Molecular Detection and Phylogenetic Analysis of the alkB Gene in Klebsiella oxytoca Strains Isolated from the Gut of Tenebrio molitor

The challenge in polystyrene disposal has caused researchers to look for urgent innovative and ecofriendly solutions for plastic degradation. Some insects have been reported to use polystyrene as their sole carbon source, and this has been linked to the presence of microbes in their guts that aid in plastic digestion. Thus, this study focuses on the molecular detection and phylogenetic analysis of the alkane-1-monooxygenase (alkB) gene in Klebsiella oxytoca strains isolated from the gut of Tenebrio molitor. The alkB gene encodes for alkane-1-monooxygenase, an enzyme involved in the oxidation of inactivated alkanes. This gene can be used as a marker to assess bacteria's ability to biodegrade polystyrene. Three bacterial strains were isolated from the guts of T. molitor mealworms and were confirmed using polymerase chain reaction (PCR) of the 16S ribosomal RNA gene. The primers used in the amplification of the 16S ribosomal RNA region were designed using NCBI, a bioinformatics tool. To detect the presence of the alkB gene in the isolated bacterial strains, a set of primers used in the amplification of this gene was manually designed from the conserved regions of the alkB nucleotide sequences of eleven bacterial species from GenBank. TCOFFE online tool was used to align the alkB sequences of the bacteria, while Jalview and ConSurf were used to view the alignment. The amplified alkB gene was then sequenced using the Sanger sequencing technique, blasted on NCBI to look for similar sequences, and a phylogenetic tree was constructed. Based on the 16S ribosomal RNA gene sequences, the isolated bacterial strains were confirmed to be Klebsiella oxytoca NBRC 102593, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca ATCC 13182. The alkB gene sequence identical to fourteen alkB gene sequences derived from Actinobacteria whole genome was detected in Klebsiella oxytoca for the first time to the best of our knowledge. The novel nucleotide sequence was published in the NCBI database under accession number OP959069. This gene sequence was found to be for the enzyme alkane-1-monooxygenase and may be one of the enzymes responsible for polystyrene degradation by the putative Klebsiella oxytoca ATCC 13182 in T. molitor.


Introduction
Tenebrio molitor is the larval form of the yellow mealworm beetle that is known as the darkling beetle [1].Te mealworms are well-known scavengers and decomposers of the earth's surface.Tey consume decaying leaves, sticks, grasses, plants, insects, and animals.Tis behaviour has been found to be benefcial to ecology because they devour organic matter that other organisms do not [2].Tese include plastic materials such as polycarbonate (PC), polystyrene (PS), and expanded polystyrene (EPS) among others.
Because of this characteristic, researchers have identifed mealworms as a possible polystyrene-degrading insect [3].
Many studies have demonstrated that the tendency to eat plastic in these insects might be associated with the digestive activity of intestinal microorganisms, which would help them with the digestion process [4].Research is therefore increasingly focusing on bacterial species isolated from plastic-degrading larvae and their potential to digest plastic on their own.Tree bacterial species from the intestines of T. molitor were reported to be good plastic biodegraders [5].Tese bacterial species were Serratia marcescens, Klebsiella oxytoca, and Pseudomonas aeruginosa.In a more recent study, three bacterial isolates identifed as Klebsiella oxytoca NBRC 102593 strain, Klebsiella oxytoca JCM 1665 strain, and Klebsiella oxytoca ATCC 13182 strain were isolated from T. molitor mealworms which were fed solely on polystyrene for 7 days [6].From this study, it was then concluded that the Klebsiella oxytoca species are among the species responsible for the biodegradability of waste-expanded polystyrene by Tenebrio molitor mealworms.
Te Klebsiella oxytoca is an enteric bacterium which is a Gram-negative member of the Klebsiella family.Recent studies have investigated the use of Klebsiella oxytoca strains as enzyme sources for plastic biodegradation, alkane hydroxylases (AHs) being the key enzyme [7].Monooxygenases are the most important enzymes in the alkane hydroxylase system.Tus far, the integral-membrane alkane monooxygenase (alkB)-related AHs are the most common AHs found in both Gram-negative and Gram-positive bacteria [8].
Te aim of this study was to investigate the possible presence of the alkane-1-monooxygenase (alkB) gene responsible for polystyrene degradation, in Klebsiella oxytoca strains isolated from the gut of T. molitor.

Isolation of Klebsiella oxytoca Strains from T. molitor's
Gut.A total number of 50 Tenebrio molitor mealworms were fed with polystyrene as the sole carbon source for seven days and then sterilized by immersing them in 75% alcohol for one minute.Te mealworms were immersed in saline water (0.85%) for one minute.Saline water was used to remove the alcohol and kill the mealworms by osmosis as it contains salt.Surgical blades and forceps were used to remove the entire gut of the worms.Te guts were placed in a sterile Petri dish, and the midguts were drawn out and placed in a centrifuge tube containing 5 ml of saline solution.A Dounce homogenizer was then used to homogenize the sample for 10 minutes.Te gut cell suspension was collected and inoculated into a fask containing 100 ml of polystyrene emulsion and liquid carbon-free basal medium (LCFBM).Te fask that served as the control contained polystyrene emulsion and LCFBM only.Te fasks were then incubated on a rotary shaker at 120 rpm at 30 degrees for 28 days.After 28 days, the solution was spread on a plate with modifed polystyrene agar.Te plate was incubated for 24 hours at 30 degrees to increase the number of bacteria present in the culture.Colonies from the plate were collected based on morphology and subcultured on fresh polystyrene agar.Te quadrant streak method was used to obtain pure colonies.

Designing of Primers. Confrmation of isolation of
Klebsiella oxytoca strains, Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca NBRC 102593 was done using polymerase chain reaction (PCR).Primers that amplify the 16S ribosomal RNA gene in these bacterial strains were designed using the sequences of the 16S ribosomal RNA gene for the three bacterial strains.Te sequences used to design the primers were previously reported [6].To design the primers, the NCBI primer blast tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi) was used.Each of these sequences was uploaded to the NCBI Primer blast tool.A PCR product size of 70 up to a thousand base pairs was specifed.Te melting temperature of each primer was set to a minimum of 50 °C and a maximum of 63 °C with an optimum of 60 °C, and the maximum melting temperature diference between the 2 primers was set to 3 °C.Te GC content for the primers was set between 45% and 60%.More than ten sets of primers were returned after blasting.A pair for each strain that had the right characteristics of appropriate primers was selected.An ofine tool, SnapGene viewer (https://www.snapgene.com/snapgene-viewer), was used for in silico PCR to note the expected amplicon sizes.Lyophilized primers were ordered from Inqaba Biotec and were diluted to a stock concentration of 100 mM using the protocol provided for each primer.Te stock solutions were further diluted to make a working solution of 10 mM using nuclease-free sterile water.Table 1 shows the sequences used to design the primers.

Amplifcation of the 16S Ribosomal RNA Gene by PCR.
Conventional PCR was carried out using the designed primers to amplify the 16S ribosomal RNA gene in each of the 3 isolates obtained.To prepare the DNA template for the PCR, single colonies were either picked from the culture plates and crushed in 10 μL of nuclease-free water, and the crushed samples were incubated at 95 °C for 10 minutes and centrifuged at 13000 rpm for two minutes or cultured overnight in LB broth, using 1 μL of the overnight culture as a template.Each of the isolate suspensions was used as a DNA template for amplifcation using all the designed primer sets in 3 diferent reactions to determine which of the strains was Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca NBRC 102593.PCR reactions (10 μL) were prepared by mixing 5 μL of One Taq ® Quick-Load ® 2X Master Mix with Standard Bufer supplied by New England Biolabs (https://www.neb.com/M0486) with 1 μL each of forward and reverse primers, 2 μL nuclease free water, and 1 μL of the template DNA.Cycling conditions for conventional PCR for all the primers were as follows: one cycle of the initial denaturation at 95 °C for 10 minutes followed by 30 cycles of 95 °C for 30 seconds, primer annealing at 55 °C for 30 seconds, extension at 72 °C for 30 seconds, and a fnal extension at 72 °C for 10 minutes, and samples were cooled to 4 °C.

Gel Electrophoresis to View PCR Results
. Te SnapGene tool was used in in silico gel electrophoresis.Diferent conditions were specifed for the in silico gel to determine the optimum gel electrophoresis conditions.Guided by the in silico gel, a 1% agarose gel was prepared by dissolving 1 gram of agarose in 100 ml of 1 X TAE bufer and microwaving the mixture for 2 minutes with intermittent mixing.Te agarose solution was allowed to cool to about 50 °C before 3 μL of 2 Te Scientifc World Journal ethidium bromide was added.Te solution was then poured into a gel tank and left to set for 15 minutes.Amplicons and the molecular weight marker were loaded into each well on the agarose gel. 100 bp and 1 kb genomic ladders were used in gel electrophoresis for PCR reaction with JCM primers and NBRC primers, respectively.Gel electrophoresis was done for 20 minutes at a voltage of 100 A. A UV transilluminator was used for the visualization of the band pattern on the agarose gel.

Molecular Detection of the Alkane 1-Monooxygenase
(alkB) Gene in Each Strain.Eleven alkB gene sequences from eleven diferent bacterial strains from GenBank were selected for designing the primers.Te accession numbers of the selected sequences are given in Table 2. Tese sequences were then aligned using TCOFFE (https://www.ebi.ac.uk/Tools/msa/tcofee/).To view the alignment, Jalview, an ofine tool, and ConSurf (Te ConSurf Server (tau.ac.il)), an online tool, were used.Tese tools made it easy to determine the conserved and variable regions of the alignment clearer.Primers were designed from the conserved regions to have a GC content between 45% and 60%, T m between 60 °C and 62 °C, and length between 18 and 24 bp.Te forward primer was designed from bases 210 to 230 of the alignment.Te reverse primer was designed from bases 540 to 520 of the alignment.SnapGene was used to note the attachment of the primers to the template.Te designed primers were named Deg-r for the reverse and Deg-f for the forward.Lyophilized Te Scientifc World Journal primers were ordered from Inqaba Biotec, dissolved and diluted to a stock concentration of 100 mM using the protocol provided for each primer.Te stock solutions were further diluted to make a working solution of 10 mM using nuclease free sterile water.

Selection of Other alkB
Targeting Primers.To add to the set of primers designed, two more sets were selected from the literature [9].Tese primers were successful in amplifying the gene in 49% of the bacteria in the study.Te selected primers were Alkb-f and Alkb-r and Alk-BFB and Alk-BRB.PCR products were separated by 1% agarose gel electrophoresis and visualized.A 100 bp genomic ladder was used in gel electrophoresis for PCR reaction with Alk-BFB and Alk-BRB, and Deg-f and Deg-r. 1 kb genomic ladder was used in gel electrophoresis for a PCR reaction with Alkb-f and Alkb-r.Gel electrophoresis was done for 20 minutes at a voltage of 100 A. A UV transilluminator was used for visualization of the agarose gel.

Sequencing and Analysis of the PCR Products.
Te products of the genes that were successfully amplifed were sent to a local company called Biotech Research Institute for sequencing using the Sanger sequencer.A DNA sequence reader CHROMAS (https://chromas.software.informer.com/2.5/)was downloaded and used to view and analyse the sequence.Te analysis included identifcation and trimming of low-quality bases and manual base calling of degenerate bases to obtain a good sequence.
Te cleaned sequence was saved in FASTA format in a word document.Te FASTA sequence was blasted on NCBI against nucleotide sequences in GenBank.

Phylogenetic Analysis.
AlkB gene sequences from 14 diferent bacteria were chosen from GenBank.Tese sequences were used for phylogenetic analysis in MegaX.Te evolutionary relationship in MegaX was inferred by using the maximum likelihood method and Tamura-Nei model.

Isolation of the Polystyrene-Degrading Klebsiella oxytoca
Strains from T. molitor's Gut.Te colonies obtained after culturing Tenebrio's gut cell suspension in modifed polystyrene agar were collected based on the morphology of the Klebsiella oxytoca strains and subcultured on fresh polystyrene agar using the quadrant streak method to obtain pure colonies.Tree diferent isolates were identifed, and these were reported as samples 1, 2, and 3 in Figure 1.Sample 1 colonies were round, cream, and shiny.Tese colonies were the smallest as compared to the other colonies in samples 2 and 3. Sample 2 colonies were white and elongated.Sample 3 colonies were whitish and were the biggest among all the 3 colonies.

Designing of Primers.
To confrm that the isolated bacteria were Klebsiella oxytoca strains, primers that amplify the 16S ribosomal RNA gene in the three Klebsiella strains were designed using the sequences of the 16S ribosomal RNA gene.Primers that amplify the 16S ribosomal RNA region of Klebsiella oxytoca NBRC 102593 were designed to give a product of 247 base pairs.Te GC content of the forward and the reverse primers for this primer set was 55% and 60%, respectively.Te melting temperatures were 59.96 °C and 60.53 °C, respectively.Te self-complementarity score of the forward and reverse primers was 4 and 5, and the 3′ complementary score was 0 and 3. Primers that amplify the 16S ribosomal RNA region of Klebsiella oxytoca ATCC 13182 strain were designed to give a product of 329 base pairs.Te GC content of the forward and the reverse primers for this primer set was 52.6% and 55%, respectively.Te melting temperatures were 57.6 °C and 59.9 °C, respectively.Te self-complementarity score of the forward and reverse primers was 4 and 3, and the 3′ complementary score was 2 and 3. Primers that amplify the 16S ribosomal RNA region of Klebsiella oxytoca JCM 1665 were designed to give a product of 262 base pairs.Te GC content of the forward and the reverse primers for this primer set was 60% and 50%, respectively.Te melting temperatures were 60.6 °C and 61.46 °C, respectively.Te self-complementarity score of the forward and reverse primers was 4 and 6, and the 3′ complementary score was 3 and 1.Table 3 shows the forward and the reverse primer sequences designed to amplify the 16S ribosomal RNA of the Klebsiella oxytoca NBRC 102593 strain, Klebsiella oxytoca JCM 1665 strain, and Klebsiella oxytoca ATCC 13182 strain.

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Amplifcation of the 16S Ribosomal RNA Gene by PCR.
Conventional PCR was carried out using the designed primers to amplify the 16S ribosomal RNA gene in each of the 3 isolates obtained.Each of the isolates was amplifed using all the 3 designed primer sets to determine which of the strains was Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca NBRC 102593.
Figure 2 shows amplifcation of the three Klebsiella oxytoca strains with JCM-specifc primers on 1.5% agarose gel.Te JCM primers which were used in this reaction to amplify Klebsiella oxytoca JCM 1665 strain had an expected product size of 262 base pairs.Te gel shows a band at an approximate size of 260 base pairs in well number 2 which was loaded with sample 1. Tus, sample 1 was identifed as Klebsiella oxytoca JCM 1665.Only primer dimers above 100 bp mark were observed in wells 5 and 6 (loaded with samples 2 and 3, respectively).
Figure 3 shows amplifcation of Klebsiella oxytoca NBRC 102593 strain with NBRC-specifc primers on a 1.5% agarose gel when 1 μL of LB broth overnight culture was used as the DNA template and when single colonies were picked from overnight culture plates and used as a DNA template for sample 2. Te results show that sample 2 was identifed as Klebsiella oxytoca NBRC 102593 strain since all 4 lanes show a band of approximately 247 base pairs, which was the expected product size with NBRC primers.Te results also show that the two forms of the template DNA work in a PCR reaction in the same way.
Amplifcation of the three Klebsiella oxytoca strains with ATCC-specifc primers was also carried out and the expected product size of 337 base pairs was observed in the well loaded with sample 3.
Tus, from the results obtained from the amplifcation of the 16S ribosomal RNA gene by PCR, the isolated samples were identifed as Klebsiella oxytoca JCM 1665  Te Scientifc World Journal strain-sample 1, Klebsiella oxytoca NBRC 102593 strain-sample 2, and Klebsiella oxytoca ATCC 13182 strain-sample 3.

Designing of Oligonucleotide Primers Tat Amplify alkB
Gene.To detect the presence of the alkB gene in each of the three isolates, primers that amplify the alkB gene were designed manually.Table 3 shows the forward and the reverse (Deg-f and Deg-r degenerate) primer sequences designed to amplify the alkB gene in the three Klebsiella oxytoca strains.Te primers that amplify the alkB gene in Klebsiella oxytoca strains were designed to give a product size of 338 base pairs.Te forward primer had a T m of 42.81 °C, a GC content of 50%, a self-complementary score of 4 and 3′ complementary score of 0. Te reverse primer had a T m of 47.69 °C, a GC content of 58.82%, a self-complementary score of 6 and 3′ complementary score of 0.

Oligonucleotide Primers Chosen from Literature Tat
Amplify the alkB Gene.To increase the chances of amplifying the alkB gene in the three isolates, two more primer sets were selected from the literature to add to the one which was designed to make them 3 primer sets.Te sequences of the 2 sets of primers selected from the literature are shown in Table 3. Te selected sets of primers were AlkB-f and AlkB-r degenerate and Alk-BFB and Alk-BRB.

Amplifcation of the alkB Gene by PCR in Klebsiella oxytoca
Strains. Figure 4 shows the results of the PCR amplifcation of the three Klebsiella oxytoca strains with AlkB-f and AlkB-r primers on 1.5% agarose gel.Amplifcation was observed in lane 6 as shown by a band on the gel at approximately 550 base pairs, an expected product size of the alkB gene with AlkB-f and AlkB-r primers.Lane 6 contained sample 3.However, no bands were observed in other lanes (2)(3)(4)(5) confrming no amplifcation of the gene in samples 1 and 2. Sample 3 had already been identifed as Klebsiella oxytoca ATCC 13182 by amplifcation of 16S using PCR.
Figure 5 shows the result of the PCR amplifcation of the three Klebsiella oxytoca strains with Alk-BFB and Alk-BRB degenerate primers and Deg-f and Deg-r primers on 1.5% agarose gel.Tese primers could not amplify the gene since no bands were observed in the lanes.Te gels show primer dimers only which are just above the 100 base pair mark and no band at the expected level on the gel.
Tus, results from amplifcation of the alkB gene by PCR showed that only sample 3 which had been identifed as Klebsiella oxytoca ATCC 13182 strain contained this alkB gene.6 Te Scientifc World Journal

Sequencing of the Gene and Sequence Analysis
7.1.Gene Sequencing.Te PCR product from the Klebsiella oxytoca ATCC 13182 strain was sequenced to confrm if the amplifed gene was the expected alkB gene.

Sequence Analysis.
Te query nucleotide sequence, which is the alkB sequence in Klebsiella oxytoca, was then blasted against nucleotide sequences in GenBank in search for homology, and the results obtained from NCBI are shown in Table 4. Te frst column of the table shows the sequence accession number for the whole genome of the Te Scientifc World Journal bacteria in the alignment, the third column shows the percentage identity of the query sequence, and the last column shows a link to the exact region within the genome of the bacteria that was involved in the alignment which is the alkB gene in all ten genomes.Te alkB sequence in Klebsiella oxytoca ATCC strain was found to have more than 95% similarity with the alkB gene sequences in these 10 bacteria species.
For phylogenetic analysis, a phylogenetic tree was constructed using the maximum likelihood method and Tamura-Nei model.Figure 6 shows the constructed phylogenetic tree.
Te novel sequence was translated and BLASTp analysis was conducted on the NCBI database.High similarity was observed with alkB genes from other bacterial species.A novel nucleotide sequence was published on the NCBI database under accession number OP959069.

Discussion
Many studies have shown that the tendency of insects like T. molitor to consume plastic may be linked to the digestive activity of intestinal microorganisms, which aids in the digestion process [10].To see if the mealworms' proclivity to eat plastic was related to gut microorganisms, the gut cell suspension was cultured on a polystyrene emulsion, which provided the only carbon source for the microorganisms present.Because the polystyrene emulsion provided the nutrients, only microorganisms capable of feeding on polystyrene and thus responsible for polystyrene degradation were able to grow on the culture plates.Te microorganisms isolated from the guts of Tenebrio molitor were confrmed to be Klebsiella oxytoca strains which have previously been isolated using the polymerase chain reaction (PCR) method [6].Primers that amplify the 16S ribosomal RNA region in each of the isolates were designed to confrm that the bacteria were of Klebsiella oxytoca strains Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca NBRC 102593 using the provided sequences [6].
To determine the presence of the gene (alkB) responsible for plastic degradation, degenerate primers that amplify the alkB gene in bacterial isolates from mealworm guts were designed manually by selecting eleven alkB gene sequences from eleven diferent bacterial strains from GenBank.TCOFFE was used to align the sequences, while Jalview, an ofine tool, and ConSurf, an online tool, were used to view the alignment.After aligning eleven alkB gene sequences with TCOFFE, the conserved regions were discovered.Although 99% of the bases in the conserved regions aligned well, some sequences within the conserved regions contained single-nucleotide polymorphisms.Tese SNPs resulted in degenerate bases in the primer sequences.Because of the degenerate bases, the manufactured primers had a melting temperature range rather than a specifc temperature as with specifc primers and this resulted in the application of touchdown PCR for the amplifcation of the gene.
To increase the chances of amplifying the alkB gene in the three isolates, two more primer sets were selected from the literature to add to the one which was designed to make them 3 primer sets [9].Tese two selected primer sets were successful in amplifying the alkB gene in 49% of bacteria.Te selected primers were AlkB-f and AlkB-r and Alk-BFB and Alk-BRB.
Diferent primers were selected because it has been reported that several bacteria have multiple alkane hydroxylases that have been shown to potentially broaden the host strain's n-alkane range [11,12].For example, the coexistence of AlkB and CYP153 was discovered in Dietzia sp.DQ12-45-1b, as well as the coexistence of multiple alkane hydroxylases in Amycolicicoccus subfavus DQS3-9A1T13 [13].Some Rhodococcus strains were found to have multiple alkB homologous genes with varying substrate ranges and induction styles.Distribution of the alkB and CYP153 genes in the genomes of all the bacteria that had been deposited on GenBank has been reported.Multiple copies of alkB and CYP153 homologous genes were found in 20% and 36% of the alkB-containing genomes and CYP153-containing genomes, respectively.In Rhodococcus erythropolis SK121 and Parvibaculum lavamentivorans DS-1, up to six alkB homologous genes and fve CYP153 homologous genes were discovered.Te sequence similarities between these multiple copies of genes within one genome ranged from 27.7% to 99.7% for alkB genes and 42.4% to 100% for CYP153 genes [14].
Te results from the gel in Figure 6 show that sample 3 had the alkB gene since a band was observed at 550 bp which was the expected product size with specifc primers used.Te other samples 1 and 2 did not show a band of amplifcation.Tis however does not necessarily mean that these bacteria do not have the alkB gene as it has been reported that a single bacterial strain can contain up to six copies of a single gene with sequence similarity as low as 28% [14].Tus, there is a 50% chance that the gene is present, but maybe the available copy could not be amplifed with this primer set used.A phylogenetic tree was constructed using the maximum likelihood method and Tamura-Nei model in MEGA X. Te results show that the alkB gene from Klebsiella oxytoca ATCC 13182 strain and all the bacteria in the tree share one common ancestor, but this is more closely related to the gene in Acinetobacter.Te gene was expected to align with the alkB gene in Klebsiella strains because of the involvement of Klebsiella oxytoca in this study, but it only aligned closely with the alkB gene in Acinetobacter and other bacteria.Tis implies that no work has been done on this gene in Klebsiella species, resulting in the gene's deposition in databases such as GenBank.Te query sequence only aligned with one alkane-1-monooxygenase gene, and the rest of the alignments were between the gene and a region in the bacteria's whole genome.Tis could also be explained by the lack of research on this gene in bacteria.
In addition, the use of degenerate primers can result in negative results.Degenerate primers can make optimizing PCR assays difcult because only a limited number of primer molecules in a degenerate primer mixture are complementary to the template; the melting temperature (Tm) of primer sequences can vary signifcantly; and the sequences of some primers can be complementary to those of others.All these issues can cause the PCR to fail, leading to the conclusion that the gene is not present when it is.A set of degenerate primers was used to amplify the alkB gene in three Gordonia species, and negative results were obtained until specifc primers were designed and used in the PCR reaction, after which the results were positive [15].Te other Klebsiella oxytoca strains in this study lacked the alkB gene, as evidenced by a negative PCR.However, one can only be certain of this conclusion after using specifc primers [16].

Conclusions
Te work in this study outlines the discovery of the alkb gene from a Klebsiella oxytoca strain that was isolated from the gut of the mealworms of T. molitor which can feed and survive sorely on polystyrene.Te polystyrene-degrading ability of the mealworms of T. molitor is possibly linked to bacteria of Klebsiella oxytoca strains that are found in the guts of the mealworms.Since the alkB gene has been linked to the degradation of polystyrene by Klebsiella oxytoca strains, the results provided in this study paves a way for further studies to investigate the mutual relationship between Klebsiella oxytoca strains and T. molitor in the degradation of plastic and using it as a source of carbon.Alkane-1-monooxygenase enzyme may be one of the main enzymes responsible for the degradation of polystyrene by Klebsiella oxytoca ATCC 13182 strain in T. molitor.9.1.Recommendations.Te authors recommend that activity tests for alkB enzyme be carried out to categorically say that the alkB enzyme is a key enzyme in the degradation of polystyrene.

Figure 1 :
Figure1: Overnight cultures of the bacteria isolated from the midgut of the T. molitor worms.A is the control which is the culture medium only, and B is the isolated bacteria on the culture media, and 1, 2, and 3 are the pure colonies obtained after the quadrant streak method.

Figure 2 :Figure 3 :
Figure2: Amplifcation of the three Klebsiella oxytoca strains with JCM-specifc primers on 1.5% agarose gel.Lane 1:100 bp ladder, lane 2: sample 1 which was identifed as Klebsiella oxytoca JCM 1665, lane 3: positive control which is the master mix with 16S ribosomal RNA primers plus the DNA template, lane 4: negative control which is the PCR master mix without the DNA template, and lanes 5 and 6 had samples 2 and 3, respectively.

Table 1 :
DNA sequences that were used to design 16S ribosomal RNA gene-specifc primers for the 3 bacterial strain.

Table 2 :
Te species name and strain and the accession numbers of the alkB gene sequences used for the alignment.
Gene.To detect the presence of the alkB gene in the bacteria of Klebsiella strains, touchdown colony PCR was performed on all the samples against each of the 3 primer sets, Alkb-f and Alkb-r, Alk-BFB and Alk-BRB, and the designed Deg-f and Deg-r.Cycling conditions for Deg-f and Deg-r were as follows: one cycle of 95 °C for 10 minutes, 20 cycles of 95 °C for 2 minutes, 55 °C for 1 minute (annealing), and 72 °C for 30 seconds, reducing the annealing temperature by 0.5 °C each cycle, and 20 cycles of 95 °C for 2 minutes, 45 °C for 1 minute (annealing) and 72 °Cfor 30 seconds, and fnal extension at 72 °C for 10 minutes and fnal hold at 4 °C∞.For Alkb-f and Alkb-r primer sets, the cycling conditions were as follows: one cycle of 95 °C for 10 minutes, 20 cycles of 95 °C for 2 minutes (denaturation), 65 °C for 1 minute (annealing), and 72 °C for 30 seconds (extension), reducing the annealing temperature by 0.5 °C for 10 minutes and fnal hold at 4 °C∞.

Table 3 :
Forward and the reverse primer sequences designed to amplify the 16S ribosomal RNA of the Klebsiella oxytoca NBRC 102593 strain, Klebsiella oxytoca JCM 1665 strain, and Klebsiella oxytoca ATCC 13182 strain and alkB gene in Klebsiella oxytoca strains.

Table 4 :
Similarity of the alkB sequence in Klebsiella oxytoca ATCC with the alkB gene sequences in 10 selected bacteria.