Rapid start-up and stable maintenance of the mainstream nitritation process based on the accumulation of free nitrous acid in a pilot-scale two-stage nitritation-anammox system

This work has been financed by the European Commission (EU) through the LIFE project ZERO WASTE WATER (LIFE19 ENV/ES/000631), the Waterworks 2014 Cofounded Call (Water JPI/Horizon) through the Pioneer_STP (PCIN-2015-022 MINECO (AEI)/ID 199 (UE)) and by the Spanish Government (AEI) through GRANDSEA (CTM2014-55397-JIN), TREASURE (CTQ2017-83225-C2-1-R) and ECOPOLYVER (PID2020-112550RB-C21 and PID2020-112550RB-C22) projects. Alba Pedrouso also acknowledges the Xunta de Galicia (Spain) for her post-doctoral fellowship (ED481B-2021-041). Authors from the USC belong to the Galician Competitive Research Group (GRC D431C-2021/37). Authors gratefully acknowledge the staff of the WWTP for their support.Two-stage partial nitritation (PN) and anammox (AMX) systems showed promising results for applying auto-trophic nitrogen removal under mainstream conditions. In this study, a pilot-scale (600 L per reactor) two-stage PN/AMX system was installed in a municipal wastewater treatment plant (WWTP) provided with a high-rate activated sludge (HRAS) system for organic carbon removal. The PN/AMX system was operated without tem-perature control (ranging from 11 to 28 degrees C) and was subjected to the same variations in wastewater charac-teristics as the WWTP (22 to 63 mg NH4+- N/L). The developed strategy is simple, does not require the addition of chemicals and is characterised by short start-up periods. The PN process was established by applying a high hydraulic load and maintained by in situ accumulated free nitrous acid (FNA) of 0.015-0.2 mg HNO2-N/L. Based on pH value, a controlled aeration strategy was applied to achieve the target nitrite to ammonium ratio in the effluent (1.1 g NO2--N/g NH4+-N) to feed the AMX reactor. Although NOB were not fully washed out from the system, nitrite accumulation remained (>99 %) stable with no evidence of NOB activity. In the AMX reactor, an overall nitrogen removal efficiency of 80 % was achieved. Regarding effluent quality, 12 +/- 3 mg TN/L was obtained, but 5 mg NO3--N/L was already in the HRAS effluent. The relative abundance of NOB showed a strong negative correlation with the FNA concentration, providing a good strategy for establishing PN under main-stream conditions.European Commission (EU) through the LIFE project ZERO WASTE WATER LIFE19 ENV/ES/000631Waterworks 2014 Cofounded Call (Water JPI/Horizon) through the Pioneer_STP (MINECO (AEI)/(UE)) ED481B-2021-041Spanish Government (AEI) 199, CTM2014-55397-JIN, CTQ2017-83225-C2-1-R, PID2020-112550RB-C21Xunta de Galicia PID2020-112550RB-C22PCIN-2015-02

Simplified engineering design towards a competitive lipid-rich effluents valorization

Medium- and long-chain fatty acids and glycerol contained in the oily fraction of many food-industry effluents are excellent candidates to produce biobased high-value triacylglycerides (TAGs) and polyhydroxyalkanoates (PHAs). The typical process configuration for TAGs recovery from lipid-rich streams always includes two steps (culture enrichment plus storage compounds accumulation) whereas, for PHAs production, an additional pretreatment of the substrate for the obtainment of soluble volatile fatty acids (VFAs) is required. To simplify the process, substrate hydrolysis, culture enrichment, and accumulation (TAG and PHA storage) were coupled here in a single sequencing batch reactor (SBR) operated under the double growth limitation strategy (DGL) and fed in pulses with industrial waste fish oil during the whole feast phase. When the SBR was operated in 12 h cycles, it was reached up to 51 wt % biopolymers after only 6 h of feast (TAG:PHA ratio of 50:51; 0.423 CmmolBIOP/ CmmolS). Daily storage compound production was observed to be over 25% higher than the reached when enrichment and accumulation stages were carried in separate operational units. Increasing the feast phase length from 6 to 12 h (18 h cycle) negatively affected the DGL strategy performance and hence system storage capacity, which was recovered after also extending the famine phase in the same proportion (24 h cycle). Besides, the carbon influx during the feast phase was identified as a key operational parameter controlling storage compounds production and, together with the C/N ratio, culture selection. The different cycle configurations tested clearly modulated the total fungal abundances without no significant differences in the size of the bacterial populations. Several PHA and TAG producers were found in the mixed culture although the PHA and TAG productions were poorly associated with the increased relative abundances (RAs) of specific operational taxonomic units (OTUs).Spanish Government (AEI, Spain) through the TREASURE project CTQ 2017-83225-C2-1-R operative program FSE Galicia 2014-2020European Commissio

Revealing the dissimilar structure of microbial communities in different WWTPs that treat fish-canning wastewater with different NaCl content

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This research was supported by the Spanish Government (Spanish Ministry of Science and Innovation) through TREASURE-TECHNOSALT (CTQ2017-83225-C2-1-R) and TREASURE-MICROSALT (CTQ2017-83225-C2-2-R) projects. The authors Alba Roibas-Rozas and Anuska Mosquera-Corral belong to the Galician Competitive Research Group GRC-ED431C 2017-29 and to the CRETUS Strategic Partnership (ED431E 2018/01) . All these programmes are co-funded by FEDER (UE) . Funding for open access charge: Universidad de Granada/CBUA.Studies that characterize the microbial communities in wastewater treatment plants (WWTPs) are numerous, yet similar studies in industrial WWTPs treating fish-canning effluents are limited. The microbial communities in samples of 4 fish-canning WWTPs that operated under different NaCl concentrations were investigated by qPCR and partial 16S rRNA gene Illumina sequencing. The absolute abundances of key microbial populations (Total Bacteria, Archaea and Fungi, ammonium oxidizing bacteria (AOB), Mycolata, Candidatus Microthrix, Ca. Accumulibacter and Ca. Competibacter) presented statistical differences among the WWTPs. The NaCl concentration negatively affected the absolute abundance of Bacteria and Fungi, filamentous, and phosphate (PAO) and glycogen (GAO) accumulating bacteria, while AOB and Ca. Microthrix populations were statistically higher in the WWTP with higher NaCl contents. On the other hand, the main bacterial operational taxonomic units (OTUs) were classified as members of Kouleothrix (Chloroflexia, Chloroflexi) and Tetrasphaera (Actinomycetia, Actinobacteria), family Beijerinckiaceae (Alphaproteobacteria, Proteobacteria), order Betaproteobacteriales (Gammaproteobacteria, Proteobacteria), Sphingobacteriales (Sphingobacteriia, Bacteroidetes) and Frankiales (Actinobacteria, Actinobacteria), class Anaerolineae (Chloroflexi), phylum Chloroflexi and Bacteria_unclassified. The structure of the bacterial community was highly dissimilar among the 4 WWTPs, as the identities of the dominant OTUs differed significantly among them. Therefore, the individual characteristics of the different WWTPs, mainly NaCl concentration, were responsible for the narrow assemblage of the bacterial communities. Different OTUs belonging to the phyla Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes and Proteobacteria were revealed as salttolerant. Taking into account these results, NaCl content was an important driver of the abundance of microbial populations and the bacterial community structure in the analysed industrial facilities.Spanish Government (Spanish Ministry of Science and Innovation) CTQ2017-83225-C2-1-R CTQ2017-83225-C2-2-REuropean CommissionGalician Competitive Research Group GRC-ED431C 2017-29CRETUS Strategic Partnership ED431E 2018/0

Dynamics of PHA-Accumulating Bacterial Communities Fed with Lipid-Rich Liquid Effluents from Fish-Canning Industries

This research was supported by the Spanish Government (Spanish Ministry of Science and Innovation) through the TREASURE-MICROSALT (CTQ2017-83225-C2-2-R) and ECOPOLYVER-MACROPOLYVER (PID2020-112550RC22) projects.The biosynthesis of polyhydroxyalkanoates (PHAs) from industrial wastes by mixed microbial cultures (MMCs) enriched in PHA-accumulating bacteria is a promising technology to replace petroleum-based plastics. However, the populations’ dynamics in the PHA-accumulating MMCs are not well known. Therefore, the main objective of this study was to address the shifts in the size and structure of the bacterial communities in two lab-scale sequencing batch reactors (SBRs) fed with fish-canning effluents and operated under non-saline (SBR-N, 0.5 g NaCl/L) or saline (SBR-S, 10 g NaCl/L) conditions, by using a combination of quantitative PCR and Illumina sequencing of bacterial 16S rRNA genes. A double growth limitation (DGL) strategy, in which nitrogen availability was limited and uncoupled to carbon addition, strongly modulated the relative abundances of the PHA-accumulating bacteria, leading to an increase in the accumulation of PHAs, independently of the saline conditions (average 9.04 wt% and 11.69 wt%, maximum yields 22.03 wt% and 26.33% SBR-N and SBR-S, respectively). On the other hand, no correlations were found among the PHAs accumulation yields and the absolute abundances of total Bacteria, which decreased through time in the SBR-N and did not present statistical differences in the SBR-S. Acinetobacter, Calothrix, Dyella, Flavobacterium, Novosphingobium, Qipengyuania, and Tsukamurella were key PHA-accumulating genera in both SBRs under the DGL strategy, which was revealed as a successful tool to obtain a PHA-enriched MMC using fish-canning effluents.Spanish Government (Spanish Ministry of Science and Innovation) CTQ2017-83225-C2-2-R PID2020-112550RC2

Structure of fungal communities in sequencing batch reactors operated at different salinities for the selection of triacylglyceride-producers from a fish-canning lipid-rich waste stream

Oleaginous fungi natively accumulate large amounts of triacylglycerides (TAG), widely used as precursors for sustainable biodiesel production. However, little attention has been paid to the diversity and roles of fungal mixed microbial cultures (MMCs) in sequencing batch reactors (SBR). In this study, a lipid-rich stream produced in the fish-canning industry was used as a substrate in two laboratory-scale SBRs operated under the feast/famine (F/F) regime to enrich microorganisms with high TAG-storage ability, under two different concentrations of NaCl (SBR-N: 0.5 g/L; SBR-S: 10 g/L). The size of the fungal community in the enriched activated sludge (EAS) was analyzed using 18S rRNA-based qPCR, and the fungal community structure was determined by Illumina sequencing. The different selective pressures (feeding strategy and control of pH) implemented in the enrichment SBRs throughout operation increased the abundance of total fungi. In general, there was an enrichment of genera previously identified as TAG-accumulating fungi (Apiotrichum, Candida, Cutaneotrichosporon, Geotrichum, Haglerozyma, Metarhizium, Mortierella, Saccharomycopsis, and Yarrowia) in both SBRs. However, the observed increase of their relative abundances throughout operation was not significantly linked to a higher TAG accumulation.Spanish Government CTQ2017 - 83225-C2 - 1- R CTQ2017 - 83225-C2 - 2-R PID-2020 - 112550RB-C2

454-Pyrosequencing Analysis of Bacterial Communities from Autotrophic Nitrogen Removal Bioreactors Utilizing Universal Primers: Effect of Annealing Temperature

Identification of anaerobic ammonium oxidizing (anammox) bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for the Bacteria domain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing) of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON) and three full-scale bioreactors (anammox, CANON, and DEMON), was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature). The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C) and hence a range of annealing temperatures of 44–49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed

Rapid start-up and stable maintenance of the mainstream nitritation process based on the accumulation of free nitrous acid in a pilot-scale two-stage nitritation-anammox system

Two-stage partial nitritation (PN) and anammox (AMX) systems showed promising results for applying autotrophic nitrogen removal under mainstream conditions. In this study, a pilot-scale (600 L per reactor) two-stage PN/AMX system was installed in a municipal wastewater treatment plant (WWTP) provided with a high-rate activated sludge (HRAS) system for organic carbon removal. The PN/AMX system was operated without temperature control (ranging from 11 to 28 °C) and was subjected to the same variations in wastewater characteristics as the WWTP (22 to 63 mg NH4+- N/L). The developed strategy is simple, does not require the addition of chemicals and is characterised by short start-up periods. The PN process was established by applying a high hydraulic load and maintained by in situ accumulated free nitrous acid (FNA) of 0.015–0.2 mg HNO2-N/L. Based on pH value, a controlled aeration strategy was applied to achieve the target nitrite to ammonium ratio in the effluent (1.1 g NO2–-N/g NH4+-N) to feed the AMX reactor. Although NOB were not fully washed out from the system, nitrite accumulation remained (>99 %) stable with no evidence of NOB activity. In the AMX reactor, an overall nitrogen removal efficiency of 80 % was achieved. Regarding effluent quality, 12 ± 3 mg TN/L was obtained, but 5 mg NO3–-N/L was already in the HRAS effluent. The relative abundance of NOB showed a strong negative correlation with the FNA concentration, providing a good strategy for establishing PN under mainstream conditionsThis work has been financed by the European Commission (EU) through the LIFE project ZERO WASTE WATER (LIFE19 ENV/ES/000631), the Waterworks 2014 Cofounded Call (Water JPI/Horizon) through the Pioneer_STP (PCIN-2015-022 MINECO(AEI)/ID 199 (UE)) and by the Spanish Government (AEI) through GRANDSEA (CTM2014-55397-JIN), TREASURE (CTQ2017-83225-C2-1-R) and ECOPOLYVER (PID2020-112550RB-C21 and PID2020-112550RB-C22) projects. Alba Pedrouso also acknowledges the Xunta de Galicia (Spain) for her postdoctoral fellowship (ED481B-2021-041). Authors from the USC belong to the Galician Competitive Research Group (GRC D431C-2021/37)S

Biodegradation of olive washing wastewater pollutants by highly efficient phenol-degrading strains selected from adapted bacterial community

The bacterial community of an olive washing water (OWW) storage basin was characterized, by both cultivation and cultivation-independent methods. PCR-TGGE fingerprints analysis of different samples, taken along the olive harvesting season, revealed important variations of the bacterial community structure showing rapid establishment of prevalent bacterial populations. Several bacteria, isolated from OWW, were cultivated, in media containing increasing amounts of polyphenols, in order to select high phenol-degrading strains for the effluent pollutants reduction. Strains PM3 and PM15, affiliated to Raoultella terrigena and Pantoea agglomerans by 16S rRNA gene sequencing, were selected and used for OWW biological treatment under batch conditions in shake flasks cultures. The OWW content of phenols, BOD5, COD and colour, was reduced by 93, 91, 89 and 62%, respectively, permitting effluent disposal and/or reuse with no additional treatments.This research was supported by European project Algatec (FP7/SME/2008/1/232331)

Utilización de Rhizobium leguminosarum bv. Viceae cepa Z25 como inoculante para el cultivo de plantas leguminosas

Número de solicitud: 9502149La utilización de Rhizobium leguminosarum bv. viceae cepa Z25 como inoculante para el cultivo de plantas leguminosas representa un nuevo microorganismo simbiótico de las plantas leguminosas, caracterizado por su infectividad, efectividad y capacidad bacteriocinogénica. La expresión constitutiva de la actividad bacteriocinogénica de la cepa Z25, bajo condiciones ambientales muy diversas en cuanto a disponibilidad de nutrientes (triptona-extracto de levadura, D-manitol, D-fructuosa, D-sorbitol, D-trehalosa) y dentro de un rango muy amplio de temperaturas de incubación (4-37ºC), así como la eficiencia de la simbiosis que establece con Vicia faba, son características favorables para la utilización de la cepa Z25 como inoculante eficaz y competitivo aplicable a los cultivos de Vicia faba y otras plantas leguminosas pertenecientes al mismo grupo de inoculación cruzada

Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR

The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs). For an accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides); ampC, blaTEM, blaSHV, and mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARGs’ biodiversity than those of several primers described to date. The adequate design and performance of the new molecular tools were validated in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980), repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs by qPCR in WWTPs and related environments.Funding for open access publishing: Universidad de Granada/CBUA.ECORESISTOME project (A-RNM-62-UGR20) funded by Consejería de Universidad, Investigación e Innovación de la Junta de Andalucia (Spain), and by “ERDF A way of making Europe”Predoctoral fellowship of the Carolina Foundation 2021Aid in the Hiring of Doctoral Research Personnel Program of the Andalusian Government for postdoctoral contrac