Astroecology 
A branch of astrobiology - Interactions of microorganisms and plants with solar system soils in meteorites and planetary materials, in relation to the origins of life and future terraforming.

Planetary bioresources and astroecology.
1. Plant and algal microcosm bioassays of martian and meteorite soils
M. N. Mautner, Icarus 2002, 158, 72-86

Michael N. Mautner, Soil, Plant and Ecological Sciences Division, Lincoln University, Lincoln, New Zealand, and Department of Chemistry, University of Canterbury, Christchurch 8002, New Zealand m.mautner@solis1.com

Corresponding address:
Michael N. Mautner
Soil
Plant and Ecological Sciences Division
Lincoln University
Tel.: (64) (3) 325-2811Fax: (64) (3) 325-3607
Email: m.mautner@solis1.com

This material has been submitted to Academic Press for possible publication. Copyright may be transferred without notice, after which this version may no longer be accessible.

Figure 1. Plant tissue cultures of Asparagus officinalis in meteorite and soil extracts, all supplemented with 5 mM NH4NO3 and 3% sucrose. Scale divisions 1 mm, small ticks 0.5 mm units.

Figure 1c Murchison Meteorite
Figure 1d DAG476 Mars Meteorite
Figure 1a Water Blank
Figure 1b Hawaii Lava Mars simulant

Figure 2. Benthic algal growth on powder of Dar al Gani 476 Mars meteorite supporting a mixed algal population of filamentous blue-green and single-cell green algae populations. Scale as in figure 4 below.

Figure 2 Dar al Gani 476

Figure 3. Algal population observed in meteorites and soil extracts supplemented by 0.5 mM NH4NO3, after 32 days of growth. The spacing between the counting chamber lines in the Allende culture is 0.2 mm, and the other figures are on the same scale.

Figure 3a Allende Meteorite
Figure 3b  Hawaii Lava Mars simulant
Figure 3c Murchison Meteorite
Figure 3d DAG476 Mars Meteorite

Figure 4. Algal population growth curves in meteorite and simulant extracts. (D) DaG 476, (x) Hawaii lava Mars simulant, (*) Murchison, (¨) Allende, (&127;) deionized water.

The full paper follows below

Introduction

Rocks and soils under early planetary conditions provide resources for the origins of life and nutrients for early microorganisms. Planetary materials can also provide the resources for future space-based agriculture in space colonies and planetary terraforming. The biological fertilities of various solar system materials are of interest in these respects. Meteorites provide samples of planetary and asteroid materials for soil fertility studies.

Meteorites and similar dust or cometary materials may form highly concentrated solutions for local biogenesis on planets. These materials may also actively transport microorganisms amongst asteroids and planets (Arrhenius 1908, Crick and Orgel 1973, Chyba and McDonald 1995, Mautner 1997a). In such instances, the meteorites constitute the first environments for the embedded microorganisms. The materials that can perform these functions include carbonaceous chondrites and similar interplanetary dust particles and comets (Chyba and Sagan 1992, Chyba and McDonald 1995), and Martian meteorites.

There are indications that many space materials can indeed support life. First, their mineral and organic constituents are similar to terrestrial rocks that support diverse geo-microbiology. Algae growing on meteorite dust in Greenland were observed as early as 1870 (Leslie, 1879, Maurette et al. 1986). The nutrient values of organic planetary materials were demonstrated on a synthetic terrestrial analogue, tholin (Stoker et al. 1990). The Murchison CM2 meteorite was observed to have soil fertility parameters comparable to productive terrestrial soils (Mautner 1997b, Mautner 1999). Murchison extracts were observed to support various soil microorganisms such as the oligotrophs Flavobacterium oryzihabitans and Nocardia asteroides, and experiments with Pseudomonas fluorescence showed that meteorite organics can serve as a sole carbon source (Mautner et al. 1997). Indications were also found that the Murchison materials can support the anaerobic thermophile eubacterium Thermotoga maritima and the aerobic thermophile Thermus aquaticus (H. W. Morgan, quoted in Mautner et al. 1997). In contrast, the Allende meteorite was observed to inhibit biological growth. More recently, a various carbonaceous and Mars meteorites were found to contain diverse microorganisms from terrestrial contamination (Steele et al. 2000). Martian minerals may have also supported indigenous microorganisms in the past (McKay et al. 1996).

These observations suggest that planetary materials have diverse biological potentials. These properties should be assayed systematically, similar to assays of agricultural soil fertility (McLaren and Cameron 1996, Beare et al. 1997). Microcosm simulations are useful as bioassays, and this method may be applied also to space-based materials. The microcosms can be inoculated with microorganisms of interest and cultured under various planetary conditions. In fact, the previous microbial experiments with tholins (Stoker et al. 1990) and with meteorites (Mautner 1997b) constituted limited planetary microcosms.

Planetary ecosystems are complex and it is desirable to apply comprehensive ecological simulations of competing microbial populations, and the mutual effects of minerals, solutions and microorganisms. These assays can be applied to small meteorite samples. Weathering effects in planetary soil development can be also simulated with small meteorite samples.

The present work illustrates some aspects of microcosm studies using Martian meteorites and analogues. The results are used to illustrate a fertility ranking of several planetary objects. Such ranking schemes may be useful in targeting searches for extraterrestrial life and in identifying space-based soil resources.

Experimental

1. Materials

The rock samples used were Martian meteorites and terrestrial analogues. The Dar al Gani 476 (DaG 476) meteorite and Elephant Morraine 79001 (EETA 79001) lithology A are both basaltic shergottites. DaG 476 contains a fined-grained pyroxene and feldspathic glass ground-mass containing also sulphides and phosphates. Both meteorites contain olivine, orthopyroxene and chromite. The DaG 476 meteorite was subject to extensive terrestrial weathering, leading to the formation of carbonates. Phosphate minerals in the shergottites include merrilite and chlorapatite. The comparative mineralogy of DaG 476 and EETA 79001 was discussed recently (Zipfel et al. 2000).

The Nakhla meteorite is a cumulate igneous rock.. Its main component is augite, with some olivine and minor other minerals (McSween and Treiman 1998). As only small amounts of Nakhla were available, we used a terrestrial analogue, the Theo’s Flow lava formation in Canada, that was described as a close mineralogical analogue of Nakhla (Friedman 1998). A further basalt sample from Timaru, New Zealand was also used. This basalt contains 65% labradorite feldspar, 25% clinopyroxenite and 10% magnetite.

Terrestrial analogues of lunar and Martian soils, distributed by NASA, were also examined. The Mars soil simulant JSC Mars-1 is a sample of lava ash from the Pu’u Nene volcano in Hawaii (Allen et al. 1998). It contains Ca-feldspar and minor magnetite, with minor olivine, augite pyroxene and glass, including highly weathered glassy matrix. It also contains nanophase ferric oxide similar to that inferred for Martian soil.

The lunar simulant JSC-1 is a glass-rich volcanic ash from the San Francisco volcanic field near Flagstaff, Arizona (McKay et al. 1983). The elemental composition is similar to Apollo 14 soil sample 14163, and contains plagioclase, clinopyroxene, orthopyroxene, olivine, magnetite, ilmenite and apatite.

A terrestrial soil agricultural soil, from the Templeton area (Udis Ustochrept, fine loamy mixd, mesic soil) in New Zealand was also used for comparison.

Two carbonaceous chondrites, Allende and Murchison, were also used. The mineralogies of both are well known (Fuchs et al. 1973, Bunch and Chang 1980) and were reviewed recently (Brierly and Jones 1998). The main component of Murchison is a phyllosilicate formed by aqueous alteration in the parent body.

 2. Extraction and Analysis

Samples of meteorites and terrestrial analogues, ground in an agate mortar, were extracted under standard sterilising conditions at 121 ^oC for 15 minutes at solid:water ratios of 1:10 w/w. Typically, 50 mg of the solid was extracted in 0.5 ml of deionized water. The extracts were analysed for cations by atomic absorption spectroscopy or ICP-MS, the latter performed by Hill Laboratories, Hamilton, New Zealand, and for anions by ion exchange chromatography. Phosphate analysis was performed by developing the solutions with malachite green reagent at 4:1 solution/reagent ratios for one hour and measuring the absorbance at 630 nm (van Veldhoven and Mannaerts 1987). All extractions were replicated 2 – 4 times at 1:1 to 1:100 solid/water ratios and extraction times of 1 – 40 days. The effects of these parameters on the extract concentrations will be reported elsewhere.

The meteorite sample sizes of 10 – 40 mg that are available per analysis are smaller than the 1 – 10 gram samples used in standard soil analysis. Because of the small samples, we had to employ soil microanalysis methods that we developed for meteorites previously (Mautner 1997b). These methods were tested on several soils from the International Soil Analysis Exchange Program. Analytical data for these samples are available for these soils from 10 – 30 laboratories world-wide. Our microanalysis data fell in the reported range.

In addition to the usual factors in the inter-laboratory scatter, the uncertainties in our data are due to the small available sample sizes and to the low concentrations of the extracted elements, often close to the detection limits of 0.1 – 1 micrograms/mL for anions and cations, and to 10 micrograms/L for phosphate. An uncertainty of ± 30% is estimated for the microanalysis measurements.

3. Plant and Algal Cultures

The plant culture media were prepared using 45 microliters of the mineral extracts described above and 5 mg of the extracted powder, which were transferred to 2 ml polythene microfuge tubes. A further 45 microliters of 10 mM NH₄NO₃ solution + 3% sucrose was added as nitrogen and carbon sources.

Algal culture media were prepared similarly by mixing equal volumes of 1:1 mineral extract with 10 mM NH₄NO₃ or with deionized water. Algal populations were counted by direct microscopic count in a hemocytometer chamber.

Plant tissue cultures were established from in vitro Asparagus officinalis, cultivar "Limbras 10" genotype ASC 69. Apical meristem shoot tips, about 1 mm long, were dissected from 4 – 8 cm plants grown on agar. It was important to use plants obtained after one month of active elongation and 3 months of arrested growth in agar media. This procedure produces plants that are depleted of nutrients and more responsive to nutrients added in the culture media. Potato plants, Solanum tuberosum cultivar Iwa were grown using a similar procedure.

Plants of Arabidopsis thaliana strain "Landsberg Erecta" were grown from seed on filter paper wetted with deionized water, to the full size achievable on the seed nutrients alone, before introduction to the culture media.

All plant cultures were grown for 20 days in closed microfuge tubes under illumination by cool white fluorescent lights with a flux of 80 m E m^-2 s^-1 using 16 h photocycles. Algal cultures were grown in similar growth chambers in vials with a punctured cap that allowed air exchange, with the vials contained in a desiccator saturated with water vapor to prevent evaporation. Some algal cultures were grown in closed 2 ml glass vials that were opened to air in sterile laminar flow cabinets for about 20 minutes once in four days to allow gas exchange with the atmosphere.

Results
1.  Extractable nutrients in Mars meteorites and analogue

a. Anions and cations. The amounts of extractable nutrients are listed in Table 1.

It is of interest to compare the nutrients from the various meteorites with each other and with terrestrial simulants and terrestrial soils.

Most materials in this study are igneous rocks that were not subject to aqueous processing, except possibly DAG 476 and the terrestrial soils including the Mars analogue JSC-MARS 1, a Hawaiian lava that experienced some weathering.

Table 1 shows that the Mars meteorites are comparable to each other and to the terrestrial analogues and soils in extractable Ca, Na and K, except the high levels of extractable Ca and sulphate in DaG 476 which may be due in part to terrestrial weathering. The Mars meteorites are higher in extractable Mg than the terrestrial Hawaii lava and Theo's Flow analogues, but comparable to the terrestrial basalt and Lunar simulant lava ash.

b. Extractable and exchangeable phosphate

Phosphate is the limiting nutrient in many terrestrial ecosystems, and we also found this to apply to nutrients in carbonaceous chondrite meteorites (Mautner et al. 1997). It is therefore important to note in Table 1 that the Mars meteorites contain significantly higher levels of soluble phosphate than any of the terrestrial analogues, terrestrial soils and carbonaceous chondrites that we examined so far.

Because of its significance, it is desirable to characterise bioavailable phosphate in more depth. A quantitative assessment is obtained by the isotope exchange kinetic method (Frossard and Sinai 1997). The method is illustrated here by results on three materials and their weathering products, and two terrestrial soils for comparison. A more extensive study will be presented elsewhere (Mautner and Sinai 2000). In the present experiments, a suspension of the solid at 1:100 solid/water ratios was allowed to equilibrate with the solution for 24 hours. A small amount of ³²PO₄^3- was then added to a soil solution. Solution samples were withdrawn 1, 10, 30 and 60 minutes after introducing the labelled tracer and were measured in a scintillation counter. In this manner, exchange between the phosphate in solution and phosphate pools in the solid is measured by the decrease of radioactivity in solution. Equation (1) expresses the decrease of radioactivity in solution as a function of time.

R(t)/R = {r(1)/R}* {t + [r(1)/R]^1/n}-n + r(¥ ) / R (1)

Here R is total introduced radioactivity, r(1) and r(¥ ) are the radioactivity remaining in solution after 1 minute and infinity, respectively, and n is a parameter describing the rate of disappearance of the radioactivity at times longer than 1 minute (Frossard and Sinai 1997).

The exchange kinetics can be used to calculate the intensity factor (concentration in solution), the quantity factor (how much nutrient can be taken up in time t), and the capacity factor. The kinetic factors reflect the division of phosphate in several pools that measure bioavailability to plants in soils: the free ions (exchangeable in 1 min.); the pool available to a root (exchangeable in 1 min. – 1 day); the pool available to a root system (exchangeable in 1 day – 3 months); and the pool that is unavailable in 3 months.

Table 2 shows data for a carbonaceous chondrite Allende, the Mars meteorite DaG 476 and a terrestrial igneous rock, Theo’s Flow, a Nakhla simulant.

The levels of soluble phosphate in the extracts show substantial variation, with DaG 476 showing particularly high levels as noted above. Buffering capacities are considered high for R/r1 > 5, and in the present case, all the three materials have low buffering capacities. Although the soluble phosphate is high in DaG 476, the longer-term exchangeable phosphate is comparable in the three materials, although low both in absolute terms and as given fraction of the total P as calculated

from E(3 m)/P_total. All the values are within the range observed in terrestrial soils. All three materials also show significant increase in the exchangeable P when subjected to planetary weathering as shown below.

2.  Mars meteorite extracts as nutrient solutions

The nutrient concentrations in the present extracts can be compared with the range of terrestrial soil solutions. Because of the 1:10 (w/w) solid/water ratios, the data in Table 1 represent solution concentrations in units of 0.1mg/mL. For comparison, the data for terrestrial soil solutions in the last two rows were converted to similar units. Concentrations in units of microgram/mL can be obtained by multiplying the values in Table 1 by 100. Note that the actual concentrations used in the plant and algal cultures are only half of those obtained from Table 1, as they were diluted by additional 10 mM NH₄NO₃ + 3% sucrose solutions in the plant tissue cultures and by 10mM NH₄NO₃ or deionized water in the algal cultures. Bearing these factors in mind, the nutrients in the Mars meteorite extracts are well within the range, mostly within a factor of 5 about the median values, of terrestrial soil solutions.

3.  The effects of planetary weathering

The atmospheres of early Earth and possibly Mars may have contained high partial pressures of carbon dioxide (Kasting and Mischna 2000). Therefore the early oceans may contain high levels of dissolved carbonic acid, that yields a pH 3.8 value under a 1 atm CO₂ atmosphere. This contributes to the weathering of rocks and can affect the adsorption/desorption of ions by altering minerals and by forming carbonates. The bioavailability of inorganic nutrients and microbial habitability may be affected significantly by planetary weathering under such conditions.

In the present study, we modelled these effects by extracting anions and cations from grounded rock and meteorite samples in water saturated by CO₂ at 1 atm. The effects of weathering and extraction in this solution on anions and cations in 24 hour extractions and the effects on phosphate availability by longer weathering in 21 day extractions were measured.

Table 1 shows the concentrations of the extracted cations and anions from weathered Allende, DaG 476 and Theo’s Flow weathered under CO₂ for 21 days . In Allende the availability of Ca, Mg and Cl, in DaG 476 Mg and in Theo’s Flow , all the materials the availability of Ca and Mg are increased increased significantly.

The most significant effects are observed on the bioavailable phosphate in Table 2, after weathering at 20 ^oC in water saturated with CO₂ for 21 days. The solution concentrations obtained from both Allende and DaG 476 increase significantly, with the DaG 476 extract showing a remarkably high concentration of over 300 ppb. On the other hand, the phosphate concentration in the Theo’s Flow extract decreases. Decreased solution values were observed also for clinopyroxenite and the Hawaii lava Martian soil analogue. Note that different relations apply in Table 1 where H₂O/ CO₂ extraction was carried out only for 24 hours and at different temperatures. The difference between the weathering effects at one day and 21 days suggests that the effects of longer weathering are due to chemical changes, probably the formation of carbonates.

With respect to phosphate, H₂O/CO₂ exposure of various rocks has variable effects on the concentrations in solution. However, the main effect is on the large increase in the fraction of total P that becomes exchangeable. Column 10 in Table 2 shows that this fraction increases from 2.2 to 57.4% in Allende, from 1.4 to 11.3% in DaG 476, and from 6.2 to 91.8% in the Theo’s Flow basalt. A very large effect is also observed in the 1 minute decay of radioactivity in solution in Allende. This may correspond to the adsorption of the introduced phosphate on carbonates formed in this material.

The observed effects show that planetary weathering in water that is in equilibrium with an atmosphere containing CO₂ at high partial pressures can increase substantially the bioavailability of nutrients, especially of phosphate. This may be significant for the survival of microorganisms in early planetary environments.

4.  Plant Bioassays

An objective of the present work was to test various plants as bioassay agents for meteorite materials. Previously, we used the responses of asparagus and potato tissue cultures with extracts of the Murchison and Allende meteorites to bioassay carbonaceous chondrites (Mautner 1997, Mautner et al. 1997). The results showed that Murchison had a nutrient effect, with optimal growth at extractions with 1:10 to 1:20 solid/water ratios. In comparison, the extracts of Allende indicated an inhibitory, possibly toxic effect. We also found that these effects could be best observed when the solutions were supplemented by NO₃-N and PO₄-P, as in their absence the yields were too small for reliable measurements.

In all the previous cultures, the scatter of the plant weight yields was substantial. The present work aimed to improve the statistics of product weight distributions by using more starved starting tissues to enhance the effects of the added nutrients. Another approach was to use arabidopsis plants grown from seed to test if seeds may yield more uniform products.

In the tissue cultures, nutrients contained in the starting meristem may decrease the effects of the added dilute rock extract media. For this reason, after one month of development, the parent plants were kept in agar for an additional three months to assure that all the nutrients in the medium were exhausted. Well formed globular apical shoot tips could be removed uniformly from each plant.

The results of Asparagus officinalis cultures are shown in Fig. 1, which compares plants grown on a water blank and on extracts of Murchison, Dag 476, and the JSC Mars 1 simulant. Table 4 shows the product weights and comparative statistics for these and some additional media.

Fig. 1 shows significant differences between the tissue cultures developed in the various media. The water blank reflects growth probably mostly due to cell enlargement, supported by the stored nutrients in the starting shoot tips. In comparison, the extract media cause tissue development specific to the media. For example, the plants in the extracts of the Hawaii lava Mars simulant and some DaG 476 products show more efficient stem development than the Murchison products. Some of the Murchison products show a partial reddening that may indicate phosphorus deficiency. The DaG 476 products show the most differentiated development and the deepest green coloration. This may correspond to the high but not toxic contents of phosphate in this extract medium.

In comparison, the cultures grown in the Allende and EETA 79001 extracts showed decreased size and a brown coloration and low fresh weights (Table 3). In the Allende extracts, that show inhibitory effects also previously (Mautner et al.), these effects may be due to toxic elements. In the EETA 79001 extracts the inhibition may be due to toxically high levels of phosphate.

The yield weights in Table 3 reflect these observed differences in plant development. The p values obtained from a Mann-Whitney non-parametric analysis shows that the yields of all the nutrient sets are statistically different from the water blank. The DaG 476 extracts and the three Mars simulants all show statistically significant increased yields, and Allende shows statistically significant inhibition. The EETA 7900 product weights are similar to those in the water blank, possibly due to compensating nutrient and inhibitory effects.

The above results compare the various nutrient sets with the water blank. A statistical analysis can also compare the various media directly with each other. For example, Table 3 shows a statistical comparison of DaG 476 with the other extracts. The DaG 476 extract shows the most significant differences from the carbonaceous meteorites and from EETA 79001, and statistical similarity to the Mars simulants. The difference between DaG 476 and EETA 79001 is particularly interesting considering their similar mineralogy, possibly due to toxically high levels of phosphate in the EETA 79001 extracts.

As in the previous carbonaceous chondrites (Mautner 1997b, Mautner et al. 1997), we also examined the effects on Solanum tuberosum potato cultures. With the present extracts, the best yields were obtained with the Hawaii lava Mars simulants. Potato plants grown in the Murchison and DaG 476 extracts gave smaller yields and a brownish coloration. The DaG 476 results may be due high phosphate levels which may be toxic in these cultures.

The results with arabidopsis plants grown from seeds were in general similar. The plants were grown from seed on filter paper wetted with deionized water, without additional nutrients. The plants, with stems and roots of about 10 mm, were introduced into the mineral extracts. The plants in the extracts of the Hawaii lava Mars simulant remained green and produced a few mm additional development. However, the plants in most of the other extracts actually yellowed and disappeared in the solutions. These preliminary results were less definitive than the tissue cultures. Nevertheless, plants grown from seeds should produce the most uniform bioassays and they merit further work.

5.  Algal Bioassays

Algae are the first colonisers in many the terrestrial environments and are also candidates for planetary terraforming, and are therefore reasonable models for bioassays. Algae are also suitable for practical reasons as their large 4 – 10 micron sizes facilitate microscopic analysis.

In the present experiments we used algae isolated from soil in Canterbury, New Zealand. Mixed algae populations were used as inoculants to allow a degree of natural selection for species suitable for bioassay. Mixed inoculating populations are also of interest to check whether a dominant species will emerge in these microcosms or if a more diverse ecosystem is established, and whether the compositions of the surviving populations are specific to the substrate media.

Two culture strategies were used. In one method, both the mineral extracts and the extracted powders were included in the culture medium. About 200 m L of mineral extracts and 20 mg of the extracted mineral powder were introduced into a cavity microscope slide partially covered with a slide cover. The cultures were kept in a closed desiccator at 100 percent humidity to prevent evaporation. In the second class of experiments, only the aqueous extracts with traces of the extracted powder were used. In the extract/powder microcosms and in one pure extract series, the medium was diluted 1:1 with 10 mM NH₄NO₃ as added nitrogen source. In the second extract series deionized water was used without added nitrate for dilution.

The inocculant cultures were grown in standard alga nutrient medium, and four cycles of centrifuging and washing with deionized water were used to eliminate residual nutrients. In both cases, the cultures were inoculated with 20 m L of mixed algal culture to yield starting populations of 10⁴ CFU/ml. The cultures contained algae that were identified microscopically to genus level as Leptolingbya sp. (filamentous blue-green), Phormidium sp. (filamentous blue-green), Chlorella sp. (green unicellualar), Chlorosarcinopsis sp. (green unicellular in aggregates), and Navicula sp. (diatom). The inoculant cultures also contained smaller flagellate bacteria of about 1 micron diameter.

In the first series, the solution and extracted residue served as a miniaturised microcosms simulating small ponds in planetary environments. We examined the growth of the algae microscopically at weekly intervals. Most of the products occurred as benthic growth of both of the filamentous blue-green algae. Aggregates of the green Chlorosarcinopsis were also associated with the mineral surfaces.

Green cells and filaments were observed to survive for at least four weeks in most extracts. The best growth, illustrated in Fig. 2, was observed with the DaG 476 materials. In contrast, in the Allende extracts growth ceased after 6 – 10 days and only empty shells were observed later, consistent with the inhibitory effects of Allende in other experiments.

The benthic growth on solids did not allow the quantitative monitoring of cell populations or biomass on the present small scales. The cultures in pure extracts were used for this purpose. Population counts were obtained using microscopic direct counts in a haemocytometer chamber using 20 m L samples withdrawn from the cultures. In all of the aqueous extracts, unlike on the mineral surfaces, the unicellular Chlorosarcinopsis. as single cells or in aggregates of 2 – 20 cells were dominant. Few filaments, of 8 – 40 m length and containing 2 – 10 cells were observed, with populations smaller by an order of magnitude. Fig. 3 shows samples of the algal populations obtained after 32 days. Notable are the relatively uniform culture of green chlorophyta in the Hawaii lava extract, compared with the species diversity in Murchison and to some extent in DaG 476, and the low population in Allende. For counting the populations, individual cells in the filaments were counted and added to the counts of the unicellular species. Although the starting inocculants contained diatoms, no diatom populations were observed after 1 – 4 weeks.

Growth curves for cultures without added nitrate are shown in Fig. 4. The total cell populations after 22 days follow a similar order as in the plant tissue cultures. Here also DaG 476 is highest, somewhat exceeding Hawaii lava and Murchison. Again, the Allende extracts give the lowest populations.

In the cultures containing added nitrate, the population levels were higher in absolute number and followed a similar order. The populations grew for about 8 days and remained approximately constant for a further 8 - 20 days. After 8 days the product populations: Allende, 1.2x10⁵; Theo’s Flow, 2.3x10⁵; Hawaii lava, 2.7x10⁵, Murchison, 3.0x10⁵, DaG 476, 10.6x10⁵ cells/mL. These relative populations are consistent with the trends in the pure extracts and in the plant tissue cultures, with a fertility order of DaG 476 > Havaiii lava, Murchison > Theo’s Flow > Allende.

In addition to the algal growth, all of the samples contained motile microorganisms of about 1 m diameter. The microbial population was approximately correlated with the algal population, at the ratio of 2 – 4 microorganisms per algal cell. The microorganisms were cultured on agar in dark. The cultures yielded bright orange colonies on nutrient agar and potato dextrose agar but not on potato dextrose agar supplemented with chloretetracyclin (auremycin) antibacterial agent. Apparently, the microorganisms are motile heterotroph bacteria supported by algal metabolites or decay products. These observations suggest that planetary materials may support a complex interactive microbial ecology.

Discussion of Results

1.   Design considerations for planetary microcosms

The design of planetary microcosms depends on the objectives of the simulation, such as the testing of planetary materials and conditions for early life and as future ecosystems. Early planetary conditions may have been reducing or dominated by carbon dioxide. On the other hand, terraforming will aim to create habitable oxygen-rich environments. Our present tests address the latter environment, but anaerobic tests are also under way.

Early planetary weathering would have occurred under atmospheres with high CO₂ partial pressures. Minerals would have weathered and nutrients would have been extracted under such conditions. The results in Table 1 and 2 show that such weathering and extraction can have significant effects on, for example, phosphate availability. This illustrates the importance of selecting appropriate planetary conditions for biological simulations.

To assess a complex planetary ecosystem, it is necessary to examine the mutual effects of atmospheric, aqueous and geological process. Such studies may be limited, however, by material requirements.

For example, the analysis of extracted nutrient cations commonly applies atomic absorption spectroscopy or ICP-MS, and for anions, ion exchange chromatography. These techniques require on the order of 1 mL solutions containing 10^-6 g of solute, usually from solids that contain them in soluble forms in concentrations of 10^-6 – 10^-3 g/g (see Table 1), ie., requiring 0.001 – 1 g mineral samples. Phosphate can be quantified reliably using colorimetry in 1 mL samples at concentrations of 10^-7 g/mL, and is usually contained at extractable levels of 10^-6 to 10^-5 g/g (see Table 1), requiring 0.01 to 0.1 g mineral samples.

For biological tests, the microcosm must contain enough nutrients to support the population. In plant bioassays, the sample plants from tissue cultures yield fresh weights about 10^-3 g, containing on the order of 1 mg/g or a net 10^-6 g of macronutrients such as Ca or K. This requires 0.001 - 1 g of mineral sample per plant, and usually 4 – 10 plants per experiment to allow statistical comparisons (see Table 3).

For microbial or algal microcosms, population levels of 10⁷ cells/mL in 1 mL cultures need to be supported. With an average algal mass of 10^-10 g/cell again a macronutrient content on the order of 10^-6 g of nutrient, extracted from 0.001 – 1 g of solid, may be required. For tests using bacteria, populations of 10⁷ cells/mL of microorganisms of

mass of 10^-13 to 10^-12 g/cell, the requirements are smaller, 10^-9 – 10^-8 g of nutrient extracted from 10^-6 – 10^-2 g of solid.

The material requirements for chemical analysis and plant and algal microcosm cultures are therefore usually 0.001 – 1 g per test. A comprehensive ecological study of a microcosm may require ten or more chemical and biological analyses and 0.01 - 10 g of material. These considerations illustrate that microcosm simulations are possible with available amounts of meteorite materials, but the experiments must be designed judiciously.

2.  Possible implications about parent body processes

It was pointed out recently that soluble meteorite components can indicate conditions on the parent body, as in the high levels of extractable NaCl in Nakhla that suggest deposition from a salty reservoir (Sawyer et al. 2000).

Similarly, the Murchison M2 meteorite is uniquely high in extractable cations and sulphate. This may reflect processes during the aqueous alteration of the parent body. The porosity of about 20% provides a high, about 10:1 solid/water (w/w) ratio when the pores are filled. This high solid/water ratio can yield concentrated solutions when the intra-pore water is melted during the aqueous alteration of the parent body and extracts the solids. The concentrated solution may move from the interior toward the surface of the parent body and evaporate, leaving a deposit of the solutes. Alternatively, the water may be incorporated into the hydrated phyllosilicate minerals during aqueous alteration, again leaving a deposit of the dissolved salts. The latter process is supported by the fact that the water content of about 10% (w/w) in the Murchison minerals is similar to the solid/water ratio of dry Murchison minerals and the water filling the pores. These considerations are consistent with a process in which the soluble salts in Murchison would have been deposited from a concentrated salty solution when the intra-pore water was incorporated into the phyllosilicates.

Notably, the shergottites DaG476 and EETA79001 do not contain the high levels of chloride that in Nakhla indicates deposition from an aqueous reservoir (Sawyer et al. 2000). However, the shergottites contain higher levels of soluble nitrate and phosphate than the terrestrial basalt analogues. The high soluble nitrate and phosphate may suggest that these materials were formed in dry Martian environments and were not exposed to water that would have leached these components.

3.  A bioassay of martian and carbonaceous chondrite materials

The present work extends the application of the microcosm technique, applied previously to carbonaceous chondrites (Mautner 1997, Mautner et al. 1997), to martian meteorites and terrestrial Mars simulants.

It is gratifying that there is a general correlation between the levels extractable limiting nutrients and the responses of the plant tissue cultures and algal cultures. These results allow a fertility ranking of the materials in Table 4, with the overall results DaG 476 (Mars meteorite) > Murchison (CM2); agricultural soils > Basalt; Hawaii lava (Mars soil simulant) > Theo’s Flow (cumulate igneous Nakhla simulant) > Allende (CV3).

Positive marks in Table 4 indicate relatively high levels of nutrients compared with terrestrial soils or positive biological effects. Zero marks indicate the absence of nutrients or of biological responses and negative marks indicate negative biological responses.

As we noted throughout, the DaG 476 Mars meteorite exhibits the highest fertility properties. The biological responses may be attributed to the high level of phosphate and the presence of nitrate, apparently in a suitable balance with the other extracted nutrients. Another interesting result is the high level of extractable phosphate in the DaG 476 and EETA 79001 Mars meteorites, which exceeds the terrestrial analogues. The extractable phosphate, both in the aqueous extracts and in the long-term exchangeable reservoirs, is further increased significantly through weathering by CO₂ solutions.

The fertility properties of the Murchison CM2 meteorite are remarkable. This material was formed under early Solar System conditions and internal hydrothermal processing altogether different from terrestrial chemical and biological weathering. Nevertheless, its organic contents, extractable nutrients, cation exchange capacity, specific surface area and bioassay responses are all comparable to fertile agricultural soils (Mautner 1999). This suggests that comets and carbonaceous asteroids have the potential to sustain microbial life and may also serve as soils for space-based agriculture.

The Theo’s Flow Nakhla analogue and the EETA 79001 Mars meteorite both have low fertility potentials but for different reasons. Theo’s Flow is an unweathered rock with low extractable nutrients. Conversely, EETA 79001 has high extractable nutrients but showed a growth inhibitory effect in the plant cultures. This may be due to too concentrated phosphate at the 1:10 solid/water ratio used in the extraction. If this is the inhibitory factor, it may be reversed in more dilute extracts.

The results illustrate that actual Mars materials may behave differently in their soil fertility and properties than mineralogically similar terrestrial analogues. This suggests that not only mineralogical but soil fertility information is also necessary for the biological modelling of planetary ecosystems.

Conclusions

Planetary materials are similar in general mineralogy to terrestrial materials, and can similarly support microbial and higher multicellular life.

Also as terrestrial soils, planetary materials display a wide range of fertility properties, ranging from the high fertility DaG 476 Mars basalt to the medium fertility of Murchison and the toxic effects of Allende. This diverse behaviour suggests that a systematic assay of planetary soil properties is necessary in the search for extraterrestrial life and space resources.

Microcosm simulations are useful for such bioassays. In these models, the biological content and physical conditions can be controlled to simulate various planetary ecologies. The present observations show that these planetary microcosms can support a complex microbial ecology. The interactions between biology, chemistry and mineralogy can be studied.

Microcosms can be constructed with available levels of meteorite materials. The data from these simulations include extractable nutrients and toxic components, exchangeable nutrient pools in the solids, effects of planetary weathering, plant responses including nutrient uptake and yield weights, and algal and microbial responses. When integrated, these data can yield correlated indicators of the fertility of planetary materials.

The data can be applied to evaluate potential targets in the search for extraterrestrial life. For example, the observations on the shergottites suggest that martian basalt flows may be useful targets for such searches, and that these materials may also be processed into agricultural soils for terraforming. The data on Murchison suggest that similar materials in carbonaceous asteroids and cometary nuclei may be able to transport and incubate microorganisms and may be applied as soils in space settlements.

In summary, microcosm models can be used for systematic bioassays and a ranking system of biological fertility for planetary materials. These applications may help in selecting targets for exobiology missions and in identifying soil resources for space settlements.

Acknowledgments. I thank Dr. Anthony J. Conner for plant tissue culture samples, Dr. Paul Broady for algal cultures and taxonomical identification, Mrs. Helene D. Mautner for algal studies, Dr. Carleton Allen and the NASA Johnson Space Center for simulated Mars and Lunar soils and samples of the EETA 79001 meteorite, Mr Gavin Robinson for the ICP-MS analysis, and Drs. Robert Sherlock and Robert Leonard for helpful discussions. This work was funded by grant LIU 901 from the Marsden Foundation, administered by the Royal Society of New Zealand.

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