Evening poster sessions

Active assimilators of soluble microbial products in wastewater anammox processes 
Rui Xiao1, Shaoyi Xu1, Huijie Lu1*
1Key Laboratory of Environment Remediation and Ecological Health, Ministry of Education, College of Environmental Resource Sciences, Zhejiang University, 310058, Hangzhou, China
* e-mail correspondence: luhuijie@zju.edu.cn
Context.
The soluble microbial products (SMP) secreted by anammox bacteria (AnAOB) supported the growth of diverse heterotrophs in the community, which actively interact with AnAOB and interfere with the carbon and nitrogen cycles. These heterotrophic bacteria mainly included fermentors, denitrifying bacteria, dissimilatory nitrate reduction to ammonium (DNRA) bacteria, accounting for up to 70% of the wastewater anammox communities. 
Gap. 
There is a lack of information on what microbes actively assimilate SMP secreted by AnAOB, as well as how they interact with each other and with AnAOB.
Aim. 
This study aimed to unravel the active assimilators of AnAOB microbial products and their metabolic interactions in wastewater anammox processes treating medium strength wastewater.
Findings. 
Fermenters Anaerolineaceae, denitrifiers Gemmatimonadaceae and Burkholderiaceae were the active utilizers of the endogenous organics produced by the predominant AnAOB Candidatus Kuenenia. Approximately 40% of the assimilated 13CO2 was secreted as UAP by AnAOB, and the yield of fermenters and denitrifiers on UAP were 0.34. These populations potentially utilized amino acids (e.g. glycine, phenylalanine), fatty acids (e.g. galactonic acid) and biotin (e.g. vitamin B12). Metabolic network between AnAOB and active heterotrophs revealed that molybdopterin cofactor (MOCO) and folate essential for anammox acetyl-CoA synthesis were likely supplied by Gemmatimonadaceae and Burkholderiaceae, and fermentative Anaerolineceae potentially provided methionine to AnAOB for ladderane synthesis.
Utilization. 
The improved knowledge onsoluble microbial products utilizers would help the optimization of anammox communities in both sidestream and mainstream towards enhanced total nitrogen removal and process stability. 
Acknowledgement. This work was funded by the Key Research and Development project in Zhejiang Province, the Fundamental Research Funds for the Central Universities.

Due to the complexity of microbiome composition and interactions, it remains challenging to uncover the bioreactor "black-box" and to predict bioreactor dynamics (e.g., anaerobic digestion) through linking the microbial community, operational factors and system performances. Previous studies have focused on abundant/functional microorganisms and community diversity. The importance of microbial interactions and potential low-abundance members may be underestimated. Microbial interactions can be modeled through network analysis using the more and more accessible 16S gene sequencing data. However, pooling all samples (systems/stages) for one network model may not be the best approach. One major gap is how to build/interpret/translate network model result to linked with engineering system prediction.



Aim. This study aims at developing network models of AD microbiome and linking the model output with operational/performance parameters.



Methods.Microbial communities from 12 lab-scale AD reactors at different stages were collected with physical-chemical variables. Five groups of communities were clustered to build separate co-occurrence networks. 



Findings. The five network models showed different topological properties. The hydrolysis efficiency correlated with Clustering Coefficient positively and Normalized Betweenness negatively. The Average Path Length correlated negatively withinfluent particulate COD and differential hydrolysis-methanogenesis efficiency. Individual OTUs' topological characteristics showed that high-ratesystem had more connectors,and low-abundance OTUs could perform central hub roles and communication roles, maintaining the stability and functionality. Our study revealed that stronger interaction is linked to higher rates/activities. The Average Path Length seems to have a global meaning in different microbiomes (activated sludge, soil), correlated negatively with substrate availability and utilization efficiency. 



Utilisation. This study provides a framework to build network models of microbiome data and extract key indicators from model outputs to indicate system performance. The model could be customized and expanded for specific systems to generate links with engineering operations/performance, which could be used for system prediction and optimization.

Context. The Alberta oil sands industry has amassed over 1 trillion litres of fluid tailings containing toxic compounds such as naphthenic acids. End-pit lakes have been proposed as a long-term reclamation strategy and Base Mine Lake (BML) is the only full-scale end-pit lake in the Canadian oil sands industry. Established in 2012, BML contains about 170 Mm3 of permanently placed fluid tailings physically sequestered below the mudline. A freshwater cap overlies the fluid tailings and water quality is expected to improve over time. The water cap supports a microbial community that is expected to biodegrade naphthenic acids and other residual hydrocarbons from tailings porewater. 
Gap. Although the phototroph community is fundamental to ecological development in BML, no research has been published on this topic yet. 
Aim. Characterize and quantify the phototroph community over time in BML from 2015-2019 and identify phototroph interactions with the microbial community. 
Methods. Sampling of the BML water cap occurred from 2015-2019. Characterization of the microbial community was completed using gene amplicon sequencing targeting the 16s, 18s, and 23s ribosomal RNA genes to respectively identify bacteria, eukaryotes, and cyanobacteria and chloroplast-bearing organisms. Phototrophic abundance was quantified using qPCR targeting the 23s rRNA gene. Phototrophic interactions have been identified using a co-occurrence network analysis based on sequencing data.
Findings. Sequencing analysis indicates major phototrophic members of the microbial community include Cryptomonasspp. (Cryptophyceae), Choricystis spp. (Trebouxiophyceae), Euglena spp. (Euglenales), and Cyanobium spp. (Synechococcales). 23s rRNA qPCR results indicate an increase in phototroph abundance from 2015-2019. Network analyses (Figure 1) indicates certain phototrophs are integral to BML's microbial community. For example, Choricystis parasitica has 13 connections in the autumn network.
Utilization. This project will contribute to research on oilsands tailings reclamation and phototroph ecology in engineered oilsands tailings environments. 

Nitrous oxide (N2O) production in autotrophic nitrogen conversion processes can be significant and requires further understanding. We operated an intermittently aerated partial nitritation/anammox (PN/A) sequencing batch reactor. Net N2O production rates increased in the aerated phase and decreased in the non-aerated phase. To put this finding in a transcriptional context, we probed the microbial community structure and function using metagenomics and metatranscriptomics analyses and targeted quantification of nitrogen cycle gene transcripts. We recovered 68 metagenome-assembled genomes (MAGs), including 4 MAGs of aerobic (AOB) and 2 MAGs of anaerobic (AnAOB) ammonium oxidation bacteria. The AOB MAGs were dominated by one MAG classified as Nitrosomonas oligotropha. During aeration, nirK expression at the AOB guild level increased ca. 20 fold and was almost exclusively associated with the dominant MAG. Furthermore, the dominant AOB MAG, unlike the other AOB MAGs, had a strong transcriptional response to the alternating redox conditions, including down-regulation of genes involved in respiration, inorganic carbon metabolism, protein synthesis, and folding, after aeration was turned off. No changes in the transcript levels of narG, nirS, norB, and nosZ were detected at the community level upon the transition to the aerated phase. A MAG classified as Brocadia caroliniensis dominated the AnAOB MAGs. Differences in the transcriptional response to the switches between the oxic and anoxic conditions imposed by the intermittent aeration strategy were also found among the AnAOB. The dominant AnOAB MAG did not show any significant change in gene regulation, while the minor AnAOB MAG down-regulated respiration and folding pathways in the aerated phase. These results suggest that nitrifier denitrification drives N2O production in intermittently-aerated PN/A reactors and highlight that variability in the transcriptional response of members within the same guild may shape reactor microbial community dynamics and performance.

Global population will reach 10 billion by 2050, threatening food security and fresh water resources due to discharges of organic micropollutants (OMPs). Circular solutions enable efficient resource use, but pose risks due to OMP recirculation. This study presents a membrane-aerated bioreactor (MABR) that enables simultaneous nutrient recovery as microbial protein and OMPs oxidation using aerobic methanotrophs. The MABR treated synthetic secondary effluent spiked with 2 µg/L of several OMPs and influent nitrate and phosphate levels were 51 mgN/L and 11 mgP/L, respectively. Methane was supplied at three C/N ratios (mgCH4/mgN), including 11, 22 and 5.5. Methane and oxygen were supplied through different membranes at an optimal ratio of 40:60 v/v. Solid retention time was 3 days. After reaching steady state, the reactor was operated in batch for 24h to assess OMP degradation kinetics. Microbial protein was analyzed after reaching steady state. Table1 summarizes the results. The MABR removed more than 6.2 mgN/d/L and 1.1 mg-P/d/L, showing good performance as a polishing step for nutrient removal. Higher methane supply rates yielded better biodegradation for all OMPs. Thus, treatment with aerobic methanotrophs can sustain safer water reuse for irrigation. Biomass productivity and methane yields reached levels comparable to conventional fermenters growing the same methanotrophic enrichment using bubbling and explosive atmospheres. Off-gas monitoring found methane levels never were above low explosive limits. Protein content was similar to other studies, with little sensitivity towards methane supply, suggesting that OMP biodegradation can be enhanced without compromising protein accumulation. Furthermore, the protein to N ratio was lowest during the starvation period. Thus, the methanotrophs grown at feast conditions are suited both as good microbial feeds and OMP biodegraders. Given current restrictions, methanotrophs cannot substitute traditional proteins, but instead used as biostimulants. They contain amino acids, e.g. proline, that are plant growth promoters.

High rate activated sludge (HRAS) systems, which are aerobic, are used for redirecting organics for energy recovery. The HRAS works at low sludge retention time (SRT), very short hydraulic retention time (HRT) and a high food to microorganism ratio (F/M). Effluents from these systems have a low carbon level which makes them suitable for partial nitritation anammox (PN/A) treatment. However, unsuccessful nitrite oxidizing bacteria suppression and low ammonia concentrations prevent the implementation of mainstream PN/A in cold climates. Thus, high rate denitrification system is proposed as an alternative for simultaneous carbon recovery and nitrate removal as an A.stage, where the B.stage would be a nitrifying reactor whose clarified effluent is recycled to the A.stage for denitrification (mimicking pre-denitrification/nitrification). This study focuses on the A.stage, evaluating process stability at low SRTs, relating settleabilty to floc morphology and assessing the energy and nutrient recovery potential as biogas and microbial protein, respectively. 
Denitrification rates ranged between 0.77-1.33 gNO3N/gTSS/d, higher than reported for the pre-anoxic zones for domestic wastewater (0.04-0.25 gNO3N/gVSS/d). Phosphate removal averaged around 50%. Sludge volume index (SVI30) was below 50 ml/g, which is lower than for aerobic HRAS systems (typically ranging 50-300 ml/g), suggesting good sludge settling characteristics. Decreasing SRTs led to decreasing floc size, without compromising sludge sedimentation. The sludge yield ranged between 0.41-0.67 gTSS/gCOD, similar to aerobic HRAS. Highest biomethane yield, 686±82 mlCH4/gVS, was observed at SRT 3 days, which is higher than yields for secondary sludge. Protein content was highest at the lowest SRT, 26.8% of dry weight, in the range for aerobic HRAS. Protein contained all amino acids, although their balance was far from those of fish meal or soymeal proteins. Given the low protein yield and the poor amino acid balance, biogas production seems a better option for sludge valorization.

Biodesulfurization (BD) under haloalkaline conditions removes H2S from gas streams and converts it to elemental sulfur by using haloalkaliphilic sulfur-oxidizing bacteria (SOB). In this process, gas is first absorbed in an absorber and oxidized into sulfur in an aerated bioreactor. Recently, the BD process improved with an anoxic bioreactor placed between the absorber and the oxic bioreactor to minimize the unwanted production of sulfate and thiosulfate1. However, microbial activities affect the product selectivity directly, and therefore it is needed to study the microbiology involved in the updated process line-up.
This study aimed to analyse the diversity of the microbial community in the BD process by determining the active players and study their ecophysiology. For this, biomass was obtained from six different month-long operational runs of a pilot-scale BD plant, each corresponding to different operational conditions. A complementary approach of DNA and RNA-based 16SrRNA gene amplicon sequencing was used to determine the active and inactive fraction of the microbial community of each run.
16SrRNA-based amplicon sequence analyses with QIIME2 indicated significant differences in the active and non-active microbial populations, suggesting that only part of the population is active. The composition of the active community was consistent but differentially abundant in all six operations. Contrarily, the composition of the non-active fraction varied among runs. The microbial community composition also changed within the duration of each run. There was no difference in microbial diversity in the different sections of the system. 
The remarkable differences in active and non-active bacterial populations indicate that RNA-based microbial composition analysis is essential to determine the actual key players and their activity in the haloalkaline BD process. The next step is to determine the correlations with essential process parameters and use the information to better understand and control the bioprocess.

Energy-efficient nitrogen removal in activated sludge (AS) treatment requires process control strategies that limit the oxidation of ammonia at nitrite. Previous reports have revealed the adaptation of nitrite-oxidizing bacteria (NOB) to various out-selection strategies in AS, likely due to their high metabolic versatility and diversity. Thus, it is crucial to illuminate the biokinetic mechanisms driving NOB adaptation to out-selection pressures. Current wastewater treatment process models do not account for functional diversity, which limits their ability to capture the ecophysiology of co-existing NOB populations. A process modeling framework that resolves functional degeneracy is therefore needed. This study combined genome-centric metagenomic abundance data with AS process modelling to capture the biokinetic characteristics of physiologically diverse and co-existing NOB. A lab-scale mainstream AS system was operated with continual treatment of 20% return activated sludge with 200 mg-N/L free ammonia (FA) in a side-stream flow. A genome-centric metagenomic approach was used to recover metagenome-assembled genomes (MAGs), and metabolic reconstruction was performed on the MAGs to assess their genomic potentials. A genome-centric process model was formulated in AQUASIM by integrating highly-paralleled respirometry, in-situ reactor monitoring, and MAG abundance data over a 100-day operational period. Routine exposure of the AS community to FA in a side-stream reactor led to transient nitrite accumulation in the mainstream and a regime change in Nitrospira NOB lineages. Genome annotation indicated that certain Nitrospira MAGs could use formate as an electron donor, and also had the potential for reactive oxygen species degradation and osmolyte production, to aid their survival in the side-stream FA-exposure. The genome-centric process model resolved the biokinetics of two Nitrospira lineages, and better predicted reactor nitrogen dynamics than a conventional AS model.

Context. The performance of activated sludge systems is dependent on the growth kinetics of core microbial community (Saunders et al. 2016). To date, the design of many wastewater treatment plants (WWTPs) in the tropics are adopting default growth kinetic parameters from the temperate climate regions, which may be suboptimal for the WWTPs' operation.



Gap. Past study by Song et al. (2021) suggested that WWTPs in the tropics may harbor a diverse core nitrifying community, such as the presence of Nitrospira at higher relative abundances than WWTPs situated at higher latitudes. Yet, the significance of latitudinal diversity gradient is inconclusive (Wu et al. 2019). Further investigations are warranted to study the core nitrifying community in the tropics.



Aim. This study applied 16S rRNA amplicon sequencing to elucidate the core nitrifying community of activated sludge processes in the tropics.



Methods. We collected wastewater and sludge samples from eight WWTPs in the Greater Kuala Lumpur region, Malaysia, from December 2020 to January 2021. We extracted the DNA from the sludge and sequenced the 16S rRNA amplicons using IonTorrent Personal Genome Machine.



Findings. Most WWTPs removed the total chemical oxygen demand (TCOD) and ammoniacal nitrogen (NH4+-N) adequately (Table 1). Interestingly, WWTPs 3 and 6 operated at low-dissolved-oxygen (low-DO) condition (< 1 mg L-1) achieved low effluent NH4+-N and nitrate nitrogen (NO3--N). The active nitrification in low-DO condition may indicate a diverse core nitrifying community in the tropics. For instance, Nitrospira affiliated with complete ammonia oxidizers was previously reported in low-DO reactors below 1 mg L-1 at warm temperatures (How et al. 2020). The sequencing is ongoing to reveal the unique nitrifying community in the tropics.

Utilization. This study emphasizes that WWTPs in the tropics should adopt nitrifiers' growth parameters specific to their microbial assembly to achieve a more optimal operation.

Context. Decentralized highstrength wastewater treatment can be accomplished in microbial electrochemical cells (MEC), in which anode-respiring bacteria (ARB) degrade organic matter to respire electrons to an electrode (anode), generating an electrical current that can be used for producing electricity or other beneficial chemicals.
Gap. Maximizing electrical current production from oxidized organics and preventing diversion to undesirable end products, such as methane, via understanding and engineering the associated microbial consortium, is important for MEC application.
Aim. To study the conversion of primary sludge-based synthetic blackwater (PS-BW) to quantify the proportion of electrons from COD recovered as electrical current vs. methane.
Methods. We operated five, two-chamber MEC; two were fed a 475 mg N/L ammonia PS-BW to simulate a combined blackwater (human excrement and urine combined, referred to as high-ammonia), two were fed a 40 mg N/L ammonia PS-BW to simulate a source separated blackwater (referred to as low-ammonia), and one was an inoculum-only control. We measured current, methane, and volatile fatty acid concentrations over time and performed 16S rRNA sequencing to identify the taxa contributing to the suspension and biofilm communities.
Findings. COD conversion to electrical current was 22.6% for the high-ammonia condition and 22.8% for the low-ammonia condition. Volatile fatty acids did not accumulate. Methane production was limited, less than 1% COD conversion to methane, but in simultaneously studied biochemical methane potential tests COD conversion to methane was 21.4% and 27.2% in the high- and low-ammonia conditions, respectively. These findings suggest the PS-BW inherently limited methane production in the MEC. The suspension microbial community consisted of a mix of fermentative bacteria and hydrogenotrophic methanogens, including Bacteroides, Bellilinea, Clostridium, and Methanobrevibacter. The biofilm communities were dominated by Geobacter.
Utilization. Maximizing electron recovery from blackwater will enable widespread implementation of MEC for decentralized treatment.

Context. The anaerobic digesters have potential to produce 14,000 TWh from currently available feedstocks, corresponding to 6-9 % of the world's energy consumption. 



Gap. While the gas-producing microbiota are well described and understood, little is known about the hydrolysing microorganisms, although this step represents one of the biggest limitations during the anaerobic digestion.



Aim and Methods. We examined an anaerobic digester reactor fed with cellulose at the metabolic level using the approach of retrieving simultaneous information on the identity of the microorganisms (metagenomics assemblies) and the activity from the 13C-labelled proteins (Protein-SIP) after incubation with 13C-labelled cellulose.



Findings. The metagenome conceived 17,155 variants of carbohydrate-active enzymes, corresponding to 238 different enzymes, 6 of these were directly associated to cellulose degradation and were linked to 756 scaffolds in the metagenome. The enzymes cover cellulase, cellobiose phosphorylase, cellodextrin phosphorylase, cellulose 1,4-beta-cellobiosidase (non-reducing end), cellulose 1,4-beta-cellobiosidase (reducing end), and cellobiose dehydrogenase. These enzymes were affiliated with four different phyla: Actinobacteria, Euryachaeota, Firmicutes, and Proteobacteria. 
A total of 2,075 peptides were identified, and overall 20 peptides were found to have assimilated labelled carbon from degradation of 13C-cellulose. These peptides were binned to the metagenome and revealed that the majority affiliated with organisms representing the phylum Firmicutes.
The 13C isotopic profiling during the start of incubation showed that CH4 and CO2 are the main products of cellulose degradation. After two days of incubation the isotopic ratio was 0.116, corresponding to the isotopic ratio fed to the reactor (10 %).



Utilization. The combined approach of with metagenomics and metaproteomics using 13C-labelled compounds has been successfully shown to provide new insight into the most active cellulose degrading communities in anaerobic digesters and provide a unique approach to couple function to identity.

Influence of Microbial Community and Biofilm Thickness in the Removal of Organic Micro Pollutants through a Membrane Aerated Biofilm Reactor


Sanchez-Huerta C. 1,*, Fortunato L.1, Leiknes T. 1, Hong P.-Y. 1
1 King Abdullah University of Science and Technology (KAUST), Water Desalination and Reuse Center (WDRC), Thuwal 23955-6900, Saudi Arabia


* e-mail correspondence: claudia.sanchezhuerta@kaust.edu.sa



Context. Presence of organic micropollutants (OMPs) in natural water bodies is an increasing concern due to the risk their occurrence represents for ecosystems and human health. Nitrification has been linked to biotransformation of OMPs via co-metabolism by ammonia monooxygenase enzyme. Membrane aerated biofilm reactor (MABR), which supports simultaneous nitrification-denitrification, is suggested to enhance OMPs biotransformation through their unique stratified bacterial community and substrate gradient formed within biofilm thickness.



Gap. Little is known regarding the correlation between MABR biofilm thickness, bacterial consortium development, and OMPs removal.



Aim. This study aims to find an optimal MABR biofilm thickness that allows nitrification and high biotransformation of selected OMPs while exploring functional microbial community



Methods. Lab-scale Zeelung MABR reactors are fed with municipal wastewater spiked with 13 OMPs, including pharmaceuticals and endocrinal disrupting compounds, at 50 µg L-1. Optical coherence tomography is used to monitor biofilm thickness. OMPs quantification is performed through Gas Chromatography-Mass Spectrometry. Characterization of microbial community is investigated through 16S rRNA gene-based amplicon sequencing.



Findings. MABR is highly efficient for nitrification-denitrification process, even at biofilm thickness of 0.2±0.16 µm. An increase in biofilm thickness impacted positively the biotransformation of selected OMPs (acetaminophen, triclosan, ethinyl estradiol, estrone, bisphenol-A, ibuprofen and, naproxen). Resulting in an increase of OMP biotransformation efficiency along with nitrification-denitrification rates. 



Utilization. Results will help establish operational parameters that promote the development of microbial communities supporting OMPs biotransformation.






Fig. 1 Biofilm thickness vs COD and ammonia removal (right); and OMPs removal (left)