Journal of Cleaner Production 268 (2020) 122272

Contents lists available at ScienceDirect

Journal of Cleaner Production

journal homepage: www.elsevier.com/locate/jclepro

Green application and toxic risk of used diaper and food waste as
growth substitute for sustainable cultivation of oyster mushroom
(Pleurotus ostreatus)
Nyuk Ling Ma a, b, **, 1, Shing Ching Khoo a, b, 1, Wanxi Peng a, 1, Chia Min Ng b,
Chin Hoe Teh c, Young-Kwon Park d, Su Shiung Lam e, a, *
a
Henan Province Engineering Research Center for Biomass Value-added Products, School of Forestry, Henan Agricultural University, Zhengzhou, 450002,
China
b
Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia
c
Bruker Malaysia Sdn Bhd, 11900, Bayan Lepas, Pulau Pinang, Malaysia
d
School of Environmental Engineering, University of Seoul, Seoul, 02504, Republic of Korea
e
Pyrolysis Technology Research Group, Pyrolysis Technology Research Group, Institute of Tropical Aquaculture and Fisheries (AKUATROP), Universiti
Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Used diaper and food waste form a major proportion of wastes piled up in landfill. This study aims to
Received 27 August 2019 reduce used diaper and food wastes (comprising coffee waste, sugarcane bagasse, banana skin, eggshell)
Received in revised form via converting them into a unique formulated growth substrate, providing an alternative of growth
5 May 2020
medium in mushroom cultivation. The growth media showed high content of cellulose (27%), hemi-
Accepted 14 May 2020
Available online 18 May 2020
cellulose (16%) and high nutrients with nitrogen (15,779 mg/kg), phosphorus (867 mg/kg) and potassium
(7,758 mg/kg), promoting nearly two-fold mushroom production (73 g) compared to commercial media
Handling editor: CT Lee (37 g). The mushroom cultivation also led to a 40% reduction in weight of growth substrate, confirming
its efficient utilization for growing mushroom while simultaneously leading to waste reduction. The
Keywords: mushroom harvested showed similar metabolite profile to that cultured by commercial media via
Cultivation profiling using nuclear magnetic resonances. The results indicated similar biological composition and no
Mushroom accumulation of undesired toxic and biological components that might be derived from the waste-
Diaper formulated substrate, thus providing assurance that it is safe to be used for sustainable mushroom
Waste
cultivation.
Metabolomics
© 2020 Elsevier Ltd. All rights reserved.

1. Introduction
There are about 1.3
109 tons (t) of food waste and 2
1010
pieces (pcs) of used diaper produced yearly due to urbanization and
rapid increase of human population (FAO, 2017). Food waste and
used diaper had been ranked at the top and third place in the total
of solid waste in the landfill, respectively (Sheila, 2016). One-third
* Corresponding author. Henan Province Engineering Research Center for
Biomass Value-added Products, School of Forestry, Henan Agricultural University,
Zhengzhou, 450002, China.
** Corresponding author. Henan Province Engineering Research Center for
Biomass Value-added Products, School of Forestry, Henan Agricultural University,
Zhengzhou, 450002, China.
E-mail addresses: nyukling@umt.edu.my (N.L. Ma), lam@umt.edu.my (S.S. Lam).
1
These authors contributed equally to this paper and acted as first authors.
of global food production had been turned into wastes. This has
raised environmental issues and caused many landfills to have
exceeded their carrying capacity and resulted in massive green-
house gas emissions (Chen and Lo, 2016). Even though almost 90%
of food waste is biodegradable and can be recycled for production
of bio-fertilizer and compost, in reality, the food wastes are mixed
with other types of solid wastes and make the recycling process
difficult to the extent of nearly impossible (Abdullah and Chin,
2010). Dumping and piling the food waste in landfill generate
methane, a major source of greenhouse gas, if it is not properly
managed (Lee et al., 2017).
The invention of disposable diapers brings convenience to par-
ents. The downside is that it also generates massive wastes up to a
stage that cause difficulties to the disposal of the waste (Espinosa-
Valdemar et al., 2014). A baby can use more than 8,000 pcs of

https://doi.org/10.1016/j.jclepro.2020.122272
0959-6526/© 2020 Elsevier Ltd. All rights reserved.
2 N.L. Ma et al. / Journal of Cleaner Production 268 (2020) 122272
disposable diapers from birth to potty training, leading to pro-
duction of 2 t of slow-to-degrade used diapers that are expected to
take up to 500 years (y) to be fully decomposed in landfill (Jesca and
Junior, 2015). Hence, efforts should be made to divert food waste
and used diaper from landfilling via conversion into value-added
products.
Studies have been performed to convert used diapers (Khoo
et al., 2018) and food wastes (Mahmood et al., 2019) into value-
added products for application as compost and soil fertilizer. This
provides the motivation for transforming used diaper and food
waste largely accumulated in the landfill into a unique growth
substrate for mushroom cultivation. The aim was to reduce these
wastes while simultaneously utilize the organic components as
nutrients for growing mushrooms. Oyster mushroom (Pleurotus
ostreatus), a common edible mushroom, was selected for this
study. The fruit bodies of mushroom are developed from the
germination of spore followed by the formation of mycelium. The
organic components of the growth substrate would be decomposed
into smaller molecules that can be absorbed for the growth and
development of the fruit bodies along the growth period. Therefore,
the mushroom is deemed to be a good candidate with the potential
to naturally degrade, transform, and utilize organic components
from waste as nutrients for its fruit body formation.
The growth substrate formulated from used diaper and food
waste is expected to possess three advantages based on the natural
enzymatic biodegradation mechanism in mushroom e 1) supply of
nutrients comprising nitrate (N), phosphate (P), and potassium (K)
derived from food waste and urine in the used diaper, 2) the cel-
lulose core of used diaper acting as platform for spreading and
growth of mycelium, and 3) waste reduction from the degradation
action of cellulose by the lignocellulolytic enzyme produced during
mushroom cultivation (Espinosa-Valdemar et al., 2015). Baby di-
apers, made up of 70% cellulose wood pulps, can potentially provide
a rich source of cellulose to be consumed by mushrooms for growth
and mycelium spreading (Khoo et al., 2018). Oyster mushroom is
classified as lignocellulolytic fungi which can perform lignocellu-
lose degradation (Miles and Chang, 2004). In nature, mushroom is
often found as saprophytes that feed on lignocellulosic material,
where the cellulose is reported to be converted into glucose from
the smallest repetitive unit of cellulose called cellobiose (Pathak,
2017).
Food wastes such as coffee ground, sugarcane bagasse, eggshell,
and banana were selected as raw material for formulating the
mushroom growth substrate. The main reason is to transfer the
nutrients present in the wastes to enrich the growth substrate.
There were reports on the nutrient profiles of coffee ground with
high hemicellulose content (39.1%) and high carbon to nitrogen (C/
N) ratio of up to 60.46 according to Ballesteros et al. (2014), banana
with high K of about 0.78 g/kg by Mohapatra et al. (2010), eggshell
with high calcium (about 33%) by Ray et al. (2017), sugarcane
bagasse with high cellulose and hemicellulose content at 38.59%
and 27.89% respectively by Masarin et al. (2011). In addition, these
food wastes were selected as they are more accessible for collection
and can be obtained in large quantity in many places.
There have been studies on mushroom cultivation using
different growth substrates such as commercially used sawdust,
rice straw (Saskiawan et al., 2016), and tea wastes (Yang et al.,
2016). However, so far, only two studies have been reported on
the use of diaper as substrate for mushroom cultivation (Espinosa-
Valdemar et al., 2011, 2015). Espinosa-Valdemar et al., 2011
explored the feasibility of diaper in mushroom cultivation of Pleu-
rotus ostreatus and they subsequently investigated the use of diaper
waste and gardening wastes such as withered leaves, grass, and
wheat straw as co-substrate for mushroom cultivation (Espinosa-
Valdemar et al., 2015). In this study, an improved method of
mushroom cultivation via the use of a growth substrate formulated
from the combination of used diapers and food wastes (e.g., coffee,
banana, sugarcane bagasse, and eggshell) to maximize mushroom
cultivation. The findings of this study advocate the concept of
recycling waste into a useful resource for mushroom cultivation
while simultaneously serves as a promising alternative to mush-
room farmers with respect to increased mushroom production and
generation of income.
Following the formulation of the growth substrate from the
used diaper and food waste, it was evaluated for its performance in
growing oyster mushroom in mushroom farm. The growth and
yield of oyster mushrooms were observed in different stages of
growth, such as mycelium running stage, primordial development
stage, and fruiting body development stage. The growth substrate
was examined for its weight reduction during mushroom cultiva-
tion as an evaluation of the utilization of the organic components
for mushroom growth. The growth substrate was also examined for
its contents of mineral, cellulose, hemicellulose, and lignin to
correlate the utilization of these as nutrients (carbon source) for
growing mushroom. In addition, metabolomic profiling of the
harvested mushroom was performed using nuclear magnetic res-
onances (NMR) to assess the presence of toxic or undesired bio-
logical elements that might derive from the waste-formulated
substrate.
2. Material and methods
2.1. Mushroom source
Oyster mushroom in spawning condition (containing enriched
content of mycelium) was obtained from S.R Mushroom Sdn. Bhd.,
Terengganu, Malaysia. The mushroom spawn was kept at 4  C for
long term storage.
2.2. Used diaper and food waste source
Used diapers with only urine discharged from kids below two
years olds were collected from Taska Juhani, Terengganu, Malaysia.
The cellulose pulp in the diaper (diaper core) is deemed to be the
primary carbon source that is beneficial for mushroom growth. The
diaper core was collected and then autoclaved for 15 min at 121  C.
The pre-treatment process was followed by the drying process in
oven at 90  C for 2e3 days to make sure it is entirely dried for long
term storage. The food wastes comprised of coffee ground collected
from local Starbucks coffee cafe, eggshell collected from local res-
taurants as well as banana skin and sugarcane bagasse collected
from local fruit juice stall. These food wastes were individually
dried in oven at 70  C for 2 days. Then, the dried food waste was
blended using an electronic blender into powder form and kept for
the subsequent formulation of blocks of growth substrate.
2.3. Formulation of growth substrate in block
Used diaper core and food wastes were mixed at different
weight ratios to form the mushroom growth substrate (Table 1).
The pH of the growth substrate was adjusted to pH 5.8 ± 0.8 by
adding a small amount of calcium carbonate powder (Khan et al.,
2013). The moisture level of the growth substrate was maintained
at 75 ± 5% (Koncsag et al., 2012). The resulting growth substrate
was packed into a polyethylene cylindrical bag with a final weight
of 600 g, and the experiments were performed in triplicates for
each treatment. All the substrate blocks were autoclaved at 121  C
for 15 min and kept in a clean environment.
 N.L. Ma et al. / Journal of Cleaner Production 268 (2020) 122272
Table 1
Formulation of growth substrate block in different weight ratios of used diaper core to food waste.
Treatment Formulation
T1 (control) Commercial mushroom cultivation block (control) e sawdust: rice bran: lime powder (Weight ratio-100:1:1)
T2 Fully DC
T3 DC: CG: SCB: BS: ES (Weight ratio- 5: 2: 1: 1: 1)
T4 DC: CG: SCB: BS: ES (Weight ratio- 6: 1: 1: 1: 1)
T5 DC: CG: SCB: BS: ES (Weight ratio- 7: 1: 1: 1: 1)
Notes: DC ¼ diaper core, CG ¼ coffee waste, SCB ¼ sugarcane bagasse, BS ¼ banana skin and ES ¼ eggshell.
2.4. Mushroom cultivation on the growth substrate
The formulated growth substrate was applied and tested for
growing mushroom in a mushroom cultivation house of a mush-
room farm nearby Universiti Malaysia Terengganu, Malaysia. The
mushroom cultivation began with the addition of 5 spatulas
(approximately 1.5 g) of mushroom spawn into each growth sub-
strate. The growth substrates were then kept in an enclosed envi-
ronment between temperatures 28 and 31  C. Humidity was
maintained at 70% by sprinkling water on the floor of the mush-
room cultivation house. When primordia was first appeared, the
humidity of mushroom house were increased to 90e95% by
sprinkling water to the surrounding environment of the substrate
blocks, and also the wall and floor of the mushroom cultivation
house three times a day (Baysal et al., 2003). After the primordia
formation, it took about one week for full fruiting of the mushroom.
Once the fruiting bodies of the mushroom were formed and fully
matured, they were then harvested for further analysis.
2.5. Growth and yield of mushroom
The growth of mushrooms started with the spreading of the
mycelium network until the mycelium fully colonized the whole
substrate block. The emerge of nodules or pinhead-like structure
called primordia was then developed. The primordia then enlarged
to form a prominent roundish structure of fruiting body. The
growth performance of mushrooms was observed based on pa-
rameters comprising mycelium running rate (MRR), duration (days)
of complete colonization of substrate block, duration (days) taken
for stimulation to primordia initiation, and duration (days) taken
for primordia initiation to harvest, average total number of
primordia, average number of fruiting body, average number of
effective fruiting body, dimension of fruiting body (cm), biological
yield (g) and economic yield (g).
MRR is an important parameter as it represents the length of the
mycelium running (growing) vertically per day. It was measured
and recorded for every five days from day 1 of cultivation until full
colonization of mycelium throughout the whole growth substrate.
The diameter of the pileus (cm), the thickness of the pileus (cm),
the diameter of the stipe (cm) and the length of the stipe (cm) of
every single fruiting body were measured using a slide caliper. The
dimension of fruiting body is the key parameter to access the
performance of mushroom growth.
2.6. Weight reduction of the substrate block
The reduction of weight is an indicator that the mushroom had
absorbed and degraded the organic components from the diaper
and food waste for its growth. After harvesting the mushroom, the
substrate blocks were weighed to calculate the weight reduction in
each block with the formula as stated in (Eq. (1)).
3
IW  FW
Weight reduction ð%Þ ¼ x 100 (1)
IW
where IW is the initiation weight of growth substrate and FW is the
final weight of growth substrate.
2.7. Analysis of lignin, cellulose, hemicellulose, and nutrient content
Lignin, cellulose, and hemicellulose content of growth sub-
strates were analyzed based on the protocol by Badu et al. (2011). 1
g of the growth substrate block was weighed and put into muffle
furnace at 500  C for 5 h to form ash. The ash was then added with
2 ml of concentrated hydrochloric acid (HCl) and then dried on a
hot plate until complete evaporation of the HCl. The process was
followed by the addition of 10 ml of 20% nitric acid (HNO3) into the
mixture and placed in the water bath for 1 h. The mixture was then
diluted with distilled water to make up a volume of 100 ml of
extracted liquids. The mineral contents of the extracted fluids were
analyzed and quantified by Inductively Coupled Plasma - Optical
Emission Spectrometry (ICP-OES) (Optima 8300, PerkinElmer,
United States) (Liu and Han, 2011).
2.8. Metabolomics analysis
Mushroom grown on different growth substrates were har-
vested and frozen in liquid nitrogen before it was freeze-dried
overnight. For metabolites extraction, 500 mg of dried sample
was crushed and ground using mortar and pestle. The sample was
extracted with extraction solvent formed by a mixture of 5 ml of
water-methanol solution (1:1 ratio) and 5 ml of chloroform. The
mixture was then vortexed and centrifuged. The down layer of
organic chloroform phase was carefully collected into glass vial and
was completely dried up using speed vacuum and stored at e 80  C
(Ma et al., 2018). Prior to Nuclear Magnetic Resonance (NMR)
analysis by Bruker DRX-400 Advance (400 MHz) spectrometer
(Bruker, Fremont, CA) machine, the extracted metabolites were
dissolved in 600 ml of 99.8% chloroform-d and transferred into a
6 mm NMR tube for organic metabolites detection. NMR sample
acquisition was carried out according to the method by Ma et al.
(2019).
2.9. Statistical analysis
All data were subjected to analysis of variance (ANOVA) using
Statistical Package for Social Science (SPSS) Version 20. The differ-
ences among means were tested by the Tukey’s HSD test in SPSS.
The spectra obtained from NMR analysis were pre-processed using
MestReNova software (Mnova) and continued with an unsuper-
vised chemometric analysis involving principal component analysis
(PCA) (Bellettini et al., 2016) using SIMCA 13þ software (Ma et al.,
2015).
4 N.L. Ma et al. / Journal of Cleaner Production 268 (2020) 122272

3. Result and discussion

3.1. Growth performance of mushroom with respect to the

development of mycelium, primordia and fruiting body

The MRR and duration taken for full mycelium colonization

throughout the whole growth substrate are presented in Table 2
and Fig. 1(a). All treatments from waste-formulated growth sub-
strates showed higher MRR and shorter period taken for full colo-
nization than the control (commercial growth substrate), except for
T2. From the observation, the use of only diaper (100% diaper core)
as growth substrate for T2 resulted in having high water content,
and it was lack of other useful minerals compared to the growth
substrate formulated with food waste, leading to poor aeration that
could promote the spreading of others microbe instead of the
mycelium (Igathinathane et al., 2008). The diaper core was made
from superabsorbent polymer (SAP), which is known to have high
water absorption and retention abilities (Wambui et al., 2015). High
water content in the growth substrate was likely to have resulted in
low ventilation inhibiting perspiration, causing the mycelium to
shrink and difficult to spread in a watery environment (Shen et al.,
2008). In the presence of high water content, the hydrogen ions and
hydroxide ions in the water molecules could form ionic bonding
with the compounds in the growth substrate, hence causing the
growth substrate to densify and become less porous, and this, in
turn, limited the oxygen transfer from mycelium to the mushroom
fruiting bodies (Bellettini et al., 2016b). The mycelium expansion
activities were limited under this condition and even ceased during
the long-term exposure to high moisture content.
T3 growth substrate formulated using the used diaper, coffee
waste, sugarcane bagasse, banana skin, and eggshell at the ratio of
Fig. 1. Development and growth of oyster mushroom with respect to the mycelium
5:2:1:1:1, showed the fastest MRR and fastest full mycelium colo- spreading rate, and the morphological structure of mushroom growth: 1a) mycelium
nization of the substrate block. The result corroborates with the running rate at Day 20 via treatment by T1-T5 as mentioned in Table 1; 1b) shows (1)
higher mushroom production observed in T3 (Table 3). The dura- the emergence of primordia in T3 marked with blue circle and (2) the fruiting body
tion from stimulation to the initial primordial formation in T3 (3 ready to be harvested; 1c) the dimension of fruiting body is measured and recorded
according to the diameter of (a) pileus, (b) thickness of pileus, (c) length of stipe and
days) was significantly shorter than that observed for control (6
(d) diameter of stipe using a slide caliper. (For interpretation of the references to colour
days). Following the primordial initiation, the mushroom became in this figure legend, the reader is referred to the Web version of this article.)
bigger and ready to be harvested. The average number of fruiting
body, effective fruiting bodies obtained, and the average fresh
weight obtained by T3 were relatively higher compared to that utilization of wastes such as coffee ground, diaper core and banana
obtained by other growth substrates (Table 3). Even though the skin as part of the ingredients of the growth substrate provided
diameter of the pileus and its thickness were not significantly high carbon and nitrogen content to enhance mushroom produc-
different in all treatment, the diameter and length of the stipe in T3 tion (Ballesteros et al., 2014). In mushroom cultivation, C/N ratio is a
were significantly different to that obtained by commercial growth crucial factor for mycelium colonization and the formation of the
substrate (Table 4). All the measurements of the mushroom parts fruiting body. This is because mushroom consumes a significant
provide comparative data to analyze the mushroom growth per- amount of N as the main nutrient source to promote growth (Miles
formance and mushroom production in different treatment. and Chang, 2004).
Biological yield can be defined as the weight of the whole cluster
of fruiting bodies, while economic yield is the overall weight of the
3.2. 2. Weight reduction and lignocellulose contents
fruiting bodies, excluding the lower hard portion of the mushroom.
T3 growth substrate showed a superior yield of mushroom among
The weight of the growth substrates was measured after the
the treatments with different growth substrates, producing the
mushroom was harvested and the results are presented in Fig. 2. T3
highest average fresh weight of mushroom. It was likely that the
growth substrate formulated at a ratio of 5:2:1: 1:1 of diaper core,

Table 2
MRR and duration taken for full mycelium colonization throughout the whole growth substrate.

Treatments MRR (cm/day) Duration taken for Full Mycelium Colonization (days)

T1 (control) 0.45 ± 0.04 a 32 ± 2 a

T2 No spreading No spreading
T3 0.53 ± 0.05 b 26 ± 3 b
T4 0.51 ± 0.05 a, b 28 ± 3 a, b
T5 0.46 ± 0.03 a 31 ± 2 a

Data presented are means ± SD, n ¼ 3, and means in the same column with same letters are not significantly different at p  0.05 according to
Tukey’s HSD test.
 N.L. Ma et al. / Journal of Cleaner Production 268 (2020) 122272 5

Table 3
Primordial development period from stimulation to initiation and then from initiation to harvest, the average number of primordial, fruiting bodies and effective fruiting
bodies harvested, and the average fresh weight of individual fruiting bodies.

Treatments Primordial Development Period (Days) Average number harvested Average fresh weight of individual fruiting bodies
(g)
From Stimulation to From Initiation to Primordial Fruiting Effective fruiting
Initiation Harvest bodies bodies
b a a a a a
T1 6±2 4±1 19 ± 3 9±5 5±4 9±3
(control)
T2 N/A N/A N/A N/A N/A N/A
T3 3 ± 1a 3±1 a
25 ± 3 a
11 ± 2 b 8±2 b
11 ± 1 b
T4 5±2b 4±1 a
21 ± 2 a
9 ± 1 ab 6±1 ab
9 ± 1 ab
T5 4±2a 3±0 a
18 ± 5 a
7±4a 5±3 a
10 ± 1 ab

Data presented are means ± SD, n ¼ 3, means with the same column followed by the same letters are not significantly different at p  0.05 according to Tukey’s HSD test. N/A
represent data not available.

Table 4
Average dimension of the fruiting body of oyster mushroom, and its biological yield and economic yield.

Treatments Diameter of pileus (mm) Thickness of pileus (mm) Diameter of stipe (mm) Length of stipe (mm) Biological yield (g) Economic yield (g)

T1 (control) 67 ± 11a 4±0 a

11 ± 1 ab
43 ± 4 a
39 ± 24 a
37 ± 24 a

T2 N/A N/A N/A N/A N/A N/A

a a b b
T3 80 ± 9 5±0 12 ± 4 53 ± 5 84 ± 16 c* 72 ± 14 c*
a a b b
T4 76 ± 4 4±0 12 ± 1 55 ± 1 55 ± 7b 45 ± 6b
a a a b
T5 69 ± 4 4±1 10 ± 1 58 ± 4 49 ± 23 b 42 ± 20 b

*Highest production yield among the treatments. Data presented are means ± SD, n ¼ 3, means with the same column followed by the same letters are not significantly
different at p  0.05, according to Tukey’s HSD test. N/A represents data not available.

Fig. 2. Weight reduction of substrate block after the harvested of mushroom fruit body. Different small letters indicate significantly different at p < 0.05 according to Tukey’s HSD
test, n ¼ 3.

coffee waste, sugarcane bagasse, banana skin, and eggshell showed
the highest percentage of weight reduction (40%) compared to
other growth substrate blocks. The higher weight reduction in-
dicates the efficient utilization of the growth substrate by the
mushroom for growth while simultaneously serves as an indicator
of the degrading efficiency of the mushroom in reducing the used
diaper and food waste, representing a beneficial feature leading to
both waste utilization and reduction. The mechanisms behind the
waste utilization and reduction could be derived from the
cellulose-hydrolyzing enzymes and cellulase produced during the
cultivation of the mushroom, which have an important role in
cellulose-degrading activity (Duhan and Gulati, 2017). Espinosa-
Valdemar et al. (2011) also found that the weight of growth sub-
strate (diaper added with grape waste) was reduced after two
harvest cycles of mushroom.
The weight reduction of the growth substrate is closely related
to the lignocellulosic content in the growth substrate (Fig. 3). T3
showed a higher content of lignin and hemicellulose but lower
content of cellulose compared to that of control (Fig. 3). Biocon-
version of lignin, cellulose, and hemicellulose to glucose was likely
to have occurred during mushroom cultivation. This was supported
by Zhang et al. (2019) in their mushroom cultivation study on
Agaricus bisporus, recording a degradation and conversion of
lignocellulosic content (14e22%) to glucose. The transformation of
lignocellulosic components into simple sugar molecules can act as
nutrient for the growth of mycelium and fruiting bodies.
Cellulose biomass can be converted into glucose-containing
compounds through the synergistic action of the cellulolytic
enzyme while hemicellulose can be converted into sugars such as
xylose, galacturonic acid and glucuronic acid (Pathak, 2017). The
presence of high content of cellulose and hemicellulose can be an
indicator that there are more sugar available for conversion into
carbon sources for mushroom growth when these lignocellulosic
substrates are digested (Mussatto and Teixeira, 2010). This explains
the higher yield of mushroom shown by T3 growth substrate
compared to the commercial growth substrate since the higher
contents of holocellulose (a combination of hemicellulose and
cellulose) in overall as observed in T3 was likely to have boosted up
the growth and subsequently the yield of mushroom. These find-
ings corroborate the beneficial role of cellulose and hemicellulose
derived from the waste materials in supplying additional nutrients
for the growth of the mycelium and mushroom. Oyster mushroom
6 N.L. Ma et al. / Journal of Cleaner Production 268 (2020) 122272
Fig. 3. Lignocellulose content in the T3 growth substrate compared to control. The different letters at the top of the bars indicate significantly different at p < 0.05, according to
Tukey’s HSD test, which indicates that T1 (Control) is significantly different from T3 with respect to cellulose, lignin, and hemicellulose content.
Table 5
Nutrient content of T3 growth substrate and control (commercial growth substrate).
Type of nutrients Nutrient Content (mg/kg)
T1 (Control)
N 1,157
P 206
K 1,065
Mg 409
Ca 1,836
Na 94
Fe 765
Zn 24
Cu 85
Notes: N ¼ nitrogen, P ¼ phosphorus, K ¼ potassium, Mg ¼ magnesium,
Ca ¼ calcium, Na ¼ sodium, Fe ¼ iron.
Zn ¼ zinc, Cu ¼ copper.
is classified as one of the lignocellulolytic fungi which perform
lignocellulose degradation (Miles and Chang, 2004). Thus, it was
likely to have broken down the cellulose, hemicellulose, and lignin
present in the growth substrate formed by the diaper and food
waste during the cultivation of this mushroom.
3.3. Nutrient profile of growth substrate
Apart from providing high content of lignin and hemicellulose
for the growth and development of the oyster mushroom, the
formulation of T3 also showed higher content of nutrients
compared to that of control (Table 5). In particular, the macronu-
trients needed for the mushrooms to grow, especially the N, P, K,
were significantly higher in T3 compared to the control (commer-
cial mushroom cultivation block). The nutrient value of N, P, K in T3
growth substrate are 15,779 mg/kg, 867 mg/kg and 7,758 mg/kg
respectively compared to commercial growth substrates with
N ¼ 1,157 mg/kg, P ¼ 206 mg/kg and K ¼ 1,065 mg/kg. This cor-
roborates the higher yield of mushroom from the use of the T3
growth substrate due to its higher nutrient content (Table 5).
In addition, the nutrient content of growth substrate also
significantly affected the mycelium growth performance and
mushroom yield. Royse et al. (2004) revealed that the increase in
nutrient level at high rates can boost the mycelial growth by
providing more energy and thus increase the overall growth of the
fruiting bodies. In addition, the utilization of different ratios of the
waste substrate by the fungus (Pleurotus ostreatus) was closely
related by its capacity to secrete digestive enzymes. The digestive
enzymes involved include hydrolytic enzymes (cellulase, tannase,
and xylanase) and also oxidative enzymes (laccase, ligninase, and
manganese peroxidase), which play a major role in degradation of
T3
lignocellulosic substrates (Bellettini et al., 2016). Hence, the
formulation ratio in T3 might be a promising composition for the
15,779
optimal enzyme activities to be functioned in the growth substrate.
867
7,758 It is further supported by the study by Kuforiji and Fasidi (2008)
853 that the high nutrient level of the substrate with a higher content
25,302 of enzyme showed a higher growth rate in the fruiting bodies.
674
To further boost the growth performance of the mushroom,
768
263 optimization of the growth substrate could be performed as future
20 study. It has been reported that biochar could improve the myce-
lium spreading rate and also the yield of oyster mushroom (Nam
et al., 2018). The motivation behind might be due to the role of
biochar in improving the plant-soil nutrient attraction by acting as
housing that could provide many porous holes in which the nu-
trients from wastes can be attached to (Liew et al., 2019; Xiong
et al., 2017). Therefore, biochar can be a promising, supplemen-
tary ingredient to enhance the utilization of the growth substrate in
mushroom cultivation.
3.4. Metabolite profiling
Fig. 4(a) shows that the peaks of spectra acquired from T3 are
similar to the NMR spectra shown by the control. Moreover, the PCA
score plot in Fig. 4(b) showed only one single cluster, confirming
that there is no variation between the metabolome of both mush-
room samples. The result demonstrates that the metabolomics
profiles of mushroom cultivated by T3 are highly similar to the
control and no extra peaks were observed indicating similar bio-
logical composition and no accumulation of undesired toxic or
biological components that might have derived from the use of
waste materials to form the growth substrate, thus providing an
assurance that the T3 growth substrate is safe to be used for
mushroom cultivation.
4. Conclusions
Used diaper and food waste were successfully converted into a
unique, formulated growth substrate, consisting of 27% cellulose
and 16% hemicellulose, leading to improved growth of oyster
mushroom (P. ostreatus). The application of this formulated growth
substrates resulted in an increase of mushroom production up to
two-fold compared to that shown by commercial growth substrate.
NMR metabolite profiling showed that the substrate is safe to be
 N.L. Ma et al. / Journal of Cleaner Production 268 (2020) 122272
Fig. 4. Metabolomic profiling of the harvested oyster mushroom using 1H-NMR. (a) 1H NMR spectra (600.13 MHz) of metabolites obtained from three biological replicates of the
mushroom cultivated by T3 growth substrate and T1 (control); the data were analyzed with MestReNova (Mnova), (b) PCA score plot of mushroom cultivated from T3 growth
substrate and the control. Black squares represent the control, whereas red dots represent T3. (For interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
used for mushroom cultivation with no detection of toxic or un-
desired biological elements that might derive from the waste-
formulated substrate. These offer alternative to commercial
growth substrate in mushroom production, and also the suitability
of oyster mushroom for use as a biological agent to reduce used
diaper and food, while simultaneously maximizing the utilization
and recycling potential of waste in mushroom industry and further
reducing the waste in landfill. Future study can be conducted by
optimizing the different growth factors such as pH, moisture con-
tent, and C/N ratio of this kind of waste-derived growth substrate in
order to further maximize the mushroom yield, particularly on the
application of this technique to formulate growth substrate in
cultivation mushroom species with medical value.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
CRediT authorship contribution statement
Nyuk Ling Ma: Conceptualization, Data curation, Funding
acquisition, Project administration, Resources, Supervision, Vali-
dation, Writing - review & editing. Shing Ching Khoo: Data cura-
tion, Formal analysis, Investigation, Writing - original draft, Writing
- review & editing. Wanxi Peng: Funding acquisition, Supervision.
Chia Min Ng: Data curation, Formal analysis, Investigation, Meth-
odology, Writing - original draft. Chin Hoe Teh: Methodology,
Validation, Visualization. Young-Kwon Park: Writing - review &
editing. Su Shiung Lam: Conceptualization, Data curation, Funding
acquisition, Project administration, Resources, Supervision, Vali-
dation, Writing - review & editing.
Acknowledgments
The authors gratefully acknowledge the Ministry of Education
Malaysia for the financial support under FRGS research grant (Vot
59482). The authors also acknowledge Henan Agricultural Univer-
sity for the financial, facility, and technical support throughout this
research under a Research Collaboration Agreement (RCA) and
Golden Goose Research Grant (Vot 55191) with Universiti Malaysia
Terengganu, and Taska Juhani (Kid Nursery) Terengganu for
providing the used diaper.
7
References
Abdullah, N., Chin, N., 2010. Simplex-centroid mixture formulation for optimised
composting of kitchen waste. Bioresour. Technol. 101 (21), 8205e8210.
Badu, M., Twumasi, S.K., Boadi, N.O., 2011. Effects of lignocellulosic in wood used as
substrate on the quality and yield of mushrooms. Food Nutr. Sci. 2, 780e784.
Ballesteros, L.F., Teixeira, J.A., Mussatto, S.I., 2014. Chemical, functional, and struc-
tural properties of spent coffee grounds and coffee silverskin. Food Bioprocess
Technol. 7 (12), 3493e3503.
Baysal, E., Peker, H., Yalinkiliç, M.K., Temiz, A.J.B.T., 2003. Cultivation of oyster
mushroom on waste paper with some added supplementary materials. Bio-
resour. Technol. 89 (1), 95e97.


Bellettini, M.B., Fiorda, F.A., Maieves, H.A., Teixeira, G.L., Avila, S., Hornung, P.S.,
Júnior, A.M., Ribani, R.H., 2016. Factors affecting mushroom Pleurotus spp. Saudi
J. Biol. Sci. 26 (4), 633e646.
Chen, Y.C., Lo, S.L., 2016. Evaluation of greenhouse gas emissions for several
municipal solid waste management strategies. J. Clean. Prod. 113, 606e612.
Duhan, K., Gulati, R., 2017. Review article on lignocellulosic biomass as substrate for
Pleurotus (Oyster mushroom) cultivation. Int. J. Technol. Res. Sci. 2 (3), 137e141.
Espinosa-Valdemar, R.M., Turpin-Marion, S., Delfín-Alcala
, I., Va
zquez-
Morillas, A.J.W.m., 2011. Disposable diapers biodegradation by the fungus
Pleurotus ostreatus. Waste Manag. 31 (8), 1683e1688.
Espinosa-Valdemar, R.M., Sotelo-Navarro, P.X., Quecholac-Pina, X., García-
Rivera, M.A., Beltr
an-Villavicencio, M., Ojeda-Benítez, S., V
azquez-Morillas, A.,
2014. Biological recycling of used baby diapers in a small-scale composting
system. Resour. Conserv. Recycl. 87, 153e157.
Espinosa-Valdemar, R., V
azquez-Morillas, A., Ojeda-Benítez, S., Arango-Escorcia, G.,
Cabrera-Elizalde, S., Quecholac-Pin ~ a, X., Velasco-Pe

rez, M., Sotelo-Navarro, P.J.S.,
2015. Assessment of gardening wastes as a co-substrate for diapers degradation
by the fungus Pleurotus ostreatus. Sustain. Times 7 (5), 6033e6045.
FAO, 2017. FAOstat- Food and Agriculture Data accessed. http://www.fao.org/
faostat/en/#home. (Accessed 29 May 2019).
Igathinathane, C., Womac, A., Pordesimo, L., Sokhansanj, S.J.B.t., 2008. Mold
appearance and modeling on selected corn stover components during moisture
sorption. Bioresour. Technol. 99 (14), 6365e6371.
Jesca, M., Junior, M., 2015. Practices regarding disposal of soiled diapers among
women of child bearing age inpoor resource urban setting. J. Nurs. Health Sci. 4
(4), 63e67.
Khan, M.W., Ali, M.A., Khan, N.A., Khan, M.A., Rehman, A., Javed, N.J.P.J.B., 2013.
Effect of different levels of lime and pH on mycelial growth and production
efficiency of oyster mushroom (Pleurotus spp.). Pak. J. Phytopathol. 45 (1),
297e302.
Khoo, S.C., Phang, X.Y., Ng, C.M., Lim, K.L., Lam, S.S., Ma, N.L., 2018. Recent tech-
nologies for treatment and recycling of used disposable baby diapers. Process
Saf. Environ. Protect. 123, 116e129.
Koncsag, C.I., Eastwood, D., Collis, A.E., Coles, S.R., Clark, A.J., Kirwan, K., Burton, K.,
2012. Extracting valuable compounds from straw degraded by Pleurotus
ostreatus. Resour. Conserv. Recycl. 59, 14e22.
Kuforiji, O., Fasidi, I.J.B.T., 2008. Enzyme activities of Pleurotustuber-regium (Fries)
Singer, cultivated on selected agricultural wastes. Bioresour. Technol. 99 (10),
4275e4278.
Lee, U., Han, J., Wang, M., 2017. Evaluation of landfill gas emissions from municipal
solid waste landfills for the life-cycle analysis of waste-to-energy pathways.
J. Clean. Prod. 166, 335e342.
Liew, R.K., Chai, C., Yek, P.N.Y., Phang, X.Y., Chong, M.Y., Nam, W.L., Su, M.H.,
Lam, W.H., Ma, N.L., Lam, S.S., 2019. Innovative production of highly porous
carbon for industrial effluent remediation via microwave vacuum pyrolysis plus
sodium-potassium hydroxide mixture activation. J. Clean. Prod. 208,
1436e1445.
8 N.L. Ma et al. / Journal of Cleaner Production 268 (2020) 122272
Liu, K., Han, J.J.B.T., 2011. Changes in mineral concentrations and phosphorus profile
during dry-grind processing of corn into ethanol. Bioresour. Technol. 102 (3),
3110e3118.
Ma, N.L., Aziz, A., the, K.Y., Lam, S.S., Cha, T.S., 2018. Metabolites Re-programming
and physiological changes induced in Scenedesmus regularis under nitrate
treatment. Sci. Rep. 8 (1), 9746.
Ma, N.L., Teh, K.Y., Ahmad, A., Lam, S.S., Saidon, S.A.B., Husri, M.N., Cha, T.S., 2019.
Metabolite profiling of Scenedesmus regularis using nuclear magnetic resonance
(NMR). Malays. Appl. Biol. J. 48 (1), 117e121.
Ma, N.L., Teh, K.Y., Lam, S.S., Kaben, A.M., San Cha, T.J.B.t., 2015. Optimization of cell
disruption methods for efficient recovery of bioactive metabolites via NMR of
three freshwater microalgae (chlorophyta). Bioresour. Technol. 190, 536e542.
Mahmood, A., Iguchi, R., Kataoka, R., 2019. Multifunctional food waste fertilizer
having the capability of Fusarium-growth inhibition and phosphate solubility: a
new horizon of food waste recycle using microorganisms. Waste Manag. 94,
77e84.
Masarin, F., Gurpilhares, D.B., Baffa, D.C., Barbosa, M.H., Carvalho, W., Ferraz, A.,
Milagres, A.M., 2011. Chemical composition and enzymatic digestibility of
sugarcane clones selected for varied lignin content. Biotechnol. Biofuels 4 (1),
55.
Miles, P.G., Chang, S.T., 2004. Mushrooms: Cultivation, Nutritional Value, Medicinal
Effect, and Environmental Impact, second ed. CRC Press, Boca Raton,
pp. 95e101.
Mohapatra, D., Mishra, S., Sutar, N., 2010. Banana and its by-product utilisation: an
overview. J. Sci. Ind. Res. 65, 323e329, 05.
Mussatto, S.I., Teixeira, J.A., 2010. Lignocellulose as raw material in fermentation
processes. In: Me
ndez-Vilas, A. (Ed.), Current Research, Technology and Edu-
cation Topics in Applied Microbiology and Microbial Biotechnology. Formatex
Research Center, Protugal, pp. 897e907.
Nam, W.L., Phang, X.Y., Su, M.H., Liew, R.K., Ma, N.L., Rosli, M.H.N.B., Lam, S.S., 2018.
Production of bio-fertilizer from microwave vacuum pyrolysis of palm kernel
shell for cultivation of Oyster mushroom (Pleurotus ostreatus). Sci. Total Environ.
624, 9e16.
Pathak, V.M., 2017. Review on the current status of polymer degradation: a mi-
crobial approach. Bioresour. Biorproc. 4 (1), 15.
Ray, S., Barman, A.K., Roy, P.K., Singh, B.K., 2017. Chicken eggshell powder as dietary
calcium source in chocolate cakes. Pharma Innov. J. 6 (9), 1e4.
Royse, D., Rhodes, T., Ohga, S., Sanchez, J.J.B.T., 2004. Yield, mushroom size and time
to production of Pleurotus cornucopiae (oyster mushroom) grown on switch
grass substrate spawned and supplemented at various rates. Bioresour. Technol.
91 (1), 85e91.
Saskiawan, I., Hasanah, N., Shimomura, N., 2016. Cultivation of Pleurotus ostreatus
using sorghum-supplemented spawn on various substrates. Mushroom Sci.
Biotechnol. 23 (4), 179e182.
Sheila, S.P., 2016. Soiled Diapers Stuck in Landfills. http://www.thestar.com.my/
metro/community/2016/05/23/soiled-diapers-stuck-in-landfills-recycling-is-
the-way-forward-says-expert/. (Accessed July 2019).
Shen, Q., Liu, P., Wang, X., Royse, D.J.J.B.T., 2008. Effects of substrate moisture
content, log weight and filter porosity on shiitake (Lentinula edodes) yield.
Bioresour. Technol. 99 (17), 8212e8216.
Wambui, K.E., Joseph, M., Makindi, S.J., 2015. Soiled diapers disposal practices
among caregivers in poor and middle income urban settings. J. Sci. Res. Publ. 5
(10), 154e163.
Xiong, X., Iris, K., Cao, L., Tsang, D.C., Zhang, S., Ok, Y.S., 2017. A review of biochar-
based catalysts for chemical synthesis, biofuel production, and pollution con-
trol. Bioresour. Technol. 246, 254e270.
Yang, D., Liang, J., Wang, Y., Sun, F., Tao, H., Xu, Q., Zhang, L., Zhang, Z., Ho, C.T.,
Wan, X., 2016. Tea waste: an effective and economic substrate for oyster
mushroom cultivation. J. Sci. Food Agric. 96 (2), 60e684.
Zhang, H.L., Wei, J.K., Wang, Q.H., Yang, R., Gao, X.-J., Sang, Y.-X., Cai, P.-P., Zhang, G.-
Q., Chen, Q.J., 2019. Lignocellulose utilization and bacterial communities of
millet straw based mushroom (Agaricus bisporus) production. Sci. Rep. 9 (1),
1151.