Changes in Microbial Biofilm Communities during Colonization of Sewer Systems.

The coexistence of sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) in anaerobic biofilms developed in sewer inner pipe surfaces favors the accumulation of sulfide (H2S) and methane (CH4) as metabolic end products, causing severe impacts on sewerage systems. In this study, we investigated the time course of H2S and CH4 production and emission rates during different stages of biofilm development in relation to changes in the composition of microbial biofilm communities. The study was carried out in a laboratory sewer pilot plant that mimics a full-scale anaerobic rising sewer using a combination of process data and molecular techniques (e.g., quantitative PCR [qPCR], denaturing gradient gel electrophoresis [DGGE], and 16S rRNA gene pyrotag sequencing). After 2 weeks of biofilm growth, H2S emission was notably high (290.7±72.3 mg S-H2S liter(-1) day(-1)), whereas emissions of CH4 remained low (17.9±15.9 mg COD-CH4 liter(-1) day(-1)). This contrasting trend coincided with a stable SRB community and an archaeal community composed solely of methanogens derived from the human gut (i.e., Methanobrevibacter and Methanosphaera). In turn, CH4 emissions increased after 1 year of biofilm growth (327.6±16.6 mg COD-CH4 liter(-1) day(-1)), coinciding with the replacement of methanogenic colonizers by species more adapted to sewer conditions (i.e., Methanosaeta spp.). Our study provides data that confirm the capacity of our laboratory experimental system to mimic the functioning of full-scale sewers both microbiologically and operationally in terms of sulfide and methane production, gaining insight into the complex dynamics of key microbial groups during biofilm development.

Anaerobic treatment of swine manure under mesophilic and thermophilic temperatures: Fate of veterinary drugs and resistance genes.

The effect of anaerobic treatment of swine manure at 35 °C (mesophilic) and 55 °C (thermophilic) on methane production, microbial community and contaminants of emerging concern was investigated. Pasteurization pretreatment and post treatment was also investigated in combination with anaerobic treatment at 35 °C. Specific methane production (SMP), 26 pharmaceutical compounds (PhACs) and five antibiotic resistance genes (ARGs) (qnrS, tetW, ermB, sul1 and blaTEM) were evaluated. Mesophilic treatment resulted in the highest SMP regardless of whether pasteurization was applied. Marbofloxacin was the most abundant antibiotic in swine manure. In general, all groups of PhACs showed higher removals under thermophilic temperatures as compared to mesophilic. In general, pasteurization pretreatment followed by mesophilic anaerobic digestion provided the highest removals of ARGs. Finally, the genera Streptococcus, Clostridium and Pseudomonas which contain pathogenic species, were present in the swine manure. Streptococcus, which was the most abundant, was decreased during all the treatments, while the others only decreased under certain treatments.

Could nitrite/free nitrous acid favour GAOs over PAOs in enhanced biological phosphorus removal systems?

Enhanced biological phosphorus removal (EBPR) normally occurs together with nitrogen removal in wastewater treatment plants (WWTPs). In recent years, efforts have been devoted to remove nitrogen via the nitrite pathway (oxidation of ammonia to nitrite and reduction of nitrite to nitrogen gas without going through nitrate), reducing the requirement for carbon and oxygen in the plant. However nitrite and free nitrous acid (FNA), the protonated species of nitrite, have been shown to cause EBPR deterioration under certain concentrations. This study provides a direct comparison between the different levels of FNA inhibition in the aerobic processes of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) by reviewing the studies published in this area. Also, new data is presented assessing the FNA effect on the anaerobic metabolism of these two groups of bacteria. Overall, FNA has shown inhibitory effects on most of the processes involved in the metabolism of PAOs and GAOs. However, the inhibition-initiation levels are different between different processes and, even more importantly between the two groups. In general, PAOs appear to be more affected than GAOs at the same level of FNA, thus giving GAOs competitive advantage over PAOs in EBPR systems when nitrite is present.

Assessing the occurrence of pharmaceuticals and antibiotic resistance genes during the anaerobic treatment of slaughterhouse wastewater at different temperatures.

This study investigates the effect of psychrophilic, mesophilic and thermophilic temperatures on the anaerobic treatment of slaughterhouse wastewater, in terms of biogas production, occurrence of 30 pharmaceutical compounds of veterinary use, 4 antibiotic resistance genes (ARGs) which provide resistance to tetracyclines (tetW), fluoroquinolones (qnrS), macrolide-lincosamide-streptogramin (ermB) and sulfonamides (sul1) antibiotics, as well as class I integron-integrase gene (intI1), related to horizontal gene transfer. The highest methane yield was obtained at a mesophilic temperature (35 °C) (323 mL CH4/g TCOD) followed by the yield obtained at thermophilic temperature (53 °C) (242 mL CH4/g TCOD). Regarding pharmaceuticals, chlortetracycline, oxytetracycline, tilmicosin, and lincomycin were the most abundant in the slaughterhouse wastewater, being detected predominantly in the solid phase (with median concentrations >200 µg/kg dry weight). On the other hand, ciprofloxacin, ofloxacin, norfloxacin, lincomycin and ibuprofen were the most predominant in the anaerobic digestate regardless of the treatment temperature. Psychrophilic temperatures (21 °C) exhibited moderate to low pharmaceuticals removal, while a large fraction of them were removed at a thermophilic temperature reaching 70-90% removals for tetracycline, macrolides and one sulfonamide (sulfapyridine). The highest relative abundance of the quantified ARGs was found at 53 °C, suggesting that thermophilic temperatures normally associated with better removals of pathogens do not necessarily show better removals of antibiotic resistance genes.

Characterizing the biochemical activity of full-scale enhanced biological phosphorus removal systems: A comparison with metabolic models.

The metabolism of polyphosphate accumulating organisms (PAOs) has been widely studied through the use of lab-scale enrichments. Various metabolic models have been formulated, based on the results from lab-scale experiments using enriched PAO cultures. A comparison between the anaerobic stoichiometry predicted by metabolic models with that exhibited by full-scale sludge in enhanced biological phosphorus removal (EBPR) wastewater treatment plants (WWTPs) was performed in this study. Batch experiments were carried out with either acetate or propionate as the sole carbon source, using sludges from two different EBPR-WWTPs in Australia that achieved different phosphorus removal performances. The results support the hypothesis that the anaerobic degradation of glycogen is the primary source of reducing equivalents generated by PAOs, however, they also suggested a partial contribution of the tricarboxylic acid (TCA) cycle in some cases. The experimental results obtained when acetate was the carbon source suggest the involvement of the modified succinate-propionate pathway for the generation of poly-beta-hydroxyvalerate (PHV). Overall, the batch test results obtained from full-scale EBPR sludge with both substrates were generally well described by metabolic model predictions for PAOs.

Optimization of free nitrous acid pre-treatment on waste activated sludge.

The effectiveness of the Free Nitrous Acid (FNA) sludge treatment was tested in the range from 0 to 3.0 mg N-HNO2/L with acidified and neutral pH. 4 h pre-treatment times were used and the specific methane production (SMP) investigated. Results show that between 50 and 100 mg/L of N-NO2-/L disappeared during the FNA pre-treatment, reducing its effectiveness. A minimum level of nitrite (174 mg N-NO2-/L tested in this study), independently of pH/FNA, was necessary to assure the presence of the chemical throughout the duration of the pre-treatment. Sludge viability was compromised while WAS solubilization and SMP were enhanced with nitrite concentrations of 174 mg N-NO2-/L or higher, even at low FNA levels (<0.15 mg N-HNO2/L). Results show that acidified pH is not needed to enhance methane production, making the pretreatment more economically and environmentally attractive.

Effect of free nitrous acid pre-treatment on primary sludge at low exposure times.

The present study was undertaken to investigate the effect of different free nitrous acid (FNA) concentrations at low pre-treatment times (PTs) (1, 2 and 5h) and without pH control with mild agitation on primary sludge (PS) biodegradability and methane production (MP). Increasing PTs resulted in an increase in the solubility of the organic matter (around 25%), but not on cell-mortality (>75% in all the cases with FNA) and neither on methane generation. FNA pre-treatment at low PTs improve MP (around 16% at PT of 1h and 650mg N-NO2-/L). However, a similar improvement was found with mild agitation of PS without FNA at 2 and 5h. Taking into account the potential costs associated with the FNA pre-treatment, a mild agitation without FNA would be preferred to enhance MP in PS.

Exploring alternatives to reduce economical costs associated with FNA pre-treatment of waste activated sludge.

Recent studies have shown the effectiveness of Free Nitrous Acid (FNA) pre-treatment in enhancing sludge biodegradability and improving its methane production potential. FNA is regarded as an environmental friendly pre-treatment which can be easily applied when a source of nitrite is present in wastewater treatment plants. However, when nitrite is not available and needs to be purchased, this treatment can become less attractive due to the costs associated to nitrite. In order to overcome this possible limitation, two different strategies to optimize the use of nitrite during FNA treatment were investigated: i) Recovering NO2- after the pre-treatment is completed; and ii) Concentrating the sludge before FNA pre-treatment. Results show that recovering NO2- from the pre-treated sludge is not suitable due to the loss of soluble organic matter present in the supernatant after the pre-treatment. However, concentrating the sludge before the pre-treatment seems a good strategy to optimize the use of nitrite.

Improving the start-up of an EBPR system using OUR to control the aerobic phase length: a simulation study.

The enhanced biological phosphorus removal (EBPR) process is based on enriching the sludge with polyphosphate accumulating organisms (PAO) which are scarce in conventional non-EBPR wastewater treatment plant sludge. Hence, the start-up of EBPR systems (i.e. enriching the sludge with PAO) can be very slow and complex. A simulation study of a possible improvement of the start-up of an EBPR system in a sequencing batch reactor is presented in this work. The improvement is based on reducing the length of the aerobic phase so that it coincides with the depletion of orthophosphate from the medium. This improvement, though verified by simulation to be very successful, requires a good on-line orthophosphate sensor. To avoid this technical limitation, a link between oxygen uptake rate (OUR) measurements and orthophosphate presence is proposed. This link allows the control of the aerobic phase length with OUR as a measured variable and, consequently, a considerable improvement with respect to the conventional fixed aerobic phase length operation. An improvement of 95% in the ratio of PAO to heterotrophs and an increase of 30% in the final amount of PAO in sludge is achieved with this control strategy. The kinetic mod for simulations was a modification of the Activated Sludge Model 2d.

Assessment of free nitrous acid pre-treatment on a mixture of primary sludge and waste activated sludge: Effect of exposure time and concentration.

Free nitrous acid (FNA) has been shown to enhance the biodegradability of waste activated sludge (WAS) but its effectiveness on the pre-treatment of mixed sludge is not known. This study explores the effectiveness of four different FNA concentrations (0, 2.49, 3.55, 4.62mgN-HNO2/L) and three exposure times (2, 5, 9h) lower than the ones reported in literature (24h) on WAS characteristics and specific methane production (SMP). FNA pre-treatment reduced sludge cell viability below 10% in all cases after an exposure time of 5h, increasing the solubility of the organic matter. The treated mixed sludge was used as substrate for the biochemical methane production tests to assess its SMP. Results showed a significant increase (up to 25%) on SMP when the sludge was pretreated with the lowest FNA concentration (2.49mgN-HNO2/L) during 2 and 5h but did not show any improvement at longer exposure times or higher FNA concentrations.

Minimizing N2O emissions and carbon footprint on a full-scale activated sludge sequencing batch reactor.

A continuous, on-line quantification of the nitrous oxide (N2O) emissions from a full-scale sequencing batch reactor (SBR) placed in a municipal wastewater treatment plant (WWTP) was performed in this study. In general, N2O emissions from the biological wastewater treatment system were 97.1 ± 6.9 g N2O-N/Kg [Formula: see text] consumed or 6.8% of the influent [Formula: see text] load. In the WWTP of this study, N2O emissions accounted for over 60% of the total carbon footprint of the facility, on average. Different cycle configurations were implemented in the SBR aiming at reaching acceptable effluent values. Each cycle configuration consisted of sequences of aerated and non-aerated phases of different time length being controlled by the ammonium set-point fixed. Cycles with long aerated phases showed the largest N2O emissions, with the consequent increase in carbon footprint. Cycle configurations with intermittent aeration (aerated phases up to 20-30 min followed by short anoxic phases) were proven to effectively reduce N2O emissions, without compromising nitrification performance or increasing electricity consumption. This is the first study in which a successful operational strategy for N2O mitigation is identified at full-scale.

Response of an EBPR population developed in an SBR with propionate to different carbon sources.

The effect of different carbon sources (propionate, acetate, butyrate and glucose) on an enhanced biological phosphorus removal biomass developed with propionate as the sole carbon source was studied. Firstly, a group of different cycle studies was carried out using each substrate independently and then, another cycle study was performed with a mixture of substrates. Propionate was found to be the substrate with the highest substrate uptake rate in both sets of experiments. It was also the volatile fatty acid (VFA) which required less reducing power and less P-release to be uptaken. Four different polyhydroxyalkanoate (PHA) monomers produced during the anaerobic phase were detected, and PHB, PHV and PH2MV were quantified. Significant differences in PHA composition were obtained depending on the carbon source. The carbon recovery ratio for the anaerobic phase was also evaluated. The lowest value observed among the different cycle studies was obtained for butyrate, while the highest value was obtained for acetate.

Evaluation of process conditions triggering emissions of green-house gases from a biological wastewater treatment system.

In this study, methane (CH4) and nitrous oxide (N2O) emission dynamics of a plug-flow bioreactor located in a municipal full-scale wastewater treatment plant were monitored during a period of 10 weeks. In general, CH4 and N2O gas emissions from the bioreactor accounted for 0.016% of the influent chemical oxygen demand (COD) and 0.116% of the influent total Kjeldahl nitrogen (TKN) respectively. In order to identify the emission patterns in the different zones, the bioreactor was divided in six different sampling sites and the gas collection hood was placed for a period of 2-3 days in each of these sites. This sampling strategy also allowed the identification of different process perturbations leading to CH4 or N2O peak emissions. CH4 emissions mainly occurred in the first aerated site, and were mostly related with the influent and reject wastewater flows entering the bioreactor. On the other hand, N2O emissions were given along all the aerated parts of the bioreactor and were strongly dependant on the occurrence of process disturbances such as periods of no aeration or nitrification instability. Dissolved CH4 and N2O concentrations were monitored in the bioreactor and in other parts of the plant, as a contribution for the better understanding of the transport of these greenhouse gases across the different stages of the treatment system.

N2O and NO emissions from a partial nitrification sequencing batch reactor: exploring dynamics, sources and minimization mechanisms.

A sequencing batch reactor (SBR) was enriched with ammonia oxidizing bacteria (AOB) in order to treat synthetic reject wastewater (1 g NH4+ N/L). Partial nitrification was successfully achieved at a NH4+ -N to NO2- -N conversion rate of 98%. The emission dynamics of nitrous oxide (N2O) and nitric oxide (NO) were monitored during normal operation and under 3 different cycle configurations. An N2O peak was detected during the first 5 min of the cycle in all cases which corresponded to 60-80% of the total N2O emitted. When anoxic phases were introduced, N2O emissions were minimized but NO increased. Factors affecting the initial N2O peak were studied in a set of individual experiments. It was concluded that most of this N2O originated during settling due to biological reactions. Complete oxidation of NH4+ (or most likely hydroxylamine) as a result of sufficient aeration time can be a minimization strategy for N2O emissions in partial nitrification systems.

Effect of long-term starvation conditions on polyphosphate- and glycogen-accumulating organisms.

Endogenous processes such as biomass decay and intracellular polymers degradation of polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) were investigated. Cultures enriched in Accumulibacter (a well known PAO) or Competibacter (a well known GAO) were subjected to 21 and 26 days of alternating anaerobic/aerobic conditions respectively. The main energy source for PAOs during starvation was their intracellular polyphosphate released into the medium during the first 14 days of starvation. In contrast, GAOs used their intracellular glycogen during the 26 days of starvation. Biomass decay rates were 0.029 d(-1) for PAOs and almost negligible for GAOs. The reduction in acetate uptake rate during the starvation period, referred to as activity decay, was 0.25 and 0.047 d(-1) for PAOs and GAOs, respectively. Once wastewater was reintroduced, both populations recovered their initial substrate uptake rate after 1 day. The results obtained show that PAOs are more affected than GAOs by starvation conditions.

Nitritation versus full nitrification of ammonium-rich wastewater: comparison in terms of nitrous and nitric oxides emissions.

The processes of nitritation and full nitrification of synthetic reject wastewater were compared in terms of N2O and NO emissions. Two lab-scale sequencing batch reactors (SBR1 and SBR2) were enriched with Nitrosomonas (ammonia-oxidizing bacteria) and Nitrobacter (nitrite-oxidizing bacteria), as shown by fluorescence in situ hybridization (FISH) and high-resolution 16S rRNA tag pyrosequencing. Stable conversion of ammonium to nitrite and nitrite to nitrate was achieved in SBR1 and SBR2 respectively. Biomass from SBR2 was added in SBR1 in order to achieve full nitrification. Under nitritation, 1.22% of the converted-N was emitted as N2O, and 0.066% as NO. During the transition from nitritation to full nitrification, effluent nitrite concentrations decreased but nitrogen oxides were emitted at levels similar to the nitritation period. Gas emissions decreased sharply under full nitrification conditions (0.54% N2O-N/converted-N; 0.021% NO-N/converted-N), probably as a result of the combined effect of lower nitrite and ammonium concentrations in the bioreactor.

Stir-membrane liquid microextraction for the determination of paracetamol in human saliva samples.

BACKGROUND: In this article, stir-membrane liquid microextraction is adapted for the analysis of volume-limited biological samples, using as a model analytical problem the determination of paracetamol in human saliva by LC with UV. A three-phase microextraction mode is used for the extraction of the target analyte, taking advantage of its acid-base properties. RESULTS: All the variables involved in the extraction have been studied and optimized in depth. The method has been analytically characterized on the basis of its linearity, sensitivity and precision. The LOD is 0.5 µg/l while the repeatability, expressed as RSD, is better than 14.2 %. A tenfold preconcentration factor was obtained, which involves an absolute recovery value of 25%. Moreover, the relative recovery is very close to the 100%. CONCLUSION: Finally, the proposed method has been used to perform a PK study of paracetamol.

Stir octadecyl-modified borosilicate disk for the liquid phase microextraction of triazine herbicides from environmental waters.

In the present article, a novel extraction/stirring approach in the liquid phase microextraction context is presented. The new technique is based on octadecyl coated borosilicate disks which act as support of the organic extracting solvent thanks to hydrophobic interactions. The disk is integrated in a stirring element which favors the transference of the analytes to the extraction phase. The proposed extraction procedure has been characterized using the determination of nine herbicides in water samples by ultra performance liquid chromatography (UPLC) combined with ultraviolet (UV) detection as model analytical problem. All the variables involved in the extraction have been studied and optimized in depth and the optimized technique provides enrichment factors in the range from 79 to 839. The method has been analytically characterized on the basis of its linearity, sensitivity and precision. Limits of detection were in the range from 0.14µg/L (atrazine) to 0.56µg/L (terbumeton) with precision better than 7.3% (expressed as relative standard deviation). Relative recoveries were close to 100%, which demonstrated the applicability of the stir octadecyl-modified borosilicate disk for the proposed analytical problem.

Determining the mechanisms for aerobic granulation from mixed seed of floccular and crushed granules in activated sludge wastewater treatment.

Aerobic granulation is a novel and promising technology for wastewater treatment. However, long start-up periods required for the development of granules from floccular sludge, and the loss of biomass in this period leading to poor nutrient removal performance are key challenges. In a recent study the addition of crushed granules to a floccular sludge significantly reduced the start-up period, and also maintained the nutrient removal performance during granulation. In this study, we examined the mechanisms responsible for the fast granulation from a mixture of floccular and granular sludges. Fluorescent microbead particles (4 µm diameter) were successfully applied to differentially label the surfaces of floccular and crushed granular aggregates. Labelled flocs and crushed granules were added to a laboratory scale wastewater treatment reactor, and the granule formation process was monitored using confocal laser scanning microscopy over an 80 day period. Flocs were observed to attach to the surface of the seeding granules, resulting in reduced biomass washout during granulation. This mechanism not only reduces the granulation period, but also maintains the nutrient removal performance of the reactor. The results indicate that the granules acted as nuclei for floccular particle attachment, which accelerated granule formation.