Respirometric Assays Research Articles

An efficient and high-throughput method for the evaluation of mitochondrial dysfunction in frozen brain samples after traumatic brain injury.

Mitochondrial function analysis is a well-established method used in preclinical and clinical investigations to assess pathophysiological changes in various disease states, including traumatic brain injury (TBI). Although there are multiple approaches to assess mitochondrial function, one common method involves respirometric assays utilizing either Clark-type oxygen electrodes or fluorescent-based Seahorse analysis (Agilent). However, these functional analysis methods are typically limited to the availability of freshly isolated tissue samples due to the compromise of the electron transport chain (ETC) upon storage, caused by freeze-thaw-mediated breakdown of mitochondrial membranes. In this study, we propose and refine a method for evaluating electron flux through the ETC, encompassing complexes I, II, and IV, in frozen homogenates or mitochondrial samples within a single well of a Seahorse plate. Initially, we demonstrate the impact of TBI on freshly isolated mitochondria using the conventional oxidative phosphorylation protocol (OxPP), followed by a comparison with ETC analysis conducted on frozen tissue samples within the context of a controlled cortical impact (CCI) model of TBI. Additionally, we explore the effects of mitochondrial isolation from fresh versus snap-frozen brain tissues and their storage at -80°C, assessing its impact on electron transport chain protocol (ETCP) activity. Our findings indicate that while both sets of samples were frozen at a single time point, mitochondria from snap-frozen tissues exhibited reduced injury effects compared to preparations from fresh tissues, which were either homogenized or isolated into mitochondria and subsequently frozen for later use. Thus, we demonstrate that the preparation of homogenates or isolated mitochondria can serve as an appropriate method for storing brain samples, allowing for later analysis of mitochondrial function, following TBI using ETCP.

Susceptibility of the Different Oxygen-Sensing Probes to Interferences in Respirometric Bacterial Assays with Complex Media.

Respirometric microbial assays are gaining popularity, but their uptake is limited by the availability of optimal O2 sensing materials and the challenge of validating assays with complex real samples. We conducted a comparative evaluation of four different O2-sensing probes based on Pt-porphyrin phosphors in respirometric bacterial assays performed on standard time-resolved fluorescence reader. The macromolecular MitoXpress, nanoparticle NanO2 and small molecule PtGlc4 and PtPEG4 probes were assessed with E. coli cells in five growth media: nutrient broth (NB), McConkey (MC), Rapid Coliform ChromoSelect (RCC), M-Lauryl lauryl sulfate (MLS), and Minerals-Modified Glutamate (MMG) media. Respiration profiles of the cells were recorded and analyzed, along with densitometry profiles and quenching studies of individual media components. This revealed several limiting factors and interferences impacting assay performance, which include probe quenched lifetime, instrument temporal resolution, inner filter effects (mainly by indicator dyes), probe binding to lipophilic components, and dynamic and static quenching by media components. The study allowed for the ranking of the probes based on their ruggedness, resilience to interferences and overall performance in respirometric bacterial assays. The 'shielded' probe NanO2 outperformed the established MitoXpress probe and the small molecule probes PtGlc4 and PtPEG4.

Performance assessment of the two oxygen sensor based respirometric platforms with complex media and in selective bacterial assays

Two common platforms currently used in respirometric microbial assays were evaluated comparatively with a panel of selective and differential media specific for E. coli, including the MacConkey (MC), M-Lauryl Sulfate (MLS), Minerals Modified Glutamate (MMG) and Rapid Coliform ChromoSelect (RCC) broths. The first automated platform is based on a soluble O2 probe MitoXpress, standard microwell plate substrate and time-resolved fluorescence reader detection. While operating stably in MC and MMG, it showed prominent interferences and instability in RCC and MLS media, due to phenol red dye and the surfactant largely attenuating the probe signal. The second platform, based on the longwave emitting solid-state sensor coatings deposited at the bottom of disposable assay vials and read with a handheld sensor reader Piccolo2. While having lower sample throughput and automation, this platform demonstrated stable operation in all the media, because its sensor dye PtBP is effectively shielded from interference by sample and media components making the assays stable and robust. Calibration functions for E.coli enumeration show linear relationship with measured Threshold Time (TT, hours): Log (CFU/mL) = a - b*TT, with a ranging 7.24–7.73 h-1 and b ranging 0.49–0.77 for the different selective media. These results help to choose the optimal respirometry platform for large-scale applications such as selective microbial assays and culturomics.

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays.

Most of the cell's energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions inany given organ or tissue. Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Ecotoxicity and bioremediation potential assessment of soil from oil refinery station area

PurposeThe aim of the present study was to evaluate the toxicity and biodegradation potential of oil hydrocarbons contaminated soil samples obtained from different depths at an oil refinery station area. An approach involving chemical, microbiological, respirometry and ecotoxicity assessment of soil polluted by oil hydrocarbons was adopted, in order to determine the biodegradability of pollutants and ecotoxicological effects of natural attenuation strategy.MethodsThe ecotoxicity of soil samples was evaluated using an ostracod test kit and a seed germination test. The results of the phytotoxicity assay were expressed as a percentage of seedling emergence and as the relative yield of fresh and dry biomass compared to control plants. The intrinsic biodegradation potential of the contaminated soil was examined using a Micro-Oxymax respirometer. Intrinsic biodegradation rates were estimated from the slopes of linear regressions curves plotted for cumulative O2 uptake. The obtained values were then entered in the mass balance equation for the stoichiometric reaction of hydrocarbon decomposition and converted per kg of soil per day.ResultsAlthough the tested contaminants were biodegradable in the respirometric assay, they were slightly to moderately toxic to plants and extremely toxic to ostracods. The noxious effects raised with the increased concentration of contaminants. The monocotyledonous oat was more tolerant to higher concentrations of oil hydrocarbons than the other test plants, indicating its greater suitability for soil reclamation purposes.ConclusionBy assessing phytotoxicity and effect on ostracod mortality and progress of soil self-decontamination process, proper approach of reclamation of demoted area can be provided.

Hydrothermal carbonization liquor as external carbon supplement to improve biological denitrification in wastewater treatment

This study investigates the suitability of a sequencing batch reactor (SBR) for treating the liquor (HTCL) derived from hydrothermal carbonization (HTC) of sewage sludge in addition to the influent municipal wastewater. The SBR was fed for 80 days with an increasing mixing ratio HTCL/municipal wastewater. HTCL was used as external carbon source to enhance the denitrification process in the water line of the wastewater treatment plant (WWTP). The feeding of the HTCL caused an overloading in the COD and TKN concentration of the biological treatment equal to, respectively, 25% and 8% (Period II), 50% and 16% (Period III) and 100% and 32% (Period IV). Results revealed that good efficiencies of COD removal were obtained during the entire experimental test, accounting up to 91% in Period IV. Moreover, the removal of TN was improved increasing from 28% in the reference period (Period I), where no HTCL was fed, to 33%, 47% and 64% respectively in Period II, III and IV. Thus, an improvement in the denitrification process was obtained when HTCL was fed to the biological reactor in addition to the influent wastewater. The implementation of the SBR system was supported by nitrate uptake rate (NUR) tests and respirometric assays to evaluate the nitrate removal after the addition of HTCL and the fraction of readily biodegradable COD (RBCOD) and soluble biodegradable COD (sCODbio) on the total COD of the HTCL.

Interactions and Cytotoxicity of Human Neurodegeneration- Associated Proteins Tau and α-Synuclein in the Simple Model Dictyostelium discoideum.

The abnormal accumulation of the tau protein into aggregates is a hallmark in neurodegenerative diseases collectively known as tauopathies. In normal conditions, tau binds off and on microtubules aiding in their assembly and stability dependent on the phosphorylation state of the protein. In disease-affected neurons, hyperphosphorylation leads to the accumulation of the tau protein into aggregates, mainly neurofibrillary tangles (NFT) which have been seen to colocalise with other protein aggregates in neurodegeneration. One such protein is α-synuclein, the main constituent of Lewy bodies (LB), a hallmark of Parkinson’s disease (PD). In many neurodegenerative diseases, including PD, the colocalisation of tau and α-synuclein has been observed, suggesting possible interactions between the two proteins. To explore the cytotoxicity and interactions between these two proteins, we expressed full length human tau and α-synuclein in Dictyostelium discoideum alone, and in combination. We show that tau is phosphorylated in D. discoideum and colocalises closely (within 40 nm) with tubulin throughout the cytoplasm of the cell as well as with α-synuclein at the cortex. Expressing wild type α-synuclein alone caused inhibited growth on bacterial lawns, phagocytosis and intracellular Legionella proliferation rates, but activated mitochondrial respiration and non-mitochondrial oxygen consumption. The expression of tau alone impaired multicellular morphogenesis, axenic growth and phototaxis, while enhancing intracellular Legionella proliferation. Direct respirometric assays showed that tau impairs mitochondrial ATP synthesis and increased the “proton leak,” while having no impact on respiratory complex I or II function. In most cases depending on the phenotype, the coexpression of tau and α-synuclein exacerbated (phototaxis, fruiting body morphology), or reversed (phagocytosis, growth on plates, mitochondrial respiratory function, Legionella proliferation) the defects caused by either tau or α-synuclein expressed individually. Proteomics data revealed distinct patterns of dysregulation in strains ectopically expressing tau or α-synuclein or both, but down regulation of expression of cytoskeletal proteins was apparent in all three groups and most evident in the strain expressing both proteins. These results indicate that tau and α-synuclein exhibit different but overlapping patterns of intracellular localisation, that they individually exert distinct but overlapping patterns of cytotoxic effects and that they interact, probably physically in the cell cortex as well as directly or indirectly in affecting some phenotypes. The results show the efficacy of using D. discoideum as a model to study the interaction of proteins involved in neurodegeneration.

Mechanistically driven identification of novel structural alerts for mitochondrial toxicity

Mitochondrial toxicity is a problem of growing concern in modern society, resulting in several serious adverse health effects including cardiac failure, hepatotoxicity, and neurodegenerative disorders. Adverse outcome pathways provide models for structuring a mechanistic understanding of toxicology, but most existing in silico models for prediction of mitochondrial toxicity do not account for the mechanism of action of potential toxicants. In a recent study by Hallinger et al., the Seahorse respirometric assay provides experimental data relating to the mechanism of action of several mitochondrial toxins. This makes possible, for the first time, the development of structural alerts for mitochondrial toxicity linked to mechanisms. By using a maximal common substructure searcher and Bayesian statistical analysis, we have discovered 11 alerts associated with different mechanisms of action. Eight of these are completely novel. By incorporating the mechanisms into the structural alert, more information can be gained about the molecular initiating events involved and build toward more complete adverse outcome pathways for mitochondrial toxicity.

Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors

Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10–260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.

Isolation of Mitochondria from Retinal Pigment Epithelial Cell Cultures and an Application of High-Resolution Respirometric Assay (XFe96 Seahorse Assay).

Intracellular Ca2+ is strictly regulated to maintain optimal levels for function of cellular organelles as well as mitochondrial respiratory signaling at the tricarboxylic acid cycle and electron transport chain level. Optimal Ca2+ concentration for these processes vary between cell types. Furthermore, exposure of mitochondria to sustained, elevated levels of Ca2+ induces mitochondrial Ca2+ overload and damage to mitochondrial oxidative phosphorylation and ATP production. Isolated mitochondria are widely used to study mitochondrial physiology and drug effects on mitochondrial metabolism and respiratory function. However, isolated mitochondria are easily damaged during the mitochondrial isolation process. The present article describes a mitochondrial isolation method using Ca2+-chelation to minimize mitochondrial damage. We follow up the isolation process with an application that requires an optimized buffer Ca2+ concentration: the characterization of their respiratory function using a high-resolution respirometric assay.

Comparative assessment of purified saponins as permeabilization agents during respirometry

Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed in biological applications for transferring hydrophilic molecules through cell membranes. Widely used commercial saponins include digitonin and saponins from soap tree bark, both of which constitute complex mixtures of little definition. We assessed the permeabilization power of pure saponins towards cellular membranes in an effort to detect novel properties and to improve existing applications.In a respirometric assay, we characterized half-maximal permeabilization of the plasma membrane for different metabolites, of the mitochondrial outer membrane for cytochrome C and the full solubilization of mitochondrial inner membrane protein complexes.Beyond the complete list as repository for the field, we highlight several findings with direct applicability. First, we identified and validated α-chaconine as alternative permeabilization agent in respirometric assays of cultured cells and isolated synaptosomes, superior to digitonin in its tolerability for mitochondria. Second, we identified glycyrrhizic acid to form exceptionally small pores impermeable for adenosine diphosphate. Third, in a concentration dependent manner, tomatine proved to be able to selectively permeabilize the mitochondrial outer, but not inner membrane, allowing for novel states in which to determine cytochrome C oxidase activity.In summary, we provide a list of the permeabilization properties of 18 pure saponins. The identification of two saponins, namely tomatine and chaconine, with direct usability in improved or novel cell biological applications within this small subgroup demonstrates the tremendous potential for further functional screening of pure saponins.

Assessment of a fast method to predict the biochemical methane potential based on biodegradable COD obtained by fractionation respirometric tests

The biochemical methane potential test (BMP) is the most common analytical technique to predict the performance of anaerobic digesters. However, this assay is time-consuming (from 20 to over than 100 days) and consequently impractical when it is necessary to obtain a quick result. Several methods are available for faster BMP prediction but, unfortunately, there is still a lack of a clear alternative. Current aerobic tests underestimate the BMP of substrates since they only detect the easily biodegradable COD. In this context, the potential of COD fractionation respirometric assays, which allow the determination of the particulate slowly biodegradable fraction, was evaluated here as an alternative to early predict the BMP of substrates. Seven different origin waste streams were tested and the anaerobically biodegraded organic matter (CODmet) was compared with the different COD fractions. When considering adapted microorganisms, the appropriate operational conditions and the required biodegradation time, the differences between the CODmet, determined through BMP tests, and the biodegradable COD (CODb) obtained by respirometry, were not significant (CODmet (57.8026 ± 21.2875) and CODb (55.6491 ± 21.3417), t (5) = 0.189, p = 0.853). Therefore, results suggest that the BMP of a substrate might be early predicted from its CODb in only few hours. This methodology was validated by the performance of an inter-laboratory studyconsidering four additional substrates.

Respirometric Screening and Characterization of Mitochondrial Toxicants Within the ToxCast Phase I and II Chemical Libraries.

Mitochondrial toxicity drives several adverse health outcomes. Current high-throughput screening assays for chemically induced mitochondrial toxicity typically measure changes to mitochondrial structure and may not detect known mitochondrial toxicants. We adapted a respirometric screening assay (RSA) measuring mitochondrial function to screen ToxCast chemicals in HepG2 cells using a tiered testing strategy. Of 1042 chemicals initially screened at a singlemaximal concentration, 243 actives were identified and rescreened at 7 concentrations. Concentration-response data for 3 respiration phases confirmed activity and indicated a mechanism for 193 mitochondrial toxicants: 149 electron transport chain inhibitors (ETCi), 15 uncouplers and 29 adenosine triphosphate synthase inhibitors. Subsequently, an electron flow assay was used to identify the target complex for 84 of the 149 ETCi. Sixty reference chemicals were used to compare the RSA to existing ToxCast and Tox21 mitochondrial toxicity assays. The RSA was most predictive (accuracy = 90%) of mitochondrial toxicity. The Tox21 mitochondrial membrane potential assay was also highly predictive (accuracy = 87%) of bioactivity but underestimated the potency of well-known ETCi and provided no mechanistic information. The tiered RSA approach accurately identifies and characterizes mitochondrial toxicants acting through diverse mechanisms and at a throughput sufficient to screen large chemical inventories. The electron flow assay provides additional confirmation and detailed mechanistic understanding for ETCi, the most common type of mitochondrial toxicants among ToxCast chemicals. The mitochondrial toxicity screening approach described herein may inform hazard assessment and the in vitro bioactive concentrations used to derive relevant doses for screening level chemical assessment using new approach methodologies.

Impact of digestate and its fractions on mineralization of 14C-phenanthrene in aged soil.

The impact of whole digestate (WD) and its fractions (solid [SD] and liquid [LD]) on 14C-phenanthrene mineralization in soil over 90d contact time was investigated. The 14C-phenanthrene spiked soil was aged for 1, 30, 60 and 90d. Analysis of water-soluble nitrogen, phosphorus, total (organic and inorganic) carbon, and quantitative bacterial count were conducted at each time point to assess their impact on mineralization of 14C-phenanthrene in soils. Indigenous catabolic activity (total extents, maximum rates and lag phases) of 14C-phenanthrene mineralization were measured using respirometric soil slurry assay. The soil amended with WD outperformed the SD and LD fractions as well as showed a shorter lag phase, higher rate and extent of mineralization throughout the study. The digestates improved (P<0.05) the microbial population and nutritive content of the soil. However, findings showed that spiking soil with phenanthrene generally reduced the growth of microbial populations from 1 to 90d and gave a lower nutritive content in comparison with the non-spiked soil. Also, soil fertility and bacteria count were major factors driving 14C-phenanthrene mineralization. Particularly, the non-phenanthrene degraders positively influenced the cumulative mineralization of 14C-phenanthrene after 60d incubation. Therefore, the digestates (residue from anaerobic digestion) especially WD, which enhanced 14C-phenanthrene mineralization of the soil without minimal basal salts medium nor additional degraders should be further exploited for sustainable bioremediation of PAHs contaminated soil.

Modelling N2O dynamics of activated sludge biomass: Uncertainty analysis and pathway contributions

Nitrous oxide (N2O) is a potent greenhouse gas emitted during biological wastewater treatment. A pseudo-mechanistic model describing three biological pathways for nitric oxide (NO) and N2O production was calibrated for mixed culture biomass from an activated sludge process using laboratory-scale experiments.The model (NDHA) comprehensively describes N2O producing pathways by both autotrophic ammonium oxidizing bacteria and heterotrophic bacteria. Extant respirometric assays and anaerobic batch experiments were designed to calibrate endogenous and exogenous processes (heterotrophic denitrification and autotrophic ammonium/nitrite oxidation) together with the associated net N2O production. Ten parameters describing heterotrophic processes and seven for autotrophic processes were accurately estimated (variance/mean < 25%). The model predicted accurately NO and N2O dynamics at varying dissolved oxygen, ammonium and nitrite levels, and was validated against an independent set of experiments with the same biomass.In aerobic ammonium oxidation experiments the nitrifier denitrification and heterotrophic denitrification estimated pathway contributions increased at high nitrite and low oxygen concentrations; while the nitrifier nitrification pathway showed the largest contribution at high dissolved oxygen levels. The uncertainty of N2O emissions during model calibration is commonly overlooked, which limits the confidence of model-based mitigation strategies. Here we show that the precision of the estimated parameters resulted in a low uncertainty of the N2O emission factors during aerobic ammonium oxidation at DO ≈ 2.0 mg/L (1.2 ± 0.1%) and DO ≈ 0.5 mg/L (4.6 ± 0.6%).

Microbiological evaluation of combined advanced chemical-biological oxidation technologies for the treatment of cork boiling wastewater

This paper contains a multidisciplinary approach that will contribute to design and properly evaluate a treatment line for complex biorecalcitrant wastewaters. To demonstrate this approach a specific industrial wastewater (cork boiling wastewater, CBW) was used. A treatment line based on a coagulation-flocculation step followed by an Advanced Oxidation Process (AOP) (solar photo-Fenton) and combined with an aerobic biological system was evaluated. Applied microbiological techniques: optical microscopy, plate count, DNA extraction and qPCR, indicated that some communities disappeared after the activated sludge adaptation period to the partially treated wastewater, while communities that did not disappear were damaged: 2-log reduction of total heterotrophic bacteria (THB) and a decrease in DNA concentration from 200 ng/μL to 65 ng/μL were observed. Therefore, chemical and microbiological results obtained along the set of experiments, suggested the inefficiency of the combined treatment option between solar photo-Fenton and advanced aerobic biological systems for CBW. This led to the necessity of applying solar photo-Fenton without combining with biotreatment and with the objective of improving the effluent quality enough for being reused in the own industry. Toxicity tests, based on different organisms (after coagulation-flocculation followed by solar photo-Fenton), showed increase on acute toxicity (from 46% to 71% by respirometric assays) and the development of chronic toxicity (from 21–29% to 83–90% also measured by respirometric assays), made evident the incompatibility of this type of wastewater with a biological treatment even after the application of an AOP.

Critical analysis of decay rate measurement methodology

In modeling studies, estimation of microorganisms kinetic parameters set is a key element for proper model operation and predictability. Nitrification process is very often, a crucial element of the wastewater treatment systems as bacteria responsible for ammonium and nitrite oxidation are slow growing microorganisms, making whole nitrification process vulnerable to external factors i.e. temperature, inhibition and load fluctuations. Growth and decay rate of nitrifiers decide about amount nitrifying biomass in the wastewater treatment plants, thus the nitrification efficiency. Paper presents analysis of the decay rate (ba) estimation methodology based on respirometric assays measuring the oxygen uptake rate (OUR). Evaluation of this simple and cheap method was made based on decay estimation tests performed on sludge samples from side-stream partial nitritation reactor treating reject water from digested sludge dewatering. Database obtained from these tests were analyzed to evaluate the impact of respirometric assay duration on calculated decay rate values. 11 time ranges were selected for the performed analysis. Calculated ba values were compared showing the optimal test duration between 5–6 hours, while test shorter than 2 hours resulted in unsatisfactory ba outcome.

Determination of the intrinsic kinetic parameters of ammonia-oxidizing and nitrite-oxidizing bacteria in granular and flocculent sludge

The different oxygen affinities of ammonia-oxidizing (AOB) and nitrite-oxidizing bacteria (NOB) are often used to define the operational strategy to achieve partial nitritation (PN) required before the anammox (AMX) process. For this purpose, apparent kinetic parameters are mainly used in the case of granular sludge, which can lead to errors when defining the operational conditions to obtain only nitritation (avoiding nitratation). In the present study, a mathematical methodology is proposed to determine the intrinsic kinetic parameters of AOB and NOB in granular sludge based on data obtained by respirometric assays. Additionally, the oxygen affinity constant (KO2) and maximum specific rate (rmax) of flocculent and granular sludge sample, produced under mainstream and sidestream conditions were determined at various temperatures (15, 20 and 30 °C). The results show that for granules, the intrinsic KO2 and rmax values were lower and higher, respectively, than the apparent values. Furthermore, the KO2 values for flocs and granules at all of the tested temperatures were lower for NOB than for AOB. The values obtained for the kinetic parameters indicated that it is impossible to maintain partial nitritation by only controlling the dissolved oxygen concentration.

DCPIP and Respirometry Used in the Understanding of Ametryn Biodegradation

In view of an environmental scenario of degradation of the terrestrial ecosystem due to the indiscriminate use of organic compounds, it is necessary to use complementary techniques to evaluate the remediation processes of contaminated soils. Thus, the objective of this work was to evaluate the biodegradation of ametryn herbicide, which is applied in sugarcane crops to control broadleaf weeds. The colorimetric assay was carried out by oxidation of 2.6-dichlorophenol-indophenol (DCPIP) in order to quantify the biodegradation of organic compounds present in the soil, in a short period of time. Another method used was the quantification of CO2 from the microbial respiration responsible for the metabolization of organic compounds in soil. The biodegradation of the ametryn herbicide was evaluated in soil, with the addition of microbial consortium as bioaumentation and addition of surfactant, as a form of microbial stimulation. It was observed that the addition of bioaumentation and biostimulation favored the increased metabolism of ametryn, reducing its residence time in the environment. The colorimetric assay is simple to perform, bringing rapid results. The respirometric assay is simple to perform, however, it has had long-term results. Thus, the use of a colorimetric assay as the primary evaluation of the biodegradation process of organic compounds and the use of the respirometric assay for the long-term evaluation of the metabolization process is suggested. Therefore, the use of complementary methods helps in understanding the process of biodegradation of organic compounds in the environment, as well as in the combination of factors that favor the mitigation of contaminated sites.