sustainability

Article
Utilization of Agro-Industrial Wastes for the Production of
Quality Oyster Mushrooms
Morzina Akter 1 , Riyadh F. Halawani 2 , Fahed A. Aloufi 2 , Md. Abu Taleb 2 , Sharmin Akter 1
and Shreef Mahmood 1, *

1 Department of Horticulture, Hajee Mohammad Danesh Science and Technology University,

Dinajpur 5200, Bangladesh; [email protected] (M.A.); [email protected] (S.A.)
2 Department of Environmental Science, Faculty of Meteorology, Environment and Arid Land Agriculture,
King Abdulaziz University, Jeddah 21589, Saudi Arabia; [email protected] (R.F.H.);
[email protected] (F.A.A.); [email protected] (M.A.T.)
* Correspondence: [email protected]; Tel.: +880-1712289013

Abstract: The objective of this study was to utilize agro-lignocellulosic wastes for growing oyster
mushroom which become problematic for disposal. Pleurotus ostreatus was cultivated on five agro-
industrial wastes: rice straw (RS), wheat straw (WS), corncobs (CC), saw dust and rice husk @ 3:1
(SR) and sugarcane bagasse (SB). Approximately 500 g sized polypropylene bags (20.32 × 30.48 cm)
were used for each substrate. The SR significantly improved the number of fruiting body (27.80), size
of the fruiting body (5.39 g), yield (115.13 g/packet), ash and shortened the days for stimulation to
primordial initiation and harvest (9.2 days). The maximum percentage of visual mycelium growth
with the least time (15.0 days) to complete the mycelium running was found in SB, whereas the highest





 biological efficiency value (56.5) was calculated in SR. The topmost value of total sugar (33.20%) and
Citation: Akter, M.; Halawani, R.F.; ash (10.87 g/100 g) were recorded in WS, whereas the utmost amount of protein (6.87 mg/100 g) and
Aloufi, F.A.; Taleb, M.A.; Akter, S.; total polyphenolics (196.88 mg GAE/100 g) were detected from SB and SR, respectively. Overall SR
Mahmood, S. Utilization of gave the highest amount of the fruiting body with the topmost polyphenols and ash, moderate protein
Agro-Industrial Wastes for the and total sugar, and secured maximum biological efficiency too. The results demonstrate that saw
Production of Quality Oyster dust with rice husk could be used as an easy alternative substrate for oyster mushroom cultivation.
Mushrooms. Sustainability 2022, 14,
994. https://doi.org/10.3390/
Keywords: oyster mushroom; agro-industry; wastes; productivity; quality
su14020994

Academic Editors: Nallapaneni

Manoj Kumar, Md. Ariful Haque and
Sarif Patwary 1. Introduction
Received: 1 December 2021 Developing countries such as Bangladesh suffer much from a food insecurity problem,
Accepted: 13 January 2022 mainly due to inadequate and imbalanced diet intake. The problem is further compounded
Published: 17 January 2022 by the rapid growth of the population in the country. As a consequence, people, espe-
cially children and women, are experiencing chronic malnutrition problems. Mushrooms
Publisher’s Note: MDPI stays neutral
could substantiate this malnutrition problemto some extent, as edible mushrooms are rich
with regard to jurisdictional claims in
sources of protein, vitamins, minerals and also contain a number of secondary plant metabo-
published maps and institutional affil-
lites [1–4]. Bangladesh is an agrobased country and various agroindustries generate a large
iations.
amount of lignocellulosic byproducts annually that are worthy of being transformed. Some
of these byproducts are used as feeds for livestock and the compost industry, and some are
still treated as waste. These wastes are mainly burned for cooking purposes or disposed
Copyright: © 2022 by the authors. into surrounding environments, leading to various environmental problems. However,
Licensee MDPI, Basel, Switzerland. these agro-industrial wastes can potentially be used in cultivating mushrooms, which, in
This article is an open access article turn, contribute to minimizing malnutrition problems and could reduce the environmental
distributed under the terms and pollution [5]. In addition, such uses helplandless and marginal farmers to increase their
conditions of the Creative Commons income through intensive indoor farming and create employment opportunities, especially
Attribution (CC BY) license (https:// for unemployed youth and women folk. These actions would directly impact Sustainable
creativecommons.org/licenses/by/ Development Goals.
4.0/).

Sustainability 2022, 14, 994. https://doi.org/10.3390/su14020994 https://www.mdpi.com/journal/sustainability

Sustainability 2022, 14, 994 2 of 10

Edible mushrooms are saprophytic fungi and have the ability to degrade lignocel-
lulosic materials by their extensive enzymes [6]. Among the edible mushrooms, Pleu-
rotusostreatus is ranked first in Bangladesh because of its adaptability in local climatic
conditions and ability to grow on a wide range of substrates [5]. Different studies also
reported the potential uses of various agro-industrial residues, including cotton waste,
wheat straw, sawdust, rice straw, sugarcane bagasse, and corncobs, in mushroom cultiva-
tion [7–9]. In Bangladesh, rice straw is usually used as a substrate to cultivate mushrooms;
however, its demand is increasing day by day because of the expansion of cattle farming.
The availability of sufficient rice straw all year round, in all parts of the country, is also
uncertain. Therefore, the potentiality of other agro-industrial wastes, such as wheat straw,
rice husk, corn cob, and sugarcane bagasse, etc., needs to be evaluated to identify options
that are cost effective, and can provide a better yield and quality of mushroom. Proper use
of these agro-industrial wastes as substrates for mushroom cultivation could improve the
economic status of the farmers, contribute to alleviating nutritional problems and would
reduce environmental pollutions. In this context, the present study has been undertaken to
evaluate the productivity and quality of oyster mushrooms using different locally produced
agro-industrial wastes.

2. Materials and Methods

2.1. Location and Treatments
The present investigation was carried out both at the Laboratories of the Horticulture,
and Food Processing and Preservation, Hajee Mohammad Danesh Science and Technology
University (HSTU), Bangladesh. The single factor experiment consisted of five treatments,
i.e., different types of substrates: rice straw (RS), wheat straw (WS), corn cobs (CC), mixture
of 75% saw dust and 25% rice husk (SR), and sugarcane bagasse (SB).

2.2. Collection and Preparation of Substrates

Rice straw, wheat straw, corn cobs and sugarcane bagasse were collected locally from
the Agricultural Farm, Parbatipurupazila, Dinajpur and saw dust from Horticulture Center,
Dinajpur. All the substrates were chopped (2 cm length), except the mixture of saw dust
and rice husk, and immersed in water for about 24 h to achieve 65–70% moisture. The next
day, after removing excess water from the substrates, all those substrates were boiled for 1 h
and cooled. The pasteurized substrates were cooled and used for mushroom cultivation.

2.3. Preparation of Spawn Packet

Packets of spawn were prepared separately with polypropylene bags (20.32 × 30.48 cm)
with each type of the substrates. Firstly, a layer of the prepared substrates was placed into
a polypropylene packet and, afterwards, approximate 125 g of the cultured mother spawn
was spread on the outer side of the substrate. Depending on the type of substrates, the
weight of the spawn packet was approximately 500 g. The spawning process was repeated
again following the same procedure, and the top most layer of the spawn was covered with
the minimum amount of substrate. The neck of the packet was covered with a heat resistant
plastic neck and plugged with cotton. Afterward, the neck was covered with brown paper
by placing a rubber band to hold it in place. All the packets were placed on the floor of the
laboratory with the necessary hygienic measures.

2.4. Cultivation of Spawn Packets and Harvest of Fruiting Bodies

When all packets were covered by mycelium, then the cotton plug, brown paper,
rubber bands were removed. In the case of saw dust mixed with rice husk packets, the
upper position of both sides of the plastic packet were cut into a “D” shape with a sharp
knife. However, in the cases of the other substrates, four (4) cuts were made in a rectangular
(5 × 1 cm) shape. After removing the plastic sheet, the substrate of the cut surface was
scraped to remove the thin whitish mycelium layer. The packets were placed separately
on the floor of the culture room and covered with a brown paper. High humidity was
Sustainability 2022, 14, 994 3 of 10

maintained in the culture room by spraying water thrice daily. The light in the culture room
was totally cutoff, but the ventilation was maintained throughout the culture time. The
humidity and temperature of the culture room was recorded at 3h intervals. Harvesting
was performed as the fruiting bodies came out from the cut surface of the packet and
attained the maximum size.

2.5. Parameters Recorded

2.5.1. Physical Parameters
Parameter Procedure of Measurement
This was measured for each substrate before the
mycelium surrounded the packet. It was noted at 4, 8, 12
Percent (%) visual mycelium
and 15 days after inoculation (DAI) and the percentages
were estimated with the observation of the naked eye.
This indicates the days required from inoculation to the
completion of the running of the mycelium. When the
Days required to complete the
whole spawn packet turned white with the growth of
mycelium running in spawn
the mycelium, then it was noted as the indication of the
completion of the mycelium running of spawn.
The days required from cutting the spawn packet to
Days required from stimulation to the
primordia initiation were recorded and measured at
primordia initiation
both the first and the second flush.
The time (days) required from stimulation to harvesting
Days required from stimulation to
was counted as the sum of days required from
harvest
stimulation to harvesting and was recorded.
W-developed fruiting bodies were considered as
Number of effective fruiting bodies effective fruiting bodies which were counted and
per packet (NFBP) expressed in number per packet. However, the tiny
fruiting bodies were not counted.
The diameter and length of the stalk of each fruiting
body was recorded from the top to the base of the stalk
Diameter and length of stalk (cm)
using an electric digital caliper (Model: Guanglu,
China).
The cap diameter and thickness over one gram (wt.) was
Diameter and thickness of cap (cm): measured using an electric digital slide caliper (Model:
Guanglu, China).
The individual weight of each fruiting body (IWFB) was
measured without removing the lower hard portion.
Individual and total weight of The weight of all fruiting bodies per packet was
fruiting bodies per packet weighed without the lower hard and dirty portions. The
weight was measured using an electric balance (Model:
PA 214, USA).
The following formula was used to calculate the
biological efficiency [10].
Biological efficiency
Biological efficiency (%) =
Weight of fresh mushrooms harvested per packet
Weight of dry substrate per bag × 100
For determining ash, 1 g of each fruiting body was taken
into a crucible. The crucible was placed in a muffle
Ash content furnace for 6 h at 600 ◦ C. Then, total ash was calculated
using the following equation [11]:
Weight of Ash
Ash content (g/100 g) = Weight of Sample taken × 100
Sustainability 2022, 14, 994 4 of 10

2.5.2. Total Sugar Content (mg/100 g fw)

The total soluble sugar content of each fruiting body was determined by using the
colorimetric method [12]. For this, firstly, 2 mL previously extracted of supernatant was
diluted with 1 mL phenol solution (5%). Subsequently, 5 mL of H2 SO4 (95.5%) was added to
the samples. The testtubes were then allowed to stand for 10 min and vortexed for 30 s. The
test tubes were kept in a water bath at room temperature for 20 min for color development.
Finally, the absorbance was recorded using a UV-VIS spectrophotometer (PG Instrument
Ltd., Bristol, UK) at a wavelength of 490 nm. The standard curve for the total soluble sugar
determination was constructed by using glucose solutions whose concentrations ranged
between 0 to 0.25 mg/mL.

2.5.3. Protein Content (mg/100 g of Fresh wt.)

The protein concentrations were determined using the colorimetric method [13].
Coomassie Brilliant Blue G-250 (0.04 mg/mL) and ortho-phosphoric acid (85%) were
used as protein reagent in the assay. One gram of afresh sample was taken for preparing the
extraction solution [14]. The fresh sample was extracted in 5 mL of 100 mM Tris-HCl (pH
7.5) using a homogenizer (Model: VELP Scientifica, Usmate Velate, Italy). After vigorously
vortexing, the mixture was kept in a refrigerator at 4–5 ◦ C for one hour and afterward
centrifuged at 5300 rpm for 15 min at 4 ◦ C. One hundred microliters (100 µL) of the su-
pernatant was mixed with 1400 µL distilled water, to which previously prepared 1.5 mL
Bearden solution was added. After vortexing, the absorbance was recorded at 595 nm
by using a UV/VIS spectrophotometer (PG Instrument Ltd., Bristol, UK). The content of
protein in the sample was calculated using bovine serum albumin (BSA, Sigma-Aldrich,
Saint Louis, MO, USA) as the standard.

2.5.4. Total Phenolics (mg GAE/100 g of Fresh wt.)

The total phenolic compounds in the fruiting body were estimated by Folin-Ciocalteu
reagent (FC) and the colorimetric method [15]. The extraction was performed using 1 g
fresh sample [16]. The mushroom tissue was extracted in 4 mL methanol (80%) containing
2.7% HCl (37%), shaken for 2 h on an orbital shaker (200 rpm) at room temperature and
centrifuged at 5300 rpm for 15 min at 4 ◦ C. The extraction procedure was repeated again
and the supernatants were combined for the total phenolic assay. Three hundred microliters
(300 µL) of the extract was diluted with 2.25 mL of Folin-Ciocalteu reagent and 2.25 mL of
sodium carbonate solution (60 g/L), respectively. The samples were vortexed and left for
90 min at room temperature. After incubation, the absorbance was recorded at 765 nm by
using a UV/VIS spectrophotometer. Then, the content of total phenolics was quantified
from a standard curve of gallic acid.

2.6. Statistical Analyses

The study was designed as a complete randomized design (CRD), with five treatments
and each having five replicates. Data were subjected to analysis of variance (ANOVA) with
the Statgraphics Plus Version 2.1 statistical program [17]. Comparisons of the treatment
means was performed by the Fisher’s Least Significant Difference (Lsd) test at 5% level of
significance.

3. Results
3.1. Growth and Development of Mycelia and Fruiting Body
In general, the growth of mycelia in various substrates increased with the passage of
time and notable variation (p ≤ 0.05) was found among the substrates in different days after
inoculation (DAI), except at 4 DAI (Table 1). At 16 DAI, the maximum growth was recorded
in SB (100) and WS (97.0), while the lowest growth was in SR (46.6%). The same substrate
(SB) also took the fewest days (15.0) from the day of inoculation to complete the mycelium
running, but WS needed the most days (38.2) to complete the mycelium running. It was
also observed that SR required significantly fewer days (2.6) from stimulation to primordia
Sustainability 2022, 14, 994 5 of 10

initiation in the first flush while in the second flush, the fewest days were required in WS
(12.8). SR also required significantly fewer days (6.6 days) from stimulation to harvest in
the first flush but in the second flush, fewer days were required (20.6 days) in WS (Table 2).
In contrast, the WS, RS, SB and CC substrates took 8.0, 10.8, 13.0 and 13.4 days, respectively,
for stimulation to harvest in the first flush, while in the second flush SB took the most days
(31.2 days) and no harvest was possible in the CC substrate after the second flush.

Table 1. Percent visual mycelium of oyster mushroom on different agro-industrial waste substrates.

Percent (%) Visual Mycelium at Different DAI

Substrates
4 8 12 15
Rice straw 8.80 NS 22.80 c 31.40 b 66.00 b
Wheat straw 9.20 21.40 c 31.00 b 97.00 a
Corn cob 10.60 27.20 b 58.60 a 60.00 c
Saw dust with
9.20 16.20 d 31.80 b 46.60 d
rice husk
Sugarcane
10.80 31.60 a 60.00 a 100.00 a
bagasse
Lsd 2.01 3.72 3.90 3.86
CV (%) 15.67 11.82 6.95 3.96
NS Nonsignificant; the means with the same letter(s) in a column do not differ significantly as per Lsd test
(p ≤ 0.05).

Table 2. Growth of mycelium and number of fruiting bodies of oyster mushroom on different
agro-industrial waste substrates.

Number of Fruiting Bodies Per

Complete First Flush (Days) Second Flush (Days)
Packet
Mycelium
Substrates Running in Stimulation to Stimulation to
Stimulation to Stimulation to
Spawn Primordial Primordial 1st Flush 2nd Flush
Harvest Harvest
Initiation Initiation
Rice straw 19.20 c 6.00 b 10.80 b 20.40 b 25.00 b 12.20 c 12.00 a
Wheat straw 16.60 d 4.60 c 8.00 c 12.80 c 20.60 c 13.60 b 12.20 a
Corn cob 20.60 b 7.80 a 13.40 a - - 8.80 d -
Saw dust with
38.20 a 2.60 d 6.60 d 18.80 b 26.60 b 21.20 a 6.60 b
rice husk
Sugarcane
15.00 e 5.40 bc 13.00 a 25.40 a 31.20 a 12.00 c 5.80 b
bagasse
Lsd 1.10 0.85 0.79 1.95 2.66 1.31 1.10
CV (%) 3.82 12.27 5.78 7.54 7.69 7.30 9.01
The means with the same letter(s) in a column do not differ significantly as per Lsd test (p ≤ 0.05).

3.2. Number, Size and Yield of Fruiting Bodies, and Biological Efficiency
The number of fruiting bodies per packet (NFBP) varied from 8.80 to 21.20 among the
substrates (Table 2). The highest NFBP was counted in SR (21.20), followed by WS (13.6),
RS (12.20) and SB (12.0), while the lowest was in CC (8.80) in the first flush whereas in the
second flush, the highest NFBP was recorded in WS (12.20) and RS (12.0). When combining
the NFBP of both the first and second flushes, then the highest NFBP was obtained from
SR (21.2 + 6.6 = 27.8) and the lowest from CC (8.80). Regarding size, the highest diameter
and length of stalk was measured in the substrate SR (1.34 and 0.41 cm) in the first flush
but in the second flush, WS produced the longest stalk and maximum diameter (1.37 and
0.29 cm) (Table 3). In all cases, the lowest length and diameter of stalk was measured in
SB. Similar to the stalk, the SR substrate also had the highest diameter and thickest cap
(2.01 and 0.28 cm), while the lowest was recorded in CC (1.60 and 0.16 cm) in the first flush.
It was also noted that the variations in thickness of cap among the substrates were not
significant in the second flush.
Sustainability 2022, 14, 994 6 of 10

Table 3. Size of fruiting body on different agro-industrial waste substrates.

First Flush (cm) Second Flush (cm)

Substrates Size of Stalk Size of Cap Size of Stalk Size of Cap
Length Diameter Diameter Thickness Length Diameter Diameter Thickness
Rice straw 1.04 bc 0.36 b 2.00 a 0.22 b 1.03 d 0.24 bc 1.66 c 0.19 NS
Wheat straw 1.13 b 0.39 ab 2.00 a 0.20 b 1.37 a 0.29 a 1.82 b 0.18
Corn cob 0.99 c 0.25 c 1.60 c 0.16 c - - - -
Saw dust with rice husk 1.34 a 0.41 a 2.01 a 0.28 a 1.21 b 0.26 ab 1.91 a 0.17
Sugarcane bagasse 0.89 d 0.22 c 1.84 b 0.20 b 1.12 c 0.21 c 1.68 c 0.17
Lsd 0.10 0.05 0.09 0.03 0.06 0.04 0.05 0.02
CV (%) 9.26 9.58 3.74 9.52 3.79 12.65 2.53 9.62
NS Nonsignificant; the means with the same letter(s) in a column do not differ significantly as per Lsd test

(p ≤ 0.05).

The IWFB and total weight of fruiting bodies per packet ranged from 2.22 to 5.39 g
and 27.68 to 115.13 g, respectively; no harvest was possible in CC substrate at second flush
(Table 4). In both flushes, the highest IWFB was measured in the substrate SR (142.58 g)
followed bythe second highest in WS (127.36 g) and the lowest IWFB was in the CC
substrate (27.68 g). In the first flush, the highest number of fruiting bodies per packet
was harvested from SR (115.13 g) but in the second flush WS yielded the highest fruiting
body (62.29 g). In all cases, a moderate yield was obtained from RS (107.90 g) and SB
(64.41 g), and the lowest was from CC (27.68 g). Regarding the biological efficiency of
oyster mushroom, it was significantly influenced by the substrates, with high SR (56.5)
performing the best followed by WS (48.3), RS (38.8) and SB (30.4) and lowest value (20.5)
was obtained from CC.

Table 4. Yield and biological efficiency of oyster mushroom on different agro-industrial waste
substrates.

First Flush (g) Second Flush (g) Biological

Substrates Individual Weight Weight of Fruiting Individual Weight Weight of Fruiting Efficiency
of Fruiting Body Bodies Per Packet of Fruiting Body Bodies Per Packet (%)

Rice straw 4.69 b 60.32 c 3.86 b 47.58 b 38.80 c

Wheat straw 4.73 b 65.07 b 4.59 a 62.29 a 48.30 b
Corn cob 2.75 d 27.68 e - - 20.50 e
Saw dust with rice husk 5.39 a 115.13 a 4.02 b 27.45 c 56.50 a
Sugarcane bagasse 3.60 c 47.61 d 2.22 c 16.80 d 30.40 d
Lsd 0.30 4.81 0.29 6.52 7.49
CV (%) 5.28 5.83 6.09 12.62 10.43
The means with the same letter(s) in a column do not differ significantly as per Lsd test (p ≤ 0.05).

3.3. Quality of Fruiting Body

The quality of mushroom depends on its biochemical constituents. The biochemical
constituents studied in this study varied significantly (p ≤ 0.05) among the substrates
(Table 5). The content of ash ranged from 6.94 to 10.87 g/100 g in the fruiting bodies and
the highest amount of ash was determined in the fruiting bodies grown on both WS (10.87)
and SR (10.05) substrates, and the lowest was from SB (6.94 g/100 g). Regarding total
sugar, the maximum sugar was detected in the fruiting bodies grown on WS substrate
(22.41), which were statistically identical with RS (21.78). On the contrary, both CC and SB
substrates had the minimum total sugar statistically, while mushroom grown on the SR
substrate contained moderate total sugar (18.65 mg/100 g). The maximum protein content
in the fruiting body was in SB substrate which was statistically similar with RS, SR and
CC whereas the minimum from the WS (Table 5). The concentration of total polyphenols
ranged from 109.59 to 196.88 mg and varied significantly (p ≤ 0.05) among the substrates
compared. The maximum concentration of polyphenols was extracted in the fruiting body
Sustainability 2022, 14, 994 7 of 10

obtained from SR (196.88 mg), whereas the lowest from SB (109.59 mg). The RS, WS and
CC substrates gave statistically similar amounts of total polyphenols.

Table 5. Biochemical constituents of oyster mushroom on different agro-industrial waste substrates.

Ash (g/100 Total Sugar Protein Polyphenols

Substrates
g) (mg/100 g) (mg/100 g) (mg GAE/100 g)
Rice straw 9.02 b 21.78 a 6.12 b 165.48 b
Wheat straw 10.87 a 22.41 a 5.67 c 152.68 b
Corn cob 8.97 b 16.65 c 6.33 b 156.90 b
Saw dust with rice husk 10.05 a 18.65 b 6.48 b 196.88 a
Sugarcane bagasse 6.94 c 16.23 c 6.87 a 109.59 c
Lsd 0.85 1.48 0.37 16.23
CV (%) 4.05 7.51 3.66 14.88
The means with the same letter(s) in a column do not differ significantly as per Lsd test (p ≤ 0.05).

4. Discussion
The variation in the growth and development of mycelia and fruiting bodies with
different substrates might be due to the composition of different substrates. The proper
amount of alpha-cellulose, hemi-cellulose and lignin enhance the growth and develop-
ment of mycelia whereas the presence of polyphenolic compounds retard the growth and
development of mycelia [18]. The higher mycelia growth and spawn running in SB may
be due to the availability of a higher level of nutrients at the beginning of inoculation.
Although the lowest growth of mycelia was recorded in SR substrate, it took the fewest
days from stimulation to harvest. The content of cellulose and lignin in SR might favor
the growth of fruiting body. The present findings are in accordance with a previous study
where authors [19] reported that sawdust amended with paddy straw provided suitable
conditions for spawn running. The slower growth and development of fruiting bodies in
CC might be due to the presence of a higher level of nitrogen and/or polyphenols, which
inhibit the growth and development of mycelia. In other studies, the rapid growth and
development of the mycelia of king oyster mushroom (Pleurotuseryngii) on CC and milky
mushroom (Calocybeindica) on WS have been reported more than other substrates [7,8]
which might be due the variation in the chemical composition of substrates and the different
species of mushroom used in the study. In this study, oyster mushrooms produced the
maximum number of fruiting bodies on the SR substrate, which might be due to the fact
that this mixture contains comparatively higher amounts of cellulose, hemicelluloses and
lignin, which might favor the growth and development of oyster mushrooms in the present
study [20,21]. The favorable conditions of the SR substrate enhanced the growth of fruiting
bodies and thereby produced the biggest stalk and cap. Similar findings were also reported
earlier [22]; however, some other studies showed variations in the size of stalk and cap of
fruiting bodies, which might be due to the variation in the strains of oyster mushrooms, as
well as different substrates and growing conditions [23,24].
In the present investigation, SR produced the highest IWFB followed by WS, which
might be due to their larger size of stalk and cap. On the contrary, RS and SB substrates
gave moderate IWFB; this is logical, as these substrates yielded a medium size of stalk and
cap. However, the lowest value of IWFB was obtained from the CC substrate because of
the characteristics that contribute to the lowest yield value. From the results of the present
experiment, it is evident that SR yielded the highest number of fruiting bodies (first harvest
+ second harvest) over other substrates. The reason for this may be the physical nature and
high cellulose, hemicelluloses and lignin of the SR substrate, which were suitable for the
oyster mushroom cultivation. The present result is in close proximity with an earlier study,
where the authors opined that maximum yield, biological efficiency and the number of
fruiting bodies of oyster mushrooms was obtained from sawdust [20]. The lowest value
of all yield contributing parameters in the second flush could be linked with a lower
availability of simpler carbon at the first flush while leaving few carbon compounds for the
Sustainability 2022, 14, 994 8 of 10

subsequent flushes [25]. Biological efficiency is used to assess the efficiency of substrate
bioconversion into fruiting bodies [9]. From an economic point of view, BE value should
be over 50% [9]. In this study, only the SR substrate exceeded a 50% level of BE, as this
substrate yielded the highest fruiting bodies per packet. Oyster mushrooms grown on a
CC substrate have a much lower BE than earlier studies [9,26,27], the reason for this may
be that the adverse C:N ratio retarded the growth of the mycelium, thereby influencing the
overall yield and BE. However, similar BE for RS, WS and SB substrates have been reported
earlier by several authors [28–31].
Mushrooms grown on SR and WS have higher levels of ash, which might be due
to the fact that they accumulated minimum moisture in their fruiting bodies and similar
values of ash have been reported by several authors [7,10,25,32]. In this study, the amount
of total sugar was detected in the range of 16.23 to 22.41 mg/100 g, while protein values
ranged from 5.67 to 6.87 mg/100 g. The lower value of total sugars and protein content
in the fruiting bodies might be due to the different protocols used for protein estimation
and also most of the authors quantified the carbohydrate and protein content on the dry
weight basis not on fresh weight basis. The significant differences in total sugar content
in mushrooms may possibly be due to the C:N and various chemical composition of the
substrates [33].Since the WS and RS substrates are rich in carbohydrate and fiber, as a result
their fruiting bodies are also found to be rich in sugars. This is in conformity with several
reports [10,25,32], where WS and RS produced carbohydrate rich mushrooms. It was also
observed that the fruiting bodies grown on the SB substrate contained the highest amount of
protein, which might be due to the availability of higher levels of nitrogen in this substrate.
A similarly higher level of protein in the mushroom has also been reported by authors [33].
Polyphenolics are strong antioxidant compounds and, in this study, mushroom grown
on SR substrate exhibited the highest amount of total ployphenols than other substrates.
However, insufficient literature related to the polyphenol content in mushroom is available
to make a conclusive statement on mushroom polyphenol in relation to different substrates.

5. Conclusions
Among the substrates used in this study, sugarcane bagasse exhibited faster mycelia
growth and time from inoculation to mycelium running than other substrates; however,
this did not correspond with time from stimulation to primordial initiation and stimulation
to harvest, size, yield and quality of mushroom. In all cases, rice straw and corncob
substrates showed slower growth and also gave poor yield compared to other substrates.
In some cases, wheat straw performed better than sawdust with rice husk but, due to
moderate yield and slower mycelium running rate, it may not be economical for small scale
cultivation. Based on the present results, it is apparent that most of the yield contributing
characteristics and biological efficiency were better in sawdust with the rice husk substrate.
In addition, the highest concentration of polyphenols and moderate amount of total sugar
and protein were detected from the same substrate. Therefore, saw dust in combination
with rice husk (3:1) can be used as an alternative source for the small scale cultivation of
oyster mushrooms.

Author Contributions: The work was conducted as a collaboration among all the authors. Authors
M.A. and S.M. designed the experiment and M.A., S.M. and R.F.H. analyzed the data. M.A. and S.A.
prepared the visualization and S.A., F.A.A. and M.A. organized the first draft of the manuscript. Au-
thor M.A.T., S.M. and F.A.A. wrote the manuscript and S.A., R.F.H. and F.A.A. edited the manuscript,
and M.A.T., R.F.H. and F.A.A. were responsible for fund acquisition. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was partially funded by the Ministry of Science and Technology, Bangladesh.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Sustainability 2022, 14, 994 9 of 10

Data Availability Statement: Mother culture of Pleurotus ostreatus was used in this study which was
kindly provided by the Horticulture Center, Department of Agricultural Extension, Dinajpur 5200,
Bangladesh.
Acknowledgments: Authors are expressing their appreciation to the Ministry of Science and Tech-
nology, Bangladesh for the partial financial support to complete the research project.
Conflicts of Interest: The authors declare that they have no conflict of interest.

References
1. Hirano, R.; Sasamoto, W.; Matsumoto, A.; Itakura, H.; Igarashi, O.; Kondo, K. Antioxidant ability of various flavonoids against
DPPH radicals and LDL oxidation. J. Nutr. Sci. Vitaminol. 2001, 47, 357–362. [CrossRef]
2. Khan, M.A.; Khan, L.A.; Hossain, M.S.; Tania, M.; Uddin, M.N. Investigation on the nutritional composition of the common
edible and medicinal mushrooms cultivated in Bangladesh. Bangladesh J. Mushroom 2009, 3, 21–28.
3. Kalač, P. A review of chemical composition and nutritional value of wild-growing and cultivated mushrooms. J. Sci. Food Agric.
2013, 93, 209–218. [CrossRef]
4. Valverde, M.E.; Hernándea-Pérez, T.; Paredes-López, O. Edible mushrooms: Improving human health and promoting quality life.
Int. J. Microbial. 2015, 2015, 376387. [CrossRef]
5. Ferdousi, J.; Riyadh, J.A.; Hossain, M.I.; Saha, S.R.; Zakaria, M. Mushroom production benefits, status, challenges and opportuni-
ties in Bangladesh: A review. Annu. Res. Rev. Biol. 2019, 34, 1–13. [CrossRef]
6. Kumla, J.; Suwannarach, N.; Sujarit, K.; Penkhrue, W.; Kakumyan, P.; Jatuwong, K.; Vadthanarat, S.; Lumyong, S. Cultivation of
mushrooms and their lignocellulolytic enzyme production through the utilization of agro-industrial waste. Molecules 2020, 25,
2811. [CrossRef]
7. Sardara, H.; Alib, M.A.; Anjuma, F.N.; Hussaina, S.; Naza, S.; Karimd, S.M. Agro-industrial residues influence mineral elements
accumulation and nutritional composition of king oyster mushroom (Pleurotus eryngii). Sci. Hort. 2017, 225, 327–334. [CrossRef]
8. Sardar, H.; Anjum, M.A.; Nawaz, A.; Naz, S.; Ejaz, S.; Ali, S.; Haider, S.T. Effect of different agro-wastes, casing materials and
supplements on the growth, yield and nutrition of milky mushroom (Calocybe indica). Folia Hort. 2020, 32, 115–124. [CrossRef]
9. Sardar, H.; Anjum, M.A.; Nawaz, A.; Ejaz, S.; Ali, M.A.; Khan, N.A.; Nawaz, F.; Raheel, M. Impact of various agro-industrial
wastes on yield and quality of Pleurotus sajor-caju. Pak. J. Phytopathol. 2016, 28, 87–92.
10. Prasad, S.; Rathore, H.; Sharma, S.; Tiwari, G. Yield and proximate composition of Pleurotus florida cultivated on wheat straw
supplemented with perennial grasses. Indian J. Agric. Sci. 2018, 88, 91–94.
11. Raghuramulu, N.; Madhavan, N.K.; Kalyanasundaram, S. A Manual of Laboratory Techniques. National Institute of Nutrition; Indian
Council of Medical Research: Hyderabad, India, 2003; pp. 56–58.
12. Dubois, M.K.A.; Giles, J.K.; Hamilton, P.A.; Smith, F. Colorimetric method for determination of sugars and related substances.
Anal. Chem. 1956, 28, 350–356. [CrossRef]
13. Bearden, J.C. Quantitation of submicrogram quantities of protein by an improved protein-dye binding assay. Biochim. Biophys.
Acta (BBA)-Protein Struct. 1978, 533, 525–529. [CrossRef]
14. McCown, B.H.; Beck, G.E.; Hall, T.C. Plant leaf and stem proteins. Extraction and electrophoretic separation of the basic,
water-soluble fraction. Plant Physiol. 1968, 43, 578–582. [CrossRef] [PubMed]
15. Singleton, V.L.; Rossi, J.A. Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am. J. Enol. Vitic.
1965, 16, 144–158.
16. Velioglu, Y.S.; Mazza, G.; Gao, L.; Oomah, B.D. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain
products. J. Agric. Food Chem. 1998, 46, 4113–4117. [CrossRef]
17. STSC. Statgraphics Users Guide; STSC: Spencer, MA, USA, 1987.
18. Wang, D.; Sakoda, A.; Suziki, M. Biological efficiency and nutritional value of Pleurotus ostreatus cultivated on spent beer grain.
Bioresour. Technol. 2001, 78, 293–300. [CrossRef]
19. Khan, A.M.; Khan, S.M. Studies on the cultivation of Oyster mushroom Pleurotus ostreatus on different substrates. Pak. J. Phytopath
2001, 13, 140–143.
20. Shah, Z.A.; Asar, M.; Ishtiaq, M. Comparative study on cultivation and yield performance of oyster mushroom on different
substrates (wheat straw, leaves, saw dust). Pak. J. Nutr. 2004, 3, 159–160.
21. Dos Santos, R.M.; Neto, W.P.F.; Silverio, H.A.; Martins, D.F. Cellulose nano crystals from pineapple leaf, a new approach for the
reuse of this agro-waste. Ind. Crops Prod. 2013, 50, 707–714. [CrossRef]
22. Zhang, R.; LiXiu, J.H.; Fadel, J.G. Oyster mushroom cultivation with rice and wheat straw. Bioresour. Technol. 1998, 82, 277–284.
[CrossRef]
23. Khlood, A.; Ahmad, A. Production of oyster mushroom (Pleurotus ostreatus) on olive cake agro-waste. Dirasat Agric. Sci. 2004, 32,
64–70.
24. Sarker, N.C.; Hossain, M.M.; Sultana, N.; Mian, I.H.; Karim, A.J.M.S.; Amin, S.M. Performance of Different Substrates on the
growth and Yield of Pleurotus ostreatus (Jacquin ex Fr.) Kummer. Bangladesh J. Mushroom 2007, 1, 44–49.
25. Adenipekun, C.O.; Omolaso, P.O. Comparative study on cultivation, yield performance and proximate composition of Pleurotus
pulmonarius Fries. (Quelet) on rice straw and banana leaves. World J. Agric. Sci. 2015, 11, 151–158.
Sustainability 2022, 14, 994 10 of 10

26. Rofiqah, U.; Kurniawan, A.; Aji, R.W.N. Effect of temperature in ionic liquids pretreatment on structure of lignocellulose from
corncob. J. Phys. Conf. Ser. 2019, 1373, 012018. [CrossRef]
27. Pointner, M.; Kuttner, P.; Obrlik, T.; Jager, A.; Kahr, H. Composition of corncobs as a substrate for fermentation of biofuels. Agron.
Res. 2014, 12, 391–396.
28. El-Tayeb, T.S.; Abdelhafez, A.A.; Ali, S.H.; Ramadan, E.M. Effect of acid hydrolysis and fungal bio treatment on agro-industrial
wastes for obtainment of free sugars for bioethanol production. Braz. J. Microbiol. 2012, 43, 1523–1535. [CrossRef] [PubMed]
29. Limayema, A.; Ricke, S.C. Lignocellulosic biomass for bioethanol production: Current perspectives, potential issues and future
prospects. Prog. Energy Comb. Sci. 2012, 38, 449–467. [CrossRef]
30. Motte, J.C.; Trably, E.; Escudié, R.; Hamelin, J.; Steyer, J.P.; Bernet, N.; Delgenes, J.P.; Dumas, C. Total solids content: A key
parameter of metabolic pathways in dry anaerobic digestion. Biotechnol. Biofuels 2013, 6, 164. [CrossRef]
31. Zainudin, M.H.M.; Rahman, N.A.; Abd-Aziz, S.; Funaoka, M.; Shinano, T.; Shirai, Y. Utilization of glucose recovered by phase
separation system from acid-hydrolysed oil palm empty fruit bunch for bioethanol production. Sci. Pertanika J. Trop. Agric. 2012,
35, 117–126.
32. Pardo-Giménez, A.; Pardo, J.E.; Dias, E.S.; Rinker, D.L.; Caitano, C.E.C.; Zied, D.C. Optimization of cultivation techniques
improves the agronomic behavior of Agaricus subrufescens. Sci. Rep. 2020, 10, 8154. [CrossRef]
33. Adedokun, O.M.; Akuma, A.H. Maximizing agricultural residues: Nutritional properties of straw mushroom on maize husk,
waste cotton and plantain leaves. Nat. Res. 2013, 4, 534–537. [CrossRef]