Alternate: Bioplastic Production from Salty Food Waste

1.Introduction High salinity food wastes management has been a challenge because it is inhibitory to conventional biological processes such as anaerobic digestion or composting [1]. Haloferax mediterranei (HM) is a hyperhalophilic microorganism capable of producing polyhydroxyalkanoate (PHA) from wastewater with salinity in the range of 7-30% [2]. Such a high salinity environment also protects HM from culture contamination by other indigenous organisms that came with the wasted materials [3]. Moreover, the high osmotic pressure in HM cells also allow the cell wall to burst in fresh water and thus offer low energy and chemical free PHA recovery [4]. Hence, this study demonstrated an innovative pathway enabling the bioconversion of high salinity food wastes to PHA by taking advantage of HM. The outcomes of this study include: (1) confirmed the positive effect of high salinity food waste on volatile fatty acids (VFAs) production; (2) verified that unrefined VFAs can be directly used as feedstocks for high PHA productivity; (3) proved that by fresh water can be used to open the cell walls of HM for high PHA recovery without chemical addition. Key words: Haloferax mediterranei; Polyhydroxyalkanoate; Volatile fatty acid; Food waste 2.Materials and Methods VFA production Although HM does not require VFAs to provide PHA, the PHA yield is always higher when VFAs were used as a feedstock [2]. For that reason, dark fermentation (DF) was employed to ferment high salinity food waste collected from Quasar Energy Group [5]. The inoculum was collected from an anaerobic DF reactor running over one year at University of Maryland, College Park. The food waste to inoculum ratio was 1:2 or 4:1 (by VS). The fermenter was operated with 20-day retention time at 35 ±1oC with pH adjustment of 7. PHA production The wild-type strain HM (ATCC 33500) was used throughout the study. The cells were firstly activated in ATCC 1176 medium [6]. Batch reactors were cultivated in an incubator shaker controlled at 37 oC and 150 rpm. After 48 hours incubation, the cell density measured at optical density (OD) 600 nm reached 0.5 in the triplicate flasks. The activated culture was distributed into 30, 5 ml sterile tubes with 15% glycerol (v/v) for long term storage at -80 °C. To check if there is any contamination, activated culture was spread in triplicate agar plates and incubated for another 144 hours at 37 °C. Only the characteristic pink colonies of HM strain grew in the plates (Figure 1), which confirmed there was no contamination during the HM activation process. To understand the optimized conditions for HM strain to produce PHA using VFAs as carbon source in high salinity digestate, activated HM strain was inoculated in high salinity digestate. OD and suspended solid (SS) were tested to evaluate cell growth. The culture was cultivated in the incubator shaker until the OD reached a plateau. 30 45 ml culture was collected for PHA extraction and quantified by gas chromatograph (GC) [7]. 3.Results 3.1 VFA production from high salinity food waste Food waste with salinity ranging from 0 to 150 g/L were inoculated with food to inoculum ratio of 1:2 and 4:1(by VS) to understand the effects of salinity and inoculum ratio on VFA yields. As can be seen from Figure 2, VFA yield increased with food waste salinity and increased with the food to inoculum ratio. The greatest VFA yield of 43 g/L was achieved in food waste with salinity as high as 150 g/L and food to inoculum ratio of 4:1. Therefore, it is important discovery from this experiment that high salinity food waste actually favors VFA production probably due to its inhibition of methanogenesis as reported previously [1]. 3.2 PHA production from high salinity digestate DF digestates were diluted for 2, 2.5, 3, 4.5, and 9 times with DI water to investigate the dilution effect on PHA yield when salinity was maintained at 156 g/L NaCl. As can be seen from Figure 3, the greatest PHA yields of 60% PHB and 5% PHV were obtained at 2 times dilution when digestate COD was around 9 g/L. In contrast, HM did not grow in raw digestate probably because of inhibition. Further dilution of the digestate proportionally decreased PHA yield, which is expected because of the loading rate decrease. The PHA yields of 65% from HM were much higher than from mixed culture (e.g. 30%) [8]. Moreover, PHV yields of 5% from HM higher than that in mixed culture ( ‰ˆ0% PHV) makes it more commercially valuable since higher HV content results in more elasticity of the plastic material [7, 9]. 3.2 PHA recovery in fresh water HM cells were harvested in centrifuge and then soaked in fresh water for 48 hours for PHA recovery, during which the water was refreshed for 10 times. A comparison was made between two groups with and without stirring. Results showed that the PHA recovery efficiencies 94 % and 95%, respectively, with and without stirring. Hence, it can be concluded that stirring is not important for PHA recovery in fresh water. 4.Conclusions It was concluded from this study that high salinity in food wastes actually favor VFA production in DF. HM cells can effectively utilize VFAs in the two times diluted digestates for PHA production. Soaking HM cells in fresh water can offer PHA recovery efficiency as high as 95% without chemical addition.