Tenebrio molitor and its gut bacteria growth in polystyrene ( PS ) presence as the sole source carbon

Although polystyrene (PS) is considered a non-biodegradable material, recent work has shown the degradation capacity of this material by microorganisms, especially those that are part of the natural microbiota of the digestive tract of some invertebrates. The present work sought to evaluate the growth of the larva of the mealworm (Tenebrio molitor) and its bacteria, using PS as the sole source of carbon. In this way it was possible to demonstrate the consumption of PS plates by the larva, found in holes and tunnels in the material, however, nutritionally it is not enough for the larva to gain biomass, notably reducing its size and time survival. Similarly, bacteria isolated from the digestive tract of T. molitor presented the ability to generate biofilms o n P S s heets, g enerating changes ( cracks, holes, etc.) in them, which were observed under scanning electron microscopy (SEM), indicating the possible use of this material as a carbon source for its growth.


Introduction
Plastic is one of the most used materials in modern human societies. A derivative of oil, plastic has a light weight and is resistant, flexible, and relatively inert. These features make it ideal as a raw material for bags, packaging, construction material, thermal and electrical insulation products, etc. Annually, 250 million tons of plastic are produced in the world and 40 % of which is destined to the production of packaging (Cardoso et al., 2017). However, only 9.5 % is recycled or reused. The remaining reaches landfills and dumps (Zalasiewicz et al., 2016). Most of the massively consumed plastic serves its purpose in a short time and, depending on the complexity of the material, its degradation time can be short or long. This leads to an accumulation of plastic in aquatic systems and soils that negatively impact the biosphere and generate foci of contamination leading to public health issues (Barnes et al., 2009). Polystyrene (PS) is a plastic that, given its versatility and low cost, has been used in a wide range of consumer products, such as insulation materials, food packaging, cutlery and kitchen utensils, among others. In Colombia, PS is produced at a capacity of 110 000 tons per year and is the third most-produced petroleum-derived plastic, after polyvinyl chloride and propylene polymers (Acoplásticos, 2017). The expanded form of polystyrene is perhaps the most produced PS type thanks to its low density, hygroscopic properties, and innocuousness, making it a sought-after supply for the production of packaging items and construction and insulation materials (Ferrándiz-Mas & García-Alcocel, 2013). However, these characteristics also make it a highly resistant material to microbial degradation.
Taking into account the complexity of this material and its accumulation in ecosystems, methods of thermal decomposition have been proposed. However, these techniques, besides being expensive, generate large quantities of dioxins and aromatic compounds that magnify the damage to the environment (Desmet et al., 2005;Moqadam et al., 2015). Different studies have focused on the biological degradation of plastics, reporting on the capacity of soil invertebrates to use these materials as food, observing partial or complete transformation (Bombelli et al., 2017;Yang et al., 2015a). Similarly, the ability of various microorganisms to use plastic as the sole source of carbon has been evaluated. Such is the case of Orr et al., (2004), who evaluated the degradation capacity of polyethylene by a strain of the fungus Rhodococcus ruber, observing an 8 % biodegradation of this material. Likewise, Velasco et al., (1998) studied a molecular biology of Pseudomonas sp. and the the enzymes involved in the transformation of styrene to phenylacetate. Similarly, Yang et al., (2015b) established that the bacterium Exiguobacterium sp. from the digestive tract of the mealworm, or Tenebrio molitor beetle larvae, has the ability to degrade PS.
In the present study, we build on the body of work on PS degradation by the gut bacteria of T. molitor larvae. We sought to test the hypothesis the that the gut bacteria of T. molitor larvae have the ability to use PS as sole source of carbon, as has been reported in other studies, however, this ability does not varies in relation to geographic location were mealworm is studied.Specifically, we evaluated the growth capacity of T. molitor and the bacteria isolated from its digestive tract in the presence of PS as the sole carbon source, to establish the role of these microbiota in the utilization of the PS and to evaluate whether this capacity is generalized for all the species, regardless of the geographic location of the organism.

Materials and Methods
Environmental adaptation of the T. molitor larvae A total of 100 individual T. molitor larvae (14 days old) were used. Mealworms were collected in the locality of in Nilo, department of Cundinamarca (Colombia) (altitude of 336 m.a.s.l. and annual average temperature of 26.5 • C) and transported alive in a cardboard box covered with insulating materials to facilitate the mealworms acclimation to temperature and weather conditions in the city of Bogota (altitude of 2630 m.a.s.l. and annual average temperature of 14 • C), where the experiments were carried out. Once in the laboratory, the mealworms were left to acclimate to the new environmental conditions for one week. Then, they were placed in a glass container of 13×15×15cm (H × L × W) with a supply of 500 g of wheat flour as a source of food. During this period, room temperature was recorded daily (23 • C -26 • C) and light was kept at a minimum due to the mealworms photophobic behavior.

Evaluation of PS intake
After the acclimation week, a total of 60 T. molitor larvae were separated in groups of 15 individuals and deposited in four glass containers of 10x10x15 cm (H x L x W). The acclimation conditions, previously described, were maintained for the first group hence becoming the control group; whereas the other three groups were fed with and an expanded PS plate of 5x9x1 cm of known weight (1.5 g). During 4 months and on a biweekly basis, the change in mass of control and treatment larvae were recorded with an analytical balance. The mass of the PS plates in the treatment groups was likewise assessed. In parallel, one PS plate of 4x7x1 cm (1.0 g) was placed in fith glass container to track the effect of the environmental conditions on the mass of the PS plate.
The mealworms that were not used in the experiments were kept until reaching their imago or adult stage, and were then used to evaluate their capacity to ingest PS as a food source. To do this, a total of 15 T. molitor adults were placed in contact with a PS plate of 5x9x1 cm (1.5 g) which was visually inspected for two weeks to find changes in its surface that were signs of ingestion by the beetles.

Isolation and identification of the T. molitor gut bacteria
After the fourth month, the digestive tracts of the T. molitor larvae, fed on PS plates, were excised under a stereoscope and sterile conditions with the help of a scalpel. The obtained digestive tract contents were subjected to serial dilutions up to 10 −7 , and the last three dilutions were sown on nutritive agar (agar-agar 15.0 g/l, beef extract 3.0 g/l, and gelatin peptone 5.0 g/l). These sowings were incubated at (30 • C) , in the presence and absence of oxygen, to promote the growth of aerobic and anaerobic bacteria. To obtain pure cultures from each strain, multiple peals were made on a Petri dish until a single colonial morphology was observed.
Subsequently, each bacterial isolate was coded and biochemically characterized through TSI, MRVP, Citrate, Urease, Catalase, Oxidase, and motility tests as well as Gram staining. Additional isolate identification was performed with molecular barcoding via 16S rRNA sequencing and subsequent comparison of the obtained sequences with databases such as RDP, Greengenes, and NCBI RefSeq / RNA. All bacterial isolates were tested for growth on PS (see the following section) and cryopreserved for future research. Adult T. molitor microbiota was isolated, grown, and tested following the same procedure above.
Evaluation of the growth of bacteria on PS as the sole carbon source To evaluate the growth of mealworm gut bacteria isolates on PS, 0.1 ml of each resuspended isolate were cultured at 30 • C for 3 weeks in plates containing minimal salt medium (K H 2P O4 0.7 g/l, K2H P O4 0.7 g/l, M g SO4*7H 2O 0.7 g/l, N H 4N O3 1.0 g/l, NaCl 0.005 g/l, F e SO4*7H 2O 0.002 g/l, Z nSO4*7H 2O 0.002 g/l, M nSO4*H 2O 0.001 g/l), agar (15 g/l), and PS sheets. Each PS sheet was prepared from 2.3 g of expanded PS dissolved in a benzene solution and recovered after solvent evaporation. During these 3 weeks, the formation of colonies on the agar and PS sheet surfaces was monitored. The plates showing colonial growth were observed under a scanning electron microscope (SEM) at magnifications of 17500X , 18000X , 22000X , 26500X , 29000X and 31000X to evaluate changes in the material imputable to microbial growth.

Results and Discussion
PS intake by T. molitor larvae All glass containers with T. molitor larvae and PS plates had signs of PS consumption. The PS plates exhibited holes and tunnels, where the T. molitor larvae were found (Fig. 1A). Also, the mass of the plates decreased in the presence of the larvae (Fig. 2). In contrast, in the container without larvae, the PS plate did not reveal any changes in its morphology or weight (Fig. 1B). Mealworms fed on wheat flour steadily gained biomass throughout the experiment. Whereas those fed only on PS tended to maintain or decrease their biomass despite evidence of plate consumption (Fig. 2). Our observations indicate that T. molitor larvae were capable of consuming PS, generating distinguishable changes in the plates. However, PS was of little nutritional value to these larvae as indicated by the presence of smaller-size surviving individuals at the end of the experiment. Since PS is not composed of organic molecules, it lacks the carbohydrates and proteins, otherwise found in wheat flour, necessary for larval growth (Catalán et al., 2011). Yang et al., (2018) observed a biomass loss in T. molitor larvae fed on PS with a survival rate of 85 % after 32 days of incubation. These values are similar to those found in the present work, with a 100 % survival rate observed at day 60 ( Fig. 2). Allegedly, mealworms are capable of consuming PS, managing to survive for a while. Yet, PS alone does not provide the necessary energy for biomass generation because the surviving larvae lost weight and shrunk. been affected because of cannibalism. The complete or partial disappearance of mealworm bodies is a likely sign of cannibalism. This behavior can be triggered when the relative humidity or moisture content of foods is low (Ichikawa & Kurauchi, 2009). Since PS is a hygroscopic material, it may not help meet mealworm water needs.
T. molitor larvae have gained the attention of the scientific community due to their capacity to consume PS. However, it is not yet clear whether adults retain this capacity. In this work, T. molitor adults were allowed to feed on PS plates only, but after 15 days of incubation, none of the PS plate surfaces exhibited significant changes. The apparent inability of adults to feed on PS could be attributed to larval-to-adult jaw structure changes (Wilson, 1971), preventing adults from breaking and ingesting PS. Alternatively, changes in intestinal microbiota composition through the life stages of T. molitor (Wynants et al., 2017) may lead to the adult's inability to process and transform PS.

Larval and adult T. molitor gut bacteria
Nine morphologically different bacterial strains were isolated from T. molitor digestive tracts. Five, of these isolates, were obtained from larvae and the remaining four from adults. Each isolate received a numerical code preceded by the letters G (for larval origin) or E (for adult origin). Table 1. shows the morphological and biochemical characteristics of the nine isolates. All of the isolates had bacillary form, and five of them were Gram-negative. In general, the isolates were facultative, fermenting bacteria, with negative mobility and urease test results. Furthermore, the sequencing of their respective 16S ribosomal RNA regions revealed that two of the strains from larval guts were members of the genus Bacillus, whereas the other three larval gut isolates belonged to the genus Stenotrophomonas. Similarly, two of the bacterial isolates from adult guts belonged to the genus Bacillus, and the remaining two, to the genera Pantoea and Erwinia (Table 1). The observed differences in the bacteria inhabiting the digestive tracts of larval and adult T. molitor could be related to the loss of the ability of the adult to use PS as a source of food. Yang et al., (2015b) were the first to identify the bacteria in the digestive tract of T. molitor larvae involved in the transformation of PS. The authors obtained a total of 13 bacterial strains, chiefly Enterobacteriaceae, and determined that a member of the genus Exiguobacterium was the main responsible for the degradation of PS. In the present study, however, the most common isolates belonged to the genus Bacillus. This contrast highlights the effect of the diet on the composition of gut microbiota in insects of the same species. In the previously mentioned study, the acclimation diet of the mealworms consisted of oat flakes, whereas in the present work, it was wheat Gram-positive bacterium, lactose or sucrose fermenter with gas production. Positive indole or catalase, with motility.

3G
Cells with bacillary morphology that generate white colonies, punctate with full edge.
Gram-negative bacterium, fermenter of the 3 sugars with gas production. Positive citrate and catalase with motility.

5G
Cells with bacillary morphology that generate yellow colonies, punctate with full edge.
Gram-negative bacterium, fermenter of the 3 sugars with gas production. Positive VP and oxidase with motility.

8G
Cells with bacillary morphology that generate white colonies, circular with full edge Gram-positive bacterium, glucose or sucrose fermenter with no gas production. Positive catalase and oxidase with motility.

10G
Cells with bacillary morphology that generate white colonies, punctate with full edge.
Gram-negative bacterium, non-glucose fermenter, positive citrate and catalase with motility.

11E
Cells with bacillary morphology that generate yellow colonies, punctate with full edge.
Gram-negative bacterium, lactose fermenter or sucrose with no gas production. Positive VP, citrate and catalase with motility.

12E
Cells with bacillary morphology that generate white colonies, circular with wavy edge.
Gram-negative bacterium, lactose or sucrose fermenter with no gas production, with motility.

13E
Cells with bacillary morphology that generate white colonies, circular with wavy edge.
Gram-positive bacterium, glucose or sucrose fermenter with no gas production. Positive catalase with motility.

14E
Cells with bacillary morphology that generate white colonies, circular with wavy edge.

Bacilus anthracis
Bacterial growth on PS as the sole carbon source Fig. 3 shows the growth of one of the isolates on PS, as the sole carbon source. The isolates formed colonies around, above, or below the PS sheets. Turbidity and darkening of the mineral medium were also signs of bacterial growth. The observed morphologies differed from those expressed by the strains isolated in nutritive agar. Isolates, revealing growth on PS sheets, were observed under a scanning electron microscope (SEM) (Fig. 4). Three of these isolates, namely  Bacillus anthracis, Bacillus sp, and Stenotrophomonas sp. formed tunnels, cracks, and cellular aggregates on the PS sheets. None of these changes were observed in the control sample (PS sheet without microorganism). Multiple SEM-assisted works have documented bacterial-driven transformations of PS and other plastics (Ho et al., 2018;Sekhar et al., 2016;Skariyachan et al., 2016;Yang et al., 2015a;Yang et al., 2015b;Yoshida et al., 2016). These transformations start with biofilm generation, on the material's surface, followed by material deterioration because of the enzymatic activity of the microorganisms.
The metabolic pathways involved in the transformation of PS are still unclear. Nevertheless, the oxidation of styrene to phenylacetate, via the citric acid cycle, is common among different microorganisms (Ho et al., 2018). Bacteria of genus Bacillus stand out for their capacity to transform styrene to phenylacetate. Bacillus monocultures or consortia have been studied in different PS degradation experiments (Asmita et al., 2015;Atiq et al., 2010;Kiatkamjornwong et al., 1999). Our results highlight the importance of Bacillus in the degradation of PS. Taking into account that four out of nine isolates obtained from the intestinal tract of T. molitor belonged to the genus Bacillus and two of them elicited signs of PS degradation, namely forming biofilms on the material and triggering its wearing.
Finally, although PS sheet wearing was not evaluated with bacteria isolated from adult T. molitor digestive tracts, there is evidence that bacteria of the genus Pantoea, present in the adult gut isolates in our study, have the capacity to degradate plastics such as low-density polyethylene (Skariyachan et al., 2016). Additionally, taking into account that our T. molitor adults also have Bacillus in their intestinal flora, it is likely that T. molitor can digest PS. But, due to the structural changes in their jaws, it may not be possible for them to ingest it.

Conclusions
In light of our results, we can conclude that T. molitor larvae can use PS as food. Though, PS alone cannot meet larval nutritional needs. Larvae feeding only on PS for extended periods reduced their body size and commenced exhibiting cannibalistic behaviors. Furthermore, this use of PS by T. molitor larvae is conceivable due to the presence of bacteria in their digestive tract with the ability to grow with this polymer as their sole source of carbon. Likewise, the larval capacity seems not to reamin in adults, which could be attributed to larval-to-adult jaw changes, preventing from breaking and ingesting PS. Alternatively, changes in intestinal microbiota composition through the life stages of T. molitor may lead to the adult inability to process and trasnform PS.

Miguel A. Ballen-Segura
Ph.D. and MSc with emphasis in fundamental and applied ecology. currently works at the School of Exact Sciences and Engineering, Sergio Arboleda University as professor and researcher, developing projects related to use of microorganisms as remediation tools and their role in high mountain aquatic systems.

Paula M. Peña-Pascagaza
Environmental engineer with knowledge in bioprocesses, microbiology, geographic information systems, environmental study and monitoring. Successfully develop accompaniment and advice related to environmental areas such as the development of research and guidelines, management of relevant legislation, processing before competent environmental authorities and application of software. Highly effective skills such as adaptation, recursiveness, results orientation and analytical capacity.

Nathalia A. López-Ramírez
Is an enthusiastic and innovative Environmental Engineer who has experience in Health, Safety and Environment (HSE) aspects. She has developed post-consumption programmes for multinational companies and has knowledge about waste management, transport and handling of chemical substances and continuous improvement process.