食品残渣（甘蔗渣、香蕉皮、咖啡渣、蛋壳和茶渣）和尿布废料中木质素，半纤维素和纤维素含量的检测

将食物肥料生物质和尿布废料转化为医用蘑菇-灵芝的可持续生产Valorisation of biomass and diaper waste into a sustainable production of the medical mushroom Lingzhi Ganoderma lucidumChemosphere 286 (2022) 131477Contents lists available at ScienceDirect S.C. Khoo et al.Chemosphere 286 (2022) 131477 Chemosphere journal homepage： www.elsevier.com/locate /chemosphere 将食物肥料生物质和尿布废料转化为医用蘑菇-灵芝的可持续生产 Valorisation of biomass and diaper waste into a sustainable production of the medical mushroom Lingzhi Ganoderma lucidum Shing Ching Khoo a b ，1， Nyuk Ling Ma cal ， Wan Xi Peng ， Kah Kei Ng ， Meng Shien Goh，Hui Ling Chen ， Suat Hian Tan ， Chia Hau Lee ， Vijitra Luang-In‘， Christian Sonne 8a ，* a Henan Province International Collaboration Lab of Forest Resources Utilization， School of Forestry， Henan Agricultural University， Zhengzhou， 450002， China Faculty of Science and Marine Environment， University Malaysia Terengganu， Kuala Nerus， 21030， Terengganu， Malaysia Eco-Innovation Research Interest Group， Faculty of Science and Marine Environment， University Malaysia Terengganu， 21030， Kuala Nerus， Terengganu， Malaysia “ Facutly of Industrial Sciences & Technology， Universiti Malaysia Pahang， Gambang， 26300， Pahang， Malaysia e School of Chemical and Energy Engineering， Faculty of Engineering， Universiti Teknologi Malaysia， 81310， Skudai， Johor， Malaysia Natural Antioxidant Innovation Research Unit， Department of Biotechnology， Faculty of Technology， Mahasarakham University， Khamriang， Kantarawichai， Mahasarakham， 44150， Thailand 8 Department of Bioscience， Aarhus University， Arctic Research Center (ARC)， Frederiksborgvej 399， PO box 358， DK-4000， Roskilde， Denmark ARTICLEINFO ABSTRACT 固废 Handling Editor： Derek Muir Keywords：Waste management Ganoderma lucidum Metabolomics Circular economic Diaper Food waste Global solid waste is expected to increase by at least 70% annually unti l year 2050. The mixture of solid waste including food waste from food industry and domestic diaper waste in landfil l s is causing environmental and human health i ssues. Neverthe l ess， food and diaper waste containing high lignocellulose can easily degrade using lignocellulolytic enzymes thereby converted into energy for the development and growth of mushroom.Therefore， t his study explores the potent i a l of recycling biomass waste f rom coffee ground， banana， eggshell， tea waste， sugarcane bagasse and sawdust and diaper waste as raw material for Lingzhi mushroom (Ganoderma lucidum) cultivation. Using 2% of diaper core with sawdust biowaste leading to the fastest 100% mushroom mycelium spreading completed in one month. The highest production yield i s 71.45 g mushroom； this represents about 36% production biological efficiency compared to only 21% as in commercial substrate. The high mushroom substrate reduction of 73% reflect the valorisation of landf i ll waste. The metabolomics profi l ing showed that the Lingzhi mushroom produced is of high quality with a high content of triterpene being the bioactive compounds that are medically important for t reating assorted disease and used as health supplement.In conclusion， our study proposed a potential resource management towards zero-waste and circular bioeconomy for high profitable mushroom cul t ivation. 1. Introduction Global waste is expected to increase from 2 to more than 3 billion tons dur i ng the period 2016-2050 (The W o rld Ba n k ，2018a ). According to the statistics recorded by the World Bank， the distribution of solid wastes decreases as follows； food wastes (44%) > paper (17%)> plastic (12%)> glass (5%)> rubber and wood materials (2% each) (T h e World B a n k ， 2018b). Around 20 bi l lion diapers are disposed yearly in the United States (US) encountering about 3.5 mil l ion tons of solid waste (W ri g ht ， 2018). Data from Malaysia shows that each child use about 6000-9600 diapers during its first 2.5 years， which equal 1.7 million tons of diaper waste annually (Kho o e t a l.， 2019； Shei la， 2016). In a number of Europe and Asia countries， diaper waste is incinerated (K im an d Ki m ， 2018). Aside from diaper waste， about 1.3 billion tons of edible food is wasted word-wide each year (D e Cl e r cq et al .， 2018). Diaper and food waste disposal and incineration necessitate exten-sive spaces， which are frequently opposed by the surrounding commu-nity whereas the incineration through full oxidative combustion at high temperature ranged from 900 to 1000 °C emit toxic compounds such as Dioxin and PAHs as well as greenhouse gases (H ai k a l ， 2016； Mo y a et al .，2017). The incineration system is more complex and expensive process compared to landfill therefore often used in developed countries (Kh o o * Corresponding author. Henan Province International Collaboration Lab of Forest Resources Utilization， School of Forestry， Henan Agricultural University，Zhengzhou， 450002， China. E-mail address： c s@b io s.a u .dk (C. Sonne). first authors： These authors contributed equally in preparation of this manuscript. h t tps：//d oi .or g/10.1016/j .c hemosp h e r e.2021.131477 Received 8 March 2021； Received in revised form 21 June 2021； Accepted 6 July 2021 Available online 10 July 2021 0045-6535/C 2021 The Authors. Published by Elsevier Ltd. This i s an open access article under the CC BY license (http：//c r ea ti vecom m ons.org/l icenses/by/4.0/). e t a l .， 2019)， example of advance incineration system are sanitary landfi l l and modern waste incineration (A li et al .， 2021； S adi an d A r a-b k o o h s a r ， 2019； Seib e r t e t al.， 2019). Sanitary landfill with speci f ic features such as methane gas recovery well and leachate collection pipes had been developed to minimise the environmental issues (I m r o n et a l .，2019； M il l i s ， 2017). Modern waste incinerator systems are vary among countries such as in Japan， Sweden， Finland and Italy， the system are designed to manage disposed waste in the same time regenerate methane gases as free energy by-product (Ade k o m aya an d M aj o zi ， 2020；D on g e t a l.， 2018； Fal co n e a nd De R o s a， 2020； Ritchi e an d Ros er， 2020). Sustainable waste management using naturally-occurring microbes for waste degeneration have been explored but unfortunately the pro-cess i s slow and inefficient while pos i ng a zoonotic risk (G upt a et a l.，2018； T o r r e n t e-V e l asq u ez et al.， 2020). Macro-fungus such as mush-rooms are new alternatives for efficient waste degradation as enzymes degrade recalci t rant macromolecules into many smaller fragments which essentia l to be use in the growth and development of mushroom (G u p ta e t al.， 2018； Z a v a r z ina et a l .， 2018). Among cultivated mush-room， Lingzhi mushroom (G.lucidum)) represents one of the high-commercial value mushrooms found in more than 100 Chinese medical products (Li e t al.， 2016). The Lingzhi contains bioactive com-pounds， including polysaccharides， peptidoglycans and t riterpenes as well as high nutritional values such as amino acids， fibre， low in fat， and vitamins B1， B2， B12， C， D， and E (N a k a ga wa et al .， 2018； W ang e t a l .，2018). The species is rare in the wild and given thei r high demands growing the market for in situ cultivation using wood logs and substrates bags (M e h t a et a l.， 2014； R ol i m e t al.， 2014). The production of fruiting bodies in the wood logs produce a better quality but requirement of about six months to culture (Li et a l .， 2016). Therefore， the substrate bag cultivation is more favourable as mushroom hyphae grows faster and harvest of fruit i ng bodies happen more quickly (Li e t a l .， 2016). Since biomass from the industry or agricultural waste contain high lignocel-lulose sources， including cellulose， hemicellulose， and lignin i t is suit-able as source for Lingzhi cultivation (Zho u ， 2017). Nevertheless，feasibility of diaper waste and food waste as growth substrate for val-orisation of medical mushroom and its quality and safety production have never been studied before. Therefore， this study offer an oppor-tunity to increase the profi t margin of farmers and creat i ng a circular economic for sustainable mushroom industry. 2. Methodology 2.1. Activation of old Lingzhi mycelium stock on agar media The mycelium of Lingzhi obtained from Gano Farm Sdn. Bhd.， Tan-jung Sepat， Malaysia was cultured in the potato dextrose agar (PDA)inside the laminar airflow. The mycelium allowed to develop on PDA in within 5-7 days in an incubator at 30 ±2°C. 2.2. Preparation of mother spawn 2.3. Collection of food waste and diaper waste The food wastes such as sugarcane bagasse (SB)， banana skin (BK)，coffee grounds (CG)， eggshell (ES)， and tea waste (TW) are collected from local restaurants and allowed to dry in an oven preheat a t 80°C for 48 h to completely remove its water content (M a e t a l .， 2019). The raw waste materials are then blended into powder form with a conventional blender. The diaper wastes are collected from a kid nursery， Taska Juhani from Gong Badak， Terengganu. This study only use disposable diapers contained urine without faeces of kids below 2 years old. For hygienic purposes， autoclave process of diaper wastes under 121 °C for 15 min is conducted to kill microbe before diaper core is separated from the outer layer plastic wool and dried subsequently for 3 days at 90°C in the oven (Ma et al .，2019). 2.4. Chemical composition and nutrient profile of the food waste and diaper waste The chemical composition of food and diaper wastes for element carbon (C)， hydrogen (H)， nitrogen (N) and sulphur (S) were analysed by using element analyzer (Flash Smart ， Thermo-Fisher Scienti f ic， United States) according to the protocol as reported in L am et al. (2019). Each waste samples were milled into smaller size using mortar and f iltered through a 125 um size sieve. The milled samples were dried at 105 °C to completely remove moisture. The waste samples are then loaded into furnace at 1000 °C with oxygen and Helium gas (He) as carrier gas.Quantitative detection of C， H and S contents in weight percent are obtained from the selective infrared absorption detectors， where the N contents was measured by thermal conductivity detector (L am et al.，2018). The lignocellulosic contents such as lignin， hemicellulose and cel -lulose are al l analysed using Fibre-therma l analyser (Gerhardt Analytical System， Germany). About 1 g of milled waste sample is weighted and put into Gerhardt system fibre bag and loaded into the Fibre-thermal ma-chine (F e t twe i s a n d Ku hl ， 2015). Inductively Couple Plasma-Optical Emission Spectrometer (ICP-OES)(7300 DV，PerkinElmer PE Optima， America) is used for nutrient profiles analysis phosphorus (P)， potassium (K)， magnesium (Mg)， manganese (Mn)， copper (Cu)， zinc (Zn)， calcium (Ca)， iron (Fe)， sodium (Na)，and baron (B). About 1 g of each waste material is added to 2 mL of concentrated HCl in a crucible and allowed to evaporate on a hot plate.The dried samples in the crucibles were dissolved in 10 mL of 20% nitric acid and put in the water bath for 1 h. The mixture is diluted with 100mL distilled water prior loading into the ICP-OES analysis (7300 DV，Perkin El). 2.5. Preparation of mushroom growth substrate Different ratio of waste compos i tion substrates were prepared as in (Tabl e 1). To prepare the growth substrate with waste baby diaper formulation， the dried diaper cores were immersed in water to allow complete water absorption. The substrates were mixed and packed in triplicate with final weight of 600 g per block (T a bl e l a and b). The pH is adjusted to 6.8±0.4 with addition of calcium carbonate by using soil pH meter (Takemura Denki Seisakusho， Japan) and the moisture is main-tained a t 55%-60% (Kh a n et a l .， 2013). All substrate blocks were then sterilized in autoclave machine at 121 °C for 60 min. 2.6. Inoculation of mother spawn onto growth substrate The mother spawn of Lingzhi was transferred onto the growth sub-strate block and the mouth of substrate block was covered with a cap for l i mited air exchanged to support mycel i um spreading and prevent i ng other microbe contamination. The mushroom house was cleaned by 70% alcohol to prevent the occurrence of contamination by fungi or a. Growth substrate plus waste baby diaper formulation Diaper (g/kg) Coffee ground (g/kg) Banana skin (g/kg) Eggshell (g/kg) Sugarcane bagasse (g/kg) Other substrates (g/kg) TA 500 200 100 100 100 一 TB 530 100 60 110 110 tea waste TC 550 一 30 120 180 120 tea waste TD 380 200 90 130 110 一 TE 250 100 50 50 50 500 commercial substrate block D4 Commercial growth substrate +5%(50 g/kg) of diaper b. Growth substrate formulated with food and diaper waste. bacteria from the environment. The Lingzhi substrate blocks were kept on the shelf of the mushroom house， the temperature was maintained at 25-29°C and the humidity was maintained within 70-80% by spraying water once a day on the surrounding of mushroom house and constant l y checked using a digital hygrometer and thermometer probe. 2.7. Growth performance and yield of Lingzhi mushroom The growth performances of Lingzhi were observed by its cumulative mycel i um spreading rate (CMSR)， duration to complete colonization，durat i on to harvest and total mushroom yield (Si n d h u et al.， 2016). The cumulative mycelium spreading rate was observed in a weekly basis for 6 weeks and calculated using the formulation in Eq. (1)： Duration to complete colonization is counted as day needed to ach-ieve ful l mycelium colonization starting from the zero day of mother spawn inoculation. Once full mycelium colonization is achieved，removing the cover cap of substrate block to allow the emerge of primordia. The Lingzhi fruiting bodies were harvested once the colour of fruiting bodies turn reddish. The duration to harvest was calculated as the total days taken from the zero day of spawn inoculation to harvest.Total mushroom yield is the total weight of Lingzhi mushroom harvested once they reached maturation. The diameter， thickness and circumfer-ence of mushroom frui ti ng bodies from di f ferent substrate blocks were measured. Weighting the remaining substrate blocks after harvest the mushroom to calculate the weight reduction of every block. The bio-logical efficiency of the fresh Lingzhi mushroom was calculated as in Eq.(2). 2.8. Analysis of bioactive compounds from Lingzhi mushroom The fresh Lingzhi samples were harvested and deep-frozen in liquid nitrogen and immediately freeze-dried in Freeze Dry System (Lab-concoTM Benchtop 208/230v 60 Hz Model， Fisher Scientific， United State). For metabolites extraction， crushing about 500 mg of Lingzhi using mortar and pestle and extracting using 100% methanol and water in 1：1 ratio for polar metabolites， while 99% absolute chloroform (ACS graded， Sigma Aldrich) is used for organic metabolite extraction. The mixture was vortex prior centr i fugation process at 4000 rpm for 20 min， the upper layer and lower layer samples were withdrawn carefully and kept in a separated vial. The samples were stored in -80°C fridge prior to metabolomic detection. 2.9. Gas chromatography mass spectrometry (GC-MS) acquisition The volatile extracts from chloroform were further analysed with online-linked GC-MS. The analysis was performed using Agilent GC-MSTM system linked with mass selective detector (6890 N+5975C，Agilent Co.， Ltd， USA). An elastic quartz capillary column HP-5MS (30m x 250 pm x0.25 um) was used i n the system with high purity he l ium as carrier gas at flow rate of 1 mL/min. Setting the temperature program from 50 °C and slowly increasing to 250 °C at the rate of 10 °C/min. The injection temperature was set on 250 °C and then continue raise the temperature to 280°C at a rate of 5°C/min. The program scan mass of MS was ranged from 30 to 600 atomic mass unit (amu) with the ioni -zation current of 150 uA electron ionization and 70eV of ionization voltage. The program setting for the quadrupole and ion source tem-perature were 150 °C and 230°C respectively. 2.10. Liquid chromatography mass spectrometry (LC-MS)-QTOF acquisition Phytochemical analysis of Lingzhi extracts were carried out using auto sample AcquityUPLC system equipped with a binary solvent attach to a photodiode-array (PDA) detector. The column used i s Acquity UPLC BEH C8 (2.1×100 mm， 1.7 um particle size) coupled to a quadrupole time-of-flight (QTOF) mass spectrometer (Vion IMS LCQTOF MS， Wa-ters， United State) equipped with electrospray ionization (ESI). Mobile phases are acidi f ied water (0.2% acetic acid， v/v) plus acetoni t rile with flow rate setting at 0.4 mL/min throughout all the gradients. The experiment temperature is set to 40 °C and the injection volume is 2 uL.The operating parameters are as below： source temperature of 120°C，capillary voltage of 2.0 kV (ESI +)， desolvation gas flow of 800 L/h under desolvation temperature of 550C. In Ms mode， trap collision energy of low energy function is set at 4 eV， while ramp trap col l ision energy of high energy function is set at 10-40 eV. Spectral data are acquired by full scan in a mass range of m/z 50-1000. Data acquisition and processing are referred to elementa l compositions of the precursors，the most rational molecular formula was sought in chemical databases such as Traditional Medicine Library (UNIFI 1.7) (waters， Manchester，U.K.). 2.11. Weight reduction of growth substrate The weight reduction of growth substrat e is an indicator to reflect the biodegradation rate of biomass materials from the food waste and diaper waste. The initial weight of growth substrate before mycelium inocu-lation and the f i nal weight of growth substrate after harvesting the mushroom’s fruiting bodies are measured and calculated by using Eq (3). 2.12. Statistical analysis Statistical Package for Social Science (SPSS) software (SPSS Version 23， IMB Worldwide， USA) was used for the initia l statistical analysis. In the SPSS software， all the triplicate data for each treatment obtained in the experiments such as mineral and lignocellulose content of waste materials， mycelium spreading rate， mushroom yield and size were firstly checked for descriptive normal i ty test and then the data were reported as mean ± standard deviation (SD). The data were normally distributed when the skewness and kurtosis statistical data from descriptive test fal l within z value=± 1.96 (F a ruk， 2019)， where the skewness and kurtosis statistical data is calculated from each variable in the formula of mean/standard errors. The normally distributed data were then tested with one-way analysis of variance (ANOVA) for discrimination analysis. In the ANOVA analysis， the homogeneity vari-ance test and Post Hoc Test using equal variances assumed Tukey's HSD，where n = 3 for each parameter were conducted. The treatments that fall in the different homogenous subset group and Tukey’s HSD Post Hoc Test with p < 0.05 denoted for significantly difference among the sub-strate treatment. The spectra obtained from GC-MS analysis were pre-processed for baseline and phrasing correction using MestReNova software (Mnova)and principal component analysis (PCA) was performed in SIMCA 13+software (M a e t a l .， 2020). 3. Results and discussion 3.1. Chemical composition and nutrient profile of waste material Lignocellulose and nutrient profile analysis of waste materials is shown in T a bl e 2. The diaper waste also recorded high contents of minerals in both diaper and food wastes (Ta ble 2). The high lignocel-lulose content of diaper cores suggests a potentia l for mushroom cult i-vation through lignocellulase enzymatic reactions (K o ut r o tsi os et al.，2019). Previous studies report that the high lignocellulose content in cardboard and chopped office paper promote denser mycelium biomass and higher oyster mushroom yields (M a n deel e t a l .， 2005). S halahud di n e t a l . (2018) report a high NPK content in mushroom substrates signif-icantly shortens mycelium-spreading rate and enhance the growth of fruiting bodies of oyster mushroom Pleurotus ostreatus. One of the basic criteria for a good mushroom substrate is the content of carbohydrate and N to support the growth of mushroom (O gu nde l e et a l .， 2014).Substrates with high nutrition content shorten the mycelium-spreading period due to a faster colonization of the whole mushroom substrate compared to those with low nutritional value (Sofi e t a l .， 2014).Contrarily， deficiency of nutri t ional value could be the cause of poor mycel i a densi t ies and high risk of fungus contamination. Excess N value in mushroom substrate results in high lignin degradation lead to mycelium inhibition (Bel l e t t i ni e t a l .， 2019). 3.2. Mycelium spreading performance Mycelium in substrate blocks containing diaper waste (D1-D4) is signi f icantly faster in term of colonization (3-4 week) compared to the control (5 week) (F ig .1a). As high humidity >80% inhibit the growth of mycel i um and promote microbial contamination， the use of diaper waste containing superabsorbent polymers (SAP) hydrogels controls water content thereby promoting Lingzh i growth (I sla m et al.， 2017). Another study shows that adding SAP improves seedling and root growth of Populus euphratica and corncob (El -Re h i m e t al.， 2004). In agricul t ure，SAP enhance plant growth through improved soil permeability and density and boosting the infiltration rate of water and i ncreasing aera-tion and reducing irritation frequency of soil (S ha hidia n e t al .， 2010).The D3 and D4 substrates significantly shortens the period for mycelium to achieve full colonization and maturation period to harvest i .e. within 59 days (F ig . 1b). This is approximately 1-4 weeks shorter when comparing the harvest period of G. l ucidum with previous studies and hence supplying diaper waste substrate with 4-5% of diapers as in D3and D4 substrate could promote the growth of G. lucidum mushroom (G u r un g e t al .， 2012； P eksen an d Y aku po g l u ， 2009). The TE substrate significantly boost mycelium spreading compared to the control and other substrate formulation (F i g . 1a). The highest Mineral， elemental compound and lignocel l ulose contents of waste collec t ed from various type of waste. The data are presented in means ± SD (n=3). Type of Wastes Diaper core Coffee ground Eggshell Banana skin Tea waste Sugarcane bagasse Mineral contents (mg/kg) Nitrogen (N) 23，997±826 27，683±650 21，510±373 10，143±1712 5168±1236 1332±176 Phosphorus (P) 2428±130 895±21 2837±196 973±68 4186±1318 607±137 Potassium (K) 40.516±3371 4139±252 6688±587 734±537 3698±368 2120±85 Sodium (Na) 91±12 76±15 45，938±4504 913±358 453±64 29±8 Magnesium (Mg) 1308±103 1082±35 325±25 2410±127 2015±163 260±12 Calcium (Ca) 1463±35 895±133 957±82 70±63 8456±806 418±39 Iron (Fe) 33±2 39±7 130±67 122±92 211±22 40±24 Zinc (Zn) 26±2 11±2 10±2 9±1 32±1 22±1 Copper (Cu) 3±0 17±1 2±1 1±0 20±4 4±0 Manganese (Mn) 159±8 27±2 2±1 1±1 930±74 30±1 Baron(B) 24±5 7±0 3±1 1±1 4.67±1 -3±0 Elemental analysis (%) Carbon (C) 36.85±5 50.59±1 11.53±1 32.5±6 43.55±3 39.87±0 Hydrogen (H) 5.68±1 7.4±1 0.44±0 4.39±1 5.55±0 5.43±1 Nitrogen (N) 1.11±0 2.12±0 0.86±0 2.39±1 2.25±2 0.15±0 Sulphur (S) 0 0 0 0 0 0 Lignocellulose contents (%) Hemicellulose 14.46±3.5 34.43±7.23 N/A 16.38±2.81 18.79±0.84 21.51±0.72 Cellulose 66.69±7.45 23.58±0.27 N/A 18.40±0.32 22.04±2.47 30.40±3.42 Lignin 4.59±0.68 29.59±2.82 N/A 23.60±0.75 24.36±3.25 14.16±9.6 Others 14.26±1.28 12.60±2.75 N/A 41.62±3.02 34.81±0.21 33.93±4.84 b. Treatment Mycelium spreading in six weeks (%) Day to complete colonization (days) Day to harvest (days) C 100±0.00a 35±1C 73±2° D1 100±0.00a 33±0b，c 73±1C D2 100±0.00a 29±0a，b 66±1b D3 100±0.00a 24±0a 59±0a D4 100±0.00a 25±0a 59±0a TA 49.37±1.42b N/A N/A TB 42.16±10.08b N/A N/A TC 100±0.00a 43±3d 87±0d TD 97.67±3.33a 53±1e 91±1d TE 100±0.00a 29±1 a，b 71±1C Fig.1. Growth. a) Cumulative mycelium spreading rate of G. lucidum cultivation i n di f ferent substrate blocks. b) The growth performance of mushroom (mycel i um spreading rate， day to complete colonization and day to harvest). All data are presented i n means ± SD， n =3 and the alphabetic letters or * denotes for the sig-nificant different at p ≤ 0.05. mycelium rate of TE among all food waste derived substrates was probably due to the addition of the sawdust in the substrate compos i-tion. Sawdust is well known for the best substrate used in the mushroom cultivation and i t had been commercially used by most of the mushroom developers (Li a ng et al.， 2019；Ogund e le et al.， 2014). Contradict result was observed in formula TA and TB (no sawdust were added in the media formulation)， the mycelium spreading was observed terminated after 3 weeks so no output was recorded (Fi g . 1 a and 1b). This phe-nomenon might probably be related to unfavourable environment caused by excess nutrients that change the pH of subtract block (Simoni c e t al .， 2008； Tang e t a l .， 2009). The acidi f ication of substrate is critically inf l uenced by the available minerals and affecting the mycelial density (Ann a et a l.， 2004). According to T a n g et a l . (2009)， the valubale metabolite from medica l Lingzhi mushroom is inhibited at pH below 3.Moreover， high nitrogen fertilizers might cause alteration of the meta-bolic composi t ion of the mycelium biomass and hence cease the myce-l i um spreading process (S p er l in g et al .， 2019). The yield performance of Lingzhi cultivated using different substrate blocks. The data are presented i n means ± SD (n =3) and showed significant difference at p<0.05. Treatment Diameter of fruiting bodies (cm) Thickness of fruiting bodies (cm) Circumference of fruiting bodies (cm) Mushroom Yield (g) Biological efficiency (%) C 9.3±1 b，c，d 2.4±0° 25.8±6° 42.00±5 b，c 21.01±6b，c D1 11.2±0a，b 3.5±1a，b 27.2±13 58.70±2a，b 29.12±28，b D2 12.5±0a 4.1±1a，b 28.5±4 71.45±4° 36.01±3 D3 8.7±0 b，c，d 4.6±1b 23.3±3° 43.96±2b，c 21.98±1 b，c D4 7.7±0cd 3.7±1a，b 19.5±4a，b 36.43±115d 18.22±5 b.c，d TA N/A N/A N/A N/A N/A TB N/A N/A N/A N/A N/A TC 6.8±0d 2.5±0a.b 23.8±1° 31.01±3cd 15.44±3c.d TD 4.7±2d 3.4±1a，b 13.2±2 17.43±6“ 8.76±5 TE 10.0±2a，b，c 3.3±1a，b 22.5±2a，b 48.4±2a.b 24.20±21 b，c D2 substrate significantly yield more Lingzhi mushroom biomass compared to control with an average weight of 71.45 g per substrate block (T able 3). Even though the biomass mushroom harvest from D3and D4 substrate blocks are faster than for D2， the growth potential of D3 and D4 is only 50% of D2. Higher biological efficiency in mushroom cultivation is the efficiency of mushroom production in relative to the mushroom substrate used. It is a critical parameter for mushroom farmers to check on the overal l efficiency and manufactural benef i t in mushroom production (S h i e l d s ， 2018). The highest biological efficiency is seen in substrate block D2 (36%)， which is significantly higher compared to commercial control substrate blocks that only recorded 21.01% growth. The biological efficiency comparison to other substrate such as 27.5% for poplar substrate (J a nd a ik e t al.， 2013)， 10% for wheat bran (R as ha d e t a l .， 2019) and 5% malt substrate (A zi zi e t a l .，2012) had further proved that D2 with sawdust and diaper core substrates are much productive . Overall， D2 was the most optimal growth substrates for mushroom growth in thi s study after consideration of all the growth aspects in term of mycelium spreading rate， shape and size of mush-room， mushroom yield， and biological eff i ciency. 3.4. Principle component analysis (PCA) PCA is an unsupervised technique that used to reduce the dimen-sionality of high dimensional datasets such as mass spectra obtained from GC-MS analysis， in the meant i me preserve the original distribution structure that showed inherent relationship to the original dataset (Pac k t ， 2019). In this study， PCA performed to give an overview inter-pretation of metabolite profile of Lingzhi fruiting bodies cultivated in di f ferent substrate blocks (F ig . 2). The 2D score scatter plot reveals that the Lingzhi metabolic prof i le through t he cul ti vation i n subtract blocks C， D2， D3， D4 and TE are quite similar (Fi g. 2a). The 3D PCA score scatter plot show that C， D2， D3 and D4 are closely clustered indicating that adding diaper waste leads to the production of secondary metabo-l i tes similar to the control (F ig. 2b). The stacking spectra of D2 being the most efficient growth substrate show no extra suspicious peaks indi-cating that using diaper waste does not cause any production of un-wanted secondary toxins thereby being safe as human consumption (Fi g . 2c). In addition， the consistency in metabolites production throughout three different batches of cultivation reflect D2 as a suitable growth media for Lingzhi production. 3.5. Metabolomic profiling of triterpenes Lingzhi mushrooms contains various bioactive compounds including a wide range of polysaccharides， triterpenes， nucleosides， minerals and trace elements (T a of i q e t a l .， 2017). Nevertheless， the incons i stency and low quantity of bioactive compounds i s a challenge to the industry due to di ff erent production methods used including type of substrates and different quality of mushroom strain used especially the repeated sub-culturing mycelium and mushroom spawn are commonly seen in the mushroom industry (G a l or et a l .， 2011； H ap u a r a chchi e t a l.， 2018). By using UPLC-QTOF-MS Systems to profil i ng the t riterpenes metabolites，at least 113 and 101 type of triterpenes metabol i tes detected from methanol and chloroform extract of D2 fruit body，respectively， which is slight ly higher than found for methanol (100) and chloroform (78) in the fruiting body of control cultivated from commercial block. This is probably due to adding of diaper waste provide huge amount of degraded cellulose for fruit body formation. The quality of triterpenes metabolites in control and D2 were identified and compared showing that D2 is an ideal substrate formulation for the cultivation of Lingzhi mushroom (Fi g. 3， T ab le 4). Differences in triterpene functional groups including ganoderic acid (GAs)， ganederic acid， lucidenic acid， poricoic acid， and saponin deriv-ative triterpene among C and D2 extracts have been detected (T a ble 4 & Sup p l ement ary Materi a l 1). These triterpenes have remarkable thera-peutic effects that i ncludes treatment of prostate cancer， fatigue syn-drome and hepat i t i s among other (Liang e t a l.， 2019). There are about 14 type of GAs detected in extracts of Lingzhi mushroom fruiting bodies seen in T abl e 4. GAs are classified based on their functional group including acetyl， carbonyl and hydroxyl group found i n A， C2， U，V and X type were only detected in control treatment whereas GAs types such as B， Ma， R and were detected in D2 treatment (Gil l e t a l.， 2018)(T a bl e 4). The variation of t riterpene compounds detected i n control and D2 might probably due to the metabol i te changes in D2 substrate with high nitrogen content seem to alter the bonding formation which trigger the different functional groups i n ganoderic acid (Zhu et a l.， 2019). This high nitrogen contents influence nitrogen metabolism genes which is an important transcription factor for the biosynthesis of GAs (Z hu e t al.，2019). GAs are the most significant triterpenoid produced from Lingzhi with antihepatotoxic， having antihypertensive properties， suppress prol i feration of breast cancer cells (L iu e t al .， 2009； Xu et a l.， 2010； Z h ou et a l .， 2006). GAs had reported to be useful in inhibiting the formation of tumor cells such as in colorectal carcinoma cel l (HCT-116 cells) human hepatocellular cell (HuH-& cells)， HepG2/ADM cells， and breast cancer cells (L i et al.， 2005； L iu e t al.， 2015； Yang et al .， 2018). To date， GAs are still mainly extracted from Lingzhi fruiting bodies (D on g e t al.， 2019；Re n et al .， 2020). Therefore， the detection of a wide variety of GAs in D2cultivation show a sustainable production of these natural compounds. 3.6. Waste reduction of mushroom substrate Upon the completion of the mushroom cultivation， D2 showed the highest weight reduction of subtract block about 70% which indirectly reflected about 70% of waste reduction during the bio-degradation process in mushroom cultivation (F ig. 4). It is estimated that more than 104.2 billion tons of waste subtract block will be generated from mushroom i ndustry which wil l be managed through dumping to the landf i l l in 2026 (K hoo et a l .， 2020)， hence the high waste reduction achieved in this study represent that 70% reduction to landfill . The accumulation of spent mushroom substrate in landfills have been re-ported to cause severe environmental issue such as the release of greenhouse gas (In n o c e n t i et al .， 2017) and breeding ground for i nsect pests (Na jafi e t a l.， 2019). Therefore， the D2 cultivation using 2% of diaper core with sawdust is an alternative to current cultivation method that clearly reduces the impact to environment. In addition to this， the spent substrate emushroom blocks could also be used in plywood/bio-board production thereby creating a zero-waste circular--economy mushroom industry (Kho o e t a l .， 2020). 4. Conclusion This study shows the potential valorisation of recycling food and diaper waste to generate Lingzhi mushroom growth sustaining zero-waste production. The addi t ion of diaper core up to 5% had resulted in higher mycelium spreading rate. The substrate D2 owned highest mushroom yield among the growth substrate， which indicate that addition of 2% diaper cores can act as good moisture control agent and nutrient booster for Lingzhi mushroom cultivation. Furthermore， the metabolite profiling from LCMS and GCMS proven that the high quality of Lingzhi produced for market demand. The market for powder Lingzhi is expected to increase at 8% from 2021 to 2027 but the output is reduced by the COVID-19 pandemic and shortage of workers. Therefore，the introduction of this method could provide site income for farmers while providing impressive 73% of waste reduction while reducing the pressure of solid waste generated to landfil l and signi f icantly contribute to the pos i tive impact to our environment . Therefore， using mushroom cultivation as waste management strategy is a smart move as it is completely green and effective to degrade the recalcitrant plant cells that required chemica l pre-treatment or otherwise need a long period to degrade in landfill. This formulation has also been applied in a C a 13.5 Methyllucidenate 3e6 Q(10.17) Ganoderic acid H 2e6 (9.10 & 9.42) Poricoic acid Ganoderic acid le6 E(8.03) Ganoderenic B(11.26) acid C (7.12) 0 一 上上 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 Retention time (mins) Fig.3. Comparison of metabolite profi l ing of triterpene of Lingzhi in control (a， c) and D2 (b，d). a&b： chromatogram of methanol extract and c&d： chromatogram of chloroform extract detected by using UPLC-QTOF -MS System. Table 4Comparison of ganoderic acid (GAs) in Lingzhi harvested from Control and D2 by UPLC-QTOF-MS System. Identified Triterpene compounds Rt (min) Measured m/z Adducts Molecular formula Extract types CC CM DC DM Ganoderic acid Ganoderic acid A 6.49 517.32 +H C30H44O7 一 √ 一 Ganoderic acid B 9.16，11.26 539.30，539.30 +Na，+Na C30H44O7 √ Ganoderic acid C2 7.12 557.31 +H，+Na，+K C30H46Og 一 一 Ganoderic acid G 7.60 555.29 +H，+Na，+K C30H44O8 一 √ Ganoderic acid H 9.10， 9.52，9.58，9.42 573.31，573.31，573.31，595.29 +H， +H， +H，+Na C32H44O9 一 √ Ganoderic acid MA 10.38，11.43，11.49 573.38，573.38，573.38 +H， +H，+H C34H52O7 一 一 √ Ganoderic acid R 12.91 577.35 +H，+Na C34H50O6 一 Ganoderic acid S 12.11，13.38 453.34，453.34 +H，+Na，+H，+Na C30H44O3 √ √ Ganoderic acid U 12.06 473.36 +H C30H4gO4 一 √ Ganoderic acid V 11.15 529.35 +H C32H48O6 一 一 Ganoderic acid X 12.06 513.35 +H C32H48O5 一 Ganoderic acid α 5.58，8.74 575.3238，597.30 +H，+Na，+K C32H46O9 √ √ Ganoderic acid β 8.03，8.26 501.32，501.32 +H， +H C30H4406 一 Ganoderic acide 7.49 517.31 +H C30H44O7 √ Notes： CM denotes for the control from methanol extract； CC denotes for the control from chloroform extract； DM denotes for the D2 from methanol extract； DC denotes for the D2 f rom chloroform extract. Symbol √denotes the detection of compounds whereas symbol- denotes that data i s not avai l able. horticulture， where growth has been detected in some explants (e.g.orchid). However ， other variable elements (e.g. pH， temperature) still need to be optimised. Credit author statement Shing Ching Khoo： Writing - original draft preparation and main Project administration. Nyuk Ling Ma： Conceptualization， wri t ing and editing. Wanxi Peng： Resources or Funding acquisition， Validation and Visualization. Kah Kei Ng， Meng Shien Goh & Hui Lin Chen： Project administration involved in experimental Formal analysis and Software Formal analysis. Suat Hian Tan & Vij i tra Luang-In： Resources and Investigation. Chia Hau Lee： Metabolomic Formal analysis. Christian Sonne： Conceptualization， review， data Visualization and edi t ing. Declaration of competing interest The authors declare that they have no known competing financial interests or persona l relationships that could have appeared to i nfluence the work reported in this paper. Acknowledgement The authors would also like to thank Ministry of Higher Education Malaysia for the financia l support under Fundamental Research Grant Scheme (FRGS) (FRGS/1/2018/TK10/UMT/02/2， Vot 59512). Appendix A. Supplementary data Supplementary data to this article can be found online at ht t p s ：//do i .org /10.1016/j .ch e mo s phe r e.2021.131477. References A d e kom aya， O .， M a joz i， T.， 2020. I n c i n er at ion an d ene r gy reco v ery fro m waste m a t e r ia l s： asses sme nt of en v iro nm en t al impac t of e m i t ted g as e s. Eng. Ap pl. Sci . Res.47，458-464. Anna， Rosl i ng， Lindahl ， Bjorn D.， Taylor， Andy F.S.， Finlay， Roger D.， 2004. Mycelial growth and substrate ac i di f icat i on of ectomycor r hiza l fungi in response to di f ferent minerals. 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