Improving polyhydroxyalkanoates production in phototrophic mixed cultures by optimizing accumulator reactor operating conditions

The authors would also like to acknowledge the Fundacao para a Ciencia e a Tecnologia (Portugal) for funding through SFRH/BPD/101642/2014. co-financed by ERDF under PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728).Polyhydroxyalkanoates (PHAs) production with phototrophic mixed cultures (PMCs) has been recently proposed. These cultures can be selected under the permanent presence of carbon and the PHA production can be enhanced in subsequent accumulation steps. To optimize the PHA production in accumulator reactors, this work evaluated the impact of 1) initial acetate concentration, 2) light intensity, 3) removal of residual nitrogen on the culture performance. Results indicate that low acetate concentration (<30 CmM) and specific light intensities around 20 W/gX are optimal operating conditions that lead to high polyhydroxybutyrate (PHB) storage yields (0.83 ± 0.07 Cmol-PHB/Cmol-Acet) and specific PHB production rates of 2.21 ± 0.07 Cmol-PHB/Cmol X d. This rate is three times higher than previously registered in non-optimized accumulation tests and enabled a PHA content increase from 15 to 30% in <4 h. Also, it was shown for the first time, the capability of a PMC to use a real waste, fermented cheese whey, to produce PHA with a hydroxyvalerate (HV) content of 12%. These results confirm that fermented wastes can be used as substrates for PHA production with PMCs and that the energy levels in sunlight that lead to specific light intensities from 10 to 20 W/gX are sufficient to drive phototrophic PHA production processes.authorsversionpublishe

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

Impact of CO2 concentration and light exposure on process performance

Funding Information: This research was financed by national funds from FCT-Fundação para a Ciência e a Tecnologia , I.P., in the scope of the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. Publisher Copyright: © 2023 The AuthorsThe utilization of non-aerated microalgae-bacterial consortia for phototrophic biological nutrient removal (photo-BNR) has emerged as an alternative to conventional wastewater treatment. Photo-BNR systems are operated under transient illumination, with alternating dark-anaerobic, light-aerobic and dark-anoxic conditions. A deep understanding of the impact of operational parameters on the microbial consortium and respective nutrient removal efficiency in photo-BNR systems is required. The present study evaluates, for the first time, the long-term operation (260 days) of a photo-BNR system, fed with a COD:N:P mass ratio of 7.5:1:1, to understand its operational limitations. In particular, different CO2 concentrations in the feed (between 22 and 60 mg C/L of Na2CO3) and variations of light exposure (from 2.75 h to 5.25 h per 8 h cycle) were studied to determine their impact on key parameters, like oxygen production and availability of polyhydroxyalkanoates (PHA), on the performance of anoxic denitrification by polyphosphate accumulating organisms. Results indicate that oxygen production was more dependent on the light availability than on the CO2 concentration. Also, under operational conditions with a COD:Na2CO3 ratio of 8.3 mg COD/mg C and an average light availability of 5.4 ± 1.3 W h/g TSS, no internal PHA limitation was observed, and 95 ± 7%, 92 ± 5% and 86 ± 5% of removal efficiency could be achieved for phosphorus, ammonia and total nitrogen, respectively. 81 ± 1.7% of the ammonia was assimilated into the microbial biomass and 19 ± 1.7% was nitrified, showing that biomass assimilation was the main N removal mechanism taking place in the bioreactor. Overall, the photo-BNR system presented a good settling capacity (SVI ∼60 mL/g TSS) and was able to remove 38 ± 3.3 mg P/L and 33 ± 1.7 mg N/L, highlighting its potential for achieving wastewater treatment without the need of aeration.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

A breakthrough in outdoor pilot-scale operation

This work was supported by national funds from FCT - Fundação para a Ciência e a Tecnologia , I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB . Likewise, the INCOVER project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement n° 689242 ). J.R.A. also acknowledges the financial support of FCT - Fundação para a Ciência e a Tecnologia through the Ph.D. grant DFA/BD/8201/2020. Publisher Copyright: © 2023 The Author(s)The versatile capacity of purple phototrophic bacteria (PPB) for producing valuable bioproducts has gathered renewed interest in the field of resource recovery and waste valorisation. However, greater knowledge regarding the viability of applying PPB technologies in outdoor, large-scale systems is required. This study assessed, for the first time, the upscaling of the phototrophic polyhydroxyalkanoate (PHA) production technology in a pilot-scale system operated in outdoor conditions. An integrated system composed of two up-flow anaerobic sludge blanket (UASB) reactors (for fermentation of wastewater with molasses), and two high-rate algal ponds retrofitted into PPB ponds, was operated in a wastewater treatment plant under outdoor conditions. UASB's adaptation to the outdoor temperatures involved testing different operational settings, namely hydraulic retention times (HRT) of 48 and 72 h, and molasses fermentation in one or two UASBs. Results have shown that the fermentation of molasses in both UASBs with an increased HRT of 72 h was able to ensure a suitable operation during colder conditions, achieving 3.83 ± 0.63 g CODFermentative Products/L, compared to the 3.73 ± 0.85 g CODFermentative Products/L achieved during warmer conditions (molasses fermentation in one UASB; HRT 48 h). Furthermore, the PPB ponds were operated under a light-feast/dark-aerated-famine strategy and fed with the fermented wastewater and molasses from the two UASBs. The best PHA production was obtained during the summer of 2018 and spring of 2019, attaining 34.7 % gPHA/gVSS with a productivity of 0.11 gPHA L−1 day−1 and 36 % gPHA/gVSS with a productivity of 0.14 gPHA L−1 day−1, respectively. Overall, this study showcases the first translation of phototrophic PHA production technology from an artificially illuminated laboratory scale system into a naturally illuminated, outdoor, pilot-scale system. It also addresses relevant process integration aspects with UASBs for pre-fermenting wastewater with molasses, providing a novel operational strategy to achieve photosynthetic PHA production in outdoor full-scale systems.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

Ammonia impact on the selection of a phototrophic - chemotrophic consortium for polyhydroxyalkanoates production under light-feast / dark-aerated-famine conditions

Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. Likewise, the INCOVER project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement n° 689242 ). J.R.A. also acknowledges the financial support of FCT - Fundação para a Ciência e a Tecnologia through the Ph.D. grant DFA/BD/8201/2020 . Publisher Copyright: © 2023 The AuthorsPhototrophic polyhydroxyalkanoate (PHA) production is an emerging technology for recovering carbon and nutrients from diverse wastewater streams. However, reliable selection methods for the enrichment of PHA accumulating purple phototrophic bacteria (PPB) in phototrophic mixed cultures (PMC) are needed. This research evaluates the impact of ammonia on the selection of a PHA accumulating phototrophic-chemotrophic consortium, towards the enrichment of PHA accumulating PPB. The culture was operated under light-feast/dark-aerated-famine and winter simulated-outdoor conditions (13.2 ± 0.9 °C, transient light, 143.5 W/m2), using real fermented domestic wastewater with molasses as feedstock. Three ammonia supply strategies were assessed: 1) ammonia available only in the light phase, 2) ammonia always present and 3) ammonia available only during the dark-aerated-famine phase. Results showed that the PMC selected under 1) ammonia only in the light and 3) dark-famine ammonia conditions, presented the lowest PHA accumulation capacity during the light period (11.1 % g PHA/g VSS and 10.4 % g PHA/g VSS, respectively). In case 1), the absence of ammonia during the dark-aerated-famine phase did not promote the selection of PHA storing PPB, whereas in case 3) the absence of ammonia during the light period favoured cyanobacteria growth as well as purple sulphur bacteria with increased non-PHA inclusions, resulting in an overall decrease of phototrophic PHA accumulation capacity. The best PHA accumulation performance was obtained with selection under permanent presence of ammonia (case 2), which attained a PHA content of 21.6 % g PHA/g VSS (10.2 Cmmol PHA/L), at a production rate of 0.57 g PHA/L·day, during the light period in the selection reactor. Results in case 2 also showed that feedstock composition impacts the PMC performance, with feedstocks richer in more reduced volatile fatty acids (butyric and valeric acids) decreasing phototrophic performance and leading to acids entering the dark-aerated phase. Nevertheless, the presence of organic carbon in the aerated phase was not detrimental to the system. In fact, it led to the establishment of a phototrophic-chemotrophic consortium that could photosynthetically accumulate a PHA content of 13.2 % g PHA/g VSS (6.7 Cmmol PHA/L) at a production rate of 0.20 g PHA/L·day in the light phase, and was able to further increase that storage up to 18.5 % g PHA/g VSS (11.0 Cmmol PHA/L) at a production rate of 1.35 g PHA/L·day in the dark-aerated period. Furthermore, the light-feast/dark-aerated-famine operation was able to maintain the performance of the selection reactor under winter conditions, unlike non-aerated PMC systems operated under summer conditions, suggesting that night-time aeration coupled with the constant presence of ammonia can contribute to overcoming the seasonal constraints of outdoor operation of PMCs for PHA production.publishersversionpublishe

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

Development of image analysis methodologies to quantify intracellular PHA, polyphosphate and glycogen within wastewater treatment plants

In wastewater treatment plants (WWTP), enhanced biological phosphorus removal (EBPR) processes are performed by mixed cultures containing polyphosphate (PAO) and glycogen accumulating organisms (GAO). In these processes, it is of crucial importance to monitor the intracellular metabolism, namely glycogen, polyhydroxyalkanoates (PHA) and polyphosphate (polyP) inclusions, to determine its efficiency. However, traditional monitoring, carried out through off-line chemical analyses, is laborious and time-consuming. Therefore, there is a clear need to develop new techniques to promptly quantify these intracellular polymers, with image analysis emerging as a promising tool. The use of staining methodologies with specific fluorescent dyes is widespread in EBPR research, including Nile blue for PHA and DAPI for polyP. Although rarely applied in EBPR studies, Aniline blue is a fluorescent stain that can be used for glycogen determination. Furthermore, these fluorescent stains have generally been employed for qualitative rather than quantitative analysis. Therefore, this study aim focused on developing fluorescence-based staining methodologies for glycogen, and on acquisition, processing and image analysis procedures for PHA, polyP and glycogen. Image analysis data was then correlated with traditional analytical data by multivariable statistics. Regarding the determination of the glycogen intracellular concentration, results have been promising, presenting a good correlation (R2 of 0.915) between analytical and image analysis data. The staining and image analysis procedures for the determination of the intracellular concentration of PHA and polyP are currently being optimized. This study will provide a quantitative means to assess PAO and GAO metabolic activity in-situ in WWTP, facilitating the optimisation of these processes