Bioconversion of glycerol into polyhydroxyalkanoates through an atypical metabolism shift using Priestia megaterium during fermentation processes: A statistical analysis of carbon and nitrogen source concentrations.

Polyhydroxyalkanoates (PHA) are bioplastics which are well known as intracellular energy storage compounds and are produced in a large number of prokaryotic species. These bio-based inclusions are biodegradable, biocompatible and environmental friendly. Industrial production of, short chain and medium chain length PHA, involves the use of microorganisms and their enzymes. Priestia megaterium previously known as Bacillus megaterium is a well-recognized bacterium for producing short chain length PHA. This study focuses to characterize this bacterium for the production of medium chain length PHA, and a novel blend of both types of monomers having enhanced properties and versatile applications. Statistical analyses and simulations were used to demonstrate that cell dry weight can be derived as a function of OD600 and PHA content. Optimization of growth conditions resulted in the maximum PHA production as: 0. 05 g. g-x. H-1, where the rate of PHA production was 0.28 g L-1. H-1 and PHA concentration was 4.94 g. L-1. This study also demonstrated FTIR to be a semi quantitative tool for PHA production. Moreover, conversion of scl-PHA to mcl-PHA with reference to time intermissions using GC-FID are shown.

Multi-omics phenotyping of the gut-liver axis reveals metabolic perturbations from a low-dose pesticide mixture in rats.

Health effects of pesticides are not always accurately detected using the current battery of regulatory toxicity tests. We compared standard histopathology and serum biochemistry measures and multi-omics analyses in a subchronic toxicity test of a mixture of six pesticides frequently detected in foodstuffs (azoxystrobin, boscalid, chlorpyrifos, glyphosate, imidacloprid and thiabendazole) in Sprague-Dawley rats. Analysis of water and feed consumption, body weight, histopathology and serum biochemistry showed little effect. Contrastingly, serum and caecum metabolomics revealed that nicotinamide and tryptophan metabolism were affected, which suggested activation of an oxidative stress response. This was not reflected by gut microbial community composition changes evaluated by shotgun metagenomics. Transcriptomics of the liver showed that 257 genes had their expression changed. Gene functions affected included the regulation of response to steroid hormones and the activation of stress response pathways. Genome-wide DNA methylation analysis of the same liver samples showed that 4,255 CpG sites were differentially methylated. Overall, we demonstrated that in-depth molecular profiling in laboratory animals exposed to low concentrations of pesticides allows the detection of metabolic perturbations that would remain undetected by standard regulatory biochemical measures and which could thus improve the predictability of health risks from exposure to chemical pollutants.

New model development for qualitative and quantitative analysis of microbial polyhydroxyalkanoates: A comparison of Fourier Transform Infrared Spectroscopy with Gas Chromatography.

FT-IR spectroscopy is a non-destructive testing technique that requires minimal sample preparation time and allows the rapid characterization of structural features of complex, polymeric material. This technique has been frequently used in the past for the detection of PHA (Polyhydroxyalkanoates) but rarely for their quantification. In this work, by using cluster and discriminant statistical analysis of FT-IR data, different models are proposed for rapid identification of PHA monomers produced under different growth conditions by bacterial strains, and for their semi quantification. The results on the ability to produce large amounts of PHA (of 21 strains) in different environmental conditions of medium, substrates and deficiency of nutrients are presented. The spectral data analysis gives qualitative and semi quantitative information about the PHA produced in the samples. Models are proposed to test a large number of cultural conditions of strains and substrates in the field of screening and for identifying best conditions of PHA production in lab scale bioreactor and on industrial scale.

Use of Shotgun Metagenomics and Metabolomics to Evaluate the Impact of Glyphosate or Roundup MON 52276 on the Gut Microbiota and Serum Metabolome of Sprague-Dawley Rats.

BACKGROUND: There is intense debate on whether glyphosate can inhibit the shikimate pathway of gastrointestinal microorganisms, with potential health implications. OBJECTIVES: We tested whether glyphosate or its representative EU herbicide formulation Roundup MON 52276 affects the rat gut microbiome. METHODS: We combined cecal microbiome shotgun metagenomics with serum and cecum metabolomics to assess the effects of glyphosate [0.5, 50, 175mg/kg body weight (BW) per day] or MON 52276 at the same glyphosate-equivalent doses, in a 90-d toxicity test in rats. RESULTS: Glyphosate and MON 52276 treatment resulted in ceca accumulation of shikimic acid and 3-dehydroshikimic acid, suggesting inhibition of 5-enolpyruvylshikimate-3-phosphate synthase of the shikimate pathway in the gut microbiome. Cysteinylglycine, γ-glutamylglutamine, and valylglycine levels were elevated in the cecal microbiome following glyphosate and MON 52276 treatments. Altered cecum metabolites were not differentially expressed in serum, suggesting that the glyphosate and MON 52276 impact on gut microbial metabolism had limited consequences on physiological biochemistry. Serum metabolites differentially expressed with glyphosate treatment were associated with nicotinamide, branched-chain amino acid, methionine, cysteine, and taurine metabolism, indicative of a response to oxidative stress. MON 52276 had similar, but more pronounced, effects than glyphosate on the serum metabolome. Shotgun metagenomics of the cecum showed that treatment with glyphosate and MON 52276 resulted in higher levels of Eggerthella spp., Shinella zoogleoides, Acinetobacter johnsonii, and Akkermansia muciniphila. Shinella zoogleoides was higher only with MON 52276 exposure. In vitro culture assays with Lacticaseibacillus rhamnosus strains showed that Roundup GT plus inhibited growth at concentrations at which MON 52276 and glyphosate had no effect. DISCUSSION: Our study highlights the power of multi-omics approaches to investigate the toxic effects of pesticides. Multi-omics revealed that glyphosate and MON 52276 inhibited the shikimate pathway in the rat gut microbiome. Our findings could be used to develop biomarkers for epidemiological studies aimed at evaluating the effects of glyphosate herbicides on humans. https://doi.org/10.1289/EHP6990.

Impact of Antibiotics on Efficacy of Cry Toxins Produced in Two Different Genetically Modified Bt Maize Varieties in Two Lepidopteran Herbivore Species, Ostrinia nubilalis and Spodoptera littoralis.

The insecticidal crystal proteins from Bacillus thuringiensis (Bt) are widely-used biopesticides that are used both as Bt spore-crystal preparations in sprayable formulations and as activated toxins in genetically modified (GM) plants. Models for their modes of action have been proposed but many issues remain unresolved. Among those is the role of commensal gut bacteria in target insect death: previous studies showed that antibiotics attenuate the toxicity of Bt sprays. We tested whether antibiotics interfere with the effects of GM plant-produced Bt toxins in larvae of two Lepidopteran species, the European corn borer Ostrinia nubilalis and the cotton leafworm Spodoptera littoralis. The larvae were reared on artificial diet with or without antibiotics and, thereafter, fed two varieties of Bt GM maize in comparison to conventional non-Bt maize leaves sprayed with antibiotic solution and/or with a Bt formulation. Antibiotics significantly reduced or delayed the toxicity of Cry toxins, although to a lesser extent than previously reported for Bt-sprays. This supports the hypothesis that Cry toxins induce mortality by themselves in the absence of Bt bacteria and spores, and of commensal gut bacteria. However, larvae that were not treated with antibiotics died faster and at a higher rate which was further compounded by plant variety and species sensitivity. These findings support a hypothesis that toxicemia alone can inflict significant mortality. However, in the absence of antibiotics, the gut bacteria likely enhance the Cry toxin effect by inflicting, additionally, bacterial septicemia. This has important implications in field situations where antibiotic substances are present-e.g., from manure of animals from conventional production systems-and for ecotoxicological testing schemes of Bt toxins and nontarget organisms that are often using artificial diets enriched with high concentrations of antibiotics.

Sex-dependent impact of Roundup on the rat gut microbiome.

A growing body of research suggests that dysbiosis of the gut microbiota induced by environmental pollutants, such as pesticides, could have a role in the development of metabolic disorders. We have examined the long-term effects of 3 doses of the Roundup(R) herbicide (made of glyphosate and formulants) on the gut microbiota in male and female Sprague-Dawley rats. A total of 141 bacteria families were identified by a 16S sequencing analysis approach. An OPLS-DA analysis revealed an increased Bacteroidetes family S24-7 and a decreased Lactobacillaceae in 8 out of the 9 females treated with 3 different doses of R (n = 3, for each dose). These effects were confirmed by repetitive sequence-based PCR fingerprinting showing a clustering of treated females. A culture-based method showed that R had a direct effect on rat gut microbiota. Cultivable species showed different sensitivities to R, including the presence of a high tolerant or resistant strain identified as Escherichia coli by 16S rRNA sequencing. The high tolerance of this E. Coli strain was explained by the absence of the EPSPS gene (coding glyphosate target enzyme) as shown by DNA amplification. Overall, these gut microbiome disturbances showed a substantial overlap with those associated with liver dysfunction in other studies. In conclusion, we revealed that an environmental concentration of R (0.1 ppb) and other two concentrations (400 ppm and 5,000 ppm) have a sex-dependent impact on rat gut microbiome composition and thus warrants further investigation.

FTIR spectroscopy of whole cells for the monitoring of yeast apoptosis mediated by p53 over-expression and its suppression by Nigella sativa extracts.

p53 over expression in yeast results in cell death with typical markers of apoptosis such as DNA fragmentation and phosphatidylserine externalization. We aimed to substitute/supplement classical fluorescent techniques (TUNEL, Annexin V, ROS detection) usually used to detect biochemical changes occurring during yeast apoptosis mediated by p53 over expression and the effect of anti-apoptotic purified molecules from Nigel (Nigella sativa) extracts on these same yeasts by the label free technique of FTIR spectroscopy. The comparison of the entire IR spectra highlighted clear modifications between apoptotic p53-expressing yeasts and normal ones. More precisely, DNA damage was detected by the decrease of band intensities at 1079 and 1048 cm-1. While phosphatidylserine exposure was followed by the increase of νsCH2 and νasCH2 bands of unsaturated fatty acids that were exhibited at 2855 and 2926 cm-1, and the appearance of the C = O ester functional group band at 1740 cm-1. In a second step, this FTIR approach was used to estimate the effect of a purified fraction of the Nigel extract. The modulation of band intensities specific to DNA and membrane status was in agreement with apoptosis supression in presence of the Nigel extracts. FTIR spectroscopy is thus proven to be a very reliable technique to monitor the apoptotic cell death in yeast and to be used as a means of evaluating the biomolecules effect on yeast survival.

Impact of carbon source and variable nitrogen conditions on bacterial biosynthesis of polyhydroxyalkanoates: evidence of an atypical metabolism in Bacillus megaterium DSM 509.

Twenty bacterial strains were examined on their ability to produce polyhydroxyalkanoates (PHA) from different carbon sources under rich and depleted nitrogen conditions. Preliminary experiments with glucose as sole carbon source allowed to select PHA producing bacteria using FTIR spectroscopy. They were further tested with eight additional carbon substrates including organic, fatty acids or sugars. PHA content and monomer composition of four chosen strains (Pseudomonas putida mt-2, Bacillus megaterium DSM 90 and DSM 509, Corynebacterium glutamicum DSM 20137) were assessed by gas chromatography techniques for two cultural conditions: during growth phase on a mineral medium (MM) and after transfer of cells on a fresh MM without nitrogen (MM-N). For several carbon substrates, substantial amounts of PHA (up to 53% of the cell dry weight: CDW) were already obtained in MM for C. glutamicum DSM 20137 and the two B. megaterium strains; after transfer in MM-N, PHA contents remained constant except for B. megaterium DSM 509 where PHA production increased whatever the carbon source. P. putida mt-2 synthesized PHA under deprived nitrogen conditions. Highest PHA accumulation reached 48 and 77% of CDW with octanoic acid as substrate in B. megaterium DSM 90 and P. putida mt-2, respectively. Surprisingly, an atypical metabolic shift was observed for B. megaterium DSM 509 cultivated with nearly all unrelated carbon sources: whereas short chain length PHA (scl-PHA) were synthesized in MM, medium chain length PHA (mcl-PHA) were produced after transfer of cells into MM-N supplemented with the same substrate.

Quantitative and qualitative variability of the caseinolytic potential of different strains of Pseudomonas fluorescens: implications for the stability of casein micelles of UHT milks during their storage.

Pseudomonas fluorescens grows at low temperature and produces thermo-resistant protease(s) that can destabilize UHT (Ultra High Temperature) milk during its storage. The consequences of contamination of microfiltered milk with 9 strains of P. fluorescens on the stability of the corresponding UHT milk during storage had been investigated in this study. The strains were classified in two groups according to their ability to destabilize UHT milk. For the group of highly destabilizing strains, sedimentations of UHT milks, low values to phosphate test and the presence of aggregates were observed. Zeta potential and hydration of casein micelles decreased, whereas non casein nitrogen (NCN) and non protein nitrogen (NPN) contents increased. The analyses of NCN fraction by liquid chromatography coupled to mass spectrometry indicated that the different casein molecules were hydrolyzed in a similar way for the destabilizing strains suggesting that the same enzyme was implicated. For the group of slightly or not destabilizing strains no visual and biochemical alteration were found. This study showed that destabilization of UHT milk by P. fluorescens was highly variable and strain-dependent.

Effects of Roundup(®) and glyphosate on three food microorganisms: Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus.

Use of many pesticide products poses the problem of their effects on environment and health. Amongst them, the effects of glyphosate with its adjuvants and its by-products are regularly discussed. The aim of the present study was to shed light on the real impact on biodiversity and ecosystems of Roundup(®), a major herbicide used worldwide, and the glyphosate it contains, by the study of their effects on growth and viability of microbial models, namely, on three food microorganisms (Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus) widely used as starters in traditional and industrial dairy technologies. The presented results evidence that Roundup(®) has an inhibitory effect on microbial growth and a microbicide effect at lower concentrations than those recommended in agriculture. Interestingly, glyphosate at these levels has no significant effect on the three studied microorganisms. Our work is consistent with previous studies which demonstrated that the toxic effect of glyphosate was amplified by its formulation adjuvants on different human cells and other eukaryotic models. Moreover, these results should be considered in the understanding of the loss of microbiodiversity and microbial concentration observed in raw milk for many years.

FT-IR spectroscopy for rapid differentiation of Aspergillus flavus, Aspergillus fumigatus, Aspergillus parasiticus and characterization of aflatoxigenic isolates collected from agricultural environments.

In agricultural areas, Aspergillus flavus, Aspergillus fumigatus and Aspergillus parasiticus are commonly identified in various feedstuffs and bioaerosols originated from feed handling. Some isolates belonging to these fungal species could produce mycotoxins and constitute a risk factor for human and animal health. In this study, Fourier-transform infrared spectroscopy was used for a rapid detection and characterization of 99 isolates collected from agricultural areas. The results showed a first cluster corresponding to strains previously attributed to the A. fumigatus group according to current taxonomic concepts, and a second cluster divided in 2 groups around reference strains of A. flavus and A. parasiticus species. The toxigenic capacity of isolates was evaluated by high performance liquid chromatography coupled to mass spectrometry. In the A. flavus group, only 6 strains of A. parasiticus and 4 strains of A. flavus were able to produce aflatoxins on culture media. FT-IR spectroscopy, respectively, allowed the differentiation of non-toxigenic and toxigenic A. flavus and A. parasiticus isolates at 75 and 100%. Discrimination between toxigenic and non-toxigenic A. fumigatus was not possible because all of the isolates produced at least one mycotoxin.

Sulfato/thiosulfato reducing bacteria characterization by FT-IR spectroscopy: a new approach to biocorrosion control.

Sulfato and Thiosulfato Reducing Bacteria (SRB, TRB) can induce corrosion process on steel immersed in seawater. This phenomenon, called biocorrosion, costs approximatively 5 billion euros in France each year. We provide the first evidence that Fourier Transformed InfraRed (FTIR) spectroscopy is a competitive technique to evaluate the sulfurogen flora involved in biocorrosion in comparison with time consuming classical identification methods or PCR analyses. A great discrimination was obtained between SRB, TRB and some contamination bacteria known to be present in seawater and seem to be able to reduce sulfate under particular conditions. Moreover, this preliminary study demonstrates that FTIR spectroscopic and genotypic results present a good correlation (these results are confirmed by other data obtained before or later, data not shown here). The advantages gained by FTIR spectroscopy are to give information on strain phenotype and bacterial metabolism which are of great importance in corrosion processes.