DNA Takes Over on the Control of the Morphology of the Composite Self-Organized Structures of Barium and Calcium Silica–Carbonate Biomorphs, Implications for Prebiotic Chemistry on Earth

: The origin of life is associated with the existing environmental factors of the Precambrian Era of the Earth. The minerals rich in sodium silicates, in aluminum and in other chemical elements, such as kaolinite, were among the factors present at that time. Kaolinite is an abundant mineral on our planet, which indicates that it possibly had an essential role in the origin of the first blocks that constructed life on Earth. Evidence of this is the cherts, which are rocks with a high concentration of silica that retain the vestiges of the most ancient life on our planet. There are also inorganic structures called biomorphs that are like the cherts of the Precambrian, which take on a morphology and crystalline structure depending on the chemical molecules that make up the reaction mixture. To evaluate the interaction of kaolinite with DNA, the objective of this work is to synthesize biomorphs in the presence of kaolinite and genomic DNA that comes from a prokaryote and a eukaryote microorganism. Our results show that the difference between the prokaryote DNA and the eukaryote DNA favors the morphology and the crystalline phase of the calcium silica–carbonate biomorphs, while in the case of the barium silica–carbonate biomorphs, the environmental factors participate directly in the morphology but not in the crystalline phase. Results show that when a mineral such as kaolinite is present in genomic DNA, it is precisely the DNA that controls both the morphology and the crystalline phase as well as the chemical composition of the structure. This fact is relevant as it shows that, independently of the morphology or the of size of the organism, it is the genomic DNA that controls all the chemical elements toward the most stable structure, therefore allowing the perpetuation, conservation and maintenance of life on our planet (since the origin of the genomic DNA in the Precambrian Era to the present day).


Introduction
Traveling through the history of life is a never-ending journey that can lead to many different paths.The very origin of Earth is frequently associated with the presence of microorganisms as the only existing form of life [1].Currently, it is postulated that before the existence of microorganisms, there must have been a primitive organism with a primary metabolism [2].However, various physical, chemical and atmospheric conditions at that time and space must have participated in the formation of that pre-cell [3,4].Within these elements, banks of sand formed by minerals and accumulations of water were found.These sandbanks were rich mainly in sodium silicates, calcium, aluminum and in potassium which react with the carbon dioxide (CO 2 ) present in water, favoring the lowering of CO 2 considerably down to levels far below to those found at present [5].Kaolin is found in the group of minerals that contain high amounts of silicates.These include kaolinite, dickite, nacrite, halloysite, antigorite, chamosite, chrysotile and cronstedtite [6,7].Kaolinite is of special geologic relevance because can be found abundantly in soils and hydrothermally eroded rocks [8,9].The geologic origin of kaolinite has been inferred through studies that include geomorphologic context, the analysis of trace elements and the associated mineralogy.Proof of hydrothermal context, or at least of the origin of acid-base on Earth, is when kaolinite is found associated with jarosite, alunite and opaline silica [10,11].Similar phases to kaolinite have also been observed on the surface of Mars [12][13][14][15][16].The fact that kaolinite is an abundant mineral on our planet that has also been found in similar phases on Mars indicates that this mineral possibly played an essential role in the origin of the first building blocks of life on Earth [17][18][19].Evidence of this is the cherts, which are rocks rich in silica that preserve remnants of more ancient life on our planet [20][21][22][23].Although some groups are opposed to accepting that Precambrian cherts are evidence of the first forms of life in the world [24][25][26][27][28][29], no other evidence of the origin of life has either been identified or reported.There are also inorganic structures called biomorphs that are morphologically and chemically similar to the microfossils identified in the Precambrian cherts, presenting epitaxial nucleation and growth [23,[30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47].Due to the characteristics shared between the cherts and the biomorphs, our work group has proposed that biomorphs are the remains of the cherts, though not as a fossil hallmark, and that they were the first inorganic framework where the formed protobiomolecules were concentrated, preserved, aligned and duplicated to give rise to the pre-cell [35,48].We have previously evaluated the effect of various clays, ions and biomolecules independently, in dissolution in the synthesis of biomorphs [34,35,37,48], as well as the implication of various ions and biomolecules together with some clay in dissolution [3,4].Recently, it has been shown that biomorphs, if obtained on a mineral, interact with the molecules of the mineral and the mixture of the synthesis of the biomorph [49].Nevertheless, what has not been evaluated during the synthesis of the biomorphs is the way in which minerals rich in aluminum silicates such as kaolinite interact with a unique biomolecule such as DNA.Knowing this interaction will favor the comprehension of the way in which, being in dissolution in this first stage of the Earth, the mineral and the biomolecule could interact to give rise to the pre-cell formed by an inorganic and an organic part.For the purpose of evaluating the interaction between kaolinite and DNA in the formation of the pre-cell, the objective of this work is to synthetize biomorphs in the presence of kaolinite and DNA found both in a prokaryote microorganism and in a eukaryote microorganism.Our results showed that the difference between the prokaryotic genomic DNA and the eukaryotic DNA favored the morphology and the crystalline phase of the calcium silica-carbonate biomorphs.However, in the case of the barium silica-carbonate biomorphs, the environmental factors influenced the morphology directly but not the crystalline phase.Results show that when a mineral such as kaolinite is found in the presence of genomic DNA, it is the DNA, not the mineral, that controls the morphology, the crystalline phase and the chemical composition of the structure.This is a relevant fact as it shows that, independently of the morphology and of the size of the organism, it is the genomic DNA that controls all the chemical elements toward the most stable structure, allowing the perpetuation, conservation and maintenance of life on our planet (since the origin of the genomic DNA in the Precambrian Era until our current time).

Extraction of DNA
The microorganisms Escherichia coli and Candida albicans were used for the extraction of DNA.Below we describe the methodology that we carried out for obtaining the DNA that was unique to each species as the first was a bacterium and the second a yeast.

E. coli
The E. coli strain JM109 was grown in 10 mL of liquid Luria-Bertani medium for 16 h at 37 • C with orbital stirring at 180× g.The cellular culture was centrifuged at 3000× g for 10 min to precipitate the cells and the supernatant was discarded.The protocol previously described by the work group [35] was followed, according to the One-4-all genomic DNA kit from Bio Basic (Markham, Ontario, Canada), which is briefly described below.First, 300 µL of the lysis solution was added to the cellular pellet (20 mM Tris-Cl, pH 8.0, 2 mM sodium EDTA, 1.2% Triton X-100, lysozyme to 20 mg/mL), 180 µL of ACL buffer and 20 µL of proteinase K.The mixture was incubated for 60 min at 56 • C. The mixture was then centrifuged for 10 min at 12,000× g, and the supernatant was recovered.Then, 200 µL of 96% RNAse-and DNAse-free ethanol was added.The mixture was transferred to an EZ-10 column provided by the supplier where, by adding various solutions, the elimination of RNA and proteins was carried out, to allow for the DNA to precipitate.It was centrifuged at 14,000× g for 1 min and the ethanol was decanted carefully, allowing the total evaporation of ethanol for 15 min at room temperature.The DNA was hydrated with 50 µL of water free from nucleases for 1 h at 65 • C. The DNA was stored at −20 • C for later use in the synthesis of the biomorphs.

C. albicans
The original culture of C. albicans for obtaining DNA was carried out in 10 mL of liquid YPD medium (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose), which was incubated all night (approximately 16 h) at 28 • C with constant 220× g stirring.The protocol that was followed has been reported previously [35] and is described succinctly below.The culture of C. albicans was centrifuged at 3000× g for 10 min.The cellular pellet was lysed using four cycles of freezing-defrosting using liquid nitrogen.After the cellular break-up, 600 µL of a urea solution (urea 7 M, NaCl 0.35 M, Tris-HCl 50 mM pH 8.0, EDTA 20 mM, N-laurylsarcosine 1%) was mixed with the aid of a vortex for 2 min and was left to rest at room temperature for 30 min.At the end of this time, 600 µL of a solution of isoamyl phenol-chloroform-alcohol (25:24:1) was added and mixed by vortex for 2 to 10 min and centrifuged at 12,000× g for 10 min.The aqueous phase was recovered, and this step was repeated several times until the organic phase was no longer visible.Then, the DNA was precipitated by adding an equal volume of isopropanol to the supernatant from the previous step, centrifuging at 14,000× g for 10 min and discarding the supernatant.The pellet was resuspended in 500 µL of 70% cold ethanol, mixed in the vortex and centrifuged at 12,000× g for 10 min.The supernatant was discarded, and the pellet was left to dry on absorbent paper at room temperature.Finally, the DNA was hydrated with 50 µL of sterile water free of nucleases at 65 • C for 1 h.To eliminate RNA, 1 µL of RNAse (20 mg/mL) was added and incubated at 37 • C for 20 min.The genomic DNA obtained was used for the formation of the biomorphs.

Visualization of DNA in Agarose Gel
The DNAs obtained from E. coli and C. albicans were visualized in an 0.8% denatured agarose gel [50].The 1 kb molecular weight marker was used (New England Biolabs; Ipswich, MA, USA).The migration of DNA through the agarose gel was carried out at 80 volts for 30-45 min.Once the electrophoresis had concluded, the gel was stained with 0.1% ethyl bromide and the bands were visualized with UV light (Gel Doc XR System, version 2.0.Bio-Rad; Hercules, CA, USA).

Spectrophotometer Analysis
The concentration of the DNAs was estimated using the NanoDrop 2000 spectrophotometer with a 260 nm wavelength (Thermo Fisher Scientific, Inc., Waltham, MA, USA).Purity was determined with the A 260 /A 280 and A 260 /A 230 relationships [51,52].

Biomorph Synthesis
The calcium or barium biomorph synthesis was carried out using the gas diffusion method adapted by our work group [37,42,53] where glass slides for all conditions were used: 5 mm long, 5 mm wide and 1 mm thick.The glass slide was placed in a crystallizing cell with a final volume of 200 µL.Synthesis was carried out in two different atmospheric conditions: one emulating the Precambrian Era (5% CO 2 , 50 • C) and the second at current temperatures and CO 2 (STP).All the reagents used were from Sigma-Aldrich (St. Louis, MO, USA).The formation of the biomorphs was allowed for 24 h.The experiments were carried out in triplicate.
The synthesis mixtures were carried out as follows:

Control Biomorphs
The mixture for the synthesis of the control biomorphs contained 1000 ppm sodium metasilicate, 20 mM calcium or barium chloride, and the pH was adjusted to 11.0 with sodium hydroxide (NaOH).

Biomorphs with Kaolinite and Genomic DNA
The mixture for the synthesis of the calcium or barium silica-carbonate biomorphs with DNA and kaolinite contained 2 ng of genomic DNA of E. coli, or of C. albicans, 4200 ppm of kaolinite, 1000 ppm sodium metasilicate, calcium or barium chloride 20 mM.The pH of the mixture was adjusted to 11.0 with NAOH.

Characterization of the Biomorphs
The morphology of the biomorphs was visualized by scanning electron microscope.The chemical composition and the crystalline structure of the biomorphs were determined by Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR).

Scanning Electron Microscopy (SEM)
The description of the synthesized biomorph morphology under the various conditions was carried out using a TESCAN scanning electron microscope (Brno, Kohoutovice, Czech Republic) model VEGA3 SB, with a secondary electron detector (SE) from 10 to 20 kV in high-vacuum conditions (work distance of 10 mm).Previous to the SEM analyses a sputter coater system (version 3.0) was used to perform a gold coating of 30 s (West Chester, Pennsylvania, USA.SPI Module Sputter Coater and Vaccum Base with Pump 110 V 50/60 Hz).

Raman Spectroscopy
The chemical analysis and structural determination of the biomorphs were carried out according to the methodology described previously by the work group.The Raman spectra were obtained using a Raman WiTec Alpha300RA spectrophotometer coupled to a confocal microscopy system (WITec GmbH, Ulm, Germany) and 532 nm laser light excitation originated from a NY:YVO4 green laser.The incident laser beam with a power of 14.4 mW focused using 20×, 50× and 100× (Zeiss, Germany) objectives, 0.4, 0.75 and 0.9 NA, respectively.The Raman map was obtained using 0.03 s of integration time.Processing and analysis of the data were carried out using WITec Project Version 5.1 software [37].

Fourier Transform Infrared Spectroscopy (FTIR)
The characterization of the biomorphs was additionally carried out by means of Fourier transform infrared spectroscopy (FTIR).This technique was carried out in a Nicolet iS50R Thermo Scientific (Waltham, MA, USA) spectrometer, equipped with an attenuated total reflectance (ATR) diamond crystal accessory (Smart-iTX), as has been carried out previously by the work group [15][16][17][18][19][20][21][22].Spectra acquisitions were collected with 32 scans, 4 cm −1 of spectral resolution, in the range of 525 to 4000 cm −1 .The data processing and analysis were performed with the OriginPro version 2021 software.

The Difference between the Prokaryotic and the Eukaryotic DNA Favors the Morphology and Crystalline Phase of the Silica-Carbonates of Calcium
Once the calcium silica-carbonate biomorphs were obtained, whether in the presence of kaolinite or DNA, the morphology that they adopted was visualized by SEM.The control biomorphs obtained in current temperature and CO 2 atmospheric conditions (STP) presented polycrystalline aggregate morphology (Figure 1A).In the case of the biomorphs produced in the conditions that emulate the Precambrian, a polycrystalline arrangement was also found, as well as druse type spheres (Figure 1B).This type of morphology observed in the control biomorphs has been found when synthetizing biomorphs of this type in atmospheric conditions [48].The druse morphology was identified in the morphology of the calcium silica-carbonate biomorphs with E. coli DNA in STP conditions (Figure 1C) while the biomorphs synthesized with DNA from E. coli in the Precambrian conditions presented druse morphology (Figure 1D).In the presence of DNA from C. albicans in STP conditions, biomorphs with druse morphology were obtained, as well as with DNA from E. coli.This type of morphology observed in the control biomorphs has been found when synthetizing biomorphs of this type in atmospheric conditions [48].The druse morphology was identified in the morphology of the calcium silica-carbonate biomorphs with E. coli DNA in STP conditions (Figure 1C) while the biomorphs synthesized with DNA from E. coli in the Precambrian conditions presented druse morphology (Figure 1D).In the presence of DNA from C. albicans in STP conditions, biomorphs with druse morphology were obtained, as well as with DNA from E. coli.
In conditions that emulate the Precambrian, biomorphs with a long thin leaf shape were visualized (Figure 1F).The morphologies identified in the biomorphs in the presence of genomic DNA of E. coli or of C. albicans are found to be in accord with the morphology found with the genomic DNA of these microorganisms [37].These results also show the way in which the genomic DNA molecules direct the inorganic molecules to adopt morphologies that emulate flowers or leaves, as previously reported [37].However, they also show that regardless of the origin of the genomic DNA (whether from a prokaryote or eukaryote), biomorphs emulate morphologies of living organisms, as we know them today.Biomorphs whose synthesis was carried out in STP conditions in the presence of kaolinite and the DNA of E. coli presented a morphology characteristic to that of a flower (Figure 1G).Meanwhile, the biomorphs that were obtained with these two components, but under conditions that emulate those of the Precambrian, were found as aggregates with the morphology of small flowers (Figure 1H).However, when using the mixture for the synthesis of the C. albicans DNA with kaolinite, in STP conditions, we observed a polycrystalline arrangement emulating an aggregate of leaves (Figure 1I).With this same mixture for synthesis, but in conditions that emulated the Precambrian, we observed a morphology emulating thin long leaves (Figure 1J), very similar to those observed with this DNA but without kaolinite (Figure 1F).The biomorph morphologies obtained both with DNA and with kaolinite and genomic DNA produced morphologies that emulate organisms such as leaves and flowers.This shows that the pre-cell or pioneer organism indeed must have been formed with one part mineral and a second part organic, just as Wächtershäuser proposed (2006) [2].This union that took place from the first era of Earth, part mineral, with organic molecules, was what allowed the formation of the pioneer organism that gave rise to life.From then on, and up to the present, in the structure of living organisms, both parts co-exist, the mineral and the organic [54].
To find out the chemical composition as well as the crystalline phase of the synthesized calcium silica-carbonate biomorphs, these were analyzed with Raman spectroscopy and FTIR.For the control biomorph in STP conditions, Raman spectra identified bands at 157, 282, 712, 1086, 1750 cm −1 (Figure 2a(ii); Table 1), and by FTIR the bands were at 712.1, 871.1, 1399.9 cm −1 (Table 1).
The spectra of the biomorphs in both conditions of synthesis (STP and Precambrian), in the presence of E. coli DNA with kaolinite showed bands by Raman spectroscopy at 157, 282, 712, 1085, 1086 cm −1 (Figure 2e(ii),f(ii); Table 1).Bands found for these biomorphs by FTIR were at 544.0, 595.0, 626.0, 680.7, 797.5, 913.4,939.0, 1008.3,1031.0,1386.9, 1407.0,2980.7,3482.6,3619.6 and 3690.8 cm −1 (Table 1).These bands of the biomorphs synthesized with Earth 2024, 5 299 E. coli and kaolinite DNA also correspond to calcite.These data show that in the presence of genomic E. coli DNA with kaolinite, the polymorph obtained from the silica-carbonate biomorphs is calcite as the main component of the crystallites.It has been reported that calcite is the most frequent polymorph identified in the hard and support structures of organisms [55-61], being also the most thermodynamically stable polymorph, followed by aragonite and vaterite [62] (Carteret et al., 2009).Biomineralization is the chemical process that occurs during the synthesis of the biomorphs, through which the CaCO 3 polymorphs are obtained on the surface or the internal part of the biomorphs.This is a mechanism that continues to occur in organisms [54,[63][64][65][66][67].It has been reported that biomineralization occurs practically in all forms of life, from bacteria to superior organisms, reporting, therefore, the formation of more than 60 different biological minerals [68,69].When analyzing biomorphs with C. albicans genomic DNA by Raman spectroscopy and synthesized in both atmospheric conditions (STP and Precambrian), bands were identified at 111, 157, 282, 470, 711, 1086, 1480, 1760 and 2911 cm −1 (Figure 3c,d; Table 1).While by FTIR, bands were determined at 580, 711, 792.6, 871.20, 1073.1, 1387.4,1406.2 cm −1 (Table 1).These bands correspond to the calcite and aragonite polymorphs as the main components of the crystallites inserted in the silica-carbonate surface.
phologies that emulate flowers or leaves, as previously reported [37].However, they also show that regardless of the origin of the genomic DNA (whether from a prokaryote or eukaryote), biomorphs emulate morphologies of living organisms, as we know them today.Biomorphs whose synthesis was carried out in STP conditions in the presence of kaolinite and the DNA of E. coli presented a morphology characteristic to that of a flower (Figure 1G).Meanwhile, the biomorphs that were obtained with these two components, but under conditions that emulate those of the Precambrian, were found as aggregates with the morphology of small flowers (Figure 1H).However, when using the mixture for the synthesis of the C. albicans DNA with kaolinite, in STP conditions, we observed a polycrystalline arrangement emulating an aggregate of leaves (Figure 1I).With this same mixture for synthesis, but in conditions that emulated the Precambrian, we observed a morphology emulating thin long leaves (Figure 1J), very similar to those observed with this DNA but without kaolinite (Figure 1F).The biomorph morphologies obtained both with DNA and with kaolinite and genomic DNA produced morphologies that emulate organisms such as leaves and flowers.This shows that the pre-cell or pioneer organism indeed must have been formed with one part mineral and a second part organic, just as Wächtershäuser proposed (2006) [2].This union that took place from the first era of Earth, part mineral, with organic molecules, was what allowed the formation of the pioneer organism that gave rise to life.From then on, and up to the present, in the structure of living organisms, both parts co-exist, the mineral and the organic [54].
To find out the chemical composition as well as the crystalline phase of the synthesized calcium silica-carbonate biomorphs, these were analyzed with Raman spectroscopy and FTIR.For the control biomorph in STP conditions, Raman spectra identified bands at 157, 282, 712, 1086, 1750 cm −1 (Figure 2a(ii); Table 1), and by FTIR the bands were at 712.1, 871.1, 1399.9 cm −1 (Table 1).This is a mechanism that continues to occur in organisms [54,[63][64][65][66][67].It has been reported that biomineralization occurs practically in all forms of life, from bacteria to superior organisms, reporting, therefore, the formation of more than 60 different biological minerals [68,69].When analyzing biomorphs with C. albicans genomic DNA by Raman spectroscopy and synthesized in both atmospheric conditions (STP and Precambrian), bands were identified at 111, 157, 282, 470, 711, 1086, 1480, 1760 and 2911 cm −1 (Figure 3c,d 1).These bands correspond to the calcite and aragonite polymorphs as the main components of the crystallites inserted in the silica-carbonate surface.In the case of the Raman spectra of the biomorphs in the presence of DNA with kaolinite, both in the STP and Precambrian conditions, bands were shown at 112, 156, 281, 711, 1085, 1437, 1470, 1750 and 2980 cm⁻ 1 (Figure 3e,f; Table 1).By FTIR, bands were identified at 599.4, 596.0, 576.9, 626.1).Bands correspond to the calcite and aragonite polymorphs, as well as to the biomorphs obtained with C. albicans DNA.These results obtained both with the C. albicans genomic DNA as well as with kaolinite are interesting for various reasons.The first reason to consider is that in the biomorphs that were synthesized with E. coli genomic DNA, only the calcite polymorph was obtained, while with the C. albicans DNA, two polymorphs were obtained: calcite and aragonite.Whether the DNA came from a prokaryote or from a eukaryote, the DNA is capable of controlling the inorganic molecules present in the solution so that the biomorph adopts a morphology similar to that of a living organism.It seems that the main difference is at the molecular level, since the DNA that comes from a eukaryote, while capable of directing the synthesis In the case of the Raman spectra of the biomorphs in the presence of DNA with kaolinite, both in the STP and Precambrian conditions, bands were shown at 112, 156, 281, 711, 1085, 1437, 1470, 1750 and 2980 cm −1 (Figure 3e,f; Table 1).By FTIR, bands were identified at 599.4, 596.0, 576.9, 626.1).Bands correspond to the calcite and aragonite polymorphs, as well as to the biomorphs obtained with C. albicans DNA.These results obtained both with the C. albicans genomic DNA as well as with kaolinite are interesting for various reasons.The first reason to consider is that in the biomorphs that were synthesized with E. coli genomic DNA, only the calcite polymorph was obtained, while with the C. albicans DNA, two polymorphs were obtained: calcite and aragonite.Whether the DNA came from a prokaryote or from a eukaryote, the DNA is capable of controlling the inorganic molecules present in the solution so that the biomorph adopts a morphology similar to that of a living organism.It seems that the main difference is at the molecular level, since the DNA that comes from a eukaryote, while capable of directing the synthesis toward the most stable crystalline habit, also plays an important role in the chemical composition and crystalline structure.This is possibly due to the differences between the genomic DNA of prokaryotes and eukaryotes, among which are the prokaryote DNAs that are circular chromosomes, and the eukaryote DNAs are linear chromosomes confined within a nucleus [70][71][72][73][74][75][76].The prokaryote DNA is found in the cytoplasm, while the eukaryote DNA is packed into the nucleus of the cell [70][71][72][73][74][75][76].The eukaryote genes contain introns that code for a single protein, while the prokaryote genes lack introns and are organized in operons, which code for various proteins.Prokaryote cells have only one point of origin and replication takes place in two opposing directions at the same time in the cell cytoplasm.The eukaryote cells have multiple origins and use unidirectional replication within the cell's nucleus.The number of polymerases is different in these cell types, as is the amount of DNA, which is approximately twenty-five times greater in the eukaryote cells than in the prokaryotes [70][71][72][73][74][75][76].The second reason to consider is that C. albicans genomic DNA is what directed the synthesis of the biomorphs since the same polymorphs were also obtained in the presence of kaolinite (Figure 3, Table 1).
The fact of having obtained calcite and aragonite is a result that agrees with what is found in various organisms since it has been described that the two polymorphs of CaCO 3 are the most abundant crystalline structures in organisms, all the way from microorganisms to superior organisms [55- 60,77].There are organisms with structures formed both by calcite and aragonite.For example, the pearl oyster Pinctada fucata has an outer layer formed by calcite while the inner nacreous layer is formed by aragonite [78,79].Additionally, our results show that the C. albicans DNA is what directs the crystalline structure of the biomorphs, which is also in agreement with works that have shown that macromolecules extracted from the aragonitic shell layers of some mollusks induce the formation of aragonite in vitro.The same effect is observed with the macromolecules from shell made of calcite layers that induce the formation of one of the polymorphs only.Data show that these macromolecules are responsible for the precipitation of calcite or aragonite in vivo [77].In this way, the facts obtained here favor the hypothesis that the biomorphs might have been the first crystalline structures that isolated, protected and preserved the first biomolecules that were formed on Earth.Additionally, it is shown that the formation of the calcite and aragonite polymorphs must have occurred right from the first structures that originated life.From that moment until now, it seems that inorganic structures have preserved their crystalline structure.

Environmental Factors That Participate Directly in the Morphology of the BaCO 3 Biomorphs but Not in the Crystalline Phase
The barium carbonate biomorphs are also of special relevance because biomorphs adopt morphologies associated with typical forms of life even without the presence of biomolecules [30,37,38,[80][81][82]] and, at the same time, some of their morphologies are reminiscent of fossils, which biomorphs might have very well been, though they had not been termed as such, keeping them invisible [35].The morphology, chemical composition and crystalline structure of the BaCO 3 biomorphs were studied and, in the presence of E. coli or C. albicans genomic DNA and kaolinite, the synthesis of these biomorphs was carried out.
The control barium silica-carbonate biomorphs synthesized in STP atmospheric conditions showed the aggregate that emulates the morphology of a sphere (Figure 4A).For the control biomorphs obtained in the Precambrian atmospheric conditions, the aggregate that emulates the morphology of a flower with elongated petals was observed (Figure 4B).The barium silica-carbonate biomorphs that were treated with E. coli genomic DNA in STP conditions presented polycrystalline morphology with a section that emulated the flower of the plant Taraxacum officinale (Figure 4C).In the case of the biomorphs obtained with E. coli DNA but under the atmospheric conditions that emulate the Precambrian, the morphology observed was that of flowers with short petals and leaves (Figure 4D).For the biomorphs that were synthesized with C. albicans genom druse morphology was found (Figure 4E).The biomorphs that were sy albicans genomic DNA in STP conditions presented flower with leaf mo 4F).The biomorphs produced with DNA from E. coli with kaolinite pres morphology (Figure 4G) similar to the morphology found only in the E. 4C).The biomorphs synthesized with DNA from E. coli with kaolinit conditions presented leaves, flowers and stems (Figure 4H).The biomor tained with C. albicans genomic DNA and kaolinite in STP atmosphere p stem morphology (Figure 4I) while the biomorphs obtained in Precamb For the biomorphs that were synthesized with C. albicans genomic DNA, leaf and druse morphology was found (Figure 4E).The biomorphs that were synthesized with C. albicans genomic DNA in STP conditions presented flower with leaf morphology (Figure 4F).The biomorphs produced with DNA from E. coli with kaolinite presented polycrystal morphology (Figure 4G) similar to the morphology found only in the E. coli DNA (Figure 4C).The biomorphs synthesized with DNA from E. coli with kaolinite in Precambrian conditions presented leaves, flowers and stems (Figure 4H).The biomorphs that were obtained with C. albicans genomic DNA and kaolinite in STP atmosphere presented leaf and stem morphology (Figure 4I) while the biomorphs obtained in Precambrian atmosphere from C. albicans DNA and kaolinite showed flower, leaf and stem morphology (Figure 4J).This type of morphology that emulates flowers, stems and leaves in the presence of DNA has already been found in the presence of five types of DNA [37].These results, as happened with calcium silica-carbonate biomorphs, show that the genomic DNA is what directs the morphology, the chemical composition and the crystalline structure.In addition, these morphologies that emulate various organs of plants that were identified in the barium silica-carbonate biomorphs with genomic DNA only or with kaolinite suggest that the barium, by being a chemical element present in the form of barite in the Earth's crust [83] from the first epochs on Earth, formed part of some of the first organisms.In this way, barium from that time was possibly preserved to the present day in some organisms by being a part of some of the pioneer structures, as are some species of algae, invertebrates and fish from the Irish Sea [84].When determining the crystalline structure of the control barium silica-carbonate biomorphs obtained in STP conditions by Raman spectroscopy, bands were identified at 93, 138, 154, 221, 690, 1058 cm −1 (Figure 5a(i), Table 2), and by FTIR at 549.7, 585.1, 604.3, 692.6, 768.5, 855.5, 953.2, 1059.0,1416.9, 1577.7,2980 cm −1 (Figure 5a(v), Table 2).The bands identified correspond to the polymorph of BaCO 3 , commonly known as witherite [85,86].For the biomorphs synthesized in Precambrian conditions, the witherite polymorph was also identified both by Raman spectroscopy and by FTIR (Figure 5b(i,v), Table 2).

2024, 5, FOR PEER REVIEW
Earth 2024, 5, FOR PEER REVIEW 14 The barium silica-carbonate biomorphs that were synthesized in the presence of E. coli genomic DNA in both STP and Precambrian conditions showed the witherite band spectra shown by Raman spectroscopy and FTIR (Figure 5ci,cv,di,dv, Table 2).The witherite polymorph was also identified in the biomorphs obtained with E. coli DNA and kaolinite in both evaluated atmospheric conditions (Figure 5ei,ev,fi,fv, Table 2).In the case of barium silica-carbonate biomorphs synthesized with C. albicans genomic DNA, as well as with the biomorphs with C. albicans genomic DNA and kaolinite, in both STP and Precambrian conditions, the witherite phase was identified (Figure 6, Table 2).
These results show that in the case of the barium silica-carbonate biomorphs, the crystal form that is obtained is always witherite, whether they were synthesized with a biomolecule such as DNA or in the presence of a mineral, such as kaolinite.These data agree with those from other barium silica-carbonate biomorphs obtained previously in the presence of DNA or minerals [37,48].This is because witherite is the most stable crys-  The barium silica-carbonate biomorphs that were synthesized in the presence of E. coli genomic DNA in both STP and Precambrian conditions showed the witherite band spectra shown by Raman spectroscopy and FTIR (Figure 5c(i,v),d(i,v), Table 2).The witherite polymorph was also identified in the biomorphs obtained with E. coli DNA and kaolinite in both evaluated atmospheric conditions (Figure 5e(i,v),f(i,v), Table 2).
In the case of barium silica-carbonate biomorphs synthesized with C. albicans genomic DNA, as well as with the biomorphs with C. albicans genomic DNA and kaolinite, in both STP and Precambrian conditions, the witherite phase was identified (Figure 6, Table 2).
These results show that in the case of the barium silica-carbonate biomorphs, the crystal form that is obtained is always witherite, whether they were synthesized with a biomolecule such as DNA or in the presence of a mineral, such as kaolinite.These data agree with those from other barium silica-carbonate biomorphs obtained previously in the presence of DNA or minerals [37,48].This is because witherite is the most stable crystalline phase of BaCO 3 [87].Computer studies have proven that the orthorhombic phase with the space group Pmcn is the most stable at normal temperature and pressure [87] because when the pressures are modified, other phases of the BaCO 3 are obtained [88,89].It has also been observed that when witherite is synthesized in vitro, the morphology that is obtained is dependent on the environmental conditions, such as temperature and CO2 concentration [90][91][92][93].However, this does not affect the BaCO3 crystalline phase since the orthorhombic phase is obtained from witherite.These observations are found to correlate with what we observed in our results because, as in the synthesis of the barium silica-carbonate biomorphs, the environmental conditions used, as well as the biomolecules and the mineral such as kaolinite, define the morphology they adopt (Figure 4).This has been a constant crystalline behavior that has been observed in the BaCO3 biomorphs [34][35][36][37][38][39][40].These results altogether indicate on the one hand that the barium silica-carbonate biomorphs, as we have proposed, might be one of the first structures where the protomolecules that gave rise to the pioneer organism were protected, aligned, duplicated and preserved [34,35].On the other hand, it was a big advantage that BaCO3 could adopt one or the other morphology, depending on the environmental conditions.These conditions perhaps favored the existence of various morphologies of the pioneer organisms where, possibly, some organisms have conserved the morphology or mimicked it, as well as some part of the chemical composition [34,35].This is based on the fact that today there are organisms which contain chemical elements that differ from the majority of organisms; for example, chemical elements such as barium (Ba), strontium (Sr), arsenic (As), cadmium (Cd), wolframium (W), among others, have been identified [94][95][96].So, our results allow the inference that possibly some of the pioneer organisms that existed in the Precambrian Era may have been conserved up to the present, while other organisms evolved more complex and functional forms whereas others inevitably perished.It has also been observed that when witherite is synthesized in vitro, the morphology that is obtained is dependent on the environmental conditions, such as temperature and CO 2 concentration [90][91][92][93].However, this does not affect the BaCO 3 crystalline phase since the orthorhombic phase is obtained from witherite.These observations are found to correlate with what we observed in our results because, as in the synthesis of the barium silica-carbonate biomorphs, the environmental conditions used, as well as the biomolecules and the mineral such as kaolinite, define the morphology they adopt (Figure 4).This has been a constant crystalline behavior that has been observed in the BaCO 3 biomorphs [34][35][36][37][38][39][40].These results altogether indicate on the one hand that the barium silica-carbonate biomorphs, as we have proposed, might be one of the first structures where the proto-molecules that gave rise to the pioneer organism were protected, aligned, duplicated and preserved [34,35].On the other hand, it was a big advantage that BaCO 3 could adopt one or the other morphology, depending on the environmental conditions.These conditions perhaps favored the existence of various morphologies of the pioneer organisms where, possibly, some organisms have conserved the morphology or mimicked it, as well as some part of the chemical composition [34,35].This is based on the fact that today there are organisms which contain chemical elements that differ from the majority of organisms; for example, chemical elements such as barium (Ba), strontium (Sr), arsenic (As), cadmium (Cd), wolframium (W), among others, have been identified [94][95][96].So, our results allow the inference that possibly some of the pioneer organisms that existed in the Precambrian Era may have been conserved up to the present, while other organisms evolved more complex and functional forms whereas others inevitably perished.

Conclusions
Even when minerals are considered a fundamental part of the origin of life on Earth due to their contribution in the formation of the first inorganic structures in which biomolecules have been conserved and perpetuated, our results show that when a mineral such as kaolinite is found in the presence of genomic DNA, it is precisely the DNA, not the mineral, that directs the morphology, the crystalline stage and the chemical composition of the structure.This is a relevant fact as it shows how, independently of the morphology and of the size of the organism, it is the DNA that directs all the chemical elements towards the most stable structure, allowing for the perpetuation, conservation and maintenance of life on our planet (since the origin of the genomic DNA in the Precambrian Era to the present day).The fact that the morphology of barium silica-carbonate biomorphs changes depending on the environmental conditions whereas their crystalline structure remains stable possibly indicates that, in a first stage in the origin of the proto-cell, the barium silica-carbonate biomorphs may have formed part of the inorganic structure.As time went on and the chemical composition of the Earth changed, these inorganic structures might have replaced or might have incorporated other elements, such as calcium, so that calcium might have replaced barium in the structure of the organisms.This opens a wide range of possibilities regarding morphologies that have arisen in the organisms that we know today.

Figure 2 .
Figure 2. Identification by Raman spectroscopy of the polymorphs obtained in the calcium silicacarbonate biomorphs synthesized in STP and Precambrian conditions.(a,b) Control.(c,d) DNA from E. coli.(e,f) DNA from E. coli and kaolinite.(i) SEM micrograph; (ii) Raman spectra; (iii) Raman map.

Figure 3 .
Figure 3. Characterization by Raman spectroscopy of the polymorphs synthesized in the calcium silica-carbonate biomorphs, in STP and Precambrian conditions.(a,b) Control.(c,d) DNA from C. albicans.(e,f) DNA from C. albicans, kaolinite.

Figure 3 .
Figure 3. Characterization by Raman spectroscopy of the polymorphs synthesized in the calcium silica-carbonate biomorphs, in STP and Precambrian conditions.(a,b) Control.(c,d) DNA from C. albicans.(e,f) DNA from C. albicans, kaolinite.

Table 1 .
Identification through Raman and IR spectroscopy of the polymorphs of calcium silicacarbonate biomorphs.

Table 2 .
Identification through Raman spectroscopy and IR spectroscopy of the barium silicacarbonate biomorphs.