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

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

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

Up-scale challenges on biopolymer production from waste streams by Purple Phototrophic Bacteria mixed cultures: A critical review

Financial support from the Regional Government of Madrid through the project S2018/EMT-4344 BIOTRES-CM is gratefully acknowledged. D. Puyol wishes to thank the Spanish Ministry of Economy for the Ramon y Cajal grant. J. FThe increasing volume of waste streams require new biological technologies that can address pollution concerns while offering sustainable products. Purple phototrophic bacteria (PPB) are very versatile organisms that present a unique metabolism that allows them to adapt to a variety of environments, including the most complex waste streams. Their successful adaptation to such demanding conditions is partly the result of internal polymers accumulation which can be stored for electron/energy balance or as carbon and nutrients reserves for deprivation periods. Polyhydroxyalkanoates, glycogen, sulphur and polyphosphate are examples of polymers produced by PPB that can be economically explored due to their applications in the plastic, energy and fertilizers sectors. Their large-scale production implies the outdoor operation of PPB systems which brings new challenges, identified in this review. An overview of the current PPB polymer producing technologies and prospects for their future development is also provided.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

Multicentric Genome-Wide Association Study for Primary Spontaneous Pneumothorax

Despite elevated incidence and recurrence rates for Primary Spontaneous Pneumothorax (PSP), little is known about its etiology, and the genetics of idiopathic PSP remains unexplored. To identify genetic variants contributing to sporadic PSP risk, we conducted the first PSP genome-wide association study. Two replicate pools of 92 Portuguese PSP cases and of 129 age- and sex-matched controls were allelotyped in triplicate on the Affymetrix Human SNP Array 6.0 arrays. Markers passing quality control were ranked by relative allele score difference between cases and controls (|RASdiff|), by a novel cluster method and by a combined Z-test. 101 single nucleotide polymorphisms (SNPs) were selected using these three approaches for technical validation by individual genotyping in the discovery dataset. 87 out of 94 successfully tested SNPs were nominally associated in the discovery dataset. Replication of the 87 technically validated SNPs was then carried out in an independent replication dataset of 100 Portuguese cases and 425 controls. The intergenic rs4733649 SNP in chromosome 8 (between LINC00824 and LINC00977) was associated with PSP in the discovery (P = 4.07E-03, ORC[95% CI] = 1.88[1.22-2.89]), replication (P = 1.50E-02, ORC[95% CI] = 1.50[1.08-2.09]) and combined datasets (P = 8.61E-05, ORC[95% CI] = 1.65[1.29-2.13]). This study identified for the first time one genetic risk factor for sporadic PSP, but future studies are warranted to further confirm this finding in other populations and uncover its functional role in PSP pathogenesis

Review: Feeding conserved forage to horses: recent advances and recommendations

The horse is a non-ruminant herbivore adapted to eating plant-fibre or forage-based diets. Some horses are stabled for most or the majority of the day with limited or no access to fresh pasture and are fed preserved forage typically as hay or haylage and sometimes silage. This raises questions with respect to the quality and suitability of these preserved forages (considering production, nutritional content, digestibility as well as hygiene) and required quantities. Especially for performance horses, forage is often replaced with energy dense feedstuffs which can result in a reduction in the proportion of the diet that is forage based. This may adversely affect the health, welfare, behaviour and even performance of the horse. In the past 20 years a large body of research work has contributed to a better and deeper understanding of equine forage needs and the physiological and behavioural consequences if these are not met. Recent nutrient requirement systems have incorporated some, but not all, of this new knowledge into their recommendations. This review paper amalgamates recommendations based on the latest understanding in forage feeding for horses, defining forage types and preservation methods, hygienic quality, feed intake behaviour, typical nutrient composition, digestion and digestibility as well as health and performance implications. Based on this, consensual applied recommendations for feeding preserved forages are provided

Formation of lipofuscin-like autofluorescent granules in the retinal pigment epithelium requires lysosome dysfunction

Funding Information: Supported by Funda??o para a Ciência e Tecnologia (FCT) ? Portugal co-funded by FEDER under the PT2020 Partnership Agreement (to MCS, including project PTDC/MED-PAT/30385/2017, iNOVA4Health-UIDB/04462/2020, research infrastructure PPBI-POCI-01-0145-FEDER-022122, M-ERA.NET 2/0005/2016), Boehringer Ingelheim (to MCS), Fight for Sight UK (to MCS), Wellcome Trust grant number 212216/Z/18/Z/ (to CEF). MJH was funded by Moor-fields Eye Charity with the Bill Brown 1989 Charitable Trust PhD studentship 538158, MLS was funded by FCT-CEECIND/01536/2018, ACF was funded by FCT PhD studentship (PD/BD/135503/2018). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund) and this article is supported by the LYSOCIL project funded by the European Union?s Horizon 2020 programme under grant agreement No. 811087. Funding Information: Supported by Fundação para a Ciência e Tecnologia (FCT) – Portugal co-funded by FEDER under the PT2020 Partnership Agreement (to MCS, including project PTDC/MED-PAT/30385/2017, iNOVA4Health-UIDB/04462/2020, research infrastructure PPBI-POCI-01-0145-FEDER-022122, M-ERA.NET 2/0005/2016), Boehringer Ingelheim (to MCS), Fight for Sight UK (to MCS), Wellcome Trust grant number 212216/Z/18/Z/ (to CEF). MJH was funded by Moor-fields Eye Charity with the Bill Brown 1989 Charitable Trust PhD studentship 538158, MLS was funded by FCT-CEECIND/01536/2018, ACF was funded by FCT PhD studentship (PD/BD/135503/2018). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund) and this article is supported by the LYSOCIL project funded by the European Union’s Horizon 2020 programme under grant agreement No. 811087. Publisher Copyright: Copyright 2021 The AuthorsPURPOSE. We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers. METHODS. We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing. RESULTS. AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation. CONCLUSIONS. We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.publishersversionpublishe