Effects of high doses of zearalenone on some antioxidant enzymes and locomotion of Tenebrio molitor larvae (Coleoptera: Tenebrionidae).

The mealworm Tenebrio molitor L. (Coleoptera: Tenebrionidae) feeds on wheat bran and is considered both a pest and an edible insect. Its larvae contain proteins and essential amino acids, fats, and minerals, making them suitable for animal and human consumption. Zearalenone (ZEA) is the mycotoxin most commonly associated with Fusarium spp. It is found in cereals and cereal products, so their consumption is a major risk for mycotoxin contamination. One of the most important effects of ZEA is the induction of oxidative stress, which leads to physiological and behavioral changes. This study deals with the effects of high doses of ZEA (10 and 20 mg/kg) on survival, molting, growth, weight gain, activity of antioxidant enzymes superoxide dismutase (SOD) and glutathione S-transferase (GST), and locomotion of mealworm larvae. Both doses of ZEA were found to (i) have no effect on survival, (ii) increase molting frequency, SOD, and GST activity, and (iii) decrease body weight and locomotion, with more pronounced changes at 20 mg/kg. These results indicated the susceptibility of T. molitor larvae to high doses of ZEA in feed.

Long-lasting insecticidal activity in plants driven by chlorogenic acid-loaded metal-organic frameworks.

Metal-organic frameworks (MOFs) possess a variety of interesting features related to their composition and structure that make them excellent candidates to be used in agriculture. However, few studies have reported their use as delivery agents of agrochemicals. In this work, the natural polyphenol chlorogenic acid (CGA) was entrapped via simple impregnation in the titanium aminoterephthalate MOF, MIL-125-NH2. A combination of experimental and computational techniques was used to understand and quantify the encapsulated CGA in MIL-125-NH2. Subsequently, CGA delivery studies were carried out in water at different pHs, showing a fast release of CGA during the first 2 h (17.3 ± 0.3% at pH = 6.5). In vivo studies were also performed against larvae of mealworm (Tenebrio molitor), evidencing the long-lasting insecticidal activity of CGA@MIL-125-NH2. This report demonstrates the potential of MOFs in the efficient release of agrochemicals, and paves the way to their study against in vivo models.

Mammalian neurotoxins, Blarina paralytic peptides, cause hyperpolarization of human T-type Ca channel hCav3.2 activation.

Among the rare venomous mammals, the short-tailed shrew Blarina brevicauda has been suggested to produce potent neurotoxins in its saliva to effectively capture prey. Several kallikrein-like lethal proteases have been identified, but the active substances of B. brevicauda remained unclear. Here, we report Blarina paralytic peptides (BPPs) 1 and 2 isolated from its submaxillary glands. Synthetic BPP2 showed mealworm paralysis and a hyperpolarization shift (-11 mV) of a human T-type Ca2+ channel (hCav3.2) activation. The amino acid sequences of BPPs were similar to those of synenkephalins, which are precursors of brain opioid peptide hormones that are highly conserved among mammals. However, BPPs rather resembled centipede neurotoxic peptides SLPTXs in terms of disulfide bond connectivity and stereostructure. Our results suggested that the neurotoxin BPPs were the result of convergent evolution as homologs of nontoxic endogenous peptides that are widely conserved in mammals. This finding is of great interest from the viewpoint of the chemical evolution of vertebrate venoms.

Nano/microparticles in conjunction with microalgae extract as novel insecticides against Mealworm beetles, Tenebrio molitor.

The intensive use of insecticides in global agricultural production has attracted much attention due to its many adverse effects on human health and the environment. In recent years, the utilization of nanotechnology has emerged as a tool to overcome these adverse effects. The aim of this work was to test different microparticles (zinc oxide (ZnO MPs) and silicon dioxide microparticles (SiO2 MPs)), and silver nanoparticles (Ag NPs) and to study their toxicity on a model organism, Tenebrio molitor. A comprehensive comparative study, which included more than a thousand mealworms divided into nine separate groups, was conducted. In addition to pure nano/microparticle solutions, the effect of particles mixed with the microalgae extract Chlamydomonas reinhardtii was also observed. Pure Ag NPs and SiO2 MPs resulted in larval mortality of more than 70% compared to that of pure ZnO MPs, in which the mortality rate was approximately 33%. A mixture of the algal extract with zinc oxide microparticles resulted in mortality that was double compared to that observed with pure ZnO MPs. In parallel, atomic absorption spectrometry (AAS) was used to determine the difference in the concentration of trace elements in the bodies of dead and live larvae.

Metabolic fate and toxicity reduction of aflatoxin B1 after uptake by edible Tenebrio molitor larvae.

The use of insects as food and feed is gaining more attention for ecological and ethical reasons. Despite the high tolerance of edible yellow mealworm (Tenebrio molitor) larvae to aflatoxin B1 (AFB1), the metabolic fate of the toxin along with its toxic potential in the insect is uncertain. The present study aimed at investigating the AFB1 mass balance and the metabolite formation in a feeding trial with AFB1-contaminated grain flour. T. molitor larvae tolerated the AFB1 level of 10,700 µg/kg in the feed, however, weight gain was decreased by 15% over a 4-weeks feeding period. The investigation of the phase I metabolite pattern revealed the formation of AFM1 and a novel presumably monohydroxylated compound in larvae extracts that was not formed by reference incubation with rat, bovine or porcine liver microsomes. Mass balance quantification of ingested AFB1 revealed that 87% of the initial toxin remain undetected in larval body or residue. Analysis of histone H2Ax phosphorylation in human liver cells as a surrogate for genotoxicity showed that extracts from exposed larvae did not exhibit an elevated toxic potential. Although toxicological uncertainties remain due to the undetected transformation products, the resulting mutagenicity of the edible larvae appears to be low.

The impact of polystyrene consumption by edible insects Tenebrio molitor and Zophobas morio on their nutritional value, cytotoxicity, and oxidative stress parameters.

The objective of this study was to determine of nutritional value, in vitro cytotoxicity, and oxidative stress parameters in cells of selected insect species (Tenebrio molitor and Zophobas morio) after 30 days of Styrofoam consumption. Furthermore, part of our research is also a consumer survey on the willingness to eat insects fed with Styrofoam (EPS 80). Mealworms fed with Styrofoam were determined to have higher protein (48.66 ± 0.92%) and ash content (4.81 ± 0.22%) with reduced fat (24.05 ± 0.55%) and carbohydrate content (2.95 ± 0.15%) than insects with a conventional diet (48.66 ± 0.92, 2.82 ± 0.12, 43.74 ± 0.77, and 4.78 ± 0.18, respectively) while in the case of superworms, no significant difference in nutrient composition was observed. Moreover, Styrofoam has no influence on the health status of gut cells in examined insects. Additionally, in studied concentrations of insects extracts standardized for protein replacement of the traditional insect diet with polystyrene foam did not increase the cytotoxic properties.

Selenium accumulation in protein fractions of Tenebrio molitor larvae and the antioxidant and immunoregulatory activity of protein hydrolysates.

Although numerous types of organisms have been used to enrich selenium, a low-cost and efficient organism is yet to be identified. This study aimed to develop a new means of selenium enrichment using Tenebrio molitor larvae. Our results indicated that the total selenium content in larvae was increased 83-fold to 54.21 ± 1.25 µg/g, and of this content, organic selenium accounted for over 97% after feeding the larvae with 20 µg/g of sodium selenite. Selenium was distributed unequally in the protein fraction with following order: alkali-soluble protein-bound selenium (36.32%) > salt-soluble protein-bound selenium (19.41%) > water-soluble protein-bound selenium (17.03%) > alcohol-soluble protein-bound selenium (3.21%). Additionally, 81% of the selenium within the soluble proteins was distributed in subunits possessing molecular weights of <40 kDa. After hydrolysis by alcalase, the protein hydrolysate of selenium-enriched larvae possessing 75% selenium recovery exhibited stronger antioxidant and immunoregulatory activities than those of regular larvae.

Liquid Chromatography-Tandem Mass Spectrometry for the Simultaneous Analysis of 353 Pesticides in the Edible Insect Tenebrio molitor Larvae (Mealworms).

Tenebrio molitor larvae (mealworm) is an edible insect and is considered a future food. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a novel method for simultaneous analysis of 353 target analytes was developed and validated. Various sample preparation steps including "quick, easy, cheap, effective, rugged, and safe" (QuEChERS) extraction conditions, number of acetonitrile-hexane partitions, and dispersive-solid phase extraction (dSPE) sorbents were compared, and the optimal conditions were determined. In the established method, 5 g of homogenized mealworms was extracted with acetonitrile and treated with QuEChERS EN 15662 salts. The crude extract was subjected to three rounds of acetonitrile-hexane partitioning, and the acetonitrile layer was cleaned with C18 dSPE. The final solution was matrix-matched and injected into LC-MS/MS (2 µL). For target analytes, the limits of quantitation (LOQs) were ≤10 µg/kg, and the correlation coefficient (r2) of calibration was >0.990. In recovery tests, more than 90% of the pesticides showed an excellent recovery range (70-120%) with relative standard deviation (RSD) ≤20%. For more than 94% of pesticides, a negligible matrix effect (within ±20%) was observed. The analytical method was successfully applied and used for the detection of three urea pesticides in 4 of 11 mealworm samples.

Relative expression of microRNAs, apoptosis, and ultrastructure anomalies induced by gold nanoparticles in Trachyderma hispida (Coleoptera: Tenebrionidae).

The extensive use of nanomaterials generates toxic effects on non-target species and the ecosystem. Although gold nanoparticles (Au-NPs) are generally expected to be safe, the recent study contains conflicting data regarding their cytotoxicity in the darkling beetles Trachyderma hispida. The study postulated cellular perturbation in the ovarian tissue of the beetles induced by a sublethal dose of Au-NPs (0.01 mg/g). When compared with the controls, a significant inhibition in the activities of the antioxidant enzymes selenium-dependent (GPOX) and selenium-independent (GSTP) glutathione peroxidases (GPx) was observed in the treated beetles. The study proposed microRNAs (miRNA-282 and miRNA-989) as genotoxic markers for the first time, reporting a significant suppression in their transcriptional levels in the treated beetles. Furthermore, TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and flow cytometry assays (annexin V-Fitc) indicated a significant increase in ovarian cell apoptosis in the treated beetles. Additionally, an ultrastructure examination revealed pathological changes in the ovarian cells of the treated beetles. The resulting anomalies in the present study may interrupt the fecundity of the beetles and lead to the future suppression of beetle populations.

Dietary zinc enrichment reduces the cadmium burden of mealworm beetle (Tenebrio molitor) larvae.

The industrial production of Tenebrio molitor L. requires optimized rearing and processing conditions to generate insect biomass with high nutritional value in large quantities. One of the problems arising from processing is a tremendous loss in mineral accessibility, affecting, amongst others, the essential trace element Zn. As a feasible strategy this study investigates Zn-enrichment of mealworms during rearing to meet the nutritional requirements for humans and animals. Following feeding ZnSO4-spiked wheat bran substrates late instar mealworm larvae were evaluated for essential micronutrients and human/animal toxic elements. In addition, growth rate and viability were assessed to select optimal conditions for future mass-rearing. Zn-feeding dose-dependently raised the total Zn content, yet the Znlarvae/Znwheat bran ratio decreased inversely related to its concentration, indicating an active Zn homeostasis within the mealworms. The Cu status remained stable, suggesting that, in contrast to mammals, the intestinal Cu absorption in mealworm larvae is not affected by Zn. Zn biofortification led to a moderate Fe and Mn reduction in mealworms, a problem that certainly can be overcome by Fe/Mn co-supplementation during rearing. Most importantly, Zn feeding massively reduced the levels of the human/animal toxicant Cd within the mealworm larvae, a technological novelty of outstanding importance to be implemented in the future production process to ensure the consumer safety of this edible insect species.

Solanum Nigrum Fruit Extract Increases Toxicity of Fenitrothion-A Synthetic Insecticide, in the Mealworm Beetle Tenebrio Molitor Larvae.

Synthetic insecticides are widely used for crop protection both in the fields and in the food stored facilities. Due to their toxicity, and assumptions of Integrated Pest Management, we conducted two independent experiments, where we studied the influence of Solanum nigrum unripe fruit extract on the toxicity of an organophosphorus insecticide fenitrothion. In the first variant of the experiment, Tenebrio molitor larvae were fed with blended fenitrothion (LC50) and the extract in four concentrations (0.01, 0.1, 1 and 10%) in ratio 1:1 for 3 days. In the second variant, a two-day application of fenitrothion (LC40) was preceded by a one-day extract treatment. The first variant did not show any increase in lethality compared to fenitrothion; however, ultrastructure observations exhibited swollen endoplasmic reticulum (ER) membranes in the midgut and nuclear and cellular membranes in the fat body, after application of blended fenitrothion and extract. An increased amount of heterochromatin in the fat body was observed, too. In the second variant, pre-treatment of the extract increased the lethality of larvae, decreased the level of glycogen and lipids in the fat body and disrupted integrity of midgut cellular membranes. S. nigrum extract, applied prior to fenitrothion treatment can be a factor increasing fenitrothion toxicity in T. molitor larvae. Thus, this strategy may lead to decreased emission of synthetic insecticides to the environment.

Acetamiprid Affects Destruxins Production but Its Accumulation in Metarhizium sp. Spores Increases Infection Ability of Fungi.

Metarhizium sp. are entomopathogenic fungi that inhabit the soil environment. Together, they act as natural pest control factors. In the natural environment, they come into contact with various anthropogenic pollutants, and sometimes, they are used together and interchangeably with chemical insecticides (e.g., neonicotinoids) for pest control. In most cases, the compatibility of entomopathogens with insecticides has been determined; however, the influence of these compounds on the metabolism of entomopathogenic fungi has not yet been studied. Secondary metabolites are very important factors that influence the fitness of the producers, playing important roles in the ability of these pathogens to successfully parasitize insects. In this study, for the first time, we focus on whether the insecticide present in the fungal growth environment affects secondary metabolism in fungi. The research revealed that acetamiprid at concentrations from 5 to 50 mg L-1 did not inhibit the growth of all tested Metarhizium sp.; however, it reduced the level of 19 produced destruxins in direct proportion to the dosage used. Furthermore, it was shown that acetamiprid accumulates not only in plant or animal tissues, but also in fungal cells. Despite the negative impact of acetamiprid on secondary metabolism, it was proofed to accumulate in Metarhizium spores, which appeared to have a stronger infectious potential against mealworm Tenebrio molitor, in comparison to the insecticide or the biological agent alone.

Development and Biomass Composition of Ephestia kuehniella (Lepidoptera: Pyralidae), Tenebrio molitor (Coleoptera: Tenebrionidae), and Hermetia illucens (Diptera: Stratiomyidae) Reared on Different Byproducts of the Agri-Food Industry.

The aim of this study was to evaluate five agro-industrial byproducts (apricots, brewer's spent grains, brewer's spent yeast, feed mill byproducts including broken cereal grains, and hatchery waste including eggshell debris, fluff, infertile eggs, dead embryos, and egg fluids) or mixtures thereof as food diets of Ephestia kuehniella (Zeller), Tenebrio molitor (L.), and Hermetia illucens (L.). Eleven out of 26 tested combinations allowed the first instar larvae to reach the adult stage. Results showed that bioconversion parameters and biomass composition can vary depending on the diet composition, especially in the case of E. kuehniella and H. illucens, whose nutritional requirements seem more complex than those of T. molitor. Tenebrio molitor was able to develop in almost all byproducts. However, only when T. molitor was fed with suitable mixtures of byproducts the development parameters were similar to those obtained with the standard diet. The best results in terms of bioconversion parameters were obtained by feeding H. illucens with a diet including dried brewer's spent grain, feed mill byproducts and brewer's spent yeast. The larvae of these three species can be considered interesting from a nutritional point of view, because of their high protein and fat content. However, the fatty acids profile of H. illucens larvae, with high proportions of saturated fatty acids, seems less healthy for human consumption compared with those of E. kuehniella and T. molitor.

Individual variation in cardiotoxicity of parotoid secretion of the common toad, Bufo bufo, depends on body size - first results.

Anurans secrete a wide diversity of toxins from skin glands to defend themselves against predators and pathogens. Bufonids produce potent poison in parotoid macroglands located in the postorbital region. Parotoid secretion is a rich source of bioactive compounds with cardiotoxic, cytotoxic and hemolytic activity. Poison content and toxicity may vary between species, populations, and among conspecifics inhabiting the same area. In the present paper, we pre-analyzed the individual variation in cardiotoxicity of parotoid extract of common toads (Bufo bufo Linnaeus, 1758) and impact of body mass (BM), snout to vent length (SVL), and body condition (BC) of toad on the poison toxicity. We hypothesized that large toads produce poison with higher cardiotoxicity than smaller ones. Parotoid extract was fractionated by reverse phase chromatography, and then in vitro physiological bioassays were carried out on the semi-isolated hearts of the mealworm beetle (Tenebrio molitor Linnaeus, 1758) to determine cardiotoxicity of the whole poison and separated fractions. Generalized linear mixed models were used to determine effects of BM, SVL, and BC on the poison toxicity. We recorded significant changes in the insect heart contractility after treatment with the whole poison and separated fractions. We found an individual variation in cardiotoxicity of the parotoid extract which was explained by the body size of toad. Poison of smaller toads displayed a negative, whereas poison of larger toads positive, chronotropic effect on the heart contractility. Thus, we conclude that the effectiveness of parotoid secretion in repelling predators may vary depending on the toad individual size.

Synthesis and Studies of the Inhibitory Effect of Hydroxylated Phenylpropanoids and Biphenols Derivatives on Tyrosinase and Laccase Enzymes.

The impaired activity of tyrosinase and laccase can provoke serious concerns in the life cycles of mammals, insects and microorganisms. Investigation of inhibitors of these two enzymes may lead to the discovery of whitening agents, medicinal products, anti-browning substances and compounds for controlling harmful insects and bacteria. A small collection of novel reversible tyrosinase and laccase inhibitors with a phenylpropanoid and hydroxylated biphenyl core was prepared using naturally occurring compounds and their activity was measured by spectrophotometric and electrochemical assays. Biosensors based on tyrosinase and laccase enzymes were constructed and used to detect the type of protein-ligand interaction and half maximal inhibitory concentration (IC50). Most of the inhibitors showed an IC50 in a range of 20-423 nM for tyrosinase and 23-2619 nM for laccase. Due to the safety concerns of conventional tyrosinase and laccase inhibitors, the viability of the new compounds was assayed on PC12 cells, four of which showed a viability of roughly 80% at 40 µM. In silico studies on the crystal structure of laccase enzyme identified a hydroxylated biphenyl bearing a prenylated chain as the lead structure, which activated strong and effective interactions at the active site of the enzyme. These data were confirmed by in vivo experiments performed on the insect model Tenebrio molitur.

Anticryptococcal activity of hexane extract from Spondias tuberosa Arruda and associated cellular events.

Cryptococcosis is an opportunistic systemic mycosis whose treatment is limited to three drugs. In this work, we evaluated the antifungal activity of a hexane extract (HE) from Spondias tuberosa leaves against Cryptococcus neoformans and Cryptococcus gattii. Minimal inhibitory concentrations (MIC) were determined, and putative mechanisms were evaluated by flow cytometry. In addition, an in vivo infection assay was performed using Tenebrio molitor larvae. Treatment with HE inhibited the growth of standard and clinical isolates of C. neoformans and C. gattii (MICs ranging from 0.78 to 3.12mg/mL), significantly (P<0.05) increased mitochondrial superoxide anion levels, and induced mitochondrial membrane depolarization, loss of lysosomal membrane integrity, and phosphatidylserine externalization. The mean survival time of C. gattii-infected T. molitor larvae significantly (P<0.05) increased from 1.225 days in control to 3.067 and 3.882 days in HE-treated groups (78 and 156mg/kg, respectively). In conclusion, HE showed anticryptococcal activity, induced mitochondrial and lysosomal damage in yeast cells, and exhibited anti-infective action against C. gattii in T. molitor larvae.

SPME-GC/MS Analysis of Volatile Compounds Contained in the Insect Larvae of Tenebrio molitor and Leptinotarsa decemlineata before and after Using Insecticides.

The larvae of two harmful insect species Tenebrio molitor and Leptinotarsa decemlineata were analyzed. The insects were sprayed with insecticides containing the active substances cyfluthrin and deltamethrin (T. molitor), and thiamethoxam and acetamiprid (L. decemlineata). The sprayed insect larvae were left for 24, 48 and 72 h. Samples were then prepared using SPME fiber to identify the volatile compounds contained in the larvae. The determinations were made by gas chromatography coupled with mass spectrometry (GC/MS). Chemical compounds were found in the analyzed samples: alkanes, aldehydes, ketones, esters, terpenes, fatty acids. In the samples prepared from T. molitor larvae, more than 20 % of aldehydes and 8-41 % of alkanes were determined. In the samples from L. decemlineata, fatty acids were 8-65 % depending on the length of time after applying the insecticide.

Timing of sub-lethal insecticide exposure determines parasite establishment success in an insect-helminth model.

Environmental toxicants are pervasive in nature, but sub-lethal effects on non-target organisms and their parasites are often overlooked. Particularly, studies on terrestrial hosts and their parasites exposed to agricultural toxicants are lacking. Here, we studied the effect of sequence and timing of sub-lethal exposures of the pyrethroid insecticide alpha-cypermethrin on parasite establishment using the tapeworm Hymenolepis diminuta and its intermediate insect host Tenebrio molitor as a model system. We exposed T. molitor to alpha-cypermethrin (LD20) before and after experimental H. diminuta infection and measured the establishment success of larval tapeworms. Also, we conducted in vitro studies quantifying the direct effect of the insecticide on parasite viability. Our results showed that there was no direct lethal effect of alpha-cypermethrin on H. diminuta cysticercoids at relevant concentrations (LD10 to LD90 of the intermediate host). However, we observed a significantly increased establishment of H. diminuta in beetles exposed to alpha-cypermethrin (LD20) after parasite infection. In contrast, parasite establishment was significantly lower in beetles exposed to the insecticide before parasite infection. Thus, our results indicate that environmental toxicants potentially impact host-parasite interactions in terrestrial systems, but that the outcome is context-dependent by enhancing or reducing parasite establishment depending on timing and sequence of exposure.

Synthesis and biological activity of new derivatives with the preserved carane system.

Terpenoid derivatives, which contain a preserved carane system in their structure, exhibit a broad spectrum of biological activities. Among them, we can distinguish insecticides, structures with pharmacological application etc. In the presented paper, the substrate - (-)-cis-caran-trans-4-ol was transformed using the reactions of typical organic synthesis to obtain novel derivatives. Most importantly, bromolactone ((-)-(1R,4R,6S)-2'-(bromomethyl)-4,7,7-trimethylspiro[bicyclo[4.1.0]heptan-3,3'-furan]-5'(4'H)-one) with the preserved carane system was synthesized. This bromolactone was tested for antifeedant activity against the lesser mealworm, Alphitobius diaperinus Panzer, and peach potato aphid (Myzus persicae). In addition, its moderate antibacterial activity was observed against the Bacillus subtilis strain (with Minimal Inhibitory Concentration of 200 µg/mL).

Changes in the gut microbiome and enzymatic profile of Tenebrio molitor larvae biodegrading cellulose, polyethylene and polystyrene waste.

Recent studies have demonstrated the ability of mealworm (Tenebrio molitor) for plastic degradation. This study is focused on changes in microbiome structure depending on diets. Microbial community obtained from oat and cellulose diet formed similar group, two kinds of polyethylene formed another group, while polystyrene diet showed the highest dissimilarity. The highest relative abundance of bacteria colonizing gut was in PE-oxodegradable feeding, nevertheless all applied diets were higher in comparison to oat. Dominant phyla consisted of Proteobacteria, Bacteroides, Firmicutes and Actinobacteria, however after PS feeding frequency in Planctomycetes and Nitrospirae increased. The unique bacteria characteristic for cellulose diet belonged to Selenomonas, while Pantoea were characteristic for both polyethylene diets, Lactococcus and Elizabethkingia were unique for each plastic diet, and potential diazotropic bacteria were characteristic for polystyrene diet (Agrobacterium, Nitrosomonas, Nitrospira). Enzymatic similarity between oatmeal and cellulose diets, was shown. All three plastics diet resulted in different activity in both, digestive tract and bacteria. The enzymes with the highest activity were included phosphatases, esterases, leucine arylamidase, ß-galactosidase, ß-glucuronidase, α-glucosidase, ß-glucosidase, chitinase, α-mannosidase and α-fucosidase. The activity of digestive tract was stronger than cultured gut bacteria. In addition to known polyethylene degradation methods, larvae may degrade polyethylene with esterase, cellulose and oatmeal waste activity is related with the activity of sugar-degrading enzymes, degradation of polystyrene with anaerobic processes and diazotrophs.