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Key words: Mushroom cultivation, substrate supplementation, Hericium erinaceus, organic acids, cellulose based substrates, ascorbic acid, malic acid, pyruvate, lipids

The efficient utilization of cellulose based agricultural waste products for the generation of high-quality mushroom fruiting bodies represents a value-add bioresource recovery component to many agricultural practices and provides sustainable expansion of circular agri-centric based economies (Ayodele and Okhuoya 2007, Ozcaria-Fermossele et al., 2018, Win and Ohga 2018, Harada 2023). Concordantly, an increasing list of mushrooms are progressively becoming in-demand as their nutritional, gourmet and medicinal properties are being recognized (Stamets 2011). In as such, agricultural cellulosic waste-products are often poor in nitrogen thus necessitating supplementation (Mkhize et al., 2018) if an optimal yield of product is to be achieved. This commonly requires the addition of a nitrogen source, for example rice or wheat bran, to improve the carbon to nitrogen (C:N) ratio (Salama et al., 2019, Thrang et al., 2020, Stamets 2011, reviewed by Moore et al., 2008). This supplementation has been correlated with improved yields, optimized biological efficiency, and thus forms the basis of some commercial supplements (Royse and Sanchez 2017, Pardo-Giménez et al., 2020). Unfortunately, with increased nutrient supplementation comes the risk of fostering mold growth (Mkhize et al., 2018), in addition to incurring higher costs for the additive.

Ideally, a basal solid state associated supplement should promote the prodigious growth of mycelia enabling rapid and though substrate colonization, foster primordium formation, and support a high yield of visually well-formed and suitably sized fruiting bodies without apparent blemishes to detract consumer selection. The addition of calcium salts, particularly in the form of calcium carbonate and/or calcium sulfate, to the basal substrate has been shown to significantly support and augment mycelial growth (Singh et al., 2018) and fruiting body production in multiple species (Lu et al., 1973, Tabata and Ogura 2003; Royse and Sanchez-Vazquez 2003, Kumar et al., 2011, Nguyen et al., 2018, Singh et al., 2018). Hence these additives have been a common and necessary core supplement to basal substrates (Stamets 2011). Other functional additives such as grass chaff was shown to contain adenosine which exhibited a notable fructification enhancing effect at low concentrations (Domondon et al., 2004) and can also augment mycelial growth (Zhang et al., 2010), while saponin derivatives can stimulate mushroom fruiting (Magae 1999).

Hericium erinaceus represents a valuable medicinal species whose market appeal as a gourmet mushroom is flourishing. The cultivation characteristics of this species have been investigated, but many substrate additives examined have been typically additional, or alternative, agricultural waste associated materials such as sugarcane bagasse and other cellulosic-based agricultural byproducts augmenting the primary basal hardwood or softwood sawdust substrate. Unlike rapid colonizers such as Pleurotus species, Hericium moderately consolidates its cellulosic substrate, and this slower colonization may permit the opportunity for concomitant contamination with mold species such as Trichoderma spp. and other undesirable weed fungi if excessive rich supplementation is used.

In assessing substrate additives, many possibilities exist and can be represented as bulk organic type additives, namely complex cellulosic based components (bran, bagasse, leaf debris, etc., (Thrang et al., 2020)), but may also be supplementary low concentration additives such as lipids (Schisler 1967, Wardle and Schisler 1969, Li et al., 1992), seed meals (Schisler and Sinden 1965), amino acids (He and Suzuki 2003) or other organic and inorganic components (Rzymski et al., 2016, Salama et al., 2019, Postemsky et al., 2018). Inorganic additives such as magnesium, manganese, copper, zinc and selenium have been investigated with varied success depending on the species investigated (Estrada and Royse 2006, Rzymski et al., 2016, Kumar et al., 2018, Nhung et al., 2019), while some inorganic additives can abolish fruiting body formation entirely (i.e., LiCl, Chang et al., 2019). Ideally, a substrate supplement would not negatively impact fruit body flavor or appearance, be safe, inexpensive, and be required at a concentration that both economizes production costs but promotes mycelium growth and mushroom fruiting body formation. Complementing yield would be the equalization, or preferably, the synchronization of substrate colonization and subsequent primordium formation resulting in prodigious fruiting bodies with potential augmentation of nutritional content (i.e., elevated zinc as shown by Poursaeid et al., 2015), or desirable secondary metabolites (Wu et al., 2021), with a predicted periodicity for crop harvests.

In this investigation, a variety of organic acids, edible oil lipids and other additives (amino acids, glucose, starch, etc.) were used to assess the impact on solid state Hericium erinaceus primordium emergence as an indicator that the colonizing mycelium is prepared for fruit body formation, the time to mushroom collection, and the first flush mushroom weight just prior to spore formation. In doing so, a model system was developed whereby the emergence of Hericium erinaceus primordium through a cellulose filter was utilized to indicate that sufficient resources have been attained to signal the mycelial commitment to the morphogenic sequalae to support fruit body formation (Moor et al., 2008).

Wood sawdust-based substrate was prepared in a manner essentially outlined by Stamets 2011, that was comprised of equal proportions of softwood (predominantly spruce and pine) and mixed hardwood sawdust to which was added a final concentration of 10% wheat bran, 2% each of calcium carbonate and calcium sulfate, and sufficient water to attain a moisture saturation level that was close to, but not exceeding the holding capacity, of the moisturized substrate. Typically, a batch of basal substate was prepared by adding 1800 g blended sawdust, 200 g wheat bran, 20 g CaCO3, 20 g CaSO4 and 4000 g water unless otherwise noted. This basal substrate exhibited a pH of 6.5. Water soluble additives such as organic acids, were dissolved directly into the water component of the basal substrate. Non-water-soluble components such as seed, or vegetable oils were added to the rehydrated and blended sawdust substrate and thoroughly mixed to equally disperse the additive. The blended substrate was divided among 500 mL glass jars to a mass of approximately 374±10.1 g with mild compression to compact the rehydrated components.

Organic acid supplementation
Organic acids as represented by malic acid, citric acid, calcium pyruvate, and ascorbic acid were added to the water prior to rehydrating the wood/bran and inorganic components. The final concentrations of each was 559 μM malic acid, 260 μM citric acid, 233.4 μM or 466.8 μM calcium-pyruvate and 142 μM ascorbic acid. The free acids were sufficient to lower the pH of the basal mix to 5.5 whereas the calcium salts of the organic acids did not influence the pH of the basal substrate, remaining at 6.5.

Seed or Vegetable Oil Supplementation
For all seed and vegetable oil supplementation studies, 414 μL of oil per 100 g basal moistened substrate and mixed thoroughly to ensure homogeneous distribution which equated to 25 mL supplement per batch of substrate.

Additional Supplements
To evaluate the effect of additional supplements, L-glutamine, L-glutamate, L-leucine and glucose, the compounds were thoroughly dissolved in water before adding to the dry basal substrate components to a concentration of 2.94 mM, 2.92 mM, 3.81 mM and 6.94 mM, respectively. Corn starch was thoroughly dispersed in water to a concentration of 1% weight per volume before adding to the dry basal substrate components.

Assessing primordium formation and fruiting body formation.
To assess the readiness of the colonizing mycelium for fruiting body initiation, Hericium primordium emergence through a cellulose filter was monitored daily and scored as emerged when the primordium attained an approximate minimum size of 0.5 x 0.5 cm. Thereupon the culture vessel was covered with a humidity retaining polypropylene bag to maintain moisture and misted once daily to ensure a high humidity environment for the developing fruiting body. The temperature in the cultivation room was maintained at 23oC until the fruiting body was developed, harvested and weighted.

A basal substrate comprising of calcium carbonate and calcium sulfate additives yielded an approximate pH of 6.5 and conferred a colonization period of 49.46±11.08 days (n=64) and exhibited a broad spread in Hericium erinaceus primordium emergence time. While the acidic forms of organic acids at the quantities added changed the pH of the substrate mix to approximately 5, pH alone did not impact the rate of primordium emergence between acidic and calcium salt forms of the organic acid. Interestingly, ascorbic acid was the only organic acid that significantly improved the primordium emergence time, reducing emergence time significantly to 32.4±8.7 days (p =0.0013, n=7). In contrast, the only calcium salt of an organic acid, calcium-malate, reduced the colonization period to 35.5±3.83 days (p=0.039, n=12). A combination of acidic and calcium salt derivatives of organic acids appeared to synergistically shorten primordium emergence time significantly to 25.1±2.4 days (p≤0.001, n=11; Figure 2A). Only the organic acid combination influenced the mushroom maturation rate (Figure 2B), enabling fruiting body collection to be attained at 19.1 days (p≤0.05) rather than 22.75 days for the basal substrate alone.

Several organic acid supplements were able to influence the collected mushroom weight. These included the individual organic acids, malic acid and ascorbic acid, increasing the first harvest mushroom weight from 28.96 grams (no additives) to 37.2 grams (p=0.029) and 39.6 grams (p=0.016) for malic acid and ascorbic acid, respectively. Calcium citrate was able to improve the yield weight in a manner similar to malic acid, conferring a harvest weight of 37.58 grams (p=0.01). Combined organic acids, namely malic acid, citric acid, ascorbic acid and calcium pyruvate, significantly improved the harvest weight to 33.1 grams (Figure 2 C), but the supplement performance, although better than the no additives control, was less than either the malic acid, ascorbic acid and calcium citrate conditions. 

The addition of various seed or vegetable oils to the basal substrate exerted both negative and positive impacts on primordium emergence. For example, coconut oil (61.3±19.8 days, p=0.002) and rice bran oil (71.9±38.7 days, p=0.013) supplementation significantly slowed primordial emergence relative to the control condition (49.5±11.6 days) as shown in Figure 3A. None of the oil supplements assessed influenced the Hericium fruiting body development time (Figure 3B), but canola and safflower oil addition did moderately increase the collected mushroom weight from 29.0±12.3 grams for the un-supplemented condition to 33.2±4.1 grams (p=0.027) and 35.2±12.4 grams (p≤0.001), respectively (Figure 3C).

A series of additional additives reflected by various amino acids (glutamine, glutamate and leucine), corn starch and glucose, were assessed (Figures 3A _endash_ 3C). Both corn starch and glucose addition significantly slowed Hericium primordium emergence to 62.6±12.0 days (p≤0.001) and 54.3±15.8 (p≤0.001) days respectively, relative to the no-additive control (49.46±11.68 days, Figure 4A). Similarly, the addition of corn starch to the basal supplement also extended the period to fruiting body collection to 29.8±7.3 days relative to the non-supplemented control (22.75±12.47 days, p=0.008, Figure 4B).

The use of primordium emergence through a cellulose filter presents a useful model to investigate the use of basal media supplementation and examine the effects of such additives on Hericium erinaceus fruiting body formation. This system enables the direct evaluation of primordium emergence at a period that is suitable for the expanding mycelium mass independent of the colonization period. Typically, for in-jar or vented bag cultivation, the cultivator determines the period when a culture vessel is opened to initiate fruiting (Sakamoto 2018). Conventional approaches enable this once the substrate is fully colonized, but this emergence assessment system allows for fruitification that is deemed most suitable by the species, which may or may not be when full substrate colonization is achieved. Emergence through the filter approach effectively uncouples substrate colonization from fruiting body emergence, and it is possible that particular additives may accelerate fruitification. This effect was notable in this study with the addition of ascorbic acid, calcium malate and various combined organic acids. It was also noted, particularly for combined organic acids, that the variability in primordium emergence was decreased enabling better synchronization for fruiting body production. Combined organic acids also shortened the period for mushroom harvesting and moderately increased mushroom weight. Interestingly, the concentration of organic acids added was quite low suggesting that the additives are not simply being utilized as a supplemental carbon source and may have a signaling effect on the mycelium. In fact, ascorbic acid addition (1 mM) has been shown to augment antioxidant enzyme systems in Hypsizygus marmoreus, increase mycelial mass and regulate fruiting body development (Chen et al., 2020). A similar effect was noted for primordia formation in Pleurotus osteratus, whereby exogenously added ascorbic acid improved mycelium growth rate and hastened fruiting body formation (Qi et al., 2021); findings similar to this study for Hericium erinaceus. Interestingly, pyruvate appears to protect fungi from oxidative stress by acting as a reactive oxygen scavenger in Metarhizium robertsii (Zhang et al., 2017, 2018), likely acting in a manner similar to ascorbic acid as was noted in Pleurotus ostreatus (Yan et al., 2020) and likely explains the improved effect of increasing pyruvate supplement levels (the 2x pyruvate condition), or combining various organic acids. This is supported by the observation that malic acid is also capable of activating antioxidant enzyme systems in plants exposed to cadmium stress (Guo et al., 2017, Mousavi et al., 2022). Other organic acids such as succinic, citric and lactic acids supported mycelial growth of Coriolus versicolor (Jo et al., 2010), while acetate stimulated growth of Agaricus bisporus mycelium (Lehrian et al., 1976), likely by a similar mechanism. Notably, the added acidic forms of organic acids reduced the pH from approximately 6.5 to 5.5 in the basal substrate. This range does not appear to significantly modify mycelium growth as demonstrated by Nguyen et al. (2018), and may be favorable for Hericium erinaceus (Gonkhom et al., 2022), although strain dependence has been noted (Imtiaj et al., 2008). But note that for other basidiomycetes, such as Agaricus bisporus, a pH of approximately 7.5 was preferred (Wood 1976). Various fungi are noted to accumulate various tricarboxylic acid intermediates as a response to stress (reviewed by Goldberg et al., 2006), so supplementation in the basal media may support the various aspects of the Hericium life cycle culminating in fruiting body production yield.

Specific lipid supplementation to the basal media significantly reduced the period to primordial emergence which included canola, pumpkin and safflower oils. Other lipids had either a neutral effect, for example, avocado, corn or olive oils, or were negatively impacting as in the example of coconut or rice bran oils. This effect for Hericium erinaceus is concordant with other mushroom species as various lipids have been shown to promote mycelium growth (Lehrian et al., 1976, Schisler and Volkoff 1977, Song et al., 1989, Li et al., 1992), while shorter chain lipids, such as those from coconut oil, were noted as being inhibitory (Song et al., 1989). Saturated lipids such as those derived from palm oil augmented mycelial biomass and polysaccharide yield (Ahmad et al., 2018), which is notable as high concentrations of palmitic acid were found to augment Hericium mycelium growth and pinhead formation (Kang et al., 2002). It is advisable that different oils be tested for the species or interest, for example in this study olive oil did not impact primordial emergence but has been shown to augment mycelial growth for Grifola frondosa (Hsieh et al., 2005) and Ganoderm lucidum species (Yang et al., 2000), while rice bran oil promoted mycelial growth for Cordyceps sinensis (Chau et al., 2018). Also notable in this study were the favorable influence of canola and safflower oils on fruiting body weight, possibly related to the degree of unsaturated fatty acids which have been shown to support the growth and aerobic metabolism of the yeast Saccharomyces cerevisiae (Walenga and Lands 1975).

Also explored was the addition of various amino acids: glutamine, glutamate and leucine; which did not appear to influence emergence, time to fruiting body collection or fruiting body weight, at the concentrations evaluated. In contrast, amino acids (Asn, Gln, Gly) were shown to influence protein content, pilus diameter and fruit body numbers of Agaricus bisporus at 150 and 300 mg/L, which may influence the C:N ratio of the substrate (Hesami et al., 2014). A similar observation was made for Lentinus tuberregium, whose mycelial biomass was augmented by glycine (Manjunathan and Kaviyarasan 2010). The opposite effect was noted in non-basidiomycetes, whereby Ross and Bremner (1971) evaluated leucine as a nitrogen source, and noted that perithecia of Venturia inaequalis did not form with this supplement as well as with high concentrations of nitrogen. Additionally, perithecia did not form in media containing high concentrations of sugars, while low levels of calcium carbonate were favorable as noted for other species.

Added sugars were shown to negatively impact Agaricus bisporus mycelium growth (Wood 1976) but augment mycelium growth for Calocybe indica and Cordyceps militaris (Dang et al., 2018, Singh et al., 2018). A notable decrease in the primordium emergence period for Hericium erinaceus was observed when basal media was supplemented with glucose or corn starch, but a significant increase in fruiting body yield was obtained for the glucose additive. This effect may be concentration dependent as the direct effect of supplementing sawdust substrates with glucose at levels of 1 _endash_ 3% was investigated by Siwulski et al. (2009) and was shown to positively impact mycelium growth rate and fruiting body yield on both beech and pine based sawdust substrates. This trend was noticed, but not significantly so in this study, and may simply reflect the lower level of glucose used (approximately 0.25% versus upwards of 3% in the Siwulski study).

Conclusions

Various additives to a basal wood sawdust-based substrate have been shown to significantly decrease the time to Hericium erinaceus primordium emergence and impact fruiting body weight. Particularly notable were specific organic acids (ascorbic acid, calcium malate) when used as individual additives, or when combined (malic acid, ascorbic acid, citric acid and calcium pyruvate). Oils such as avocado canola, pumpkin and safflower were positively impacting on primordium emergence, while coconut and rice bran oils slowed emergence. Only canola oil increased the fruiting body weight at harvest. The addition of corn starch and glucose slowed primordium emergence without any significant improvement to time to harvest or fruiting body weight, Overall select low-cost supplement selection, particularly organic acids and lipids, as demonstrated, can influence cultivation characteristics for Hericium erinaceus and improve harvest yields.

Conflict of Interest
The author declares no conflict of interest.

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