Characterization of polystyrene as feedstock, mealworms and mealworm frass
Biodegradation and depolymerization of ingested polystyrene were assessed by visual observation of both the polystyrene feedstock and frass. The hollows observed on polystyrene in groups 2 and 3 show that the mealworms were feeding themselves with polystyrene, hence, the mealworms were able to survive when they were fed with polystyrene foam as their sole carbon diet as shown in Figure 1.
Overall, there were visual changes that occurred to the polystyrene feedstocks after they were consumed by the mealworms which involved roughening of the surface, creation of holes and cracks. The general changes are shown in Figure 2.
The frass egested by Tenebrio molitor mealworms confirmed biodegradation of polystyrene as it contained very tiny residues of polystyrene as shown in figure 3.
The decrease in mass of the polystyrene as feedstock also confirmed that the mealworms were depending on polystyrene as their sole carbon diet. Thus, the mass loss was recorded on daily basis and results are shown in Figure 4. The results show the decrease in mass of the polystyrene from the initial mass of 1.5 g over seven days in groups 2 and 3. There was a 40 % decrease in the mass of polystyrene for group 2 mealworms and a 33.33 % decrease for group 3.
The number of the larvae that were still surviving from both groups were recorded after seven days as shown in Table 1. The larvae that developed into pupae were also recorded and then collected and placed in one container containing corn flour and carrots.
Table 1: Number of surviving larvae and pupae after seven days
These results show that out of the 100 mealworms that were placed in each group, only 10 died from both groups 1 and 2 and only 15 from group 3. The results also show that the mealworms fed with polystyrene only, slowly developed into pupae.
The percentage survival rate of the mealworms from the three different groups was presented in the form of a line graph as shown in Figure 5. The control group which had mealworms fed on cornflour and carrots and the second group with mealworms fed on polystyrene and carrots had a survival percentage rate of 90 %. However, the third group that had mealworms fed on polystyrene only had a percentage survival rate of 85 % which was slightly lower than the percentage survival rates of both group 1 and group 2.
Some pupae further developed into adults (darkling beetles). Figure 6 shows the collected pupae and some darkling beetles.
Isolation of the polystyrene-degrading bacteria from Tenebrio molitor’s gut
Colonies were observed in the culture plate (figure 7) and this shows that the bacteria in the guts of Tenebrio molitor was isolated.
After obtaining the bacteria that was present in the enrichment culture, five colonies were selected and collected as different isolates basing on cell morphology which included the shape, colour and size. The selected colonies were streaked on a fresh polystyrene modified agar plate for sub-culturing that was done repeatedly to get pure colonies as shown in Figure 8.
The gram stain procedure was carried out and the results show that all the isolates were gram-negative as they all stained pink to red (figure 9)
After confirming the viability of all the five isolates, each of the isolates was cultured in nutrient broth to be used for DNA extraction. Successful isolation of the DNA was confirmed by agarose gel electrophoresis as shown in figure 10. The isolates numbers 1-5 are showing clear bands which explains the presence of DNA in all the isolated samples. The high molecular weight DNA obtained confirmed that the DNA isolated is genomic DNA.
16S rRNA gene PCR
Each sample of DNA obtained from the 5 isolates was subjected to 16S rRNA gene amplification. The 16S amplicons obtained after amplification with an expected band length of 1500 bp – 1550 bp are shown in Figure 11. These results show that the 16S rRNA genes from all the isolates were successfully amplified.
M13 RAPDs PCR
The DNA of the 5 isolates from the gut of Tenebrio molitor gut were also subjected to M13 RAPD-PCR to investigate the phylogenetic relationship of these isolate. The RAPD-PCR results revealed that isolates numbers 1, 2 and 3 were identical. The results for the amplification of the bacterial DNA using the M13 primers are shown in figure 12.
The phylogenetic tree was then constructed from the M13 RAPD-PCR, using dendroUPGMA. The results, shown in Figure 13, indicate s that isolates 1,2 and 3 are identical.
Identical isolates 1, 2 and 3 were regarded as one sample and hence this reduced the total number of 16S amplicons from 5 to 3.
Identification of the bacteria based on the nucleotide sequences obtained
The 3 different 16S amplicons were sequenced directly with the 27F primer using the Sanger method. The nucleotide sequences in the form of chromatograms for the three isolates are shown in Figure 14. The nucleotide sequences in the form of chromatograms for the three isolates were exported to fasta files and the bases were generated for each isolate. Bases obtained for each isolate are reported in additional file 1.
Strain identification by the Basic Local Alignment Search Tool (BLAST) showed that isolate number 1 was Klebsiella oxytoca strain ATCC 13182. Since isolate number 1 was identical to isolates numbers 2 and 3, isolates 2 and 3 were also identified to be Klebsiella oxytoca strain ATCC 13182. Isolates numbers 4 and 5 were identified as Klebsiella oxytoca JCM 1665 and Klebsiella oxytoca NBRC 102593, respectively.
Table 2: Percentage similarity of tested strains against representative species in the BLAST search
The phylogenetic tree was then constructed from the obtained sequences using MEGAX software with the Neighbour Joining algorithm as shown in Figure 15.
The maximum Composite Likelihood method for the determination of the evolutionary distances among the isolates gave a phylogenetic tree in Figure 16. The results show a relationship among isolated bacteria and closely related species of the genus Klebsiella.