Identification and Characterization of Digestive Enzymes and Intestinal Microorganisms that Degrade Polystyrene in Tenebrio Molitor Larvae

Plastic has been produced since 1950s and it has been also widely used successfully as a fundamental material. However, it is now considered as a major reason of environmental hazard because its non-biodegradable property. More specifically, non-biodegradable plastic (NBP) mean a high-molecular-carbon-polymers that does not break down in a natural condition by biological processes. In recent decades, various research groups have been conducted to develop a solution for the biodegradation of non-biodegradable plastics such PE, PP, PS, PET and PVC. Recent research indicated that mixed-microbial can be used for digestion of NBPs. Bacterial strains have been isolated from environment or living-organisms and their ability of biodegradation was confirmed that convert into low-molecular-weight organics or mineralize to CO₂. In this study, up-regulated enzymes were identified when insects were feed with NBPs instead bran. Microorganisms were also isolated from various insects and characterized that could biodegrade NBPs. To isolate NBP-digesting microorganisms, the larvae of <i>Galleria mellonella, Tenebrio molitor Linnaeus, Plodia interpunctella </i>and<i> Sitotroqa cerealella </i>were collected from environment and NBPs (polyethylene (PE), polystyrene (PS), and polypropylene (PP)) were produced instead bran. It is identified that <i>T. molitor</i> larvae had a potential for consuming PS comparing other insects. In order to identify the intestinal digestive enzymes and intestinal microorganisms of <i>T. molitor</i> involved in PS biodegradation, we divided them into 5 groups according to the diet. The survival rate was higher in the group of 'PS fed only' comparing with 'starvation'. This indicated that larvae have an ability that can convert energy source from digestion of PS. Intestinal microorganisms were excluded by feeding antibiotics to confirm the PS digestion ability only by digestive enzyme. Frass were characterized with various analytical approaches (such as GPC, FTIR, NMR and TGA) and result indicated that there were no significant differences in PS degradation ability between the presence or absence of intestinal microorganisms. To identify the digestion enzymes of PS with or without treatment of antibiotics, total mRNA was isolated from intestine and sequenced. Lipase3, Phospholipase A11, and Peroxiredoxin-6 were up-regulated that is known as relating fatty acid-digestion. In addition, Calcium uniporter protein mitochondrial, Sulfate permease chloroplast and Sialin were also up-regulated that regulate cell bioenergetics. These results indicate that intestinal enzymes are related in the process of PS digestion process and cellular-energetics. Furthermore, metagenome analysis of the intestinal microflora was performed at 5-day intervals to confirm the change in the microbials community of<i> T. molitor</i> according to PS feeding. It is confirmed that increasing and decreasing-bacterial strains over time and intestine was isolated to identify digesting-microorganisms. Isolated intestine was crushed and cultured in various medium condition including LB, NB, BHI, TSB and GPY. Isolated microorganisms were cultured in an inorganic medium with PS (as an only carbon source). As a result, 6 types of bacterial strains were successfully identified: <i>Bacillus</i> sp., <i>Bacillus mycoides, Bacillus cereus, Enterobacter xiangfangensis, Enterobacter cancerogenus </i>and <i>Enterococcus gallinarum</i><i>.</i> Our result indicates intestinal bacteria and enzymes are relating in the process of digestion of PS and cellular-energetics.