Life on Earth can grow on extraterrestrial organic carbon

The universe is a vast store of organic abiotic carbon that could potentially drive heterotrophy on habitable planets. Meteorites are one of the transporters of this carbon to planetary surfaces. Meteoritic material was accumulating on early Earth when life emerged and proliferated. Yet it is not known if this organic carbon from space was accessible to life. In this research, an anaerobic microbial community was grown with the CM2 carbonaceous chondrite Aguas Zarcas as the sole carbon, energy and nutrient source. Using a reversed 13C-stable isotope labelling experiment in combination with optical photothermal infrared (O-PTIR) spectroscopy of single cells, this paper demonstrates the direct transfer of carbon from meteorite into microbial biomass. This implies that meteoritic organics could have been used as a carbon source on early Earth and other habitable planets, and supports the potential for a heterotrophic metabolism in early living systems.


Microbial use of extraterrestrial organic carbon
The transfer of the isotopic signature from carbon within the meteorite into an anaerobic microbial community was observed.O-PTIR spectroscopy results showed that microorganisms can use extraterrestrial organic carbon as the carbon source and incorporate it into their proteins.Figure 1 shows the spectral region from 1500 to 1760 cm −1 , which illustrates vibrational modes associated with proteins (amide I and II regions, 1500-1700 cm −1 ) and lipids (1720-1800 cm −1 ).Peaks between 1580 and 1700 cm −1 are associated with the amide I vibrational mode from bacteria grown on 12 C-and 13 C-containing carbon sources.Bacteria that are 13 C-labelled in the starting culture (from growth on 13 C-labelled sodium acetate) exhibit an amide I peak at 1616 cm −1 , which corresponds to amide I vibrations where the carbonyl (C=O) is labelled with 13 C 22 .After transferring the labelled bacteria to microcosms with Aguas Zarcas, containing 1.77 + 0.03% carbon with a δ 13 C value of -0.91 ± 0.25 ‰, a shift was observed in the amide I peak position from 1616 ( 13 C) to 1657 cm −1 ( 12 C).The same 12 C carbonyl peak was observed in Control B, in which the 13 C-labelled bacteria from the starting culture were transferred to microcosms containing 12 C-sodium acetate as the sole carbon supply.When the starting culture was transferred to microcosms containing 13 C-labelled sodium acetate (Control A), there was no shift in the amide I peak, which remained corresponding to 13 C (1616 cm −1 ).In Control C, where the starting culture was transferred to microcosms containing no carbon source, the bacteria retained an amide I peak corresponding to 13 C (1616 cm −1 ).The principal component analysis (PCA) scores plot (Fig. 2) shows a clear separation between bacteria grown in 12 C-media (on Aguas Zarcas and Control B) and 13 C-media (starting culture, Control A and C) along the principal component 1 (PC1) axis.By evaluating the PCA loadings (Supplementary Fig. S1), it was concluded that this PC1 separation pattern is mainly based on the amide I peak.Fourier transform infrared (FT-IR) spectroscopy was used to analyse non-biological samples.No amide I peak was observed in the meteoritic material nor the minimal medium in Control A and B (Supplementary Fig. S2).Infrared spectroscopy requires drying the samples before analysis due to the strong contribution of water in the infrared spectrum, therefore spectral data from non-biological Control C showed no vibrational modes (data not shown) as this sample contained solely water.

Microbial growth on extraterrestrial organic carbon
Bacterial growth of the community was observed in microcosms with Aguas Zarcas, as well as the three controls, although growth was slower in the Aguas Zarcas-containing samples (Supplementary Table S1).
The bacterial community composition is shown in Fig. 3.The starting culture mainly contained Pseudomonadaceae (94-100%), as well as other families at low abundance (e.g.Caulobacteraceae, Carnobacteriaceae, Corynebacteriaceae and Moraxellaceae).After 14 days of growth on Aguas Zarcas, the community composition had shifted significantly.Next to the mainly abundant Pseudomonadaceae (62-91%), a variety of less abundant families was present (e.g.Bacillaceae, Beijerinckiaceae, Burkholderiaceae, Carnobacteriaceae, Clostridiales Family XI, Enterobacteriaceae and Micrococcaceae).Control A, B and C all mainly contained Pseudomonadaceae (98-100%), as well as a few other families at low abundance (e.g.Burkholderiaceae, Micrococcaceae, Sphingomonadaceae).The pH of the microcosms was measured before inoculation and 14 days after inoculation ("before" and "after" respectively in Supplementary Table S2).A significant difference between the groups was found (ANOVA: F-statistic (15,32) = 29.78;p-value < 0.001).A post-hoc Tukey test showed that the pH of the microcosms containing Aguas Zarcas -both at the start of the experiment and 14 days after incubation-was significantly higher than in all other conditions (p-values < 0.001).The pH of Control C significantly increased throughout the experiment (p-value = 0.0041), while the pH of none of the other samples significantly changed throughout the experiment.

Discussion
On early Earth, substantial organic material was being delivered to the surface of the planet in meteoritic material 1, 3,5 .Questions persist as to whether this organic material could have allowed a heterotrophic origin of life, and whether after the origin of life, this extraterrestrial material could be a source of organic molecules to power an early heterotrophic biosphere on Earth as well as on other young planets.This research has shown that bacteria can use organic carbon from the Aguas Zarcas carbonaceous chondrite for cell growth, investigated by combining reverse stable isotope labelling with infrared spectroscopy.This was done by focusing on the incorporation of 12 C or 13 C into bacterial biomass as evidenced by vibration shifts in the carbonyl (C=O) stretches in amide I from proteins.The results showed that the amide I peak of 13 C-labelled bacteria (from growth on 13 C-labelled sodium acetate as the sole carbon source) shifted the 13 C amide I peak at 1616 cm −1 in the starting culture to the 12 C amide I peak at 1657 cm −1 after growth on Aguas Zarcas, demonstrating the incorporation of carbon (from 12 C substrates) from the meteorite into the bacteria.The same shift was observed in control bacteria after growth on 12 C-sodium acetate (Control B).On the other hand, after growth on 13 C-labelled sodium acetate (Control A), and in the control in which the bacteria were transferred to conditions without a carbon source (Control C), the bacteria retained their 13 C labelling, showing a 13 C amide I peak at 1616 cm −1 .The latter result shows that the 13 C amide I peak is not lost during starvation, nor that there was any 12 C-carbon contamination in the experimental setup that could have caused the shift to 1657 cm −1 as observed in the Aguas Zarcas samples.These results show that the sole 12 C amide I peak in the Aguas Zarcas containing samples originates from the meteoritically-associated carbon as the carbon source.
Carbonaceous chondrites on early Earth could thus have provided microorganisms with a substrate, as well as biologically available organics such as nucleobases, amino acids and polycyclic aromatic hydrocarbons [9][10][11][12]23 . Thebiologically available organics in carbonaceous chondrites would locally provide concentrated solutions that could be used by early life 15 .An increase in biodiversity by the presence of extraterrestrial carbon and nutrient sources could have occurred, as well as favouring the growth of heterotrophs or iron and sulphur reducers 24 .
The microbial community composition after growth on Aguas Zarcas mainly consisted of Pseudomonadaceae, as in the starting culture.Some Pseudomonadaceae species are facultative anaerobes, for example the Pseudomonas and Rhizobacter genus, which can use nitrate as an alternative electron acceptor 25 .Carbonaceous chondrites including Aguas Zarcas contain water-soluble nitrate, albeit in low amounts 15,26,27 .Furthermore, Pseudomonas species can degrade organic recalcitrant compounds 25 , which are highly abundant in carbonaceous chondrites.Although microorganisms on early Earth were likely to be different from present-day microorganisms, similarities may exist.For example, Pseudomonas can anaerobically perform arginine deiminase, a pathway that is thought to be a primitive remnant from early life on Earth 25,28 .
Next to Pseudomonadaceae, Aguas Zarcas also supported the growth of bacterial families that were not observed in the starting culture or the controls (e.g.Bacillaceae, Beijerinckiaceae, Clostridiales Family XI, Enterobacteriaceae and Steroidobacteraceae).However, these families are assumed to also have been present in very low concentrations in the starting culture.Due to the low biomass in the microcosms, the sequence depth was 5,463 reads.This could mean that families with a low abundance in the inoculum could have been undetected using the 16S rRNA sequencing analysis.The rare biosphere is often underrepresented due to detection limitations, but microbial communities are known to be skewed towards a few dominant species and a high number of rare species [29][30][31] .Although some of these families could potentially be contaminants, the fact that these families were not observed in any of the controls limits the chance of laboratory contamination as the source of these families.In addition, no bacterial contamination was observed on Aguas Zarcas, which was tested by incubation of the meteoritic powder and by colony-forming unit (CFU) counting (data not shown).
Although microbial incorporation of carbon from Aguas Zarcas was shown, the microbial community grew slower than in the control experiments.A potential reason for this is that the pH of Aguas Zarcas containing microcosms was higher than the other microcosms, including the starting culture.Another explanation could be that Aguas Zarcas created different, and potentially less favourable, geochemical and ionic environmental conditions in the microcosms compared to the starting culture and the controls, for example by the leaching of water-soluble material or ions from the silicate matrix.
Although Aguas Zarcas is considered to be one of the most pristine carbonaceous chondrites, recently, an analysis of the meteorite fragments found terrestrial organic contaminant molecules 32 .This is usually the case for all material that makes contact with the Earth's atmosphere and surface.Although terrestrial organic contamination of Aguas Zarcas was observed 32 , the total number of detected organics by Tunney et al. was not as extensive as in other research 8,33 .For example, only two amino acids were discovered in one of the five specimens, while previously a wide range of amino acids was detected in Aguas Zarcas 34 .In addition, the organic content of Aguas Zarcas shows bulk similarities with the Murchison CM2 chondrite 7,25 , for example in the aliphatic amines and monocarboxylic acids 8,26 .
The contaminants observed by Tunney et al. were grouped into five categories: agricultural products, fuels, pesticides, pharmaceuticals and plastics 32 .Many of these compounds are not easily degradable 35,36 .Therefore, it is unlikely that the microorganisms in the present study will have avoided meteoritic carbon and preferentially consumed the terrestrial contaminants.
Unfortunately, any meteorite that has landed on Earth is subject to some potential contamination.The material used in this study is considered relatively pristine.However, in order to eliminate terrestrial contamination completely, we suggest the repetition of this experiment with freshly collected material returned from carbonaceous asteroids, for example, material obtained from the asteroid Bennu in the NASA OSIRIS-REx samplereturn mission.
The results of this research show that the theory that meteoritic organics are too heterogeneous for microbial use 19 seems unlikely since we have shown that heterotrophy driven by extraterrestrial organic carbon can be a www.nature.com/scientificreports/viable mode of energy acquisition on early Earth.Like the early Earth, the habitability of other young, terrestrial planets could also benefit from the influx of external organics 5 .These beneficial results would be even more pronounced on planets with limited biologically available organics on the surface, and the influx of meteoritic material could thus significantly increase the habitability of a planet.Further, these results indicate that the universe is a potentially vast store of accessible organic carbon to drive heterotrophy.The results also show that organic carbon produced in protoplanetary discs could power an early biosphere on other young planets.The recent discovery of molecular precursors to complex organic materials by the James Webb Space Telescope (JWST) shows that the production of organic molecules is widespread in early planet-forming regions 37 .Finally, our results show that anaerobic microbial communities can be used to access and metabolically transform carbonaceous asteroidal material in future human space settlement schemes.Carbon-rich extraterrestrial resources could potentially be used as feedstock in anaerobic bioreactors for the production of end products such as rocket propellant and plastics to support a sustainable human presence in space.Here, we have demonstrated the proof-of-concept that organic material and the associated silicate matrix in carbonaceous chondrites are not toxic to anaerobic organisms and are metabolically accessible to power growth, which is the first step in such schemes.Synthetic biology might be used to engineer organisms using the pathways we have demonstrated here to carry out these useful metabolic transformations.

Microbial community
An anoxic, environmental microbial community was sampled from pond sediment of Blackford Pond, Edinburgh, UK according to Waajen et al. 16 .Pond sediment was transported to the laboratory, where the microorganisms were grown anaerobically in microcosms containing the CM2 carbonaceous chondrite Cold Bokkeveld according to Waajen et al. 16 .After three transfers over the course of six months, the stable anaerobic community was stored in 25% glycerol at − 80 °C for further use.

Meteoritic material
After the initial establishment of a stable community by the CM2 carbonaceous chondrite Cold Bokkeveld, the CM2 carbonaceous chondrite Aguas Zarcas was used for all further experiments.Aguas Zarcas was an observed fall of a highly brecciated 38 , clay-and organic-rich 39 CM2 carbonaceous chondrite in 2019 in Costa Rica.The used specimen has been collected pre-rain, thereby minimizing terrestrial contamination and weathering 39 .Due to the short terrestrial residence time, Aguas Zarcas is one of the most pristine extraterrestrial materials on Earth.Aguas Zarcas has low terrestrial organic contamination 26 , but like any meteorite that has been in contact with Earth's surface, it is not completely free from terrestrial organics 32 .Aguas Zarcas is rich in hydrocarbons, carboxylic acids, dicarboxylic acids and macromolecular organics, and is similar to the Murchison CM2 chondrite 8 ; but contains relatively low levels of ammonia, amino acids and amines 8,26 .
The carbon content of the meteorite was analysed using an Elemental Analyser-Isotope Ratio Mass Spectrometry (EA-IRMS).Samples of ≤ 40 mg powdered meteorite in tin capsules were loaded into an auto-sampler on a Europa Scientific elemental analyser.Samples were dropped into a furnace in an oxygen-rich environment, and heated from 1000 to ~ 1700 °C.A helium stream transported produced gases over combustion catalyst wires with chromium trioxide and copper oxide to oxidise hydrocarbons and silver wool to remove sulphur and halides.The resulting gases N 2 , NO x , H 2 O, O 2 and CO 2 were passed through a reduction stage of pure copper wires at 600 °C to remove O 2 and convert NO x species to N 2 .Water was removed using a magnesium perchlorate chemical trap.A packed column gas chromatograph at 100 °C separated CO 2 from N 2 .The resulting CO 2 chromatographic peak was then ionised and accelerated by the ion source of the Europa Scientific 20-20 IRMS.A magnetic field separated gas species of different mass, while the isotopomers of CO 2 at m/z 44, 45 and 46 were measured using a Faraday cup collector array.

Preparation of anaerobic cultures
Microcosms were prepared in 12.5 mL glass serum bottles with butyl rubber stoppers.Glassware was made organic-free as described in Waajen et al. 16 following Eaton et al. 40 .Butyl rubber stoppers were washed with common detergent and copiously rinsed with distilled water.Then, the stoppers were boiled three times for 5 min in Invitrogen™ UltraPure™ DNase/RNase-Free Distilled Water and air-dried.
Microcosms were made containing 5 mL M9 medium (3 g/L KH 2 PO 4 , 7 g/L Na 2 HPO 4 , 1 g/L NH 4 Cl, 0.5 g/L NaCl, 0.12 g/L MgSO 4 , 0.011 g/L CaCl 2 ) with 3.47 g/L of either regular CH 3 COONa or double 13 C-labelled CH 3 COONa (double labelled being that both 12 C atoms were replaced with 13 C atoms in the molecule; also denoted as [ 13 C 2 ]-CH 3 COONa) as the sole carbon source and sterile Invitrogen™ UltraPure™ DNase/RNase-Free Distilled Water.In addition, microcosms were prepared containing 0.5 g powdered Aguas Zarcas or Cold Bokkeveld in 5 mL sterile Invitrogen™ UltraPure™ DNase/RNase-Free Distilled Water.The meteorites were handled under sterile conditions, broken into pieces with a heat-sterilised chisel and powdered with an organic-free mortar and pestle.These had been sterilised by heating to 550 °C for 6 h in a Carbolite 1100 °C Chamber Furnace.Microcosms were made anoxic by flushing with sterile N 2 gas.
Vol:.( 1234567890 C).These communities were incubated for 14 days, except for the communities on powdered Aguas Zarcas (1), which were incubated for 33 days due to their slower growth rate.Non-biological controls of each of these conditions were also performed, where the four conditions above were incubated in the absence of the microbial community.
Microbial growth was tested by colony-forming unit (CFU) counts on anoxic LB agar plates (adapted from DSM 381 (DSMZ; Deutsche Sammlung von Mikroorganismen und Zellkulturen): 10.0 g/L tryptone; 5.0 g/L yeast extract; 5.0 g/L NaCl; 20 g/L agar; pH 7.0) at room temperature.Agar plates were plated by single plate-serial dilution spotting (SP-SDS) 41 .SP-SDS of the communities in microcosms were conducted both immediately after incubation and 14 days after incubation.The communities on Aguas Zarcas were also spotted with SP-SDS 33 days after inoculation.
All experiments were carried out in triplicate, except for the non-biological control with Aguas Zarcas, which was conducted once due to limitations in the amount of material.Prior pilot experiments in triplicate showed no contamination of microbial growth on Aguas Zarcas (data not shown).

pH
The pH of the microcosms was measured prior to inoculation and 14 days after inoculation using a Jenway 3510 pH meter (Cole-Parmer, Staffordshire, UK) and InLab Semi-Micro-L pH electrode (Mettler Toledo Ltd, Leicester, UK).
An ANOVA and post-hoc Tukey test were performed to compare the pH of the microcosms prior to inoculation to 14 days after inoculation; the pH of the different conditions prior to inoculation to each other; and the pH of the different conditions 14 days after inoculation to each other.

O-PTIR and FT-IR
After the incubation period, anoxic aliquots were shipped to the University of Liverpool, where biological samples were prepared for optical photothermal infrared (O-PTIR) spectroscopy according to Lima et al. 22 .Samples were washed three times by repeatedly centrifuging the cells for 10 min at 5000×g at room temperature using a benchtop Eppendorf microcentrifuge 5424R (Eppendorf Ltd., Cambridge, U.K.), discarding the supernatant and resuspending the samples in 2 mL deionised water.Five microliters of each cell suspension were spotted on CaF 2 substrates and left to dry in a desiccator at room temperature.
A mIRage infrared microscope (Photothermal Spectroscopy Corp., Santa Barbara, USA) with a tunable fourstage QCL device and a continuous wave 532 nm laser probe beam was used to acquire O-PTIR measurements using the single-point and imaging mode.Using reflection mode, spectral data were collected using a 40 ×, 0.78 NA, and 8 mm working distance Schwarzschild objective.A spectral region of 800-1800 cm −1 was analysed with 2 cm −1 spectral resolution and 50 scans per spectrum to obtain single-point spectral data.Single frequency images were collected at a 500 nm step size by tuning the QCL device to the frequencies corresponding to amide I of labelled and unlabelled cells (1616 and 1657 cm −1 respectively) in order to find the exact location of bacterial cells.PTIR Studio software provided by the manufacturers was used for instrument control and data collection.
Non-biological samples were analysed using Fourier transform infrared (FT-IR) spectroscopy.Twenty microliters of non-biological samples were transferred to 96-well silicon substrates (Bruker Ltd, Coventry, UK) and dried prior to data collection.FT-IR data were acquired in the mid-IR range (4000-600 cm −1 ), with 64 spectral co-adds and using an FT-IR spectrometer (Bruker Invenio, Bruker Ltd, Coventry, UK).
All spectra were pre-processed by baseline correction, smoothed via a Savitzky-Golay filter using a polynomial of second order in an 11-point window (approximately 22 cm −1 in size), vector-normalised and meancentered.After pre-processing, spectra were cut from 1500 to 1760 cm −1 and the data were subjected to principal component analysis (PCA) using Quasar version 1.7.0 42,43 .All collected data were processed using MATLAB software version 2011a (Mathworks Inc., Natwick, USA), and all code used for these analyses is available via GitHub (https:// github.com/ Biosp ec/).

16S rRNA gene amplicon sequencing and analysis
DNA of biological samples 14 days after incubation was extracted using the DNeasy PowerSoil Pro Kit (QIAGEN GmbH, Germany).Additionally, a negative control containing no community sample was examined.Extracted DNA concentration was measured using a Qubit 3 Fluorometer (Life Technologies).Extracted DNA was stored at − 20 °C and shipped on dry ice to the Research and Testing Laboratories (RTLGenomics, Texas, USA) for amplicon sequencing.

Figure 1 .
Figure 1.Microbial uptake of organic material from the carbonaceous chondrite Aguas Zarcas.Optical photothermal infrared (O-PTIR) spectroscopy spectra of biological samples with the amide I peaks indicated by dashed lines.The bacteria in the starting culture, Control A (containing 13 C-labelled sodium acetate) and C (containing no carbon source) have an amide I peak associated with 13 C (the carbonyl vibrations are at 1616 cm −1 ).Bacteria on Aguas Zarcas and in Control B (non-labelled sodium acetate) have an amide I peak associated with 12 C (carbonyl vibrations are at 1657 cm −1 ).

Figure 2 .
Figure 2. Bacterial differentiation based on the stable isotope of the carbon source.Principal component analysis (PCA) of optical photothermal infrared (O-PTIR) spectroscopy spectra from 1500 to 1760 cm −1 of biological samples.Principal component 1 (PC1) separates bacteria grown on a 12 C carbon source (Bacteria on Aguas Zarcas and Control B) from bacteria grown on a 13 C carbon source or without an external carbon source (Starting culture, Control A and Control C).See PCA loading plots in Supplementary Fig. S1.

Figure 3 .
Figure 3. Bacteria growing on the carbonaceous chondrite Aguas Zarcas.Stacked barplot of the abundance of bacterial families present in the starting culture (SC), Aguas Zarcas containing microcosms 14 days after inoculation (Aguas Zarcas), Control A (containing 13 C-labelled sodium acetate), Control B (non-labelled sodium acetate) and Control C (containing no carbon source).Samples 1, 2 and 3 are biological replicates. https://doi.org/10.1038/s41598-024-54195-6 )The cryostock of the stable anaerobic community was used as the starting culture for the experiment.Three anoxic microcosms containing 5 mL M9 with [ 13 C 2 ]-CH 3 COONa were inoculated with 20 μL of the cryostock.These microcosms were incubated for approximately two weeks, shaking at 150 rpm at room temperature (20-25 °C) after which they were transferred to fresh microcosms with [ 13 C 2 ]-CH 3 COONa.This process was repeated twice, after which the community was transferred to the following anoxic microcosms in triplicate: (1) Containing 0.5 g powdered Aguas Zarcas and 5 mL sterile Invitrogen™ UltraPure™ DNase/RNase-Free Distilled Water;(2)