Cultivating Sporeless Pleurotus ostreatus (Pearl Oyster) Mushrooms on Alternative Space-Based Substrates under Elevated Carbon Dioxide

A sporeless strain of the fungus P. ostreatus, the pearl oyster mushroom, was used in this study and was procured as production spawn grown on millet grain (strain SPX, Sylvan America, Inc., Kennet Square, PA). This sporeless strain was selected to eliminate potential contamination concerns of space vehicles and surface habitats from spore dispersal. Additionally, this sporeless strain was selected to mitigate the potential risk of allergic reactions that might arise in a subset of the population after prolonged exposure. A pure culture of the SPX strain was also procured and subcultured on potato dextrose agar (PDA, Fisher Scientific, Pittsburg, PA) and maintained for additional studies requiring a pure fungal culture.

Three ISS waste materials—100% cotton t-shirt rags and handkerchiefs, inedible biomass from several viable space crops, and astronaut food packaging plastic waste—along with two standard mushroom substrates of wheat straw pellets and soy hull pellets (MushroomMediaOnline, Rock Valley, IA) were used as substrates for the cultivation of SPX (Figure 1). The wheat and soy hull pellets (Figure 1A) came ready to use, but the waste substrates had to be slightly modified. The cotton t-shirt rags [Model # S-9980, Uline, Pleasant Prairie, WI] (Figure 1B) and handkerchiefs [15″ × 15″; SKU # 816594701, EEEKit, Walmart] (Figure 1C) were manually cut into small square pieces. The astronaut food packaging plastic waste came sealed with food, and thus, the food contents (Figure 1D) were removed from the packaging, leaving only the plastic waste made from composite polymers containing nylon, ethylene-vinyl alcohol copolymer (EVOH), and low-density polyethylene (LDPE) and minor food residues. The plastic was then manually cut into 1-cm square pieces [MB 110LHS, Winpak, Winnipeg, MB, Canada] (Figure 1E). The inedible biomass included root and leaf residues from several potential space crops including tomatoes, chili peppers, melons, cucumbers, lettuce, and leafy greens that were harvested, dried at 70°C for >48 h, and then blended into smaller pieces (Figure 1F).

Figure 1.

(A) Wheat straw and soy hull pellets mixed together. (B) Cotton t-shirt rags torn into strips as well as cut into small square pieces. (C) Handkerchiefs cut into small square pieces. (D) Astronaut food packaged in a rehydratable pouch made from a composite polymer containing nylon, EVOH, and LDPE. (E) Food packaging plastic waste (with food residue) cut into 1-cm square pieces. (F) Inedible biomass from various crops that were dried and ready to be processed.

For combining substrates into optimal recipes, the processed substrates were designated as either a primary carbon source or a primary nitrogen source (Bellettini et al., 2019; Osunde et al., 2019). The designated carbon sources were straw pellets, cotton clothing and handkerchiefs, and plastic waste, and the designated nitrogen sources were soy hull pellets and inedible plant biomass. Carbon- and nitrogen-designated substrates were paired into seven different recipes listed in Figure 2. Each substrate was weighed out by dry weight according to the seven recipe ratios (Table 1). For these trials, 4″ × 4″ × 15″ autoclavable, gusseted, 65-micron-thick polypropylene mushroom grow bags equipped with a 0.2-micron filter disc to allow gas exchange and prevent contamination were used [Field and Forest Products, Cat # SFDB10, Peshtigo, WI]. Each mushroom grow bag consisted of 227 g (dry weight) of total substrate and 454 mL (water weight) of added water to achieve a moisture content (MC) of 66.67% (Equation 1). MC=wet weight−dry weightwet weight→water weightwet weight→water weightwater weight+dry weight {\rm{MC}} = {{\left( {{\rm{wet}}\;{\rm{weight}} - {\rm{dry}}\;{\rm{weight}}} \right)} \over {{\rm{wet}}\;{\rm{weight}}}} \to {{{\rm{water}}\;{\rm{weight}}} \over {{\rm{wet}}\;{\rm{weight}}}} \to {{{\rm{water}}\;{\rm{weight}}} \over {\left( {{\rm{water}}\;{\rm{weight}} + {\rm{dry}}\;{\rm{weight}}} \right)}}

Equation 1: Moisture content of substrate where adding equal parts water and substrate would achieve a moisture content of 50%.

Figure 2.

List of substrate recipes and their carbon/nitrogen ratios inside their respective grow bags before sterilization. (A) Recipe 1—70/30 Straw/Soy. (B) Recipe 2—70/30 Cotton/Soy. (C) Recipe 3—70/30 Straw/Inedible. (D) Recipe 4—70/30 Cotton/Inedible. (E) Recipe 5—50/50 Cotton/Inedible. (F) Recipe 6—100 Cotton. (G) Recipe 7—40/30/30 Cotton/Inedible/Packaging. (H) Aerial view of sterilized and inoculated grow bags inside a dark spawn run container.

Each bag was shaken and mixed prior to hydrating to ensure homogeneity. All substrate preparation was performed within the mushroom grow bags to minimize loss of material. Once the substrate was saturated and no free water remained, each bag was folded over, autoclaved for 1 h at 14.5 psi at 121°C via a 20-min exposure liquid cycle, and allowed to cool overnight in a BSLII safety hood. Three bags (replicates) for each recipe were prepared in this manner.

Inoculation of the substrate with the SPX spawn was performed underneath a BSLII safety hood. An inoculation rate of 5% was used based on the wet weight of the substrate after autoclaving. Grain spawn was added at once and mixed throughout. Once all bags were inoculated, each one was sealed using a laboratory heat sealer and placed into a storage container for incubation.

Following inoculation, the sealed cultivation bags were placed vertically in a dark, well-ventilated, opaque storage container at 24°C ± 1°C to allow the fungus to colonize the substrate. This is known as the spawn run. The spawn run was allowed to develop for 28 days. Temperature and humidity data were monitored during the spawn run using a Govee device [Model # B5041, Govee, Hong Kong]. Each bag was scored for % colonization every other day using a 5-point rating scale: 1 = 0–5% mycelial colonization, 2 = 6–30% colonization, 3 = 31–69% colonization, 4 = 70–94% colonization, 5 = 95–100% colonization. Photographs were also taken every other day and at inoculation and harvest to visually record mycelium colonization.

Once the 28-day spawn run was completed, the bags were then transferred into a customized cultivation chamber (Supplementary Figure 1) to initiate fruiting. This was done by slicing several small holes in the growth bags to release the buildup of CO₂ inside the bags and create outlets for mushrooms to develop from. The chamber was subjected to the following conditions: 24°C ± 1°C, relative humidity at 85–95%, CO₂ concentration at 1200–1500 ppm, and a photoperiod of 10 h/day with a light density at 15–350 lux. Increased relative humidity, decreased CO₂ concentration, and increased light exposure can act as stimulants for fruiting initiation. Temperature can also be modified but was not carried out for this study. Temperature, relative humidity, and CO₂ data were monitored using a Govee device [Model # B5041, Govee] and an Autopilot Desktop CO₂ Monitor & Data Logger [SKU# APCEM2, Hydrofarm, Shoemakersville, PA]. Internal chamber temperature was maintained by ambient laboratory temperature of 24°C ± 1°C; relative humidity was increased to 85–95% via standing water, manual misting, and placement of wet rags near fruiting bags; and CO₂ was passively released through the filtered lid. No active (energy-required) environmental controls were used in the cultivation chamber. After 14 ± 1 days, mushrooms were harvested, fresh weight was recorded, and food safety analyses were conducted.

To investigate SPX’s ability to colonize substrate from agar plates, standard PDA petri plates were incubated at room temperature for 15 days. Three 4″ × 4″ × 15″ bags [Field and Forest Products, Cat # SFDB10, Peshtigo, WI] filled with 1 lb of 70/30 wheat/soy substrate were each inoculated with SPX mycelium that colonized the entirety of a standard PDA petri plate as a single layer. The spawn run was allowed to proceed for 23 days before fruiting was initiated. After 10 days, mushrooms from each of the three bags were harvested and fresh weights were recorded.

Microbiological food safety analysis was conducted on each recipe that resulted in mushroom growth. From each recipe, 25 g of sample was collected and placed in a sterile blender bag containing 225 mL of tryptic soy broth to dilute and enrich possible bacterial growth. The bags were placed in a bag mixer and samples were homogenized for 2 min. Sample extracts were plated onto selective media for Escherichia coli (E. coli)/coliforms and Staphylococcus aureus (S. aureus) [E. coli/coliform and Staph Express Petrifim, 3M, St. Paul, MN] according to manufacturer instructions.

Screening for Salmonella sp. was done by incubating the buffered peptone water (BPW) with the macerated sample at 35°C for 24 h followed by selective enrichment in which 1 mL of incubated extract was transferred into 5 mL of Rappaport-Vassiliadis (RV) broth [Thermo Fisher Scientific, Waltham, MA, USA] and incubated for 24 h at 35°C. The broths were then streaked onto Hektoin-Enteric selective media for Salmonella and incubated at 35°C for 24–48 h.

For the enumeration of aerobic bacteria, BPW from the sample mix was serially diluted in sterile BPW and 100 mL of appropriate dilutions was plated and spread onto duplicate plates of tryptic soy agar (TSA). Plates were incubated at 30°C for 48 h for bacteria followed by colony enumeration.

Total coliform, S. aureus, and E. coli were tested using the following methods from the Association of Official Analytical Chemists (AOAC): Total coliform/E. coli (AOAC 991.14) and Staph. Express System (AOAC 2003.07, AOAC 2003.08, and AOAC 2003.1). Methods for food safety screening and enumeration were adapted from the FDA bacteriological analytical manual [FDA U.S. Food and Drug Administration, 2018].

Because of dimensional limits in the designed fruiting chamber, the experiment was conducted as a randomized complete block design (RCBD) with blocks (n = 3) represented as experimental runs in series and treatments represented by substrate recipes (n = 7). One-way analysis of variance (ANOVA) was conducted on the yield and biological efficiency results using R Studio.

Data from yield and microbiological counts (log-transformed) were compared using a one-way ANOVA followed by Tukey’s multiple comparisons tests using GraphPad Prism version 10.0.0 for Windows [GraphPad Software, San Diego, CA, USA].

Mycelium colonization occurred on all seven substrate recipes across the 28-day spawn runs. Data revealed that recipe 1 and recipe 2 colonized faster and more completely than all other substrates, with recipe 2 performing the best (Figure 3; Supplementary Figures 2, 3, 4, and 5; and Supplementary Data). The colonization coverage for recipe 3 (70/30—Straw/Inedible) in the first 14 days across trials was slow, but by day 28, colonization was near complete. Recipe 4 (70/30—Cotton/Inedible) started with fast colonization and slowed near the middle of the grow-out but rapidly increased towards the end of the spawn run. Recipe 5 (50/50 - Cotton/Inedible), Recipe 6 (100 - Cotton), and Recipe 7 (40/30/30 - Cotton/Inedible/Plastic) had moderate colonization in the first 14 days of colonization but had the lowest colonization by the conclusion of the spawn run. No signs of contamination or unwanted growth occurred in any of the recipes during the colonization stage. Regardless of substrate or rate and coverage during the spawn run, all seven recipes experienced >60% colonization.

Figure 3.

(A) Examples of the colonization rating scale of SPX mycelium during the spawn run on wheat and soy pellets (control). (B) Detailed characteristics of the colonization coverage scale. (C) Graph of the average mycelium colonization scale for all seven substrate recipes in all three replicates. Error bars represent the standard deviation across the three replicates. Detailed colonization ratings can be found in the Supplementary Data.

Although all substrate recipes experienced colonization, only recipes 1–4 successfully fruited while recipes 5–7 showed no signs of fruiting (Figure 4). In terms of fresh weight, recipe 2 performed best, with fresh weight yield averaging 136.58 g (Figure 5A). Recipes 1, 3, and 4 had average fresh weight yields of 96.86 g, 57.71 g, and 39.57 g, respectively. One-way ANOVA with Tukey’s correction showed that recipe 2 with average yields of 136.58 g was not significantly different from recipe 1 but was significantly higher (P < 0.05) than those of recipes 3 and 4 (Figure 5B).

Figure 4.

Fruiting representation on day of harvest for Rep 1. (A) Recipe 1. (B) Recipe 2. (C) Recipe 3. (D) Recipe 4. (E) Recipe 5. (F) Recipe 6. (G) Recipe 7. (H) Aerial view of mushroom grow bags inside the cultivation chamber prior to harvest.

Figure 5:

Comparative yield of mushrooms and biological efficiency from various waste substrates. (A) Graphical representation of the average fresh weight (g) harvested from each substrate recipe that fruited. Error bars indicate the standard deviation from all three replicates. (B) One-way ANOVA with Tukey’s correction for the average fresh weights from recipes 1 to 4. (C) Graphical representation of the average biological efficiency percentage from each substrate recipe that fruited. Error bars indicate the standard deviation from all three trials. (D) One-way ANOVA with Tukey’s correction for the calculated biological efficiency from recipes 1 to 4.

In addition to fresh weight yield, the biological efficiency was calculated (Figure 5C). Biological efficiency (Equation 2) was utilized since not all the substrates had the same density; however, it indicates the same trends as harvest yield since each recipe contained 227 g of dry substrate, making it a common denominator. Biological Efficiency %=Weight of fresh mushroom fruiting bodiesWeight of dry substrate×100 {\rm{Biological}}\;{\rm{Efficiency}}\;\% = {{{\rm{Weight}}\;{\rm{of}}\;{\rm{fresh}}\;{\rm{mushroom}}\;{\rm{fruiting}}\;{\rm{bodies}}} \over {{\rm{Weight}}\;{\rm{of}}\;{\rm{dry}}\;{\rm{substrate}}}} \times 100

Equation 2: Biological efficiency equation where the numerator equals harvest yield of each recipe, and the denominator equals 227 g of dry substrate.

The calculated biological efficiency trends reflected that of fresh weight. Recipe 2 performed best, with biological efficiency percentages averaging 60.17% (Figure 5C). Recipes 1, 3, and 4 had average percentages of 42.67%, 25.42%, and 17.43%, respectively (Figure 5C and Supplementary Data). However, one-way ANOVA with Tukey’s correction showed that none of the recipes were statistically significant from another (Figure 5D).

The first signs of fruiting differed for each substrate recipe and the fruiting stage lasted between 13 and 15 days (Table 2). In Rep 1, recipe 2 showed the first signs of fruiting followed by recipes 1, 3, and 4. These results were indicative of the final yield for each recipe as the recipe that fruited first subsequently produced the most yield. In Rep 2, recipe 1 showed the first signs of fruiting followed by recipes 2, 3, and 4. In Rep 3, the order was similar to Rep 2, except recipe 4 unexpectedly fruited second, with recipes 2 and 3 following it. However, unlike the trend in Rep 1, Rep 2’s largest and smallest yielding recipes both fruited last, while Rep 3’s largest and smallest yielding recipes both fruited in the middle of the set (Table 2). Early fruiting indications recorded were the number of pins and clusters from each substrate recipe in each of the replicates. Recipes 1 and 2 produced the most clusters and pins while recipes 3 and 4 produced just over half as many (Supplemental Data).

SPX subcultures on PDA plates were used for 70/30 wheat/soy substrate inoculation to see if fruiting could be achieved without the use of grain spawn (Figure 6). All bags produced more than 100 g of mushrooms with an average yield of 164.39 g and an average biological efficiency of 36.24%.

Figure 6.

Comparative yield analysis of SPX on 70/30 Straw/Soy 1lb substrate bags when inoculated with mycelium from a single layer of fully colonized standard PDA plates.

Screening for selected food-borne pathogens yielded negative results in mushrooms grown in all substrates (Figure 7). Aerobic bacterial plate counts (APC) ranged from 1650 to 6.15 × 10⁵ CFU per gram in mushrooms from recipe 4. Counts from mushrooms from all other recipes fell within this range (Figure 7). There were no significant differences in bacterial counts between recipes.

Figure 7.

Bacterial APC on TSA from mushrooms grown on four different substrates. Substrates were mixed 70/30 for each combination (recipes 1–4). Bars represent minimum and maximum values. Mean is shown by +, n = 3.

The results generated from this investigation explore the feasibility of using ISS-generated waste to produce mushrooms, thereby reducing the amount of upmass needed for additional food production. Although this process is still very early in the pipeline, data from this investigation suggest that it is feasible to generate food from ISS waste. A comparison of the fresh weight harvest from recipes 1–4 shows that cotton textiles are a promising waste substrate for oyster mushroom cultivation. Moreover, recipe 2 (70/30—Cotton/Inedible) performed better than recipe 1 (70/30—Straw/Soy), which contains straw, a standard in the commercial mushroom industry, rather than cotton textiles. Although recipes 3 (70/30—Straw/Inedible) and 4 (70/30—Cotton/Inedible) yielded some mushrooms, the production was much less than in recipes 1 and 2. Recipes 1 and 2 contain soy hulls as the nitrogen component whereas Recipes 3 and 4 contain inedible plant biomass, but the same carbon components. This suggests that the inedible plant biomass does not function as a nitrogen component as well as soy hulls. However, these results do demonstrate that cotton textiles can be utilized as a substrate for oyster mushroom production in elevated CO₂ environments and can perform better than traditional substrate like straw. Despite recipes 5–7 not fruiting, >60% colonization is a promising finding and warrants further investigation into optimizing the substrate recipe ratios for oyster mushroom production.

Colonization rates among substrates revealed a similar trend to mushroom yields. Strong colonization was achieved with recipes 1 and 2. Close inspection of the bags during the spawn run suggests that recipes 4, 5, and 7 could have had areas with reduced mycelium colonization where inedible biomass was over-saturated or not properly mixed. Similarly, recipe 2 had areas with reduced mycelium colonization where soy was over-saturated. Recipes 1, 3, and 6 performed better with homogeneous colonization, as recipes 1 and 3 had more particulate substrates while recipe 6 only consisted of one substrate. Comparing the strong colonization coverage of recipe 6 to the weak colonization coverage of recipe 5 indicates that some type of middle ground is likely to be effective. Recipe 7’s weak colonization coverage was hindered by the buildup of unabsorbed water and led to eventual pockets of mild contamination once fruiting initiation was exposed to the external environment; however, the presence of any colonization at all is still promising and warrants an opportunity to reinvestigate with a reduced moisture content to accommodate for the use of impermeable plastic waste. Additionally, ensuring adequate homogeneity and reducing particulate size in recipes with various-sized substrate pieces could prevent the oversaturation of nitrogen in isolated areas and increase substrate colonization amounts for each recipe.

The subcultures of SPX cultivated on PDA provided a baseline growth timeline for SPX spawn run and its ability to fully colonize a substrate without the use of grain spawn (Figure 6). The elimination of grain spawn for mushroom production greatly simplifies procedures relevant to space applications because grain spawn violates a weight penalty, may act as a potential source of contamination, and may be a particulate matter safety concern. The results of successful colonization and production using agar spawn offers promising preliminary data for more simplistic SPX mushroom production cycles that are still within a 35-day growth period (not including the 12-day agar incubation time).

Bacterial counts between 5.2 and 12.5 log CFU per gram have been reported on white button mushrooms after harvest (Rossouw and Korsten, 2017), and counts have been shown to increase significantly after harvest and during storage (Siyoum et al., 2016). Another study reported counts between 5.0 and 5.3 log CFU per gram in oyster mushrooms, the species variety tested in this work (Wang et al., 2017). All bacterial counts in our study fell below 5.8 log CFU per gram (6.15 × 10⁵), lower than counts found in other studies on commercially grown and market mushrooms (Venturini et al., 2011; Schill et al., 2021; Ban et al., 2022). While crops grown for astronaut consumption have no specific microbiological standards, comparison to the NASA standards for non-thermal stabilized foods is taken into consideration (Perchonok et al., 2012; Khodadad et al., 2020). No E. coli, Salmonella, or S. aureus were found in the mushrooms we tested, which meets the NASA standards and indicates a safe product for consumption. The bacterial loads in the mushrooms tested in this work are higher than the NASA standard of <2 × 10⁴ CFU per gram for APC (Perchonok et al., 2012); however, these values are for processed, non-thermo-stabilized foods and do not apply to freshly grown products. A comparison of market fresh and stored products is more relevant for the evaluation of the food safety of these mushrooms grown in different substrates.

This study investigates a sporeless cultivar of P. ostreatus, addresses the weight penalty of substrates by utilizing existing ISS waste streams, and demonstrates the ability to colonize substrate from low volumes of agar spawn. However, it fails to address key challenges to mushroom growth in space such as understanding the impacts of higher CO₂ and lower RH environments on fruiting; overcoming these harsh ISS growth conditions through engineering controls or environmental manipulation; accounting for mushroom respiration and competition for oxygen between crop and crew; addressing substrate sterilization, saturation, and preparation challenges in microgravity; and understanding the full impact of microgravity, including the lack of convection, on mycelium and fruiting body development.

The average CO₂ level on board the ISS is approximately 3000 ppm while the RH level ranges from 40% to 75% (IECLSSIS, 2019). Although this environment should not impede mycelium growth and colonization of contained substrates, it will have an impact on fruiting and mushroom development (Lin et al., 2022). The morphology of the SPX mushrooms grown in this study differed from the morphology of standard oyster mushrooms, exhibiting trumpet-like cap growth and stem elongation. This distinction in morphology is likely due to the elevated CO₂, which was approximately 1200 to 1500 ppm during this investigation. Mushroom morphology and yield can be affected by various environmental conditions such as CO₂, RH, substrate moisture content, temperature, airflow, and lighting (Jang et al., 2003; Whitmore, 2024). Thus, more work needs to be dedicated to cultivating SPX in elevated CO₂ and reduced RH environments, utilizing gradients of both CO₂ and RH to investigate the effects of these environmental parameters on mushroom yield, morphology, physiology, and nutrition. Once better understood, engineering controls to accommodate these adverse environmental conditions can be tested, such as increasing the RH around the fruiting body or decreasing the temperature of the growth chamber.

O₂ consumption is another unique challenge as the majority of space crop and bioregenerative life support studies in the past have dealt with photosynthetic organisms that do not consume significant cabin oxygen (Zabel et al., 2016). Mushrooms respire as humans do and they will be competing with astronauts for oxygen, which is already a limited resource in an abnormally elevated CO₂ environment (Cronyn et al., 2012). A lower consumption of O₂ has been achieved when interplanting mushrooms with plants, and with additional experimentation and optimization, a stable polyculture system could be implemented that eliminates the buildup of both oxygen and carbon dioxide (Jung, 2017; Hamed et al., 2021; Gutierrez-Jaramillo et al., 2021; Gellenbeck et al., 2023). This is particularly applicable to current closed unmanned plant production system concepts, as plant-produced oxygen is not utilized by the crew and creates the concern of O₂ buildup as a potential fire hazard (Morrow et al., 2016). More investigation needs to be done on the aerobic rate and CO₂ production of oyster mushrooms over their entire life cycle so that the species can be modeled and compared to other living organisms. If the aerobic rate is similar to a human, it is more problematic than if it is similar to mice, organisms already present on the ISS.

Substrate sterilization, preparation, and saturation will likely be more difficult in microgravity. However, certain aspects such as saturation and mixing could potentially be assisted by microgravity via engineering controls like water injection ports and sealable bags. Sterilization is energy intensive and a capability currently lacking on board the ISS. However, sterilization requirements can be greatly reduced for oyster mushrooms due to their aggressive growth characteristics (Sekan et al., 2019, Sánchez, 2009). This is especially true when paired with a substrate such as cotton textiles that inhibit the growth of competing microbial contamination. Chemical sterilization treatments such as 0.3 % hydrogen peroxide, formaldehyde, or hydrated lime (calcium hydroxide) exist that are less energy intensive but slightly less proficient (Cotter, 2014; Shrestha et al., 2021). Additionally, novel sterilization methods such as the use of cold plasma have been investigated and show promise for future space applications (Agun et al., 2021; Engeling, 2021; Gott et al., 2022). Thus, further investigations on techniques, technology, and practices will need to be conducted to address these challenges.

Fluid physics in space is a difficult challenge in space crop production and ample work has been devoted to overcoming it (Jones and Or, 1998; Monje et al., 2003; Porterfield et al., 2003; Heinse, 2015). The advantage of mushrooms is that they are simplified biological systems that do not require frequent fluid transport via evapotranspiration like plants do. They require a one-time saturation and gradually lose water over time. However, the margin of error for added water could be critical as hypoxia is a common concern for plants in space due to lack of seepage and oxygen diffusion via convective airflow (Stout et al., 2001; Poulet et al., 2016). Lack of convection should not pose a challenge for mycelium colonization but could pose a significant challenge for fruiting. This might be resolved with engineering controls such as forced convection. Ultimately, growth experiments both terrestrially and in simulated or real microgravity are needed to further investigate these potential challenges and their respective solutions.

The primary objective of this study was to utilize the aggressive growth characteristics, high biological efficiency, and short growth period of oyster mushrooms to produce a nutritious food crop in space from otherwise unutilized waste in a minimally controlled environment. Oyster mushrooms are a good source of nutrients currently lacking in the astronaut diet, specifically the vitamin B complex, antioxidants, and fatty acids (Raman et al., 2020; Tang et al., 2021; Effiong et al., 2024), and have medicinal uses due to their anti-carcinogenic, anti-inflammatory, anti-hypercholesteremic, anti-viral, and immune-stimulating properties (Patel et al., 2012; Golak-Siwulska et al., 2018; Mishra et al., 2021). Numerous studies have demonstrated oyster mushrooms as a valuable crop for both nutritional and medicinal purposes. However, the nutritional content of these mushrooms when grown under space-relevant conditions or waste streams is an important aspect to investigate to ensure oyster mushrooms’ potential for supplementing a space crew’s diet. Nutritional qualities of most interest include protein and other proximates (fiber such as beta-glucan), antioxidants, vitamins (both fat and water soluble, with an emphasis on the vitamin B complex), and amino acids such as ergothioneine.

Secondly, when the mycelium colonizes the waste substrates, it breaks down the complex organic waste components into smaller molecules. The ability to recover bioavailable nutrients from the SPX spent waste substrate is another crucial analysis needed to complete the picture of mushrooms as a component of the BLiSS cycle. Much work has demonstrated oyster mushroom’s ability to breakdown substrates, recover nutrients, and utilize spent substrate for many applications (Sakellari et al., 2019; Koutrotsios et al., 2020; Mortada et al. 2020; El-Ramady et al., 2022; Martín et al., 2023). However, this same ability needs to be investigated for SPX and the waste substrates it colonizes. Elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS) for elemental isotopes such as potassium, magnesium, calcium, sodium, and various minerals; molecular analysis via ion chromatography (IC) for anions and cations such as sulfates, phosphates, nitrates, nitrites, and ammonia; and total organic carbon (TOC) for analysis of decomposed carbon should all be assessed.

Current and past NASA initiatives, such as the Trash-to-gas project and the Biomass Production Chamber (BPC), have investigated recovering nutrients from waste streams for plant cultivation. Trash-to-gas works to reduce, reuse, or recycle space-derived waste streams via thermal degradation, and one of their recent initiatives is using the ash residue that results from their gasification processes, combined with inedible biomass, to act as a growth substrate for plants. The BPC, which operated successfully from 1988 to 2000 under NASA’s Controlled Ecological Life Support System (CELSS) program, grew various crops hydroponically via the nutrient film technique (NFT) and explored the ability to recover nutrients from the produced inedible biomass via physical leaching or biological methods and feed it back to plants (Mackowiak et al., 1996, 1997; Wheeler et al., 1996; Lunn et al., 2017). Since pearl oyster mushrooms are a primary decomposer and a white rot fungus, they have the potential to degrade some of these currently utilized waste streams more effectively or with less energy or pretreatment, while simultaneously producing a highly nutritional food output during the degradation process. Therefore, understanding these processes of recovering bioavailable nutrients from the spent mushroom substrate for further plant cultivation will enable a more efficient way to recycle waste and ensure more sustainable BLiSS systems.

Thirdly, the innovative use of fungi to enable mycoremediation and mycofabrication has diversified its utilization for space-based applications. P. ostreatus has been used as a green biotechnology for remediating soil and water pollutants such as heavy metals, petroleum solid wastes, industrial wastes, pharmaceuticals, and pesticides (El-Ramady et al., 2022; Kapahi and Sachdeva, 2017). Additionally, owing to their excellent waste recycling capabilities and diverse growth conditions, oyster mushrooms (and their spent substrate) have played a key role in soil generation and biofermentation through the production of enzymes, biofertilizers, biomass, bioethanol, and animal feeds (El-Ramady et al., 2022; Kapahi and Sachdeva, 2017). Oyster mushrooms have even been used for the mycofabrication of mycelium-based composites, although this application is less common (Attias et al., 2019; Kohphaisansombat et al., 2023). When we look beyond the limits of oyster mushrooms, however, fungi have been considered for producing mycelial materials to construct extraterrestrial habitats and structures due to their self-replicating, flexible, insulating, non-volatile, fire-retardant, regolith-compatible properties and for secreting glues or plastics to form biocomposites (Rothschild et al., 2019; Brandić Lipińska et al., 2022). The fungus kingdom encompasses an enormous diversity of taxa with varied ecologies, life cycle strategies, and morphologies, and oyster mushrooms represent only a small sample of these versatile end uses and products. Much work can be done in exploring this entirely new kingdom’s application and viability to space.