Diclofenac biotransformation in the enhanced biological phosphorus removal process.

Diclofenac is a pharmaceutical active compound frequently detected in wastewater and water bodies, and often reported to be persistent and difficult to biodegrade. While many previous studies have focussed on assessing diclofenac biodegradation in nitrification and denitrification processes, this study focusses on diclofenac biodegradation in the enhanced biological phosphorus removal (EBPR) process, where the efficiency of this process for diclofenac biodegradation as well as the metabolites generated are not well understood. An enrichment of Accumulibacter polyphosphate accumulating organisms (PAOs) was operated in an SBR for over 300 d, and acclimatized to 20 µg/L of diclofenac, which is in a similar range to that observed in domestic wastewater influents. The diclofenac biotransformation was monitored in four periods of stable operation and linked to the microbial community and metabolic behaviour in each period. Nitrification was observed in two of the four periods despite the addition of a nitrification inhibitor, and these periods were positively correlated with increased diclofenac biodegradation. Interestingly, in two periods with excellent phosphorus removal (>99%) and no nitrification, different levels of diclofenac biotransformation were observed. Period 2, enriched in Accumulibacter Type II achieved more significant diclofenac biotransformation (3.4 µg/gX), while period 4, enriched in Accumulibacter Type I achieved lower diclofenac biotransformation (0.4 µg/gX). In total, 23 transformation products were identified, with lower toxicity than the parent compound, enabling the elucidation of multiple metabolic pathways for diclofenac biotransformation. This study showed that PAOs can contribute to diclofenac biotransformation, yielding less toxic transformation products, and can complement the biodegradation carried out by other organisms in activated sludge, particularly nitrifiers.

The storage compounds associated with Tetrasphaera PAO metabolism and the relationship between diversity and P removal.

In enhanced biological phosphorus removal (EBPR), Tetrasphaera can potentially be an abundant and important polyphosphate accumulating organism (PAO), however ongoing questions remain concerning its storage compounds, phosphorus (P) removal capabilities and metabolic behaviour. This study investigated each of these points in an enriched Tetrasphaera culture (95% biovolume). The enriched Tetrasphaera culture fermented amino acids, while also converting and storing diverse amino acids as aspartic and glutamic acid within cells. Subsequent intracellular consumption of these two amino acids during the aerobic phase supports their importance in the metabolism of Tetrasphaera. Polyhydroxyalkanoate (PHA) cycling was also observed in this study, in contrast to some previous studies on Tetrasphaera. While exhibiting anaerobic phosphorus release and aerobic uptake, the highly enriched Tetrasphaera culture was unable to completely remove phosphorus in sequencing batch reactors (SBR) cycles, with an average removal efficiency of 72.3 ± 7.8%. This is unlike a previous study containing both Tetrasphaera (70%) and Accumulibacter (22%), which regularly performed complete phosphorus removal under otherwise similar operational conditions, at efficiencies of > 99%. Notably, the phylodiversity of organisms belonging to Tetrasphaera was substantially different in the present work, consisting mainly of organisms within Clade 2, likely impacting PHA cycling. These results suggest that the contribution of Tetrasphaera towards P removal is highly dependent on the composition of its Clades within this microbial group and an observed higher abundance of Tetrasphaera in WWTPs does not necessarily imply overall higher P removal. This study improves our understanding of the role of Tetrasphaera within EBPR systems and key factors impacting its metabolism.

Sludge retention time impacts on polyhydroxyalkanoate productivity in uncoupled storage/growth processes.

The process involving mixed microbial cultures (MMCs) and waste-based substrates emerged as an alternative solution to reduce the market price of polyhydroxyalkanoates (PHAs). The selection of an efficient MMC that displays a significant PHA accumulation potential and a high growth rate is considered a key factor for the MMC PHA production feasibility. This study used a pilot plant to investigate the dynamics of growth vs storage in a mixed culture fed with fermented fruit waste under uncoupled carbon and nitrogen feeding. Varying sludge retention times (SRTs) (2 and 4 d) and organic loading rates (OLRs) (from 2.6 to 14.5 gCOD.L-1.d-1) were imposed for this purpose. Results showed that, regardless of the OLR imposed, cultures selected at lower SRT grew faster and more efficiently using stored PHA. However, they had inferior specific storage rates and accumulation capacity, resulting in lower PHA productivity. Additionally, the polymer storage yield was independent of the SRT, and was directly linked with the abundance of putative PHA-storers in the MMC. The high PHA productivity (4.6 ± 0.3 g.L-1.d-1) obtained for the culture selected at 4 d of SRT was 80% above that obtained for the lower SRT tested, underlining the importance of achieving a good balance between culture growth and accumulation capacity to increase the viability of the PHA-producing process from wastes.

Accumulibacter diversity at the sub-clade level impacts enhanced biological phosphorus removal performance.

Accumulibacter is a well-known group of organisms, typically considered to be polyphosphate accumulating organisms (PAOs), but potentially capable of glycogen accumulating organism (GAO) metabolism under limiting influent phosphate levels. Metabolic features of Accumulibacter are typically linked to its phylogenetic identity at the Type or clade level, though it is unclear the extent to which Accumulibacter diversity can correlate with its capacity to perform P removal. This paper investigates the fine-scale diversity of Accumulibacter and its link with enhanced biological phosphorus removal (EBPR) performance under various operating conditions, to understand the conditions and community structure leading to successful and unsuccessful EBPR operation. For this purpose, the organic carbon feeding rate and total organic carbon concentration were varied during three distinct operational periods, where influent phosphate was never limiting. Accumulibacter was always the dominant microbial group (>80% of all bacteria according to quantitative fluorescence in situ hybridisation - FISH) and low levels of Competibacter and other GAOs were consistently observed (<15% of all bacteria). Steady state was achieved in each of the three periods, with average phosphorus removal levels of 36%, 99% and >99%, respectively. Experimentally determined stoichiometric activity supported the expression of a mixed PAO/GAO metabolism in the first steady state period and the typical PAO metabolism in the other two steady state periods. FISH quantification and amplicon sequencing of the polyphosphate kinase (ppk1) functional gene indicated that Accumulibacter clade IIC was selected in the first steady state period, which shifted to clade IA after decreasing the carbon feeding rate in steady state period 2, and finally shifted back to clade IIC in the third steady state period. Fine-resolution Ppk-based phylogenetic analysis revealed three different clusters within Accumulibacter clade IIC, where clusters IICii and IICiii were linked to poor EBPR performance in period 1, and cluster IICi was linked to good EBPR performance in period 3. This study shows that the deterioration of EBPR processes through GAO activity at non-limiting P concentrations can be linked to organisms that are typically classified as PAOs, not only to known GAOs such as Competibacter. Intra-clade phylogenetic diversity within Accumulibacter showed that some clusters actually behave similarly to GAOs even without influent phosphate limitation. This study highlights the need to closely re-examine traditional interpretations regarding the link between the microbial community composition and identity with the performance and metabolism of EBPR systems.

Valorization of raw brewers' spent grain through the production of volatile fatty acids.

This work is focused on production of volatile fatty acids (VFA) through anaerobic digestion (AD) using raw (without pre-treatment) brewers' spent grain (BSG) as feedstock. VFAs are by-products from AD of organic wastes with wide potential industrial application in bioplastic production. A long term fed batch stirred-tank reactor was operated and the impact of three hydraulic retention times (HRT) and two organic loading rates (OLR) on VFA production was assessed. Results showed clearly that AD of raw BSG is possible without a pre-treatment step. The maximum volumetric VFA productivity of 91.3 ± 9.1 mgCODL-1 h-1 and VFA concentration of 24.9 ± 2.6 g L-1 were obtained for 16 days of HRT and 16 gTSinL-1d-1 of OLR. This is the highest value of VFA concentration so far reported for BSG. Propionic, acetic and butyric acids were the main VFAs produced. Community identification by FISH and its correlation with process parameters was performed by principal component analysis.

Phosphorus and ammonium removal characteristics from aqueous solutions by a newly isolated plant growth-promoting bacterium.

An indigenous plant growth-promoting bacterium isolated from Peganum Harmala rhizosphere in the arid ecosystem was found to solubilize and accumulate phosphates. This isolate was identified as Pseudomonas sp. (PHR6) by partial 16S rRNA gene sequence analysis. Controlled batch experiments on nutrients removal by this isolate in mineral medium showed relatively high efficiencies after 24 h of aerobic incubation with average values of 117.59 and 335.38 mg gVSS-1 for phosphorus (P-PO4) and nitrogen (N-NH4), respectively. Furthermore, the strain performed heterotrophic nitrification ranging from 48.81% to 84.24% of the total removed nitrogen. On the other hand, the experimental results showed that a short idle period (24 h) significantly enhanced P accumulation (up to 95%) and N assimilation (up to 50%) of the total removed amounts. However, long idle period (20 days) revealed firstly aerobic phosphorous release phase succeeded by another removal one within 24 h of incubation. Overall, the idle treatment enhances P removal efficiency from the mineral liquid medium without significant effects on N-NH4 removal performance. The isolated strain showed also significant nutrient removal ability from synthetic wastewater providing an accumulated fraction of 98% from the total removed phosphorus amount. This study highlights the potential contribution of the selected rhizobacterium PHR6 to both environmental nutrient recycling and pollution control especially regarding phosphorus.

Performance of a two-stage anaerobic digestion system treating fruit pulp waste: The impact of substrate shift and operational conditions.

Food and beverage industry wastes present high amounts of organic matter, which may cause water quality degradation if not treated. Two-stage anaerobic digestion is a promising and efficient solution for the treatment of this type of wastes whilst producing bioenergy. The composition of fruit pulp waste varies throughout the different harvesting seasons, which may impact the process performance. In this study, a two-stage anaerobic digestion system was operated to assess the effect of substrate shift from peach to apple pulp wastes (obtained from a fruit juice company) on the microbial community activity and performance. During acidogenesis, the sugar conversion was higher than 95% for all operational conditions tested, obtaining a degree of acidification up to 89%. Principal Component Analysis was used to evaluate the relationship between the initial fermentation state of the residues in each operational condition and the obtained effluent. Methanogenic activity resulted in high organic carbon consumption (89%) and high methane productivities, achieving a maximum of 4.33 [Formula: see text] for peach waste influent. Overall, the results showed that the microbial community activity was not affected by the substrate shift, converting the sugars into biogas rich in methane (>70% CH4). Microbial analysis showed that the communities present in the acidogenic and methanogenic reactors were highly enriched in bacteria and archaea, respectively. The observed stability of the process, also demonstrated in pilot scale, confirmed the robustness of the process and thus, was suitable for implementation in companies producing seasonally different fruit wastes in a continuous operation.

The link of feast-phase dissolved oxygen (DO) with substrate competition and microbial selection in PHA production.

Polyhydroxyalkanoates (PHAs) are biobased and biodegradable polyesters with the potential to replace conventional plastics. Aeration requires large amounts of energy in PHA production by mixed microbial cultures (MMCs), particularly during the feast phase due to substrate uptake. The objective of this study was to investigate the impact of DO concentrations on microbial selection, substrate competition and PHA production performance by MMCs. This represents the first study investigating DO impact on PHA production while feeding the multiple volatile fatty acids (VFAs) typically encountered in real fermented feedstocks, as well as the substrate preferences at different DO levels. Efficient microbial cultures were enriched under both high (3.47 ± 1.12 mg/L) and low (0.86 ± 0.50 mg/L) DO conditions in the feast phase containing mostly the same populations but with different relative abundance. The most abundant microorganisms in the two MMCs were Plasticicumulans, Zoogloea, Paracoccus, and Flavobacterium. Butyrate and valerate were found to be the preferred substrates as compared to acetate and propionate regardless of DO concentrations. In the accumulation step, the PHA storage capacity and yield were less affected by the change of DO levels when applying the culture selected under low DO in the feast phase (PHA storage capacity >60% and yield > 0.9 Cmol PHA/Cmol VFA). A high DO level is required for maximal PHA accumulation rates with the four VFAs (acetate, propionate, butyrate and valerate) present, due to the lower specific uptake rates of acetate and propionate under low DO conditions. However, butyrate and valerate specific uptake rates were less impacted by DO levels and hence low DO for PHA accumulation may be effective when feed is composed of these substrates only.

Dynamic change of pH in acidogenic fermentation of cheese whey towards polyhydroxyalkanoates production: Impact on performance and microbial population.

Polyhydroxyalkanoates (PHA) are a sustainable alternative to conventional plastics that can be obtained from industrial wastes/by-products using mixed microbial cultures (MMC). MMC PHA production is commonly carried out in a 3-stage process of acidogenesis, PHA culture selection and accumulation. This research focused on the possibility of tailoring PHA by controlling the acidogenic reactor operating conditions, namely pH, using cheese whey as model feedstock. The objective was to investigate the impact that dynamically varying the acidogenic pH, when targeting different PHA monomer profiles, had on the performance and microbial community profile of the anaerobic reactor. To accomplish this, an anaerobic reactor was continuously operated under dynamic pH changes, ranging from pH 4 to 7, turning to pH 6 after each change of pH. At pH 6, lactate and acetate were the dominant products (41-48% gCOD basis and 22-44% gCOD basis, respectively). At low pH, lactate production was higher while at high pH acetate production was favoured. Despite the dynamic change of pH, the fermentation product composition at pH 6 was always similar, showing the resilience of the process, i.e. when the same pH value was imposed, the culture produced the same metabolic products independently of the history of changes occurring in the system. The different fermentation product fractions led to PHAs of different compositions. The microbial community, analysed by high throughput sequencing of bacterial 16S rRNA gene fragments, was dominated by Lactobacillus, but varied markedly when subjected to the highest and lowest pH values of the tested range (4 and 7), with increase in the abundance of Lactococcus and a member of the Candidate Division TM7. Different bacterial profiles obtained at pH 6 during this dynamic operation were able to produce a consistent profile of fermentation products (and consequently a constant PHA composition), demonstrating the community's functional redundancy.