. Their analy­ses of the hy­dro­gen (δ²H) and oxy­gen (δ¹⁸O) iso­tope com­po­si­tion of the xylem sap of H. squa­ma­tum re­vealed that in the drier sum­mer sea­son, and un­like dur­ing spring with a more bal­ance com­po­si­tion, the con­tri­bu­tion of gyp­sum crys­tal­liza­tion wa­ter ac­counted for a whop­ping 70–90% of the wa­ter in the xylem sap of the plants while the con­tri­bu­tion of free top soil wa­ter de­creased to 0–10%. How­ever, these re­searchers could not un­am­bigu­ously iden­tify the min­ers re­spon­si­ble, that is, whether the dig­ging was done by the plant or by its as­so­ci­ated root mi­croor­gan­isms (in­clud­ing ar­bus­cu­lar and ecto-my­c­or­rhizae.)

2  When Chroococ­cid­iop­sis dig for wa­ter in gyp­sum, they leave be­hind an­hy­drite as Huang et al. have con­vinc­ingly shown. What the au­thors do not men­tion ex­plic­itly is that, with the H₂O gone, the den­sity of gyp­sum (2.32 g/cm³ for CaSO₄·2H₂O) in­creases to 2.96 g/cm³ in an­hy­drite (CaSO₄), thus leav­ing ac­tual space for the bac­te­r­ial colony/biofilm to ex­pand by growth. Drilling holes with­out me­chan­i­cal aids, and with­out dis­pos­ing of the rub­ble by trans­porta­tion but by pro­mot­ing a phase tran­si­tion in the min­er­als, that is, their re‑crystallization is rather for­eign to the imag­i­na­tion of min­ing en­gi­neers.

Fig­ure 5. Left: Close-up of a cal­cite chip heav­ily in­fested with M. tes­tarum BC008. Sin­gle green fil­a­ments can be ob­served deep in the crys­tal ma­trix. No­tice the re­precipitation of a yel­low, light-scat­ter­ing mi­crite layer close to the surfa­ce. Right: In­ferred na­ture and distri­bution of trans­porter com­po­nents (leg­end at bot­tom), and the fate of Ca²⁺ and CO₃²⁻ af­ter cal­cite dis­so­lu­tion. Source

3  Min­ing en­gi­neers would prob­a­bly ap­pre­ci­ate how an­other cyanobac­terium, one that is only dis­tantly re­lated to Chroococ­cid­iop­sis, per­forms min­ing. Mastigo­coleus tes­tarum BC008 is a fil­a­men­tous, true‑branching, hete­rocystous cyanobac­terium, and con­sid­ered an euendo­lith, that is, an or­gan­ism that pen­e­trates ac­tively into the in­te­rior of rocks form­ing tun­nels that con­form roughly with the shape of its body (men­tioned here in STC by guest au­thor Jeff Mar­low.) Fer­ran Gar­cia-Pichel and co­workers from the School of Life Sci­ences, Ari­zona State Uni­ver­sity, Tempe AZ, found in a study from 2010 that this cyanobac­terium bores into cal­cite (CaCO₃) un­der lab con­di­tions, with ac­tive pho­to­syn­the­sis pro­vid­ing the re­quired en­ergy, ATP. As de­picted schemat­i­cally in Fig­ure 5 (right), the lead­ing cell in a fil­a­ment, the tip of the drill so to speak, ex­ports H⁺ ions dur­ing pho­to­syn­the­sis, which sol­u­bi­lize the car­bon­ate, CO₃²⁻, from the cal­cite, re­sult­ing in an in­crease in the con­cen­tra­tion of free Ca²⁺ ions. The Ca²⁺ ions are taken up and, dri­ven by ATP con­sump­tion, trans­ported from the tip 'up­wards' to the lag­ging cell via a hand­over by P‑type Ca²⁺ AT­Pase trans­porters in the chain of fil­a­men­tous cells (they found in buried fil­a­ments two such Ca²⁺ AT­Pase trans­porters over­ex­pressed two- to ten-fold in com­par­i­son to non-bor­ing cells.) The lag­ging cell de­posits the rub­ble, Ca²⁺, as mi­crite (re­crys­tal­lized form of lime mud) on a dump at the en­trance to the shaft (Fig­ure 5, left). Doesn't this sound much more like "clas­si­cal min­ing" than what Chroococ­cid­iop­sis is do­ing with gyp­sum?

Mi­cro­bi­ol­o­gists are left with the un­sur­pris­ing in­sight that they have to look closely at each sys­tem, at each cyanobac­terium in­di­vid­u­ally, if they want to un­der­stand where, why, and how they go about "min­ing." And do­ing this would not be a side-track since it is be­com­ing in­creas­ingly clear that cyanobac­te­ria − for a long time thought to be mainly busy with pho­to­syn­the­sis − make up a con­sid­er­able pro­por­tion of the deep-sub­sur­face mi­cro­biota, this life on the dark side.