Correction BIOPHYSICS AND COMPUTATIONAL BIOLOGY, MICROBIOLOGY Correction for “Mechanism of water extraction from gypsum rock by desert colonizing microorganisms,” by Wei Huang, Emine Ertekin, Taifeng Wang, Luz Cruz, Micah Dailey, Jocelyne DiRuggiero, and David Kisailus, which was first published May 4, 2020; 10.1073/pnas.2001613117 (Proc. Natl. Acad. Sci. U.S.A. 117, 10681–10687). The authors note that on page 10682, right column, first full paragraph, second sentence, and on page 10686, left column, second full paragraph, first sentence, “the listed size of the mineral coupons used were off by an order of magnitude. In our original manuscript, we stated that the dimensions of the mineral coupons were: 0.5 mm × 0.8 mm × 0.5 mm. The correct size is: 5 mm × 8 mm × 0.5 mm.” Published under the PNAS license. First published November 9, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2021524117 www.pnas.org PNAS | November 24, 2020 | vol. 117 | no. 47 | 29989 CORRECTION Downloaded by guest on May 10, 2021 Downloaded by guest on May 10, 2021 Downloaded by guest on May 10, 2021 Downloaded by guest on May 10, 2021 Downloaded by guest on May 10, 2021 Downloaded by guest on May 10, 2021 Downloaded by guest on May 10, 2021 Downloaded by guest on May 10, 2021 Downloaded by guest on May 10, 2021
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Correction
BIOPHYSICS AND COMPUTATIONAL BIOLOGY, MICROBIOLOGYCorrection for “Mechanism of water extraction from gypsumrock by desert colonizing microorganisms,” by Wei Huang, EmineErtekin, Taifeng Wang, Luz Cruz, Micah Dailey, JocelyneDiRuggiero, and David Kisailus, which was first published May 4,2020; 10.1073/pnas.2001613117 (Proc. Natl. Acad. Sci. U.S.A. 117,10681–10687).The authors note that on page 10682, right column, first full
paragraph, second sentence, and on page 10686, left column,second full paragraph, first sentence, “the listed size of the mineralcoupons used were off by an order of magnitude. In our originalmanuscript, we stated that the dimensions of the mineral couponswere: 0.5 mm × 0.8 mm × 0.5 mm. The correct size is: 5 mm ×8 mm × 0.5 mm.”
Mechanism of water extraction from gypsum rock bydesert colonizing microorganismsWei Huanga, Emine Ertekinb, Taifeng Wangc, Luz Cruzc, Micah Daileyb, Jocelyne DiRuggierob
,and David Kisailusa,c,d,1
aDepartment of Chemical and Environmental Engineering, University of California, Riverside, CA 92521; bDepartment of Biology, Johns Hopkins University,Baltimore, MD 21218; cMaterials Science and Engineering Program, University of California, Riverside, CA 92521; and dDepartment of Materials Science andEngineering, University of California, Irvine, CA 92697
Edited by Donald E. Canfield, Institute of Biology and Nordic Center for Earth Evolution, University of Southern Denmark, Odense M., Denmark, and approvedMarch 30, 2020 (received for review January 28, 2020)
Microorganisms, in the most hyperarid deserts around the world,inhabit the inside of rocks as a survival strategy. Water is essentialfor life, and the ability of a rock substrate to retain water isessential for its habitability. Here we report the mechanism bywhich gypsum rocks from the Atacama Desert, Chile, provide waterfor its colonizing microorganisms. We show that the microorganismscan extract water of crystallization (i.e., structurally ordered) from therock, inducing a phase transformation from gypsum (CaSO4·2H2O) toanhydrite (CaSO4). To investigate and validate the water extractionand phase transformation mechanisms found in the natural geologi-cal environment, we cultivated a cyanobacterium isolate on gypsumrock samples under controlled conditions. We found that the cyano-bacteria attached onto high surface energy crystal planes ({011}) ofgypsum samples generate a thin biofilm that induced mineral disso-lution accompanied by water extraction. This process led to a phasetransformation to an anhydrous calcium sulfate, anhydrite, whichwas formed via reprecipitation and subsequent attachment and align-ment of nanocrystals. Results in this work not only shed light on howmicroorganisms can obtain water under severe xeric conditions butalso provide insights into potential life in even more extreme envi-ronments, such as Mars, as well as offering strategies for advancedwater storage methods.
microorganisms | water extraction | anhydrite | gypsum | phasetransformation
Water plays many roles in organismal function: It is not onlycritical for metabolic processes but also acts as a structural
component in materials and tissues (1, 2). Against all odds, evenin the driest places on Earth where nothing grows, microorgan-isms were found to colonize lithic (rock) substrates as a lastrefuge for life (3, 4). By filtering out UV irradiation and en-hancing access to water, the rock provides protection and sta-bility to an unexpected diversity of microbial taxa, includingcyanobacteria, actinobacteria, Chloroflexus, and proteobacteria(4, 5). Such assemblies of endolithic (within rock) microorgan-isms have been found in the Atacama Desert in Chile, one of thedriest and oldest deserts on Earth (6–8) and an analogous en-vironment to Mars (9). The aridity index (AI) of the AtacamaDesert, the ratio of the average water supply and potentialevapotranspiration, can be as low as 0.0075 (10), whereas an AIthreshold of 0.05 is used to defined hyperarid deserts (11). In thehyperarid core of the desert, records of air relative humidity(RH) show extensive periods below 60% RH (SI Appendix, Fig.S1), illustrating the scarcity of water. It is important to note that0.585 water activity (aw; 58.5% RH) is the lower limit at whichmetabolic activity was detected (12). Thus, understanding howmicroorganisms acquire water under extreme xeric stress couldprovide insights into potential life on past, or present, Mars, andalso help in the development of new technologies for waterstorage and acquisition (13, 14). Xeric stress is defined here as alack of water (desiccation) that produces biochemical, metabolic,physical, and physiological stress (1). The range of water activity
for the onset of xeric stress varies from 0.91aw to 0.585aw,depending the microorganisms (1, 12).One type of mineral commonly found in the Atacama Desert
is gypsum (7), a hydrated calcium sulfate (CaSO4·2H2O). Whilethis substrate contains other minerals, such as sepiolite, that canpotentially alleviate xeric stresses due to its porous structure andwater absorption and retention abilities (8, 15), the water foundwithin the gypsum is crystalline, with up to 20.8% of the totalmass stored within its lattice. Thus, it is reasonable to surmisethat gypsum can act as a source of water for organisms livingunder extreme xeric stress (16). In fact, it was determined that ashallow-rooted plant, Helianthemum squamatum, lives on gyp-sum and extracts water from the rock during dry summers innortheastern Spain (17). However, the mechanism by which thiswater is exacted from gypsum as well as its resulting effect on therock remains unidentified.The transitions between the various phases of geologic calcium
sulfate minerals—gypsum, bassanite, and anhydrite—has beenstudied (15, 18–20). It is known that gypsum may lose some or allof its structural or “crystalline” water and subsequently transformsto either a hemihydrite phase, bassanite (CaSO4·1/2H2O), or ananhydrous phase, anhydrite (CaSO4), in different environments(18, 22). In addition, gypsum, which has been found in evaporiticminerals in the upper crust of Earth, can undergo a reversibletransformation to anhydrite after dehydration−hydration cyclingunder certain geochemical conditions (18). Phase diagrams ofCaSO4 and water have been developed to show effects of tem-perature and pressure on the solubility and stability of differentphases (15, 19). Gypsum is the thermodynamically stable phase
Significance
This research provides an in-depth analysis of how microor-ganisms are able to survive in the world’s driest non-polarplace, the Atacama Desert, Chile. We show that these organ-isms extract water from gypsum rocks in this desert, enablingthese colonizing microorganisms to sustain life in this extremeenvironment. We believe the results in this work could not onlyshed light on how microorganisms can obtain water undersevere xeric conditions, but also provide insights into potentiallife in even more extreme environments, such as Mars, as wellas offer strategies for advanced water storage methods.
Author contributions: W.H., J.D., and D.K. designed research; W.H., E.E., T.W., L.C., andM.D. performed research; W.H., E.E., J.D., and D.K. analyzed data; and W.H., E.E., J.D., andD.K. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Published under the PNAS license.1To whom correspondence may be addressed. Email: [email protected].
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2001613117/-/DCSupplemental.
below 40 °C, but is unstable with increasing temperature, trans-forming to the anhydrite phase (19). The kinetics of thesegypsum–anhydrite phase transformations are controlled by addi-tional environmental conditions such as acidity and ionic strength,which could potentially affect the hydrogen bonds between watermolecules and sulfate ions inside gypsum crystals. For example,the presence of H+ can facilitate the extraction of water of crys-tallization (i.e., structurally ordered) inside the gypsum crystals byforming H3O
+, which increases the mineral solubility (20).Here, we use a combination of microscopy and spectroscopy to
characterize gypsum samples from both geological and labora-tory environments, revealing the processes by which colonizingmicroorganisms obtain water from their substrate, and theresulting effect on the rock. We report that gypsum rockstransform to anhydrites because of the loss of water of crystal-lization in a process induced by microorganisms. The findings inthis study will provide insight toward mechanisms of survival fororganisms living in extreme environments and thus have poten-tial for use in identifying water storage sources for extraterres-trial exploration or habitation.
Microorganisms Living in the Gypsum RocksObservation of gypsum rock collected in the Atacama Desertshowed a green colonization zone under the surface (white arrowin Fig. 1A), indicating the presence of photosynthetic microor-ganisms (7). Microcomputed tomography (μ-CT) images(Fig. 1B) uncovered microbial colonies inside the rock. In ad-dition, the CT scans revealed the pores present within the rockmatrix, and the microorganisms colonizing them, as previouslyreported (6–8). Further observations provided by scanningelectron microscopy (SEM) (Fig. 1 C and D) showed that themicroorganisms had a preferable attachment to specific crystalfacets of gypsum. Raman spectroscopy and mapping (SI Ap-pendix, Fig. S2) and SEM micrographs (SI Appendix, Fig. S3)confirmed that the microbial cells assembled primarily on the{011} planes of gypsum. It is likely that these specific planeshave a rougher surface (23), which enables stronger adhesion,but may also facilitate accelerated water access (i.e., via en-hanced dissolution kinetics) (24, 25). Cyanobacteria-like cellswere identified from morphological features in the microbialcolonies (Fig. 1D). As a summary of our observations of thegypsum rocks, a schematic is presented (Fig. 1E) of the micro-organisms colonizing rocks under xeric stresses (1).To further investigate the microbe−substrate interface, we
applied a combination of elemental and structural analyses to
the colonized gypsum rocks. Specifically, elemental informationfrom the rock was obtained using energy-dispersive X-ray spec-troscopy (EDS) and mapping. In addition to the main elementsin gypsum (S, Ca, O, and C), Si, Al, Mg, Na, and Fe were alsofound (Fig. 2 A–C), most likely from sepiolite, a clay mineralpreviously found in gypsum from the Atacama Desert (SI Ap-pendix, Fig. S4) (6, 8). Structural information, specifically, thephases existing in the gypsum rocks, were identified using X-raydiffraction (XRD) in areas with and without the microorganisms(Fig. 2D). Interestingly, anhydrite was observed in areas popu-lated with microorganisms, while substrate areas without mi-croorganisms consisted only of gypsum. Fourier transforminfrared spectroscopy (FTIR) maps were acquired in the areaswith microorganisms to further verify the existence of anhydritephase (21, 26). The resulting spectrum shows a reduction in theintensity of peaks representative of water of crystallization(Fig. 2E, highlighted in orange), suggesting a transformation toanhydrite in that region. FTIR mapping (Fig. 2 F and G) furthervalidated the existence of an anhydrite phase around the gypsumphase. These results indicate that the microorganisms are likelyresponsible for the transformation of gypsum to the anhydritephase. In previous studies, it has been shown that gypsum cantransform to anhydrite by losing its water of crystallization whenannealing at 440 K (27). Thus, it is plausible that microorganismscan also drive this transformation by extracting the water theyrequire for survival. To test this hypothesis, culture experimentswere performed in a laboratory under controlled conditions.
Gypsum as a Source of Water for MicroorganismsGypsum rock samples collected in the Atacama Desert wereused as substrate in culture experiments with a cyanobacteriumstrain, previously isolated from similar samples (Fig. 3A). Gyp-sum coupons (0.5 × 0.8 × 0.5 mm average size pieces of gypsumrocks) were subjected to two different culture conditions: 1)cyanobacteria in “dry conditions” (defined as only adding theinoculum to the substrate and leaving it to dry during the in-cubation period) and 2) cyanobacteria in “wet conditions” (de-fined as adding culture medium to the substrate during theincubation period, in addition to the inoculum). At a 30-d in-cubation period, cells on, and within, the gypsum coupons had abright green color, indicating the presence of photosyntheticpigments (Fig. 3B). The presence and distribution of the cya-nobacteria within the substrate were further validated by thecoexistence of carbon and nitrogen, by EDS mapping (Fig. 3C)and SEM imaging (Fig. 3D). All of the gypsum coupons
Fig. 1. Microorganisms live within gypsum rocks from the Atacama Desert. (A) Photo of gypsum rock samples. The green color indicated with a white arrowshows the area colonized by microorganisms. (B) μ-CT images of gypsum rocks, highlighting the microorganisms living within. The yellow and red colorsrepresent microorganism colonies inside the rock. (C and D) SEM images of gypsum. The extracellular matrix surrounding cyanobacteria cells in gypsumsamples is indicated in green in D. (E) Diagram of the microbe colonization and their location in the gypsum rock. UV, ultraviolet.
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harbored cyanobacteria cells; however, due to the difference inthe culture conditions, the final composition of the gypsumsubstrate was different. XRD reveals that anhydrite was presentin gypsum coupons cultured in “dry conditions,” but was not
found in gypsum without microorganisms (negative control) orthose cultured under hydrated conditions (i.e., in a liquid me-dium; Fig. 3E). This suggests that the “dry conditions” promotedthe extraction of water by cyanobacteria from the gypsum rock,
Fig. 2. Structure and chemistry of gypsum rocks sample from the Atacama Desert. (A) Optical microscopy of a thin cross-section of the gypsum sample withblack impurities within the rocky matrix. (B) Energy-dispersive X-ray (EDX) mapping on gypsum rock samples. The mapping area is indicated with red box in A.The white area is gypsum, while the black area shows Si, Al, Mg, O, and Fe. (C) Average spectrum (counts per second [cps] versus energy) of the EDX mappingin B. (D) XRD of gypsum samples in the areas with and without microbes. Anhydrite phase is observed in areas with microbes. (E) FTIR mapping and spectrumsof area with microbes in gypsum rocks. The highlighted area, also shown in the Inset, from 3,000 cm−1 to 3,800 cm−1 indicates the presence of water of crystallizationpeaks in the gypsum crystals. (F) Optical microscopy image shows the mapping area. (Scale bar, 100 μm.) (G) FTIR map of peak intensity at 3,400 cm−1, indicating thewater of crystallization in the gypsum crystals. Blue area represents the anhydrite phase, while green and yellow color indicate the existence of gypsum.
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which leads to its transformation to anhydrite. These resultscorroborate our observations of gypsum rocks collected from theAtacama Desert. More importantly, we found that the XRDpeak intensity of the anhydrite phase was greater in couponscultured with a higher number of cyanobacteria cells, furthervalidating our hypothesis on the role of microorganisms in thephase transformations observed in gypsum.SEM analysis of the cultured gypsum also showed the pres-
ence of an extracellular material forming a biofilm-like matrixthat covered the cyanobacteria (Fig. 3D and SI Appendix, Fig.S5) (28). FTIR analysis of gypsum coupons cultured with highconcentrations of cyanobacteria showed significantly more in-tense absorption bands for C=O and O−H, indicating the exis-tence of higher concentrations of organic material. Here, it islikely that this organic is rich in carboxylic acid moieties (Fig. 3F)
(26, 29, 30). The presence of organic acids in the biofilm sur-rounding the cyanobacteria cells was also confirmed by Ramanspectroscopy (Fig. 3G) (31–33). In addition, subsequentRaman maps highlight the phase transformation from gypsumto anhydrite (Fig. 3H, pure gypsum and Fig. 3I, a mixture ofgypsum and anhydrite). Based on these results, it is likely thatthe organic acids in the biofilm reacted and etched the gypsumrock, releasing water in its lattice to the cyanobacteria. As thebacteria grow, they produce more organic acid and thus extractadditional water that induces further transformation of gyp-sum. Similarly, bacterial biofilms on tooth surfaces have alsobeen verified to contain acids (i.e., lactic acid) that could leadto the dissolution of calcium phosphate and the decay of toothenamel (34, 35).
Fig. 3. Cyanobacteria culture on gypsum samples. (A) Schematic of cyanobacteria cultured in dry and liquid media. (B) Optical microscopy image of gypsumsamples showing colony of cyanobacteria (green color) on the gypsum after culture experiments. (C) EDS mapping of cyanobacteria cultured on the gypsum.(D) SEM images of gypsum sample after culture, showing a porous structure and attachment of cyanobacteria (green color) on the surface. Biofilm is foundsurrounding the cyanobacteria. (E) XRD of gypsum rock control (black curve; i.e., not exposed to microbes), samples cultured in low concentration (LC) andhigh concentration (HC) of cyanobacteria in both dry and liquid medium environments. Diffraction peaks labeled with black squares represent the anhydritephase, while those labeled with stars are from gypsum. (F) FTIR of samples in the control group and the cyanobacteria cultures at low and high concen-trations. Specific absorption bands, representing organic acids, are found on the surfaces of the gypsum samples with cyanobacteria cultures. (G) Ramanspectroscopy of gypsum samples cultured with a high concentration of cyanobacteria. Both gypsum and anhydrite are detected on the sample surface. Red and bluespectra represent two different areas indicated in I. Absorption from water in gypsum is marked with a yellow band. (H and I) Mapping of gypsum and anhydritephases from Raman spectroscopy. (H) Optical micrograph shows the mapping area (black box) used in I. (I) Gypsum (red) and anhydrite (blue) phase map.
10684 | www.pnas.org/cgi/doi/10.1073/pnas.2001613117 Huang et al.
Mechanisms of Gypsum−Anhydrite Phase TransformationIn order to better understand the transformation of gypsum toanhydrite by these microorganisms, SEM and transmissionelectron microscopy (TEM) micrographs of both gypsum andanhydrite crystals at different stages of transformation were ac-quired. Based on our observations, we describe the gypsum–
anhydrite phase transformation in four sequential stages (Fig. 4).Initially, microorganisms attach and form a biofilm onto {011}planes of gypsum particles (Fig. 4A and SI Appendix, Fig. S1).Prior to any interaction with microbes, the gypsum particles aresingle crystalline (as observed by TEM in Fig. 4B). The biofilmthat is coating the gypsum (Fig. 4 A and C and SI Appendix, Fig.S4) contains organic acids (19) that induce mineral dissolution,enabling the extraction of water that can be acquired by themicroorganisms. Observation of the surface of the gypsumsamples reveals a porous structure decorated with biofilmbridges, which is suggestive of the driving force behind mineraldissolution (Fig. 4C). High-resolution TEM (HRTEM) imagingand selected area electron diffraction (SAED) (Fig. 4 D and H,Inset, respectively) show anhydrite nanocrystals precipitatedrandomly near the surface of the dissolving gypsum, which sug-gests that a gypsum−anhydrite phase transformation is occur-ring. The phase transformation of gypsum to anhydrite has twoprocesses: dissolution of gypsum and precipitation of anhydrite,which can be described as
CaSO4 · nH2O↔Ca2+(aq) + SO2−4(aq) + nH2O,
where n is the hydration number (19, 36, 37). Thus, the phasetransformation is determined by the solubility of the differentphases in this specific environment, and anhydrite has beenproved to be a more stable phase and has lower solubility (19,20). The acidic environment created by the microorganisms aswell as the extraction of water can increase and facilitate thephase transformation. As additional anhydrite is formed, these“primary” nanocrystals attach, via short-range alignment, to formhierarchically assembled mesocrystals (Fig. 4 E and F). Misalign-ment of crystal planes from two adjacent nanocrystals formedduring an oriented attachment process is observed (SI Appendix,Fig. S6). Additional assembly (Fig. 4 G and H) of these primaryparticles yields larger anhydrite particles. A long-range align-ment of these nanocrystals along the [002] direction is observed(Fig. 4H), with the interfaces of these aligned primary particlesclearly present (see HRTEM micrograph in Fig. 4 H, Inset).A schematic of the cyanobacteria-induced gypsum−anhydrite
phase transformation mechanism is illustrated in Fig. 4I. Mi-croorganisms attached preferentially to the {011} planes in theoriginal gypsum samples found in the Atacama Desert (Fig. 1Cand SI Appendix, Fig. S5). This is also observed in the controlledcyanobacteria culture experiments (Fig. 4A). Examination of thecrystal structure of gypsum (SI Appendix, Fig. S7) shows that thewater within the mineral was exposed at the {011} planes, but isshielded by {010} planes (38), which may explain the preferentialcolonization of these organisms. Upon losing the water of crys-tallization, the monoclinic gypsum crystals become unstable andtransform to orthorhombic anhydrite crystals. The relatively in-soluble anhydrite (i.e., under acidic conditions) subsequentlyprecipitates as anhydrite nanocrystals near the surfaces of gyp-sum (20). As this phase transformation proceeds, the anhydritenanocrystals appear to align and attach to each other in an or-dered manner forming mesocrystals, suggesting that additionalgrowth occurs via a nonclassical pathway (39, 40). This growthmechanism is different from classical crystal growth pathways,which typically occur via monomer-by-monomer addition (40).This oriented attachment of primary particles provides a meansto reduce the free energy of the system without Ostwald ripen-ing, yielding larger crystals. This process has also been observed
in gypsum crystal growth mechanisms in synthetic environments(41). The surfaces of the final anhydrite crystals are rough, high-lighted by numerous interfaces from the nanocrystals (42, 43).
ConclusionsEndolithic microorganisms in the Atacama Desert have adaptedto their extremely dry environment by using their rocky substrate,such as gypsum, not only to protect themselves from extremesolar irradiance but also as a source of water. Microorganisms,such as cyanobacteria, that inhabit these rocks extract water in-corporated within the gypsum lattice (water of crystallization),resulting in the concurrent phase transformation to anhydrite.Organic acids were found in the biofilms surrounding these mi-croorganisms. Subsequent etching occurred on high-energycrystal planes of gypsum, releasing water to the microorgan-isms. Experiments under controlled conditions with cyanobac-teria cultures grown on native gypsum rock samples, alsocollected in the Atacama Desert, validated the aforementionedobservations from the geological environment. Specifically, cul-turing cyanobacteria on the gypsum rocks in a dry environmentresulted in a phase transformation from gypsum to anhydrite,with the extent of phase transformation directly correlated to thenumber of cells in the culture. No phase transformation wasobserved under hydrated culture conditions, indicating that wa-ter extraction from rock only occurs in environments wherewater is scarce. Analysis of this phase transformation revealed aspecific pathway that involves the dissolution of gypsum phase byorganic acids in the microbial biofilms with subsequent pre-cipitation of anhydrite nanocrystals, which then grow by particleattachment. The results from the current study not only provideinsight into specific interactions between microorganisms and min-erals but may also offer potential strategies for water storage tech-nologies in extreme environments, including extraterrestrial habitats.
Materials and MethodsGypsum Rocks Collection. Colonized gypsum rocks were collected in theAtacama Desert, Chile (GPS coordinates: 20°43’S, 069°58’W; 944 m above sealevel) in March 2018. Samples were stored in sterile Whirlpack bags in a darkand at room temperature (∼25 °C) before further processing.
Materials Characterization.SEM and EDS. Gypsum samples collected from the Atacama Desert werethawed in air for 24 h before SEM/EDS characterization. Gypsum rocks werefractured with a chisel and then sputter coated with Pt/Pd. Fracture surfaceswere then imaged using a field emission SEM (MIRA3 GMU; TESCAN) op-erated at 20 kV. For EDS analysis, gypsum samples were first embedded inepoxy (Epofix Cold-Setting Embedding Resin; Electron Microscopy Sciences),polished flat, and evaluated using an SEM (MIRA3 GMU; TESCAN) operatedat 20 kV and a Quantax 400 EDS system equipped with dual xFlash 6 SSDdetectors (Bruker).Powder XRD. Areas with and without green colonies in the gypsum rocks wereisolated and ground into fine powders. An X-ray diffractometer (Empyrean;PANalytical) with Cu-Kα radiation with a generator voltage of 45 kV andtube current of 40 mA was used. The scan range (2θ) was from 10° to 70°.FTIR. Gypsum rock samples were embedded in epoxy. An ultramicrotome(RMC MT-X; Boeckeler Instruments) was used to provide smooth samplesurfaces for FTIR mapping. A 70 × 70 μm map was created using an FTIRspectrometer (Cary 600 series; Agilent Technologies) with an attenuatedtotal reflection germanium crystal.Raman spectroscopy.Gypsum coupons, after culturing with a high-concentrationcyanobacteria, were examined by Raman spectroscopy performed with aWITec confocal Raman microscope fitted with a thermoelectrically cooledcharged-coupled device camera and a broadband UHTS300 spectrometer inthe visible to near-infrared range, coupled with a MIRA3-TESCAN SEM. Thespectra were collected using a Zeiss 100× objective in the SEM chamber undervacuumwith a 532-nm laser. A 40 × 40 μmmap was obtained on the surface ofthe gypsum coupon.TEM. Embedded gypsum samples sectioned with an ultramicrotome (RMCMT-X; Boeckeler Instruments) to get electron transparent sections (∼70 nm).A Field Electron and Ion Company (FEI) Tecnai12 (operated at 120 KV) and an
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FEI Titan Themis 300 (operated at 300 KV; Thermo Fisher Scientific) were usedto obtain bright-field TEM and HRTEM images, respectively.
Cyanobacteria Isolation, Culture, and Analysis. A cyanobacterium isolate wasobtained by incubating ground colonized gypsum collected in the AtacamaDesert (6) in BG11 liquid medium (44, 45) for 5 wk at 25 °C, under 24 μmolphotons·m−2·s−1 of white light. Colonies were isolated from 1% agar (wt/vol)BG11 medium, and the isolate G-MTQ-3P2 was identified as Chroococci-diopsis sp. using 16S ribosomal RNA gene sequencing.
Gypsum coupons (0.5 mm × 0.8 mm × ∼0.5 mm thick) were prepared usinga diamond saw. For culture experiments, coupons were sterilized by auto-claving (20 min at 121 °C) and placed in 96 well plates. One hundred mi-croliters of cyanobacteria culture (isolate G-MTQ-3P2), grown in BG11medium, were used to inoculate each gypsum coupon at two cell densities,
105 cells/mL or 108 cells/mL. Controls were inoculated in a sterile BG11 me-dium; 96-well plates were incubated at 25 °C under 24 μmol photons·m−2·s−1
of white light or in the dark for 30 d, either under “wet” or “dry” condi-tions. Under “wet conditions,” BG11 medium was added periodically tokeep a thin liquid layer on top of each coupon. Under “dry conditions,” noadditional medium was added during the incubation period. One samplefrom each condition was ground into a fine powder for XRD (Empyrean;PANalytical). SEM, TEM, and FTIR characterization was performed on theremaining samples, based on the aforementioned methods.
Data Availability.All data that support the findings of this study are self-inclusive.
ACKNOWLEDGMENTS. We thank Dr. Krassimir Bozhilov at University ofCalifornia, Riverside for help with TEM analysis. This work was supported by
Fig. 4. Mechanisms of gypsum−anhydrite phase transformation. The process is described in four stages. Stage I: microorganisms attach on the gypsumcrystals and form a biofilm. (A and B) SEM and TEM images highlight the gypsum crystals. Confirmation of single crystalline gypsum provided through TEMand SAED (Inset) is shown in B. Stage II: (C and D) gypsum dissolution and water extraction with subsequent anhydrite nanocrystal precipitation. (C) A porousstructure is observed at the periphery of the gypsum particles, indicating their dissolution. (D) Anhydrite nucleation on the surface of gypsum crystals. SAEDanalysis (Inset) provides evidence for the random distribution of the anhydrite nanocrystals. Stage III: (E and F) Anhydrite crystal growth. (E) SEM image showslarge, faceted, anhydrite particles on the surface of gypsum. (F) Bright-field TEM demonstrates the short-range alignment of anhydrite nanocrystals, sug-gesting particle attachment. The SAED pattern (Inset) indicates alignment of the nanocrystals during the attachment process. Stage IV: (G and H) Completionof the gypsum-anhydrite phase transformation. (G) SEM image of anhydrite particles, highlighting surface faceting. (H) Bright-field TEM image and SAEDindicate a preferential alignment along the [002] direction. The blue box (Inset) shows the interfaces between nanocrystals observed through HRTEM, in-dicated with yellow circles. (Scale bar, 5 nm.) (I) Summary and schematic of microorganism induced gypsum–anhydrite phase transformation. Microorganismsattach and form biofilms on the {011} planes of gypsum crystals; gypsum dissolves, and water extraction occurs. Based on the crystal structure of gypsum, thewater of crystallization layer is exposed to the {011} planes, but not to the {010} planes. As the single crystalline gypsum dissolves and loses water of crys-tallization, 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.
10686 | www.pnas.org/cgi/doi/10.1073/pnas.2001613117 Huang et al.
funding from NASA (Grant NNX15AP18G) to J.D. and the Army ResearchOffice (ARO) (Grant W911NF-18-1-0253) to D.K. and J.D. Also, D.K.
acknowledges funding from ARO (Grants W911NF-16-1-0208 and W911NF-17-1-0152).
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