Cultivation of Oyster Mushroom Pleurotus Ostreatus on Date-Palm Leaves Mixed with Other Agro-Wastes in Saudi Arabia

Saudi Journal of Biological Sciences (2014) xxx, xxx–xxx

King Saud University Saudi Journal of Biological Sciences

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE Cultivation of oyster mushroom Pleurotus ostreatus on date-palm leaves mixed with other agro-wastes in Saudi Arabia

Kholoud M. Alananbeh *, Nahla A. Bouqellah, Nadia S. Al Kaﬀ

Biology Department, Science College, Taibah University, Al Madinah Al Munawwarah, Saudi Arabia

Received 26 May 2014; revised 29 July 2014; accepted 6 August 2014

KEYWORDS Abstract Promoting the use of agricultural waste is one of the newly prepared water and environ- Oyster mushroom; ment friendly agriculture strategies in the Kingdom of Saudi Arabia (KSA). The objective of this Date palm; research was to study the efficiency of cultivating oyster mushroom (Pleurotus ostreatus) on date Earliness; palm wastes mixed with other agricultural wastes available in KSA. Four agricultural wastes were Proximate analysis; mixed with date palm leaves at different ratios, with two supplements and three spawn rates were Agro wastes; used. Wheat straw mixed with date palm at ratio of 25 (date palm): 75 (agro-waste) showed the best Saudi Arabia results in most of the parameters measured. Corn meal was superior over wheat bran as a supplement in all treatments. Parameter values increased with the increase of the spawn rate of P. ostreatus. Treatments with date palm leave wastes contained higher carbohydrates and fibers. No significant differences were found among the fruiting bodies produced on the different agro- wastes studied for the different proximates analyzed. Analyses of metal concentration showed that potassium was the highest in all the treatments tested followed by Na, Mg, Ca, and Zn. This is the first study that reported the success of growing oyster mushroom on date palm leaf wastes mixed with other agro-wastes obtainable in KSA. ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introduction

Mushrooms are ﬂeshy fruiting bodies (Alexopoulos et al., 1996) * Corresponding author. Address: Biology Department, Science that are considered one of the delicious fruits, and are com- College, Building 21-Room Sb27, Taibah University, Madinah, Saudi monly produced worldwide (Madbouly and Al-Hussainy, Arabia. Tel.: +966 550040471. 1996). They are a rich source of carbohydrates, proteins, vita- E-mail address: [email protected] (K.M. Alananbeh). mins, and minerals (Ananbeh, 2003). Mushrooms grow on Peer review under responsibility of King Saud University. decayed organic matters rich in lignin, cellulose, and other com- plicated carbohydrates. Large quantities of agro-industrial wastes that are produced worldwide often cause environmental and health Production and hosting by Elsevier http://dx.doi.org/10.1016/j.sjbs.2014.08.001 1319-562X ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.

Table 1 Regression analysis for the different parameters used for Pleurotus ostreatus cultivation. Source of variation DF* 1st flush 1st flush yield 1st flush FB 1st flush FB AvWt FPFPFPF P Regression 88 6.64 0.000 19.42 0.000 6.02 0.000 1.51 0.004 Experiment 1 1.14 0.287 7.40 0.007 2.61 0.107 4.38 0.037 Waste 2 149.47 0.000 478.22 0.000 115.92 0.000 9.66 0.000 Ratio 4 17.48 0.000 39.51 0.000 16.49 0.000 5.64 0.000 Supplement 1 0.01 0.913 45.72 0.000 8.60 0.004 0.00 0.978 Spawn 2 0.84 0.433 25.13 0.000 6.12 0.002 1.56 0.212 Replicate 2 0.11 0.897 2.11 0.122 1.19 0.305 0.45 0.635 Waste*ratio 8 11.65 0.000 45.40 0.000 13.83 0.000 0.85 0.557 Waste*supplement 2 1.89 0.153 8.25 0.000 2.61 0.075 0.66 0.517 Waste*spawn 4 0.43 0.791 2.01 0.091 0.83 0.506 0.60 0.660 Ratio*supplement 4 1.39 0.238 6.75 0.000 2.74 0.028 1.27 0.281 Ratio*spawn 8 3.30 0.001 1.52 0.149 0.58 0.792 0.98 0.448 Supplement*spawn 2 2.50 0.083 0.09 0.910 2.47 0.085 0.68 0.507 Waste*ratio*supplement 8 4.84 0.000 3.04 0.002 2.14 0.031 1.29 0.249 Waste*ratio*spawn 16 1.32 0.178 0.83 0.657 0.76 0.730 0.67 0.829 Ratio*supplement*spawn 8 0.58 0.795 1.82 0.072 1.15 0.331 2.86 0.004 Waste*ratio*supplement*spawn 16 0.76 0.730 0.47 0.960 1.76 0.034 0.89 0.586 Error 451 Total 539 DF 2nd flush 2nd flush yield 2nd flush FB 2nd flush FB AvWtà FPFPFPF P Regression 88 5.05 0.000 8.13 0.000 4.69 0.000 1.50 0.005 Experiment 1 1.16 0.283 0.03 0.857 4.56 0.033 10.76 0.001 Waste 2 76.20 0.000 148.89 0.000 45.72 0.000 9.97 0.000 Ratio 4 12.54 0.000 7.09 0.000 4.99 0.001 0.44 0.778 Supplement 1 2.34 0.127 44.40 0.000 30.72 0.000 0.29 0.586 Spawn 2 1.05 0.351 43.82 0.000 31.17 0.000 0.10 0.903 Replicate 2 0.07 0.935 5.75 0.003 4.06 0.018 1.68 0.188 Waste*ratio 8 11.53 0.000 14.56 0.000 6.14 0.000 1.12 0.350 Waste*supplement 2 0.61 0.546 0.33 0.718 1.97 0.141 0.23 0.791 Waste*spawn 4 0.30 0.898 0.92 0.454 2.37 0.051 0.26 0.901 Ratio*supplement 4 11.97 0.000 3.91 0.004 3.67 0.006 0.25 0.910 Ratio*spawn 8 1.85 0.066 3.33 0.001 4.07 0.000 1.90 0.058 Supplement*spawn 2 0.59 0.557 2.29 0.102 2.52 0.081 1.58 0.206 Waste*ratio*supplement 8 3.32 0.001 2.92 0.003 1.51 0.152 0.87 0.546 Waste*ratio*spawn 16 0.96 0.497 1.31 0.187 0.97 0.493 0.86 0.612 Ratio*supplement*spawn 8 1.96 0.050 1.70 0.096 2.26 0.022 0.58 0.794 Waste*ratio*supplement*spawn 16 1.23 0.242 1.24 0.234 2.19 0.005 2.53 0.001 Error 451 Total 539 DF Earliness Total yield BE Total FB FPFPFPF P Regression 88 7.32 0.000 13.61 0.000 13.60 0.000 7.45 0.000 Experiment 1 1.35 0.247 4.20 0.041 4.12 0.043 6.03 0.014 Waste 2 147.08 0.000 185.12 0.000 184.76 0.000 151.87 0.000 Ratio 4 25.80 0.000 34.21 0.000 34.11 0.000 11.46 0.000 Supplement 1 0.29 0.594 48.98 0.000 49.21 0.000 26.72 0.000 Spawn 2 1.80 0.166 39.26 0.000 39.44 0.000 21.49 0.000 Replicate 2 0.06 0.942 4.22 0.015 4.22 0.015 1.34 0.264 Waste*ratio 8 12.97 0.000 48.63 0.000 48.58 0.000 15.97 0.000 Waste*supplement 2 2.81 0.061 2.99 0.052 2.94 0.054 3.29 0.038 Waste*Spawn 4 0.27 0.900 0.70 0.589 0.72 0.577 1.06 0.374 Ratio*supplement 4 2.09 0.081 14.32 0.000 14.34 0.000 3.73 0.005 Ratio*spawn 8 2.90 0.004 0.78 0.621 0.78 0.617 0.94 0.486 Supplement*spawn 2 4.52 0.011 0.86 0.425 0.82 0.441 0.64 0.527 Waste*ratio*supplement 8 6.31 0.000 6.66 0.000 6.66 0.000 1.81 0.074 Waste*ratio*spawn 16 1.39 0.144 0.85 0.622 0.85 0.623 0.67 0.826 Ratio*supplement*spawn 8 0.66 0.726 1.19 0.306 1.19 0.301 0.99 0.445

Table 1 (Continued) Source of variation Waste*ratio*supplement*spawn 16 0.77 0.722 0.69 0.797 0.70 0.792 1.99 0.010 Error 451 Total 539 * DF: degrees of freedom. First ﬂush fruiting bodies’ average weight. à Second ﬂush fruiting bodies’ average weight.

problems (Garg and Gupta, 2009). In addition, the ever- was found to be the highest in palm leaves compared to other growing need of cheap nutritious food, and the lack of protein parts tested (Abdulhadi and Hassan, 2013). in developing countries led to the development of the mush- The previous mushroom production studies conducted in room cultivation industry (Sivaprakasam and Kandasawmy, Saudi Arabia or in Iraq did not test the possibility of mixing 1981; Levanon et al., 1993; Yildiz et al., 1997; Croan, 2000; the date palm agro wastes with other agro wastes as media Zervakis et al., 2001). to grow mushrooms. The current study hypothesized that The Oyster mushroom Pleurotus ostreatus is characterized using date palm leaves alone or mixed with other agro wastes by its rapid growth on agro-wastes such as olive cake, tomato will not be efficient in producing oyster mushroom. Thus, the tuff, pine needles, wheat straw, banana leaves (Ananbeh and objective of this research was to study the efficiency of convert- Almomany, 2005, 2008 Al-Momany and Ananbeh, 2011), leaf ing date palm leaf wastes mixed with other agricultural wastes of hazelnut (Yildiz et al., 1997), cotton waste (Oh et al., 2000), obtainable in KSA into value added product with high protein maize stover (Fanadzo et al., 2010), palm oil (Rizki and content by growing the mushroom species P. ostreatus. Tamai, 2011) and other wastes. The individual specimens of the oyster mushroom often grow in layers on top of each other 2. Materials and methods (Hoglov, 1999). Mushroom production technology is not well-established in 2.1. Substrate preparation Saudi Arabia. Very few studies have been conducted to produce mushrooms and other basidiomycetes (Al-Qarawi et al., Leaves of date palms were collected from the local farms in Al 2013; Khanaqa, 2006). Different agro wastes are available in Madinah Al Munawwarah, in KSA. Leaves were ground using huge amount in Saudi Arabia such as windbreak residues, cit- a date-palm leaf grinder. Other agricultural wastes including rus and olive trees pruning, green landscapes, vegetables and wheat straw, saw dust, and boobialla Myoporum serratum fruits, date trees (Sadik et al., 2010), boobialla leaves, sawdust, leaves (cultivated as ornamentals in streets and parks) were and wheat straw. Most of these residues are utilized in an mixed with date palm leaves at different ratios. Five ratios inefficient way, but some were successfully used as compost from each agro-waste and date palm leaves were used: 0: (Al-Barakah et al., 2013; Sadik et al., 2010). 100%, 25%: 75%, 50%: 50%, 75%: 25%, and 100%: 0% Date palm (Phoenix dactylifera) is an important ancient (date palm: agro-waste). Two supplements, wheat bran and monocotyledonous plant in the Kingdom of Saudi Arabia. It corn meal, were added to the mixture at 5% of the dry weight. is composed of trunk, leaves, seeds, and fruits. Date palm Gypsum was also added for all the treatments at 5% of the leaves are 100–250 cm long and may reach up to 4–7 m in substrate dry weight. The agro-wastes, supplements, and length (Al-Gboori and Krepl, 2010). Date palm parts are gypsum were mixed thoroughly and 500 g was placed in 5 obligatory by-products of date production and their main L-heat-tolerant plastic bags. The mixture was sterilized chem- use concentrated to improve soil organic content, animal feed, ically by adding water with 750 ppm formaldehyde (Hussain and manifested feedstock such as cellulose and hemicelluloses. et al., 2002), and incubated for 18 h outdoor where the ambi- High percentage of cellulose and lignin is found in different ent temperature ranged between 40 C and 45 C. parts of the date palms, bases and midribs, leaflets, fruit stalks, and spathe. These parts can be used as a substrate to grow 2.2. Fungal culture nutritious product with low cost such as oyster mushroom. Moreover, date palm leaves contain minerals such as N, P, K, Ca, Mg, and Fe (Abo Hassan and Bacha, 1982). The fungus P. ostreatus (Jacquin: Fries) P. Kummer was used Media made from a mixture of different portions of date in this study. The strain was purchased from the White Button palm seeds and leaves were evaluated for cultivating P. ostre- Establishment in Al-Salt, Amman, Jordan. The fungus was atus mycelium (Al-Qarawi et al., 2013). They found that the obtained as ready spawn grown on wheat seeds. mycelial growth incubated for eight days was the highest at a mixture of 80% seeds and 20% leaves. Another study was con- 2.3. Inoculation, incubation, and culture conditions ducted to evaluate two sterilization methods, chemical and heat, of different date palm parts to produce oyster mushroom Three spawn rates were used (5%, 7.5%, and 10% of the sub- (Abdulhadi and Hassan, 2013). Their results showed no signif- strate dry weight) for inoculation. Inoculated bags were then icant differences in the total mushroom production among the placed inside a cold room at 20–25 C under 80–95% relative different parts used, and the preference of using heat versus humidity with complete darkness during the first three weeks chemical methods in sterilization. Moreover, protein content until the substrate was completely colonized with mycelium.

Figure 1 Main effects of the factors used in studying oyster mushroom production on three agro-wastes with different mixing ratios, supplements, and spawning rates. BZ: boobialla leaves, SD: sawdust, WS: wheat straw.

Bags were punctured (+shape) using clean knife from four total, and biological efﬁciency (percentage yield of fresh mush- sides to facilitate primordial initiation. room over the dry weight of the substrate).

2.4. Harvesting and parameters 2.5. Proximate analysis

Mushrooms were harvested from the substrate when the caps Two treatments from waste*ratio were used for proximate got fully mature and before the fruiting bodies start to curl analysis. Analytical measurements included crude protein up. The clusters of the mushrooms were weighed, and several determined by Bradford method (Bradford, 1976), crude fat parameters were evaluated: the length of each phase of the fun- determined by estimation of total lipids method (Van gus production cycle (first and second flush), earliness (the time Handel, 1985), crude fiber (Amendig Annex 1, 1992), and car- elapsed between the day of inoculation and the day of the first bohydrates using the phenol–sulfuric acid method (DuBois harvest), fruiting bodies number per flush and in total, the et al., 1956). Residual formaldehyde was also measured for average weight of individual basidiomata per flush and in seven treatments, each was a mixture of multiple fruiting total, the average yield for each treatment per flush and in bodies (EMEA, 2002).

Table 2 Proximate analysis for the mushroom fruiting body and the agro-waste mixtures used for oyster mushroom cultivation. Treatment* Ratio Proximate analysis Fiber (%) Lipid (%) Protein (g/100 g) Carbohydrate (g/100 g) Fruiting bodies SD-Ctrl 0:100 10.15 4.90 1.30 14.15 DP:SD 25:75 11.75 4.50 2.80 26.15 DP:SD 50:50 13.00 7.65 1.75 18.65 DP:SD 75:25 10.50 4.70 2.00 19.35 DP-Ctrl 100:0 12.30 3.60 1.90 15.45 WS-Ctrl 0:100 11.90 5.85 1.90 19.90 DP:WS 25:75 14.20 5.55 1.90 22.85 DP:WS 50:50 12.00 5.40 2.65 28.30 DP:WS 75:25 10.75 3.60 1.95 22.40 BL-Ctrl 0:100 11.15 5.50 1.45 14.35 DP:BL 25:75 14.50 2.45 1.95 19.55 DP:BL 50:50 11.00 2.25 2.05 24.40 DP:BL 75:25 15.00 2.80 2.00 22.50 Agro wasteà SD – 9.00 0.25 2.40 4.00 DP – 12.00 0.70 9.60 3.30 BL – 5.00 0.28 4.20 2.00 WS – 15.00 0.71 8.00 3.80 * BL: boobialla leaves, DP: date palm, SD: sawdust, WS: wheat straw. The values for proximate analysis are an average of two replicates. à The proximate analysis was performed on each waste.

2.6. Mineral analysis average weight (Table 1). However, among the five ratios used, 0:100 ratio had 34.5, 11, 270%, and 135 g for pin appear- Similar to the proximate analysis, minerals and heavy metals ance time, fruiting bodies/bag, biological efficiency, and total including Ca, Cr, Cu, Fe, K, Mn, Ni, Pb, Zn, Na, and Mg were yield/bag, respectively, followed by the 25 (date palm): 75 measured. (agro-waste) treatment which had 35 d for pin appearance time, 15 fruiting bodies/bag, 250% biological efficiency, and 2.7. Experimental design and data analysis 120 g total yield/bag. The ratio 100 (date palm): 0 (agro-waste) had the highest earliness time (37 d) and the highest fruiting body average weight/bag (8.2 g). Completely randomized design was used to arrange the treat- The supplement added to the substrates had significant val- ments. Three agro-wastes (wheat straw, saw dust, and boobia- ues for all parameters except for the second flush average lla leaves) * five ratios (0: 100%, 25%: 75%, 50%: 50%, 75%: weight parameter. Corn meal gave higher number of fruiting 25%, and 100%: 0% (date palm: agro-waste)) * two supple- bodies (14), yield (120 g), and biological efficiency ments (wheat bran and corn meal) * three spawning rates (245%) compared to wheat bran supplement which had (5%, 7.5%, and 10% of total dry weight in each bag) * three 11, 100 g, 200%, respectively (Fig. 1). replicates were used. The experiment was repeated twice and The used spawn rate showed significant P-values for the 1st the data were analyzed using Minitab16 Statistical Software and 2nd flush yields, 2nd flush fruiting body numbers, total (State College, PA: Minitab, Inc. (www.minitab.com) at 95% yield, BE, and total fruiting bodies (Table 1). In general, all level of confidence. parameter values had a direct relationship with the spawning rate except for earliness and 1st flush harvesting date. 3. Results The interactions between the different factors studied varied in their significance (Table 1), however, the 3.1. Parameters tested waste*ratio*supplement was the only interaction that showed significance in most of the parameters tested (Table 1). The Regression analysis showed significant P-values in the models interaction of 25 (date palm): 75 (agro-waste) ratio, corn meal for all the parameters tested (Table 1). In all parameters mea- supplement, and 10% spawning rate was the best combination sured, the agro-waste type showed significant P-values for most of the parameters tested (figures not shown). (Table 1) with wheat straw being the best waste used among most of the variables measured (Fig. 1) (Appendix 1). For 3.2. Proximate analysis example, it had the highest biological efficiency (300%), total yield (>140 g), total number of fruiting bodies (18), and fruit- Wheat straw and date palm were rich in fibers and protein ing body average weight per bag (8.5 g) (Fig. 1). content (Table 2). All agro wastes had <1% lipid, and their The ratio of the mixed agro-wastes was also significant in carbohydrate content ranged between 2.00 and 4.00 g/100 g all the parameters except for the second flush fruiting body (Table 2). P. ostreatus fruiting bodies were rich in

Table 3 Analysis of variance for the proximate analysis of mushrooms produced from three agro-wastes. Source of variation DF Proximate Protein Lipids Fibers Carbohydrates FP FP FP F P Treatment* 2 0.52 0.780 2.70 0.127 0.92 0.435 1.50 0.279 Ratio 4 1.14 0.375 1.19 0.386 1.06 0.436 5.13 0.024 Interaction 8 0.72 0.673 2.03 0.113 2.30 0.078 0.81 0.606 * Three agro wastes were studied: boobialla leaves, wheat straw, and date palm. The three agro-wastes were mixed at ﬁve ratios with date palm: 0:100, 25:75, 50:50, 75:25, and 100:0 (date palm: agro-waste).

Table 4 Mineral analysis for the different agro-waste mixtures used for oyster mushroom cultivation. Waste* Ratio Mineral concentration (mg/L) Ca Na Mg Cr Cu Fe K Mn Ni Pb Zn Fruiting bodies SD-Ctrl 0:100 10.430 22.050 16.930 0.000 1.700 0.000 146.650 0.161 0.001 0.000 9.261 DP:SD 25:75 4.005 11.210 15.140 0.000 0.000 0.000 172.600 0.086 0.000 0.000 0.271 DP:SD 50:50 10.610 13.260 17.030 0.000 0.000 0.000 147.700 0.117 0.000 0.000 0.381 DP:SD 75:25 5.700 31.220 16.810 0.060 0.000 0.000 172.500 0.085 0.000 0.068 9.463 DP-Ctrl 100:0 6.320 18.730 17.570 0.000 0.000 0.000 121.250 0.092 0.005 0.000 6.536 WS-Ctrl 0:100 7.920 34.770 16.630 0.000 0.000 0.000 60.990 0.087 0.002 0.036 17.530 DP:WS 25:75 4.350 43.950 16.580 0.000 0.000 0.018 62.910 0.085 0.004 0.115 17.254 DP:WS 50:50 2.240 41.140 15.970 0.000 0.000 0.000 58.950 0.069 0.004 0.145 19.004 DP:WS 75:25 7.930 27.140 16.710 0.000 0.000 0.310 58.280 0.085 0.000 0.017 6.897 BL-Ctrl 0:100 17.990 38.870 25.370 0.000 0.000 0.000 65.710 0.707 0.000 0.000 0.171 DP:BL 25:75 14.760 36.790 24.790 0.000 0.000 0.000 59.610 0.122 0.001 0.000 0.220 DP:BL 50:50 15.350 27.280 22.010 0.000 0.000 0.000 110.410 0.116 0.260 0.000 0.182 DP:BL 75:25 21.350 21.930 19.390 0.000 0.000 0.000 128.750 0.138 0.001 0.000 0.409 Agro wasteà SD – 0.000 0.000 0.000 0.000 0.000 0.000 0.158 0.000 0.000 0.000 0.000 DP – 111.400 6.022 34.920 0.036 0.024 5.615 149.600 0.535 0.004 7.686 0.395 BL – 800.900 13.950 265.100 0.000 0.006 15.07 32.930 2.376 0.000 19.190 0.156 WS – 77.760 16.680 19.130 0.037 0.035 8.003 142.800 0.472 0.000 10.300 0.195 * BL: boobialla leaves, DP: date palm, SD: sawdust, WS: wheat straw. The values for mineral analysis are an average of two replicates. à The proximate analysis was performed on each waste.

Agro wastes had a high level of calcium by themselves. Generally, lignocellulosic materials are low in their protein Boobialla leaves had 800 mg/L of calcium which is eight times content, and they require additives to provide them with differ- more than date palm and wheat straw. Similarly, Mg and Fe ent minerals such as phosphorus, potassium, and nitrogen, and were the highest in boobialla leaves. Potassium was high in thus, enhance mushroom production (Mangat et al., 2008). its amount in both date palm and wheat straw. Moreover, For that reason, supplementation is an important step in DP, WS, and BL have high Pb values. On the other hand, saw- enhancing oyster mushroom production (Estrada et al., dust was the poorest among the different agro-wastes in its 2009), however, the supplement ratio should not be high due content of the different minerals analyzed (Table 4). to the possibility of yield reduction (Fanadzo et al., 2010), contamination possibility (Yildiz et al., 2002), and increase in the bed temperature, and possibility of mycelium killing 4. Discussion (Upadhyay et al., 2002). Different supplements were used to enhance oyster mushroom production including cottonseed P. ostreatus is an easily cultivable mushroom that colonizes hull (Fanadzo et al., 2010), wheat bran (Yildiz et al., 2002; various crop residues as substrates. Pleurotus spp. are able to Al-Momany and Ananbeh, 2011), olive mill waste (Ruiz- degrade and convert lignocellulosic compounds into protein- Rodrı´guez et al., 2010), cotton seed and soya bean rich biomass (Mamiro and Mamiro, 2011), and help in manag- (Upadhyay et al., 2002), rice bran, and maize powder (Alam ing agro-wastes whose disposal has become a problem (Das et al., 2010). Wheat bran is recommended over other supple- and Mukherjee, 2007). It demonstrated higher colonization ments. During mycelium growth, wheat bran elemental com- rates, improved earliness, and sporophore yield on different position decreases in its carbon content and increases in agro wastes compared to other cultivated mushroom genera nitrogen and oxygen content, suggesting a preferential degra- (Philippoussis et al., 2001). Its cultivation has increased greatly dation by the fungal mycelium for certain polysaccharides than during the last few decades throughout the world (Chang, the others, and accumulation of proteins in the substrate 1999) due to its ability to adapt varied agro-climatic conditions (Locci et al., 2008). Moreover, cellulose: lignin and carbon: (Jandaik and Goyal, 1995). In addition to that, P. ostreatus nitrogen ratios are found to be positively correlated to myce- growth on different agricultural wastes can provide more food lium growth and yield for Pleurotus spp., respectively and decrease different crop residues (Nwokoye et al., 2010). (Philippoussis et al., 2001; Rizki and Tamai, 2011). Carbon: Sterilization is an important step for mushroom cultivation. nitrogen ratio plays an important role in spawn running Previous studies tested different sterilization techniques includ- (Naraian et al., 2009). In the current study, both supplements, ing hot water, autoclave, formalin and bavistin (Hussain et al., wheat bran and corn meal, gave good results for the different 2002). In that study, sterilizing with formalin and bavistin and measurements; however, corn meal was superior over wheat autoclave found to have better spawn running, pin head and bran in the results of total yield and biological efficiency. No fruiting body formation, and yield. In another study, disinfec- previous articles studied the effect of using corn meal as tion of the substrate was conducted by 0.1% KMnO4 plus 2% supplement in oyster mushroom cultivation. In a previous formalin solution in hot water which resulted in a 42.6% study by Alam et al. (2010), maize powder supplement gave increase in yield of Pleurotus sajor caju over control (Banik the maximum biological and economical yield as compared and Nandi, 2004). In the current study, formalin was used at to other supplements for Calocybe indica mushroom. The 750 ppm and the treated substrates were left for the next day result could be attributed to the ability of corn meal supple- and exposed to the sun light for about 10 h with an ambient ment to promote different enzymes’ (cellulose (s), hemicellu- temperature of 55 C. Among the total number of bags used, lase (s), and laccases) secretion, which are important in the only two bags were contaminated with Trichoderma sp. sug- degradation of cellulose, hemicelluloses and lignin, respectively gesting the success of using the combination of formalin and (Jackson, 1978; Rajarathnam et al., 1986), more than wheat the high temperature of Saudi Arabia in sterilizing the sub- bran. On the other hand, Alam et al. (2010) found that increas- strate. Experiments must be conducted to compare between ing the maize powder is directly related to the mushroom pro- the high temperature, the formalin, and the interaction duction but to a certain point (30%), where the yield started to between them on the pasteurization of substrates. Residual decrease after that. Similarly, for wheat bran, increasing the formaldehyde was not found in the fruiting bodies resulted percentage has a negative effect on quality and BE (Shen from different treatments suggesting the possibility of its deg- and Royse, 2002). radation and the safety of its use in pasteurization of the In the current study, three levels of spawning: 5, 7.5, and substrates. 10% were used, and the different measurements such as yield, The heat-tolerant plastic bags (oven bags) used in this BE, and total fruiting bodies increased as the percentage study were chosen to suit the temperature conditions in the increased. Similar results were obtained with Upadhyay et al. region. The internal temperature of the bags are expected to (2002), where the addition of cotton seed cake gave the highest be higher than the ambient ones, thus, increasing the pasteur- mushroom yield at 10%. Increasing spawning rate shortens ization level of the substrates, and improving the spawn mycelial colonization time, primordia formation, and the time running, yield, and giving contamination-free media. Previous to first crop of mushroom (Yang et al., 2013). The increased study revealed that polyethylene bags resulted in higher level of nutrient available in spawn at higher rates would biological efficiency compared to pottery, plastic trays, and provide more energy for mycelial growth and development polyester net (Mandeel et al., 2005). In addition to good pas- (Royse et al., 2004). teurization results, plastic bags are preferable because they Many studies have been conducted to test the ability of retain the relative humidity within the substrate during spawn P. ostreatus to grow on different agro wastes. Examples of running time, thus, preventing them from drying as compared those agro wastes include rice straw, wheat straw and cotton to other methods. wastes (Hussain et al., 2002; Pant et al., 2006), olive mill waste

(Ruiz-Rodrı´guez et al., 2010; Al-Momany and Ananbeh, plants were collected was irrigated from raw water, while the 2011), maize stover, thatch grass (Fanadzo et al., 2010), palm boobialla leaves collected from streets are irrigated from pri- oil (Rizki and Tamai, 2011), weed plants (Das and Mukherjee, mary and secondary-treated waste waters in Al Madinah 2007), chopped office papers, cardboard, and plant fibers region. Thus, these plants can be studied for their phytoreme- (Mandeel et al., 2005), sawdust, banana leaves and tomato tuff diation possibility. (Ananbeh and Almomany, 2008), biogas residual slurry man- Mushrooms are rich in proteins, carbohydrates, fats, miner- ure (Banik and Nandi, 2004), and jute waste products als, vitamins, and amino acids (Chang and Buswell, 1996; (Basak et al., 1996). Certain agro wastes proved to give higher Jiskani, 2001; Adejumo and Awosanya, 2005). In the current yields than others. For example, wheat straw was found to be study, potassium was the highest in its concentration com- superior over other agro wastes in colonization rates and pared to other nutrients, followed by Mg, Ca, and Zn. This production (Philippoussis et al., 2001; Pant et al., 2006; could be due to the high content of K in the agro-wastes used Fanadzo et al., 2010) which is confirmed in the current study. for mushroom cultivation. K is essential for several enzymatic Moreover, mixing agro wastes at different ratios enhances the reactions and it is found in plenty in P. ostreatus (Wang et al., productivity of P. ostreatus (Yildiz et al., 2002; Ananbeh and 2001). High K: Na ratio found in Pleurotus spp. makes it ideal Almomany, 2008; Al-Momany and Ananbeh, 2011). In the for patients suffering from heart and hypertension diseases. As current study, wheat straw was also superior in mushroom previously mentioned, substrates have effect on mushroom production followed by boobialla leaves and sawdust regard- properties (Oyetayo and Ariyo, 2013). Our results were in less of the ratio used. On the other hand, date palm alone agreement with Patil et al. (2010). However, other studies were (100:0 treatment) had the least earliness time and the highest not agreeing with our results. Oyetayo and Ariyo (2013) who fruiting body average weight/bag. Moreover, the 25 (date measured proximate and mineral analysis for P. ostreatus cul- palm): 75 (agro-waste) gave the highest total yield, total fruit- tivated on different agro-wastes, found that phosphorus had ing bodies, and biological efficiency compared to the other the highest value in all the minerals analyzed. ratios. Thus, it is recommended to mix date palm leaves with In the current study, carbohydrates were found to be the wheat straw at this ratio to get good mushroom yield and bio- highest nutrient followed by fibers. Previous studies showed logical efficiency. Moreover, the residue of the different agro high carbohydrates and fiber content in P. ostreatus fruiting wastes that remains at the end of cultivation can be mixed with bodies as well (Patil et al., 2010; Oyetayo and Ariyo, 2013). soils as fertilizers, and thus, is still considered beneficial. On the other hand, protein content found to be less than what Boobialla plant is widely cultivated in Saudi Arabia as an have been previously reported (Chirinang and Intarapichet, ornamental, and its leaf residues are of no use. Although it 2009; Oyetayo and Ariyo, 2013). Patil et al. (2010) reported had the least values in some of the measurements, it can still that high protein content in mushrooms is due to the decom- be used for mushroom cultivation, and thus, its residue in a position of total carbohydrate, cellulose, hemicellulose, and beneficial way. Interestingly, boobialla leaves had the highest fiber during inoculation stage. Ca, Mg, and Fe contents as compared to the other agro-wastes In this study, only two flushes were harvested. Earliness used, and the content of Ca and Mg was also the highest in all took 35 ± 2 days and it varied based on agro waste, ratio, sup- the treatments that involved this agro-waste. This is the first plement, and spawn rate. The first flush was after 7 ± 2 days time this agro-waste is studied in oyster mushroom cultivation. of pin head appearance based on the different factors involved. Previous studies reported the success of using different weeds The first flush had the highest yield as compared to the second (Das and Mukherjee, 2007) and wood (Tisdale et al., 2006) flush. This is not surprising, since after the first flush, the pro- as substrates for oyster mushroom cultivation. duction reduced drastically due to either nutrient reduction or Substrates that are used in cultivating mushrooms have toxic substance accumulation that are unfavorable for fruiting effect on the chemical, functional, and organoleptic character- (Upadhyay et al., 2002). Mushroom yield and BE are directly istics of mushrooms (Oyetayo and Ariyo, 2013). Protein related to strain, growth conditions, and substrate nutrition content and other nutrients were found to vary in the mush- (Upadhyay et al., 2002). room fruiting bodies when grown on different agro-wastes The contaminated bags with Trichoderma were excluded (Michael et al., 2011). For that, it is of importance to know from the analysis because no fruiting bodies were obtained. the chemical composition of the substrates before being used The contamination occurred in the beginning of the spawning in mushroom cultivation (Patil et al., 2010). The four agro- period, thus, all parameters were affected. Previous study on wastes studied here are variable in their content of fibers, pro- contaminants of Agaricus blazei showed no effect on the differ- teins and minerals. Date palm was rich in fiber, protein, and in ent parameters studied because it was introduced into previ- some minerals like Ca, Mg, K, and to some extent in Fe. These ously colonized composts (de Andrade et al., 2007). contents make date palm one of the favorable agro-wastes for In conclusion, wheat straw mixed with date palm at ratio of mushroom production. 25 (date palm): 75 (agro-waste) showed the best results in most Certain heavy metals were found in certain amounts in of the parameters measured with 35 d for pin appearance time, P. ostreatus fruiting bodies, and in some of the agro-wastes like 15 fruiting bodies/bag, 250% biological efficiency, and 120 g date palm and boobialla leaves. The presence of heavy metals total yield/bag. Additionally, substrate supplemented with in the white-rot fungi substrate is an important factor because corn meal was superior over wheat bran in all treatments, with it affects the biodegradation process and growth of the fungus higher number of fruiting bodies (14), yield (120 g), and bio- (Baldrian, 2003; Baldrian and Gabriel, 2003) through affecting logical efficiency (245%). Moreover, parameter values the activities of cellulolytic and hemicellulolytic enzymes increased with the increase of the spawn rate of P. ostreatus. (Baldrian and Gabriel, 2003). Possible heavy metal contami- Proximate analysis showed that the oyster mushroom fruiting nant of the date-palm and boobialla leaves came from the irri- bodies were rich in carbohydrate followed by fibers, lipids, and gation water used, where the field from which the date palm protein. In case of minerals, potassium had the highest value

Please cite this article in press as: Alananbeh, K.M. et al., Cultivation of oyster mushroom Pleurotus ostreatus on date-palm leaves mixed with other agro-wastes in Saudi Arabia. Saudi Journal of Biological Sciences (2014), http://dx.doi.org/10.1016/j.sjbs.2014.08.001 Cultivation of oyster mushroom Pleurotus ostreatus on date-palm leaves 9 compared to the other minerals tested. The use of date palm Ananbeh, K.M., Almomany, A.R., 2005. Production of oyster alone or mixed as a substrate for cultivating oyster mushroom mushroom Pleurotus ostreatus on olive cake agro waste. Dirasat was successful, thus, the different agro wastes can be utilized in Agric. Sci. 32, 64–70. a beneficial manner. Ananbeh, K., Almomany, A., 2008. Production of Oyster mushroom (Pleurotus ostreatus) on tomato tuff agrowaste. Dirasat Agric. Sci. 35, 133–138. Acknowledgements Baldrian, P., 2003. Interactions of heavy metals with white-rot fungi. Enzyme Microb. Technol. 32, 78–91. 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