Let’s break for a moment with interstellar issues to finish up a story I first covered at the beginning of the year. In 2022, members of the Interstellar Research Group led by Doug Loss began exploring the biological side of establishing a human presence on Mars. By ‘biological,’ what the team was looking at was how to create soil as opposed to regolith, soil with the microbial components needed to produce crops for human consumption on Mars. Alex Tolley wrote the idea up in MaRMIE: The Martian Regolith Microbiome Inoculation Experiment. Today’s post is the finalized document that has grown out of this effort, an attempt to foster further research by offering a framework for experiment. While the IRG lacks the means of executing these experiments itself, it offers this paper as a contribution to planetary studies to connect with those who can.
by Alex Tolley and Doug Loss*
* Contact: Doug Loss at email@example.com
The proposed designs for the settlement of Mars include various approaches for local food production. Food will most likely be based on traditional terrestrial crops to ensure that a variety of cuisines can be cooked for the well-being of the settlers. To farm on Mars, as well as provide an environment for plants and trees, will require establishing soils using the Martian regolith. The presence of (per)chlorates at levels toxic to plants and humans requires remediation of the regolith to remove the (per)chlorates. Prior work indicates that there is a knowledge gap in how to remediate the regolith to make it ready to support various crops for Martian agriculture. We propose a framework of experiments to help bridge the gap between the state of the regolith on the surface and the initial stages of soil creation.
With the renewed interest in settling Mars, there has been considerable attention on how to feed a base crew and its subsequent expansion into a larger settlement population. Unlike a human presence in low Earth orbit (LEO) and on the Moon where travel times are sufficiently short that food can be provided in regular shipments from Earth, the long 6 to 9-month, low-energy journeys to Mars that have 2-year gaps between flights, suggest that using local Martian resources to grow food would be a better option, both from economic and safety perspectives.
It should be noted that the flight times with current rocket transport technology are similar to that of the sailing ships traveling from England to the Botany Bay colony in Australia in the late 18th century. The resupply ship arrived 2 years later, with the colony starving from inadequate food supplies and an inability to successfully farm. Local food production on Mars would ensure that adequate, high-nutrition foods are available and avoid any supply problems from Earth.
The lower ambient light levels on Mars are sufficient for photosynthesis for a large range of plants from unicellular algae to many terrestrial crops . Additional light if needed can be supplied with mirrors or artificial lighting. The question then becomes what sort of plants should be cultivated? The simplest plants, such as cyanobacteria, have been proposed as they have short lifecycles and rapid growth, requiring small production areas and a few basic nutrients. However, anecdotal evidence from supplying astronauts to the ISS indicates that food quality is a very important factor in astronaut well-being [46,47,48]. Experiments with celebrity chef-developed meals have proven the popularity of meals that are similar to those on Earth and that are tasty, not just nutritious. For crew and settlers staying for long periods on Mars with minimum 2-year rotations, foods that can be prepared with different cuisines to be cooked by a base chef or personally would seem to be preferable. Cyanobacteria and algal species may grow quickly and be technically nutritious. However, algae is not a completely nutritious diet and only Spirulina  has been shown to be useful as a meal supplement, used for less than 1% of the diet, and therefore should be considered as feedstuffs for animals and as soil amendments.
Growing conventional food hydroponically  is often mooted as the means to grow conventional crops. It has the advantage of having a pedigree of experience in terrestrial farms as well as experimental success in space. Hydroponic food production can be carefully controlled which makes it attractive to those of technical expertise. However, hydroponics requires substantial inputs of nitrogen and phosphorus which are usually applied directly from external sources, and not all plants can be successfully grown hydroponically. In addition, an expansion for a growing settlement will require either transporting equipment from Earth or finding ways to manufacture at least the simple components locally on Mars. A more attractive approach has been to try growing conventional crops in the Martian regolith. Experiments using regolith simulants  have shown that given added nutrients and light, a number of common terrestrial leafy crops can be grown.
The advantage of using the Martian regolith as a medium to grow conventional crops is that it provides the needed anchorage and potentially water retention medium used by terrestrial plants. Martian agriculture would work like terrestrial agriculture which is done in a greenhouse. On Mars, the atmosphere and temperature would be controlled to maximize crop growth and it is feasible that some animal species might be transported to produce the high-protein foods. For example, fish eggs could be transported and herbivorous fish species such as Tilapia could feed on the algae and convert it for human consumption. However, it should be noted that soils are not simple, but include ecosystems with a large number of species including bacteria, fungi, and animals from annelid worms to insects..
Despite the research done to date, there are considerable gaps in our knowledge concerning how Martian agriculture should proceed. The Martian environment is very cold, and dry, with a thin atmosphere around 0.1% of Earth’s, composed mainly of carbon dioxide with a little nitrogen. While aqueous algal growth experiments have been done in conditions that approximate some of the Martian conditions, it is not known which conditions must be tightly
controlled for good growth of the algae. For complex plants that are to be grown in either regolith or hydroponically, what partial pressure of CO2 in the atmosphere and at what pressure is needed to ensure healthy growth? Crops grow in different soils on Earth, from near desert sandy soils for millet to rich dark loams and different acidities for different crops. We take for granted the quality of terrestrial soils, but on Mars, the regolith is considered sterile, with no organic carbon content to retain water and provide an environment for soil organisms.
Given that these conditions can be evaluated on Earth, the big gap in our knowledge is the issue of remediation of the toxic levels of (per)chlorates in the Martian regolith. All of the various experiments on growth conditions assume that none of these toxic compounds are present. Powdered terrestrial rocks and more carefully constructed Mars Regolith Simulants are free of (per)chlorates and therefore experiments on plant growth assume the (per)chlorates are removed. With (per)chlorate levels that are far higher than any found naturally on Earth, they are at levels found around sites that manufacture munitions where the compound is used as an oxidant. The US EPA has guidelines for the remediation of soils contaminated by (per)chlorates .
Soils can vary, with plants varying in requirements for water, nutrients, soil carbon, soil organisms, pH, climate, and weather conditions. Nutrients and organic carbon will need to be added, as well as soil organism inoculants to improve the regolith to become a soil capable of good crop production.
To get an agricultural food system working, which factors are critical? How best to detoxify the regolith? How best to amend its properties? Which crops are best suited and at which stages?
A low-mass approach is to employ bacteria that can metabolize (per)chlorates and grow locally. (Per)chlorate metabolizing organisms are proteobacteria of which there are more than 40 species known. Dechloromonas and Azospira genera appear to be ubiquitous on Earth. They have different pH tolerances and some can function in acidic conditions as low as pH 5  The Martian regolith has up to 1% of (per)chlorate  which is far higher than any uncontaminated place on Earth. (Per)chlorate reduction only occurs in anaerobic conditions . This suggests that regolith remediation may need to be kept isolated from the crop-growing areas. Experiments with Moorella sp show that these bacteria can grow on a variety of reduced carbon sources, optimally at neutral pH and warm temperatures (40-70C) . None of the experiments have tested the (per)chlorate-reducing metabolic rates and growth of the various potential bacterial inoculants under conditions between Mars and human habitation, such as lower atmospheric pressure, gas composition, and water requirements. As these bacteria need a carbon source, how would that source be provided by chemical means or by biological carbon fixation?
There is considerable interest in using cyanobacteria as carbon-fixing microorganisms. These hold promise to weather the regolith, release nitrogen and phosphorus for growth, create organic carbon to improve water retention, and allow a richer variety of solid organisms that may be needed for crop growth. These cyanobacteria have been tested in a variety of conditions to determine how they will fare under conditions closer to that on the surface of Mars. Resting states of cyanobacteria suitable for transport from Earth indicate that UV exposure is not tolerated, although survival in a vacuum is good . Cyanobacteria do require a lot of water to suspend the rock dust and particles, In CO2-dominant atmospheres, full terrestrial pressure reduces growth, partly because of the lowered pH of the aqueous media, while 100 mbar appeared more favorable. Temperatures need to be maintained between 15 and 30C. Most important is the finding that cyanobacteria cannot survive in (per)chlorate-contaminated conditions, requiring its removal before growth . Extensive testing of bacteria has shown that while a few can survive down to the 7 mbar of the Martian atmosphere, most require at least 25 mbar. Nitrogen-fixing bacteria can fix atmospheric nitrogen to as low as 1 mbar, but the 2.8% of nitrogen in the Martian atmosphere would require increasing the total local atmospheric pressure 50x.
From this prior work, it is clear that there is a difficulty in remediating the Martian regolith from its toxic state to a soil suited for crop growth. (Per)chlorate-reducing bacteria require reduced carbon sources with nitrogen and phosphorus for growth to detoxify the regolith. Ideally, this could be supplied by cyanobacteria that fix the CO2 in the atmosphere and can release the nitrogen and phosphorus from the regolith. The cyanobacteria can also provide the organic carbon in the soil to support crop growth. However, these cyanobacteria cannot tolerate the toxic (per)chlorates. Lastly, both the (per)chlorate-reducing bacteria and the cyanobacteria need to grow in aqueous conditions with the regolith particles separated to allow rapid microorganism growth. The regolith would then need to be drained and allowed to dry out before being suited to most crop growth, although rice might be able to grow in a “paddy field” of regolith that has settled. This suggests that there may need to be separate areas for removing the (per)chlorates, supplying needed nutrients for the (per)chlorate-reducing bacteria, by cyanobacteria growing in pre-treated regolith.
The following outline experiments are suggested to fill the gaps in treating the Martian regolith to make it suited for growing crops for the Martian settlement.
The exposed Martian regolith is both too cold and dry, as well as relatively airless, for bacteria to detoxify the (per)chlorates. Ideally, the detoxification would take place in optimal growth conditions for the bacteria. Given that maintaining atmospheric composition and pressure, as well as water and humidity conditions, incurs a mass penalty, it is important to determine what are the factors that can be reduced towards Mars’ conditions to reduce this cost. This will help decide whether the detoxification process must be carried out in a greenhouse suited to growing conventional crops, or whether simpler management of the regolith is sufficient. Other questions are also evident, such as the level of detoxification necessary before crops can be successfully grown in the treated regolith.
This suggests several experiments to test for these factors:
1. Composition of Bacterial inoculant
There are many known terrestrial (per)chlorate-metabolizing bacteria, e.g. Dechloromonas that can metabolize oxygenated chlorines. All are anaerobes and therefore may function with the existing composition of the Martian atmosphere. Questions to be considered are:
a. Should the inoculant be a single species or multiple?
b. Do other species need to be included to create a viable ecosystem, or are single-species populations both sufficient and effective?
2. Atmospheric pressure
Mars’s atmosphere is about 0.7% of that of Earth. While too low to support crop plants, how much pressure is needed for bacterial growth to be maintained? Unlike plants, the bacteria are aquatic, and therefore the needed atmospheric pressure need only be sufficient to prevent water from boiling off. In a sealed reactor, water vapor will provide the needed pressure to maintain the equilibrium. As the bacteria are anaerobes, the regolith would seem likely to be processed in separate areas from the crop plants, with the detoxified regolith then added to the agricultural area in the greenhouse to increase the cultivation area.
3. Atmospheric composition
Mars’ atmosphere is primarily CO2 with a little N2. This is not suitable for crop plants, but how much of a factor is this for the bacteria? Combined with atmospheric pressure, what composition is needed for the bacteria? For example, does the nitrogen partial pressure need to be increased to supply the needed nitrogen for bacterial growth, perhaps in combination with nitrogen-fixing bacteria in the inoculant, or just added as ammonia or nitrate? [c.f. Item 1 concerning species in the inoculant.]
Lastly, bacteria need wet conditions to grow and multiply. How wet does the regolith need to be for the bacteria? Do the bacteria survive and grow in an aqueous slurry, or would high humidity conditions be sufficient, saving water resources needed elsewhere?
To test these, experiments will need to be set up in conditions to test these various requirements, most probably in containers to maintain the conditions. It is assumed that surface UV and ionizing radiation do not need to be tested as simple shielding will be sufficient to mitigate these factors.
These experiments are primarily devoted to extending the existing work done on (per)chlorate removal by bacteria [2,10,13,22], extending prior work. If the regolith detoxification and preparation for traditional crops is to be the goal, the regolith will need additional preparation for crops, including nitrogen, phosphorus, and carbon supplements. Inoculants may be required to allow nitrogen-fixing bacteria to grow in association with the root nodules of crops like green beans. Prior experiments [30,34,40] with cyanobacteria have demonstrated the extraction of nitrogen from the regolith, suggesting this approach to fertilize the crop plants after the regolith has been cleared of the toxic chlorate and (per)chlorate.
A stretch goal might include gene splicing experiments to extend the capabilities of some microbial species. Can the (per)chlorate-reducing genes be added to cyanobacteria removing the need for the bacterial species? Conversely, can genes to extract the nitrogen and phosphorus from the regolith be inserted into the bacteria? Can the (per)chlorate genes be edited so that the oxygen is liberated safely in the organism, allowing the (per)chlorate to become an oxygen source for the Martian settlement? Suggestions as to possible ideas have been mooted [40, 49].
To start processing Martian regolith for food production on Mars, there is a substantial gap in our knowledge on getting this process underway in the volumes needed compared to the small-scale lab experiments. Firstly, the regolith must be detoxified to remove the (per)chlorates. While the lab experiments demonstrate that various species of bacteria can metabolize the (per)chlorates, there are two limitations. Firstly, the regolith needs to be in powdered form to expose the surfaces to the bacteria and be turned into an aqueous environment for the bacteria to survive. How wet the slurry needs to be is unknown and therefore the water requirements are also unknown. Secondly, the sterile regolith provides no useful food supplies for the bacteria to grow. How to supply the nutrients and from what source needs to be determined. Terrestrial starter kits may be inadequate for bulk regolith processing.
Cyanobacteria have been demonstrated in the lab to be able to fix the atmospheric CO2 and grow while extracting the needed nitrogen, phosphorus, and trace elements from the regolith, but only after the (per)chlorates have been removed.
Terrestrial crops are yet another step away as they need detoxified regolith, fertilizers, and organic carbon in the “soil” to grow successfully, suggesting that both the (per)chlorate-metabolizing bacteria and the cyanobacteria must preprocess the regolith.
While the bacterial cultures grow in aqueous conditions, terrestrial crops do not and are therefore subject to even more critical issues of the surrounding atmosphere: pressures, and composition.
Currently, it appears as if the regolith can be prepared by iteratively starting with (per)chlorate-metabolizing bacteria, followed by cyanobacteria to grow and produce the needed food for a larger amount of regolith to be detoxified so that large volumes of regolith can be prepared for conventional crops to be grown. Once the regolith has been prepared it is turned over to the agronomists to determine how best to provide the conditions and associated organisms to cultivate crops to feed the base crew or settlers. While hydroponics is favored for supplying small populations with food, more conventional agriculture using local resources including the regolith seems more likely to be the preferred approach once large settlements start to appear.
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