Chemical and Enzymatic Valorisation of Confectionery Waste into Biofuel: An Application of Circular Economy

Chemical Enzymatic of Confectionery An ABSTRACT Waste oil was extracted from lollipop effluent stream using a mixture of organic solvents viz. n-hexane and ethyl acetate. Lollipop effluent samples found to contain ~ 10-18 wt.% oil. Subsequently, the oil was subjected to biodiesel synthesis under solvent free condition using chemical and enzymatic catalysts. Among the base catalysts used, KOH catalyzed reaction gave highest biodiesel yield (99 %) in 20 minutes. Furthermore, nine different lipases were screened as catalysts for biodiesel preparation from lollipop effluent oil. The screening experiments revealed that Novozyme-435 was best among the lipases which gave 94 % biodiesel in 18 h at 40 0 C.


INTRODUCTION
Confectionery industries contribute immensely towards global food supply (Henricus, 1980). These industries are major enterprises across the world and still expanding rapidly. Liquid and solid wastes generated from confectionery industries contain substantial amount of lipids and carbohydrates. These are non-edible in nature and needs to be managed from the circular economy perspective (Karmee, 2016a(Karmee, , 2016b(Karmee, , 2017(Karmee, , 2018a(Karmee, , 2018bKarmee et al., 2015Karmee et al., , 2018. Thus, these wastes can be valorised for biofuel production. In this context, majority of the world's energy needs are met by burning natural gas, petroleum based fuels and coal. This is leading to depletion of fossil fuels and simultaneously causing environmental degradation. Therefore, there is a need for development of alternative energy resources. In addition, energy starved nations are also exploring renewable energy sources as an option to become energy independent. Along this line, biofuels are extensively researched and recognized as a potential source of energy. In the above context, biodiesel is a commercially available fuel primarily synthesized from fats, oils and greases. Thus far, industrial biodiesel production is largely focussed on use of many edible feedstocks. However, use of edible feedstocks is creating several challenges including food vs fuel debate. In addition, feedstocks are also increasing the biodiesel cost. To circumvent these limitations, non-edible waste feedstocks viz. food waste, sewage sludge and organic waste are currently employed as low-cost or no-value resources for biofuel production (Karmee and Chadha, 2005;Karmee and Lin, 2014). In line with this, confectionery products and their wastes contain oils, syrups, nuts, candied fruits and colour powders. Confectionery industrial effluent streams containing oils and carbohydrates can be directly valorised for bioenergy production (Figure 1).
In this paper, biodiesel was prepared from a lollipop effluent stream. In the initial stage, oil from lollipop effluent stream was extracted using n-hexane and ethyl acetate. Approximately, 10-18 wt.% oil was recovered from lollipop effluent streams. Moisture from the obtained oil was removed using sodium sulphate (Na2SO4). Subsequently, for biodiesel production chemical (KOH, CaO and Ca(OH)2) and biocatalysts (lipases) were used under solvent free condition. Biodiesel preparation via lipase catalysis is advantageous since it is substrate specific, moisture tolerant, recyclable and operates under mild experimental conditions (Karmee 2015(Karmee , 2016a(Karmee , 2016b(Karmee , 2017. Initially, a series of commercially available lipases were screened for biodiesel production from confectionery waste oil. Immobilized lipase from Candida antartica lipase-B (CAL-B) was found to be suitable. Further optimization of reaction parameters were carried out using CAL-B. Key parameters including feedstock to alcohol molar ratio, temperature, and time course of reaction were optimised. Finally, reusability experiments for the Novozyme-435 was performed.

Enzymes, Chemicals and Instruments
Feedstock used in this experiment was procured from a local lollipop unit located in North-West province, South Africa. KOH, CaO, Ca(OH)2, methanol, diethyl ether and deuterated chloroform (CDCl3) of analytical grades were purchased from Sigma-Aldrich and Associated Chemical Enterprises (ACE), Johannesburg, South Africa. Mucor miehei, Pseudomonas cepacia, Rhizopus delemar, Geotrichum candidum, Candida rugosa, Caprica papaya, Porcine pancreas-II, Pseudomonas fluorescence and Candida antarctica lipase-B (Novozyme-435) lipases were obtained from Sigma-Aldrich, Amano Enzyme Inc., Nagoya, Japan and from local enzyme suppliers of South Africa. Waste oil to biodiesel conversion (%) was calculated from 1 H NMR (Nuclear Magnetic Resonance) using a Bruker instrument (600 MHz) located at the Faculty of Natural Sciences, Laboratory for Analytical Services, North West University, Potchefstroom, South Africa.

Preparation of Biodiesel from Lollipop Effluent Using Base Catalysts (KOH, CaO and Ca(OH)2
A reaction mixture containing waste oil (1 g) and methanol (480 l) was prepared in a round bottom flask. To this mixture base catalyst (1 wt.%) was added at 60 0 C. The reaction was performed at 1:10 molar ratio of waste oil to methanol for 90 min. The reaction was monitored using 1 H NMR.

Screening of Different Lipases for Biodiesel Production from Lollipop Effluent
Nine lipases as stated in the earlier section were used during the screening experiments. For the screening experiments a molar ratio 1: 4 (oil to methanol) was used. The reaction mixture comprising methanol (189 μl), oil (1 g) and lipase (0.1 g, 10 wt%) was stirred at 40 0 C for 6 h. After that, the mixture was diluted using diethyl ether (2 ml x 2). Subsequently, lipase was filtered and air dried. The biodiesel was placed in a 50 ml round bottomed flask and dried at 70 0 C for 30 min under vacuum. The product was analyzed using 1 H NMR.

Optimization of Reaction Temperature
A moral ratio of 1:4 (oil to methanol) was used for the experiments, which consists of a mixture of methanol (1:4,189 μl), feedstock (1g) and biocatalyst (Novozyme-435) (0.1 g, 10 wt.%). Reactions were performed at 30 0 C, 40 0 C, 50 0 C and 60 0 C to determine the maximum biodiesel yield. Each of these experiments was carried out for 6 h.

Two-step Methanol Addition Process for Biodiesel Production
A two-step methanol addition process was followed to maximize the yield of biodiesel. The mixture was comprised of methanol (1:4, 189 μl), oil (1 g) and biocatalyst (0.1 g, 10 wt. %). The initial time duration was 6 h at 40 0 C. Subsequently, after 6 h of duration an additional amount of methanol (47 μl) was added to the reaction flask through septum. Further, the reaction was allowed to continue until 36 h. All the obtained samples were analyzed using 1 H NMR to quantify the biodiesel.

Confectionary Waste Utilization
Different solid and liquid effluents are generated in confectionery industries, which are known to contain organic substances (Beal and Raj, 2000;Das et al., 2013;El-Kassas et al., 2015;Lafitte-Trouque and Forster, 2000). These wastes are generally composed of biodegradable materials such as sugar, sweetener, casein, oil, milk, food colouring and flavouring agents. These are considered no-value resources as these are discarded without any further applications. Confectionery wastes can be converted into aquatic feeds, carbon richsources, bioenergy and value added products (Beal and Raj  Das et al., 2013;El-Kassas et al., 2015;Lafitte-Trouque and Forster, 2000). In this context, South Africa, has many confectionery industries such as lollipop factories that produce sizeable quantities of wastes, which can be valorised into biofuels, chemicals and biopolymer (García et al., 2011;Genc and Ozbay, 2015;Gough et al., 2013;Johnstone-Robertson, 2017;Lunghi and Burzacca, 2004;Pilarska et al., 2019;Ranade et al., 1989;Ruggeri et al., 2013;Tsakona et al., 2016) (Table 1). Similarly, using advanced valorisation technologies the ~ 13% organic waste generated in South Africa can be converted into biofuels, chemicals, materials and other high value products (Ekelund and Nyström 2007; NOWCS South Africa 2013; Environment outlook 2013). (Figure 2). Environmental friendly management of confectionery wastes is significant from waste management and circular economy context (Miah et al., 2018) (Figure 3).

Biodiesel Production from Lollipop Effluent via Base Catalysis
Food industries generate significant amounts of waste during processing, production, packaging, and storage (Sohair et al., 2008). South Africa generates large quantities of organic food wastes which needs technical valorisation (Greben and Oelofse, 2009;Oelofse and Nahman, 2013). During the course Dual anaerobic co-digestion CH4 Chocolate waste water AD Biogas and COD reduction Confectionery waste water Anaerobic pretreatment and aerobic treatment CH4 and reduced COD levels and water for irrigation Confectionery waste water Sequential two-stage anaerobic treatment Decrease in COD levels Confectionery waste water Fermentation Clean water with reduced COD, BOD, FOG, TSS, and odors. Confectionery waste water Aerobic treatment using trickling filter Decrease in COD levels Figure 2. Distribution of different types of wastes generated in South Africa (Ekelund and Nyström, 2007;Environment outlook, 2013;NOWCS South Africa, 2013) of this work, lollipop industry waste stream was valorised for biofuel production. Initially oil from the lollipop effluent was extracted using n-hexane and ethyl acetate (95v: 5v) as a solvent. Up to 10-18 wt.% of oil was recovered from confectionery waste samples. After extraction, the recovered oily portions were combined and concentrated via a roatary evaporator. To remove trace amount of moisture in the oily fraction it was dried over sodium sulphate and stored for further use.
The waste oil isolated from the lollipop effluent was subjected to base catalysed biodiesel preparation. The KOH, CaO and Ca(OH)2 catalysed (1 wt. %) reactions were performed at 1:10 molar ratio of waste oil to alcohol at 60 0 C in a two necked round bottom flask equipped with heating bath, condenser and magnetic stirrer (Figure 4). Among all base catalysts, high conversion (99%) of biodiesel was observed in 20 min during KOH catalysed reaction (Figure 5).

Biodiesel Production from Lollipop Effluent Using Lipases
Lipases are widely used enzymes for applications in energy, food, beverage, detergent, leather, pharmaceutical and paper industries. Under physiological conditions, lipases catalyse hydrolysis of triglycerides to glycerol and free fatty acids. However, in non-conventional media lipase catalyses esterification and transesterification reaction. As mentioned in Figure 6, nine different lipases were screened for biodiesel production from waste oil derived from lollipop effluent stream. The biodiesel yield was in the range of 4-34% ( Figure  6). During screening it was observed that Candida antarctica lipase-B (Novozyme-435) gave 34% biodiesel yield (Figure 6).
Novozyme-435 was applied for further optimization of reaction conditions. The time course study of biocatalytic biodiesel production was carried out for 36 h. However, at 18 h, a maximum 94% biodiesel yield was achieved (Figure 7). Therefore, 18 h is considered to be the optimum reaction time. The reusability of the biocatalyst (Novozyme-435) was studied at 1:5 molar ratio and 40 0 C. It was found that, after 14 th reaction cycle, 72 % biodiesel yield was obtained. Around 24% decrease in biodiesel yield was observed after the 14 th reaction cycle (Figure 8).
Base catalysed reaction is faster as compared to the enzyme catalysed reactions. During base catalysis methoxide is formed; whereas, during lipase catalysed reaction enzyme-  . Potential of lipases for biodiesel production from confectionery waste oil substrate complex is formed. For the base catalysed reaction optimum conditions are: 1:10 molar ratio (substrate to alcohol), 60 0 C and 20 min (reaction time). In contrast, for the lipase catalysed reaction, optimum conditions are: 1:5 molar ratio (substrate to alcohol), 40 0 C and 18 h.

CONCLUSIONS
Liquid effluents from confectionery industries are potential feedstocks for biofuel production. In this work, both chemical (base) and biocatalysts (lipases) were used for biodiesel preparation from confectionery waste oil. KOH gave 99% biodiesel yield in 20 min; whereas, Novozyme-435 catalyzed reaction produced 94% biodiesel in 18 h.