Recycling of the biomass waste defatted almond meal as a novel nutritional supplementation for cultivated edible mushrooms

Defatted almond meal (DAM) is an useless biomass waste obtained after oil extraction. The substrate designed for mushroom cultivation is achieved through a controlled composting process from agricultural by-products (chicken manure and wheat straw). This work shows the potential of DAM as efficient compost supplement for culturing the species Agaricus bisporus (J.E. Lange) Imbach and Pleurotus ostreatus (Jacq.) P. Kumm. Supplementation during A. bisporus cultivation results in larger mushrooms with a firmer texture and higher dry matter and protein contents in comparing with the non-supplemented substrate. In P. ostreatus, supplementation at a dosage of 15 g kg provided a yield improvement up to 31.8%, compared to the control without supplement. The supplementation with DAM supposed equivalent or better yield than the commercial supplements. Therefore, the technique developed assessed good agronomic potential for application of DAM at the commercial scale in P. ostreatus cultures, adding value to a worthless organic by-product.


Introduction
Several alternative methods can be applied to extract the oil contained inside the almond (Marzouki, Piras, & Rosa, 2008;Mezzomo, Martínez, & Ferreira, 2009).Among them, extraction using pressure is the best option because it yields high quality oils at an affordable price (Da, Hongzhi, Li, Hui, & Qiang, 2014;Sena-Moreno et al., 2015).The by-product obtained from this process is a DAM, which has high protein content, minerals, dietary fibre and substances with antioxidant capacity (Martínez, Marín, Gili, Penci, & Ribotta, 2017), although it does not have an extensive application.
The lipid, protein, carbohydrate and mineral fractions are the main components in the almond seed.Additionally, a large group of compounds called phytochemicals are present; these compounds are found in low quantities but play a significant role in the almond quality.The percentages of these fractions differ based on the variety, type of culture and geographic origin (Yada, Lapsley, & Huang, 2011;Yada, Huang, & Lapsley, 2013;Kodad, Estopañán, Juan, Socias, & Company, 2014a;Kodad et al., 2014b).Yada et al. (2011) reported that the total protein Acta Scientiarum.Agronomy, v. 40, e39341, 2018 (considering a nitrogen conversion factor of 5.18) values ranged between 14 and 61 g for 100 g of raw almonds, whereas the soluble sugar ranges were between 1.8 and 7.6 g for 100 g and between 2.5 and 12 g for 100 g of raw nuts.
The almond is the most important nut cultured in the world in terms of its commercial production.Most of the world's production is concentrated in three regions, which include California, the Mediterranean and Central Asia/Middle East (Food and Agriculture Organization of the United Nations [FAO], 2017).The almond tree is a very significant crop primarily due to its high adaptability to extremely dry conditions, such as the conditions that occur in the Mediterranean basin.In this region, the maintenance of the almond as a crop and the genetic reserve of the species is important since the vegetal material presents a very high variability (Homet-Gutierrez, Schupp, & Gómez, 2015).
The mushrooms use nutritional supplements directly to increase the yield without influencing the quality.The yield increase can range between 10 and 20% and occasionally may be higher.The types of nutrients, the best moment for the application, the cost of the supplement and the economic benefits should be analysed in advance (Naraian et al., 2009;Royse & Chalupa, 2009;Royse, 2010;Sánchez, 2010).
Cereal grains and oilseeds, which are widely used as supplements for mushroom composting, contain variable proportions of the three basic nutritional requirements for mushrooms (carbohydrates, proteins and lipids) (Sánchez, 2010;Arce-Cervantes et al., 2015).Most commercialised supplements use raw materials based on vegetables rich in protein, among them soybean meal is the main ingredient (Zied et al., 2011;Arce-Cervantes et al., 2015).Zervakis et al. (1996) also studied the effect of olive mill residues on the production of two Pleurotus species.Recently, the viability of the use of defatted pistachio meal for the supplementation of substrates for the cultivation of A. bisporus and Pleurotus ostreatus was also successfully validated (Pardo-Giménez et al., 2016).
Worthy of note is that the technology based on supplementation is highly attractive since its application stimulates yield while improving the quality, shelf life and preventing disease occurrence (Wheeler & Wach, 2006).The supplementation equilibrates the nitrogen content and the C/N ratio, increasing the production yields and even provides benefits to the nutritional content of the harvested basidiomes (Zied et al., 2011;Pardo-Giménez, Zied, Álvarez-Ortí, Rubio, & Pardo, 2012).Since both the cereal straw and sawdust, agricultural by-product that are commonly used as base materials for the production of mushroom substrates, are poor nitrogen sources; nitrogen depletion in the substrate during fruiting (when most of the nitrogen is used for mycelial growth) is unsuitable and limits the yields.An immediate consequence of this situation is a drastic drop in production after the first flush, which may be caused by nutrient depletion, among other factors.
The supplementation technique shows high potential to correct this issue.
Agaricus and Pleurotus are the two most cultivated genera worldwide, and Europe is the second largest producer after China (Royse, 2014), in a global market valued over 4,700 $ billion only considering the species Agaricus bisporus (Sonnenberg et al., 2011).Mushrooms are recognised as a very healthy source of nutrients and its consumption has significantly increased within the last years (Roncero-Ramos, Mendiola-Lanao, Pérez-Clavijo, & Delgado-Andrade, 2017).
The aim of the present study was to investigate the viability to recycle an interesting by-product, DAM, as a novel nutritional substrate supplement for the mushroom industry of the species Agaricus bisporus and Pleurotus ostreatus.

Characterization of substrates and supplements employed
A commercial A. bisporus Phase III compost based on wheat straw and chicken manure inoculated with the Amycel XXX  (Amycel, Watsonville, CA, USA) mycelium (thick white hybrid) and provided by Champinter S.C.L. (Villamalea, Albacete, Spain) was used as substrate for the A. bisporus production.Euroveen  (Euroveen BV, Grubbenvorst, The Netherlands), which is a commercial mixture of Dutch origin, was used as the casing layer.The thickness of the casing was 3.5 cm.
The variety Marcona was used in this study, which is the most cultivated in Spain in upland farming.The almonds were acquired from the Manchega Almond Cooperative (Cooperativa Manchega de la Almendra) of Villamalea.Shelled almonds were placed on trays and the DAM was obtained following the procedure described by Pardo-Giménez et al. (2016).Untreated substrates and commercial supplements designed for use in A. bisporus (Promycel 600, Amycel, Watsonville, CA, USA) and P. ostreatus (Promycel Pleurotus, Amycel, Watsonville, CA, USA) cultures were used as negative and positive controls respectively.
The following parameters were determined for the physical, chemical and biological characterisation of the substrate, the casing materials and t h e supplements: humidity, pH, electrical conductivity, total nitrogen content, raw material and ashes, C:N ratio, crude fibre, crude fat, nitrogen-free extract, cellulose, hemicellulose, lignin and neutral detergent-soluble fibres, real density, apparent density, total porosity and water retention capacity, and the presence of nematodes, mites and competitor moulds, as described by Pardo-Giménez et al. (2012) and Pardo-Giménez et al. (2016).

Research layout
Two trials in crop were performed (the first for A. bisporus and the second for P. ostreatus) using a random block design with six replicates.For A. bisporus, boxes containing 10 kg of compost with 70 kg m -2 of load density were used.A total of 30 boxes were evaluated, corresponding to the six replicates per treatment of the three doses (5, 10, and 15 g kg -1 ), the nonsupplemented controls and the controls using the commercial A. bisporus supplement (10 g kg -1 ) assayed.For P. ostreatus, transparent pierced polyethylene bags with a 6 kg substrate capacity were used; four orifices (25 mm in diameter) were uniformly distributed on the lateral surface of each bag.Similarly, a total of 30 bags were used corresponding to six replicates per treatment of the three doses used (5, 10, and 15 g kg -1 ), the non-supplemented controls and the commercial P. ostreatus supplement (8 g kg -1 ) assayed.

Crop cycle
The mushroom crop cycle was conducted in a climate-controlled growing room at the Mushroom Research, Experimentation and Service Centre (CIES, Quintanar del Rey, Spain).The room is supplied with automatic systems for humidification, heating/cooling and recirculation/external ventilation to control temperature, relative humidity and the carbonic anhidric ratio as recommended by the spawn supplier.Peat-based casing was applied the same day as the filling; then, the routine insecticide (diflubenzuron 25%, 3.8 g m -2 ) and antifungal (prochloraz-Mn 46%, 0.47 g m -2 ) treatments were applied for A. bisporus production.The casing was ruffed 8 days after application, and fruiting was induced 24h later.Three flushes were harvested along a crop cycle of 37 days.
An air-conditioned greenhouse tunnel that was similarly equipped with humidification, heating/cooling, recirculation/external ventilation and lighting systems was employed for growing P. ostreatus.The culture cycle was performed under the temperature, relative humidity, carbonic anhidric ratio and lighting conditions recommended by the spawn manufacturer.The substrate was incubated for 17 days with neither external ventilation nor lighting; then, fruiting was induced using ventilation, lighting and decreasing temperature and relative humidity, during a crop cycle of 70 days.

Mushroom harvest and quality parameters
For A. bisporus, harvesting was performed daily at the optimal commercial development stages.Mushroom picked were daily weighed and counted.Additionally, the production was separated into two groups based on their sizes as follows: thick (≥ 40 mm) and medium (15-40 mm) for A. bisporus crop.The production was determined from the yield by surface unit, considering the load density of the compost and its humidity content.The unit weight of the mushrooms was determined from the obtained yield and the number of harvested mushrooms.A second estimation of the size (expressed as the diameter of the cap in mm) was determined using the non-linear regression curves previously determined from the measurement of the diameter and the weight of the mushrooms corresponding to the three flushes of the chosen variety of commercial mycelium.The earliness was expressed as the time from the application of the casing until the harvesting of the first flush considering the daily relative production of the harvesting (Pardo-Giménez, Pardo-González, & Zied, 2011).
On the day of the maximum harvest of each flush, mushrooms of uniform size and maturity were chosen for the assessment of other quality parameters.The colour, dry matter content and texture of the mushrooms were determined as described by Pardo-Giménez et al. (2016).For P. ostreatus, mushroom harvest was performed daily at the optimal commercial development stages.The number of harvested mushrooms was determined by counting during the whole crop cycle.The amount of mushrooms produced daily was weighed per each bag to calculate the yield.The yield provided per unit area was assessed by taking into account the load density of the substrate in the bags and the humidity content.The others quantitative (earliness) and qualitative (dry matter, colour and firmness) were analysed using the same methods.

Statististics
The data obtained were processed by one-way analysis of variance (ANOVA).The Fisher's least significant difference (LSD) test, at 5% probability, was employed to establish significant differences between the mean values.The analysis was performed using the software package Statgraphics Plus software, v. 4.1 (Statistical Graphics Corp., Princeton, NJ, USA).

Result Characterization of composts and supplements employed
Table 1 shows the determined composition of the different composts and supplements used in the crop trials.Among the supplements used for Agaricus, it is remarkable the lower protein content of the almond meal (420.0 g kg -1 ) compared to the control Promycel 600 (510.4 g kg -1 ).Higher crude fibre (200.4 vs 87.9 g kg -1 ) and much higher crude fat (248.3 vs 31.4 g kg -1 ) contents have been detected.Additionally, we highlight a lower total carbohydrate content (273.8 vs 393.9 g kg -1 ).The higher hemicellulose, cellulose and lignin contents represent a lower neutral detergentsoluble fibre content (373.1 vs 564.6 g kg -1 ).
The supplement of DAM shows a remarkable higher crude fat content (248.3 g kg -1 ) compared to the reference Promycel Pleurotus (38.1 g kg -1 ) used for Pleurotus (Table 1).Notably, the lower nitrogen-free extractive (73.4 vs 350.2 g kg -1 ), total carbohydrate (273.8 vs 478.4 g kg -1 ) and neutral detergent-soluble fibre contents (373.1 vs 548.9 g kg -1 ) should be highlighted; the latter effect was due to the higher hemicellulose content and, particularly, higher lignin content.

Agaricus bisporus yield and quality
Although no significant differences were found for most of the quantitative production parameters, some findings should be highlighted (Figure 1, Table 2).The non-supplemented substrates provided lower unit weights of the mushrooms (18.7 g) than the supplemented substrates, although the difference was not significant.However, larger unit weights were obtained for the mushrooms produced after the addition of almond meal regardless of the dose.Variations caused by the dose applied did not allow us to obtain conclusions.
Regarding the yields, a larger production (28.12 kg m -2 ) was obtained with the substrate supplemented with almond meal at a dose of 10 g kg -1 , although the differences were neither significant (Figure . 1).Values followed by different superscript letters within a column are significantly different at p ≤ 0.05 (LSD Fisher's test); NS: non-supplemented control, PRO600: Promycel 600 control, DAM: deffated almond meal, D5: supplement amount 5 g kg -1 , D10: supplement amount 10 g kg -1 , D15: supplement amount 15 g kg -1 .All the supplemented substrates registered yield increases in the first flush, and even more significant for almond meal at a dose of 10 g kg -1 , which was the only parameter that showed significant differences.However, the behaviour found in the subsequent flushes did not follow a pattern associated with the applied supplement dose.
The distribution of the production by sample size increased the yield of thick mushrooms with the supplemented substrates, albeit not significantly.This result is related to the finding that the diameter of the mushrooms is the largest for the supplemented substrates with similar unit weight, resulting in larger diameter mushrooms produced with the substrate supplemented with almond meal at a 15 g kg -1 dose (40.0 mm), although the difference was not significant.
The only significant differences found regarding the qualitative parameters concern the dry matter content of button mushrooms.The nonsupplemented substrate produced mushrooms with a low dry matter content (71.3 g kg -1 ), which was significantly below the content of the mushrooms produced with substrate supplemented with the highest dose of almond meal (75.6 g kg -1 ) (Table 3).
In fact, considering the relation between the dry matter content and the texture, a lower puncture force and compression energy have also been obtained with mushrooms using a non-supplemented substrate (19.01N and 132.7 mJ, respectively) (Table 2).Similarly, higher breaking strength and compression energy were obtained for mushrooms produced with the substrate supplemented with the highest dose of almond meal (20.71N and 164.7 mJ, respectively), although the differences were not significant.Neither significant differences were found regarding the colour of the carpophores (Table 2).
Regarding the mushroom mycochemical characteristics (Table 3), no significant differences were found in any of the analysed parameters.Despite the lack of significant differences, supplementation increased the protein content of the carpophores with the increase in the almond meal dose, which could be explained by the additional contribution of the supplementation with nitrogen-rich materials.For the remaining parameters, supplementation resulted in an increase in the crude fibre content and decreases in the total carbohydrate, nitrogen-free extractive and ash contents, although the differences were not outstanding.

Pleurotus ostreatus yield and quality
Regarding productivity, all supplemented substrates showed important increases compared to the non-supplemented substrate (26.47 kg m -2 ), with a significant increase up to 34.90 kg m -2 observed with the substrate supplemented with DAM at the 15 g kg -1 dose, achieving a maximum increment of 31.8% (Figure 1).All recorded values were considered outstanding compared with the usual values obtained with commercial culture.Regarding the behaviour of the production by flush, all supplemented substrates provided higher productivity values than the non-supplemented substrates for the first and second flushes.Larger increases were found for the first flush, where the differences were significant compared to the control except for the 5 g kg -1 dose.For the second flush, only the differences obtained with the highest dose were significant.In addition to leading the greatest significant increase recorded, the maximum dose resulted in a more uniform temporal production distribution considering the higher yields reached during the second and third flushes.Taking into account the days elapsed until the last harvesting, the difference was not significant even though the supplemented substrates provided larger production value rates than the non-supplemented substrate (between 1.93 and 1.96 kg dt -1 day -1 vs 1.63 kg dt -1 day -1 ).No significant differences were found regarding the unit weight and earliness.
The qualitative parameters defining the colour and texture (firmness) did not show significant differences, although the dry matter content of the carpophores differed significatly (Tables 3 and 4).The lowest records corresponded to the nonsupplemented substrate (75.9 g kg -1 ) and were significantly lower than the results for the substrates supplemented with the 10 and 15 g kg -1 doses (83.2 and 86.0 g kg -1 , respectively).Thus, the dose increases translated to 9.6 and 13.3% respectively, in the dry matter content of the harvested mushrooms (Table 3).Promycel Pleurotus control, DAM: deffated almond meal, D8: supplement amount 8 g kg -1 , D5: supplement amount 5 g kg -1 , D10: supplement amount 10 g kg -1 , D15: supplement amount 15 g kg -1 .
The supplementation tended to produce significant decreases in the carpophore water content similar to the observations with the A. bisporus.It also induced an increase in the protein content in all cases.This is even larger when the applied almond meal dose rises, reaching a maximum of 176.2 g kg -1 for the supplementation with 15 g kg -1 , which was significantly greater than the non-supplemented control.On the contrary, the addition of supplements decreases the fat values significantly.For the remaining parameters, supplementation improved the crude fibre content and reduced the total carbohydrate, nitrogen-free extractive and ash content, similar to the results obtained for A. bisporus, although the differences observed were not significant (Table 3).Compared to A. bisporus, mushrooms from the genus Pleurotus showed lower protein and mineral (ash) content and a higher carbohydrate content and energy value.

Discussion
There is controversy regarding the nutrient content within supplements used for mushroom growing.In mushroom compost, wheat straw in the major carbon source while chicken litter is the major nitrogen source, but frequently the colonized compost (Phase III compost used for A. bisporus cultivation) is supplemented with sterile protein rich nutrients (Vos et al., 2017).According to Arce-Cervantes et al. (2015), A. bisporus needs a simultaneous source of oil and protein in order to increase yields, and that with a source of polysaccharides it is possible to formulate a supplement equivalent to the traditional one based on crushed soybeans (25% protein and 18% oil).Dahlberg (2004) suggested that carbon metabolism is also important in the mushroom growth process.Ingredients containing cellulose and hemicellulose are preferred as supplements because formulations based on simple sugars or starch are subjected to the attack of microorganisms or pathogenic moulds such as Trichoderma.Moreover, base ingredients with high carbohydrate contents are cheap and easily available which is an added value.
The supplements used in this research showed high fibre, fat, hemicellulose and lignin content and low carbohydrate, and resulted in equal and even higher yield than commercial supplements consecrated in the market.Therefore, our results contradict the broadly accepted theory that supplements used in the production of mushrooms must necessarily present both high protein and high carbohydrate content.
Larger sizes are usually preferred by farmers due to the fresh market demand and the reduction of labour costs in harvesting; however, medium sizes may be preferable by facilities prepared for mechanical harvesting and canned industry.Typically, high yields are associated with high fruiting rates.Thus, when the number of harvested mushrooms increases, the mushroom size decreases (Pardo-Giménez et al., 2012).This statement is consistent with our results.Besides, the mushroom dry matter content is a parameter of special interest because it directly reflects the quality and commercial lifetime.Mushrooms with a high dry matter content, most likely the button mushroom cultivated with DAM supplementation, show a firmer texture, a higher nutritional value and a lower sensitivity to microbial spoilage (Diamantopoulou & Philippoussis, 2015).
Considering the cost of the substrate and the supplement, in addition to the increase in production registered, a substantial improvement in the benefits can be achieved by incorporating the by-product defatted almond meal as a nutritional supplementation for enhancing mushroom composition and yield.

Conclusion
The by-product defatted almond meal can be successfully recycled for enhancing mushroom production and quality on compost application.The species P. ostreatus had a higher response than A. bisporus in terms of yield.The quality of mushrooms improved after supplementation.Doses of DAM between 10 and 15 g kg -1 of fresh substrate show good potential for application at the commercial scale in P. ostreatus primarily due to the expected production increases, with direct incidence on the benefits and cost-effectiveness of the mushroom industry.Complementary the recycling of an useless by-product contributes to the sustainability of the agriculture and to optimize the biomass generated from a relevant agronomical activity base on almonds trees.
Supplementary material (Table S1) collects the main quantitative production parameters along the crop cycle.

Table 1 .
Physical-chemical characteristics of the various substrates and supplements used.

Table 2 .
Results obtained for the main qualitative production parameters assessed in Agaricus bisporus mushrooms from the various treatments.

Table 3 .
Mycochemical characterization of Agaricus bisporus and Pleurotus ostreatus harvested from the various treatments.

Table 4 .
Results obtained for the main qualitative production parameters assessed in oyster mushrooms from the various treatments.