Long term operation of a phototrophic biological nutrient removal system: Impact of CO2 concentration and light exposure on process performance.

The 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.

Impact of biogenic substrates on sulfamethoxazole biodegradation kinetics by Achromobacter denitrificans strain PR1.

Pure cultures have been found to degrade pharmaceutical compounds. However, these cultures are rarely characterized kinetically at environmentally relevant concentrations. This study investigated the kinetics of sulfamethoxazole (SMX) degradation by Achromobacter denitrificans strain PR1 at a wide range of concentrations, from ng/L to mg/L, to assess the feasibility of using it for bioaugmentation purposes. Complete removal of SMX occurred for all concentrations tested, i.e., 150 mg/L, 500 µg/L, 20 µg/L, and 600 ng/L. The reaction rate coefficients (kbio) for the strain at the ng/L SMX range were: 63.4 ± 8.6, 570.1 ± 15.1 and 414.9 ± 124.2 L/g[Formula: see text]·day), for tests fed without a supplemental carbon source, with acetate, and with succinate, respectively. These results were significantly higher than the value reported for non-augmented activated sludge (0.41 L/(g [Formula: see text]·day) with hundreds of ng/L of SMX. The simultaneous consumption of an additional carbon source and SMX suggested that the energetic efficiency of the cells, boosted by the presence of biogenic substrates, was important in increasing the SMX degradation rate. The accumulation of 3-amino-5-methylisoxazole was observed as the only metabolite, which was found to be non-toxic. SMX inhibited the Vibrio fischeri luminescence after 5 min of contact, with EC50 values of about 53 mg/L. However, this study suggested that the strain PR1 still can degrade SMX up to 150 mg/L. The results of this work demonstrated that SMX degradation kinetics by A. denitrificans PR1 compares favorably with activated sludge and the strain is a potentially interesting organism for bioaugmentation for SMX removal from polluted waters.

Polyhydroxyalkanoates production in purple phototrophic bacteria ponds: A breakthrough in outdoor pilot-scale operation.

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.

The phototrophic metabolic behaviour of Candidatus accumulibacter.

The phototrophic capability of Candidatus Accumulibacter (Accumulibacter), a common polyphosphate accumulating organism (PAO) in enhanced biological phosphorus removal (EBPR) systems, was investigated in this study. Accumulibacter is phylogenetically related to the purple bacteria Rhodocyclus from the family Rhodocyclaceae, which belongs to the class Betaproteobacteria. Rhodocyclus typically exhibits both chemoheterotrophic and phototrophic growth, however, limited studies have evaluated the phototrophic potential of Accumulibacter. To address this gap, short and extended light cycle tests were conducted using a highly enriched Accumulibacter culture (95%) to evaluate its responses to illumination. Results showed that, after an initial period of adaptation to light conditions (approximately 4-5 h), Accumulibacter exhibited complete phosphorus (P) uptake by utilising polyhydroxyalkanoates (PHA), and additionally by consuming glycogen, which contrasted with its typical aerobic metabolism. Mass, energy, and redox balance analyses demonstrated that Accumulibacter needed to employ phototrophic metabolism to meet its energy requirements. Calculations revealed that the light reactions contributed to the generation of, at least more than 67% of the ATP necessary for P uptake and growth. Extended light tests, spanning 21 days with dark/light cycles, suggested that Accumulibacter generated ATP through light during initial operation, however, it likely reverted to conventional anaerobic/aerobic metabolism under dark/light conditions due to microalgal growth in the mixed culture, contributing to oxygen production. In contrast, extended light tests with an enriched Tetrasphaera culture, lacking phototrophic genes in its genome, clearly demonstrated that phototrophic P uptake did not occur. These findings highlight the adaptive metabolic capabilities of Accumulibacter, enabling it to utilise phototrophic pathways for energy generation during oxygen deprivation, which holds the potential to advance phototrophic-EBPR technology development.

Segregated flux balance analysis constrained by population structure/function data: the case of PHA production by mixed microbial cultures.

In this study we developed a segregated flux balance analysis (FBA) method to calculate metabolic flux distributions of the individual populations present in a mixed microbial culture (MMC). Population specific flux data constraints were derived from the raw data typically obtained by the fluorescence in situ hybridization (FISH) and microautoradiography (MAR)-FISH techniques. This method was applied to study the metabolic heterogeneity of a MMC that produces polyhydroxyalkanoates (PHA) from fermented sugar cane molasses. Three populations were identified by FISH, namely Paracoccus sp., Thauera sp., and Azoarcus sp. The segregated FBA method predicts a flux distribution for each of the identified populations. The method is shown to predict with high accuracy the average PHA storage flux and the respective monomeric composition for 16 independent experiments. Moreover, flux predictions by segregated FBA were slightly better than those obtained by nonsegregated FBA, and also highly concordant with metabolic flux analysis (MFA) estimated fluxes. The segregated FBA method can be of high value to assess metabolic heterogeneity in MMC systems and to derive more efficient eco-engineering strategies. For the case of PHA-producing MMC considered in this work, it becomes apparent that the PHA average monomeric composition might be controlled not only by the volatile fatty acids (VFA) feeding profile but also by the population composition present in the MMC.

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

Phototrophic 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.

Long-term operation of a reactor enriched in Accumulibacter clade I DPAOs: performance with nitrate, nitrite and oxygen.

The microbiology of denitrifying enhanced biological phosphorus removal systems has been a subject of much debate. The question has centred on the affinities of different types of Candidatus Accumulibacter PAOs, type I and type II, towards different electron acceptors such as oxygen, nitrate and nitrite. This study used a propionate anaerobic/anoxic/aerobic lab-scale sequencing batch reactor where a microbial culture was successfully enriched in Accumulibacter type I organisms (approx. 90%). The culture was able to take up phosphorus using nitrate, nitrite and oxygen as electron acceptors, although experiments with oxygen led to the fastest P removal rate. The phosphorus uptake to nitrogen consumed ratio (P/N ratio), when using both nitrate and nitrite, was shown to be affected by pH in the range of 7-8.2, achieving higher values for lower pH values (7.0-7.5). The effect of pH on P removal seems to follow a similar trend for both nitrate and nitrite. To our knowledge, this is the first study where the impact of pH in the phosphate removal stoichiometry using the three most significant electron acceptors is shown for such a high enrichment in Accumulibacter type I.

Multivariate analysis of the transport in an ion exchange membrane bioreactor for removal of anionic micropollutants from drinking water.

The present study focuses on investigating the effects of biological compartment conditions on the transport of nitrate and perchlorate in an Ion Exchange Membrane Bioreactor (IEMB). In this hybrid process, the transport depends not only on the membrane properties but also on the biological compartment conditions. The experiments were planned according to the Plackett-Burman statistical design in order to cover a broader range of experimental conditions, under which a previously developed mechanistic transport model was not able to predict correctly the transport fluxes of the target pollutants. Using Principal Component Analysis, it was possible to identify not only the concentrations of target (nitrate and perchlorate) and of major driving counter-ion (chloride) but also those of some biomedium components (e.g. ammonia, ethanol and sulphate) as variables that affect the transport rate of micropollutants across the membrane. These conclusions are based on the loadings of the two first principal components that describe 84% of the data variance. The present study also revealed that the hydraulic retention time and the hydrodynamic conditions in the biocompartment have a minor contribution to the micropollutants transport. The results obtained are important for process optimization purposes.

Real-time monitoring of membrane bioreactors with 2D-fluorescence data and statistically based models.

The application of membrane bioreactors (MBR) for wastewater treatment is growing worldwide due to their compactness and high effluent quality. However, membrane fouling, mostly associated to biological products, can reduce MBR performance. Therefore, it is important to monitor MBRs as close to real-time as possible to accelerate control actions for maximal biological and membrane performance. 2D-fluorescence spectroscopy is a promising on-line tool to simultaneously monitor wastewater treatment efficiency and the formation of potential biological fouling agents. In this study, 2D-fluorescence data obtained from the wastewater and the permeate of a MBR was successfully modelled using projection to latent structures (PLS) to monitor variations in the influent and effluent total chemical oxygen demand (COD). Analysis of the results also indicated that humic acids and proteins highly contributed to the measured COD in both streams. Nevertheless, this approach was not valid for other performance parameters of the MBR system (such as influent and effluent ammonia and phosphorus), which is usually characterised through a high number of analytical and operating parameters. Principal component analysis (PCA) was thus used to find possible correlations between these parameters, in an attempt to reduce the analytical effort required for full MBR characterisation and to reduce the time frame necessary to obtain monitoring results. The 3 first principal components, capturing 57% of the variance, indicated and confirmed expected relationships between the assessed parameters. However, this approach alone could not provide robust enough correlations to enable the elimination of parameters for process description (PCA loadings ≤ 0.5). Nevertheless, it is possible that the information captured by 2D-fluorescence spectroscopy could replace some of the analytical and operating parameters, since this technique was able to successfully describe influent and effluent total COD. It is thus proposed that combined modelling of 2D-fluorescence data and selected performance/operating parameters should be further explored for efficient MBR monitoring aiming at rapid process control.

Achieving nitrogen and phosphorus removal at low C/N ratios without aeration through a novel phototrophic process.

Conventional wastewater treatment technologies for biological nutrient removal (BNR) are highly dependent on aeration for oxygen supply, which represents a major operational cost of the process. Recently, phototrophic enhanced biological phosphorus removal (photo-EBPR) has been suggested as an alternative system for phosphorus removal, based on a consortium of photosynthetic microorganisms and chemotrophic bacteria, eliminating the need for costly aeration. However, wastewater treatment plants must couple nitrogen and phosphorus removal to achieve discharge limits. For this reason, a new microalgae-bacterial based system for phosphorus and nitrogen removal is proposed in this work. The photo-BNR system studied here consists of a sequencing batch reactor operated with dark anaerobic, light aerobic, dark anoxic and idle periods, to allow both N and P removal. Results of the study show that the photo-BNR system was able to remove 100% of the 40 mg N/L of ammonia fed to the reactor and 94 ± 3% of the total nitrogen (Influent COD:N ratio of 300:40, similar to domestic wastewater). Moreover, an average of 25 ± 9.2 mg P/L was simultaneously removed in the photo-BNR tests, representing the P removal capacity of this system, which exceeds the level of P removal required from typical domestic wastewater. Full ammonia removal was achieved during the light phase, with 67 ± 5% of this ammonia being assimilated by the microbial culture and the remaining 33 ± 5% being converted into nitrate. The assimilated P corresponded to 2.8 ± 0.23 mg P/L, which only represented, approximately, 1/9 of the P removal capacity of the system. Half of the nitrified ammonia was subsequently denitrified during the dark anoxic phase (50 ± 24%). Overall, the photo-BNR system represents the first treatment alternative for N and P from domestic wastewater with no need of mechanical aeration or supplemental carbon addition, representing an alternative low-energy technology of interest.

Polyhydroxyalkanoates production from fermented domestic wastewater using phototrophic mixed cultures.

Phototrophic mixed cultures (PMC) have been found to be a promising technology to produce polyhydroxyalkanoates (PHA), however, work performed thus far has focussed mainly on the use of synthetic feedstocks and operational conditions that differ from those expectable in full-scale processes. The goals of this work were to study, for the first time, the capability of PMCs to produce PHA using real fermented domestic wastewater as feedstock under mixing/light/temperature conditions that are naturally found in outdoor open systems. Various operational strategies were evaluated in this study to increase PHA productivity, namely the poly(3-hydroxybutyric-co-3-hydroxyvaleric) copolymer (PHBV) by PMC systems. Two lab-scale photobioreactors were operated in parallel, with transient illumination (12 h light/12 h dark) and subjected to feedstock fluctuations under two culture selection strategies that best suit the oxidative conditions of high rate algal ponds (HRAPs) which are commonly applied in wastewater treatment plants (WWTP). Under a permanent carbon feast regime (selection strategy 1), the PMC became highly enriched in phototrophic purple bacteria (PPB), and two complementary conditions that can improve the selection of PHA accumulating bacteria were discovered: phosphate cycling, where 20% PHA/VSS (86HB:14HV in a C-mol basis) with a light phase productivity of 0.23 g PHA/L•d_light phase was attained; and transitioning from selection under low organic loading rate (OLR) to high OLR where 17.6% PHA/VSS (60HB:40HV in C base) with a light phase productivity of 0.18 g PHA/L•d_light phase was achieved. Under a feast and famine regime (selection strategy 2), a PMC consortium of microalgae and PPB was obtained, and a multiple pulse feeding strategy during the first hours of the light phase in the selector reactor led to a 26.1% PHA/VSS (36HB:64HV in C base) content, with a productivity of 0.26 g PHA/L•d_light phase and 0.52 g PHA/L•d_feast phase. An accumulation test under higher light intensity led to 30.8% PHA/VSS (85HB:15HV on a C-mol basis) with a productivity of 2.67 g PHA/L•d, along the 8 h of accumulation.

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

The 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.

Analysis of 65 pharmaceuticals and personal care products in 5 wastewater treatment plants in Portugal using a simplified analytical methodology.

Pharmaceuticals and personal care products (PPCPs) are becoming increasingly recognised as important micropollutants to be monitored in wastewater treatment plants (WWTPs), since WWTP effluents represent an important point source to natural aquatic systems. In this study, the abundance of 65 PPCPs was analysed in 5 Portuguese WWTPs during the spring and autumn. Due to the fact that analytical approaches normally used to quantify the abundance of these compounds are labour intensive and require various specific procedures, this study proposes a set of simplified analytical methods for the quantification of pharmaceutically active compounds (PhACs) and polycyclic musks in liquid and sludge samples. The analytical methods were validated using influent wastewater matrices, showing comparable limits of detection and quantification as literature values for most PPCPs, with the exception of the estrogenic compounds. The PhAC concentrations detected in the WWTP survey were in the range of 0.050-100 µg L(-1) in the influent and up to 50 µg L(-1) in the effluent, where the non-steroidal anti-inflammatory drugs (NSAIDs) were the most abundant and frequently detected group. Some musks were detected up to 11.5 µg L(-1) in the influent and 0.9 µg L(-1) in the effluent, and adsorbed in the sludge up to 22.6 µg g(-1).

Influence of feeding strategies of mixed microbial cultures on the chemical composition and microstructure of copolyesters P(3HB-co-3HV) analyzed by NMR and statistical analysis.

NMR spectroscopy was applied for quantitative and qualitative characterization of the chemical composition and microstructure of a series of poly(3-hydroxybutyrate-co-3-hydoxyvalerate) copolymers, P(3HB-co-3HV), synthesized by mixed microbial cultures at several different feeding strategies. The monomer sequence distribution of the bacterially synthesized P(3HB-co-3HV) was defined by analysis of their high-resolution 1D (13)C NMR and 2D (1)H/(13)C HSQC and (1)H/(13)C HMBC NMR spectra. The results were verified by employment of statistical methods and suggest a block copolymer microstructure of the P(3HB-co-3HV) copolymers studied. Definitive distinction between block copolymers or a mixture of random copolymers could not be achieved. NMR spectral analysis indicates that the chemical composition and microstructure of the copolymers can be tuned by choosing a correct feeding strategy.

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

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 ±â€¯0.07 Cmol-PHB/Cmol-Acet) and specific PHB production rates of 2.21 ±â€¯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.

The effect of seed sludge on the selection of a photo-EBPR system.

The phototrophic-enhanced biological phosphorus removal system (photo-EBPR) was recently proposed as an alternative photosynthetic process to conventional phosphorus removal. Previous work showed the possibility of obtaining a photo-EBPR system starting from a culture already enriched in polyphosphate accumulating organisms (PAOs). The present work evaluated whether the same could be achieved starting from conventional activated sludge. A sequencing batch reactor inoculated with sludge from a wastewater treatment plant (WWTP) was fed with a mixture of acetate and propionate (75%:25%) and subjected to dark/light cycles to select a photo-EBPR system containing PAOs and photosynthetic organisms, the oxygen providers for the system. The results showed that it is possible to obtain a photo-EBPR system starting from a WWTP sludge, although the process is slower than when started with a sludge already enriched in PAOs. After 15 days of operation, the system could remove 60 ± 2 mg-P/L of phosphorus (approximately 67% of the concentration at the end of dark period) in the light period, from which 13 ± 1 mg-P/L was removed during the phase without external air supply. These results indicate that a photo-EBPR system can be obtained independently of the seed sludge initially used, provided that a suitable operating strategy is implemented, i.e. by imposing conditions that favour the growth and coexistence of PAOs and photosynthetic microorganisms.

Kinetic and metabolic aspects of Defluviicoccus vanus-related organisms as competitors in EBPR systems.

A reactor was successfully enriched (90% as shown by Fluorescence in situ Hybridization) in Defluviicoccus vanus-related organisms presenting a Glycogen Accumulating Organisms (GAO) phenotype. Initial batch tests were performed using anaerobic/aerobic conditions to assess the capacity of different carbon sources utilization frequently abundant in wastewater: acetate, propionate, butyrate, valerate and glucose. Acetate and propionate were totally consumed in the anaerobic phase as well as butyrate and valerate, though these last ones with a very low consumption rate. All substrates were converted to polyhydroxyalkanoates (PHA). Glucose had a very slight anaerobic consumption but failed to disclose a typical GAO phenotype. In aerobic conditions, again all carbon sources were readily consumed except for glucose, with acetate and propionate having the higher consumption rates. Therefore, glucose seems not be used by this type of organisms. Acetate and propionate consumption rates indicated that these GAOs could reveal good competition advantages in EBPR systems where these carbon sources are available, especially propionate. Volatile Fatty Acid (VFA) uptake in aerobic phase and consequential PHA production indicate these organisms as possible candidates for PHA production.

Burkholderia thailandensis as a microbial cell factory for the bioconversion of used cooking oil to polyhydroxyalkanoates and rhamnolipids.

The present work assessed the feasibility of used cooking oil as a low cost carbon source for rhamnolipid biosurfactant production employing the strain Burkholderia thailandensis. According to the results, B. thailandensis was able to produce rhamnolipids up to 2.2 g/L, with the dominant congener being the di-rhamnolipid Rha-Rha-C14-C14. Rhamnolipids had the ability to reduce the surface tension to 37.7 mN/m and the interfacial tension against benzene and oleic acid to 4.2 and 1.5 mN/m, while emulsification index against kerosene reached up to 64%. The ability of B. thailandensis to accumulate intracellular biopolymers, in the form of polyhydroxyalkanoates (PHA), was also monitored. Polyhydroxybutyrate (PHB) was accumulated simultaneously and consisted of up to 60% of the cell dry weight. PHB was further characterized in terms of its molecular weight and thermal properties. This is the first study reporting the simultaneous production of polyhydroxyalkanoates and rhamnolipids by the non-pathogen rhamnolipid producer B. thailandensis.

Biosynthesis of silver nanoparticles and polyhydroxybutyrate nanocomposites of interest in antimicrobial applications.

This study deals with the optimization and scaling up of the production of poly(3-hydroxybutyrate), PHB, nanocomposites containing biosynthesized silver nanoparticles (AgNPs) to generate materials with antimicrobial performance. First, a comparative study of the chemical and biological synthesis of AgNPs during the fermentation process of Cupriavidus necator at shake flask-scale was carried out. These experiments demonstrated the inherent capacity of C. necator to reduce the silver salt and produce AgNPs without the need for adding a reducing agent and, that the method of synthesis (with or without reducing agent) affects the dispersion of the AgNPs and their antimicrobial performance. Finally, the process was scaled-up to a 10Liters bioreactor and the relevant physical properties of the PHB-AgNPs nanocomposites pressed into films were determined. From the characterization work, the AgNPs were found to be well dispersed and distributed into the polymer matrix, having a maximum frequency of particles with average diameter of 76-95nm. Moreover, the presence of AgNPs did not cause any effect on the thermal properties of the biopolymer, although a slight reduction in crystallinity was seen. The developed materials presented a strong antimicrobial activity against the food-borne pathogens Salmonella enterica and Listeria monocytogenes, which makes them potentially suitable for active coatings and packaging applications. Complete biodisintegration of the samples occurred during composting conditions within the first 40days. Interestingly, the presence of the AgNPs did not impair the profile of biodegradation of the microbial polymer.

The impact of operational strategies on the performance of a photo-EBPR system.

A novel Phototrophic - Enhanced Biological Phosphorus Removal (Photo-EBPR) system, consisting of a consortium of photosynthetic organisms and polyphosphate accumulating organisms (PAOs), was studied in this work. A sequencing batch reactor was fed with a mixture of acetate and propionate (75%-25%) and subjected to dark/light cycles in order to select a photo-EBPR system containing PAOs and photosynthetic organisms, the latter likely providers of oxygen to the system. The results from the selection period (stage 1) showed that the photo-EBPR culture was capable of performing P release in the dark and P uptake in the presence of light, under limited oxygen concentrations. During the optimization period, the aeration period, which was initially provided at the end of the light phase, was gradually reduced until a non-aerated system was achieved, while the light intensity was increased. After optimization of the operational conditions, the selected consortium of photosynthetic organisms/PAOs showed high capacity of P removal in the light phase in the absence of air or other electron acceptor. A net P removal of 34 ± 3 mg-P/L was achieved, with a volumetric P removal rate of 15 ± 2 mg-P/L.h, and 79 ± 8% of P removal from the system. Also, in limiting oxygen conditions, the P uptake rate was independent of the PHA consumption, which demonstrates that the organisms obtained energy for P removal from light. These results indicated that a photo-EBPR system can be a potential solution for P removal with low COD/P ratios and in the absence of air, prospecting the use of natural sunlight as illumination, which would reduce the costs of EBPR operation regarding aeration.