Bacterial Bioassays Research Articles

Cosmetic nanomaterials in the environment: nano-zinc oxide and zinc-influence on soil microorganisms

This study aimed to investigate the impact of water-containing sunblock products with different residual quantities of bulk ZnO or ZnO nanoparticles (NPs) on soil microorganisms using a bioassay toxicity experiment. The two forms of ZnO were studied at different concentrations ranging from 0 to 10 mg L−1, and leachates obtained from the water disposal during a handwashing simulation experiment were also evaluated, along with raw sunblocks containing both bulk ZnO and ZnO NPs (at 50% and 100%). The key characteristics of each type of ZnO material were analyzed using multiple analytical techniques, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and ultraviolet–visible spectroscopy (UV–Vis). The outcomes revealed that the stability of ZnO nanoparticles was considerably high, with the highest dissolution rate estimated after 36 h as 0.19% of the sunblock's overall ZnO NPs concentration. The different forms of pure ZnO used in the bacterial bioassay demonstrated that the Zn concentration of 10 mg L−1 exhibited the largest inhibition zone area compared to the other treatments. The disc diffusion bioassay findings confirmed that ZnO NPs are active components with greater toxicity than bulk ZnO. These results demonstrated that the antimicrobial effect was exclusively due to the nano-specific influence at higher concentrations. However, additional research is needed to understand better the environmental effects of different types of ZnO particles disposed of by sunblock users. Examining how such substances react in actual environmental conditions is crucial, as they contain various diverse ingredients that may cause varying reactions compared to bulk ZnO particles.

Functionalized Magnetite Nanoparticles: Characterization, Bioeffects, and Role of Reactive Oxygen Species in Unicellular and Enzymatic Systems.

The current study evaluates the role of reactive oxygen species (ROS) in bioeffects of magnetite nanoparticles (MNPs), such as bare (Fe3O4), humic acids (Fe3O4-HA), and 3-aminopropyltriethoxysilane (Fe3O4-APTES) modified MNPs. Mössbauer spectroscopy was used to identify the local surrounding for Fe atom/ions and the depth of modification for MNPs. It was found that the Fe3O4-HA MNPs contain the smallest, whereas the Fe3O4-APTES MNPs contain the largest amount of Fe2+ ions. Bioluminescent cellular and enzymatic assays were applied to monitor the toxicity and anti-(pro-)oxidant activity of MNPs. The contents of ROS were determined by a chemiluminescence luminol assay evaluating the correlations with toxicity/anti-(pro-)oxidant coefficients. Toxic effects of modified MNPs were found at higher concentrations (>10-2 g/L); they were related to ROS storage in bacterial suspensions. MNPs stimulated ROS production by the bacteria in a wide concentration range (10-15-1 g/L). Under the conditions of model oxidative stress and higher concentrations of MNPs (>10-4 g/L), the bacterial bioassay revealed prooxidant activity of all three MNP types, with corresponding decay of ROS content. Bioluminescence enzymatic assay did not show any sensitivity to MNPs, with negligible change in ROS content. The results clearly indicate that cell-membrane processes are responsible for the bioeffects and bacterial ROS generation, confirming the ferroptosis phenomenon based on iron-initiated cell-membrane lipid peroxidation.

Impact of test conditions on the bacterial bioassay in the presence of TiO2 nanoparticles

To assess the fate and behavior of engineered nanoparticles in the aquatic environment, it is crucial to conduct appropriate bacterial bioassay procedure. However, test conditions of bacterial bioassay are limitedly investigated; especially in the field of environmental media chemistry or aging of nanoparticles which interfere the procedure. For this purpose, TiO2 nanoparticles were treated with different test conditions by keeping them in 1% and 100% seawater for one day and 20 days. The bacterial bioassay of treated nanoparticles towards gram-negative (Pseudomonas aeruginosa) and gram-positive (Staphylococcus aureus) bacteria were examined, as well as the mechanism by biochemical responses of bacteria and physicochemical properties of TiO2 nanoparticles. The viability response of tested bacteria reduced remarkably in seawater concentration, while treatment (aging) duration of tested nanoparticles slightly affected the viability response of tested bacteria. Moreover, key events which affected the bioassay response were changed with the treatment duration and media concentration. The results also showed that aging duration and concentration of the media influenced the main physicochemical properties due to the alterations in surface from aging media compared to unaged ones.

A microbial sensor platform based on bacterial bioluminescence (luxAB) and green fluorescent protein (gfp) reporters for in situ monitoring of toxicity of wastewater nitrification process dynamics

To avoid the upset of nitrification process in wastewater treatment plants, monitoring of influent toxic chemicals is essential for stable operation. Toxic chemical compounds can interfere with the biological nitrogen removal, thus affecting plant efficiency and effluent water quality. Here we report the development of fluorescence and bioluminescence bioassays, based on E. coli engineered to contain the promoter region of ammonia oxidation pathway (AmoA1) of Nitrosomonas europaea and a reporter gene (lux or gfp). The fluorescence or bioluminescence signal was measured with newly designed optical devices. The microbial sensors were tested and validated at different concentrations of nitrification-inhibiting compounds such as allylthiourea, phenol, and mercury. The signal decrease was immediate and proportional to inhibitor concentration. The developed bacterial bioassays could detect the inhibition of the nitrification process in wastewater for allylthiourea concentrations of 1 μg/L for E.coli pMosaico-Pamo-gfp and 0.5 μg/L for E.coli pMosaico-Pamo-luxAB. The results were confirmed using water from a wastewater plant, containing nitrification-inhibiting compounds.

Eco-toxicity of water, soil, and sediment from agricultural areas of Kilombero Valley Ramsar wetlands, Tanzania

ABSTRACT This study was conducted in the KVRS to; evaluate the seasonal eco-toxicity of water, soil and sediment samples; establish the suitability of using temperate biotest batteries in tropical systems; classify the toxicity of samples using Fuzzy Rules to estimate potential ecological risks. 143 water, sediment, and soil samples were collected during dry and rainy seasons in plantation areas. Pseudokirchneriella subcapitata, Aliivibrio fischeri and Arthrobacter globiformis bioassays were used to assess the toxicity of the samples. Results were categorized and classified into toxicity classes. Dry season presented a significantly higher mean inhibition of 31% than 5% shown by rainy season samples (p < 0.001) in the bacterial bioassays, indicating a lower concentration of contaminants due to flooding and increased surface runoff. A few sediment samples resulted into 100% inhibition of A. globiformis, implying organisms were physiologically inactive upon exposure to contaminants. Seventy-three percent of samples posed little or no toxic potential risk, 25% posed critical risk and 1% posed elevated critical risk, implying the KVRS ecosystem might be at risk if the extensive usage of pesticides in the area is not well managed and monitored. The temperate micro-biotests can be used in tropical systems, but with further research on suitable organisms and standardized methods.

Discovered acetylcholinesterase inhibition and antibacterial activity of polyacetylenes in tansy root extract via effect-directed chromatographic fingerprints

The knowledge about the activity of polyacetylenes was extended by their new acetylcholinesterase inhibition and antibacterial activity against plant pathogenic bacteria. For this discovery, an utmost streamlined workflow, which we consider to be of high potential in the field of natural product or superfood search was developed. It demonstrates the combined power of biological, biochemical and chemical fingerprints. Bioactive components of tansy (Tanacetum vulgare L.) root extract were profiled and identified by high-performance thin-layer chromatography hyphenated with in situ effect-directed analysis, chemical derivatizations and high-resolution mass spectrometry (HPTLC-UV/Vis/FLD-EDA-HRMS). The effect-directed profiling was performed using four bacterial bioassays including two plant pathogens, an antioxidant assay and acetyl- and butyrylcholinesterase inhibitory assays. The chromatographic, spectral and powerful mass spectrometric study of zones that exerted substantial antibacterial and/or antioxidant and/or acetylcholinesterase inhibitory effects allowed these multi-potent zones to be identified as polyacetylenes. Five polyacetylene compounds were assigned to be 2-non-1-ene-3,5,7-triynyl-3-vinyl-oxirane, 2-(2,4-hexadiynylidene)-3,4-epoxy-1,6-dioxaspiro[4.5]decane, trans- and cis-2-(2,4-hexadiynylidene)-1,6-dioxaspiro[4.5]dec-3-ene and tetradeca-2,4,6-triine-8-en-12-one. This study clearly showed the advantage of the combined use of different ionization sources, i.e. electrospray ionization via an elution-head based interface and also the Direct Analysis in Real Time interface, for HRMS analysis of compounds from the same class with very similar chromatographic behavior and polarity.

Microbial Mutagenicity Assay: Ames Test.

The Microbial mutagenicity Ames test is a bacterial bioassay accomplished in vitro to evaluate the mutagenicity of various environmental carcinogens and toxins. While Ames test is used to identify the revert mutations which are present in strains, it can also be used to detect the mutagenicity of environmental samples such as drugs, dyes, reagents, cosmetics, waste water, pesticides and other substances which are easily solubilized in a liquid suspension. We present the protocol for conducting Ames test in the laboratory.

Nutrient availability as major driver of phytoplankton-derived dissolved organic matter transformation in coastal environment

Incubation experiments were performed to examine the processing of fresh autochthonous dissolved organic matter (DOM) produced by coastal plankton communities in spring and autumn. The major driver of observed DOM dynamics was the seasonally variable inorganic nutrient status and characteristics of the initial bulk DOM, whereas the characteristics of the phytoplankton community seemed to have a minor role. Net accumulation of dissolved organic carbon (DOC) during the 18-days experiments was 3.4 and 9.2 µmol l−1 d−1 in P-limited spring and N-limited autumn, respectively. Bacterial bioassays revealed that the phytoplankton-derived DOC had surprisingly low proportions of biologically labile DOC, 12.6% (spring) and 17.5% (autumn). The optical characteristics of the DOM changed throughout the experiments, demonstrating continuous heterotrophic processing of the DOM pool. However, these temporal changes in optical characteristics of the DOM pool were not the same between seasons, indicating seasonally variable environmental drivers. Nitrogen and phosphorus availability is likely the main driver of these seasonal differences, affecting both phytoplankton extracellular release of DOM and its heterotrophic degradation by bacteria. These findings underline the complexity of the DOM production and consumption by the natural planktonic community, and show the importance of the prevailing environmental conditions regulating the DOM pathways.

Assessment of Rauvolfia nukuhivensis: from uses to biological ac-tivities and chemodiversity

Rauvolfia genus, biogeographically widespread in tropical areas, includes many species used in traditional medicine. Studies of these species showed that their uses are related to their bioactive content mostly composed by alkaloid compounds. Rauvolfia nukuhivensis, called locally “tueiao”, is an endemic species grown in Nuku-Hiva island located in Marquesas archi-pelago where the bark is used for intimate woman care [1]. Ethnopharmacological approach was adopted to assess this plant. For that purpose, firstly, we checked if the plant extract played an antiseptic role within antimicrobial effects by testing the extract and contents on bacterial and fungi strains (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus niger) and bioassay results showed moderate to low activities. Then, we supposed that traditional medicinal uses of this plant may be linked to biological activities having close relationship with secretory mechanisms regulation. This hypothesis was investigated by the ion channels inhibition related to osmotic exchanges and then, inhibiting effects on hNav1.6 currents and hERG channel were so investigated. Some alkaloidal constituents, and more especially nukuhivensiums, were shown to significantly induce a reduction of IKr amplitude (hERG current). According to these biological activities, a computational study through docking was performed in order to illustrate these results [2,3]. The chemodiversity of the alkaloids from the bark of this plant was assessed by three inte-grated approaches mainly: structural elucidation, phylogenetic analysis and a putative biosynthesis hypothesis statement. Structural elucidation by spectroscopic means (MS, NMR) led to the identification of 13 major constituents belonging to four distinct indole alkaloid skeletons (ajmalane, sarpagane, macroline and β-carboline) within six new naturally occuring compounds. As the Rauvolfia genus is well distributed in the Pacific region, we carried out a phylogenetic study (using DNA barcoding method) of Rauvolfia species, endemic or indigenous in Oceania, as a second approach, aiming to a better understanding of the occurence and distribution of these alkaloids. The very rare co-occurence of these alkaloids belonging to four different skeletons inspired to set up a putative biosynthesis of these components, as a third way of investigation of their chemodiversity

Variation in toxicity during the biodegradation of various heterocyclic and homocyclic aromatic hydrocarbons in single and multi-substrate systems

In the present study, an attempt was made to understand the variation in the toxicity during the biodegradation of aromatic hydrocarbons in single and multi-substrate system. The bacterial bioassay based on the inhibition of dehydrogenase enzyme activity of two different bacterial sp. E.coli and Pseudomonas fluorescens was used for toxicity assessment. Amongst the chosen pollutants, the highest acute toxicity was observed for benzothiophene followed by benzofuran having EC50 value of 16.60mg/L and 19.30mg/L respectively. Maximum residual toxicity of 30.8% was observed at the end during the degradation of benzothiophene. Due to the accumulation of transitory metabolites in both single and multisubstrate systems, reduction in toxicity was not proportional to the decrease in pollutant concentration. In multi-substrate system involving mixture of heterocyclic hydrocarbons, maximum residual toxicity of 39.5% was observed at the end of biodegradation. Enhanced degradation of benzofuran, benzothiophene and their metabolic intermediates were observed in the presence of naphthalene resulting in significant reduction in residual toxicity. 2 (1H) - quinolinone, an intermediate metabolite of quinoline was observed having significant eco-toxicity amongst all other intermediates investigated.

Evaluation of the properties of potassium ferrate used for water purification by luminescence bioassay

Characteristics of four natural water samples from urban and rural areas and the efficiency of a new purifying agent, potassium ferrate K2FeO4, were studied by bacterial luminescence bioassay for 30 minutes. It was revealed that two samples of water from the urban areas are toxic, while the other two samples (one from urban and one from rural environment) are nontoxic. Numerous data obtained on the increase in toxicity index with time allow reasonable conclusions to be made about the chemical nature of substances present in the test water samples. Toxic natural water samples were likely to contain heavy metals and were well purified using potassium ferrate, including via their adsorption. In nontoxic natural water samples, toxic complexes with organic compounds present in water could form at the addition of potassium ferrate. The obtained data call for further studying the properties of potassium ferrate complexes with organic compounds. Bacterial luminescence bioassay is a promising method for the rapid assessment of properties of various water sources (their integral toxicity and presumable chemical composition) and new reagents for their purification (effective concentrations, bactericidal properties, and mechanisms of interacting with heavy metals and organic substances in water).

Effect-Directed Discovery of Bioactive Compounds Followed by Highly Targeted Characterization, Isolation and Identification, Exemplarily Shown for Solidago virgaurea.

A nontargeted, effect-directed screening (bioprofiling) and a subsequent highly targeted characterization of antibacterial compounds from plant matrices is demonstrated on the example of Solidago virgaurea root extracts. The procedure comprises high-performance thin-layer chromatography (HPTLC) coupled with six bacterial bioassays including two plant pathogens, a radical scavenging assay, an acetylcholinesterase assay as well as in situ and ex situ mass spectrometric analyses. In situ mass spectra were directly recorded from the adsorbent using the Direct Analysis in Real Time interface (HPTLC-DART-MS), whereas ex situ mass spectra were recorded using an elution head-based interface (HPTLC-ESI-MS). For further bioassay-guided isolation of the main antimicrobial compounds, flash chromatographic fractionation and semipreparative high-performance liquid chromatographic purification were used and nuclear magnetic resonance data allowed the identification of the unknown antimicrobial compounds as 2Z,8Z- and 2E,8Z-matricaria esters. The discovered antibacterial activity was confirmed and specified by a luminometric assay and as minimal inhibitory concentration in the liquid phase.

Comparison of chromium III and VI toxicities in water using sulfur-oxidizing bacterial bioassays

The toxicities of Cr (III) and Cr (VI) in water were evaluated using sulfur-oxidizing bacterial (SOB) bioassays both in batch and fed-batch conditions. Two days were enough for a quick buildup of SOB consortium in the master culture reactor (MCR). At concentrations up to 100 mg L−1, Cr (III) was found to be nontoxic in both conditions, while Cr (VI) at very low concentrations (0.1–2 mg L−1) was very toxic to the SOB. Literature review suggested that the nontoxic nature of Cr (III) might be due to the absence of the iron uptake pathway in Acidithiobacillus caldus (the predominant bacteria in our reactors), which is required for Cr (III) uptake. The 2-h median effective concentration (EC50) values obtained for Cr (VI) in the batch and fed-batch tests were 2.7 mg L−1 and 1.5 mg L−1, respectively.

Autochthonous Dissolved Organic Matter Drives Bacterial Community Composition during a Bloom of Filamentous Cyanobacteria

The dynamics of dissolved organic matter (DOM) and the succession of bacterial community composition (BCC) were investigated during bloom of filamentous cyanobacteria in a mesocosm experiment conducted in the western Gulf of Finland, the Baltic Sea. The effects of labile dissolved organic carbon (glucose), inorganic nutrients (N and P) and large zooplankton (> 100 µm) on the DOM pool, bacterial production and the composition of bacterial communities were analysed over a period of ten days. In addition, the bioavailability of dissolved organic carbon (DOC) and its turnover by heterotrophic bacteria (biomass and respiration) were investigated in three one-week bacterial bioassays. Heterotrophic bacteria rapidly utilised about 25-55 % of the DOC released from the plankton community, thus assuming it to be highly labile DOC. More than half of the accumulating net DOC pool was degraded over seven days, thus assuming it to be labile. In average, labile autochthonous DOC was degraded with bacterial growth efficiency of 25%. A distinct succession of bacterial communities accompanied the supply of autochthonous DOM, with the most prominent responses occurring in a few single phylotypes of the Delta- and Gammaproteobacterial classes. About 40% of the variation in the relative shares of dominant bacterial classes could be explained by changes in the functional groups of autotrophs. Inorganic nutrient treatment proved beneficial to Deltaproteobacteria and increased bacterial production over that of other mesocosms.

Toxicity and mutagenicity of low-metallic automotive brake pad materials

Organic friction materials are standardly used in brakes of small planes, railroad vehicles, trucks and passenger cars. The growing transportation sector requires a better understanding of the negative impact related to the release of potentially hazardous materials into the environment. This includes brakes which can release enormous quantities of wear particulates. This paper addresses in vitro detection of toxic and mutagenic potency of one model and two commercially available low-metallic automotive brake pads used in passenger cars sold in the EU market. The model pad made in the laboratory was also subjected to a standardized brake dynamometer test and the generated non-airborne wear particles were also investigated. Qualitative “organic composition” was determined by GC/MS screening of dichloromethane extracts. Acute toxicity and mutagenicity of four investigated sample types were assessed in vitro by bioluminescence assay using marine bacteria Vibrio fischeri and by two bacterial bioassays i) Ames test on Salmonella typhimurium His− and ii) SOS Chromotest using Escherichia coli PQ37 strain. Screening of organic composition revealed a high variety of organic compounds present in the initial brake pads and also in the generated non-airborne wear debris. Several detected compounds are classified by IARC as possibly carcinogenic to humans, e. g. benzene derivatives. Acute toxicity bioassay revealed a response of bacterial cells after exposure to all samples used. Phenolic resin and wear debris were found to be acutely toxic; however in term of mutagenicity the response was negative. All non-friction exposed brake pad samples (a model pad and two commercial pad samples) were mutagenic with metabolic activation in vitro.

Bioluminescence‐based assays for assessing eco‐ and genotoxicity of airborne emissions

AbstractEcotoxicological tests (or bioassays) are controlled, reproducible tests where ecological responses are determined quantitatively. Due to numerous difficulties arising when airborne emissions are sampled, relatively few ecotoxicological assays have been applied. Aerosol particles are generally collected on a filter, which limits the quantity of the sample, thus also limiting the range of available test organisms. Bacterial bioassays require low sample quantity, and make a good choice when eco‐ or genotoxicity of the sample are to be determined. Of bacterial assays, the bioluminescence inhibition test has been proven applicable for assessing toxicity of airborne contaminants. In this paper diverse test protocols and their modifications are reviewed. Copyright © 2016 John Wiley &amp; Sons, Ltd.

Rapid Bioassay-Guided Isolation of Antibacterial Clerodane Type Diterpenoid from Dodonaea viscosa (L.) Jaeq.

Plant extracts are complex matrices and, although crude extracts are widely in use, purified compounds are pivotal in drug discovery. This study describes the application of automated preparative-HPLC combined with a rapid off-line bacterial bioassay, using reduction of a tetrazolium salt as an indicator of bacterial metabolism. This approach enabled the identification of fractions from Dodonaea viscosa that were active against Staphylococcus aureus and Escherichia coli, which, ultimately, resulted in the identification of a clerodane type diterpenoid, 6β-hydroxy-15,16-epoxy-5β, 8β, 9β, 10α-cleroda-3, 13(16), 14-trien-18-oic acid, showing bacteriostatic activity (minimum inhibitory concentration (MIC) = 64–128 µg/mL) against test bacteria. To the best of our knowledge, this is the first report on antibacterial activity of this metabolite from D. viscosa.

Phototransformation of sulfamethoxazole under simulated sunlight: Transformation products and their antibacterial activity toward Vibrio fischeri

Sulfamethoxazole (SMX) is a bacteriostatic antibiotic ubiquitously found in the aquatic environment. Since conventional biological wastewater treatment is not efficient to remove SMX, photolysis in natural waters can represent an important transformation pathway. It was recently shown that SMX transformation products can retain antibiotic activity. Therefore, it is crucial to better understand photochemical processes occurring in natural water just as the formation of active transformation products (TPs).During long-term SMX photolysis experiments (one week), nine TPs were identified by reference standards. Moreover, five further TPs of photodecomposition of SMX were found. For the first time, a TP with m/z 271 [M+H]+ was observed during photolysis and tentatively confirmed as 4,x-dihydroxylated SMX. The DOC mass balance clearly showed that only around 5 to 10% were mineralized during the experiment emphasizing the need to elucidate the fate of TPs. Bacterial bioassays confirmed that the mixture retains its antibiotic toxicity toward luminescence (24h) and that there is no change over the treatment time on EC50. In contrast, growth inhibition activity was found to slightly decrease over the irradiation time. However, this decrease was not proportional to the transformation of the parent compound SMX.

Development of a bacterial bioassay for atrazine and cyanuric acid detection.

The s-triazine herbicides are compounds which can disseminate into soils and water. Due to their toxic effects on living organisms, their concentrations in drinking water are legislated by WHO recommendations. Here we have developed for the first time, to the best of our knowledge, an alternative method for physicochemical quantification using two bioluminescent bacterial biosensors: E. coli SM003 for cyanuric acid detection and E. coli SM004 for both atrazine and cyanuric acid detection. The concentration of cyanuric acid detection for E. coli SM003 ranges from 7.83 μM to 2.89 mM, and for E. coli SM004 ranges from 0.22 to 15 μM. Moreover, atrazine detection by E. coli SM004 ranges from 1.08 to 15 μM. According to WHO recommendations, the cyanuric acid detection range is sensitive enough to discriminate between polluted and drinking water. Nevertheless, the detection of atrazine by E. coli SM004 is only applicable for high concentrations of contaminants.

Detection of Metal and Organometallic Compounds with Bioluminescent Bacterial Bioassays.

Chemical detection of metal and organometallic compounds is very specific and sensitive, but these techniques are time consuming and expensive. Although these techniques provide information about the concentrations of compounds, they fail to inform us about the toxicity of a sample. Because the toxic effects of metals and organometallic compounds are influenced by a multitude of environmental factors, such as pH, the presence of chelating agents, speciation, and organic matter, bioassays have been developed for ecotoxicological studies. Among these bioassays, recombinant luminescent bacteria have been developed over the past 20years, and many of them are specific for the detection of metals and metalloids. These bioassays are simple to use, are inexpensive, and provide information on the bioavailable fraction of metals and organometals. Thus, they are an essential complementary tool for providing information beyond chemical analysis. In this chapter, we propose to investigate the detection of metals and organometallic compounds with bioluminescent bacterial bioassays and the applications of these bioassays to environmental samples. Graphical Abstract.