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Effects of substrate to inoculum ratio on Phosphorus Recovery from Different Composition of Food Waste using Anaerobic Batch Digestion

Anaerobic digestion is a process by which microorganisms break down biodegradable material in the absence of oxygen. The process involves hydrolysis, acidogenesis, acedogenesis and methanogenesis stages. Anaerobic digestion of food waste has been widely investigated for biogas recovery but limited study was performed on phosphorus recovery. Substrate to inoculum ratio is a very crucial parameter in anaerobic digestion in an attempt to recover phosphorus as it highly influences the production of organic acids during acidogenesis. Therefore, this study was carried out to investigate phosphorus recovery at different ratio of substrate to inoculum, where substrates was fixed to ratio 1.0 while inoculum ratio varied to 1.5, 2.0, 2.5, 3.0, 3.5 throughout the digestion process. The main substrate used in the anaerobic digestion was food waste which was segregated into different composition namely carbohydrates rich-food waste, fiber rich-food waste and protein rich-food waste. The phosphorus recovery was performed using anaerobic batch digester at mesophilic (35±1°C) condition and pH= 6.0 for 15 days. Semi treated palm oil mill effluent (POME) was used as the inoculum to boost up the anaerobic digestion. The results indicate that substrate to inoculum ratio (1.0 : 2.0) was the optimum ratio to recover phosphorus, where protein rich-food waste shows the phosphorus recovery was about 40.8%, followed by carbohydrate rich-food waste and fibre rich-food waste with 32.8% and 26.2%, respectively. This study is very important in resources recovery from wastes as it provides information on a new strategies for phosphorus recovery from food waste.


Introduction
Food waste (FW) is one of major waste that produced globally [1]. In Malaysia, most of the FW was dumped onto the landfill, consequently lead to the air and water pollution. By way of releasing the greenhouse gases (GHGs) to the surrounding and runoff of the existed nutrients to the river which can cause the eutrophication phenomenon [2-3]. While semi-treated palm oil mill effluent (POME) was the waste that be treated in open pond. This was one alternatives method that be used in most palm oil mill for reducing the nutrients contents, before it runoff to the near river after a period of time (40 to 60 days fermentation. Besides that, it was considered as the cheap method to manage the waste. Semi-treated POME considered as the primary sludge, which claims contained of anaerobic bacteria, where the process of AD can be eventually occur. The process will produce the methane gas (CH4) which can be Anaerobic digestion (AD) is a biological process that be used in treated organic waste and at the same time can recovery the valuable of nutrients such as: phosphorus (P) [6]. The process was involved four stages which were hydrolysis, acidogenis, acedogenesis and methanogenesis [7][8]. In determination of phosphorus recovery, the stages that need to be put meticulous attention was only the hydrolysis and acidogenesis, as the process will lead to the formation of volatile fatty acids (VFAs), thus influence the removal of P from the substrate cell ( Figure 1) [9]. Heading towards to increase the P recovery, substrate to inoculum ratio as one of the factor need to be considerate in AD process. Previous study stated, the purpose of mixing within the digester is to improve the contact between substrates and microorganisms [10]. Moreover, it also claims that mixing also can improve the bacteria's population to obtain nutrients [11][12]. Additionally, reports by Dong et al. (2015) points out substrate to inoculum ratio in AD process, was also depends on the types of substrate and inoculum used. Different of both, will give different results of analysis. Due of it, this study was focus on the optimization of the inoculum ratio in order to achieve the highest of P recovery and optimum time from the digestion of FW composition (CRFW, FRFW and PRFW) and semi treated POME was acts as the inoculum.

Sample collection and preparation
FW composition was collected from a malay restaurant after lunch time which located near Kota Kinabalu City. All the FW was segregated into carbohydrates rich food waste (CRFW), fiber rich food waste (FRFW) and protein rich food waste (PRFW). The FWs was crushed to a mean size about 3-5mm using an electrical food grinder for facilitate the digestion effectively. Then, FWs composition were put in the freezer at 4 0 C for before used [8]. While, semi-treated POME collected form Lumadan mill beaufort, Sabah and it was put in an incubator at temperature 35 0 C (± 1).

Operation startup for AD (FW composition and POME)
The anaerobic digestion experimental was done using the batch reactor which represented by duran bottle with a volume of 1 L. Fifteen reactors were used in this study with differ in substrate to inocula ratio. The digestion was done for 15 days at 35 0 C (± 1) mesophilic condition. The pH value was fixed to 6.0 consider the optimum values during the preliminary analysis. Any reduction or increasing of pH throughout the test will be controlled using 1 M NaOH and 1 M HCl. Any gas was released once in three days to avoid explosion during the test. Figure 2 shows the results from CRFW experiment in determined P recovery values. From the data, it shows ratio substrates to inocula (1.0 :2.0) (CRFW-POME-SI-2.0) indicates the highest of P with 4111.14 mg/L (±0.03). Followed by CRFW-POME-SI-1.5 (1.0:1.5) and CRFW-POME-SI-2. 5    Based on the current studies above, P recovery was increase at all ratio of substrates to inoculum at early days of digestion. Then, it gradually decreased after achieved the P optimum. The statistical results show that all substrates to inoculum ratio was significantly different from each other (p<0.05). In this study, substrate (1.0) to inoculum ratio (2.0) shows the highest of P recover compared other inoculum ratio. This explained, an appropriate ratio of inoculum in AD process could contributed in higher the P recovery [12]. In this study, semi-treated POME was used as the inoculum although commonly it can be considered as the substrate [11]. Previous study had claimed that semi-treated POME physical state was slurry and it was also contained of acidogenic bacterias, thus can used in boost up the P recovery [4]. Hence, this eventually indicates the highest value of P recovery from AD of FWs composition and semi-treated POME could be due to the synergetic effects between both which directly influenced the highest value of P. As stated by Selaman, (2016), AD of different substrate in one medium could build up the synergetic effects as the substrates supply any possible missing nutrients in the process. This convince the suitability between FWs composition and semi-treated POME in recovering of P. Besides that, the data shows greater ratio of semi-treated POME (2.5, 3.0, and 3.5), recovering the lower of P compared ratio (2.0). It is possibly due to the accumulation of total volatile fatty acids (TVFAs) which eventually explain the decreasing of P yield at certain time. Similar observations were also noted by Dong et al. (2015), where the formation of TVFAs was occur at early stages of AD process. Then, its turn to accumulate after several day of fermentations due to the higher of its formation. Optimum of P recovery examined at day 5 to 12 for all data. This significantly shows the acidogenesis process was occur after day 4 of AD [11][12]. The biochemical process on how the P was produces in AD of FWs composition shown in Figure  1. While, Figure 5 provides the results obtained from the analysis of FWs composition (1.0) and inoculum (2.0) ratio Comparing of the results, it can be seen that PRFW recovery the highest of P compared by CRFW and FRFW with values 40.8%. While CRFW and FRFW values 32.8% and 26.2% respectively. Surprisingly, PRFW recovery the highest values of P, though the initial of reactive P values was lower compared other composition which been reported in previous study by Selaman, (2016). In a related report had mention that PRFW contained the better of carbon ratio that will effected the product recovery during AD process [8] [9]. So, this explained the highest value of P recovery. In contrast with FRFW, the lignin contained was harder to break down, thus effect the P removal from the substrate cell by the bacterias.

Results and Discussion
The current results indicates the acidogenesis stages was optimum examined at the day 10 of AD for all FWs composition. Several studies had claimed the short time of AD was appropriate in recovering the highest of P as the TVFAs formation was occur during the early time of digestion [7] [12]. This highlight optimum value of P recovery can be achieved in day 10 of AD between FWs composition and semi-treated POME.

Conclusion
This study suggested an optimization of substrate to inoculum ratio (1.0: 2.0) was adequate for increasing the highest P recovery throughout the AD process for all FW composition. PRFW shows the highest of P recovery about 40.8% which due to the better carbon and nitrogen ratio in the substrates. While, optimum digestion time for all substrate was on day 10. This predicts the highest of TVFAs production during the period of AD process which influence the P removal from the substrate cells. Eventually, the data shows all FW composition (CRFW, FRFW and PRFW). contained P that can be recovery through AD process.