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Tenebrio molitor: possible source of polystyrene-degrading bacteria

Tenebrio molitor: possible source of polystyrene-degrading bacteria Background: The excessive use of polystyrene as a packaging material has resulted in a rise in environmental pollu- tion. Polystyrene waste has continually increased water pollution, soil pollution and the closing of landfill sites since it is durable and resistant to biodegradation. Therefore, the challenge in polystyrene disposal has caused researchers to look for urgent innovative and eco-friendly solutions for plastic degradation. The current study focuses on the isola- tion and identification of bacteria produced by the larvae of beetle Tenebrio molitor (yellow mealworms), that enable them to survive when fed with polystyrene foam as their sole carbon diet. Materials and methods: The biodegradation of polystyrene by Tenebrio molitor was investigated by breeding and rearing the mealworms in the presence and absence of polystyrene. A comparison was made between those fed with a normal diet and those fed on polystyrene. The mealworms which were fed with polystyrene were then dissected and the guts were collected to isolate and identify the bacteria in their guts. The viability and metabolic activity of the isolates were investigated. The polymerase chain reaction (PCR) followed by sequencing was used for molecular iden- tification of the isolates. The PCR products were directly sequenced using Sanger’s method and the phylogenetic tree and molecular evolutionary analyses were constructed using MEGAX software with the Neighbour Joining algorithm. The evolutionary distances were computed using the Maximum Composite Likelihood method. Results: The decrease in mass of the polystyrene as feedstock confirmed that the mealworms were depending on polystyrene as their sole carbon diet. The frass egested by mealworms also confirmed the biodegradation of polysty- rene as it contained very tiny residues of polystyrene. Three isolates were obtained from the mealworms guts, and all were found to be gram-negative. The sequencing results showed that the isolates were Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella oxytoca JCM 1665. Conclusion: Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella oxytoca JCM 1665 maybe some of the bacteria responsible for polystyrene biodegradation. Keywords: Tenebrio molitor, Polystyrene, Mealworms, Biodegradation, Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593, Klebsiella oxytoca JCM 1665 stands to be the most widely used plastic with the scale Background of its production reaching quite a few million tons every The excessive use of durable and degradation-resistant year [2]. Polystyrene slowly fragments into nano-plas- synthetic polymers such as polystyrene as a packag- tics which are harmful and have got detrimental con- ing material has resulted in a rise in environmental pol- sequences on ecosystems, biota, and the environment lution [1]. Polystyrene, whose trade name is Styrofoam, as well on the economy and human health [3]. Residues of plastics have been found in the stomach contents of *Correspondence: rrumbie.2000@gmail.com many organisms such as earthworms, birds, turtles, dol- Department of Biotechnology and Biochemistry, University of Zimbabwe, Harare, Zimbabwe phins and whales [4]. Fish and other marine organisms © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Machona et al. BMC Biotechnology (2022) 22:2 Page 2 of 12 may continuously ingest small amounts of polystyrene the mealworms were feeding themselves with polysty- and pose a health risk when consumed by people. The rene, hence, the mealworms were able to survive by vir- long term exposure to small quantities of styrene might tue of their reserves within the 7 day test period, when cause neurotoxic effects which include fatigue, nervous - they were fed with polystyrene foam. Consumption of ness, sleeping difficulties, hematological effects which the plastic polymer was aided by the gut microbiota as involve low platelet and haemoglobin values, cytogenetic shown in Fig. 1. effects which involve chromosomal and lymphatic abnor - Overall, there were visual changes that occurred to the malities, and carcinogenic effects [5]. polystyrene feedstocks after they were consumed by the However, since the 1950s, researchers have observed mealworms which involved roughening of the surface, that yellow mealworms, which are known as the larvae of creation of holes and cracks. The general changes are Tenebrio molitor damaged plastic packaging materials [6]. shown in Fig. 2. The consumption of polystyrene foams by mealworms Figure  3 shows the frass egested by Tenebrio molitor was later on reported by students that were competing mealworms. Further work needs to be done to investigate in high school science fairs in 2003, whereby mealworms the constituents of the frass. were raised by feeding them with polystyrene foam [7]. The decrease in mass of the polystyrene as feedstock Biodegradation of polystyrene by mealworms has also also confirmed that the mealworms were consuming been confirmed by academic researchers from different polystyrene (aided by gut microbiota). Thus, the mass countries who concluded that yellow mealworms can loss was recorded on daily basis and results are shown in survive when fed with polystyrene foam as their sole car- Fig. 4. The results show the decrease in mass of the poly - bon diet [8]. Thus, the current study focuses on the isola - styrene from the initial mass of 1.5  g over seven days in tion and identification of bacteria produced by the larvae groups 2 and 3. There was a 40% decrease in the mass of beetle Tenebrio molitor, which enable them to survive of polystyrene for group 2 mealworms and a 33.33% when fed with polystyrene foam. The gut of the larvae decrease for group 3. of Tenebrio molitor may contain microorganisms that The number of the larvae that were still surviving from degrade polystyrene, hence, yellow mealworms are eco- both groups were recorded after seven days and the lar- nomically among the most important species that could vae that developed into pupae were collected and placed be used for biodegradation of polystyrene [8, 9]. in one container containing corn flour and carrots. The percentage survival rate of the mealworms from the Results three different groups was presented in the form of a line Characterization of polystyrene as feedstock, mealworms graph as shown in Fig.  5. The control group which had and mealworm frass mealworms fed on cornflour and carrots and the second Biodegradation and depolymerization of ingested group with mealworms fed on polystyrene and carrots polystyrene were assessed by visual observation of had a survival percentage rate of 90%. However, the third both the polystyrene feedstock and frass. The hollows group that had mealworms fed on polystyrene only had a observed on polystyrene in groups 2 and 3 show that percentage survival rate of 85% which was slightly lower Group 2 Group 1 Group 3 Fig. 1 The larvae of T. molitor (mealworms) reared in the presence of corn flour (Group 1), polystyrene and carrots (Group 2) and polystyrene only (Group 3) M achona et al. BMC Biotechnology (2022) 22:2 Page 3 of 12 After Before Fig. 2 The changes observed on the polystyrene feedstock before and after it was fed to Tenebrio molitor mealworms for two months the isolates were recorded as different. The three white colonies appeared different in terms of brightness hence were also recorded as different isolates. The selected col - onies were streaked on a fresh polystyrene modified agar plate for sub-culturing that was done repeatedly to get pure colonies A–E as shown in Fig. 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 (Fig. 9). DNA extraction After confirming the viability of all the five isolates, each of the isolates was cultured in nutrient broth to Fig. 3 Frass excreted by the Tenebrio molitor mealworms after being be used for DNA extraction. Successful isolation of the fed with polystyrene DNA was confirmed by agarose gel electrophoresis as shown in Fig.  10A The isolates numbers 1–5 are show - ing clear bands which explains the presence of DNA in all the isolated samples. The high molecular weight DNA than the percentage survival rates of both group 1 and obtained confirmed that the DNA isolated is genomic group 2. DNA. Each sample of DNA obtained from the 5 isolates Some pupae further developed into adults (darkling was subjected to 16S rRNA gene amplification. The 16S beetles). Figure  6 shows the collected pupae and some amplicons obtained after amplification with an expected darkling beetles. band length of 1500 bp – 1550 bp are shown in Fig. 10B. These results show that the 16S rRNA genes from all the Isolation of the polystyrene‑degrading bacteria isolates were successfully amplified. The DNA of the 5 from Tenebrio molitor’s gut isolates from the gut of Tenebrio molitor gut were also Colonies were observed in the culture plate (Fig.  7) subjected to M13 RAPD-PCR to investigate the phylo- and this shows that the bacteria in the guts of Tenebrio genetic relationship of these isolates. The RAPD-PCR molitor was isolated. results revealed that isolates numbers 1, 2 and 3 were After obtaining the bacteria that was present in the identical. The results for the amplification of the bacterial enrichment culture, five colonies were selected and col - DNA using the M13 primers are shown in Fig. 10C. lected as different isolates based on cell morphology The phylogenetic tree was then constructed from the which included the shape, colour and size. Two of the M13 RAPD-PCR, using dendroUPGMA. The results, colonies were yellow in colour whilst the other three were shown in Fig.  11, indicates that isolates 1, 2 and 3 are white. Of the two yellow colonies, one was shiny hence identical. Machona et al. BMC Biotechnology (2022) 22:2 Page 4 of 12 0246 8 0246 8 Days Days Fig. 4 Mass loss in polystyrene. A is for group 2 that was fed with polystyrene and carrots whereas B is for group 3 that was fed with polystyrene only (PS = polystyrene). All values are mean ± SD for N = 2 Identical isolates 1, 2 and 3 were regarded as one sam- ple and hence this reduced the total number. Control of 16S amplicons from 5 to 3. 100 PS+Carrots PS 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 Fig.  12. The nucleotide sequences in the form of chromatograms for the three Days isolates were exported to fasta files and the bases were Fig. 5 The survival percentage rate of the mealworms from the generated for each isolate. Bases obtained for each isolate three groups. The control had the highest survival rate followed by the group of mealworms which were fed on polystyrene and carrots are reported in Additional file  1. The 16S rRNA sequence and lastly the group that fed on polystyrene alone had the lowest generated for isolate 1, 4 and 5 had 517, 539 and 547 percentage surviving rate. All values are mean ± SD for N = 2 and bases respectively. P < 0.05 against the control for both tested samples Fig. 6 Life cycle of Tenebrio molitor completed as pupae collected from the three groups during the experiment finally turned into beetles %Survivalrate %PSmassloss %PSmassloss M achona et al. BMC Biotechnology (2022) 22:2 Page 5 of 12 a b Fig. 7 Bacteria isolated from the gut of the larvae of T. molitor. a control and b isolated bacteria Fig. 8 Pure colonies of the Tenebrio molitor’s gut microbes obtained after the quadrant streak method Strain identification by the Basic Local Alignment and 3 were also identified to be Klebsiella oxytoca strain Search Tool (BLAST) showed that isolate number 1 was ATCC 13182. Isolates numbers 4 and 5 were identified Klebsiella oxytoca strain ATCC 13182. Since isolate num- as Klebsiella oxytoca JCM 1665 and Klebsiella oxytoca ber 1 was identical to isolates numbers 2 and 3, isolates 2 NBRC 102593, respectively. Table  1 shows percentage Machona et al. BMC Biotechnology (2022) 22:2 Page 6 of 12 Fig. 9 Gram stain of the isolated Tenebrio molitor’s gut bacteria. a and b are only two of the five isolates obtained Fig. 10 A is the agarose gel electrophoresis image of isolated genomic DNA from Tenebrio molitor’s gut bacteria ran on 0.8% agarose gel and stained with ethidium bromide. M is 1 kb Plus ladder from New England Biolabs and Numbers 1–5 are the genomic DNA samples isolated from the bacterial samples, B is the agarose gel electrophoresis showing the amplification products of the 16S ribosomal RNA genes from the bacterial isolates from the gut of Tenebrio molitor. The 16S rRNA gene products were viewed on 1% agarose gel stained with ethidium bromide. M is a 50 bp ladder from Genedirex. Numbers 1–5 are the 16S amplicons and C is the agarose gel electrophoresis results after the amplification of DNA from Tenebrio molitor’s gut isolates, using the M13 primers. M is a 1 kb plus ladder from NEB. Full-length of the gel is reported in Additional file 2 similarity of tested strains against representative species The numbers above the branches are support value in the BLAST search. obtained from 1000 bootstrap replicates. The exter- The maximum Composite Likelihood method for nal nodes are representing the actual sequences that the determination of the evolutionary distances among exist today wheras internal nodes and branches that the isolates gave a phylogenetic tree in Fig.  13. The connect nodes represent the hypothetical ancestors results show a relationship among isolated bacteria and the lengths of the branches are representing the and closely related species of the genus Klebsiella. amount of change that is estimated to have occurred between a pair of nodes. M achona et al. BMC Biotechnology (2022) 22:2 Page 7 of 12 noted that the survival rate of mealworms fed with pol- ystyrene (85%) was slightly lower than those fed with a normal diet which had 90% (Fig.  5), hence, it is feasi- ble 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 depend- ing on the type of food available, and that diet affects the development time [8]. Fig. 11 Phylogenetic tree constructed from M13 RAPD-PCR results The ability of the mealworms to degrade polystyrene showing that isolates number 1, 2 and 3 are identical. Uncropped is mainly due to the role and activity of gut bacteria [9], image of the tree is reported in Additional file 2 thus, Tenebrio molitor was dissected and gut collected to isolate the bacteria that may be responsible for the Discussion Table 1 Percentage similarity of tested strains against Polystyrene recycling, as well as cleanup from the envi- representative species in the BLAST search ronment, is very expensive hence there is a need to look at a biological approach in minimizing environ- Number of Representative species Percentage isolate similarity (BLAST) mental 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 1 Klebsiella oxytoca ATCC 13182 87.70 mealworms). The results showed that these mealworms 2 Klebsiella oxytoca ATCC 13182 87.70 survived when they were fed with polystyrene for 7 days, 3 Klebsiella oxytoca ATCC 13182 87.70 and this indicates that the consumption of the polymer 4 Klebsiella oxytoca JCM 1665 99.77 plastic is aided by the gut microbiota. It has also been 5 Klebsiella oxytoca NBRC 102593 99.77 Fig. 12 The nucleotide sequences in form of chromatograms for isolates numbers 1, 4 and 5. Each peak represents a single nucleotide in the DNA sequence, and each nucleotide has a different colour, A is green, T is red, C is blue and G is black Machona et al. BMC Biotechnology (2022) 22:2 Page 8 of 12 Klebsiella oxytoca JCM 1665 NR 113341 Klebsiella oxytoca NBRC 102593 NR 114152 Klebsiella oxytoca ATCC 13182 Citrobacter gillenii CDC 4693-86 NR 041697 Klebsiella grimontii SB73 NR 159317 Klebsiella michiganensis W14 NR 118335 Pseudescherichia vulneris NBRC 102420 NR 114080 Enterobacter bugandensis 247BMC Homo sapiens NR 148649 Enterobacter hormaechei subsp. xiangfangensis xiangfangensis 10-17 NR 126208 Enterobacter ludwigii EN-119 16S ribosomal RNA NR 042349 Pantoea agglomerans JCM1236 NR 111998 Enterobacter cloacae subsp. dissolvens dissolvens ATCC 23373 NR 118011 Kosakonia quasisacchari WCHEs120001 NR 169476 Kosakonia sacchari SP1 NR 118333 60 Kosakonia pseudosacchari JM-387 Zea mays NR 135211 Citrobacter youngae GTC 1314 NR 041527 Escherichia coli NBRC 102203 NR 114042 Salmonella enterica subsp. indica indica DSM 14848 NR 044370 Citrobacter farmeri CDC 2991-81 NR 024861 Citrobacter amalonaticus LMG 7873 NR 118106 Fig. 13 Neighbor-joining tree based on 16S rRNA sequences showing the relationship between isolates number 1, 4 and other closely related species of the genus Klebsiella. The evolutionary distances were computed using the Maximum Composite Likelihood method biodegradation. The isolated bacteria were cultured in The primer set that was used was designed to amplify the a medium with polystyrene as the carbon source and near-full-length 16S rRNA gene for bacterial identifica - then plated on polystyrene modified agar so that those tion. A distinct band from each isolate from the Tenebrio able to biodegrade the polymer could grow. Based on molitor’s gut was obtained after the amplification of the morphology, size and colour, 5 colonies were picked 16S rRNA genes as shown in Fig.  10B. The PCR yielded and subcultured until pure colonies were obtained. The products of approximately 1500 bp. five colonies from the Tenebrio molitor’s gut were gram The genetic variation in the five isolates from the gut stained and all of them stained pink to red, hence, all of Tenebrio molitor was identified using random ampli - were gram-negative. fied polymorphic DNA (RAPDS), a PCR-based tech - Each of the five isolates was cultured in nutrient broth nique. The RAPD-PCR reaction distinguishes nucleotide and DNA extraction of the Tenebrio molitor’s gut bacte- sequence polymorphisms in a DNA amplification-based ria was then carried out using bacterial cells that were assay such that a single species of primer binds to the harvested in their late exponential phase, the phase that genomic DNA at two different sites on opposite strands involves multiple rounds of DNA synthesis [10]. DNA of the DNA template. It was observed that the priming was successfully isolated from the five isolates from the sites were within an amplifiable distance of each other for Tenebrio molitor’s gut using the phenol–chloroform DNA each DNA template from each of the five isolates since extraction method as confirmed by agarose gel electro - there were discrete DNA fragments that were produced phoresis which showed high molecular weight DNA through thermocylic amplification. The DNA products (Fig.  10A). The bands show that all the isolated DNA that were produced after RAPD-PCR assay were viewed were more than 10,000  bp. Generally, the size of many on agarose gel and the gel image is shown in Fig.  10C. bacterial genomes ranges from 130  kb to more than 14 The polymorphisms between isolates resulted from Mbp [11]. After isolating the genomic DNA from the five sequence differences in one or both of the primer bind - isolates, the near-full length of the 16S rRNA gene was ing sites, and this is normally shown by the presence or amplified using the universal primer set of 27F/1492R. absence of a particular RAPD band as shown in Fig. 10C. M achona et al. BMC Biotechnology (2022) 22:2 Page 9 of 12 u Th s, polymorphisms behave as dominant genetic mark - shows the relationship of the three Klebsiella oxytoca ers. In this study, there were no polymorphisms between strains that were isolated from the T.molitor gut with DNA from isolates numbers 1, 2 and 3, the DNA from closely related microorganisms. The bootstrap percent - the three isolates had the same number of scores of ages reveals the reliability of the cluster descending from RAPD bands. This revealed that isolates numbers 1, 2 every node such that the higher the number, the more and 3 were identical. However, for isolates numbers 4 reliable would be the estimate of the taxa that descend and 5, the polymorphism between them was caused by from a particular node. From the phylogenetic tree in the sequence differences in both of the primer binding Fig. 13, the nodes that had higher percentages had the fol- sites, since they did not have the same number of scores lowing taxa that descended from them and these include of RAPD bands. Citrobacter gillenni, Enterobacter hormaechei subp, Kosa- The products of RAPD-PCR shown in the gel image konia psuedosacchari and Citrobacter amalonaticus. were then used to construct a dendrogram or a phyloge- These taxa are the ones reliable in estimating their rela - netic tree to show the relationship between the isolates tionship with Klebsiella oxytoca ATCC 13182. The esti - from the T. molitor’s gut. The dendrogram was con - mated taxa could be true as there are previous studies structed based on the score for a band from each of the that observed them in the biodegradation of polystyrene. DNA from the five isolates and the dendrogram is shown Enterobacter hormaechei has previously been isolated in Fig.  11. On the phylogenetic tree, it is clear that iso- from the T.molitor [13]. lates numbers 1, 2 and 3 are identical as they are on the u Th s, in this study, the bacterial community found in same branch. Isolates numbers 4 and 5 are on different mealworms was primarily composed of the Proteobac- branches which indicates that they are different amongst teria phylum. Mostly four dominant phyla namely Pro- themselves as well as from isolates numbers 1, 2 and 3. teobacteria, Firmicutes, Tenericutes and Actinobacteria To identify the amplification products, the 16SrRNA have been obtained in other researches on mealworms gene sequencing was used in this study. The 16S rRNA fed with polystyrene [13]. Differences in results obtained gene PCR products were first purified to remove excess from this study and other researchers show that bacterial primers and nucleotides using the QiaQick PCR purifi - community composition could be completely different cation kit (Qiagen) before sequencing. The 16S rRNA among samples within the same host as observed by Jung amplicons were then sequenced with the Seqstudio who tested nine individual mealworms and noted that genetic analyser using the Sanger method. The electro - the bacterial communities were not identical across all pherograms for the fragments generated for isolates individuals [14]. This observation confirmed the theory numbers 1, 4 and 5 are shown in Fig.  12. The obtained of Cariveau who proposed that bacterial communities sequences were identified by the BLAST tool to be can be different between individuals growing in the same Klebsiella oxytoca ATCC 13182 for isolate number 1, environment [15]. The results might suggest that meal - Klebsiella oxytoca JCM 16655 for isolate number 5 and worms from different areas have a part of their microbi - Klebsiella oxytoca NBRC 102593 for isolate number 4 ome in common. In another study, differences between after comparison with sequences in the GeneBank in mealworms from diverse locations were observed and it NCBI database. The percentage identity of isolate num - was revealed that T. molitor from different regions pre - ber 1 with Klebsiella oxytoca ATCC 13182 was 87.70%, sents different microorganisms responsible for the bio - that of isolate number 4 with Klebsiella oxytoca NBRC degradation of polystyrene [16; 17]. 102593 was 97.92% and that of isolate number 5 with Klebsiella oxytoca JCM 16655 was 99.78%. Conclusions Klebsiella oxytoca is a Gram-negative, rod-shaped bac- The bacterial community found in mealworms in this terium that is closely related to K. pneumonia. However, study was primarily composed of the Proteobacteria phy- it differs from K. pneumonia in that it is indole-positive lum. The 16S ribosomal RNA gene sequencing revealed [12], as confirmed by the oxidase test which was carried that all of the five T.molitor larval gut isolates were of the out on the isolates from the mealwoms gut. It is reason- Klebsiella oxytoca species but were of different strains. able to assume that polystyrene-degrading bacteria have The isolated bacteria were Klebsiella oxytoca ATCC features present in broad families of aerobic or facultative 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella bacteria and can secrete extracellular oxidative enzymes oxytoca JCM 16655. The obtained results suggest that the that are responsible for the breaking down of polystyrene gut of T. molitor is a very good source for microorganisms polymer chains. capable of biodegrading polystyrene. However, further Obtained sequences were used to estimate the rela- work is needed to determine the capacity of the isolates tionships among taxa or sequences to construct a phy- by isolating the enzymes which may be responsible for logenetic tree as shown in Fig.  13. The phylogenetic tree plastic degradation and investigating their kinetics. Machona et al. BMC Biotechnology (2022) 22:2 Page 10 of 12 Methods as a sole source of carbon in the diet. The mealworms Mealworms were sterilized by immersing them in 75% ethanol for The mealworms (larvae of Tenebrio molitor) were 1  min followed by rinsing with sterile 0.85% saline directly purchased at their growth age of approximately water. The mealworms were then dissected to remove 5–7 instar from a local company named National Hatch the entire gut from each larva, using surgical blades Web, which is located in Harare in Zimbabwe. Based and forceps. The guts were placed in a sterile petri dish on morphology and colouration, the mealworms were and the midguts were drawn out and pooled in a 10 ml identified as Tenebrio molitor Linnaeus. Upon their centrifuge tube containing 5  ml of saline water. The arrival, the mealworms were fed with bran for two days collected samples were then homogenized for 10  min before polystyrene degradation tests were carried out. using a Dounce homogenizer. The gut tissues were removed using a pipette. The gut cell suspension was stored at 4 °C for later use as the inoculum of the poly- Extended polystyrene (EPS) feedstocks styrene-degrading bacterial enrichment. The expanded polystyrene (EPS) to be tested for bio - degradation were collected as recycle waste. The meal - Preparation of the polystyrene emulsion and agar worms were fed with EPS as feedstocks. These EPS The polystyrene emulsion for microbial degradation was feedstocks had similar densities and similar molecular prepared by dissolving EPS feedstocks in dichlorometh- weights. ane solvent at 3%. The solution was then transferred to an amber bottle containing the same volume of liquid Polystyrene biodegradation capability carbon-free medium (LCFBM) and was stored at 4 ºC for The mealworms were cultivated in polypropylene two days. After two days the polystyrene emulsion was (PP) storage containers in the laboratory and the set- placed in a fume hood overnight to volatilize the dichlo- up and methods that were used were the ones previ- romethane solvent. The polystyrene agar was prepared by ously reported [17]. The mealworms were divided and transferring 50 ml of polystyrene emulsion solution to an allocated into three groups, with different feeding Erlenmeyer flask containing 50  ml of liquid carbon-free conditions, with 100 mealworms in each group. The medium with 1.5 g of agar powder and was autoclaved at mealworms were reared for 1  week (7  days). The tem - 121 ºC for 15 min. perature was maintained at 25 ºC and humidity was maintained at 75–80% in each container. The first group was the control hence mealworms were fed with normal Isolation and identification of microorganisms feed which is cornflour and carrots, the second group The collected gut cell suspension was used as the inocu - was fed with 1.5  g of expanded polystyrene and car- lum of polystyrene-degrading bacterial culture enrich- rots and the third group was fed with 1.5 g of expanded ment. The gut cell suspension was inoculated into an polystyrene foam only. In all three groups, dead meal- Erlenmeyer flask containing polystyrene emulsion as the worms were removed whenever observed. The number sole carbon source, and liquid carbon-free basal medium of surviving larvae and pupae were recorded and poly- (LCFBM). The flask that served as the control contained styrene mass loss was monitored and calculated. polystyrene emulsion and LCFBM only. The flasks were then incubated on a rotary shaker at 120  rpm and an ambient temperature of 30  °C for 28  days. After 28  days Characterization of feedstock and frass of the incubation period, the enrichment was spread on Biodegradation and depolymerization of ingested poly- a plate with modified polystyrene agar and then incu - styrene were assessed by visual observation of poly- bated for 24  h at ambient temperature to enumerate the styrene as feedstock and the frass egested. The visual number of bacteria present in the enrichment culture. observation was done to evaluate the macroscopic Colonies from the plate were then selected based on changes such as roughening or scabrous and the crea- cell morphology (shape, colour, and size) and streaked tion of holes and cracks, on the surface structure of the on fresh polystyrene agar plates for sub-culturing. The polymer. Visual changes were used as the first indication quadrant streak method was employed to obtain pure of Tenebrio molitor larvae attack on the polystyrene poly- colonies. The pure colonies of the isolates were Gram- mer [18]. The biodegradation was also determined by stained and viewed under a light microscope for bacteria measuring the mass loss of the polystyrene feedstock. characterization. Isolation of polystyrene‑degrading bacteria Molecular identification The gut cell suspension was prepared from a total num - After obtaining pure colonies, each of the isolates ber of 50 mealworms which were fed with polystyrene was inoculated in 100  ml nutrient broth in 250  ml M achona et al. BMC Biotechnology (2022) 22:2 Page 11 of 12 conical flasks and incubated in a rotary shaker at 30 ºC sequence with organisms in the Gen Bank database for at 120 rpm. The growth of the isolates was monitored by strain identification and construction of phylogenetic measuring their optical density at 600  nm, after every tree analysis. The phylogenetic tree and molecular evo - hour. Cell suspension for DNA extraction was collected lutionary analyses were constructed using MEGAX from each isolate in its late exponential phase. software with the Neighbour Joining algorithm. The per - centage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) DNA extraction and amplification was shown next to the branches. The evolutionary dis - DNA extraction was carried out through precipita- tances were computed using the Maximum Composite tion and centrifugation using phenol–chloroform DNA Likelihood method. extracting protocol. DNA was visualised after electropho- resis at 120 V and 400 mA for 30 min on agarose gel (0.8% Supplementary Information (w/v) in TAE buffer, stained with Ethidium Bromide solu - The online version contains supplementary material available at https:// doi. tion). The concentration of DNA was determined by nan - org/ 10. 1186/ s12896- 021- 00733-3. odrop spectrophotometer. The supernatant containing the extracted DNA was used to amplify 16S ribosomal Additional file 1. Bases for isolates 1, 4 and 5. DNA segments through PCR using 16S universal primers Additional file 2. Phylogenetic tree constructed from M13 RAPD-PCR 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R results showing that isolates 1, 2 and 3 are identical. (5′-TAC GGY TAC CTT GTT ACG ACTT-3′). The PCR of the 16S rRNA gene with universal primers contained 5 µl Acknowledgements of 5X One Taq reaction buffer, 0.5  µl of 10  mM dNTPs, University of Zimbabwe Research Board (Harare, Zimbabwe) is acknowledged. 0.5  µl of 10  µM forward primer, 0.5  µl of 10  µM reverse primer, 1  µl of template DNA, 0.125  µl of Taq Polymer- Authors’ contributions OM performed all the assays reported in this study, analyzed and interpreted ase, 1 µl of 25 mM MgCl and was brought up to a final the obtained results. FC provided information on molecular identification and volume of 25 µl with ultra-pure water. The reactions were interpretation of the results and RM was the major contributor in writing the performed in a thermocycler under the following condi- manuscript. All authors read and approved the final manuscript. tions: 5 min at 95 ºC, followed by 35 cycles of 45 s at 95 Funding ºC, 40 s at 56 ºC, 2 min at 72 ºC and a final extension step Reagents used in this study was provided by the Biotechnology and Biochem- at 72 ºC for 5 min. The expected band length for the PCR istry Department at the University of Zimbabwe, Zimbabwe. product was 1500 bp. The PCR products were visualised Availability of data and materials after electrophoresis at 150 V and 400 mA for 30 min on All data generated or analysed during this study are included in this manu- agarose gel (0.8% (w/v) in TAE buffer, stained with Ethid - script [and its supplementary information files]. ium Bromide solution). The PCR products were directly sequenced with using the Sanger sequencer. The 16S Declarations rRNA sequences obtained were viewed using Chromas Ethics approval and consent to participate 2.6.6 software. The obtained sequences were subjected Not applicable. to BLAST search in NCBI database for phylogenetic Consent for publication relationship. Not applicable. Competing interests M13 RAPD‑PCR The authors declare that they have no competing interests. The M13 RAPD-PCR was carried out to investigate phy - logenetic relationship among isolates. The RAPD-PCR Received: 5 July 2021 Accepted: 29 December 2021 contained 12.5 µl of mastermix, 1 µl of the M13 forward primer, 1 µl of the reverse primer, 1 µl of DNA and 9.5 µl of ultra-pure water. The reactions were performed in a References thermocycler under the following conditions: 1  min at 1. Bandyopadhyay A, Basak GC. Studies on photocatalytic degradation of 95 ºC, followed by 40 cycles of 1 min at 95 ºC, 30 s at 38 polystyrene. 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Philos Trans R Soc B. 2009;364:2153–66. 6. Gerhardt PD, Lindgren DL. Penetration of packaging films: film materials used 507 for food packaging tested for resistance to some common stored-product insects. Calif Agr. 1954;8:3–4. 7. Sina, 2003. Mealworms can digest plastics. Accessed 08.02.2017 (in Chinese) 8. Liu C, Masri J, Perez V, Maya C, Zhao J. Growth performance and nutri- ent composition of mealworms (Tenebrio Molitor) fed on fresh plant materials-supplemented diets. Foods. 2020;151:1–10. 9. Yang Y, Yang J, Wu WM, Zhao J, Song Y, Gao L, Yang R, Jiang L. Biodegrada- tion and mineralization of polystyrene by 172 plastic-eating mealworms: part 1. Chemical and physical characterization and isotopic tests. Environ Sci Technol. 2015;49:12080–6. 10. Jaishankar J, Srivastava P. Molecular basis of stationary phase survival and applications. Front Microbiol. 2017. https:// doi. org/ 10. 3389/ fmicb. 2017. 11. McCutcheon JP, Dohlen C. An interdependent metabolic patchwork in the nested symbiosis of mealybugs. Curr Biol. 2011;21:1366–72. 12. Tang ZL, Kuo TA, Liu HH. The study of the microbes degraded polystyrene. Adv Technol Innov. 2017;2:13–7. 13. Urbanek AK, Rybak J, Wróbel M, Leluk K, Mirończuk AM. A comprehensive assessment of microbiome diversity in Tenebrio molitor fed with polysty- rene waste. Environ Pollut. 2020;262:114–281. 14. Jung J, Heo A, Park YW, Kim YJ, Koh H, Park W. Gut microbiota of Tenebrio molitor and their response to environmental change. J Microbiol Biotech- nol. 2014;24:888–97. 15. Cariveau DP, Powell JE, Koch H, Winfree R, Moran NA. Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus). ISME J. 2014;8:2369–79. 16. Wu WM, Yang J, Criddle CS. Microplastics pollution and reduction strate- gies. Front Environ Sci Eng. 2017;11:1. 17. Yang SS, Brandon AM, Xing DF, Yang J, Pang JW, Criddle CS. Progresses in polystyrene biodegradation and prospects for solutions to plastic waste pollution. Earth Environ Sci. 2018;150:1–9. 18. Nikolic V, Velickovic S, Popovic A. Biodegradation of polystyrene-graft- starch copolymers in three different types of soil. Environ Sci Pollut Res Int. 2014;21:9877–86. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. 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Tenebrio molitor: possible source of polystyrene-degrading bacteria

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Abstract

Background: The excessive use of polystyrene as a packaging material has resulted in a rise in environmental pollu- tion. Polystyrene waste has continually increased water pollution, soil pollution and the closing of landfill sites since it is durable and resistant to biodegradation. Therefore, the challenge in polystyrene disposal has caused researchers to look for urgent innovative and eco-friendly solutions for plastic degradation. The current study focuses on the isola- tion and identification of bacteria produced by the larvae of beetle Tenebrio molitor (yellow mealworms), that enable them to survive when fed with polystyrene foam as their sole carbon diet. Materials and methods: The biodegradation of polystyrene by Tenebrio molitor was investigated by breeding and rearing the mealworms in the presence and absence of polystyrene. A comparison was made between those fed with a normal diet and those fed on polystyrene. The mealworms which were fed with polystyrene were then dissected and the guts were collected to isolate and identify the bacteria in their guts. The viability and metabolic activity of the isolates were investigated. The polymerase chain reaction (PCR) followed by sequencing was used for molecular iden- tification of the isolates. The PCR products were directly sequenced using Sanger’s method and the phylogenetic tree and molecular evolutionary analyses were constructed using MEGAX software with the Neighbour Joining algorithm. The evolutionary distances were computed using the Maximum Composite Likelihood method. Results: The decrease in mass of the polystyrene as feedstock confirmed that the mealworms were depending on polystyrene as their sole carbon diet. The frass egested by mealworms also confirmed the biodegradation of polysty- rene as it contained very tiny residues of polystyrene. Three isolates were obtained from the mealworms guts, and all were found to be gram-negative. The sequencing results showed that the isolates were Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella oxytoca JCM 1665. Conclusion: Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella oxytoca JCM 1665 maybe some of the bacteria responsible for polystyrene biodegradation. Keywords: Tenebrio molitor, Polystyrene, Mealworms, Biodegradation, Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593, Klebsiella oxytoca JCM 1665 stands to be the most widely used plastic with the scale Background of its production reaching quite a few million tons every The excessive use of durable and degradation-resistant year [2]. Polystyrene slowly fragments into nano-plas- synthetic polymers such as polystyrene as a packag- tics which are harmful and have got detrimental con- ing material has resulted in a rise in environmental pol- sequences on ecosystems, biota, and the environment lution [1]. Polystyrene, whose trade name is Styrofoam, as well on the economy and human health [3]. Residues of plastics have been found in the stomach contents of *Correspondence: rrumbie.2000@gmail.com many organisms such as earthworms, birds, turtles, dol- Department of Biotechnology and Biochemistry, University of Zimbabwe, Harare, Zimbabwe phins and whales [4]. Fish and other marine organisms © The Author(s) 2022. 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:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Machona et al. BMC Biotechnology (2022) 22:2 Page 2 of 12 may continuously ingest small amounts of polystyrene the mealworms were feeding themselves with polysty- and pose a health risk when consumed by people. The rene, hence, the mealworms were able to survive by vir- long term exposure to small quantities of styrene might tue of their reserves within the 7 day test period, when cause neurotoxic effects which include fatigue, nervous - they were fed with polystyrene foam. Consumption of ness, sleeping difficulties, hematological effects which the plastic polymer was aided by the gut microbiota as involve low platelet and haemoglobin values, cytogenetic shown in Fig. 1. effects which involve chromosomal and lymphatic abnor - Overall, there were visual changes that occurred to the malities, and carcinogenic effects [5]. polystyrene feedstocks after they were consumed by the However, since the 1950s, researchers have observed mealworms which involved roughening of the surface, that yellow mealworms, which are known as the larvae of creation of holes and cracks. The general changes are Tenebrio molitor damaged plastic packaging materials [6]. shown in Fig. 2. The consumption of polystyrene foams by mealworms Figure  3 shows the frass egested by Tenebrio molitor was later on reported by students that were competing mealworms. Further work needs to be done to investigate in high school science fairs in 2003, whereby mealworms the constituents of the frass. were raised by feeding them with polystyrene foam [7]. The decrease in mass of the polystyrene as feedstock Biodegradation of polystyrene by mealworms has also also confirmed that the mealworms were consuming been confirmed by academic researchers from different polystyrene (aided by gut microbiota). Thus, the mass countries who concluded that yellow mealworms can loss was recorded on daily basis and results are shown in survive when fed with polystyrene foam as their sole car- Fig. 4. The results show the decrease in mass of the poly - bon diet [8]. Thus, the current study focuses on the isola - styrene from the initial mass of 1.5  g over seven days in tion and identification of bacteria produced by the larvae groups 2 and 3. There was a 40% decrease in the mass of beetle Tenebrio molitor, which enable them to survive of polystyrene for group 2 mealworms and a 33.33% when fed with polystyrene foam. The gut of the larvae decrease for group 3. of Tenebrio molitor may contain microorganisms that The number of the larvae that were still surviving from degrade polystyrene, hence, yellow mealworms are eco- both groups were recorded after seven days and the lar- nomically among the most important species that could vae that developed into pupae were collected and placed be used for biodegradation of polystyrene [8, 9]. in one container containing corn flour and carrots. The percentage survival rate of the mealworms from the Results three different groups was presented in the form of a line Characterization of polystyrene as feedstock, mealworms graph as shown in Fig.  5. The control group which had and mealworm frass mealworms fed on cornflour and carrots and the second Biodegradation and depolymerization of ingested group with mealworms fed on polystyrene and carrots polystyrene were assessed by visual observation of had a survival percentage rate of 90%. However, the third both the polystyrene feedstock and frass. The hollows group that had mealworms fed on polystyrene only had a observed on polystyrene in groups 2 and 3 show that percentage survival rate of 85% which was slightly lower Group 2 Group 1 Group 3 Fig. 1 The larvae of T. molitor (mealworms) reared in the presence of corn flour (Group 1), polystyrene and carrots (Group 2) and polystyrene only (Group 3) M achona et al. BMC Biotechnology (2022) 22:2 Page 3 of 12 After Before Fig. 2 The changes observed on the polystyrene feedstock before and after it was fed to Tenebrio molitor mealworms for two months the isolates were recorded as different. The three white colonies appeared different in terms of brightness hence were also recorded as different isolates. The selected col - onies were streaked on a fresh polystyrene modified agar plate for sub-culturing that was done repeatedly to get pure colonies A–E as shown in Fig. 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 (Fig. 9). DNA extraction After confirming the viability of all the five isolates, each of the isolates was cultured in nutrient broth to Fig. 3 Frass excreted by the Tenebrio molitor mealworms after being be used for DNA extraction. Successful isolation of the fed with polystyrene DNA was confirmed by agarose gel electrophoresis as shown in Fig.  10A The isolates numbers 1–5 are show - ing clear bands which explains the presence of DNA in all the isolated samples. The high molecular weight DNA than the percentage survival rates of both group 1 and obtained confirmed that the DNA isolated is genomic group 2. DNA. Each sample of DNA obtained from the 5 isolates Some pupae further developed into adults (darkling was subjected to 16S rRNA gene amplification. The 16S beetles). Figure  6 shows the collected pupae and some amplicons obtained after amplification with an expected darkling beetles. band length of 1500 bp – 1550 bp are shown in Fig. 10B. These results show that the 16S rRNA genes from all the Isolation of the polystyrene‑degrading bacteria isolates were successfully amplified. The DNA of the 5 from Tenebrio molitor’s gut isolates from the gut of Tenebrio molitor gut were also Colonies were observed in the culture plate (Fig.  7) subjected to M13 RAPD-PCR to investigate the phylo- and this shows that the bacteria in the guts of Tenebrio genetic relationship of these isolates. The RAPD-PCR molitor was isolated. results revealed that isolates numbers 1, 2 and 3 were After obtaining the bacteria that was present in the identical. The results for the amplification of the bacterial enrichment culture, five colonies were selected and col - DNA using the M13 primers are shown in Fig. 10C. lected as different isolates based on cell morphology The phylogenetic tree was then constructed from the which included the shape, colour and size. Two of the M13 RAPD-PCR, using dendroUPGMA. The results, colonies were yellow in colour whilst the other three were shown in Fig.  11, indicates that isolates 1, 2 and 3 are white. Of the two yellow colonies, one was shiny hence identical. Machona et al. BMC Biotechnology (2022) 22:2 Page 4 of 12 0246 8 0246 8 Days Days Fig. 4 Mass loss in polystyrene. A is for group 2 that was fed with polystyrene and carrots whereas B is for group 3 that was fed with polystyrene only (PS = polystyrene). All values are mean ± SD for N = 2 Identical isolates 1, 2 and 3 were regarded as one sam- ple and hence this reduced the total number. Control of 16S amplicons from 5 to 3. 100 PS+Carrots PS 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 Fig.  12. The nucleotide sequences in the form of chromatograms for the three Days isolates were exported to fasta files and the bases were Fig. 5 The survival percentage rate of the mealworms from the generated for each isolate. Bases obtained for each isolate three groups. The control had the highest survival rate followed by the group of mealworms which were fed on polystyrene and carrots are reported in Additional file  1. The 16S rRNA sequence and lastly the group that fed on polystyrene alone had the lowest generated for isolate 1, 4 and 5 had 517, 539 and 547 percentage surviving rate. All values are mean ± SD for N = 2 and bases respectively. P < 0.05 against the control for both tested samples Fig. 6 Life cycle of Tenebrio molitor completed as pupae collected from the three groups during the experiment finally turned into beetles %Survivalrate %PSmassloss %PSmassloss M achona et al. BMC Biotechnology (2022) 22:2 Page 5 of 12 a b Fig. 7 Bacteria isolated from the gut of the larvae of T. molitor. a control and b isolated bacteria Fig. 8 Pure colonies of the Tenebrio molitor’s gut microbes obtained after the quadrant streak method Strain identification by the Basic Local Alignment and 3 were also identified to be Klebsiella oxytoca strain Search Tool (BLAST) showed that isolate number 1 was ATCC 13182. Isolates numbers 4 and 5 were identified Klebsiella oxytoca strain ATCC 13182. Since isolate num- as Klebsiella oxytoca JCM 1665 and Klebsiella oxytoca ber 1 was identical to isolates numbers 2 and 3, isolates 2 NBRC 102593, respectively. Table  1 shows percentage Machona et al. BMC Biotechnology (2022) 22:2 Page 6 of 12 Fig. 9 Gram stain of the isolated Tenebrio molitor’s gut bacteria. a and b are only two of the five isolates obtained Fig. 10 A is the agarose gel electrophoresis image of isolated genomic DNA from Tenebrio molitor’s gut bacteria ran on 0.8% agarose gel and stained with ethidium bromide. M is 1 kb Plus ladder from New England Biolabs and Numbers 1–5 are the genomic DNA samples isolated from the bacterial samples, B is the agarose gel electrophoresis showing the amplification products of the 16S ribosomal RNA genes from the bacterial isolates from the gut of Tenebrio molitor. The 16S rRNA gene products were viewed on 1% agarose gel stained with ethidium bromide. M is a 50 bp ladder from Genedirex. Numbers 1–5 are the 16S amplicons and C is the agarose gel electrophoresis results after the amplification of DNA from Tenebrio molitor’s gut isolates, using the M13 primers. M is a 1 kb plus ladder from NEB. Full-length of the gel is reported in Additional file 2 similarity of tested strains against representative species The numbers above the branches are support value in the BLAST search. obtained from 1000 bootstrap replicates. The exter- The maximum Composite Likelihood method for nal nodes are representing the actual sequences that the determination of the evolutionary distances among exist today wheras internal nodes and branches that the isolates gave a phylogenetic tree in Fig.  13. The connect nodes represent the hypothetical ancestors results show a relationship among isolated bacteria and the lengths of the branches are representing the and closely related species of the genus Klebsiella. amount of change that is estimated to have occurred between a pair of nodes. M achona et al. BMC Biotechnology (2022) 22:2 Page 7 of 12 noted that the survival rate of mealworms fed with pol- ystyrene (85%) was slightly lower than those fed with a normal diet which had 90% (Fig.  5), hence, it is feasi- ble 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 depend- ing on the type of food available, and that diet affects the development time [8]. Fig. 11 Phylogenetic tree constructed from M13 RAPD-PCR results The ability of the mealworms to degrade polystyrene showing that isolates number 1, 2 and 3 are identical. Uncropped is mainly due to the role and activity of gut bacteria [9], image of the tree is reported in Additional file 2 thus, Tenebrio molitor was dissected and gut collected to isolate the bacteria that may be responsible for the Discussion Table 1 Percentage similarity of tested strains against Polystyrene recycling, as well as cleanup from the envi- representative species in the BLAST search ronment, is very expensive hence there is a need to look at a biological approach in minimizing environ- Number of Representative species Percentage isolate similarity (BLAST) mental 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 1 Klebsiella oxytoca ATCC 13182 87.70 mealworms). The results showed that these mealworms 2 Klebsiella oxytoca ATCC 13182 87.70 survived when they were fed with polystyrene for 7 days, 3 Klebsiella oxytoca ATCC 13182 87.70 and this indicates that the consumption of the polymer 4 Klebsiella oxytoca JCM 1665 99.77 plastic is aided by the gut microbiota. It has also been 5 Klebsiella oxytoca NBRC 102593 99.77 Fig. 12 The nucleotide sequences in form of chromatograms for isolates numbers 1, 4 and 5. Each peak represents a single nucleotide in the DNA sequence, and each nucleotide has a different colour, A is green, T is red, C is blue and G is black Machona et al. BMC Biotechnology (2022) 22:2 Page 8 of 12 Klebsiella oxytoca JCM 1665 NR 113341 Klebsiella oxytoca NBRC 102593 NR 114152 Klebsiella oxytoca ATCC 13182 Citrobacter gillenii CDC 4693-86 NR 041697 Klebsiella grimontii SB73 NR 159317 Klebsiella michiganensis W14 NR 118335 Pseudescherichia vulneris NBRC 102420 NR 114080 Enterobacter bugandensis 247BMC Homo sapiens NR 148649 Enterobacter hormaechei subsp. xiangfangensis xiangfangensis 10-17 NR 126208 Enterobacter ludwigii EN-119 16S ribosomal RNA NR 042349 Pantoea agglomerans JCM1236 NR 111998 Enterobacter cloacae subsp. dissolvens dissolvens ATCC 23373 NR 118011 Kosakonia quasisacchari WCHEs120001 NR 169476 Kosakonia sacchari SP1 NR 118333 60 Kosakonia pseudosacchari JM-387 Zea mays NR 135211 Citrobacter youngae GTC 1314 NR 041527 Escherichia coli NBRC 102203 NR 114042 Salmonella enterica subsp. indica indica DSM 14848 NR 044370 Citrobacter farmeri CDC 2991-81 NR 024861 Citrobacter amalonaticus LMG 7873 NR 118106 Fig. 13 Neighbor-joining tree based on 16S rRNA sequences showing the relationship between isolates number 1, 4 and other closely related species of the genus Klebsiella. The evolutionary distances were computed using the Maximum Composite Likelihood method biodegradation. The isolated bacteria were cultured in The primer set that was used was designed to amplify the a medium with polystyrene as the carbon source and near-full-length 16S rRNA gene for bacterial identifica - then plated on polystyrene modified agar so that those tion. A distinct band from each isolate from the Tenebrio able to biodegrade the polymer could grow. Based on molitor’s gut was obtained after the amplification of the morphology, size and colour, 5 colonies were picked 16S rRNA genes as shown in Fig.  10B. The PCR yielded and subcultured until pure colonies were obtained. The products of approximately 1500 bp. five colonies from the Tenebrio molitor’s gut were gram The genetic variation in the five isolates from the gut stained and all of them stained pink to red, hence, all of Tenebrio molitor was identified using random ampli - were gram-negative. fied polymorphic DNA (RAPDS), a PCR-based tech - Each of the five isolates was cultured in nutrient broth nique. The RAPD-PCR reaction distinguishes nucleotide and DNA extraction of the Tenebrio molitor’s gut bacte- sequence polymorphisms in a DNA amplification-based ria was then carried out using bacterial cells that were assay such that a single species of primer binds to the harvested in their late exponential phase, the phase that genomic DNA at two different sites on opposite strands involves multiple rounds of DNA synthesis [10]. DNA of the DNA template. It was observed that the priming was successfully isolated from the five isolates from the sites were within an amplifiable distance of each other for Tenebrio molitor’s gut using the phenol–chloroform DNA each DNA template from each of the five isolates since extraction method as confirmed by agarose gel electro - there were discrete DNA fragments that were produced phoresis which showed high molecular weight DNA through thermocylic amplification. The DNA products (Fig.  10A). The bands show that all the isolated DNA that were produced after RAPD-PCR assay were viewed were more than 10,000  bp. Generally, the size of many on agarose gel and the gel image is shown in Fig.  10C. bacterial genomes ranges from 130  kb to more than 14 The polymorphisms between isolates resulted from Mbp [11]. After isolating the genomic DNA from the five sequence differences in one or both of the primer bind - isolates, the near-full length of the 16S rRNA gene was ing sites, and this is normally shown by the presence or amplified using the universal primer set of 27F/1492R. absence of a particular RAPD band as shown in Fig. 10C. M achona et al. BMC Biotechnology (2022) 22:2 Page 9 of 12 u Th s, polymorphisms behave as dominant genetic mark - shows the relationship of the three Klebsiella oxytoca ers. In this study, there were no polymorphisms between strains that were isolated from the T.molitor gut with DNA from isolates numbers 1, 2 and 3, the DNA from closely related microorganisms. The bootstrap percent - the three isolates had the same number of scores of ages reveals the reliability of the cluster descending from RAPD bands. This revealed that isolates numbers 1, 2 every node such that the higher the number, the more and 3 were identical. However, for isolates numbers 4 reliable would be the estimate of the taxa that descend and 5, the polymorphism between them was caused by from a particular node. From the phylogenetic tree in the sequence differences in both of the primer binding Fig. 13, the nodes that had higher percentages had the fol- sites, since they did not have the same number of scores lowing taxa that descended from them and these include of RAPD bands. Citrobacter gillenni, Enterobacter hormaechei subp, Kosa- The products of RAPD-PCR shown in the gel image konia psuedosacchari and Citrobacter amalonaticus. were then used to construct a dendrogram or a phyloge- These taxa are the ones reliable in estimating their rela - netic tree to show the relationship between the isolates tionship with Klebsiella oxytoca ATCC 13182. The esti - from the T. molitor’s gut. The dendrogram was con - mated taxa could be true as there are previous studies structed based on the score for a band from each of the that observed them in the biodegradation of polystyrene. DNA from the five isolates and the dendrogram is shown Enterobacter hormaechei has previously been isolated in Fig.  11. On the phylogenetic tree, it is clear that iso- from the T.molitor [13]. lates numbers 1, 2 and 3 are identical as they are on the u Th s, in this study, the bacterial community found in same branch. Isolates numbers 4 and 5 are on different mealworms was primarily composed of the Proteobac- branches which indicates that they are different amongst teria phylum. Mostly four dominant phyla namely Pro- themselves as well as from isolates numbers 1, 2 and 3. teobacteria, Firmicutes, Tenericutes and Actinobacteria To identify the amplification products, the 16SrRNA have been obtained in other researches on mealworms gene sequencing was used in this study. The 16S rRNA fed with polystyrene [13]. Differences in results obtained gene PCR products were first purified to remove excess from this study and other researchers show that bacterial primers and nucleotides using the QiaQick PCR purifi - community composition could be completely different cation kit (Qiagen) before sequencing. The 16S rRNA among samples within the same host as observed by Jung amplicons were then sequenced with the Seqstudio who tested nine individual mealworms and noted that genetic analyser using the Sanger method. The electro - the bacterial communities were not identical across all pherograms for the fragments generated for isolates individuals [14]. This observation confirmed the theory numbers 1, 4 and 5 are shown in Fig.  12. The obtained of Cariveau who proposed that bacterial communities sequences were identified by the BLAST tool to be can be different between individuals growing in the same Klebsiella oxytoca ATCC 13182 for isolate number 1, environment [15]. The results might suggest that meal - Klebsiella oxytoca JCM 16655 for isolate number 5 and worms from different areas have a part of their microbi - Klebsiella oxytoca NBRC 102593 for isolate number 4 ome in common. In another study, differences between after comparison with sequences in the GeneBank in mealworms from diverse locations were observed and it NCBI database. The percentage identity of isolate num - was revealed that T. molitor from different regions pre - ber 1 with Klebsiella oxytoca ATCC 13182 was 87.70%, sents different microorganisms responsible for the bio - that of isolate number 4 with Klebsiella oxytoca NBRC degradation of polystyrene [16; 17]. 102593 was 97.92% and that of isolate number 5 with Klebsiella oxytoca JCM 16655 was 99.78%. Conclusions Klebsiella oxytoca is a Gram-negative, rod-shaped bac- The bacterial community found in mealworms in this terium that is closely related to K. pneumonia. However, study was primarily composed of the Proteobacteria phy- it differs from K. pneumonia in that it is indole-positive lum. The 16S ribosomal RNA gene sequencing revealed [12], as confirmed by the oxidase test which was carried that all of the five T.molitor larval gut isolates were of the out on the isolates from the mealwoms gut. It is reason- Klebsiella oxytoca species but were of different strains. able to assume that polystyrene-degrading bacteria have The isolated bacteria were Klebsiella oxytoca ATCC features present in broad families of aerobic or facultative 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella bacteria and can secrete extracellular oxidative enzymes oxytoca JCM 16655. The obtained results suggest that the that are responsible for the breaking down of polystyrene gut of T. molitor is a very good source for microorganisms polymer chains. capable of biodegrading polystyrene. However, further Obtained sequences were used to estimate the rela- work is needed to determine the capacity of the isolates tionships among taxa or sequences to construct a phy- by isolating the enzymes which may be responsible for logenetic tree as shown in Fig.  13. The phylogenetic tree plastic degradation and investigating their kinetics. Machona et al. BMC Biotechnology (2022) 22:2 Page 10 of 12 Methods as a sole source of carbon in the diet. The mealworms Mealworms were sterilized by immersing them in 75% ethanol for The mealworms (larvae of Tenebrio molitor) were 1  min followed by rinsing with sterile 0.85% saline directly purchased at their growth age of approximately water. The mealworms were then dissected to remove 5–7 instar from a local company named National Hatch the entire gut from each larva, using surgical blades Web, which is located in Harare in Zimbabwe. Based and forceps. The guts were placed in a sterile petri dish on morphology and colouration, the mealworms were and the midguts were drawn out and pooled in a 10 ml identified as Tenebrio molitor Linnaeus. Upon their centrifuge tube containing 5  ml of saline water. The arrival, the mealworms were fed with bran for two days collected samples were then homogenized for 10  min before polystyrene degradation tests were carried out. using a Dounce homogenizer. The gut tissues were removed using a pipette. The gut cell suspension was stored at 4 °C for later use as the inoculum of the poly- Extended polystyrene (EPS) feedstocks styrene-degrading bacterial enrichment. The expanded polystyrene (EPS) to be tested for bio - degradation were collected as recycle waste. The meal - Preparation of the polystyrene emulsion and agar worms were fed with EPS as feedstocks. These EPS The polystyrene emulsion for microbial degradation was feedstocks had similar densities and similar molecular prepared by dissolving EPS feedstocks in dichlorometh- weights. ane solvent at 3%. The solution was then transferred to an amber bottle containing the same volume of liquid Polystyrene biodegradation capability carbon-free medium (LCFBM) and was stored at 4 ºC for The mealworms were cultivated in polypropylene two days. After two days the polystyrene emulsion was (PP) storage containers in the laboratory and the set- placed in a fume hood overnight to volatilize the dichlo- up and methods that were used were the ones previ- romethane solvent. The polystyrene agar was prepared by ously reported [17]. The mealworms were divided and transferring 50 ml of polystyrene emulsion solution to an allocated into three groups, with different feeding Erlenmeyer flask containing 50  ml of liquid carbon-free conditions, with 100 mealworms in each group. The medium with 1.5 g of agar powder and was autoclaved at mealworms were reared for 1  week (7  days). The tem - 121 ºC for 15 min. perature was maintained at 25 ºC and humidity was maintained at 75–80% in each container. The first group was the control hence mealworms were fed with normal Isolation and identification of microorganisms feed which is cornflour and carrots, the second group The collected gut cell suspension was used as the inocu - was fed with 1.5  g of expanded polystyrene and car- lum of polystyrene-degrading bacterial culture enrich- rots and the third group was fed with 1.5 g of expanded ment. The gut cell suspension was inoculated into an polystyrene foam only. In all three groups, dead meal- Erlenmeyer flask containing polystyrene emulsion as the worms were removed whenever observed. The number sole carbon source, and liquid carbon-free basal medium of surviving larvae and pupae were recorded and poly- (LCFBM). The flask that served as the control contained styrene mass loss was monitored and calculated. polystyrene emulsion and LCFBM only. The flasks were then incubated on a rotary shaker at 120  rpm and an ambient temperature of 30  °C for 28  days. After 28  days Characterization of feedstock and frass of the incubation period, the enrichment was spread on Biodegradation and depolymerization of ingested poly- a plate with modified polystyrene agar and then incu - styrene were assessed by visual observation of poly- bated for 24  h at ambient temperature to enumerate the styrene as feedstock and the frass egested. The visual number of bacteria present in the enrichment culture. observation was done to evaluate the macroscopic Colonies from the plate were then selected based on changes such as roughening or scabrous and the crea- cell morphology (shape, colour, and size) and streaked tion of holes and cracks, on the surface structure of the on fresh polystyrene agar plates for sub-culturing. The polymer. Visual changes were used as the first indication quadrant streak method was employed to obtain pure of Tenebrio molitor larvae attack on the polystyrene poly- colonies. The pure colonies of the isolates were Gram- mer [18]. The biodegradation was also determined by stained and viewed under a light microscope for bacteria measuring the mass loss of the polystyrene feedstock. characterization. Isolation of polystyrene‑degrading bacteria Molecular identification The gut cell suspension was prepared from a total num - After obtaining pure colonies, each of the isolates ber of 50 mealworms which were fed with polystyrene was inoculated in 100  ml nutrient broth in 250  ml M achona et al. BMC Biotechnology (2022) 22:2 Page 11 of 12 conical flasks and incubated in a rotary shaker at 30 ºC sequence with organisms in the Gen Bank database for at 120 rpm. The growth of the isolates was monitored by strain identification and construction of phylogenetic measuring their optical density at 600  nm, after every tree analysis. The phylogenetic tree and molecular evo - hour. Cell suspension for DNA extraction was collected lutionary analyses were constructed using MEGAX from each isolate in its late exponential phase. software with the Neighbour Joining algorithm. The per - centage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) DNA extraction and amplification was shown next to the branches. The evolutionary dis - DNA extraction was carried out through precipita- tances were computed using the Maximum Composite tion and centrifugation using phenol–chloroform DNA Likelihood method. extracting protocol. DNA was visualised after electropho- resis at 120 V and 400 mA for 30 min on agarose gel (0.8% Supplementary Information (w/v) in TAE buffer, stained with Ethidium Bromide solu - The online version contains supplementary material available at https:// doi. tion). The concentration of DNA was determined by nan - org/ 10. 1186/ s12896- 021- 00733-3. odrop spectrophotometer. The supernatant containing the extracted DNA was used to amplify 16S ribosomal Additional file 1. Bases for isolates 1, 4 and 5. DNA segments through PCR using 16S universal primers Additional file 2. Phylogenetic tree constructed from M13 RAPD-PCR 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R results showing that isolates 1, 2 and 3 are identical. (5′-TAC GGY TAC CTT GTT ACG ACTT-3′). The PCR of the 16S rRNA gene with universal primers contained 5 µl Acknowledgements of 5X One Taq reaction buffer, 0.5  µl of 10  mM dNTPs, University of Zimbabwe Research Board (Harare, Zimbabwe) is acknowledged. 0.5  µl of 10  µM forward primer, 0.5  µl of 10  µM reverse primer, 1  µl of template DNA, 0.125  µl of Taq Polymer- Authors’ contributions OM performed all the assays reported in this study, analyzed and interpreted ase, 1 µl of 25 mM MgCl and was brought up to a final the obtained results. FC provided information on molecular identification and volume of 25 µl with ultra-pure water. The reactions were interpretation of the results and RM was the major contributor in writing the performed in a thermocycler under the following condi- manuscript. All authors read and approved the final manuscript. tions: 5 min at 95 ºC, followed by 35 cycles of 45 s at 95 Funding ºC, 40 s at 56 ºC, 2 min at 72 ºC and a final extension step Reagents used in this study was provided by the Biotechnology and Biochem- at 72 ºC for 5 min. The expected band length for the PCR istry Department at the University of Zimbabwe, Zimbabwe. product was 1500 bp. The PCR products were visualised Availability of data and materials after electrophoresis at 150 V and 400 mA for 30 min on All data generated or analysed during this study are included in this manu- agarose gel (0.8% (w/v) in TAE buffer, stained with Ethid - script [and its supplementary information files]. ium Bromide solution). The PCR products were directly sequenced with using the Sanger sequencer. The 16S Declarations rRNA sequences obtained were viewed using Chromas Ethics approval and consent to participate 2.6.6 software. The obtained sequences were subjected Not applicable. to BLAST search in NCBI database for phylogenetic Consent for publication relationship. Not applicable. Competing interests M13 RAPD‑PCR The authors declare that they have no competing interests. The M13 RAPD-PCR was carried out to investigate phy - logenetic relationship among isolates. The RAPD-PCR Received: 5 July 2021 Accepted: 29 December 2021 contained 12.5 µl of mastermix, 1 µl of the M13 forward primer, 1 µl of the reverse primer, 1 µl of DNA and 9.5 µl of ultra-pure water. The reactions were performed in a References thermocycler under the following conditions: 1  min at 1. Bandyopadhyay A, Basak GC. Studies on photocatalytic degradation of 95 ºC, followed by 40 cycles of 1 min at 95 ºC, 30 s at 38 polystyrene. 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BMC BiotechnologySpringer Journals

Published: Jan 4, 2022

Keywords: Tenebrio molitor; Polystyrene; Mealworms; Biodegradation; Klebsiella oxytoca ATCC 13182; Klebsiella oxytoca NBRC 102593; Klebsiella oxytoca JCM 1665

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