Biological Phosphate Removal Research Articles

Quantifying Glycogen in Solids at Full-Scale Enhanced Biological Phosphorous Removal Wastewater Facilities

AbstractGlycogen is a chief metabolic storage pool in bacteria performing enhanced biological phosphorous removal (EBPR) and is a potential resource for the production of bio-based fuels and chemic...

Transcriptomics investigation of thyroid hormone disruption in the olfactory system of the Rana [Lithobates] catesbeiana tadpole

Thyroid hormones (THs) regulate vertebrate growth, development, and metabolism. Despite their importance, there is a need for effective detection of TH-disruption by endocrine disrupting chemicals (EDCs). The frog olfactory system substantially remodels during TH-dependent metamorphosis and the objective of the present study is to examine olfactory system gene expression for TH biomarkers that can evaluate the biological effects of complex mixtures such as municipal wastewater. We first examine classic TH-response gene transcripts using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) in the olfactory epithelium (OE) and olfactory bulb (OB) of premetamorphic Rana (Lithobates) catesbeiana tadpoles after 48 h exposure to biologically-relevant concentrations of the THs, 3,5,3′-triiodothyronine (T3) and L-thyroxine (T4), or 17-beta estradiol (E2); a hormone that can crosstalk with THs. As the OE was particularly sensitive to THs, further RNA-seq analysis found >30,000 TH-responsive contigs. In contrast, E2 affected 267 contigs of which only 57 overlapped with THs suggesting that E2 has limited effect on the OE at this developmental phase. Gene ontology enrichment analyses identified sensory perception and nucleoside diphosphate phosphorylation as the top affected terms for THs and E2, respectively. Using classic and additional RNA-seq-derived TH-response gene transcripts, we queried TH-disrupting activity in municipal wastewater effluent from two different treatment systems: anaerobic membrane bioreactor (AnMBR) and membrane enhanced biological phosphorous removal (MEBPR). While we observed physical EDC removal in both systems, some TH disruption activity was retained in the effluents. This work lays an important foundation for linking TH-dependent gene expression with olfactory system function in amphibians.

Enhanced removal of nitrate and phosphate from wastewater by Chlorella vulgaris: Multi-objective optimization and CFD simulation

To enhance the efficiency of wastewater biotreatment with microalgae, the effects of physical parameters need to be investigated and optimized. In this regard, the individual and interactive effects of temperature, pH and aeration rate on the performance of biological removal of nitrate and phosphate by Chlorella vulgaris were studied by response surface methodology (RSM). Furthermore, a multi-objective optimization technique was applied to the response equations to simultaneously find optimal combinations of input parameters capable of removing the highest possible amount of nitrate and phosphate. The optimal calculated values were temperature of 26.3 °C, pH of 8 and aeration rate of 4.7 L·min− 1. Interestingly, under the optimum condition, approximately 85% of total nitrate and 77% of whole phosphate were removed after 48 h and 24 h, respectively, which were in excellent agreement with the predicted values. Finally, the effect of baffle on mixing performance and, as a result, on bioremoval efficiency was investigated in Stirred Tank Photobioreactor (STP) by means of Computational Fluid Dynamics (CFD). Flow behavior indicated substantial enhancement in mixing performance when the baffle was inserted into the tank. Obtained simulation results were validated experimentally. Under the optimum condition, due to proper mixing in baffled STP, nitrate and phosphate removal increased up to 93% and 86%, respectively, compared to unbaffled one.

Effect of Dissolved Oxygen on Biological Phosphorous Removal in Continuous-Flow Anoxic-Oxic Process Using Starch as Sole Carbon Source

AbstractPrevious research has demonstrated that biological phosphorus removal (BPR) could be induced by an anoxic-aerobic sequencing batch reactor (SBR). This study applies a continuous-flow anoxic...

Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased In Vivo Polyphosphate Levels.

Bacteria synthesize inorganic polyphosphate (polyP) in response to a wide variety of stresses, and production of polyP is essential for stress response and survival in many important pathogens and bacteria used in biotechnological processes. However, surprisingly little is known about the molecular mechanisms that control polyP synthesis. We have therefore developed a novel genetic screen that specifically links growth of Escherichia coli to polyP synthesis, allowing us to isolate mutations leading to enhanced polyP production. Using this system, we have identified mutations in the polyP-synthesizing enzyme polyP kinase (PPK) that lead to dramatic increases in in vivo polyP synthesis but do not substantially affect the rate of polyP synthesis by PPK in vitro These mutations are distant from the PPK active site and found in interfaces between monomers of the PPK tetramer. We have also shown that high levels of polyP lead to intracellular magnesium starvation. Our results provide new insights into the control of bacterial polyP accumulation and suggest a simple, novel strategy for engineering bacteria with increased polyP contents.IMPORTANCE PolyP is an ancient, universally conserved biomolecule and is important for stress response, energy metabolism, and virulence in a remarkably broad range of microorganisms. PolyP accumulation by bacteria is also important in biotechnology applications. For example, it is critical to enhanced biological phosphate removal (EBPR) from wastewater. Understanding how bacteria control polyP synthesis is therefore of broad importance in both the fields of bacterial pathogenesis and biological engineering. Using Escherichia coli as a model organism, we have identified the first known mutations in polyP kinase that lead to increases in cellular polyP content.

INCREASING ROLE OF MICROORGANISM WITH UBIQUINONE-9 IN DENITRIFYING PAO SLUDGE

An anaerobic-aerobic (An-Ox) sequencing batch reactor (SBR) was operated for the growth of phosphate accumulating organisms (PAOs), while an anaerobic-anoxic (An-Ax) SBR was used for the growth of denitrifying PAOs (dPAOs). Both reactors exhibited successful enhanced biological phosphorous removal (EBPR) performance. The sludge P contents in the two reactors increased from approximately 2% at the beginning of the operation to 9.1% (An-Ox SBR) and 7.3% (An-Ax SBR) after 140 d. The results of the quinone profile analysis demonstrated that the proportions of UQ-8, UQ-10, and MK-8 (H4) in the An- Ox sludge increased compared to the beginning of the operation, whereas in the An-Ax sludge, the proportion of UQ-8 was the highest and exhibited the greatest increase, and the mole fraction of UQ-9 more than doubled from 8.3% to 16.9%. Thus, the role of microorganisms with UQ-8 and UQ-9 was found to have significantly increased in the An-Ax sludge. As a result of microbial identification through the fatty acid methyl ester (FAME) analysis, Comamonas testosteroni and Pseudomonas alcaligenes, which produce the UQ-9 in the An-Ax sludge, as well as Psychrobacter immobilis, which produces UQ-8.

Comparing Bacterial Diversity in Two Full-Scale Enhanced Biological Phosphate Removal Reactors Using 16S Amplicon Pyrosequencing

Comparing Bacterial Diversity in Two Full-Scale Enhanced Biological Phosphate Removal Reactors Using 16S Amplicon Pyrosequencing

Biological nutrient removal in a continuous biofilm process

Abstract A new biological phosphorous and nitrogen removal process is developed. The process is based on biofilm on carrier elements with enhanced biological phosphorous removal and simultaneous nitrification and denitrification in a continuous process. Results from 3 years of pilot and laboratory experiments are presented with regards to removal of organic substances, phosphorous and nitrogen. This process demonstrates essential benefits and improved performance compared to other EBPR-processes in operation today. The first full scale plant was put in operation in May 2016 at Hias WWTP in Norway.

Multi-criterial evaluation of P-removal optimization in rural wastewater treatment plants for a sub-catchment of the Baltic Sea

Rural wastewater treatment plants (WWTP) are recognized as a significant phosphorous (P)-emission source in the German sub-catchments of the Baltic Sea. But enhancement of P-removal is cost intensive especially for small communities. This study proposes a concept to efficiently reduce P-emissions from WWTP by introducing low-cost non-constructional measures and assessing the resulting change of ambient water quality on a river basin scale. As case study, we choose the Warnow catchment in Mecklenburg-Vorpommern (Germany) where 76 % of WWTP-emissions originate from treatment plants without P-discharge limits. For these facilities, we evaluated two modification options: (1) optimization of enhanced biological Phosphorous removal (EBPR) in activated sludge systems (ASS) and/or (2) chemical precipitation (where EBPR is applicable). The total operational optimization potential in the Warnow catchment is about 20 %. Further improvements can only be applied by a chemical precipitation, with the drawback of increasing the wastewater disposal cost up to 6.5 euros CAP−1a−1. To prioritize relevant plants for improved P-removal we proposed two evaluation criteria: combining absolute emission, and impact on ambient water quality and costs.

Phosphorus Balance at Tartu WWTP, Estonia

Sewage sludge is usually considered as treatment residue that needs expensive treatment for removal. The treatment and handling of sludge can form 20 to 50 % from a wastewater treatment facility's costs. Sludge volumes will continue to grow worldwide with increasing population and country wealth. In a sustainable society, sludge should rather be seen as a source of energy, phosphorus and other products. Water treatment facilities are exploring technologies to utilise these values worldwide. Technologies on extracting minerals from sludge would either help offset treatment facility’s costs or even turn a profit. First step in turning sludge from a costly waste material into a profitable revenue stream should be a material balance.
 In Tartu wastewater treatment plant (120 000 pe, 28 000 m3 d-1) waste water is treated by activated sludge process with an integrated biological nitrogen and phosphorous removal. Sludge is dewatered and stabilized by windrow composting. In the treatment plant, phosphorus balance in various treatment steps was studied and potential technologies for extracting phosphorus were reviewed.

Genetic improvement of Magnetospirillum gryphiswaldense for enhanced biological removal of phosphate.

To improve its phosphate accumulating abilities for phosphate recycling from wastewater, a magnetotactic bacterium, Magnetospirillum gryphiswaldense, was genetically modified to over-express polyphosphate kinase. Polyphosphate kinase was over-expressed in the bacterium. The recombinant strain accumulated ninefold more polyphosphate from synthetic wastewater compared to original wild type. The magnetic property of the recombinant M. gryphiswaldense strain was retained. The recombinant M. gryphiswaldense can be used for phosphate removal and recovery in bioremediation.

Removal of phosphate by Staphylococcus aureus under aerobic and alternating anaerobic–aerobic conditions

ABSTRACTEutrophication of water bodies due to phosphate enrichment is an ecological problem. Phosphate is removed from wastewaters by enhanced biological phosphate removal worldwide by phosphate accumulating organism. In order to understand the process of treatment, the existing microbial community and its metabolism of phosphate removal are studied widely. This study focuses on the isolation of polyphosphate-accumulating bacteria from different environments and studying their phosphate removal capacity with different carbon supplements under varying culture conditions. The total heterotrophic bacterial population from the diverse environments showed the existence of phosphate-accumulating bacteria. Among them, Staphylococcus aureus removed 81% of phosphate in a polyphosphate-accumulating medium with storage of 93 mM polyphosphate internally. Among the different carbon sources provided, glucose induced a net specific growth rate of 0.816/d. S. aureus removed 70% of phosphate with a phosphate uptake rate of 6.29 mg PO4/g cells and a growth yield of 0.2 g cells/g glucose consumed when 1 g/L glucose was provided. Furthermore, when 2 g/L glucose was provided, 78% of phosphate was removed with a phosphate uptake rate of 13.24 mg PO4/g cells and a growth yield of 0.4 g cells/g glucose consumed under aerobic condition. S. aureus showed enhanced phosphate removal under aerobic condition in the presence of glucose.

Dynamics of microbial community structure and nutrient removal from an innovative side-stream enhanced biological phosphorus removal process

Biological phosphorous (P) and nitrogen (N) removal from municipal wastewater was studied using an innovative anoxic-aerobic-anaerobic side-stream treatment system. The impact of influent water quality including chemical oxygen demand (COD), ammonium and orthophosphate concentrations on the reactor performance was evaluated. The results showed the system was very effective at removing both COD (>88%) and NH4+-N (>96%) despite varying influent concentrations of COD, NH4+-N, and total PO43--P. In contrast, it was found that the removal of P was sensitive to influent NH4+-N and PO43--P concentrations. The maximum PO43--P removal of 79% was achieved with the lowest influent NH4+-N and PO43--P concentration. Quantitative PCR (qPCR) assays showed a high abundance and diversity of phosphate accumulating organisms (PAO), nitrifiers and denitrifiers. The MiSeq microbial community structure analysis showed that the Proteobacteria (especially β-Proteobacteria, and γ-Proteobacteria) were the dominant in all reactors. Further analysis of the bacteria indicated the presence of diverse PAO genera including Candidatus Accumulibacter phosphatis, Tetrasphaera, and Rhodocyclus, and the denitrifying PAO (DPAO) genus Dechloromonas. Interestingly, no glycogen accumulating organisms (GAOs) were detected in any of the reactors, suggesting the advantage of proposed process in term of PAO selection for enhanced P removal compared with conventional main-stream processes.

Function of Polyphosphate Kinase Gene in Biological Phosphate Removal During the Wastewater Treatment Process

This study aimed to identify the function of polyphosphate kinase gene (ppk) in phosphorus removal. With the Red system, the target DNA with the homologous short arms was amplified in the plasmid pKD4. Then the target DNA was transformed into E. coli ATCC25922 which already had the suicide plasmid pKD46 by electroporation. The plasmid pCP20 was transformed into the recombinant strains to delete the kanamycin resistance gene. With the screening by negative resistance, together with verification using positive and negative primers, the construction of ppk gene deletion strain E. coli/ppk- Kan- was confirmed. The growth characteristics of both the wild-type strain and the mutant strain were determined, and the phosphate accumulating characteristics were compared when cultured in the phosphate luxuriant medium after induced in the phosphate lacking medium. Also the phosphate accumulating characteristics of the two strains were compared after cultured in the anaerobic and aerobic alternating conditions for 5 times. The results showed that the ppk deletion strain E. coli/ppk- Kan- was successfully constructed. There was no growth difference between the mutant strain and the wild-type strain. But in the first 4 hours of log phase, the mutant strain grew faster than the wild-type strain. And 8h later, when both strains were in stationary phase, the mutant strain grew slower than the wild type, indicating that ppk affected the growth of the bacteria. Cultured in the phosphate lacking medium and the phosphate luxuriant medium, the mutant strain's ability of phosphate accumulating didn't decrease in spite of having no ppk gene. After 5 times induction, the amounts of phosphorus in both strains were about 1%-2%. The phosphate amounts in the cells did not increase with increasing inducing times. Polyphosphate or PHB was detected neither at anaerobic phase nor at the aerobic phase. It indicated that the deletion of ppk did not affect the phosphorus removal in wastewater treatment process, and the ppk gene did not show the function of phosphorus removal.

Enrichment and characterization of a psychrophilic ‘Candidatus Accumulibacter phosphatis’ culture

Enhanced Biological phosphorous removal (EBPR) processes, often operated at low temperatures, are utilised world-wide, but currently little is known regarding enrichment cultures and the characteristics of active organisms (“Candidatus Accumulibacter phosphatis” (Accumulibacter)) under psychrophilic conditions. This study assesses the long-term performance, metabolic activity, microbial community characteristics and sludge morphology in an EBPR community enriched from activated sludge at 10 °C. Long solid retention times (SRT) and low temperatures resulted in the dominance of Accumulibacter type II over type I. Despite changes in the microbial community, P-removal efficiencies did not show obvious differences and although no specific measures were implemented, the enriched Accumulibacter-PAO culture formed stable dense granules. A high level of Alginate-like exopolysaccharides (ALE) were observed, with a large number of Guluronic acid-Guluronic acid (GG) blocks derived from the biomass at 10 °C. This characteristic favors sludge granulation, increasing the mechanical strength of granules formed, which encourages solid-liquid separation and consequently, contributes to the stable operation of EBPR systems.

Bacterial microcompartment‐directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli

Processes for the biological removal of phosphate from wastewater rely on temporary manipulation of bacterial polyphosphate levels by phased environmental stimuli. In E. coli polyphosphate levels are controlled via the polyphosphate-synthesizing enzyme polyphosphate kinase (PPK1) and exopolyphosphatases (PPX and GPPA), and are temporarily enhanced by PPK1 overexpression and reduced by PPX overexpression. We hypothesised that partitioning PPK1 from cytoplasmic exopolyphosphatases would increase and stabilise E. coli polyphosphate levels. Partitioning was achieved by co-expression of E. coli PPK1 fused with a microcompartment-targeting sequence and an artificial operon of Citrobacter freundii bacterial microcompartment genes. Encapsulation of targeted PPK1 resulted in persistent phosphate uptake and stably increased cellular polyphosphate levels throughout cell growth and into the stationary phase, while PPK1 overexpression alone produced temporary polyphosphate increase and phosphate uptake. Targeted PPK1 increased polyphosphate in microcompartments 8-fold compared with non-targeted PPK1. Co-expression of PPX polyphosphatase with targeted PPK1 had little effect on elevated cellular polyphosphate levels because microcompartments retained polyphosphate. Co-expression of PPX with non-targeted PPK1 reduced cellular polyphosphate levels. Thus, subcellular compartmentalisation of a polymerising enzyme sequesters metabolic products from competing catabolism by preventing catabolic enzyme access. Specific application of this process to polyphosphate is of potential application for biological phosphate removal.

Draft Genome Sequence of Cloacibacterium normanense NRS-1 Isolated from Municipal Wastewater.

Cloacibacterium normanense is a Gram-negative bacterium recovered from untreated human wastewater. Given its high abundance in wastewater and its apparent absence in human stool, it may contribute to biological phosphate removal. Here, we perform a whole-genome sequence of C. normanense NRS-1(T) and examine particular features of this draft genome.

Nitrous oxide from moving bed based integrated fixed film activated sludge membrane bioreactors

The present paper reports the results of a nitrous oxide (N2O) production investigation in a moving bed based integrated fixed film activated sludge (IFAS) membrane bioreactor (MBR) pilot plant designed in accordance with the University of Cape Town layout for biological phosphorous removal. Gaseous and liquid samples were collected in order to measure the gaseous as well as the dissolved concentration of N2O. Furthermore, the gas flow rate from each reactor was measured and the gas flux was estimated. The results confirmed that the anoxic reactor represents the main source of nitrous oxide production. A significant production of N2O was, however, also found in the anaerobic reactor, thus indicating a probable occurrence of the denitrifying phosphate accumulating organism activity. The highest N2O fluxes were emitted from the aerated reactors (3.09 g N2ON m−2 h−1 and 9.87 g N2ON m−2 h−1, aerobic and MBR tank, respectively). The emission factor highlighted that only 1% of the total treated nitrogen was emitted from the pilot plant. Furthermore, the measured N2O concentrations in the permeate flow were comparable with other reactors. Nitrous oxide mass balances outlined a moderate production also in the MBR reactor despite the low hydraulic retention time. On the other hand, the mass balance showed that in the aerobic reactor a constant consumption of nitrous oxide (up to almost 15 mg N2O h−1) took place, due to the high amount of stripped gas.

Denitrification of nitrate and nitrite by ‘Candidatus Accumulibacter phosphatis’ clade IC

Phosphate accumulating organisms (PAO) are assumed to use nitrate as external electron acceptor, allowing an efficient integration of simultaneous nitrogen and phosphate removal with minimal organic carbon (COD) requirements. However, contradicting findings appear in literature regarding the denitrification capacities of PAO due to the lack of clade specific highly enriched PAO cultures. Whereas some studies suggest that only PAO clade I may be capable of using nitrate as external electron acceptor for anoxic P-uptake, other studies indicate that PAO clade II may be responsible for anoxic P-removal. In the present study, a highly enriched PAO clade IC culture (>99% according to FISH) was cultivated in an SBR operated under Anaerobic/Oxic conditions and subsequently exposed to Anaerobic/Anoxic/Oxic conditions using nitrate as electron acceptor. Before and after acclimatization to the presence of nitrate, the aerobic and anoxic (nitrate and nitrite) activities of the PAO I culture were assessed through the execution of batch tests using either acetate or propionate as electron donor. In the presence of nitrate, significant P-uptake by PAO I was not observed before or after acclimatization. Using nitrite as electron acceptor, limited nitrite removal rates were observed before acclimatization with lower rates in the acetate fed reactor without P-uptake and slightly higher in the propionate fed reactor with a marginal anoxic P-uptake. Only after acclimatization to nitrate, simultaneous P and nitrite removal was observed. This study suggests that PAO clade IC is not capable of using nitrate as external electron acceptor for anoxic P-removal. The elucidation of the metabolic capacities for individual PAO clades helps in better understanding and optimization of the relation between microbial ecology and process performance in enhanced biological phosphate removal processes.

Potential of hydrolysis of particulate COD in extended anaerobic conditions to enhance biological phosphorous removal.

The effect of anaerobic hydrolysis of particulate COD (pCOD) on biological phosphorous removal in extended anaerobic condition was investigated through (i) sequencing batch reactors (SBR)s with anaerobic hydraulic retention time (HRT) of 0.8, 2, and 4 h; (ii) batch tests using biomass from a full scale biological nutrient removal (BNR) plant; and (iii) activated sludge modeling (BioWin 4.1 simulation). The results from long-term SBRs operation showed that phosphorus removal was correlated to the ratio of filtered COD (FCOD) to total phosphorus (TP) in the influent. Under conditions with low FCOD/TP ratio (average of 20) in the influent, extending anaerobic HRT to 4 h in the presence of pCOD did not significantly improve overall phosphorous removal. During the period with high FCOD/TP ratio (average of 37) in the influent, all SBRs removed phosphorous completely, and the long anaerobic HRT did not have negative effect on overall phosphorous removal. The batch tests also showed that pCOD at different concentration during 4 h test did not affect the rate of anaerobic phosphorus release. The rate of anaerobic hydrolysis of pCOD was significantly low and extending the anaerobic HRT was ineffective. The simulation (BioWin 4.1) of SBRs with low influent FCOD/TP ratio showed that the default kinetics of anaerobic hydrolysis in ASM2d overestimated phosphorous removal in the SBRs (high anaerobic hydrolysis of pCOD). The default anaerobic hydrolysis rate in BioWin 4.1 (ten times lower) could produce similar phosphorous removal to that in the experiment. Results showed that the current kinetics of anaerobic hydrolysis in ASM2d could lead to considerable error in predicting phosphorus removal in processes with extended anaerobic HRT. Biotechnol. Bioeng. 2016;113: 2377-2385. © 2016 Wiley Periodicals, Inc.