Impact of Light Spectra and Substrate Composition on the Bioefficiency, Nutritional Content, and Morphology of Oyster Mushrooms

Abstract

Mushrooms are commercially cultivated in controlled environment agriculture facilities in which the parameters of temperature, humidity, and CO₂ are closely controlled. In contrast to plant production, variable lighting is generally not a parameter that mushroom producers utilize. In this study, P. ostreatus, the pearl oyster mushroom, was cultivated under one of three LED light spectra: blue (450 nm), red (625 nm), or white (broad spectrum) at an intensity of 5 μmol m⁻² s⁻¹. Substrates used for production consisted of a 70/30 mixture of straw/cottonseed or straw/mesquite bean pod, all of which were locally sourced in Arizona. Bioefficiency (BE), nutrient profile, and morphology were assessed post-production. Light spectra had no significant effect on BE, beta glucan, total amino acids, or total antioxidant content. However, red light exposure increased the number of caps per cluster by 197% and reduced cap diameter by 55%. The straw/cottonseed substrate significantly increased BE by 77% over the straw/mesquite substrate, increased levels of total protein by 9%, and increased levels of glutamic acid, arginine, and histidine by 11%, 24%, and 33% respectively. Interestingly, the straw/mesquite substrate resulted in a significant increase in total amino acid and beta glucan content over the straw/cottonseed substrate by 8 and 18%, respectively. These results illustrate how light spectra and substrate mixture can significantly impact nutritional value and production qualities of oyster mushrooms.

1. Introduction

The production of edible mushrooms worldwide increased from 37 million tons in 2015 to 50 million tons in 2023 [1]. Several species of mushrooms account for about 90% of global mushroom production, including Agaricus bisporus (button mushroom), Lentinula edodes (shiitake), Pleurotus spp. (oyster mushroom), Auricularia spp. (wood ear mushroom), Volvariella volvacea (paddy straw mushroom), and Flammulina velutipes (enokitake) [2]. The production of oyster mushrooms accounts for about 27% of total global mushroom production [3], and high yields are achieved on a variety of natural substrates and agricultural residues. Oyster mushrooms are popular with consumers due to their exceptional culinary value, but also for their healthful nutritional profile, which includes low carbohydrate and fat contents [4] and an excellent source of protein and several essential amino acids, such as glutamine, arginine, and leucine [5]. In addition, they are high in antioxidants that mitigate oxidative stress and reduce the risk of cardiovascular disorders and certain forms of cancer [6,7].

Oyster mushrooms are a wood-decaying fungus and use a variety of enzymes to break down recalcitrant substrates rich in cellulose and lignin [8]. As such, they are capable of growing on a diverse range of agricultural by-products, such as straw, wood sawdust, corn cobs, cottonseed, rice hulls, soy hulls, and many others [9]. The production of mushrooms on local agricultural by-products improves the resource use efficiency of many agricultural systems and provides economic value by producing an additional food product while reducing the amount of crop residue and food processing waste to be disposed [10]. For example, the production of a single 217 kg bale of cotton produces 336 kg of unused organic materials [11], which are ideal for the cultivation of many mushroom species. Additionally, mesquite trees (Prosopis spp.), native and abundant in southern Arizona and other desert regions, produce large amounts of edible bean pods annually at a rate of 10–50 kg tree⁻¹ year⁻¹ [12]. These pods are high in nitrogen and can provide this essential nutrient for mushroom cultivation [13,14]. However, without substantial regional and commercial use, enormous volumes of these pods are deposited in the local landfills every year [12]. The recycling of otherwise discarded materials as a mushroom substrate could improve the economic and environmental aspects of production for many cropping systems and may improve the nutritional and chemical contents of the resulting mushroom fruiting body [15,16].

Most modern commercial mushroom production occurs in controlled environment (CE) facilities where environmental variables of temperature, humidity, and CO₂ are closely monitored and controlled. Variable light spectra using light emitting diodes (LEDs) have been successfully used in CE agriculture for plant production, with notable improvements in yields, nutrition, and quality [17,18,19]. Subsequently, interest has turned to using specific wavelength LEDs in mushroom production to similarly improve yield, morphology, and nutritional aspects of the fruiting body [20,21,22]. These types of responses in plant and fungi, including P. ostreatus, are triggered by photoreceptors, such as phytochromes, cryptochromes, opsins, and white collar proteins (WC), each sensitive to different wavelengths of light and regulating various aspects of development [23]. Phytochromes respond primarily to red and far-red light, and in fungi, these influence processes of spore germination, development of reproductive structures, and the production of secondary metabolism [24]. Cryptochromes are sensitive to blue and near-UV light and in fungi, these regulate photomorphogenesis, circadian rhythms, and suppression of sexual development [25]. Although the use of LED illumination in plant production is well established, these techniques are just now being used in the mushroom industry.

Although blue light (~450 nm) has been reported to enlarge fruiting bodies or increase yields in P. eryngii [26]., P. ostreatus [21], and Ganoderma lucidum [27], the evidence base linking spectra to bioefficiency and/or morphology is sparse and often compared to a broad-spectrum white, such as fluorescent lighting. However, studies vary in species, spectral definitions, light intensity, and light duration, producing conflicting results. For example, P. ostreatues grown under blue light were identical and yielded the same amount as those grown under conventional methodologies using white fluorescent [20]. There is a gap in the knowledge of how narrow-emission light sources and substrates interact to impact production quantity and quality.

The objective of this study was to investigate the effect of light spectra and substrate composition on yield, nutrient profile, and morphology of P. ostreatus produced under standard commercial conditions. We hypothesize that (1) oyster mushroom production on the mesquite substrate will perform equally to that of the cotton substrate, and (2) blue light will increase the nutritional elements of oyster by 10% without impacting yield. Additionally, a broader goal of this research was to establish the utility of underutilized plant residues in Arizona as locally-derived substrates for local mushroom production to create a nutritionally enhanced and resource-efficient food source.

2. Materials and Methods

2.1. Fungal Culture, Spawn, and Substrate Production

The strain of pearl oyster mushroom, P. ostreatus, used in this study was obtained from Fungi Perfecti, LLC (Olympia, WA, USA) and vegetatively propagated in Petri dishes containing glucose yeast extract agar (GYEA; 10 g L⁻¹ glucose, 2.5 g L⁻¹ brewer’s yeast, and 15 g L⁻¹ agar) at 23 °C. Cultures were transferred regularly onto fresh media until needed for spawn production.

Mushroom spawn used for substrate inoculations was generated on cracked corn (O. H. Kruse, Ontario, CA, USA) purchased from a local feed store. For spawn preparation, corn was soaked in tap water overnight, rinsed 3 times in tap water, and drained for an additional 30 min before packing 1900 g into 1 L polyethylene mushroom grow bags with a 0.5 µM filter patch (Unicorn Bags, Plano, TX, USA) with the tops closed with 10 cm plastic twist-ties. Once packed, the bags were autoclaved at 121 °C for 75 min. For inoculation, 2 Petri dish plates of the P. ostreatus culture were transferred to each bag of autoclaved corn, closed again with twist-ties, and coarsely mixed. Following inoculation, the bags were incubated at room temperature (20 °C) for 2–3 weeks until fully colonized.

The production substrate consisted of either a straw/cottonseed or straw/mesquite pod mixture. Wheat straw was obtained from several local agricultural feed stores. Whole, de-linted cottonseed hulls were locally obtained from the University of Arizona’s Maricopa Agricultural Center, and the mesquite pods were locally collected by the University of Arizona campus groundskeeping services. The composition of cottonseed consisted of approximately 24.37% crude protein, 18.71% crude fiber, 5.85% ash, 24.81% lipids, and 19.3% carbohydrates [28]. The mesquite pod composition included 9.26% protein, 36.24% dietary fiber, 3.05% minerals, 1.5% lipids, and 49.95% carbohydrates [29]. The straw and mesquite pods were both processed through a landscape chipper/shredder to increase the particle surface area for hydration before packing. The substrate mixtures consisted of 70% straw and 30% cottonseed or mesquite pods (wt/wt) and were prepared in batches of 2400 g dry weight. These batches were soaked for 18 h and drained for thirty minutes to achieve a moisture content of approximately 70%. Following soaking, 1900 g of a hydrated substrate was packed into 1 L mushroom grow bags, closed tightly with 10 cm plastic twist-ties, and autoclaved at 121 °C for 75 min. Bags of the autoclaved substrate were inoculated with 100 g of grain spawn per bag, closed tightly again with twist-ties, and then mixed thoroughly. Inoculated bags were incubated at room temperature for 2–3 weeks until fully colonized.

2.2. Mushroom Production

After incubation, colonized bags were moved to shelving units in an opaque double-walled hoophouse for the fruiting stage. The temperature was maintained at 20° C using evaporative cooling, and relative humidity was maintained as 85% RH with supplemental humidification using ultrasonic atomizers (House of Hydro, Ft. Myers, FL, USA). Ventilation was supplied to maintain the CO₂ level between 400 and 700 ppm. For each substrate type, replicate bags were placed on individual shelving units surrounded by opaque barriers and illuminated by LED lights under one of three spectra: blue (450 nm), red (625 nm), or white (400–650 nm). The experiment was conducted in a 2-factorial (spectra × substrate) randomized complete block design (RCBD) with 3 blocks and 4 bags per treatment in each block. The lighting system was custom-built using tunable RGB light emitting diodes (LEDs), spaced to ensure the surface of the bags received 5 µmol m⁻² s⁻¹ of the given spectra. A spectroradiometer (PS-300, Apogee, Logan, UT, USA) was utilized to ensure the same wavelengths and photon flux densities were achieved for each trial. The spectra data is available in the Supplementary Materials. Black polyethylene sheeting was used to fully separate the lighting treatments within the hoophouse. After placement in the lighting treatment, six 2.5 cm slices were made on two opposite sides of each grow bag to allow fruiting bodies to emerge. The lights were on for the 28 days that the bags remained on the shelves.

Mushrooms were harvested as complete clusters, beginning on day 10 post-slicing and continuing daily until day 28. Only mature mushroom clusters, with most caps fully expanded but before spore drop, were harvested each day. Clusters were harvested by cutting the entire cluster off at the bag surface, and the combined cluster weight per bag was recorded. Following weighing, the number of clusters per bag was recorded, as well as the number of caps ≥ 2 cm per cluster. For each cap ≥ 2 mm, the cap diameter was determined by measuring across two axes and averaging the two values. All experiments were repeated twice.

2.3. Nutritional Analyses

To prepare mushroom samples for nutritional analysis, mushroom caps from harvested clusters samples were combined per treatment/block and dried for 3 days at 50 °C. The dried caps were coarsely ground to obtain a 100 g sample used for analysis of crude protein [30], total amino acid content and complete amino acid profile [31], and beta glucan content [32]. These analyses were performed by the University of Missouri-Columbia Analytical Services laboratory using Association of Official Analytical Collaboration (AOAC) International methodology. For each sample, analysis of total antioxidant content was performed in-house using the OxiSelect Total Antioxidant Capacity Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA).

2.4. Statistical Analysis

Harvest weight values were converted to bioefficiency (BE), which is a measure of the effectiveness of substrate utilization during mushroom production. BE was calculated as follows (Equation (1)).

$$Bioefficiency={\displaystyle \frac{{FW}_M}{{DW}_S}}\times 100$$

(1)

where FW_M is the fresh weight of the mushrooms, and DW_S is the dry weight of the substrate. The normality of data was assessed using the Shapiro–Wilk goodness-of-fit test and QQ plot. Bioefficiency data from two trials were consolidated, the means were calculated, and 2 factorial analyses of variances (ANOVAs) were performed on the treatment means. Similarly, the beta-glucan, crude protein, amino acid, total antioxidant contents, and morphological data from the two trials were pooled into a single dataset, the means were computed, and 2-factorial ANOVAs were performed. Morphology data was analyzed as a single factorial ANOVA, as the morphology dataset did not include the mesquite pod substrate data. All statistical analyses were conducted with the Statistical Analysis System (SAS v. 14.3) software, and the significance level was set at p < 0.05. A Fisher LSD t-test was employed to compare means and generate connecting letter reports.

3. Results

3.1. Impact of Light Spectra

On cotton, white LEDs resulted in a significant increase (p < 0.05) in histidine content compared to red or blue LEDs, with values of 0.86, 0.79, and 0.78 g/100 g, respectively. On mesquite, white LEDs resulted in significantly greater (p < 0.05) arginine content than red and blue, with values of 1.89, 1.71, and 1.72 g/100 g, respectively (Figure 1), and a significantly greater glutamic acid content than red and blue LEDs (4.20, 3.87, and 3.88 g/100 g, respectively). These substrate-specific outcomes were only detected with the Fisher LSD t-test; however, ANOVA showed no significant interaction between the substrate and spectra treatments, with substrate found to produce greater effects (

Table 1. Means and standard errors of the variables collected, along with the p values from the ANOVA comparing the straw/cotton and straw/mesquite substrates.

	Bio- Efficiency	Crude Protein	Total Amino Acids	Total Antioxidant Content	β-Glucan	Histidine	Glutamic Acid	Arginine
	(%)	(g/100 g)	(g/100 g)	(mM)	(g/100 g)	(g/100 g)	(g/100 g)	(g/100 g)
Cottonseed	70.4 ± 2.6	37.6 ± 0.9	26.7 ± 0.6	1.2 ± 0.0	23.0 ± 1.0	0.8 ± 0.0	4.2 ± 0.1	2.1 ± 0.1
Mesquite	39.8 ± 2.3	34.3 ± 0.6	28.8 ± 0.4	1.1 ± 0.1	27.0 ± 1.0	0.6 ± 0.0	3.8 ± 0.1	1.7 ± 0.0
p	<0.001	0.003	0.018	0.018	0.003	<0.001	<0.001	<0.001

). The spectrum of light used during the fruiting stage did not have a significant effect on the bioefficiency of either substrate. Furthermore, the light spectrum did not have a significant effect on total amino acid content, the levels of most amino acids, total antioxidant content, or beta-glucan content (

Table 2. ANOVA p-value results for the variables of block, spectra, substrate, and the interaction between substrate and spectra; significance threshold was set at p < 0.05.

Variable	Block	Spectra	Substrate	Substrate × Spectra
Bioefficiency	0.699	0.732	<0.001	0.833
Crude Protein	0.848	0.054	0.003	0.768
Total Amino Acids	0.744	0.326	0.018	0.715
Glutamic Acid	0.613	0.039	<0.001	0.264
Arginine	0.933	0.016	<0.001	0.632
Histidine	0.364	0.001	<0.001	0.404
Total Antioxidants	0.868	0.482	0.018	0.600
Beta-Glucan	0.982	0.128	0.003	0.510
Cap Diameter	0.542	<0.001
Number of Caps perCluster	0.859	<0.001

).

In contrast to most nutritional results, fruiting body morphology was dramatically altered by the spectrum of light. Red lighting resulted in nearly 3× more fruiting bodies per cluster at 8.9 caps per cluster, significantly greater than the 2.8 or 3.1 under white and blue light, respectively (Figure 2). Consequently, red light also resulted in a significantly average smaller cap diameter of 4.6 cm compared to those under blue or white light, measuring 10.1 cm and 11.1 cm, respectively (Figure 2). Importantly, this significant difference in cluster morphology did not alter the bioefficiency of mushroom production. This morphological analysis was performed only on cotton substrate, as the mesquite substrate showed reduce performance, detailed in the next section.

3.2. Effect of Substrate Composition

Most notably, the straw/cottonseed substrate resulted in a 77% increase in BE than the straw/mesquite substrate (70.4 vs. 39.8%, respectively) (Table 1). Substrate composition also had a significant effect on the nutritional properties of mushrooms. The straw/cottonseed resulted in significantly greater crude protein content (37.6 vs. 34.3 g/100 g, respectively) and significantly greater content of the specific amino acids of histidine (0.81 vs. 0.61 g/100 g, respectively), glutamic acid (4.2 vs. 3.8 g/100 g, respectively), and arginine (2.1 vs. 1.7 g/100 g, respectively). However, the straw/mesquite substrate resulted in significantly greater total amino (28.8 vs. 26.7 g/100 g, respectively) (Table 1).

The composition of substrate also had significant effects on the amounts of anti-inflammatory compounds produced. The straw/cottonseed substrate resulted in higher total antioxidants content (1.25 vs. 1.06 mM uric acid equivalents, respectively). In contrast, the straw/mesquite pod substrate resulted in significantly greater β -glucan content (27 vs. 23 g/100 g, respectively).

3.3. Statistical Summary

The condensed statistical outputs from all ANOVA analyses are presented in Table 2. Full ANOVA and LSD tables are available in the Supplementary Materials (Tables S1–S20). While a full amino acid panel was conducted, only those with significant results are presented below.

While light spectra and substrate individually impacted various measurements, there were no detectable interactions found between spectra and substrate (p > 0.05) for any of the variables measured in this study.

4. Discussion

4.1. Light Spectra

This study evaluated several responses of the oyster mushroom Pleurotus ostreatus to varied light spectra on different substrates. Fungi have light receptor proteins, phytochromes and cryptochromes, which enable them to sense red and blue light, respectively. Exposure to blue light activates cryptochrome receptors, which are known to suppress regulatory factors and may contribute to the larger cap size observed. Conversely, exposure to red light activates phytochrome receptors, which typically promote change in developmental pathways. In a study analyzing how Aspergillus nidulans reacts with light, more than 1100 genes were differentially regulated when exposed to blue, red, or far-red light [33], and 100 of them were typically red-light-induced but actually suppressed without simultaneous blue light. The complexity of fungal light responses and their impact on fruiting body composition and morphology will likely require omics-related approaches to fully understand how best to illicit the desired responses.

In other work, it was found that blue spectra increased vitamin D₂ by 20 μg/kg⁻¹ dry weight in P. ostreatus compared to natural lighting [34]; however, our findings indicate that spectra did not influence bioefficiency or many nutritional markers. Despite this, there was a significant difference in the morphological changes when grown under one of three selected spectra. These findings indicate that specific wavelengths within the blue and white spectra notably contributed to enlarging cap diameters, while red light reduced cap size during the formation of fruiting bodies. This confirms results from other researchers where blue light was found to stimulate the growth of P. ostreatus [34,35] and P. eryngii [36] with the opposite effect from red. The choice of light spectra could be manipulated to adjust P. ostreatus cluster and cap architecture to meet specific market preferences without compromising productivity.

In this study, only a low, constant intensity of lighting at 5 μmol m⁻² s⁻¹ was evaluated. Factors beyond light spectra, such as intensity and duration, could potentially impact yield, beta-glucan, and total antioxidant content. Further research exploring a broader range of lighting variables and their interactions with various fungal species and environmental conditions could provide deeper insights into optimizing nutritional content in specialty mushroom cultivation.

4.2. Importance of Substrate Selection

Crude protein and lipid content are critical to the production of Pleurotus spp. and achieving high bioefficiency. Production of Pleurotus sapidus on various straws, including soybean, rice, wheat, sorghum, millet, pigeon pea, and sunflower, revealed that the soybean straw, with the highest protein and lipid content, had the highest bioefficiency [37]. Likewise, cultivation of P. sajor caju on a straw substrate supplemented with high-protein mahua cake was found to increase yield by 75% compared to untreated straw substrate [38]. The difference in the characteristics of these two substrates have numerous outcomes, altering the bioefficiency, β-glucan, antioxidant levels, and protein content of the fruiting body. As cottonseed hulls have a greater crude protein and lipid content than mesquite pods, this study supports the idea that they are drivers in the bioefficiency of Pleurotus spp. In addition, studies have shown that substrates that result in higher mushroom yields also tend to produce mushrooms with higher nutritional protein content [38,39]. These results underscore the role of substrate selection in the final protein content of mushrooms.

Cottonseed has a significantly lower amount of carbohydrates compared to mesquite bean pods. In a trial with P. eryngii, increasing levels of carbohydrate rich olive mill solid waste mixed with eucalyptus sawdust had a positive effect on glucan production, where the yield of glucans was 29% greater than the control [40]. Similarly, in this study, the mesquite substrate, which has a higher carbohydrate content than cottonseed, produced mushrooms with a greater amount of β-glucan, which is known to reduce the risk of cardiovascular diseases [41]. The cultivation of mushrooms on various substrates also leads to differing antioxidant capacities of mushrooms. This has been shown with P. citrinopileatus [42] and P. ostreatus [43] and further confirmed with this study, highlighting the importance of substrate on the human nutritional outcomes of mushroom production.

Achieving higher levels of nutritional protein content in mushroom fruiting bodies can be facilitated by utilizing nitrogen-rich substrates with a low carbon content [44]. While the cottonseed substrate achieved a higher crude protein content, its total amino acid content was lower than the mesquite substrate. This distinction is important, as crude protein, indicative of overall protein quantity, includes all forms that may not be accessible or beneficial for human nutrition; meanwhile, total amino acids represent nutritional proteins. The small 2.1 g/100 g gain of total amino acids on the mesquite substrate may be statistically significant; however, considering its importance to human nutrition and heavily decreased bioefficiency of the mesquite substrate, the practical significance is negligible.

Individually, it was found that the amino acids, such as glutamic acid, arginine, and histidine, were higher in the cottonseed substrate and that white light can further enhance histidine and crude protein in the fruiting body. This partially aligns with previous work on P. eryngii, where it was found that white light resulted in the greatest total amino acid and crude protein content but the smallest histidine [36]. Given the market’s focus on mushrooms rich in amino acids [45], the ability to enhance amino acids content via a particular substrate and/or lighting is notable.

This study was limited by using only one constant intensity of lighting, 5 μmol m⁻² s⁻¹, and three spectrums. While this minimal light intensity elicited responses, greater intensities combined with shorter duration could potentially achieve similar or enhanced reactions with less electricity. Response of mushrooms to light and/or substrate may be species-dependent, and future work should expand to other species to see if the morphogenic responses are limited to oyster mushrooms. The protein content of the mesquite hull substrate may be limiting to mushroom cultivation; future work should investigate mesquite hull and other substrates with supplemental proteins that standardize the limiting element, providing better comparisons across spectra.

5. Conclusions

Substrate composition plays a pivotal role in the production and nutritional profile of oyster mushroom, Pleurotus ostreatus. The straw and cottonseed hull mixture increased bioefficiency by 31%, total antioxidant content by 0.19 g/100 g, and protein content of the fruiting body by 3.3 g/100 g, when compared to the mesquite substrate. However, the straw and mesquite bean pod mixture had 2.09 g/100 g greater total amino acid content and 4.06 g/100 g more β-glucan. The spectrum of light used during the cultivation stage on its own was not found to alter the bioefficiency, total amino acid, or total antioxidant content of oyster mushrooms. The use of white light on the mesquite substrate was found to increase the crude protein content of oyster mushrooms by 3.2 g/100 g, resulting in comparable levels to that observed on the cottonseed substrate, but an interaction between the substrate and light was not found to be significant. Lighting altered the fruiting body morphology, with red-light resulting in an average of 8.9 fruiting bodies per bag, three times the average for bags under white or blue treatment. The red-light fruiting bodies were also ~50% smaller in diameter than those under blue or white light, without impacting the yield.

While this study highlights some trends in P. ostreatus production associated with light spectra and substrate, it underscores the complexity of optimizing substrates and lighting for edible fungi cultivation in pursuit of the most effective strategies for enhancing crop productivity and nutrition. By further exploring the intricate interactions between spectra and substrate, cultivators can refine practices to achieve desired morphological characteristics and nutritional profiles of Pleurotus spp., advancing both agricultural efficiency and the production of nutrient-rich mushrooms.