GROWTH PERFORMANCE OF THE MUSHROOM PLEUROTUS OSTREATUS VAR. FLORIDA ON OIL PALM FRUIT BUNCH WASTE, SAWDUST AND ELEPHANT GRASS STRAW

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Increasing demand and usage of plastics and their disposal has resulted in environmental pollution burden on both land and water habitats, threatening the safety and health of wild life, aquatic life and humans (Verma et al., 2016).Plastics are non-biodegradable and difficult to eliminate from the environment; plastics are disposed into landfills and burnt causing air, water and soil pollution in Nigeria.Hence the need for recycling methods for plastics in the country.
Pleurotus spp.are the most preferred ones among the edible mushrooms due to their ability to grow quickly and productively in various lignocellulosic media (Frimpong-Manso et al., 2011;Gothwal et al., 2012).The common name for Pleurotus species is Oyster mushroom.Pleurotus is a genus in the class Basidiomycetes and a group of fungi known as white-rot fungi as they produce white mycelia (Tsujiyama and Ueno, 2013).Ofodile, I. N. et al.Pleurotus spp.are the second most cultivated worldwide and are sold in the world mushroom market (Imran et al., 2011, Royse et al., 2017;Bellettini et al., 2019).
Pleurotus ostreatus accounted for 14.2% of the total world production of edible mushroom in 1997 (Ofodile and Yusuf, 2010).The cultivation of mushroom includes selection of adequate mushroom species, preparation of quality fruiting culture, development of active spawn, preparation of substrate, mycelia spawn running, mushroom development and harvest (Ofodile and Yusuf, 2010).The species has a unique flavour, aromatic properties, rich in protein, fibre, carbohydrates and minerals (Ofodile et al., 2011;Lavelli et al., 2018).Pleurotus ostreatus showed protein contents similar to or higher than those found in many vegetables, but lower than meat and cheese contents (Mena and Manzi, 2019).Also, Pleurotus ostreatus is reported to contain attributes such as anti-viral, antibacterial, anti-tumour, anti-oxidant, anti-mutagenic, anti-parasitic and immunomudulatory (Ofodile et al 2011).Pleurotus spp.are selective degraders, degrading lignin and hemicellulose rather than cellulose.In this way, it remains exposed and can be utilised by ruminants (Cohen et al., 2002).
In Nigeria, indigenous mushrooms are harvested from forests and farmlands for sale.The need for commercial production of all edible mushrooms in Nigeria cannot be over-emphasised because of its potential contribution to agricultural production and as a source of cheap protein.Generally, agro-industrial wastes are inappropriately disposed (Sadh et al., 2018) with the consequence of threatening the environment and public health.Agro-industrial wastes are generally characterised by high biological oxygen demand (BOD) and chemical oxygen demand (COD) (Ravindran and Jaiswal, 2016) and they are also easily susceptible to bacterial contamination due to the high water content (Ravindran and Jaiswal, 2016).Therefore, their disposal is difficult.
In Nigeria oil palm mills process a large amount of oil palm generating large quantities of wastes from the fruit bunches.According to Dalino (1995) as cited by Triyono et al. (2019), for each bunch of fresh oil palm bunches, about 21% palm oil, 6-7 % palm kernel, 14-15% fiber, 5-7% shell and 23% empty bunches are obtained.The waste of oil palm fruit bunch disposed in the environment constitutes pollution.The only way to preserve edible mushroom and make them available for consumption is to cultivate them because many mushrooms eaten of old are nonexistent because of the impact of climate change (Ofodile, 2006).Therefore, to alleviate hunger and malnutrition in a world of rising food prices, cultivation of mushroom is a very reliable and profitable option.Report on the cultivation of the variety florida of P. ostreatus in Nigeria is scarce.
This work, therefore, reports the comparative study of the utilisation and the effects of some agro-industrial wastes, namely sawdust (Triplochiton scleroxylon), elephant grass (Pennisetum purpuraum) and oil palm fruit (Elaies guineensis) bunch waste on Pleurotus ostreatus var.florida

MATERIALS AND METHODS Study Location
The experiments were carried out at the Mushroom Research and Training Laboratory, Yaba College of Technology, Yaba Lagos, Nigeria.

Mycelial growth studies
Internal tissue of the carpophores was transferred onto sterile potato dextrose agar (PDA 1.5%) and incubated at 28 o C for 72 hr to prepare pure cultures that were maintained on PDA at 4 o C for further studies.Agar blocks of seven-day old culture of the mushroom were inoculated unto PDA (2%) in five replicates and incubated at 28 o C. Linear mycelia growth was measured and recorded daily for 10 days on 100 x 15 mm agar plate.

Spawn Production
Uninfected Sorghum bicolor grains (1kg) mixed with 5 g calcium carbonate and made up to 65% moisture content was prepared, divided into 500 g units, and loaded into whisky bottles.The bottles were sterilised for 1 hr at 121 o C at 15 Psi and allowed to cool.Each grain bottle was inoculated with five pieces of agar blocks (1 cm diameter) from 10-day old plate culture of P. ostreatus var.florida.After 14 days of incubation, when all bottles were fully colonised, they were used to inoculate test substrates used in growth studies.

Growth Studies
Oil palm fruit (Elaies guineensis Jacg.) bunch waste was collected from Badagry area of Lagos State.Sawdust of obeche (Triplochiton scleroxylon K. Schum.) was collected from a Sawmill in Mushin area, Lagos while the Elephant grass (Pennisetum purpureum) waste was collected from a field at Mowe area of Ogun State.Elephant grass was cleaned to obtain the straw only and the oil palm bunch waste was beaten on a hard surface to the internal fiber.The wastes were sun-dried for seven days before shredding sawdust to sizes 40-100 mesh.The grass straw was chopped to 4-5 cm lengths.Rice bran was purchased from a rice mill at Abeokuta.Calcium carbonate (CaCO3) was obtained from the chemical market at Ojota, Lagos.Moisture content in all materials was determined before use.P. ostreatus var.florida was plated on 2% PDA plates (90 mm diameter) and incubated at 25 o C for one week before use in spawn preparation.

Preparation of Substrates
Three substrates, namely oil palm fruit bunch waste (OPFBW), Sawdust (SD) and Elephant grass straw (EGS) were prepared for cultivation of P. ostreatus var.florida.Dried OPFBW (5000 g) and EGS (5000 g) wastes were pasteurised separately for 1½ h and 1h, respectively in water (100 L) mixed with calcium oxide (12.9 g).OPFBW was further boiled with fresh water (100 L) only for 1 hour.After cooling, EGS was mixed with calcium carbonate (10 g); OPFBW and EGS were separated into portions each and used for further cultivation studies.Rice bran (800 g) and CaCO3 (200 g) were appropriately mixed with SD (5000 g) and the moisture content adjusted to 64 %.The substrates OPFBW (1000 g weight) and SD (1200 g dry weight) were loaded into polyethylene bags (30 × 15 cm).EGS (1000 g weight) was packed into perforated used plastic pet bottles.Substrates were in five replicates.The bags of sawdust substrate were pasteurised for 6 hours in a locally-made steamer and allowed to cool overnight.The three substrate bags and plastic bottles were inoculated with the grain spawn (50 g) per substrate unit.
The inoculated substrates were incubated in the dark for eight weeks (SD), two weeks (OPFBW and EGS) at room temperature (20-25°C at night and 25-30°C during the day time).At the end of the incubation period, the fully colonised substrates were transferred to a fruiting room maintained at 28±2°C and 90-95% RH.Lighting was provided using sunlight coming into the fruiting house through glass windows during the day time.Aeration was achieved by opening the door to the fruiting room 30 min each, three times per day.Data collected during the fruiting experiment included the spawn run period, number of days taken until pinhead formation and number of primordial, number of days from commencement of fruit body induction to first flush, weight and number of basidiocarps per flush.The biological efficiency (BE) was calculated as the weight of fresh mushroom harvested/divided by dry weight of substrate and multiplying by 100 (Ofodile et al., 2022).

Expeerimental Design and Data Analysis
The randomised complete block design was used in the mushroom cultivation experiment.Data collected from all experiments were expressed as mean ± Standard Error of mean (SEM).Analysis of Variance (ANOVA) with Turkey's post-hoc was used for analysis at 5% level of significance.

Growth of P. ostreatus var. florida
The results of the hyphal extension of P ostreatus var.florida on potato dextrose agar is shown in Figure I.The mushroom grew well on Potato Dextrose Agar.The mycelial growth of the mushroom growth was first observed on day 2 after incubation.The mycelial growth was exponential and took 10 days to full ramification.There was significant difference in growth between day 3 and day 7. Ofodile, I. N. et al. and SD (30 days).The mushroom had a comparatively short incubation period on OPFBW.Figure 2 shows the different flushes during the cultivation of P. ostreatus var.florida.The production of mushroom differed significantly amongst the three flushes on the same substrates for OPBFW and SD but was significantly lower in the third flush for EGS.Results of the time taken for pin head emergence, number of pin heads, number and weight of carpophores, day to carpophore emergence and biological efficiency of the mushroom are shown in Table 1.The mushroom produced the highest number of pinheads on EGS.The pinheads of P. ostreatus var.florida emerged significantly faster on Oil Palm Fruit Bunch Waste (OPFBW) and Elephant Grass Straw (EGS) than on Sawdust substrate (Fig. 2).The weight of carpophore (g) by the mushroom on EGS was significantly higher than on OPFBW; the SD had the highest weight of carpophores in all the flushes.The total yield of mushroom carpophore from sawdust substrate was also significantly higher than on all the other substrates.Intervals between flushes on the three substrates (OPFBW, SD and EGS) were 14 days, 30 days and 10 days, respectively.The B.E on SD (26.72 %) was higher than on EGS while the B.E of OPFBW (4.32 %) was lower than on EGS (13.32 %) at 5% level of probability.The mushroom produced the highest number of carpophores on Elephant Grass Straw (EGS) when compared with the number of carpophores on the other two substrates (Fig. 2).The number of carpophores on OPFBW was higher than on SD.The mushroom produced carpophores on OPFBW at 14 days, 46 days on SD and 16 days on EGS after inoculation.

Substrate
Pin   (Sudirman et al., 2011) as was also observed in this study.P. ostreatus var.florida grew well on Potato Dextrose Agar and this may be as a result of the presence of glucose which has been proven to be the best carbon for the mushroom (Kuforiji and Fasidi, 1998).The effective production of spawn on Pleurotus ostreatus has been reported by Narh et al. (2011).It was observed that sorghum was an efficient medium for the preparation of mushroom seed from this study.Naraian et al. (2009) associated earlier appearance of pin head in corn straw to the high content of lignin which the fungus breaks down to release sufficient nutrients for its growth.P. ostreatus var.florida was also reported to have the fasted pin head emergence on EGS.
Pleurotus spp.have a high saprophytic ability and can successfully grow on a variety of lignocellulosic substrates (Machado et al., 2016;Iwuagwu et al., 2017), which agrees with results of this work.The formation of fruiting bodies is initiated by mycelial growth, followed by the production of primordia (El-Fallal et al., 2015).The period of mycelial growth on the different substrates in this study ranged from 8 days in OPFBW to 12 and 30 days in EGS and SD, respectively.The difference seen in the different treatments in spawn run period may be due to the composition of the substrates.Iwuagwu et al. (2017) reported that the variation in the number of days taken for the spawn to complete colonisation of a given substrate is a function of the fungus strain, growth condition and substrate type.An interval between flushes of 10 days has been reported for Pleurotus spp.cultivated on sawdust (Mandeel et al., 2005).Ten days between flushes were also reported for OPFBW and EGS.

A1 A2
B2 C2 B1 C1 Mintesnot et al. (2014) observed that the total fresh weight ranged from 377 to 665 g/kg dry substrate in the case of Pleurotus florida on different substrates, which differ from the present work.According to Jonathan et al. (2013), P. pulmonarius performed least on oil palm waste compared to coir fiber, sawdust and rice straw in flushes, weight and total yield.P ostreatus var.florida also had the least performance on oil palm fruit bunch waste in the present study.The low yield recorded by oil palm waste might be due to the complex lipid present in it.This is in agreement with the findings of Lim (1981), who reported that oil palm waste may contain complex lipids which may hinder easy access of the fungus to simpler carbon sources, thus reducing the mycelial colonisation and yield.

CONCLUSION
The study demonstrated the utilisation efficacy of Pleurotus ostreatus var.florida in oil palm fruit bunch waste, sawdust and elephant grass straw.The macrofungus performed best on sawdust although with a long incubation period.Mushroom cultivation can be leveraged on by a country like Nigeria for the recycling of used plastic bottles and other agro-industrial wastes in mitigating environmental pollution.

Fig. 1 :
Fig.1: Mean radial mycelial growth of Pleurotus ostreatus var.florida on potato dextrose agar Cultivation of P. ostreatus var.florida P. ostreatus var.florida produced the shortest spawn run period on EGS (8 days), compared to OPFBW (12 days) and SD (30 days).The mushroom had a comparatively short incubation period on OPFBW.Figure2shows the different flushes during the cultivation of P. ostreatus var.florida.The production of mushroom differed significantly amongst the three flushes on the same substrates for OPBFW and SD but was significantly lower in the third flush for EGS.Results of the time taken for pin head emergence, number of pin heads, number and weight of carpophores, day to carpophore emergence and biological efficiency of the mushroom are shown in Table1.The mushroom produced the highest number of pinheads on EGS.The pinheads of P. ostreatus var.florida emerged significantly faster on Oil Palm Fruit Bunch Waste (OPFBW) and Elephant Grass Straw (EGS) than on Sawdust substrate (Fig.2).

Fig 2 :
Fig 2: Effects of substrates on weight and total yield of harvested P. ostreatus var.florida Key: OPFBW = Oil palm bunch waste, SD= Sawdust, EGS= Elephant grass straw

Table 3 :
Growth performance of P. ostreatus var.florida on different substrates Key: OPFBW = Oil palm bunch waste; SD= Sawdust, EGS= Elephant grass straw.Substrates with different superscripts are significantly different from each other at 5% level of probability