Stringent Response of Cyanobacteria and Other Bacterioplankton during Different Stages of a Harmful Cyanobacterial Bloom.

We conducted a field study to investigate the role of stringent response in cyanobacteria and coexisting bacterioplankton during nutrient-deprived periods at various stages of bloom in a freshwater lake (Utah Lake) for the first time. Using metagenomics and metatranscriptomics analyses, we examined the cyanobacterial ecology and expression of important functional genes related to stringent response, N and P metabolism, and regulation. Our findings mark a significant advancement in understanding the mechanisms by which toxic cyanobacteria survive and proliferate during nitrogen (N) and phosphorus (P) limitations. We successfully identified and analyzed the metagenome-assembled genomes (MAGs) of the dominant bloom-forming cyanobacteria, namely, Dolichospermum circinale, Aphanizomenon flos-aquae UKL13-PB, Planktothrix agardhii, and Microcystis aeruginosa. By mapping RNA-seq data to the coding sequences of the MAGs, we observed that these four prevalent cyanobacteria species activated multiple functions to adapt to the depletion of inorganic nutrients. During and after the blooms, the four dominant cyanobacteria species expressed high levels of transcripts related to toxin production, such as microcystins (mcy), anatoxins (ana), and cylindrospermopsins (cyr). Additionally, genes associated with polyphosphate (poly-P) storage and the stringent response alarmone (p)ppGpp synthesis/hydrolysis, including ppk, relA, and spoT, were highly activated in both cyanobacteria and bacterioplankton. Under N deficiency, the main N pathways shifted from denitrification and dissimilatory nitrate reduction in bacterioplankton toward N2-fixing and assimilatory nitrate reduction in certain cyanobacteria with a corresponding shift in the community composition. P deprivation triggered a stringent response mediated by spoT-dependent (p)ppGpp accumulation and activation of the Pho regulon in both cyanobacteria and bacterioplankton, facilitating inorganic and organic P uptake. The dominant cyanobacterial MAGs exhibited the presence of multiple alkaline phosphatase (APase) transcripts (e.g., phoA in Dolichospermum, phoX in Planktothrix, and Microcystis), suggesting their ability to synthesize and release APase enzymes to convert ambient organic P into bioavailable forms. Conversely, transcripts associated with bacterioplankton-dominated pathways like denitrification were low and did not align with the occurrence of intense cyanoHABs. The strong correlations observed among N, P, stringent response metabolisms and the succession of blooms caused by dominant cyanobacterial species provide evidence that the stringent response, induced by nutrient limitation, may activate unique N and P functions in toxin-producing cyanobacteria, thereby sustaining cyanoHABs.

Probabilistic risk assessment of water distribution system in Hyderabad, Pakistan reveals unacceptable health hazards and areas for rehabilitation.

Poor water quality exacerbates multidimensional poverty in developing nations. Often centralized treatment facilities generate acceptable water quality, but the water is contaminated during distribution. Methods to assess sources of contamination in water distribution systems are lacking. A case study of two methods, human risk assessment linked to water distribution system sampling was conducted in Hyderabad, Pakistan to determine areas requiring infrastructure rehabilitation. Water samples from source water (i.e., the Indus River), treatment plant effluent and from taps in the water distribution system were analyzed by atomic adsorption spectroscopy for metals and metalloids (As, Cd, Cr, Hg, and Pb) and water quality parameters (dissolved and suspended solids, pH, conductivity, and total organic carbon). Source water exceeded acceptable drinking water levels for As, Cd, total Cr, and Pb, while the treatment plant effluent concentrations were acceptable. Concentrations of all metals and metalloids, except Hg, increased in the water distribution system post-treatment, exceeding safe drinking limits in at least one location, suggesting contamination of the water during distribution. A deterministic and a probabilistic risk assessment were conducted to evaluate two scenarios: (1) unrestricted use of piped water for all household purposes, including as drinking water and (2) restricted use of the water for purposes other than drinking in the household, including only dermal and inhalation exposure pathways. The water was deemed unsafe for unrestricted use as the sole source of drinking water by both risk assessment methods. Yet when an alternative source of drinking water was assumed and the piped water was used only for bathing and dish washing, the probabilistic risk assessment revealed acceptable health risks to the population, while the overly conservative deterministic risk assessment suggested unacceptable risks. The combined methods of water sampling, risk assessment and correlation analysis suggested areas for rehabilitation of the water distribution system in Hyderabad, Pakistan and these methods can be adopted in other developing nations to target limited funds for infrastructure rehabilitation.

Whole community transcriptome of a sequencing batch reactor transforming 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO).

Two sequencing batch reactors (SBRs) were run to bio-mineralize 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO) in lab scale settings. The reactors were shown to reproducibly biotransform these munitions under aerobic and anaerobic conditions during the operations of these SBRs. Complete removal (100% biotransformation) of DNAN (initially 17.7 ± 5.4 mg L-1) and NTO (initially 15.0 ± 7.1 mg L-1) was observed in an anaerobic SBR when Luria-Bertani (LB) broth was present. In contrast, an aerobic SBR degraded only 58 ± 22% of DNAN (initially 19.7 ± 6.2 mg L-1) and 45 ± 24% of NTO (initially 9.7 ± 6.3 mg L-1) when either LB or glucose was also added indicating that anaerobic conditions are more favorable for biotransformation of these munitions. Transcriptomic analysis of the DNAN and NTO degrading anaerobic SBR revealed upregulation of a putative nitroreductase, hydroxylaminophenol mutases, 4-hydroxylphenyl acetate associated genes, and quinone oxioreductases. Major Bacterial populations included Bacteroidales, Campylobacterales, Enterobacteriales, Pseudomonadales, Burkholderiales and Clostridiales. Results from this study can be used to inform investigation of munition degrading organisms and the functional genes responsible.

The feasibility of putrescible components of municipal solid waste for biomethane production at Hyderabad, Pakistan.

This study analyzes the feasibility of putrescible components of municipal solid waste (PCMSW) such as food waste (FW) and yard waste (YW) for methane production in Pakistan. The batch experiments have been conducted at two different inoculums to substrate ratios (ISRs) by using various inoculums under mesophilic condition. The highest methane yield of FW and YW is achieved to be 428 Nml g-1 volatile solids (VS) added and 304 Nml g-1 VS added respectively by using buffalo dung inoculum at ISR-5. While, lowest methane yield of FW and YW is obtained as 236 Nml g-1 VS added and 151Nml g-1 VS added respectively by using effluent from a continuous stirrer tank reactor as inoculum at ISR-3. The first order decay model has been introduced, which gives best fit for methane potential of PCMSW with buffalo dung inoculum. Additionally, the feasibility of PCMSW in terms of power generation potential has been analyzed. About 60.63 million m3/year energy can be generated by converting PCMSW into methane gas leading to power generation. The finding of this study concludes that the replacement of imported energy and reduction up to 1.62% in other primary energy sources would be achieved, if PCMSW are properly converted into energy through anaerobic digestion in Pakistan.

Whole-Community Metagenomics in Two Different Anammox Configurations: Process Performance and Community Structure.

Anaerobic ammonia oxidation (anammox) combined with partial nitritation (PN) is an innovative treatment process for energy-efficient nitrogen removal from wastewater. In this study, we used genome-based metagenomics to investigate the overall community structure and anammox species enriched in suspended growth (SGR) and attached growth packed-bed (AGR) anammox reactors after 220 days of operation. Both reactors removed more than 85% of the total inorganic nitrogen. Metagenomic binning and phylogenetic analysis revealed that two anammox population genomes, affiliated with the genus Candidatus Brocadia, were differentially abundant between the SGR and AGR. Both of the genomes shared an average nucleotide identify of 83%, suggesting the presence of two different species enriched in both of the reactors. Metabolic reconstruction of both population genomes revealed key aspects of their metabolism in comparison to known anammox species. The community composition of both the reactors was also investigated to identify the presence of flanking community members. Metagenomics and 16S rRNA gene amplicon sequencing revealed the dominant flanking community members in both reactors were affiliated with the phyla Anaerolinea, Ignavibacteria, and Proteobacteria. Findings from this research adds two new species, Ca. Brocadia sp. 1 and Ca. Brocadia sp. 2, to the genus Ca. Brocadia and sheds light on their metabolism in engineered ecosystems.

Biofilm control with natural and genetically-modified phages.

Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended.

Bacteriophage therapy for membrane biofouling in membrane bioreactors and antibiotic-resistant bacterial biofilms.

To demonstrate elimination of bacterial biofilm on membranes to represent wastewater treatment as well as biofilm formed by antibiotic-resistant bacterial (ARB) to signify medical application, an antibiotic-resistant bacterium and its lytic bacteriophage were isolated from a full-scale wastewater treatment plant. Based on gram staining and complete 16 S rDNA sequencing, the isolated bacterium showed a more than 99% homology with Delftia tsuruhatensis, a gram-negative bacterium belonging to ß-proteobacteria. The Delftia lytic phage's draft genome revealed the phage to be an N4-like phage with 59.7% G + C content. No transfer RNAs were detected for the phage suggesting that the phage is highly adapted to its host Delftia tsuruhatensis ARB-1 with regard to codon usage, and does not require additional tRNAs of its own. The gene annotation of the Delftia lytic phage found three different components of RNA polymerase (RNAP) in the genome, which is a typical characteristic of N4-like phages. The lytic phage specific to D. tsuruhatensis ARB-1 could successfully remove the biofilm formed by it on a glass slide. The water flux through the membrane of a prototype lab-scale membrane bioreactor decreased from 47 L/h m(2) to ∼15 L/h m(2) over 4 days due to a biofilm formed by D. tsuruhatensis ARB-1. However, the flux increased to 70% of the original after the lytic phage application. Overall, this research demonstrated phage therapy's great potential to solve the problem of membrane biofouling, as well as the problems posed by pathogenic biofilms in external wounds and on medical instruments.

The use of bottle caps as submerged aerated filter medium.

In this study, a submerged aerated filter (SAF) using bottle caps as a support medium was evaluated. The system was fed with effluent from an upflow anaerobic sludge blanket system at ETE 2-South wastewater treatment plant, under different volumetric organic load rates (VOLRs). The population of a particular nitrifying microbial community was assessed by fluorescent in situ hybridization with specific oligonucleotide probes. The system showed an average removal of chemical oxygen demand (COD) equal to 76% for VOLRs between 2.6 and 13.6 kg COD m(-3)_media.day(-1). The process of nitrification in conjunction with the removal of organic matter was observed from applying VOLRs lower than 5.5 kg COD m(-3)_media.day(-1) resulting in 78% conversion of NH4(+)-N. As the applied organic load was reduced, an increase in the nitrifying bacteria population was observed compared with total 4'-6-diamidino-2-phenylindole (DAPI) stained cells. Generally, SAF using bottle caps as a biological aerated filter medium treating wastewater from an anaerobic system showed promising removal of chemical oxygen demand (COD) and conversion of NH4(+)-N.

Investigating the viral ecology and contribution to the microbial ecology in full-scale mesophilic anaerobic digesters.

In an attempt to assess the diversity of viruses and their potential to modulate the metabolism of functional microorganisms in anaerobic digesters, we collected digestate from three mesophilic anaerobic digesters in full-scale wastewater treatment plants treating real municipal wastewater. The reads were analyzed using bioinformatics algorithms to elucidate viral diversity, identify their potential role in modulating the metabolism of functional microorganisms, and provide essential genomic information for the potential use of virus-mediated treatment in controlling the anaerobic digester microbiome. We found that Siphoviridae was the dominant family in mesophilic anaerobic digesters, followed by Myoviridae and Podoviridae. Lysogeny was prevalent in mesophilic anaerobic digesters as the majority of metagenome-assembled genomes contained at least one viral genome within them. One virus within the genome of an acetoclastic methanogen (Methanothrix soehngenii) was observed with a gene (fwdE) acquired via lateral transfer from hydrogenotrophic methanogens. The virus-mediated acquisition of fwdE gene enables possibility of mixotrophic methanogenesis in Methanothrix soehngenii. This evidence highlighted that lysogeny provides fitness advantage to methanogens in anaerobic digesters by adding flexibility to changing substrates. Similarly, we found auxiliary metabolic genes, such as cellulase and alpha glucosidase, of bacterial origin responsible for sludge hydrolysis in viruses. Additionally, we discovered novel viral genomes and provided genomic information on viruses infecting acidogenic, acetogenic, and pathogenic bacteria that can potentially be used for virus-mediated treatment to deal with the souring problem in anaerobic digesters and remove pathogens from biosolids before land application. Collectively, our study provides a genome-level understanding of virome in conjunction with the microbiome in anaerobic digesters that can be used to optimize the anaerobic digestion process for efficient biogas generation.

Anaerobic ammonia oxidation (ANAMMOX) for side-stream treatment of anaerobic digester filtrate process performance and microbiology.

A laboratory scale semi-batch fed anaerobic ammonia oxidation (ANAMMOX) reactor was operated in the lab under two different feeding operations. In the first scenario, termed as phase I, the reactor was seeded and operated with NO(2) -N added externally with the filtrate to the reactor in the ratio needed for the successful ANAMMOX. A second reactor was also initiated shortly after the start-up of the ANAMMOX to accomplish partial nitrification (nitritation reactor) to generate NO(2) -N. In phase II, the operation of the ANAMMOX reactor was switched to the mode in which case the partially nitrified effluent from the nitritation reactor was fed to the ANAMMOX reactor. In both phases, real filtrate from a local wastewater treatment plant was used as the feed. The ANAMMOX reactor sustained a loading rate (average 0.33 ± 0.03 with a max of 0.4 g N (L day)(-1) ) which is comparable with many other fed-batch reactors in the literature. Consistent total N removal (average of 82 ± 4%) could be sustained in the ANAMMOX reactor during both phases. The nitritation reactor also consistently enabled a NO(2) -N to NH(3) -N ratio of 1.2:1 which was needed for the successful operation of the ANAMMOX reactor in phase II. Sequence analysis and FISH showed that Kuenenia stuttgartiensis dominated the enriched ANAMMOX community along with several unidentified, but seemingly enriched, potential ANAMMOX strains. Microbial ecology analysis for nitritation reactor showed the dominance of Nitrosomonas europaea. In summary, this manuscript provides important information on the start-up and operation of anammox reactor with detailed investigation on microbial ecology in this reactor.

Anoxic granular activated sludge process for simultaneous removal of hazardous perchlorate and nitrate.

An airtight, anoxic bubble-column sequencing batch reactor (SBR) was developed for the rapid cultivation of perchlorate (ClO4-) and nitrate (NO3-) reducing granular sludge (GS) in this study. Feast/famine conditions and shear force selection pressures in tandem with a short settling time (2-min) as a hydraulic section pressure resulted in the accelerated formation of anoxic granular activated sludge (AxGS). ClO4- and NO3- were efficiently (>99.9%) reduced over long-term (>500-d) steady-state operation. Specific NO3- reduction, ClO4- reduction, chloride production, and non-purgeable dissolved organic carbon (DOC) oxidation rates of 5.77 ± 0.54 mg NO3--N/g VSS·h, 8.13 ± 0.74 mg ClO4-/g VSS·h, 2.40 ± 0.40 mg Cl-/g VSS·h, and 16.0 ± 0.06 mg DOC/g VSS·h were recorded within the reactor under steady-state conditions, respectively. The AxGS biomass cultivated in this study exhibited faster specific ClO4- reduction, NO3- reduction, and DOC oxidation rates than flocculated biomass cultivated under similar conditions and AxGS biomass operated in an up-flow anaerobic sludge blank (UASB) bioreactor receiving the same influent loading. EPS peptide identification revealed a suite of extracellular catabolic enzymes. Dechloromonas species were present in high abundance throughout the entirety of this study. This is one of the initial studies on anoxic granulation to simultaneously treat hazardous chemicals and adds to the science of the granular activated sludge process.

Biodegradation of insensitive munition (IM) formulations: IMX-101 and IMX-104 using aerobic granule technology.

A laboratory-scale aerobic granular sludge (AGS) sequencing batch bioreactor (SBR) was initiated in this study for the biodegradation of hazardous insensitive munition (IM) formulation constituents; 2,4-dinitroanisole (DNAN), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 1-nitroguanidine (NQ), and 3-nitro-1,2,4-triazol-5-one (NTO). Efficient (bio)transformation of the influent DNAN and NTO was achieved throughout reactor operation with removal efficiencies greater than 95%. An average removal efficiency of 38.4 ± 17.5% was recorded for RDX. NQ was only slightly removed (3.96 ± 4.15%) until alkalinity was provided in the influent media, which subsequently increased the NQ removal efficiency up to an average of 65.8 ± 24.4%. Batch experiments demonstrated a competitive advantage for aerobic granular biofilms over flocculated biomass for the (bio)transformation DNAN, RDX, NTO, and NQ, as aerobic granules were capable of reductively (bio)transforming each IM compound under bulk aerobic conditions while flocculated biomass could not, thus demonstrating the contribution of inner oxygen-free zones within aerobic granules. A variety of catalytic enzymes were identified in the extracellular polymeric matrix of the AGS biomass. 16 S rDNA amplicon sequencing found Proteobacteria (27.2-81.2%) to be the most abundant phyla, with many genera associated with nutrient removal as well as genera previously described in relation to the biodegradation of explosives or related compounds.

Effect of different types of volatile fatty acids on the performance and bacterial population in a batch reactor performing biological nutrient removal.

Different ratios of four volatile fatty acids (VFAs) were used as the primary feed to a laboratory scale biological nutrient reactor during four operational stages. The reactor performed efficiently over 500 days of operation with over 90% dissolved phosphorus and over 98% ammonium-nitrogen (NH4+-N) removal. Through in the first experimental phase, acetate and propionate were present in a significant proportion as carbon sources, the relative abundance of Candidatus Accumulibacter, a potential polyphosphate accumulating organism, increased from 10% to 57% and the Defluviicoccus genus, a known glycogen accumulating organism (GAO), decreased from 41% to 5%. Further tests indicated the presence of denitrifying phosphorus accumulating organisms (DPAO) belonging to Clade IIC, that could use nitrite as the electron acceptor during P-uptake. In general, VFAs favored the increase of the genus Defluviicoccus and Candidatus Accumulibacter. High relative abundance of Defluviicoccus did not affect the stability and the performance of the BNR process.

Retention and recycling of granules in continuous flow-through system to accomplish denitrification and perchlorate reduction.

This study employed a completely anoxic reactor and a gravity-settling design for continuously capturing and separating granules from flocculated biomass, and recycling granules back to the main reactor. The average chemical oxygen demand (COD) removal in the reactor was 98%. Average nitrate (NO3--N) and perchlorate (ClO4-) removal efficiencies of 99% and 74 ± 19% were observed, respectively. Preferential utilization of NO3- over ClO4- led to COD limiting conditions, which resulted in ClO4- in the effluent. The average granule diameter in continuous flow-through bubble-column (CFB) anoxic granular sludge (AxGS) bioreactor was 6325 ± 2434 µm, and the average SVI30/SVI1 was >90 % throughout its operation. 16s rDNA amplicon sequencing revealed Proteobacteria (68.53%-88.57%) and Dechloromonas (10.46%-54.77%) to be the most abundant phylum and the genus present in reactor sludge representing the denitrifying and ClO4- reducing microbial community. This work represents a pioneering development of CFB-AxGS bioreactor.

Integration of EBPR with mainstream anammox process to treat real municipal wastewater: Process performance and microbiology.

The mainstream application of anaerobic ammonium oxidation (anammox) for sustainable N removal remains a challenge. Similarly, with recent additional stringent regulations for P discharges, it is imperative to integrate N with P removal. This research studied integrated fixed film activated sludge (IFAS) technology to simultaneously remove N and P in real municipal wastewater by combining biofilm anammox with flocculent activated sludge for enhanced biological P removal (EBPR). This technology was assessed in a sequencing batch reactor (SBR) operated as a conventional A2O (anaerobic-anoxic-oxic) process with a hydraulic retention time of 8.8 h. After a steady state operation was reached, robust reactor performance was obtained with average TIN and P removal efficiencies of 91.3 ± 4.1% and 98.4 ± 2.4%, respectively. The average TIN removal rate recorded over the last 100 d of reactor operation was 118 mg/L·d, which is a reasonable number for mainstream applications. The activity of denitrifying polyphosphate accumulating organisms (DPAOs) accounted for nearly 15.9% of P-uptake during the anoxic phase. DPAOs and canonical denitrifiers removed approximately 5.9 mg TIN/L in the anoxic phase. Batch activity assays, which showed that nearly 44.5% of TIN were removed by the biofilms during the aerobic phase. The functional gene expression data also confirmed anammox activities. The IFAS configuration of the SBR allowed operation at a low solid retention time (SRT) of 5-d without washing out biofilm ammonium-oxidizing and anammox bacteria. The low SRT, combined with low dissolved oxygen and intermittent aeration, provided a selective pressure to washout nitrite-oxidizing bacteria and glycogen-accumulating organisms, as relative abundances of.

A Klebsiella pneumoniae NDM-1+ bacteriophage: Adaptive polyvalence and disruption of heterogenous biofilms.

Bacteriophage KL-2146 is a lytic virus isolated to infect Klebsiella pneumoniae BAA2146, a pathogen carrying the broad range antibiotic resistance gene New Delhi metallo-betalactamase-1 (NDM-1). Upon complete characterization, the virus is shown to belong to the Drexlerviridae family and is a member of the Webervirus genus located within the (formerly) T1-like cluster of phages. Its double-stranded (dsDNA) genome is 47,844 bp long and is predicted to have 74 protein-coding sequences (CDS). After challenging a variety of K. pneumoniae strains with phage KL-2146, grown on the NDM-1 positive strain BAA-2146, polyvalence was shown for a single antibiotic-sensitive strain, K. pneumoniae 13,883, with a very low initial infection efficiency in liquid culture. However, after one or more cycles of infection in K. pneumoniae 13,883, nearly 100% infection efficiency was achieved, while infection efficiency toward its original host, K. pneumoniae BAA-2146, was decreased. This change in host specificity is reversible upon re-infection of the NDM-1 positive strain (BAA-2146) using phages grown on the NDM-1 negative strain (13883). In biofilm infectivity experiments, the polyvalent nature of KL-2146 was demonstrated with the killing of both the multidrug-resistant K. pneumoniae BAA-2146 and drug-sensitive 13,883 in a multi-strain biofilm. The ability to infect an alternate, antibiotic-sensitive strain makes KL-2146 a useful model for studying phages infecting the NDM-1+ strain, K. pneumoniae BAA-2146. GRAPHICAL ABSTRACT.

Volatile fatty acid production from primary and secondary sludges to support efficient nutrient management.

Enhanced hydrolysis of sludges during fermentation is an important factor to achieve solubilization of complex carbon sources and increase the amount of soluble COD that microorganisms could use as food during biological nutrient removal processes. This research shows that a combination of mixing, bioaugmentation, and co-fermentation can be used to increase the hydrolysis of sludges and enhanced the production of volatile fatty acids (VFA). Mixing of primary sludge (PS) at 350 revolutions per minute (RPM) during fermentation increased the hydrolysis of the sludge and increased the soluble chemical oxygen demand (sCOD) by 72% compared to no mixing. Mixing also increased the production of VFA by 60% compared to no mixing conditions. PS hydrolysis was also evaluated using bioaugmentation with the bacteria Bacillus amyloliquefacients, a known producer of the biosurfactant surfactin. Results showed that bioaugmentation enhanced the hydrolysis of the PS by increasing the amount of soluble carbohydrates and soluble proteins present in the form of sCOD. Methanogenesis experiments performed with co-fermentation of decanted primary sludge (PS) and raw waste-activated sludge (WAS) at 75:25 and 50:50 ratios displayed a decreased in production of total biogas by 25.58% and 20.95% and a reduction on methane production by 20.00% and 28.76% respectively, compared to co-fermentation of raw sludges. Compared to fermentation of the sludges separately, co-fermentation of PS and WAS increased the production of VFA and it was determined that 50:50 was the optimum co-fermentation ratio for production of VFA while reducing the reintroduction of nutrients produced during the fermentation process to BNR processes.

Simultaneous anaerobic carbon and nitrogen removal from primary municipal wastewater with hydrogel encapsulated anaerobic digestion sludge and AOA-anammox coated hollow fiber membrane.

In this study, a one-stage continuous-flow membrane-hydrogel reactor integrating both partial nitritation-anammox (PN-anammox) and anaerobic digestion (AD) was designed and operated for simultaneous autotrophic nitrogen (N) and anaerobic carbon (C) removal from mainstream municipal wastewater. In the reactor, a synthetic biofilm consisting of anammox biomass and pure culture ammonia oxidizing archaea (AOA) were coated onto and maintained on a counter-diffusion hollow fiber membrane to autotrophically remove nitrogen. Anaerobic digestion sludge was encapsulated in hydrogel beads and placed in the reactor to anaerobically remove COD. During the pilot operation at three operating temperature (25, 16 and 10 °C), the membrane-hydrogel reactor demonstrated stable anaerobic COD removal (76.2 ± 15.5 %) and membrane fouling was successfully suppressed allowing a relatively stable PN-anammox process. The reactor demonstrated good nitrogen removal efficiency, with an overall removal efficiency of 95.8 ± 5.0 % for NH4+-N and 78.9 ± 13.2 % for total inorganic nitrogen (TIN) during the entire pilot operation. Reducing the temperature to 10 °C caused a temporary reduction in nitrogen removal performance and abundances of AOA and anammox. However, the reactor and microbes demonstrated the ability to adapt to the low temperature spontaneously with recovered nitrogen removal performance and microbial abundances. Methanogens in hydrogel beads and AOA and anammox on the membrane were observed in the reactor by qPCR and 16S sequencing across all operational temperatures.

Mainstream nitrogen removal from low temperature and low ammonium strength municipal wastewater using hydrogel-encapsulated comammox and anammox.

Application of partial nitritation (PN)-anammox to mainstream wastewater treatment faces challenges in low water temperature and low ammonium strength. In this study, a continuous flow PN-anammox reactor with hydrogel-encapsulated comammox and anammox was designed and operated for nitrogen removal from mainstream wastewater with low temperature. Long-term operation with synthetic and real wastewater as the feed demonstrated nearly complete ammonium and total inorganic nitrogen (TIN) removal by the reactor at temperatures as low as 10 °C. A significantly decreased nitrogen removal performance and biomass activity was observed in the reactor at 4 °C before a selective heating strategy was employed. A novel heating technology using radiation to heat carbon black co-encapsulated in the hydrogel matrix with biomass was used to selectively heat biomass but not water in the treatment system. This selective heating technology enabled nearly complete ammonium removal and 89.4 ± 4.3 % TIN removal at influent temperature of 4 °C and reactor temperature 5 °C. Activity tests suggested selective heating brought the biomass activity at influent temperatures of 4 °C and reactor temperature 5 °C to a level comparable to that at 10 °C. Comammox and anammox were consistently present in the system and spatially organized in the hydrogel beads as revealed by qPCR and fluorescence in-situ hybridization (FISH). The abundance of comammox largely decreased by 3 orders of magnitude during the operation at 4 °C, and rapidly recovered after the application of selective heating. The anammox-comammox technology tested in this study essentially enabled mainstream shortcut nitrogen removal, and the selective heating ensured good performance of the technology at temperature as low as 5 °C.

Simultaneous reduction of perchlorate and nitrate using fast-settling anoxic sludge.

Fast-settling, anoxic sludge (FAS) was cultivated and utilized in this study to simultaneously reduce elevated levels of perchlorate and nitrate in an anaerobic sequencing batch reactor (AnSBR). Average perchlorate and nitrate removal efficiencies of 96.5 ± 8.44 % and 99.8 ± 0.32 %, respectively, were achieved from an average perchlorate and nitrate loading rate of 159 ± 101 g ClO4-/m3·d and 10.8 ± 7.25 g NO3--N/m3·d, respectively, throughout long-term operation (>500-d). Batch activity tests revealed a preferential utilization of nitrate over perchlorate, where significant perchlorate reduction inhibition occurred when nitrate was present as a competing electron acceptor under carbon-limiting conditions. Specific perchlorate and nitrate reduction rates were shown to increase as the hydraulic retention time (HRT) of the AnSBR was step-wise decreased and subsequently the perchlorate and nitrate loading rates were step-wise increased. Functional, mRNA-based expression of the nitrite reductase (nirS and nirK), nitrous oxide reductase (nosZ), perchlorate reductase subunit A (pcrA), and the chlorite dismutase (cld) genes illustrated the simultaneous activity of heterotrophic denitrification and perchlorate reduction occurring throughout a complete standard reactor operational cycle, and allowed for expression trends to be documented as the HRT of the AnSBR was reduced from 5-d to 1.25-d. Nitrous oxide (N2O) production was detected as a result of incomplete denitrification, where the largest N2O production occurred at the highest nitrate loading rates investigated in this study. Thauera species were heavily enriched at a longer HRT of 5-d, but were out-competed by Dechloromonas species as the HRT of the AnSBR was step-wise reduced to 1.25-d.