Bioconversion of cassava starch by-product into Bacillus and related bacteria polyhydroxyalkanoates

Background: Unlike petroleum-based synthetic plastics, biodegradable biopolymer generation from industrial residue is a key strategy to reduce costs in the production process, as well as in the waste management, since efficient industrial wastewater treatment could be costly. In this context, the present work describes the prospection and use of bacterial strains capable to bioconvert cassava starch by-product into biodegradable polyhydroxyalkanoates (PHAs). Results: The first step of this study was the bacterial competence screening which was conducted with 72 strains covering 21 Bacillus and related species. The microorganism growth in a medium with a starch substrate was measured by an innovative MTT assay, while the ability of the bacteria to secrete amylase and produce PHA was evaluated by the Nile Red Dye method. Based on growth and potential for PHA production, four isolates were selected and identified as Bacillus megaterium by 16S rRNA sequencing. When cultivated in hydrolyzed cassava starch by-product, maximum production reached 4.97 g dry biomass/L with 29.7% of Poly-(3-hydroxybutyrate) (characterized by FTIR). Conclusions: MTT assay proved to be a reliable methodology for monitoring bacterial growth in insoluble media. Selected amylolytic strains could be used as an alternative industrial process for biodegradable plastics production from starchy residues, reducing costs for biodegradable biopolymer production and wastewater treatment operations

Genotoxicity assessment of particulate matter emitted from heavy-duty diesel-powered vehicles using the in vivo Vicia faba L. micronucleus test

International audienceDiesel exhaust particulate matter (PM) can have an impact on the environment due to its chemical constitution. A large number of substances such as organic compounds, sulfates, nitrogen derivatives and metals are adsorbed to the particles and desorption of these contaminants could promote genotoxic effects. The objective of this study was to assess the in vivo genotoxicity profile of diesel exhaust PM from heavy-duty engines. Extracts were obtained through leaching with pure water and chemical extraction using three organic solvents (dichloromethane, hexane, and acetone). The in vivo Vicia faba micronucleus test (ISO 29200 protocol) was used to assess the environmental impact of the samples collected from diesel exhaust PM. The solid diesel PM (soot) dissolved in water, and the different extracts, showed positive results for micronucleus formation. After the addition of EDTA, the aqueous extracts did not show a genotoxic effect. The absence of metals in the organic solvent extract indicated that organic compounds also had a genotoxic effect, which was not observed for a similar sample cleaned in a C18 column. Thus, considering the ecological importance of higher plants in relation to ecosystems (in contrast to Salmonella spp., which are commonly used in mutagenicity studies), the Vicia micronucleus test was demonstrated to be appropriate for complementing prokaryotic or in vitro tests on diesel exhaust particulate matter included in risk assessments

Sensitivity of different aquatic bioassays in the assessment of a new natural formicide

International audienceAgrochemicals have the potential to cause deleterious effects on living organisms and therefore they must be subjected to various (eco)toxicological studies and monitoring programs in order to protect human health and the environment. The aim of this study was to assess the ecotoxicity of a new natural formicide with a battery of three classical and three ecotox-kit tests. The former tests were performed with Aliivibrio fischeri bacteria (Lumistox test), the cladoceran Daphnia magna and Pseudokirchneriella subcapitata algae, and the latter with Thamnotoxkit F (TM) (Thamnocephalus platyurus), Ostracodtoxkit F (R) (Heterocypris incongruens) and LuminoTox (photosynthetic enzyme complexes). In the range of formicide concentrations tested (from 0.06 to 2.0 g L-1), the measurement endpoint values varied from 0.79 g L-1 for the algal test to > 2 g L-1 for the LuminoTox and Ostracodtoxkit F (R) tests. Hierarchical sensitivity ranking based on the no-observed effect concentration (NOEC) values established to assess the formicide ecotoxicity was as follows: algal growth inhibition test approximate to daphnid immobilization test approximate to bacterial luminescence inhibition test > Thamnotoxkit F (TM) > LuminoTox > Ostracodtoxkit F (R). Overall, results from the battery of bioassays showed that this formicide preparation presents low ecotoxicity as compared to the aquatic ecotoxicity of presently commercialized formicides. In conclusion, classical aquatic bioassays are more sensitive than ecotox-kit tests in the assessment and monitoring of the new natural formicide

Arsenate (As V) in water: Quantitative sensitivity relationships among biomarker, ecotoxicity and genotoxicity endpoints

International audienceIt is useful to test ecotoxicity and genotoxicity endpoints in the environmental impact assessment. Here, we compare and discuss ecotoxicity and genotoxicity effects in organisms in response to exposure to arsenate (As V) in solution. Eco(geno)toxicity responses in Aliivibrio fischeri, Lytechinus variegatus, Daphnia magna, Skeletonema costatum and Vicia faba were analyzed by assessing different endpoints: biomass growth, peroxidase activity, mitotic index, micronucleus frequency, and lethality in accordance with the international protocols. Quantitative sensitivity relationships (QSR) between these endpoints were established in order to rank endpoint sensitivity. The results for the QSR values based on the lowest observed effect concentration (LOEC) ratios varied from 2 (for ratio of root peroxidase activity to leaf peroxidase activity) to 2286 (for ratio of higher plant biomass growth to root peroxidase activity). The QSR values allowed the following sensitivity ranking to be established: higher plant enzymatic activity > daphnids approximate to cechinoderms > bacteria approximate to algae > higher plant biomass growth. The LOEC values for the mitotic index and micronucleus frequency (LOEC=0.25 mgAs L-1) were similar to the lowest LOEC values observed in aquatic organisms. This approach to the QSR of different endpoints could form the basis for monitoring and predicting early effects of pollutants before they give rise to significant changes in natural community structures

Leachates from solid wastes: chemical and eco(geno)toxicological differences between leachates obtained from fresh and stabilized industrial organic sludge

International audienceThe chemical and ecotoxicological characteristics of fresh and stabilized industrial organic sludge leachates were compared to obtain information regarding how the stabilization process can influence the ecotoxic potential of this industrial waste, which could be used for the amendment of degraded soil. Physicochemical analysis of the sludge leachates, as well as a battery of eco(geno)toxicity tests on bacteria, algae, daphnids, and higher plants (including Vicia faba genotoxicity test) and the determination of hydrolytic enzyme activity, was performed according to standard methods. The chemical comparison of the two types of leachate showed that the samples obtained from stabilized sludge had a lower organic content and higher metal content than leachates of the fresh sludge. The eco(geno)toxicological results obtained with aquatic organisms showed that the stabilized sludge leachate was more toxic than the fresh sludge leachate, both originating from the same industrial organic sludge sample. Nevertheless, phytotoxicity tests carried out with a reference peat soil irrigated with stabilized sludge leachate showed the same toxicity as the fresh sludge leachate. In the case of the industrial solid organic sludge studied, stabilization through a biodegradation process promoted a higher metal mobility/bioavailability/eco(geno)toxicity in the stabilized sludge leachate compared to the fresh sludge leachate

Ecotoxicity assessment of particulate matter emitted from heavy-duty diesel-powered vehicles: influence of leaching conditions

International audienceConcerns regarding the environmental impact of diesel exhaust particulate matter(DPM) have increased in recent years. Following emission to the atmosphere, thesefine materials can sorb many contaminants at their surface, which can subsequently bereleased, for instance, due to physico-chemical environmental changes. Thedesorption of contaminants from particulate matter will increase the environmentalpollution and can promote ecotoxicological effects. In this context, the objective of thisstudy was to assess the aquatic ecotoxicity profile of extracts of DPM obtained at twodifferent pH values. Thus, after collecting particulate matter from the diesel exhaust ofheavy engines, extracts were obtained with pure water (at pH 2.00 and 5.00) and witha mixture of three organic solvents (dichloromethane, n-hexane, and acetone). Toassess the environmental impact of DPM, the exhaust extracts were used in a batteryof aquatic bioassays including key organisms of the food chain: bacteria (Aliivibriofischeri), algae (Scenedesmus subspicatus), daphnids (Daphnia magna) and fishes(Danio rerio). The aqueous leachate at natural pH (2.0) and solvent extracts wereextremely ecotoxic, while the aqueous leachate at pH = 5.0 showed the lowestecotoxicity. The global ranking of sensitivity for the biotests tested was: daphnids >algae > bacteria > fishes. Thus, the use of this bioassay battery could improve ourunderstanding of the impact of DPM on aquatic environments, which is dependent onthe pH of the leaching process

Use of agro-industrial organic sludge amendment to remediate degraded soil: chemical and eco(geno)toxicological differences between fresh and stabilized sludge and establishment of application rates

International audienceSoil degraded by coal mining activities can be remediated by amendment with agro-industrial organic sludge. However, the environmental impacts associated with this management practice must be properly addressed. In this context, the objective of this study was to evaluate the eco(geno)toxicity of a fresh and a stabilized sludge before use in a laboratory soil remediation test. Chemical analysis of the complex mixtures (degraded soil, fresh sludge, and stabilized sludge) was carried out, as well as a battery of eco(geno)toxicity tests on microbiological enzymes (fluorescein hydrolysis), earthworms, and higher plants (including Vicia faba genotoxicity test), according to published methodologies. The results of these tests showed that fresh sludge was more toxic than sludge stabilized over 6 months toward earthworms and higher plants (lettuce, corn, and wild cabbage), while phyto(geno)toxicity tests with V. faba indicated the same genotoxicity levels for the two types of sludge. In the soil remediation simulation using different mixtures of degraded soil and stabilized sludge, the proportions of 50:50 % (dry weight basis) provided the lowest phyto(geno)toxicity effects and this mixture can be used for the revegetation of the contaminated site

Investigation of differences in sensitivity between 3 strains of Daphnia magna (crustacean Cladocera) exposed to malathion (organophosphorous pesticide)

International audienceAcute and chronic ecotoxic effects of organophosphorous insecticide malathion (Fyfanon 50 EC 500g L-1) were investigated on three strains of Daphnia magna. The nominal effective concentrations immobilizing 50% (EC50) of Daphnia after 24 and 48h were 0.53 and 0.36 mu g L-1, 0.70 and 0.44 mu g L-1, and 0.75 and 0.46 mu g L-1 for the strains 1, 2, and 3 respectively. There was an increase in malathion ecotoxicity with time of exposure as confirmed by chronic studies. In fact, after 21days of exposure, significant effects on survival (lowest observed effect concentration or LOECs) were recorded at malathion concentrations of 22, 220 and 230ng L-1 for strains 1, 2 and 3 respectively. Other endpoints were also examined, including reproduction (with different parameters), body length and embryo toxicity. ICs10 and ICs20 were calculated for these different parameters. ICs10 ranged from 4.7 to more than 220ng L-1 for the three tested strains. The most sensitive parameter was the number of neonates per adult (ICs10 = 4.7 and 10.8ng L-1 for strains 1 and 2 respectively) or the number of broods (IC10 = 10ng L-1 for strain 3). Moreover, an increase in embryo development abnormalities was observed at the two highest tested malathion concentrations. Abnormalities comprised undeveloped second antennae, curved or unextended shell spines, and curved post-abdomen spines in live neonates. Results suggest that malathion could act as an endocrine disruptor in D. magna as it interferes with development. It also induces a significant decrease in acetylcholinesterase (AChE) activities for the three strains. Both strains 2 and 3 seemed more sensitive (LOECs = 60ng L-1) than strain 1 (LOEC = 120ng L-1). Our results suggest that the AChE activity response can also be used as a biomarker of inter-strain (or inter-clonal) susceptibility (i.e. strain (or clone)-specific response)

Soil genotoxicity assessment-results of an interlaboratory study on the Vicia micronucleus assay in the context of ISO standardization

International audienceThe Vicia micronucleus assay was standardized in an international protocol, ISO 29200, "Assessment of genotoxic effects on higher plants-Vicia faba micronucleus test," for soil or soil materials (e.g., compost, sludge, sediment, waste, and fertilizing materials). The aim of this interlaboratory study on the Vicia micronucleus assay was to investigate the robustness of this in vivo assay in terms of its applicability in different countries where each participant were asked to use their own seeds and reference soil, in agreement with the ISO 29200 standard. The ISO 29200 standard protocol was adopted for this study, and seven laboratories from three countries (France, Italy, and Brazil) participated in the study. Negative and positive controls were correctly evaluated by 100 % of the participants. In the solid-phase test, the micronucleus frequency (number of micronuclei/1,000 cells) varied from 0.0 to 1.8 for the negative control (i.e., Hoagland's solution) and from 5.8 to 85.7 for the positive control (i.e., maleic hydrazide), while these values varied from 0.0 to 1.7 for the negative control and from 14.3 to 97.7 for the positive control in the case of liquid-phase test. The variability in the data obtained does not adversely affect the robustness of the protocol assessed, on the condition that the methodology described in the standard ISO 29200 is strictly respected. Thus, the Vicia micronucleus test (ISO 29200) is appropriate for complementing prokaryotic or in vitro tests cited in legislation related to risk assessment of genotoxicity potential