Benefits and drawbacks of integrating a side-stream sludge fermenter into an A2O system under limited COD conditions

Altres ajuts: acords transformatius de la UABThe implementation of a side-stream sludge fermenter (SSSF) has been identified as a possible solution to improve the performance of an anaerobic/anoxic/aerobic (A2O) configuration when treating low COD wastewater. This study systematically evaluated the effects of incorporating a SSSF into an A2O configuration (side-stream enhanced biological phosphorus removal, S2EBPR) for P/N/COD removal under a limited influent COD (CODINF) condition. The performance of the S2EBPR (with the SSSF receiving 6% of the recycled activated sludge and operating with a hydraulic retention time (HRT) of 2.4 d) and A2O were compared under the same limited CODINF (350 mg/L) condition. S2EBPR improved the amount of P removed (26.6%) under a low influent COD/P of only 26.3 compared with A2O of 32.6, and enhanced denitrification (11%) without compromising full ammonium and COD removal. However, the PLOAD to the plant increased due to the P-release in SSSF, resulting in higher effluent P concentration. The methane and energy recovery indexes were around 45% lower than those of A2O. Sequencing analysis revealed a high abundance of PAO in accordance to its higher P removal. This study represents a comprehensive evaluation of the S2EBPR configuration and provides an assessment of its suitability

The impact of biomass withdrawal strategy on the biomass selection and polyhydroxyalkanoates accumulation of mixed microbial cultures

UIDP/04378/2020 UIDB/04378/2020 LA/P/0140/2020 SFRH/BD/110673/2015The production of polyhydroxyalkanoates (PHA) by mixed microbial cultures (MMC) has been studied as an alternative to pure cultures in order to reduce the price of PHA through use of open systems and low-cost substrates, such as agro-industrial sub-products. However, the widespread applicability of this process depends on the optimization of operational factors impacting PHA productivity. This study addresses the impact of biomass withdrawal strategy on the performance of MMC selection reactors and consequently on biomass productivity and global PHA productivity. Two selection reactors were operated in parallel under similar conditions, except for the timing of biomass withdrawal, at the end of the famine phase (Reactor 1, R1) versus at the end of the feast phase (Reactor 2, R2) at an organic loading rate of 100 Cmmol.L−1.d−1 and solids retention time of 4 days. The biomass selected in both conditions had similar PHA storing capacity as shown by the similar yields of PHA per substrate obtained in the accumulation assays; however, R1 reached a higher biomass productivity (about 4-fold higher than R2). This study demonstrated that removing the excess biomass at the end of the famine phase resulted in a much higher global PHA productivity and that the key parameter affecting the global PHA productivity of the 2-stage system was the volumetric biomass productivity. Results obtained provide important insight into how MMC systems can be best operated to maximize PHA productivity.publishersversionpublishe

Dynamics of Microbial Communities in Phototrophic Polyhydroxyalkanoate Accumulating Cultures

DFA/BD/8201/2020 UIDP/04378/2020 UIDB/04378/2020 LA/P/0140/2020Phototrophic mixed cultures (PMC) are versatile systems which can be applied for waste streams, valorisation and production of added-value compounds, such as polyhydroxyalkanoates (PHA). This work evaluates the influence of different operational conditions on the bacterial communities reported in PMC systems with PHA production capabilities. Eleven PMCs, fed either with acetate or fermented wastewater, and selected under either feast and famine (FF) or permanent feast (PF) regimes, were evaluated. Overall, results identified Chromatiaceae members as the main phototrophic PHA producers, along with Rhodopseudomonas, Rhodobacter and Rhizobium. The findings show that Chromatiaceae were favoured under operating conditions with high carbon concentrations, and particularly under the PF regime. In FF systems fed with fermented wastewater, the results indicate that increasing the organic loading rate enriches for Rhodopseudomonas, Rhizobium and Hyphomicrobiaceae, which together with Rhodobacter and Chromatiaceae, were likely responsible for PHA storage. In addition, high-sugar feedstock impairs PHA production under PF conditions (fermentative bacteria dominance), which does not occur under FF. This characterization of the communities responsible for PHA accumulation helps to define improved operational strategies for PHA production with PMC.publishersversionpublishe

The impact of pH on the anaerobic and aerobic metabolism of Tetrasphaera-enriched polyphosphate accumulating organisms

Funding Information: The authors thank the Portuguese Fundaçao para a Ciência e a Tecnologia, which supports the Applied Molecular Biosciences Unit - UCIBIO, the European Commission (Water JPI project 196 (Water-Works2014 ERA-NET Co-funded Call): “Smart decentralized water management through a dynamic integration of technologies (Watintech)” and the Australian Research Council ( ARC LP190100329 ). Publisher Copyright: © 2023Members of the genus Tetrasphaera are putative polyphosphate accumulating organisms (PAOs) that have been found in greater abundance than Accumulibacter in many full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants worldwide. Nevertheless, previous studies on the effect of environmental conditions, such as pH, on the performance of EBPR have focused mainly on the response of Accumulibacter to pH changes. This study examines the impact of pH on a Tetrasphaera PAO enriched culture, over a pH range from 6.0 to 8.0 under both anaerobic and aerobic conditions, to assess its impact on the stoichiometry and kinetics of Tetrasphaera metabolism. It was discovered that the rates of phosphorus (P) uptake and P release increased with an increase of pH within the tested range, while PHA production, glycogen consumption and substrate uptake rate were less sensitive to pH changes. The results suggest that Tetrasphaera PAOs display kinetic advantages at high pH levels, which is consistent with what has been observed previously for Accumulibacter PAOs. The results of this study show that pH has a substantial impact on the P release and uptake kinetics of PAOs, where the P release rate was >3 times higher and the P uptake rate was >2 times higher at pH 8.0 vs pH 6.0, respectively. Process operational strategies promoting both Tetrasphaera and Accumulibacter activity at high pH do not conflict with each other, but lead to a potentially synergistic impact that can benefit EBPR performance.publishersversionpublishe

Metabolic modelling of polyhydroxyalkanoate copolymers production by mixed microbial cultures

Background: This paper presents a metabolic model describing the production of polyhydroxyalkanoate (PHA) copolymers in mixed microbial cultures, using mixtures of acetic and propionic acid as carbon source material. Material and energetic balances were established on the basis of previously elucidated metabolic pathways. Equations were derived for the theoretical yields for cell growth and PHA production on mixtures of acetic and propionic acid as functions of the oxidative phosphorylation efficiency, P/O ratio. The oxidative phosphorylation efficiency was estimated from rate measurements, which in turn allowed the estimation of the theoretical yield coefficients. Results: The model was validated with experimental data collected in a sequencing batch reactor (SBR) operated under varying feeding conditions: feeding of acetic and propionic acid separately (control experiments), and the feeding of acetic and propionic acid simultaneously. Two different feast and famine culture enrichment strategies were studied: (i) either with acetate or (ii) with propionate as carbon source material. Metabolic flux analysis (MFA) was performed for the different feeding conditions and culture enrichment strategies. Flux balance analysis (FBA) was used to calculate optimal feeding scenarios for high quality PHA polymers production, where it was found that a suitable polymer would be obtained when acetate is fed in excess and the feeding rate of propionate is limited to ∼0.17 C-mol/ (C-mol.h). The results were compared with published pure culture metabolic studies. Conclusion: Acetate was more conducive toward the enrichment of a microbial culture with higher PHA storage fluxes and yields as compared to propionate. The P/O ratio was not only influenced by the selected microbial culture, but also by the carbon substrate fed to each culture, where higher P/O ratio values were consistently observed for acetate than propionate. MFA studies suggest that when mixtures of acetate and propionate are fed to the cultures, the catabolic activity is primarily guaranteed through acetate uptake, and the characteristic P/O ratio of acetate prevails over that of propionate. This study suggests that the PHA production process by mixed microbial cultures has the potential to be comparable or even more favourable than pure cultures.publishersversionpublishe

Polyhydroxyalkanoate granules quantification in mixed microbial cultures using image analysis: Sudan Black B versus Nile Blue A staining

Polyhydroxyalkanoates (PHA) can be produced and intracellularly accumulated as inclusions by mixed microbial cultures (MMC) for bioplastic production and in enhanced biological phosphorus removal (EBPR) systems. Classical methods for PHA quantification use a digestion step prior to chromatography analysis, rendering them labor intensive and time-consuming. The present work investigates the use of two quantitative image analysis (QIA) procedures specifically developed for PHA inclusions identification and quantification. MMC obtained from an EBPR system were visualized by bright-field and fluorescence microscopy for PHA inclusions detection, upon Sudan Black B (SBB) and Nile Blue A (NBA) staining, respectively. The captured color images were processed by QIA techniques and the image analysis data were further treated using multivariate statistical analysis. Partial least squares (PLS) regression coefficients of 0.90 and 0.86 were obtained between QIA parameters and PHA concentrations using SBB and NBA, respectively. It was found that both staining procedures might be seen as alternative methodologies to classical PHA determination.The authors thank the Fundacao para a Ciencia e a Tecnologia (FCT) Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioEnv - Biotechnology and Bioengineering for a sustainable world", REF. NORTE-07-0124-FEDER-000048, co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. The authors also acknowledge the financial support to Daniela P. Mesquita through the postdoctoral grant (SFRH/BPD/82558/2011) and the project PTDC/EBB-EBI/103147/2008 provided by FCT

Polyphosphates and poly-β-hydroxybutyrate granules identification through quantitative image analysis in enhanced biological phosphorus removal systems

Enhanced biological phosphorus removal (EBPR) is a widely implemented technique for having the potential to cheaply and reliably remove phosphate from wastewater treatment processes, than traditional chemical methods. EBPR is performed by operating the system sequentially with anaerobic and aerobic conditions. Several studies were already performed ranging from different strategies for the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) to modeling both types of bacterial activities. Until now, slight attention has been given to the development of newer, faster, simpler, and better suited monitoring techniques for this type of system. This work is focused on the development of image analysis techniques for polyphosphates and poly-β-hydroxybutyrate granules in EBPR systems since off-line analyses are labor intensive and not able to be performed in full-scale plants. A lab-scale sequencing batch reactor fed with synthetic wastewater containing volatile fatty acids (VFAs) and orthophosphate was used. The reactor had a working volume of 4 L and was operated with a cycle time of 6 h consisting of 2 h anaerobic, 3 h aerobic, 50 min settling and decanting, and 5 min anaerobic idle periods. In each cycle, 2 L of synthetic wastewater was fed to the reactor in the first 5 min of the anaerobic period, resulting in a hydraulic retention time (HRT) of 12 h. The pH was controlled during both the anaerobic and aerobic periods around 7, and the temperature was controlled at 30 ºC in order to provide selective advantages to GAOs over PAOs. The ratio between chemical oxygen demand (COD) and P in the feed was kept at 10 (gCOD/g P). Biomass samples were collected at the end of the anaerobic and aerobic phases and fixed with phosphate buffer saline solution (PBS) and ethanol. Two fluorescence staining methods were used: (1) DAPI for poly-P identification; and (2) nile blue for poly-β-hydroxybutyrate granules. So far, promising results were achieved regarding the type of images achieved by these fluorescence staining methods and the image analysis procedures still under development

Intracellular poly-P assessment by DAPI staining and image analysis

In wastewater treatment, enhanced biological phosphorus removal (EBPR) is considered a well-established process to remove phosphate (P). EBPR is based on the activity of polyphosphate-accumulating organisms (PAOs) able to take up and store large amounts of P as intracellular (poly-P) granules. However, monitoring poly-P in mixed cultures is usually performed by a laborious and time consuming off-line chemical analysis. Thus, there is a clear need to develop new techniques to rapidly monitor these processes, such as image analysis coupled to sample staining and microscopy inspection. A lab-scale sequencing batch reactor (SBR) was fed with synthetic wastewater containing acetate and propionate as main carbon sources and an orthophosphate solution was added. A COD/P ratio of 10 mg COD mg P-PO4-1 was used to provide selective advantages to PAOs. The SBR was operated with a cycle time of 6 h: 120 min anaerobic including 5 min feed, 180 min aerobic and 60 min wasting/settling. Biomass samples were collected at the end of the aerobic stage. Bulk P concentration was determined by segmented flow analysis and total P concentration was similarly measured following acid digestion at 100oC. Intracellular poly-P concentration was determined by subtracting the bulk P from the total P. Intracellular poly-P granules were observed in epifluorescence microscopy using DAPI staining with a 25 ìg mL-1 DAPI solution. A long pass filter was used with an excitation bandpass of 365-370 nm and emission cut off at 421 nm. A specially developed program in Matlab was used for image analysis. A total of 41 samples were collected. Two thirds were fed as training data to the partial least squares (PLS) model and the remaining used for validation. Both absolute (in mg poly-P / L) and relative (in mg poly-P / g MLSS) intracellular poly-P concentrations were studied. This procedure was found to predict, at some extent, the relative intracellular poly-P concentration (real poly-P = 0.971 x predicted poly-P, R2 of 0.744). Regarding the absolute intracellular poly-P concentration, a total of 3 samples needed to be discarded in order to obtain a similar result (real poly-P = 1.005 x predicted poly-P, R2 of 0.731)

Prediction of intracellular storage polymers using quantitative image analysis in enhanced biological phosphorus removal systems

Intracellular polymers, such as polyhydroxyalkanoates (PHA) synthesized by microorganisms for energy and carbon storage, can be commercially used as biodegradable plastics in a wide range of applications. The possibility of obtaining high PHA contents (where the most common monomers found are poly-hydroxybutyrate (PHB) and poly-hydroxyvalerate (PHV)) from inexpensive inocula and raw materials emerges as a promising and commercially interesting alternative. For this purpose, mixed cultures operated under feast/famine cycles are most frequently used. PHA is an important storage polymer in the metabolism of microorganisms involved in enhanced biological phosphorus removal (EBPR) systems. It is stored by polyphosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO), as described in previous publications [1,2]. Monitoring intracellular storage polymers in bacteria is usually performed through laborious and time consuming off-line chemical analyses. Thus, there is clearly a need to develop new techniques in order to promptly monitor these processes. Image analysis techniques have the potential to be a non-invasive and rapid means of assessing the amount of different storage polymers inside microbial cells, providing the evaluation of these important biotechnological processes. The present study focuses on predicting intracellular storage polymers in EBPR systems. For that purpose, quantitative image analysis techniques were developed and partial least squares (PLS) were used to model PHA results. An EBPR fed with synthetic wastewater containing volatile fatty acids (VFAs) and orthophosphate was used. Biomass samples were collected at the end of the anaerobic and aerobic phases. Analytical PHA quantification was performed by biomass digestion and gas chromatography analysis. In the concurring image analysis methodology Nile blue was used as a fluorescence staining method for PHA granules identification. The results from image analysis allowed establishing a PHB prediction ability presenting a regression value (R2) of 0.854, a PHA prediction regression value (R2) of 0.843 and a PHV prediction regression value (R2) of 0.779. The lower prediction ability for PHV could be explained since this parameter presented only a small contribution to the overall PHA. The analysis performed based on the variable importance in the projection (VIP) established a core of three image analysis parameters (granules total area, granules total intensity and image intensity) as the most important regarding PHA, PHB and PHV prediction

Desenvolvimento de metodologias de análise de imagem para quantificar PHA, polifosfatos e glicogénio intracelular em estações de tratamento de águas residuais

O processo de remoção biológica de fósforo, em estações de tratamento de águas residuais, é um processo efetuado por culturas mistas contendo organismos acumuladores de polifosfatos (PAO) e de glicogénio (GAO). No decurso deste processo os microrganismos podem formar inclusões de glicogénio, polihidroxialcanoatos (PHA) e polifosfatos (poli-P). Neste processo, é fulcral monitorizar o metabolismo intracelular para determinar a sua eficiência. Contudo, a sua monitorização, realizada através de análise químicas em diferido, é laboriosa e morosa. Deste modo, existe uma clara necessidade do desenvolvimento de métodos mais expeditos, como metodologias de análise de imagens, para a monitorização destes polímeros intracelulares. Estas técnicas foram implementadas neste estudo, encontrando-se, no caso da determinação da concentração intracelular de poli-P, em fase de desenvolvimento dos protocolos de coloração e aquisição de imagens. Para a determinação da concentração intracelular de glicogénio, foi obtida uma boa correlação inicial. Na determinação da concentração intracelular de PHA, este estudo foca-se na otimização dos protocolos de coloração e no desenvolvimento do programa de análise de imagem