Polystyrene recycling, as well as cleanup from the environment, is very expensive hence there is a need to look at a biological approach in minimizing environmental issues it poses. This current study focused on cost-effective and affordable research on the digestion of polystyrene by the larvae of Tenebrio molitor (yellow mealworms). The results showed that these mealworms survived when they were fed with polystyrene for 7 days, and this indicates that the consumption of the polymer plastic is aided by the gut microbiota. It has also been noted that the survival rate of mealworms fed with polystyrene (85%) was slightly lower than those fed with a normal diet which had 90% (Fig. 5), hence, it is feasible that polystyrene waste can be fed to the mealworms for biodegradation. However, the rate of development of polystyrene fed mealworms to pupae was slower as compared to the control group (Table 1) even though they eventually all developed into pupae and finally into beetles. These obtained results are in line with Morales-Ramos’ who reported that the number of mealworms that survives after 7 days of feeding them varies depending on the type of food available, and that diet affects the development time .
The ability of the mealworms to degrade polystyrene is mainly due to the role and activity of gut bacteria , thus, Tenebrio molitor was dissected and gut collected to isolate the bacteria that may be responsible for the biodegradation. The isolated bacteria were cultured in a medium with polystyrene as the carbon source and then plated on polystyrene modified agar so that those able to biodegrade the polymer could grow. Based on morphology, size and colour, 5 colonies were picked and subcultured until pure colonies were obtained. The five colonies from the Tenebrio molitor’s gut were gram stained and all of them stained pink to red, hence, all were gram-negative.
Each of the five isolates was cultured in nutrient broth and DNA extraction of the Tenebrio molitor’s gut bacteria was then carried out using bacterial cells that were harvested in their late exponential phase, the phase that involves multiple rounds of DNA synthesis . DNA was successfully isolated from the five isolates from the Tenebrio molitor’s gut using the phenol–chloroform DNA extraction method as confirmed by agarose gel electrophoresis which showed high molecular weight DNA (Fig. 10A). The bands show that all the isolated DNA were more than 10,000 bp. Generally, the size of many bacterial genomes ranges from 130 kb to more than 14 Mbp . After isolating the genomic DNA from the five isolates, the near-full length of the 16S rRNA gene was amplified using the universal primer set of 27F/1492R. The primer set that was used was designed to amplify the near-full-length 16S rRNA gene for bacterial identification. A distinct band from each isolate from the Tenebrio molitor’s gut was obtained after the amplification of the 16S rRNA genes as shown in Fig. 10B. The PCR yielded products of approximately 1500 bp.
The genetic variation in the five isolates from the gut of Tenebrio molitor was identified using random amplified polymorphic DNA (RAPDS), a PCR-based technique. The RAPD-PCR reaction distinguishes nucleotide sequence polymorphisms in a DNA amplification-based assay such that a single species of primer binds to the genomic DNA at two different sites on opposite strands of the DNA template. It was observed that the priming sites were within an amplifiable distance of each other for each DNA template from each of the five isolates since there were discrete DNA fragments that were produced through thermocylic amplification. The DNA products that were produced after RAPD-PCR assay were viewed on agarose gel and the gel image is shown in Fig. 10C. The polymorphisms between isolates resulted from sequence differences in one or both of the primer binding sites, and this is normally shown by the presence or absence of a particular RAPD band as shown in Fig. 10C. Thus, polymorphisms behave as dominant genetic markers. In this study, there were no polymorphisms between DNA from isolates numbers 1, 2 and 3, the DNA from the three isolates had the same number of scores of RAPD bands. This revealed that isolates numbers 1, 2 and 3 were identical. However, for isolates numbers 4 and 5, the polymorphism between them was caused by the sequence differences in both of the primer binding sites, since they did not have the same number of scores of RAPD bands.
The products of RAPD-PCR shown in the gel image were then used to construct a dendrogram or a phylogenetic tree to show the relationship between the isolates from the T. molitor’s gut. The dendrogram was constructed based on the score for a band from each of the DNA from the five isolates and the dendrogram is shown in Fig. 11. On the phylogenetic tree, it is clear that isolates numbers 1, 2 and 3 are identical as they are on the same branch. Isolates numbers 4 and 5 are on different branches which indicates that they are different amongst themselves as well as from isolates numbers 1, 2 and 3.
To identify the amplification products, the 16SrRNA gene sequencing was used in this study. The 16S rRNA gene PCR products were first purified to remove excess primers and nucleotides using the QiaQick PCR purification kit (Qiagen) before sequencing. The 16S rRNA amplicons were then sequenced with the Seqstudio genetic analyser using the Sanger method. The electropherograms for the fragments generated for isolates numbers 1, 4 and 5 are shown in Fig. 12. The obtained sequences were identified by the BLAST tool to be Klebsiella oxytoca ATCC 13182 for isolate number 1, Klebsiella oxytoca JCM 16655 for isolate number 5 and Klebsiella oxytoca NBRC 102593 for isolate number 4 after comparison with sequences in the GeneBank in NCBI database. The percentage identity of isolate number 1 with Klebsiella oxytoca ATCC 13182 was 87.70%, that of isolate number 4 with Klebsiella oxytoca NBRC 102593 was 97.92% and that of isolate number 5 with Klebsiella oxytoca JCM 16655 was 99.78%.
Klebsiella oxytoca is a Gram-negative, rod-shaped bacterium that is closely related to K. pneumonia. However, it differs from K. pneumonia in that it is indole-positive , as confirmed by the oxidase test which was carried out on the isolates from the mealwoms gut. It is reasonable to assume that polystyrene-degrading bacteria have features present in broad families of aerobic or facultative bacteria and can secrete extracellular oxidative enzymes that are responsible for the breaking down of polystyrene polymer chains.
Obtained sequences were used to estimate the relationships among taxa or sequences to construct a phylogenetic tree as shown in Fig. 13. The phylogenetic tree shows the relationship of the three Klebsiella oxytoca strains that were isolated from the T.molitor gut with closely related microorganisms. The bootstrap percentages reveals the reliability of the cluster descending from every node such that the higher the number, the more reliable would be the estimate of the taxa that descend from a particular node. From the phylogenetic tree in Fig. 13, the nodes that had higher percentages had the following taxa that descended from them and these include Citrobacter gillenni, Enterobacter hormaechei subp, Kosakonia psuedosacchari and Citrobacter amalonaticus. These taxa are the ones reliable in estimating their relationship with Klebsiella oxytoca ATCC 13182. The estimated taxa could be true as there are previous studies that observed them in the biodegradation of polystyrene. Enterobacter hormaechei has previously been isolated from the T.molitor .
Thus, in this study, the bacterial community found in mealworms was primarily composed of the Proteobacteria phylum. Mostly four dominant phyla namely Proteobacteria, Firmicutes, Tenericutes and Actinobacteria have been obtained in other researches on mealworms fed with polystyrene . Differences in results obtained from this study and other researchers show that bacterial community composition could be completely different among samples within the same host as observed by Jung who tested nine individual mealworms and noted that the bacterial communities were not identical across all individuals . This observation confirmed the theory of Cariveau who proposed that bacterial communities can be different between individuals growing in the same environment . The results might suggest that mealworms from different areas have a part of their microbiome in common. In another study, differences between mealworms from diverse locations were observed and it was revealed that T. molitor from different regions presents different microorganisms responsible for the biodegradation of polystyrene [16; 17].
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.