Yield and Some Macro-Morphological Characters of Pleurotus pulmonarius (Fries) Quel. Fruit Bodies Cultivated on HCl-Optimized Oil Palm Bunch Substrate

Mushrooms are the reproductive structures of fleshy macro fungi which grow wild in the tropical and sub-tropical rainforest. They are capable of degrading lignin and hence are found naturally growing on different woody and non-woody agricultural residue regarded as substrates [1]. These substrate materials are usually by products from industries, households, agriculture etc, and are usually considered as wastes [2,3]. These wastes are actually resources in the wrong place at a particular time [4].

pH is an important factor for good production of oyster mushrooms. Most mushrooms grow and perform well at pH near to neutral or slightly acidic at 6.1 and 7.5 respectively [5]. In order to enhance fruit body production, Oyster mushroom growers prefer to use lime (CaCO₃) as an alkaline buffer to optimize pH of some acidic substrates [6]. On the other hand, many growers of oyster mushrooms have experienced difficulties in using oil palm bunch as substrate. Achufusi et al. [7] attempted using oil palm bunch as a substrate for the cultivation of P. ostreatus, but recorded no mycelium colonization as well as fruit body yield in the substrate and its supplementations. The substrate was contaminated by Coprinus cinereus (a competitor mushroom that contaminates mushroom beds). The pH of the substrate was later found to be high at 10.3 and was suggested as the reason for the growth of the competitor-Coprinus sp. and no yield of P. ostreatus.

In their separate investigations, Tabi et al. [8] and Lau et al. [9] also reported that P. ostreatus was unable to grow on palm empty fruit bunch (EFB) alone and this could be due to high alkaline content of the biomass which does not support mushroom growth. This investigation therefore aims to determine the most efficient means of exploiting Oil Palm Bunch as a substrate for Oyster mushroom production.

Source of culture

Pure mycelia culture (Spawn) of P. pulmonarius was obtained from the science laboratory of Plant Science and Biotechnology, Michael Okpara University of Agriculture Umudike.

Location of study

The study was conducted in the mushroom house of the department of Plant Science and Biotechnology, Michael Okpara University of Agriculture Umudike, Abia State Nigeria.

Spawn preparation/multiplication

Spawn of P. pulmonarius was prepared using sorghum grains. Sorghum grains were washed in 3 changes of water and soaked overnight. The grains were boiled in tap water for 10-15 minute using gas cooker as a local heat source. Grains was also completely drained of water before mixing with 2% (w/w) CaCO₃ and 4% CaSO₄ to optimize pH and prevent them from clumping respectively as recommended by Muhammad et al. [4,10]. Grains were subsequently packed 2/3 in heat resistant transparent bottles, tightly sealed with Aluminum foil held tight with rubber band before sterilizing in an autoclave at 121°C for 30 minutes. After sterilization, bottles were allowed to cool before they were aseptically inoculated with actively growing mycelia of P. pulmonarius by grain to grain transfer and incubated in the dark (at 27 ± 2°C) until grains were fully colonized by P. pulmonarius mycelium [11,12] (Figure 1).

Figure 1: (a) Plate 1: Oil Palm Bunch (OPB) Substrate Plate. (b) Plate 2: P. pulmonarius fruit bodies emerging from OPB.

Deterimination of pH of substrate

The pH of solution containing 5% of sample substrates in 50 ml of distilled water was determined using Jenway 3070 portable automatic temperature compensation digital pH meter, calibrated with buffer 7-4 and 10. The pH value was read on the digital scale.

Substrate preparation

1 kg of Dry OPB substrate was soaked separately in 0.1%, 0.2% and 0.3% and 0.4% HCl solutions made in 1.5 lit. tap water for 48 hrs. Another 1 kg of OPB substrate was also soaked in same amount of tap water and this served as control, according to Colavolpe et al. The pH of each HCl+OPB substrate was determined after 48 hrs of soaking. All the HCl+OPB substrate levels were packed in an improvised metallic drum (IMD) and pasteurized for 2 hrs using industrial gas cooker as a local heat source and allowed to cool over night. Various HCl+OPB substrates were made into 5 replications and packed in 2.5 liter transparent plastic buckets perforated randomly from bottom to the top.

Substrate inoculation

30 g of grain based spawn of P. pulmonarius was spread across each 200 g of OPB substrate packed in perforated transparent plastic buckets during inoculation [3]. All inoculated substrates were placed on wooden racks in the culture room and covered during spawn run. Humidity of the cropping room was optimized by constantly flooding with tap water. Substrates were regularly checked to ensure optimum moisture content prior to primordial initiation.

Measurement of morphological characters

Mushrooms growing on substrate of various percentage solutions of HCl were harvested at maturity. Stipe length and cap size were measured in centimeter using meter rule [3]. Fresh weight of individual fruit bodies was also determined using digital weighing balance.

Yield and biological efficiency

Harvested fresh fruit bodies from each 5 replicate levels of HCl treated OPB were weighed using digital weighing balance while Biological efficiency (BE%) was determined using the method of Chang and Miles.

Statistical analysis

Data obtained during the experiment were statistically analyzed using Analysis of Variance (ANOVA), mean separation and tests of significance were carried out by Duncan Multiple Range Test (DMRT) at p = 0.05 respectively [12].

Table 1 shows the effect of different concentrations of HCl acid solution on the pH of OPB and fruiting duration of P. pulmonarius. Result shows that the initial pH of OPB was found to be 9.5. Different percentage concentrations of HCl solution altered the pH of OPB in the following order: 0.1% (8.9), 0.2% (8.2), 0.3% (7.9) and 0.4% (6.2), while control was found at pH of 9.1 after soaking for 48 hrs. This shows that concentrated Hydrochloric acid simply acted as an acid buffer capable of optimizing the pH of an alkaline OPB substrate to support the growth of mushroom.

Table 1: Effect of Percentage HCl Concentrations on the pH of OPB and Fruiting Duration of P. pulmonarius.

The fruiting duration recorded in this work shows that oil palm bunch has the ability of inducing earlier fruiting of P. ostreatus and does not conform with the work of Shah et al. which recorded longer fruiting time for P. ostreatus on saw dust, wheat straws and other agro wastes combinations. Khan et al. observed relatively shorter fruiting duration when oyster mushroom on different lignocellulosic substrates. They recorded that pin head formation started 7-8 days and matured between 10-12 days of substrates inoculation. This could be that the substrates used contained more nutrients that supported early primordial formation.

During spawn run, all the HCl+OPB substrates including the control were colonized by mycelium (Sudirman et al.). Although, Coprinus cinereus, a contaminant mushroom was seen growing on control and other HCl+OPB substrates after 6 days, except 0.4% HCl+OPB. C. cinereus is a major contaminant in OPB when used in mushroom cultivation, even though pasteurization time is increased. The result also proves that C. cinereus grows better in a high alkaline medium as 0.4% HCl solution was able to reduce the high alkalinity (pH 6.2) of the OPB as well as C. cinereus contamination of the substrate.

Duration of primordia initiation shows that 0.1%+OPB took 8 days, 0.2%+OPB (9 days), 0.3%+OPB (10 days) and 0.4% OPB (11 days) to produce mushroom primordia while control took the longest duration of 12 days. This result conforms to assertion of Khan et al. which maintained that most mushrooms grow better in pH approximately near to neutral i.e., 7.0, but differ with those of Quimio; Quimio and Sardsud which noted that fruit bodies of P. ostreatus grown on various substrates emerged within 3-4 weeks after substrate inoculation. According to them, various factors such as pH, temperature, nature of substrate and method of pasteurization can determine the fruiting time of oyster mushrooms.

Table 2 shows the effect of various HCl solution concentrations on the yield and B.E (%) of P. pulmonarius fruit bodies cultivated on OPB substrate. From the result, 0.4% HCl+OPB gave the highest (900 g) fruit body yield and B.E (90.00%), followed by 0.3%+OPB (820 g), 0.2%+OPB (774 g) and 0.1%+(569 g) while control gave the lowest (493 g) with B.E of 49.30%. This result shows that as the concentration of Hydrochloric acid increased increased from 0.1- 0.4%, yield and biological efficiency of P. pulmonarius increased from 569.00 g (56.9%)-900.00 g (90.00%) while control 493.00 g (49.3%) respectively. This trend was due to Coprinus cinereus which contaminated the substrate, from control to 0.3% Hcl OPB (Achufusi). Tabi et al. and Lau et al. [9] had similar experience and reported that P. ostreatus was unable to grow on palm empty fruit bunch (EFB) alone and this could be due to high alkaline content of the biomass which does not support mushroom growth. Report of Khan et al. [5] had earlier supported this observation by stating that most mushrooms grow and perform well at pH near to neutral or slightly acidic at 6.1 and 7.5 respectively.

Table 2: Effect of Percentage HCl Solution Conc. on the Yield (gm) and Biological Efficiency (BE%) of P. pulmonarius fruit bodies on OPB substrate.

Table 3 represents the effect of percentage HCl solution concentrations on the S.L (cm), C.S (cm) and Wt.(g/kg) of P. pulmonarius fruit bodies cultivated on OPB. 0.3% HCl treated OPB substrate supported slightly high (2.77 cm) S.L than others while 0.1% gave seemingly the lowest (2.01 cm) S.L. All the percentage HCl treated substrate including control did not show a significant difference on the stipe length of P. pulmonarius fruit bodies at p>0.05 supporting the work of Okwulehie et al. [13]. Cap size (C.S) was smallest (2.49 cm) in the mushroom produced the control OPB substrate. But the largest (3.83 cm) C.S was observed in the 0.4%HCl treated OPB substrate. Individual mushroom weight shows an increased gradient from control to 0.4% HCl treated OPB and were not significant at p>0.05. Okwulehie et al. [13] observed that Pennisetum straw significantly increased the stipe length of P. ostreatus, followed by A. gayanus straw, Oryza straw. They also note that Panicum straw causes a reduction of the cap diameter. These findings revealed that substrate has significant effect on the macro-morphological characters of oyster mushrooms. Okoi et al. [14] maintained that different substrates have affect pileus diameter, stipe height and stipe girth. Apart from substrate Hydrochloric acid, Ogbo et al. reported that crude oil has significant effect macro morphological characteristics such as pileus diameter, stipe height, stipe girth, fresh weigh and weight of mushroom fruit bodies. One of the factors responsible for the production of healthy fruit bodies in mushroom industries is the absence of contaminants in the substrates [6]. Steam pasteurization may have ensured more effective pasteurization of the substrates than other techniques studied.

Table 3: Effect of Percentage HCl Solution Treated Substrate on S.L, C.S and Wt. of Fruit Bodies.

Table 4 represents the result of the relationship between S.L (cm), C.S (cm) and wt.(g/kg) of P. pulmonarius fruit bodies cultivated on HCl+OPB substrates. The result shows that there is high level of relationship between S.L and C. S at Rho=0.48** but shows no significant relationship between S.L and Wt. (0.36 ns). C.S also shows a high level significant relationship with weight of the fruit bodies (Rho=0.59**). This simply means that S.L of P. pulmonarius fruit bodies with respect to the assay was highly related to C.S while C.S does same to Wt. of the mushroom [15-20].

Table 4: Correlation Effect HCl+OPB Substrates on S.L, C.S and WT of P. pulmonarius Fruit Bodies.

In an experiment to determine the effect of HCl on OPB substrate for the cultivation of P. pulmonarius fruit bodies, it was found that HCl could be used as a suitable acid buffer to optimize the pH of alkaline-oil palm bunch (OPB) for the cultivation of P. pulmonarius fruit bodies. The pH of the raw OPB was at alkaline/pH value of 9.5 and in some cases 10.3 or more. Increase in HCl solution concentration from 0.1 - 0.4%, led to an increase in the acidity of OPB substrate from pH of 8.9 to 6.2 and increase in P. pulmonarius fruit body production while the OPB substrate used as control remained at 9.1. This shows that HCl is a good acid buffer to adjust the pH of OPB before it could be used for oyster mushroom.

Mushroom growers should harness HCl+OPB especially at 0.4% level in the cultivation of P. pulmonarius fruit- bodies. Percentage HCl solution should be increased to 0.5 or 0.6% to know if they would achieve better yield, Biological Efficiency and other growth parameters investigated in this research Further studies should aim to determine the consumption safety of P. pulmonary produced from HCl+OPB substrate. Finally, mushroom scientists should identify other cheaper alternative natural or organic acid buffer that would substitute synthetic acid. This could eliminate the irrational safety fear that may arise in the consumption of this mushroom

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