Environmental proteomics as a useful methodology for early-stage detection of stress in anammox engineered systems.

Anammox bacteria are widely applied worldwide for denitrification of urban wastewater. Differently, their application in the case of industrial effluents has been more limited. Those frequently present high loads of contaminants, demanding an individual evaluation of their treatability by anammox technologies. Bioreactors setting up and recovery after contaminants-derived perturbations are slow. Also, toxicity is frequently not acute but cumulative, which causes negative macroscopic effects to appear only after medium or long-term operations. All these particularities lead to relevant economic and time losses. We hypothesized that contaminants cause changes at anammox proteome level before perturbations in the engineered systems are detectable by macroscopic analyses. In this study, we explored the usefulness of short-batch tests combined with environmental proteomics for the early detection of those changes. Copper was used as a model of stressor contaminant, and anammox granules were exposed to increasing copper concentrations including previously reported IC50 values. The proteomic results revealed that specific anammox proteins involved in stress response (bacterioferritin, universal stress protein, or superoxide dismutase) were overexpressed in as short a time as 28 h at the higher copper concentrations. Consequently, EPS production was also increased, as indicated by the alginate export family protein, polysaccharide biosynthesis protein, and sulfotransferase increased expression. The described workflow can be applied to detect early-stage stress biomarkers of the negative effect of other metals, organics, or even changes in physical-chemical parameters such as pH or temperature on anammox-engineered systems. On an industrial level, it can be of great value for decision-making, especially before dealing with new effluents on facilities, deriving important economic and time savings.

Challenges of aerobic granular sludge utilization: Fast start-up strategies and cationic pollutant removal.

Aerobic granular sludge (AGS) is a self-aggregated microorganism consortium with pollutant removal properties. The aim of this work is to study and review the application of aerobic granules for water treatment with special focus on new applications and methodologies. Carbon-nitrogen containing pollutants are the classic targets of AGS technology. Carbon and nitrogen removal of AGS are classified as a biodegradation process. More recently, the AGS granules have been studied as sorbent materials for wastewater treatment. In particular, the sorption of cationic pollutants has been studied through biosorption and bioaccumulation mechanisms without distinguishing when one or the other process is involved. AGS conformation made them suitable for complex wastewater treatment. Indeed, several studies have demonstrated the removal of polyvalent cationic pollutants even with higher capacity than conventional sorbent materials. However, this was achieved almost exclusively for synthetic substrates, with single cation evaluation and using in some cases only qualitative measures. For successful industrial AGS application in complex substrates, it is necessary to evaluate and demonstrate the technology in real industrial conditions and reduce the currently long start-up times which limits its utility. Two new strategies have been proposed: autoinducer molecules and the production of artificial granular from common active sludge with commercial alginate. Finally, the increase of research on AGS cations assimilation properties will allow a new point of view, where granules will be materials for the recovery of valuable metals from industrial wastewater streams.

Characterization of Arsenite-Oxidizing Bacteria Isolated from Arsenic-Rich Sediments, Atacama Desert, Chile.

Arsenic (As), a semimetal toxic for humans, is commonly associated with serious health problems. The most common form of massive and chronic exposure to As is through consumption of contaminated drinking water. This study aimed to isolate an As resistant bacterial strain to characterize its ability to oxidize As (III) when immobilized in an activated carbon batch bioreactor and to evaluate its potential to be used in biological treatments to remediate As contaminated waters. The diversity of bacterial communities from sediments of the As-rich Camarones River, Atacama Desert, Chile, was evaluated by Illumina sequencing. Dominant taxonomic groups (>1%) isolated were affiliated with Proteobacteria and Firmicutes. A high As-resistant bacterium was selected (Pseudomonas migulae VC-19 strain) and the presence of aio gene in it was investigated. Arsenite detoxification activity by this bacterial strain was determined by HPLC/HG/AAS. Particularly when immobilized on activated carbon, P. migulae VC-19 showed high rates of As(III) conversion (100% oxidized after 36 h of incubation). To the best of our knowledge, this is the first report of a P. migulae arsenite oxidizing strain that is promising for biotechnological application in the treatment of arsenic contaminated waters.

The prediction of partial-nitrification-anammox performance in real industrial wastewater based on granular size.

To date, the partial nitrification-Anammox (PN-A) granular sludge size has been exclusively analyzed in synthetic substrates. In this work, different ranges of granular size of PN-A sludge were studied at low oxygen concentration using real industrial wastewater as, well as a synthetic substrate. The granular sludge was characterized by the specific nitrification activity (SNA), specific anammox activity (SAA), and granule sedimentation rate. The relative abundance of the bacterial consortium was assessed for each range of diameters through the fluorescence in situ hybridization (FISH) technique. SNA exhibits a direct association with the specific surface of granules, which proves the importance of the outer layer in the nitrification process. Even more critical, the flocculent sludge allowed the stability of the nitrifying activity. The SAA showed different performances faced the real industrial and synthetic substrates. With the synthetic substrate, the SAA decreased at higher diameter ranges, whereas with the industrial substrate, the SAA increased at higher diameter ranges. This situation is explained by the oxygen protection in the sludge maintained with industrial wastewater. The relative abundance of heterotrophic bacteria increased from 9.6 to 22%, due to the presence of organic matter in the industrial substrate. The granular sedimentation rate increased with the diameter of the granules with a linear correlation (R2 > 0.98). Thus, granular sizes can be selected through sedimentation rate control. A linear correlation between SAA and granular sludge diameter ranges was observed. With this correlation, an error of less than 11% in the prediction of SAA was achieved. The use of diameter measurement and granular sedimentation rate as routine techniques could contribute to the control and start-up of PN-A reactors. In the same sense, organic matter present in defined concentrations, can be beneficial for the granular sludge stability, and thus, for nitrogen removal.

Efficient poultry manure management: anaerobic digestion with short hydraulic retention time to achieve high methane production.

The efficient treatment or appropriate final disposal of poultry manure (PM) to avoid serious environmental impacts is a great challenge. In this work, the optimization of a 2-stage anaerobic digestion system (ADS) for PM was studied with the aim of reaching a maximal methane yield with a short hydraulic retention time (HRT). Three activities were performed: The first activity, ADS 1, consisted of evaluating the effect of the substrate concentration and the HRT on the process, with a constant organic loading rate (OLR) of 3.66 ± 0.21 gVS L-1 d-1. The second activity, ADS 2, consisted of decreasing the HRT from 9.09 to 2.74 d with a constant substrate concentration. In the third activity, ADS 3, the substrate concentration was increased from 10.09 ± 1.41 to 35.25 ± 6.20 gVS L-1 with an average HRT of 4.66 ± 0.11 d. Maximal methane yields of 0.22, 0.21, and 0.22 LCH4 gVS-1 were reached for ADS 1, ADS 2, and ADS 3, respectively, at a low HRT (3.38 to 4.66 d) and high free ammonia concentration (between 323.05 ± 56.48 and 460.93 ± 135.40 mgN-NH3 L-1). These methane yields correspond to the production of 40.36 and 42.28 cubic meters of methane per ton of PM, respectively, and a laying hen produces between 47.45 and 54.75 kg of PM per year in Chile. Finally, this is the first study of the separate and combined effects of OLR, HRT and substrate concentration on the anaerobic digestion of PM. The results demonstrate the technical feasibility of the two-stage ADS treatment of PM with a short HRT; the system tolerates variations in the total ammonia nitrogen concentration of PM throughout the year and achieves a high methane yield when the correct operational conditions are selected.

Correction to: Effluent composition prediction of a two-stage anaerobic digestion process: machine learning and stoichiometry techniques.

The original publication of this paper contains a mistake. Unfortunately, an author was inadvertently missed out, Constanza Arriagada had participated in the operation of the anaerobic digesters cited in the work and now as a PhD student, she is involved in the production of other publication.

Effluent composition prediction of a two-stage anaerobic digestion process: machine learning and stoichiometry techniques.

Computational self-adapting methods (Support Vector Machines, SVM) are compared with an analytical method in effluent composition prediction of a two-stage anaerobic digestion (AD) process. Experimental data for the AD of poultry manure were used. The analytical method considers the protein as the only source of ammonia production in AD after degradation. Total ammonia nitrogen (TAN), total solids (TS), chemical oxygen demand (COD), and total volatile solids (TVS) were measured in the influent and effluent of the process. The TAN concentration in the effluent was predicted, this being the most inhibiting and polluting compound in AD. Despite the limited data available, the SVM-based model outperformed the analytical method for the TAN prediction, achieving a relative average error of 15.2% against 43% for the analytical method. Moreover, SVM showed higher prediction accuracy in comparison with Artificial Neural Networks. This result reveals the future promise of SVM for prediction in non-linear and dynamic AD processes. Graphical abstract ᅟ.

Influence of biomass acclimation on the performance of a partial nitritation-anammox reactor treating industrial saline effluents.

The performance of the partial nitritation/anammox processes was evaluated for the treatment of fish canning effluents. A sequencing batch reactor (SBR) was fed with industrial wastewater, with variable salt and total ammonium nitrogen (TAN) concentrations in the range of 1.75-18.00 g-NaCl L-1 and 112 - 267 mg-TAN L-1. The SBR operation was divided into two experiments: (A) progressive increase of salt concentrations from 1.75 to 18.33 g-NaCl L-1; (B) direct application of high salt concentration (18 g-NaCl L-1). The progressive increase of NaCl concentration provoked the inhibition of the anammox biomass by up to 94% when 18 g-NaCl L-1 were added. The stable operation of the processes was achieved after 154 days when the nitrogen removal rate was 0.021 ± 0.007 g N/L·d (corresponding to 30% of removal efficiency). To avoid the development of NOB activity at low salt concentrations and to stabilize the performance of the processes dissolved oxygen was supplied by intermittent aeration. A greater removal rate of 0.029 ± 0.017 g-N L-1 d-1 was obtained with direct exposure of the inoculum to 18 g-NaCl L-1 in less than 40 days. Also, higher specific activities than those from the inoculum were achieved for salt concentrations of 15 and 20 g-NaCl L-1 after 39 days of operation. This first study of the performance of the partial nitritation/anammox processes, to treat saline wastewaters, indicates that the acclimation period can be avoided to shorten the start-up period for industrial application purposes. Nevertheless, further experiments are needed in order to improve the efficiency of the processes.

Modeling of the Nanoparticles Absorption Under a Gastrointestinal Simulated Ambient Condition.

Proanthocyanidins (PAs) have several bioactivities, but they are unstable in the digestive tract and possess low bioavailability. Nanoencapsulation stabilizes these compounds for oral administration. The intestinal absorption of grape seed and skin extracts, and the poly-lactic acid (PLA) nanoparticles loaded with such extracts was modeled, taking into consideration physicochemical process parameters, evaluating the PAs concentration profile in the human small intestine. Density (ρ), solubility, viscosity (µ), diffusion coefficient (D), and the global mass transfer coefficient (K) for both substrates were estimated, simulating their passing from the intestine into the blood at 37°C. For the seed and skin extracts encapsulated in PLA the physicochemical parameters were: D = 1.81 × 10^-5 and D = 5.72 × 10^-5 cm2/s; K = 3.4 × 10^-3 and K = 2.47 × 10^-4 cm/s, respectively. Lower resistance was offered by the seed extract than by skin extracts (nanoencapsulated), which was explained by differences in structural composition, and average molecular weight of the two kinds of extracts, which should be more favorable to the mass transfer in comparison to the raw extracts. The concentration profile of grape extracts in the small intestine was modeled through a pure convection model, and the encapsulated extract on PLA nanoparticles using a mixed regime model, which described the process of dissolution and absorption of the grape extracts from the intestine to the blood stream. The absorbed fraction predicted by the model was 42.7 and 24.2% for seed and skin extracts, respectively. Those values increased to 100% for both extracts after the simulation with the nanoencapsulated extracts. Consequently, extract encapsulation should produce a significant increase in intestinal absorption.

Technical and economical optimization of a full-scale poultry manure treatment process: total ammonia nitrogen balance.

A full-scale process for the treatment of 80 tons per day of poultry manure was designed and optimized. A total ammonia nitrogen (TAN) balance was performed at steady state, considering the stoichiometry and the kinetic data from the anaerobic digestion and the anaerobic ammonia oxidation. The equipment, reactor design, investment costs, and operational costs were considered. The volume and cost objective functions optimized the process in terms of three variables: the water recycle ratio, the protein conversion during AD, and the TAN conversion in the process. The processes were compared with and without water recycle; savings of 70% and 43% in the annual fresh water consumption and the heating costs, respectively, were achieved. The optimal process complies with the Chilean environmental legislation limit of 0.05 g total nitrogen/L.

Simultaneous removal of C and N from fish effluents in filter reactors: Effect of recirculation ratio on the axial distribution of microbial communities.

We simultaneously removed carbon (C) and nitrogen (N) from fish effluents in compact filter reactors operating at different recirculation ratios (RRs) (2, 10 and without recirculation) to demonstrate microbial coexistence and determine the effect of the RR on the axial bacterial stratification. We also examined the global performance of anoxic, anaerobic and aerobic processes. Microbial communities (bacteria and archaea) were analyzed using 16s rRNA amplification followed by DGGE analyses. Their banding profiles were analyzed using ecological parameters and the most representative bands were sequenced. TOC removal was larger than 98% in the three reactors. The total N removal was 48% for the RR-2 reactor, whereas in the RR-10 reactor, there was no N removal due to the absence of nitrification in the final aerobic step. Coexistence and stratification of microorganisms were observed. The microbial communities were correlated with distinct biochemical processes in each reactor fraction. The RR had a large effect on the distribution of the microbial communities. When the RR increased from 2 to 10, the stratification decreased from 60 to 30%, suggesting a close relationship between reactor performance and the presence of nitrifiers. In the RR-10 reactor, the nitrifier concentration was only 4%. Thus, in combined processes, filter reactors should operate with a moderate RR to favor bacterial stratification and improve performance.

Startup and oxygen concentration effects in a continuous granular mixed flow autotrophic nitrogen removal reactor.

The startup and performance of the completely autotrophic nitrogen removal over nitrite (CANON) process was tested in a continuously fed granular bubble column reactor (BCR) with two different aeration strategies: controlling the oxygen volumetric flow and oxygen concentration. During the startup with the control of oxygen volumetric flow, the air volume was adjusted to 60mL/h and the CANON reactor had volumetric N loadings ranging from 7.35 to 100.90mgN/Ld with 36-71% total nitrogen removal and high instability. In the second stage, the reactor was operated at oxygen concentrations of 0.6, 0.4 and 0.2mg/L. The best condition was 0.2 mgO2/L with a total nitrogen removal of 75.36% with a CANON reactor activity of 0.1149gN/gVVSd and high stability. The feasibility and effectiveness of CANON processes with oxygen control was demonstrated, showing an alternative design tool for efficiently removing nitrogen species.

Simultaneous C and N removal from saline salmon effluents in filter reactors comprising anoxic-anaerobic-aerobic processes: effect of recycle ratio.

Salmon processing generates saline effluents with high protein load. To treat these effluents, three compact tubular filter reactors were installed and an integrated anoxic/anaerobic/aerobic process was developed with recycling flow from the reactor's exit to the inlet stream in order to save organic matter (OM) for denitrification. The reactors were aerated in the upper section with recycle ratios (RR) of 0, 2, and 10, respectively, at 30°C. A tubular reactor behave as a plug flow reactor when RR = 0, and as a mixed flow reactor when recycle increases, thus, different RR values were used to evaluate how it affects the product distribution and the global performance. Diluted salmon process effluent was prepared as substrate. Using loads of 1.0 kg COD m(-3)d(-1) and 0.15 kg total Kjeldahl nitrogen (TKN) m(-3)d(-1) at HRT of 2 d, 100% removal efficiencies for nitrite and nitrate were achieved in the anoxic-denitrifying section without effect of the dissolved oxygen in the recycled flow on denitrification. Removals >98% for total organic carbon (TOC) was achieved in the three reactors. The RR had no effect on the TOC removal; nevertheless a higher efficiency in total nitrogen removal in the reactor with the highest recycle ratio was observed: 94.3% for RR = 10 and 46.6% for RR = 2. Results showed that the proposed layout with an alternative distribution in a compact reactor can efficiently treat high organic carbon and nitrogen concentrations from a saline fish effluent with OM savings in denitrification.

Microscopic modeling of País grape seed extract absorption in the small intestine.

The concentration profiles and the absorbed fraction (F) of the País grape seed extract in the human small intestine were obtained using a microscopic model simulation that accounts for the extracts' dissolution and absorption. To apply this model, the physical and chemical parameters of the grape seed extract solubility (C s), density (ρ), global mass transfer coefficient between the intestinal and blood content (k) (effective permeability), and diffusion coefficient (D) were experimentally evaluated. The diffusion coefficient (D = 3.45 × 10(-6) ± 5 × 10(-8) cm(2)/s) was approximately on the same order of magnitude as the coefficients of the relevant constituents. These results were chemically validated to discover that only the compounds with low molecular weights diffused across the membrane (mainly the (+)-catechin and (-)-epicatechin compounds). The model demonstrated that for the País grape seed extract, the dissolution process would proceed at a faster rate than the convective process. In addition, the absorbed fraction was elevated (F = 85.3%). The global mass transfer coefficient (k = 1.53 × 10(-4) ± 5 × 10(-6) cm/s) was a critical parameter in the absorption process, and minor changes drastically modified the prediction of the extract absorption. The simulation and experimental results show that the grape seed extract possesses the qualities of a potential phytodrug.

Modeling of simultaneous denitrification--anaerobic digestion--organic matter aerobic oxidation and nitrification in an anoxic-anaerobic-aerobic compact filter reactor.

A mathematical model was developed for a compact anoxic-anaerobic-aerobic filter reactor with liquid recirculation for the treatment of fishing effluents. The model includes denitrification, anaerobic digestion, aerobic carbon oxidation and nitrification steps, as well as an evaluation of the liquid gas mass transfer and pH. The model was calibrated using one experimental condition at a recycling ratio (R)=10, and was validated with R equal to 2 and 0, with an organic concentration of 554±24 mg TOCL(-1), salinity of 24 g L(-1) and hydraulic retention time (HRT) of 2 d. Carbon total removal is higher than 98%, while maximum nitrogen removal is 62% using total nitrification in the aerobic zone, due to a higher quantity of NO(x) produced which were recirculated to the anoxic zone. In the aerobic zone, simultaneous nitrification and denitrification processes occur, because the diffusion limitations cause a low oxygen penetration in the biofilm. In the anoxic-anaerobic zone, denitrification or methanogenesis inhibition by DO (caused by the recycled oxygen) is not observed.

Modeling of the denitrification/anaerobic digestion process of salmon fishery wastewater in a biofilm tubular reactor.

The literature has paid scarce attention to the modeling of the denitrification-anaerobic digestion process in packed bed biofilm tubular reactors used to treat wastewater. The present study obtained a steady-state model for industrial salmon fishery wastewater treatment in a biofilm tubular reactor, including pH as a variable and the effect of biomass on hydrolysis. The axial profile of the reactor components and process efficiency were predicted with deviations below 6%. The optimal operating zone for the process was found at hydraulic retention time (HRT)>1.5d and inlet protein concentration (S(prot,0))<3000 mgTOCL(-1). Based on our results, we concluded that the removal of organic matter and nitrogen compounds depended mainly on HRT. The effluent pH was mainly affected by the C/N ratio, where a decrease increases pH. Organic matter removal was related with the anaerobic digestion process, while denitrification influenced mostly nitrate and nitrite removal.

Operating parameters for high nitrite accumulation during nitrification in a rotating biological nitrifying contactor.

Incomplete nitrification was studied in a completely and partially submerged rotating biological contactor (RBC). In a partially submerged RBC without additional aeration, 50 to 90% nitrite accumulation (alpha) was achieved at rotation speeds (omega) of 2 to 18 min(-1). In a completely submerged RBC operating during 80 days, a higher alpha of 96% was achieved at omega = 2 min(-1). Incomplete nitrification in a completely submerged RBC at oxygen concentrations of 1.5 to 6.8 mg O2/L indicated that the mass transfer of oxygen is rate-limiting. Modeling of the completely submerged RBC predicts that the oxygen profile will not penetrate the biofilm more than 30 microm, thereby strongly limiting the nitrite-oxidizer growth and causing high nitrite accumulation. Molecular analysis (i.e., fluorescence in situ hybridization) indicated that the nitrite-oxidizers are superficially located (<200 microm) and that the ammonia-oxidizers comprise up to approximately 800 microm of the biofilm.

The effect of sodium chloride on the two-step kinetics of the nitrifying process.

Sodium chloride affects the transformation rate of several compounds in bioreactors. Most authors report a decrease in microorganism activity at increasing salt concentrations. In this work, a kinetic model that relates sodium chloride concentration with the rates of each step of the nitrification process is proposed; thus, the effect of sodium chloride concentration (0 to 60 g/L) on the nitritation and nitratation rates was separately studied. To carry out the independent study of each step, a combination of the respirometric method with sodium azide, an inhibitor of the nitratation step, was performed. The dot-blot hybridization technique with 16S rRNA-targeted probes was used to determine the ammonia-oxidizing and nitrite-oxidizing bacterial fraction, then it was possible to relate the culture's function with its biological composition. Rates of both steps were linearly reduced at increasing salt concentrations: the nitratation rate was more affected than the nitritation rate. Simulations carried out in a nitrifying sequencing batch reactor indicate that nitrite might accumulate at high salt concentrations. Sodium chloride exerts a reversible inhibition on ammonia oxidation and nitrite oxidation.

High nitrite buildup during nitrification in a rotating disk reactor.

Incomplete nitrification with high nitrite accumulation has three practical advantages: lower oxygen consumption, less need for organics for denitrification, and lower sludge production during denitrification. Nitrification leading to high nitrite formation was experimentally studied in a continuous single rotating disk reactor (RDR) and compared to a modeled continuous completely stirred tank reactor (CSTR). The results of this model show that to accumulate nitrite greater than 50% at oxygen levels higher than 3.5 mg O2/L, pH levels higher than 8.5 and 9.0 are required for a CSTR with and without cell washout, respectively. For a CSTR without cell washout at pH 7 and 1 mg O2/L, it was predicted that a nitrite accumulation less than 5% could be reached. Conversely, for a partially submerged continuous RDR without any additional aeration supply (already at pH 7 and 1.3 mg O2/L), high nitrite accumulation (more than 75%) was achieved and the influence of pH from 7 to 9 was not significant. This difference is believed to be caused by mass transfer. In addition, nitrification was observed to occur under oxygen transport limitation for a totally submerged continuous RDR.