*2.5. Material Balance for Glucose Production from 1 kg Mixed Food Waste with Crude Glucoamylase Extract*

Scheme I shows the material balance of the studied process for crude GA production from pastry waste and glucose recovery from 1 kg mixed food waste (d.b.). All the calculations are provided in the supplementary information. According to our study, hydrolysis of 1 kg mixed food waste (d.b.) could lead to 0.53 kg glucose production. Approximately, 1.4 kg pastry waste (d.b.) is required to produce sufficient amount of crude GA extract for 1 kg mixed food waste hydrolysis. Furthermore, the material balance only presents the theoretical values of the up-scaled process based on our laboratory-scale experimental data. However, upscale study is needed in order to demonstrate the process can be applied at industrial scale.

**Scheme I.** Material balance of the process in this study for processing 1 kg mixed food waste to produce glucose using crude GA extract produced from pastry waste based on the laboratory-scale experimental data.

#### **3. Experimental Section**

#### *3.1. Microorganism*

Frozen spores of the fungus *Aspergillus awamori* (ATCC 14331), were used for SSF. Spores were suspended in demineralized water and loaded into conical flasks containing cornmeal agar and incubated at 30 °C for 7 days. Harvesting of fresh spores was carried out using 10% glycerol and the number of spores was counted using a haemocytometer. Fresh spore suspension of 1 mL was diluted to the required concentration with sterile demineralized water and used immediately for SSF.

#### *3.2. Food Wastes Preparation*

Pastry waste and mixed food waste were obtained from a local Starbucks store and canteen for SSFs and enzymatic hydrolysis experiments, respectively. Once the pastry waste was collected, it was homogenized with a kitchen blender and stored at í20 °C. Pastry waste was autoclaved before subjected to SSF and mixed food waste was lyophilized before enzymatic hydrolysis.

#### *3.3. Solid State Fermentation for Glucoamylase Production*

Sterilized pastry waste of 20 g was placed in a petri dish and inoculated with 1 mL of fresh spore suspension of *Aspergillus awamori* loaded onto the surface of the substrate. For each g of substrate, 5 × 10<sup>5</sup> spores were used. The plates were incubated under static condition at 30 °C for 11 days. Whole plate content was withdrawn regularly and analyzed for GA production.

#### *3.4. Glucoamylase Extraction*

Whole content of the fermented solid in the petri dish was mixed thoroughly in a kitchen blender containing 20 mL demineralized water and the mixture was transferred into a 500 mL Duran bottle. The kitchen blender was rinsed with another portion of 20 mL demineralized water and pooled with the previously obtained mixture. The suspension was then mixed for 30 min with a magnetic stirrer at 30 °C, followed by centrifugation at 22,000 × g for 10 min. The supernatant was collected and filtered with Whitman no. 1 filter paper. The solution obtained refers to crude GA extract.

#### *3.5. Food Waste Hydrolysis for Glucose Production*

Food waste hydrolysis was conducted by adding increasing amount of crude GA extract to test tubes containing 50 mg dried food waste in 2.2 mL of 0.2 M sodium acetate (pH 5.5). The reaction mixture was incubated at 55 °C for 3 h and mixed by pipetting for every 30 min. Aliquots of samples were withdrawn regularly from the reaction mixture and mixed with 10% (w/v) trichloroacetic acid prior to glucose determination.

#### *3.6. Glucoamylase Activity*

Activity of the crude GA extract was determined using the method described by Melikoglu *et al.* [17]. Wheat flour solution of 6% (w/v) was used as substrate and it was prepared in sodium acetate (pH 5.5) and gelatinized at 80 °C for 15 min before usage. The assay was conducted by mixing 0.25 mL of 5 times diluted crude GA extract and 0.5 mL of gelatinized wheat flour solution, and incubated at 55 °C. The reaction was terminated after 10 min by adding 0.25 mL 10% (w/v) trichloroacetic acid solution to the reaction mixture. The reaction mixture was centrifuged and the glucose concentration in the supernatant was analyzed using the Analox GL6 glucose analyzer. One unit (U) of GA activity is defined as the amount of enzyme that releases 1 mol of glucose per minute under assay conditions and is expressed as U/g of dry substrate as described [12].

For determination of optimal reaction pH, the crude GA extract and the gelatinized wheat flour solutions were prepared in sodium acetate buffer from pH 3.5 to 7.5. Optimal reaction temperature determination of the crude GA extract was carried out at temperature range of 40–75 °C with 5 °C increment at optimal pH. Thermo-stability of the crude GA extract was investigated at different temperatures (55–65 °C) at optimal pH. Experiments were performed in duplicate and the standard errors were reported.

#### **4. Conclusions**

In this work, we have demonstrated the feasibility of pastry waste as feedstock for GA production. High GA yield (253.7 ± 20.4 U/g) of the crude enzyme extract was obtained without addition of nitrogen in comparison to other reported waste substrates, highlighting its potential as a feedstock for GA production in industrial scale. Under the optimal digestion conditions (pH 5.5 and 55 °C), the crude GA extract could hydrolyze mixed food waste in 1 h and generate around 53 g glucose from 100 g of mixed food waste. The work is of great significance as it shows sustainable GA production from food waste for potential municipal food waste treatment and sustainable chemicals production.

#### **Acknowledgments**

The authors acknowledge the Biomass funding from the Ability R&D Energy Research Centre (AERC) at the School of Energy and Environment in the City University of Hong Kong. We are also grateful to the donation from the Coffee Concept (Hong Kong) Ltd. For the "Care for Our Planet" campaign, as well as a grant from the City University of Hong Kong (Project No. 7200248). Authors acknowledge the Industrial Technology Funding from the Innovation and Technology Commission (ITS/323/11) in Hong Kong.

#### **Conflicts of Interest**

The authors declare no conflict of interest.

#### **References**


