Carbon Uptake And Metabolic Characteristics Of Enriched Polyphosphate Accumulating Organisms From Municipal Wastewater Treatment Plants

Enhanced biological phosphorus removal（EBPR）is economic and environmental-friendly process widely used in municipal wastewater treatment plants（WWTPs）for phosphorus removal.The existing EBPR theory are largely limited to the understanding of the utilization characteristics of typical carbon sources（i.e.,acetate,propionate and other volatile fatty acid,VFA）,by typical polyphosphate accumulating organisms（i.e.,Ca.Accumulibacter）.There is still a lack of systematic studies on the utilization and metabolic characteristics of typical PAOs（Ca.Accumulibacter）on atypical（non-VFA）carbon sources,and atypical PAOs（non-Ca.Accumulibacter）on typical and atypical carbon sources.This paper investigated the carbon uptake bioenergetics of PAOs and glycogen accumulating organisms（GAOs）in the full-scale WWTPs,precisely regulated the carbon source supply rate,and typical and atypical PAOs were enriched,physiochemical characterization of the carbon source uptake and utilization characteristics were carried out by using these highly enriched PAOs.Metagenomic and metatranscriptomics analyses were performed to systematically investigated the genomic composition and key gene transcription characteristics of these typical and atypical PAOs by using different carbon sources.Biochemical metabolic models were then rebuilt.The carbon balance of different PAOs in using different carbon source for EBPR was then systematically evaluated.This study is expected to systematically supplement and enhance the existing EBPR theory,providing theoretical bases for population regulation and carbon management in the enhanced biological phosphorus removal process in full-scale WWTPs.Firstly,the carbon uptake bioenergetics of PAOs and GAOs in full-scale WWTPs were systematically investigated.Results showed that proton motive force（PMF）was the main driving force for carbon uptake by PAOs and GAOs in full-scale WWTPs.Compared to GAOs,PAOs showed high dependency on the proton motive force at the population level.Whereas,the uptake of carbon sources（acetate,propionate,aspartate,glutamate）by GAOs was more dependent on the proton motive force generated from fumarate reductase and the reverse operation of F1/F0-ATPase coupled proton translocation.The uptake of acetate and propionate by PAOs hardly requires these two processes to generate proton motive force.Based on the above results,the precise control of the influent carbon source supply was applied as a strategy to successfully obtain highly enriched atypical PAOs,i.e.,Candidatus Thiothrix.The draft genome was reconstructed via metagenomics sequencing and assembly,which was named as Ca.Thiothrix SCUT-1.Their typical and atypical carbon source utilization characteristics were systematically investigated.Results showed that the acetate and propionate uptake rates and anaerobic phosphorus release rates were the highest,followed by aspartate,glutamate,lactate,and butyrate.The glucose uptake rate was the lowest.For different carbon sources,Ca.Thiothrix SCUT-1 showed different uptake mechanisms,metabolic pathways,intracellular reserve productions and glycogen consumption characteristics.When acetate and propionate were used as carbon sources,the ratios of PHA production to carbon source consumption was 1.32 and 0.87 C mol/C mol,respectively.However,when butyrate was used as a carbon source,the intracellular polymer production and the carbon source recovery rate was low.When aspartate was used as a carbon source,PHV is the major intracellular polymer,indicating that the metabolism of aspartate involved in the TCA cycle.When glucose was used as a carbon source,the uptake glucose was directly converted to glycogen,and TCA cycle was scarcely involved in the anaerobic glucose metabolism.The active transcription of the glucose-6-phosphate isomerase gene（a key enzyme in glycolysis）indicated that Ca.Thiothrix SCUT-1generated the required reducing power through EMP（Embden-Meyerhof-Parnas）pathway.Additional reducing power was balanced via the succinate-propionate pathway.A strain of Ca.Accumulibacter（i.e.,clade IIC SCUT-2）was enriched.Its carbon utilization characteristics towards typical（acetate）and atypical carbon sources（lactate,succinate,alanine）were investigated.Results show that the uptake of lactate and succinate were achieved through monocarboxylic acid and C₄-dicarboxylate transporter systems respectively,which are driven by the proton motive force generated via reverse operation of F1/F0-ATPase,and the reduction of fumarate to succinate by fumarate reductase where the glycolysis products of pyruvate was routed into the TCA cycle.With the co-occurrence of acetate and lactate,the two carbon sources shared the common monocarboxylate transporter protein,acetate significantly inhibited the uptake of lactate.Due to different transport systems for acetate and succinate,these two carbon sources could be taken up simultaneously.By metagenomic analysis,biochemical models of Ca.Accumulibacter SCUT-2 were reconstructed with lactate and succinate as carbon sources.Lactate and succinate uptake and anaerobic storage processes scarcely involved glycolysis.The required reducing power for the storage of these two carbon sources were mainly generated via the TCA cycle.The presence of alanine has adverse effect on EBPR systems.The metabolism of Microlunatus phosphovorus using glucose and amino acids as carbon sources were further investigated,and the metabolic model was rebuilt.Results showed that Microlunatus phosphovorus take up glucose through the phosphoenolpyruvate-glucose phosphotransferase system.Aspartate and glutamate uptake were achieved via the proton/amino acid transporter.Glucose and amino acids were taken up under anaerobic conditions and stored as glycogen via gluconeogenesis.Glutamate was oxidatively deaminized and was converted intoα-ketoglutarate via glutamate dehydrogenase,which entered in the TCA cycle.Aspartate was oxidatively deaminized and was converted into oxaloacetate and fumarate.Since the lack of phosphoenolpyruvate carboxykinase related genes,the gluconeogenesis pathway was achieved via malate dehydrogenase.Based on the above established biochemical models,the carbon balance for PAOs using different carbon sources was evaluated.Results showed that the carbon footprints for different PAOs and different carbon sources for EBPR were distinct.A majority of the intracellularly stored carbon sources were oxidized and emitted in the form of CO₂（51%～81%）.Ca.Thiothrix converted 79%intracellular carbon storage compounds into CO₂ with propionate as carbon source.Only 15%was used for biological phosphorus removal,the carbon conversion rate was low.Acetate as carbon source resulted in the highest cost for phosphorus removal,high carbon emissions and the low economic benefits.Succinate for Ca.Accumulibacter as a carbon source for biological phosphorus removal is highly advantageous,with high carbon source utilization rate,and low CO₂ emission.When Microlunatus phosphovorus uses aspartate as a carbon source,the carbon emission was 81%,showing the lowest carbon source utilization rate.The effective carbon utilization rate was high when glucose as carbon source for Microlunatus phosphovorus for EBPR,with low CO₂ emission,and the overall economic benefit was high.Therefore,under the conditions when fermentative PAOs and typical PAOs were dominant,glucose and succinate as carbon sources,respectively,could achieve the optimal environmental and economic benefits.Overall,this study provides technical solutions and theoretical guidance for carbon source selection and optimization in WWTPs.