Antimony toxicity upon microorganisms from aerobic and anaerobic environments.

Antimony (Sb) is a toxic and carcinogenic metalloid that can be present in contaminated water generated by mining operations and other industrial activities. The toxicity of Sb (III) and Sb (V) to aerobic microorganisms remains limited and unexplored for anaerobic microorganisms involved in hydrogen (H2) and methane (CH4) production. This study aimed to evaluate the toxicity of Sb (III) and Sb (V) upon aerobic and anaerobic microorganisms important in biological wastewater treatment systems. Sb (III) was more toxic than Sb (V) independently of the test and environment evaluated. Under aerobic conditions maintained in the Microtox assay, Sb (V) was not toxic to Allivibrio fischeri at concentrations as high as 500 mg/L, whereas Sb (III) caused just over 50% inhibition at concentration of 250 mg/L after 5 min of exposure. In the respirometry test, for the specific oxygen uptake rate, the concentrations of Sb (III) and Sb (V) displaying 50% inhibition were 0.09 and 56.2 mg/L, respectively. Under anaerobic conditions, exposure to Sb (III) and Sb (V) led to a decrease in microorganisms activity of fermentative and methanogenic processes. The results confirm that the microbial toxicity of Sb depends on its speciation and Sb (III) displays a significantly higher inhibitory potential than Sb (V) in both aerobic and anaerobic environments.

Continuous anaerobic oxidation of methane: Impact of semi-continuous liquid operation and nitrate load on N2O production and microbial community.

This work proves the feasibility of employing regular secondary activated sludge for the enrichment of a microbial community able to perform the anaerobic oxidation of methane coupled to nitrate reduction (N-AOM). After 96 days of activated sludge enrichment, a clear N-AOM activity was observed in the resulting microbial community. The methane removal potential of the enriched N-AOM culture was then studied in a stirred tank reactor (STR) operated in continuous mode for methane supply and semi-continuous mode for the liquid phase. The effect of applying nitrate loads of ∼22, 44, 66, and 88 g NO3- m-3 h-1 on (i) STR methane and nitrate removal performance, (ii) N2O emission, and (iii) microbial composition was investigated. Methane elimination capacities from 21 ± 13.3 to 55 ± 12 g CH4 m-3 h-1 were recorded, coupled to nitrate removal rates ranging from 6 ± 3.2 to 43 ± 14.9 g NO3- m-3 h-1. N2O production was not detected under the three nitrate loading rates applied for the assessment of potential N2O emission in the continuous N-AOM process (i.e. ∼22-66 g NO-3 m-3 h-1). The lack of N2O emissions during the process was attributed to the N2O reducing capacity of the bacterial taxa identified and the rigorous control of dissolved O2 and pH implemented (dissolved O2 values ≤ 0.07 g m-3 and pH of 7.6 ± 0.4). Microbial characterization showed that the N-AOM process was performed in absence of putative N-AOM archaea and bacteria (ANME-2d, M. oxyfera). Instead, microbial activity was driven by methane-oxidizing bacteria and denitrifying bacteria (Bacteroidetes, α-, and γ-proteobacteria).

Approaches applied to detect SARS-CoV-2 in wastewater and perspectives post-COVID-19.

Currently, SARS-CoV-2 has been detected in the influent of wastewater treatment plants (WWTP), pumping stations, manholes, sewer networks and sludge of WWTP and facilities of countries as France, Spain, Italy, Netherlands, United States, Australia, Ecuador, Brazil and Japan. Although this virus has been detected in the wastewater streams, there is no robust method for its detection and quantification in wastewater. This review compiled and analyzed the virus concentration approaches applied to detect the SARS-CoV-2, besides to provide insights about the methodology for viral concentration, limit of detection, occurrence, persistence, and perspectives post-COVID-19 related with the implications of the virus presence in wastewater. The SARS-COV-2 detection in wastewater has been related to virus concentration methods, which present different recovery rates of the virus. The most used viral concentration methods have been the polyethylene glycol (PEG) for precipitation of viral material and the ultrafiltration at molecular weight level. After viral concentration, the detection and quantification of SARS-COV-2 in wastewater are mainly via quantitative reverse transcription polymerase chain reaction (RT-qPCR), which is the clinical assay adapted for environmental purposes. Although in some experiments the positive control during RT-qPCR is running a surrogated virus (e.g., Mengovirus or Dengue virus), RT-qPCR or reverse transcription droplet digital PCR (RT-ddPCR) targeting the gene encoding nucleocapsid (N1, N2 and N3) of SARS-COV-2 are highly recommended to calculate the limit of detection in wastewater samples. Current results suggest that a rigorous methodology to elucidate the positive cases in a region from genomic copies in wastewater is needed.

Effect of transient pH variation on microbial activity and physical characteristics of aerobic granules treating 4-chlorophenol.

Chlorophenols are inhibitory compounds that can be biodegraded by aerobic granules in discontinuous processes. Many industrial wastewaters are characterized by transient pH variation over time. These pH changes could affect the overall granule structure and microbial activity during the chlorophenol biodegradation. The objective of this research was to evaluate the effects of transient pH variation on the specific degradation rate (q), granule integrity coefficient (IC), and size in sequencing batch reactors treating 4-chlorophenol (4-CP). First, aerobic granules were acclimated for efficient 4-CP degradation (>99%). The acclimated granules consisted of 55.7% of the phyla Proteobacteria and 40.6% of Bacteroidetes. The main bacteria belong to the order Sphingobacteriales (24%), as well as Amaricoccus, Acidovorax, Shinella, Rhizobium, and Flavobacterium, some of which are new genera reported in acclimated granules degrading 4-CP. Then, pH changes were applied to the acclimated aerobic granules, observing that acid pHs decreased to a greater extent the specific degradation rate (67% to 99%) than basic pHs (34% to 80%). These pH changes caused the granule disaggregation but with lower effects on the IC. The effects of pH change were mainly on the microbial activity more than the physical characteristics of aerobic granules degrading 4-CP.

Standardized protocol for determination of biohydrogen potential.

Biohydrogen production potential (BHP) depends on several factors like inoculum source, substrate, pH, among many others. Batch assays are the most common strategy to evaluate such parameters, where the comparison is a challenging task due to the different procedures used. The present method introduces the first internationally validated protocol, evaluated by 8 independent laboratories from 5 different countries, to assess the biohydrogen potential. As quality criteria, a coefficient of variation of the cumulative hydrogen production (H max) was defined to be <15 %. Two options to run BHP batch tests were proposed; a manual protocol with periodic measurements of biogas production, needing conventional laboratory materials and analytical equipment for biogas characterization; and an automatic protocol, which is run in a device developed for online measurements of low biogas production. The detailed procedures for both protocol options are presented, as well as data validating them. The validation showed acceptable repeatability and reproducibility, measured as intra- and inter-laboratory coefficient of variation, which can be reduced up to 9 %.

LC-ICP-OES method for antimony speciation analysis in liquid samples.

A method for the analysis of different species of antimony (Sb) that couples liquid chromatography with an inductively coupled plasma-optical emission spectrometry (LC-ICP-OES) system is presented. The method is simple and reliable to separate and quantify directly and simultaneously Sb(III) and Sb(V) in aqueous samples. The calibration curves showed high linearity at the three wavelengths tested. The limits of detection ranged from 24.9 to 32.3 µg/L for Sb(III) and from 36.2 to 46.0 µg/L for Sb(V), at the three wavelengths evaluated. The limit of detection for this method varied depending on the wavelength used. The lowest limit of quantification for Sb(V) (49.9 µg/L) and Sb(III) (80.7 µg/L) was obtained at a wavelength of 217.582 nm. The method sensitivity for Sb(V) was higher compared to Sb(III) at all the wavelengths considered. Samples containing different concentrations of Sb(III) and Sb(V) in three different matrices, i.e., water, basal culture medium, and anaerobic sludge plus basal medium, were analyzed. The coefficients of variation were low and ranged from 0.1 to 5.0 depending on the sample matrix. Recoveries of Sb(III) and Sb(V) were higher than 90% independently of the matrix analyzed and the wavelength used in the analysis.

Theoretical framework for the estimation of H2S concentration in biogas produced from complex sulfur-rich substrates.

A theoretical framework was developed and validated for the estimation of H2S concentration in biogas produced from complex sulfur-rich effluents. The modeling approach was based on easy-to-obtain data such as biological biogas potential (BBP), chemical oxygen demand, and total sulfur content. Considering the few data required, the model fitted well the experimental H2S concentrations obtained from BBP tests and continuous bioreactors reported in the literature. The model supported a correlation coefficient (R2) of 0.989 over the experimental data, obtaining average and maximum errors of ~ 25 and ~ 35%, respectively. The theoretical framework yielded good estimations for a wide range of experimental H2S concentrations (0.2 to 4.5% in biogas). This modeling approach is, therefore, a useful tool towards anticipating the H2S concentration in biogas produced from sulfur-rich substrates and deciding whether the installation of a desulfurization technology is required or not.

Microrespirometric determination of the effectiveness factor and biodegradation kinetics of aerobic granules degrading 4-chlorophenol as the sole carbon source.

In this study, a microrespirometric method was used, i.e., pulse respirometry in microreactors, to characterize mass transfer and biodegradation kinetics in aerobic granules. The experimental model was an aerobic granular sludge in a sequencing batch reactor (SBR) degrading synthetic wastewater containing 4-chlorophenol as the sole carbon source. After 15 days of acclimation, the SBR process degraded 4-chlorophenol at a removal rate of up to 0.9kg CODm(-3)d(-1), and the degradation kinetics were well described by the Haldane model. The microrespirometric method consisted of injecting pulses of 4-chlorophenol into the 24 wells of a microreactor system containing the SBR samples. From the respirograms obtained, the following five kinetic parameters were successfully determined during reactor operation: (i) Maximum specific oxygen uptake rate, (ii) substrate affinity constant, (iii) substrate inhibition constant, (iv) maximum specific growth rate, and (v) cell growth yield. Microrespirometry tests using granules and disaggregated granules allowed for the determination of apparent and intrinsic parameters, which in turn enabled the determination of the effectiveness factor of the granular sludge. It was concluded that this new high-throughput method has the potential to elucidate the complex biological and physicochemical processes of aerobic granular biosystems.

Microbial communities from 20 different hydrogen-producing reactors studied by 454 pyrosequencing.

To provide new insight into the dark fermentation process, a multi-lateral study was performed to study the microbiology of 20 different lab-scale bioreactors operated in four different countries (Brazil, Chile, Mexico, and Uruguay). Samples (29) were collected from bioreactors with different configurations, operation conditions, and performances. The microbial communities were analyzed using 16S rRNA genes 454 pyrosequencing. The results showed notably uneven communities with a high predominance of a particular genus. The phylum Firmicutes predominated in most of the samples, but the phyla Thermotogae or Proteobacteria dominated in a few samples. Genera from three physiological groups were detected: high-yield hydrogen producers (Clostridium, Kosmotoga, Enterobacter), fermenters with low-hydrogen yield (mostly from Veillonelaceae), and competitors (Lactobacillus). Inocula, reactor configurations, and substrates influence the microbial communities. This is the first joint effort that evaluates hydrogen-producing reactors and operational conditions from different countries and contributes to understand the dark fermentation process.

Biohydrogen production from industrial wastewaters.

The feasibility of producing hydrogen from various industrial wastes, such as vinasses (sugar and tequila industries), and raw and physicochemical-treated wastewater from the plastic industry and toilet aircraft wastewater, was evaluated. The results showed that the tequila vinasses presented the maximum hydrogen generation potential, followed by the raw plastic industry wastewater, aircraft wastewater, and physicochemical-treated wastewater from the plastic industry and sugar vinasses, respectively. The hydrogen production from the aircraft wastewater was increased by the adaptation of the microorganisms in the anaerobic sequencing batch reactor.

Performance of a single-chamber microbial fuel cell degrading phenol: effect of phenol concentration and external resistance.

The performance of a single-chamber microbial fuel cell (MFC) using wastewater containing phenol as the anodic fuel was evaluated. The evaluation was performed considering the effects of the presence of different phenol concentrations in the anodic fuel and the external resistance at which the cells were adapted. Maximum power and current densities of 49.8 mW m(-2) and 292.8 mA m(-2) were obtained, respectively. Microbial diversity on the anode surface remained relatively stable when the phenol concentration was increased. Pseudomonas sp. was the most abundant microorganism in the MFC, followed by the genus Geobacter and Shewanella. Phenol degradation was mainly conducted by bacteria present in the wastewater, and its presence did not affect the electricity generation. The operation of the MFC with a resistance different to the adaptation resistance produced lower current and power densities; however, the variation in external resistances did not adversely affect the phenol degradation.

Membrane biofouling mechanism in an aerobic granular reactor degrading 4-chlorophenol.

The membrane fouling of an aerobic granular reactor coupled with a submerged membrane in a sequencing batch reactor (SBR) was evaluated. The fouling analysis was performed by applying microscopy techniques to determine the morphology and structure of the fouling layer on a polyvinylidene fluoride membrane. It was found that the main cause of fouling was the polysaccharide adsorption on the membrane surface, followed by the growth of microorganisms to form a biofilm.

Biodegradation of 4-methylaniline in a sequencing batch reactor.

The biodegradation of the aromatic amine 4-methylaniline (4MA) using an aerobic sequencing batch reactor was evaluated. The specific degradation rate showed an exponential increase during the acclimation of the microorganisms reaching a maximal value of 34 mg 4MA/VSS/h. After the acclimation, the process showed a stable operation. A high similarity index was observed, indicating a low variation in the population diversity. During this period, the physicochemical parameters demonstrated a stable operation of the reactor.

Bacterial community structure in geographically distributed biological wastewater treatment reactors.

Current knowledge of the microbial communities within biological wastewater treatment reactors is incomplete due to limitations of traditional culture-based techniques and despite the emergence of recently applied molecular techniques. Here we demonstrate the application of high-density microarrays targeting universal 16S rRNA genes to evaluate microbial community composition in five biological wastewater treatment reactors in China and the United States. Results suggest a surprisingly consistent composition of microbial community structure among all five reactors. All investigated communities contained a core of bacterial phyla (53-82% of 2119 taxa identified) with almost identical compositions (as determined by colinearity analysis). These core species were distributed widely in terms of abundance but their proportions were virtually the same in all samples. Proteobacteria was the largest phylum and Firmicutes, Actinobacteria, Bacteroidetes were the subdominant phyla. The diversity among the samples can be attributed solely to a group of operational taxonomic units (OTUs) that were detected only in specific samples. Typically, these organisms ranked somewhat lower in terms of abundance but a few were present is much higher proportions.

Effects of short solids retention time on microbial community in a membrane bioreactor.

Effects of operating lab-scale nitrifying membrane bioreactors (MBR) at short solids retention times (SRT=3, 5 and 10d) were presented with focus on reactor performance and microbial community composition. The process was capable of achieving over 87% removal of ammonia and 95% removal of chemical oxygen demand (COD), almost regardless of SRT. The denaturing gradient gel electrophoresis (DGGE) analysis shown that bacterial communities evolved in time in a similar way at different SRT. The results of clone library analysis indicated that Betaproteobacteria was the dominant bacterial group in all the reactors but there were significant difference of species for different SRT with higher species diversity at longer SRT. Ammonia and COD removal efficiencies were not correlated with the number of bacterial species or their diversity.

Effect of starvation and shock loads on the biodegradation of 4-chlorophenol in a discontinuous moving bed biofilm reactor.

The influence of starvation (defined as the period without substrate) and shock loads on the performance of a moving bed sequencing batch reactor degrading 4-chlorophenol (4CP) were investigated. The biomass was acclimated to biodegrade 100 mg/L of 4CP, and the colonization of the packing material was followed. Two starvation periods and two shock loads were studied. The degradation capacity of the suspended and the attached biomass present on the moving bed was also evaluated. The experiments showed that, after the starvation period, the specific degradation rate decreased from 30.5 to 28.5 and 20 mg 4CP/gVSS/h, when starvation periods of 24 and 48 h were applied, respectively. When two concentration peaks of 500 and 1,050 mg/L were applied, a loss of 6% and 8% on the specific degradation rate, respectively, was also observed. The moving bed thus showed great robustness against starvation periods and shock loads. Suspended biomass presented higher specific degradation rates, but attached biomass did not generate a metabolite that is inhibitory when it accumulates.

Performance of an optimally filled discontinuous bioreactor degrading 4-chlorophenol.

The stability of a fed-batch bioreactor operated with a new control strategy (ED-TOC) during a long-term period was evaluated. The ED-TOC strategy uses solely measurements of DO concentration and volume of the reactor to control the influent flow rate, and so the substrate degradation rate oscillates around its maximal value during the whole filling phase. The bioreactor was tested for the degradation of a wastewater containing 4-chlorophenol (4CP) as a model of an inhibitory compound. The experiment was conducted twice in order to investigate whether results were reproducible. Each set of experiments consisted of two phases: acclimation of the biomass to 4-chlorophenol and operation using the ED-TOC strategy. The main conclusion is that the reactor operated by the ED-TOC strategy is robust and stable in large operation periods. Degradation efficiencies greater than 99% as 4CP and greater than 97% as COD degradation were obtained. Toxicity analysis demonstrates that the bioreactor operated by ED-TOC eliminates the toxicity in the wastewater. Community analysis by DGGE was also conducted.

Modeling for the optimal biodegradation of toxic wastewater in a discontinuous reactor.

The degradation of toxic compounds in Sequencing Batch Reactors (SBRs) poses inhibition problems. Time Optimal Control (TOC) methods may be used to avoid such inhibition thus exploiting the maximum capabilities of this class of reactors. Biomass and substrate online measurements, however, are usually unavailable for wastewater applications, so TOC must use only related variables as dissolved oxygen and volume. Although the standard mathematical model to describe the reaction phase of SBRs is good enough for explaining its general behavior in uncontrolled batch mode, better details are needed to model its dynamics when the reactor operates near the maximum degradation rate zone, as when TOC is used. In this paper two improvements to the model are suggested: to include the sensor delay effects and to modify the classical Haldane curve in a piecewise manner. These modifications offer a good solution for a reasonable complexification tradeoff. Additionally, a new way to look at the Haldane K-parameters (micro(o),K(I),K(S)) is described, the S-parameters (micro*,S*,S(m)). These parameters do have a clear physical meaning and, unlike the K-parameters, allow for the statistical treatment to find a single model to fit data from multiple experiments.

The use of fatty acid methyl esters as biomarkers to determine aerobic, facultatively aerobic and anaerobic communities in wastewater treatment systems.

The use of fatty acid methyl esters (FAME) as biomarkers to identify groups of microorganisms was studied. A database was constructed using previously published results that identify FAME biomarkers for aerobic, anaerobic and facultatively aerobic bacteria. FAME profiles obtained from pure cultures were utilized to confirm the predicted presence of biomarkers. Principal component analysis demonstrated that the FAME profiles can be used to determine the incidence of these bacterial groups. The presence of aerobic, anaerobic and facultatively aerobic bacteria in the communities, in four bioreactors being used to treat different wastewaters, was investigated by applying FAME biomarkers.

Event-driven time-optimal control for a class of discontinuous bioreactors.

Discontinuous bioreactors may be further optimized for processing inhibitory substrates using a convenient fed-batch mode. To do so the filling rate must be controlled in such a way as to push the reaction rate to its maximum value, by increasing the substrate concentration just up to the point where inhibition begins. However, an exact optimal controller requires measuring several variables (e.g., substrate concentrations in the feed and in the tank) and also good model knowledge (e.g., yield and kinetic parameters), requirements rarely satisfied in real applications. An environmentally important case, that exemplifies all these handicaps, is toxicant wastewater treatment. There the lack of online practical pollutant sensors may allow unforeseen high shock loads to be fed to the bioreactor, causing biomass inhibition that slows down the treatment process and, in extreme cases, even renders the biological process useless. In this work an event-driven time-optimal control (ED-TOC) is proposed to circumvent these limitations. We show how to detect a "there is inhibition" event by using some computable function of the available measurements. This event drives the ED-TOC to stop the filling. Later, by detecting the symmetric event, "there is no inhibition," the ED-TOC may restart the filling. A fill-react cycling then maintains the process safely hovering near its maximum reaction rate, allowing a robust and practically time-optimal operation of the bioreactor. An experimental study case of a wastewater treatment process application is presented. There the dissolved oxygen concentration was used to detect the events needed to drive the controller.