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.

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.

Medium chain length polyhydroxyalkanoates biosynthesis in Pseudomonas putida mt-2 is enhanced by co-metabolism of glycerol/octanoate or fatty acids mixtures.

The synthesis of medium chain length polyhydroxyalkanoates (mcl-PHAs) by Pseudomonas putida mt-2 was investigated under nitrogen-rich then deficient conditions with glycerol/octanoate or long-chain fatty acids (LCFAs) as carbon sources. When mixed, glycerol and octanoate were co-assimilated regardless of nitrogen availability but provided that glycerol uptake has been already triggered under non-limiting nutrient conditions. This concomitant consumption allowed to enhance mcl-PHAs accumulation (up to 57% of cell dry weight (CDW)) under both non-limiting and nitrogen deficient conditions. Octanoate then mostly drove anabolism of the polyester with 3-hydroxyoctanoate (3HO) synthesized as the main monomer (83%). If the preferred PHA precursor octanoate was supplied, glycerol was mainly involved in cell growth and/or maintenance but very little in PHA production even under nitrogen starvation. P. putida cells accumulated higher amounts of mcl-PHAs when grown on mixtures of LCFAs compared to LCFAs supplied as single substrate (25% and 9% of CDW, respectively). However, only a weak enrichment of the polyester was observed after transfer of cells in a fresh nitrogen-free medium containing the same combination of LCFAs. Some typical units within the polyester were related to the LCFAs ratio supplied in the medium indicating that tailor-made monomers could be synthesized.

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.

Efficacy and environmental acceptability of two ballast water treatment chemicals and an alkylamine based-biocide.

Ship's ballast waters transport large numbers of organisms which may become invasive in coastal regions. One option to address this problem is the use of biocides as ballast water treatment (BWT). Efficacy and environmental acceptability of three commercial active substances (the BWT biocides Peraclean(®) Ocean and Seakleen(®), and alkylamine-based biocide Mexel(®) 432/336) were tested against three bacteria species, two vegetative microalgae and one zooplanktonic larva, in 10 and 30 Practical Salinity Unit (PSU) waters. In both salinities, PeraClean(®) Ocean was the most effective biocide against bacteria causing >90% mortality at 20mg/l, compared with 50mg/l for Mexel(®) 432/336 and >500 mg/l for Seakleen(®). Regarding zooplankton, Seakleen(®) was the most effective chemical causing 90% mortality in 24h at concentrations <6 mg/l (LC90(24h)) in both salinities, compared with 23 and 26 mg/l for Mexel(®) 432/336 and 370 and 480 mg/l for PeraClean(®) Ocean in 10 and 30 PSU, respectively. Similar pattern of efficacy was obtained for microalgae in 30 PSU: effective concentrations inducing 50% growth inhibition in 4 days were ≤ 1.6 mg/l for Seakleen(®), ≤ 10.1mg/l for Mexel(®) 432/336 and ≤ 30.9 mg/l for PeraClean(®) Ocean. Our work highlighted that treated waters displayed residual toxicity after 24h still inducing mortality depending on the organism and biocide. However Mexel(®) 432/336 is the only biocide which had no impact on oyster larvae development at effective concentration. Altogether our data showed that Mexel(®) 432/336 was the only biocide displaying a broad spectrum efficacy in concentrations <50mg/l and not toxic for oyster larvae development at this concentration. However residual toxicity of treated waters for any organism should be taken into account in BWT systems utilising biocides.

Acid violet 7 and its biodegradation products induce chromosome aberrations, lipid peroxidation, and cholinesterase inhibition in mouse bone marrow.

INTRODUCTION: Acid violet 7 (AV7), mostly used in food, paper, cosmetic, and especially in textile industries, was degraded by Pseudomonas putida mt-2 at concentrations up to 200 mg/l. MATERIALS AND METHODS: In this study, toxicity of AV7, before and after biodegradation, was evaluated in vivo, in mouse bone marrow, by assessing the percentage of cells bearing different chromosome aberrations, membrane lipid peroxidation, and acetylcholinesterasic activity inhibition. The studies included same conditions for animal treatment, corresponding to increasing doses by intraperitoneal (ip) injection. RESULTS: Results indicated that AV7 showed a significant ability to induce chromosome aberrations, lipid peroxidation, and acetylcholinesterase inhibitory effect. The toxicity of AV7 increased significantly after static biodegradation with P. putida mt-2 and totally disappeared after shaken incubation. In addition, the toxicity generated by the pure azo dye and the corresponding azoreduction metabolites (4'-aminoacetanilide (4'-AA) and 5-acetamido-2-amino-1-hydroxy-3,6-naphtalene disulfonic acid (5-ANDS)) were compared. 4'-AA and 5-ANDS would be responsible of static biodegradation medium toxicity. The present study demonstrates that P. putida mt-2, incubated under aerobic condition, has a catabolism which enables it to degrade AV7, and especially to completely detoxify the dye mixture.

Mutagenicity and genotoxicity of acid yellow 17 and its biodegradation products.

Acid yellow 17 (AY17), a very important commercial azo dye used in the textile industry, was degraded by Pseudomonas putida mt-2 at a concentration of up to 200 mg/L. High-performance liquid chromatography analysis of the biodegradation media revealed the presence of 4-aminobenzensulfonic acid (4-ABS) derived from AY17 azoreduction, which attests the expression of an azoreductase by this bacterium. This amine was identified only in the medium of static incubation, which is consistent with its biotransformation under shaken incubation (i.e., aerobic conditions). The mutagenicity of AY17 and its biodegradation products was evaluated by using Salmonella typhimurium TA102 and TA104. No mutagenicity was observed in the presence or absence of a metabolic activation system (S9). In addition, the ability of tested compounds to induce DNA damage in vitro with the DNA strand scission assay was evaluated. Results showed that only static decolorization culture of AY17 showed a significant ability to induce the pKS plasmid DNA opening. The present study showed that P. putida mt-2, cultivated under aerobic conditions, was able to decolorize, and especially to detoxify, AY17.

Genotoxic and antibutyrylcholinesterasic activities of acid violet 7 and its biodegradation products.

Acid violet 7, a sulfonated azo dye was degraded by Pseudomonas putida mt-2 in mineral medium at concentrations up to 200 mg/L. The genotoxicity of AV7 and its biodegradation extracts was evaluated by using the DNA-strand scission assay. No genotoxicity was observed, even with or without exposition to UV irradiation, for biodegradation under shaking conditions, but increased significantly after biodegradation under static conditions. In addition, the ability of tested compounds to reduce human plasma butyrylcholinesterase (BuChE) activity was evaluated in vitro. Genotoxicity and anti-BuChE activity generated by the azoreduction products [4'-aminoacetanilid (4'-AA) and 5-acetamido-2-amino-1-hydroxy-3,6-naphtalene disulfonic acid (5-ANDS)] were assessed and compared with that of the parent unsubstituted amines. 4'-AA exhibited a strong genotoxicity, which was imputed to the presence of the acetoxy (COCH3) substituent on the aromatic amine; however, the presence of sulphonic groups in 5-ANDS seems to be responsible for its BuChE inhibition activity. The present study demonstrates that P. putida mt-2, incubated under aerobic conditions, has a catabolism that enables it to degrade AV7 and, especially, to detoxify the dye mixtures.

In vitro mutagenicity of Acid Violet 7 and its degradation products by Pseudomonas putida mt-2: Correlation with chemical structures.

Acid Violet 7 (AV7), a very important commercial azo dye used in the textile, food, paper and cosmetic industries, was degraded by Pseudomonas putida mt-2 at a concentration up to 200mg/l. HPLC analysis of the biodegradation media revealed the presence of either 4'-aminoacetanilide (4'-AA) or 5-acetamido-2-amino-1-hydroxy-3,6-naphthalene disulfonic acid (5-ANDS) deriving from AV7 azoreduction which attests the expression of an azoreductase by this bacterium. These amines were identified only in media of static incubation, which is consistent with their biotransformation under shaken incubation (aerobic conditions). Pure azo dye, pure azoreduction products and total lyophilized biodegradation extracts were assayed for their mutagenic properties using Ames test. Mutagenicity of AV7 even with or without the S9 metabolizing system increased significantly after static biodegradation and totally disappeared after shaken incubation. In addition, mutagenicity of pure azo reduction products of AV7 was assessed and compared with that of the parent unsubstituted amines. 4'-AA exhibited a strong mutagenicity which was imputed to the presence of the acetoxy (COCH(3)) substituent on the aromatic amine; however, the presence of sulphonic groups in 5-ANDS limited its mutagenicity.

In vitro study of DNA damage induced by acid orange 52 and its biodegradation derivatives.

Mutagenicity of acid orange 52 (AO52) and its degradation products by Pseudomonas putida mt-2 was evaluated with the use of Salmonella Typhimurium TA102 and TA104 with and without the metabolic activation system (S9). No mutagenicity was observed in the absence of S9 and in the presence of S9 for biodegradation under shaking conditions, but it increased significantly in the presence of S9 after biodegradation under static conditions. In addition, the ability of tested compounds to induce DNA damage in vitro was evaluated with the DNA strand scission assay. The toxicity generated by the pure azo dye and the corresponding azoreduction products (4-aminobenzenesulfonic acid and N,N'-dimethyl-p-phenylenediamine) were compared. We suggest that the mutagenicity mechanism of these molecules occurs through free radical generation processes. In this study, we demonstrate that P. putida mt-2 incubated under aerobic conditions undergoes catabolism that enables it to degrade AO52 completely and, especially, to detoxify the dye mixtures.

Physiological states and energetic adaptation during growth of Pseudomonas putida mt-2 on glucose.

Kinetic study of growth of Pseudomonas putida mt-2 was investigated in batch culture under aerobic conditions, on glucose as initial carbon and energy source. Cell growth was continuous and three phases were found regarding accumulation of intermediates: (1) glucose was largely converted to gluconate and 2-ketogluconate, (2) then gluconate was converted to 2-ketogluconate and (3) the latter was consumed after gluconate depletion. Examination of growth kinetics and yields showed that glucose flux was mainly oriented to oxidation reduction in the periplasm and less towards biosynthesis. Values of respiratory quotient and of CO2/biomass and O2/biomass yields were characteristic of each phase. Main enzymatic activities involved in the use of these substrates were always detected meaning that concomitant assimilation is possible. However the levels of these activities varied during growth. Membrane conversions seem to have a significant energetic contribution explaining the higher specific growth rate obtained in glucose phase compared to gluconate and 2-ketogluconate ones. This is also noticeable through the evolution of the yields Y(O2)/X and Y(CO2)/X. Although the three convergent pathways are operational and can be genetically controlled, the progression of the culture in successive phases highlights an overall level of regulation in response to the energetic needs.

Evaluation of genotoxicity and pro-oxidant effect of the azo dyes: acids yellow 17, violet 7 and orange 52, and of their degradation products by Pseudomonas putida mt-2.

Acids yellow 17, violet 7 and orange 52, very important commercial azo dyes used in the textile, food, paper and cosmetic industries, were degraded by Pseudomonas putida mt-2 at concentrations up to 100mg/l. The culture media was completely decolorized under static incubation for 60 h, this faster than under continuous shaking incubation. SOS chromotest using Escherichia coli PQ37, with and without metabolic activation (S-9 preparations), was used to assess genotoxicity potential of these dyes before and after biodegradation. None of these dyes or their metabolites was found to be genotoxic in the absence of "Araclor-Induced rat liver microsome" preparations (S-9). However, in presence of the preparation S-9, the genotoxicity of the biodegradation products was highlighted. Metabolites resulting from static cultures were more genotoxic than those obtained in shaken conditions. In addition to genotoxic effects, metabolites have shown a significant ability to induce the formation of superoxide free radical anion (O(2)(*-)). The toxicities generated by the pure azo dyes and the pure azo-reduction products (sulfanilic acid, N,N'-dimethyl-p-phenylenediamine and 4'-aminoacetanilid) were compared. These results suggest that P. putida mt-2 degrades the studied azo dyes in two steps: an azo-reduction followed by an oxygen-dependent metabolization. Some of the derived metabolites would be responsible of genotoxicity and metabolic toxicity.