Genome-centric metagenomic insights into the role of Chloroflexi in anammox, activated sludge and methanogenic reactors.

BACKGROUND: The phylum Chloroflexi is highly abundant in a wide variety of wastewater treatment bioreactors. It has been suggested that they play relevant roles in these ecosystems, particularly in degrading carbon compounds and on structuring flocs or granules. Nevertheless, their function is not yet well understood as most species have not been isolated in axenic cultures. Here we used a metagenomic approach to investigate Chloroflexi diversity and their metabolic potential in three environmentally different bioreactors: a methanogenic full-scale reactor, a full-scale activated sludge reactor and a lab scale anammox reactor. RESULTS: Differential coverage binning approach was used to assemble the genomes of 17 new Chloroflexi species, two of which are proposed as new Candidatus genus. In addition, we recovered the first representative genome belonging to the genus 'Ca. Villigracilis'. Even though samples analyzed were collected from bioreactors operating under different environmental conditions, the assembled genomes share several metabolic features: anaerobic metabolism, fermentative pathways and several genes coding for hydrolytic enzymes. Interestingly, genome analysis from the anammox reactor indicated a putative role of Chloroflexi in nitrogen conversion. Genes related to adhesiveness and exopolysaccharides production were also detected. Complementing sequencing analysis, filamentous morphology was detected by Fluorescent in situ hybridization. CONCLUSION: Our results suggest that Chloroflexi participate in organic matter degradation, nitrogen removal and biofilm aggregation, playing different roles according to the environmental conditions.

Effect of organic loading rate on the production of Polyhydroxyalkanoates from sewage sludge.

The aim of this work was to study the effect of organic loading rate on the production of Polyhydroxyalkanoates (PHA) from sewage sludge. Synthesis of PHA using sewage sludge as platform was achieved in this work. Three pilot-scale selection-sequencing batch reactors (S-SBR) were used for obtaining a culture able to accumulate PHA following a strategy of aerobic dynamic feeding (ADF) at different volumetric organic-loading-rate (vOLR): 1.3, 1.8 and 0.8 g COD L-1 d-1 for S-SBR 1, S-SBR 2 and S-SBR 3, respectively. Decreasing the vOLR enhanced the general performance of the process as for organic matter removal (from 99.2% ± 0.3% in S-SBR-3 to 92 ± 2 in S-SBR-2) while the opposite trend was recorded for PHA production (6.0 PHA % w/w in S-SBR-3 vs 13.7 PHA % w/w in S-SBR-2 at the end of the feast phase). Furthermore, indirect and direct emissions, as N2O, were evaluated during the process for the first time. Finally, three accumulation tests were performed achieving 24% w/w.

Increasing resource circularity in wastewater treatment: Environmental implications of technological upgrades.

A paradigm shift is needed in wastewater treatment plants (WWTPs) to progress from traditional pollutant removal to resource recovery. However, whether this transformation produces overall environmental benefits will depend on the efficient and sustainable use of resources by emerging technologies. Given that many of these technologies are still being tested at the pilot scale, there is a lack of environmental assessments quantifying their impacts and benefits. In particular, an integrated approach to energy and nutrient recovery can elucidate the potential configurations for WWTPs. In this study, we conduct a life cycle assessment (LCA) of emergent wastewater treatment technologies aimed at increasing resource circularity in WWTPs. We focus on increasing energy self-sufficiency through biogas upgrades and a more radical circular approach aimed at nutrient recovery. Based on a case-study WWTP, we compare its current configuration with (1) implementing autotrophic nitrogen removal in the mainstream and deriving most of the organic matter for biogas production, which increases the quality and quantity of biogas available for energy production; (2) implementing struvite recovery through enhanced biological phosphorus removal (EBPR) as a radical approach to phosphorus management, offering an alternative to mineral fertilizer; and (3) a combination of both approaches. The results show that incremental changes in biogas production are insufficient for compensating for the environmental investment in infrastructure, although autotrophic nitrogen removal is beneficial for increasing the quality of the effluent. Combined phosphorus and energy recovery reduce the environmental impacts from the avoided use of fertilizers and phosphorus and the nitrogen release into water bodies. An integrated approach to resource management in WWTPs is thus desirable and creates new opportunities toward the implementation of circular strategies with low environmental impact in cities.

Improvement of the Polyhydroxyalkanoates Recovery from Mixed Microbial Cultures Using Sodium Hypochlorite Pre-Treatment Coupled with Solvent Extraction.

The use of mixed microbial cultures (MMC) and organic wastes and wastewaters as feed sources is considered an appealing approach to reduce the current polyhydroxyalkanoates (PHAs) production costs. However, this method entails an additional hurdle to the PHAs downstream processing (recovery and purification). In the current work, the effect of a sodium hypochlorite (NaClO) pre-treatment coupled with dimethyl carbonate (DMC) or chloroform (CF) as extraction solvents on the PHAs recovery efficiency (RE) from MMC was evaluated. MMC were harvested from a sequencing batch reactor (SBR) fed with a synthetic prefermented olive mill wastewaster. Two different carbon-sources (acetic acid and acetic/propionic acids) were employed during the batch accumulation of polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from MMC. Obtained PHAs were characterized by 1H and 13C nuclear magnetic resonance, gel-permeation chromatography, differential scanning calorimetry, and thermal gravimetric analysis. The results showed that when a NaClO pre-treatment is not added, the use of DMC allows to obtain higher RE of both biopolymers (PHB and PHBV), in comparison with CF. In contrast, the use of CF as extraction solvent required a pre-treatment step to improve the PHB and PHBV recovery. In all cases, RE values were higher for PHBV than for PHB.

Recovery of polyhydroxyalkanoates (PHAs) from wastewater: A review.

Polyhydroxyalkanoates (PHAs) are biopolyesters accumulated as carbon and energy storage materials under unbalanced growth conditions by various microorganisms. They are one of the most promising potential substitutes for conventional non-biodegradable plastics due to their similar physicochemical properties, but most important, its biodegradability. Production cost of PHAs is still a great barrier to extend its application at industrial scale. In order to reduce that cost, research is focusing on the use of several wastes as feedstock (such as agro-industrial and municipal organic waste and wastewater) in a platform based on mixed microbial cultures. This review provides a critical illustration of the state of the art of the most likely-to-be-scale-up PHA production processes using mixed microbial cultures platform and waste streams as feedstock, with a particular focus on both, upstream and downstream processes. Current pilot scale studies, future prospects, challenges and developments in the field are also highlighted.

Bioplastic recovery from wastewater: A new protocol for polyhydroxyalkanoates (PHA) extraction from mixed microbial cultures.

A new protocol for polyhydroxyalkanoates (PHA) extraction from mixed microbial cultures (MMCs) is proposed. PHA-accumulating capacity of the MMC was selected in a sequencing batch reactor (SBR) fed with a synthetic effluent emulating a fermented oil mill wastewater (OMW). The highest recovery yield and purity (74 ±â€¯8% and 100 ±â€¯5%, respectively) was obtained when using NH4-Laurate for which operating conditions of the extraction process such as temperature, concentration and contact time were optimized. Best conditions for PHA extraction from MMC turned to be: i) a pre-treatment with NaClO at 85 °C with 1 h of contact time, followed by ii) a treatment with lauric acid in a ratio acid lauric to biomass of 2:1 and 3 h of contact time.

Stable long-term operation of an upflow anammox sludge bed reactor at mainstream conditions.

The efforts for implementing the anammox process at mainstream conditions with high nitrogen removal rates have gained much attention in the race for achieving an energy-positive urban wastewater treatment plant. Here, the successful and stable long-term operation of an Upflow Anammox Sludge Bed (UAnSB) reactor treating a low-strength synthetic influent amended with ammonium and nitrite for 420 days, and a nitrite-amended pre-treated real urban wastewater for 110 days at temperatures as low as 11 °C is presented. The short and long-term effects of temperature on anammox activity were assessed when the synthetic influent was treated, and the UAnSB reactor was demonstrated to be a robust reactor to confront low temperatures, typically found at mainstream conditions. In fact, a nitrogen loading rate as high as 1.8 ± 0.1 g N L-1 d-1 with 82 ± 4% of nitrogen removal was achieved at 11 °C treating the low-strength synthetic influent. Furthermore, the effect of treating a nitrite-amended pre-treated real urban wastewater at 11 °C at long-term in the UAnSB reactor was evaluated, and a stable operation was achieved with a high average nitrogen removal rate (1.2 ± 0.5 g N L-1 d-1). The relative abundance of anammox bacteria was maintained higher than 70% according to fluorescence in situ hybridization during the whole operation, being Candidatus Brocadia anammoxidans the predominant microbial species. The presence of heterotrophs in the sludge bed was surmised through heterotrophic batch tests, but anammox activity was demonstrated to be higher than heterotrophic activity, even when the synthetic influent was replaced by the nitrite-amended pre-treated real wastewater. The feasibility of operating an enriched anammox reactor at high nitrogen removal rate at long-term at mainstream conditions was demonstrated in this study.

Integrated catalytic wet air oxidation and aerobic biological treatment in a municipal WWTP of a high-strength o-cresol wastewater.

This study examines the feasibility of coupling a Catalytic Wet Air Oxidation (CWAO), with activated carbon (AC) as catalyst, and an aerobic biological treatment to treat a high-strength o-cresol wastewater. Two goals are pursued: (a) To determine the effect of the main AC/CWAO intermediates on the activated sludge of a municipal WasteWater Treatment Plant (WWTP) and (b) To demonstrate the feasibility of coupling the AC/CWAO effluent as a part of the influent of a municipal WWTP. In a previous study, a high-strength o-cresol wastewater was treated by AC/CWAO aiming to establish the distribution of intermediates and the biodegradability enhancement. In this work, the biodegradability, toxicity and inhibition of the most relevant intermediates detected in the AC/CWAO effluent were determined by respirometry. Also, the results of a pilot scale municipal WWTP study for an integrated AC/CWAO-aerobic biological treatment of this effluent are presented. The biodegradation parameters (i.e. maximum oxygen uptake rate and oxygen consumption) of main AC/CWAO intermediates allowed the classification of the intermediates into readily biodegradable, inert or toxic/inhibitory compounds. This detailed study, allowed to understand the biodegradability enhancement exhibited by an AC/CWAO effluent and to achieve a successful strategy for coupling the AC/CWAO step with an aerobic biological treatment for a high-strength o-cresol wastewater. Using 30%, as COD, of AC/CWAO effluent in the inlet to the pilot scale WWTP, the integrated AC/CWAO-biological treatment achieved a 98% of total COD removal and, particularly, a 91% of AC/CWAO effluent COD removal without any undesirable effect on the biomass.

Catalytic and non-catalytic wet air oxidation of sodium dodecylbenzene sulfonate: kinetics and biodegradability enhancement.

Wet air oxidation (WAO) and catalytic wet air oxidation (CWAO) were investigated as suitable precursors for the biological treatment of industrial wastewater containing sodium dodecylbenzene sulfonate (DBS). Two hours WAO semi-batch experiments were conducted at 15 bar of oxygen partial pressure (P(O2)) and at 180, 200 and 220 degrees C. It was found that the highest temperature provides appreciable total organic carbon (TOC) and chemical oxygen demand (COD) abatement of about 42 and 47%, correspondingly. Based on the main identified intermediates (acetic acid and sulfobenzoic acid) a reaction pathway for DBS and a kinetic model in WAO were proposed. In the case of CWAO experiments, seventy-two hours tests were done in a fixed bed reactor in continuous trickle flow regime, using a commercial activated carbon (AC) as catalyst. The temperature and P(O2) were 140-160 degrees C and 2-9 bar, respectively. The influence of the operating conditions on the DBS oxidation, the occurrence of oxidative coupling reactions over the AC, and the catalytic activity (in terms of substrate removal) were established. The results show that the AC without any supported active metal behaves bi-functional as adsorbent and catalyst, giving TOC conversions up to 52% at 160 degrees C and 2 bar of P(O2), which were comparable to those obtained in WAO experiments. Respirometric tests were completed before and after CWAO and to the main intermediates identified through the WAO and CWAO oxidation route. Then, the readily biodegradable COD (COD(RB)) of the CWAO and WAO effluents were found. Taking into account these results it was possible to compare whether or not the CWAO or WAO effluents were suitable for a conventional activated sludge plant inoculated with non adapted culture.

Enrichment of a mixed microbial culture for polyhydroxyalkanoates production: Effect of pH and N and P concentrations.

Polyhydroxyalkanoates (PHA) are biopolymers that can be an alternative against conventional plastics. The study reported herein evaluated the enrichment of a mixed microbial culture (MMC) operated under feast/famine regime and different pHs in a sequencing batch reactor (SBR) using acetate as sole carbon source to produce polyhydroxyalkanoates (PHAs). The enrichment step was evaluated at controlled pH of 7.5 and also without pH control (averaged value of 9.0). The acetate uptake rate (-qS) of both enrichments at the end of the experimental period exhibited similar behaviour being about 0.18CmolAcCmolX-1h-1 and 0.19CmolAcCmolX-1h-1 for SBR-A and SBR-B, respectively. However, the PHA-storing capacity of the biomass enriched without pH control was better, exhibiting a maximum PHA content of 36% (gPHAg-1 VSS) with a PHA production rate (qPHA) of 0.16CmolPHACmolX-1h-1. Batch experiments were performed to evaluate PHA-storing capacity of the enriched culture at different pHs and nutrients concentrations. In the pH experiments (without nutrient limitation), it was found that in the absence of controlled pH, the enriched biomass exhibited a PHA content of 44% gPHAg-1 VSS with -qS and PHA to substrate yield (YPHA/Ac) of 0.57CmolAcCmolX-1h-1 and 0.33CmolPHACmolAc-1, respectively. Regarding the experiments at variable nutrients concentration (pH ranging 8.8 to 9.2), the results indicate that the PHA content in the enriched biomass is significantly higher being around 51% gPHAg-1 VSS under nitrogen limitation. This work demonstrated the feasibility of the enrichment of a MMC with PHA storage ability without pH control. Results also suggest that better PHAs contents and substrate uptake rates are obtained without controlling the pH in the accumulation step. Finally, this work also highlights the importance of understanding the role of nutrients concentration during the accumulation step.

Long-term performance and stability of a continuous granular airlift reactor treating a high-strength wastewater containing a mixture of aromatic compounds.

Continuous feeding operation of an airlift reactor and its inoculation with mature aerobic granules allowed the successful treatment of a mixture of aromatic compounds (p-nitrophenol, o-cresol and phenol). Complete biodegradation of p-nitrophenol, o-cresol, phenol and their metabolic intermediates was achieved at an organic loading rate of 0.61 g COD L(-1)d(-1). Stable granulation was obtained throughout the long-term operation (400 days) achieving an average granule size of 2.0 ± 1 mm and a sludge volumetric index of 26 ± 1 mL g(-1) TSS. The identified genera in the aerobic granular biomass were heterotrophic bacteria able to consume aromatic compounds. Therefore, the continuous feeding regimen and the exposure of aerobic granules to a mixture of aromatic compounds make possible to obtain good granulation and high removal efficiency.

Kinetic and microbiological characterization of aerobic granules performing partial nitritation of a low-strength wastewater at 10 °C.

A granular airlift reactor enriched in ammonia oxidizing bacteria (AOB) was operated at 10 °C performing stable partial nitritation in the long-term. The reactor treated a synthetic low-strength influent during 250 days with an average nitrogen loading rate of 0.63 ± 0.06 g N L(-1) d(-1). Nitrate production was barely detected, being the average concentration in the effluent of 0.6 ± 0.3 mg N-NO3 L(-1). Furthermore, a suitable effluent for a subsequent reactor performing the anammox process was achieved. A maximum specific growth rate as high as 0.63 ± 0.05 d(-1) was determined by performing kinetic experiments with the granular sludge in a chemostat and fitting the results to the Monod model. Pyrosequencing analysis showed a high enrichment in AOB (41 and 65% of the population were identified as Nitrosomonas genus on day 98 and 233, respectively) and an effective repression of nitrite oxidizing bacteria in the long-term. Pyrosequencing analysis also identified the coexistence of nitrifying bacteria and heterotrophic psychrotolerant microorganisms in the granular sludge. Some psychrotolerant microorganisms are producers of cryoprotective extracellular polymeric substances that could explain the better survival of the whole consortia at cold temperatures.

Long-term impact of salinity on the performance and microbial population of an aerobic granular reactor treating a high-strength aromatic wastewater.

The effect of salinity over granular biomass treating a mixture of aromatic compounds (phenol, o-cresol and p-nitrophenol) was evaluated in a continuous airlift reactor. To mimic an industrial wastewater, increasing concentrations (from 2.0 to 29.0 g salts L(-1)) of a mixture of salts (MgSO4, NaCl, KCl, CaCl2 and NaHCO3) were introduced in the influent. The gradual salinity increase led to a good acclimation of the biomass obtaining complete biodegradation of the aromatic compounds and no accumulation of metabolic intermediates. However, a deterioration of the morphology of aerobic granules with a complete loss of granulation after 125 days was produced at 29.0 g salts L(-1). At that moment, anaerobic granules were added to promote granulation and after 50 days new aerobic granules were formed. These new aerobic granules remained stable for more than 100 days at the highest salinity condition with 100% removal of the mixture of aromatic compounds.

Microbial community shifts on an anammox reactor after a temperature shock using 454-pyrosequencing analysis.

To explore the changes in the microbial community structure during the recovery process of an anammox reactor after a temperature shock, the 454-pyrosequencing technique was used. The temperature shock reduced the nitrogen removal rate up to 92% compared to that just before the temperature shock, and it took 70 days to recover a similar nitrogen removal rate to that before the temperature shock (ca. 0.30 g N L(-1) d(-1)). Pyrosequencing results indicated that microbial diversity in the reactor decreased as the reactor progressively recovered from the temperature shock. Anammox bacteria were accounted as 6%, 35% and 46% of total sequence reads in samples taken 13, 45 and 166 days after the temperature shock. These results were in agreement with N-removal performance results and anammox activity measured in the reactor during the recovery process. An anammox specific primer was used to precisely determine the anammox species in the biomass samples.

Assessment of crude glycerol for Enhanced Biological Phosphorus Removal: Stability and role of long chain fatty acids.

Enhanced Biological Phosphorus Removal (EBPR) of urban wastewaters is usually limited by the available carbon source required by Polyphosphate Accumulating Organisms (PAO). External carbon sources as volatile fatty acids (VFA) or other pure organic compounds have been tested at lab scale demonstrating its ability to enhance PAO activity, but the application of this strategy at full-scale WWTPs is not cost-effective. The utilization of industrial by-products with some of these organic compounds provides lower cost, but it has the possible drawback of having inhibitory or toxic compounds to PAO. This study is focused on the utilization of crude glycerol, the industrial by-product generated in the biodiesel production, as a possible carbon source to enhance EBPR in carbon-limited urban wastewaters. Crude glycerol has non-negligible content of other organic compounds as methanol, salts, VFA and long chain fatty acids (LCFA). VFA and methanol have been demonstrated to enhance PAO activity, but there is no previous study about the effect of LCFA on PAO. This work presents the operation of an EBPR SBR system using crude glycerol as sole carbon source, studying also its long-term stability. The effect of LCFA is evaluated at short and long-term operation, demonstrating for the first time EBPR activity with LCFA as sole carbon source and its long-term failure due to the increased hydrophobicity of the sludge.

Partial nitritation and o-cresol removal with aerobic granular biomass in a continuous airlift reactor.

Several chemical industries produce wastewaters containing both, ammonium and phenolic compounds. As an alternative to treat this kind of complex industrial wastewaters, this study presents the simultaneous partial nitritation and o-cresol biodegradation in a continuous airlift reactor using aerobic granular biomass. An aerobic granular sludge was developed in the airlift reactor for treating a high-strength ammonium wastewater containing 950 ± 25 mg N-NH4(+) L(-1). Then, the airlift reactor was bioaugmented with a p-nitrophenol-degrading activated sludge and o-cresol was added progressively to the ammonium feed to achieve 100 mg L(-1). The results showed that stable partial nitritation and full biodegradation of o-cresol were simultaneously maintained obtaining a suitable effluent for a subsequent anammox reactor. Moreover, two o-cresol shock-load events with concentrations of 300 and 1000 mg L(-1) were applied to assess the capabilities of the system. Despite these shock load events, the partial nitritation process was kept stable and o-cresol was totally biodegraded. Fluorescence in situ hybridization technique was used to identify the heterotrophic bacteria related to o-cresol biodegradation and the ammonia oxidising bacteria along the granules.

Sequentially alternating pollutant scenarios of phenolic compounds in a continuous aerobic granular sludge reactor performing simultaneous partial nitritation and o-cresol biodegradation.

Industrial wastewater treatment plants must operate properly during the transient-state conditions often found in the industrial production. This study presents the performance of simultaneous partial nitritation and o-cresol biodegradation in a continuous aerobic granular reactor under sequentially alternating pollutant (SAP) scenarios. Three SAP scenarios were imposed during the operation of the granular reactor. In each one, a secondary recalcitrant compound (either p-nitrophenol (PNP), phenol or 2-chlorophenol (2CP)) were added for a short period of time to the regular influent containing only ammonium and o-cresol. Partial nitritation and o-cresol biodegradation were not inhibited by the presence of PNP or phenol and both compounds were fully biodegraded. On the contrary, the presence of 2CP strongly inhibited both processes within 2days. However, the reactor was recovered in a few days. These findings demonstrate that treatment of complex industrial wastewaters with variable influent composition is feasible in a continuous aerobic granular reactor.

Aerobic biodegradation of a mixture of monosubstituted phenols in a sequencing batch reactor.

A sequencing batch reactor (SBR) was inoculated with p-nitrophenol-degrading activated sludge to biodegrade a mixture of monosubstituted phenols: p-nitrophenol (PNP), PNP and o-cresol; and PNP, o-cresol and o-chlorophenol. Settling times were progressively decreased to promote biomass granulation. PNP was completely biodegraded. The PNP and o-cresol mixture was also biodegraded although some transitory accumulation of intermediates occurred (mainly hydroquinone and catechol). o-Chlorophenol was not biodegraded and resulted in inhibition of o-cresol and PNP biodegradation and complete failure of the SBR within a few days. The biomass had very good settling properties when a settling time of 1 min was applied: sludge volume index (SVI5) below 50 mL g(-1), SVI5/SVI30 ratio of 1 and average particle size of 200 µm.

Simultaneous nitritation and p-nitrophenol removal using aerobic granular biomass in a continuous airlift reactor.

The chemical and petrochemical industries produce wastewaters containing ammonium and phenolic compounds. Biological treatment of these wastewaters could be problematic due to the possible inhibitory effects exerted by phenolic compounds. The feasibility of performing simultaneous nitritation and p-nitrophenol (PNP) biodegradation using a continuous aerobic granular reactor was evaluated. A nitrifying granular sludge was bioaugmented with a PNP-degrading floccular sludge, while PNP was progressively added to the feed containing a high ammonium concentration. Nitritation was sustained throughout the operational period with ca. 85% of ammonium oxidation and less than 0.3% of nitrate in the effluent. PNP biodegradation was unstable and the oxygen limiting condition was found to be the main explanation for this unsteadiness. An increase in dissolved oxygen concentration from 2.0 to 4.5 mg O2 L(-1) significantly enhanced PNP removal, achieving total elimination. Acinetobacter genus and ammonia-oxidising bacteria were the predominant bacteria species in the granular biomass.

Closed-loop control of ammonium concentration in nitritation: convenient for reactor operation but also for modeling.

A mathematical biofilm model was developed to describe nitritation in aerobic granular reactor operating in continuous mode. The model includes the automatic closed-loop control of ammonium concentration in the effluent. This is integrated in a ratio control strategy to maintain the proportion between the dissolved oxygen (DO) and the total ammonia nitrogen (TAN) concentrations in the reactor effluent at a desired value. The model was validated with a large set of experimental results previously reported in the literature. The model was used to study the effect of DO and TAN setpoints on the achievement of full nitritation, as well as to establish the appropriate required range of the DO/TAN concentration ratio to be applied. Nitritation at 20 °C was tested experimentally and simulated with the model. Additionally, the importance of controlling the TAN concentration was highlighted with different scenarios, in which periodic disturbances were applied mimicking a poor control situation.