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Highly Effective Polyphosphate Synthesis, Phosphate Removal and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-copy Plasmid Strategy Xin Wang, Xiaomeng Wang, Kaimin Hui, Wei Wei, Wen Zhang, Ai-Jun Miao, Lin Xiao, and Liuyan Yang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04532 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on December 2, 2017

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Environmental Science & Technology

Highly Effective Polyphosphate Synthesis, Phosphate Removal and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-copy Plasmid Strategy

Xin Wang,† Xiaomeng Wang,† Kaimin Hui,§ Wei Wei,‡ Wen Zhang,† Aijun Miao,† Lin Xiao,† Liuyan Yang*†

†

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, 210046, PR China

§

Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for

Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing, 210046, PR China ‡

Institute of Chemistry and BioMedical Science, State Key Laboratory of Pharmaceutical

Biotechnology, School of Life Science, Nanjing University, Nanjing, 210046, PR China *

Correspondence author. Phone: +86 25 8968 0257, E-mail: [email protected]

Submitted to: Environmental Science & Technology

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Table of Contents (TOC) Art

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ABSTRACT

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Microbial polyphosphate (polyP) production is vital to phosphate removal from wastewater.

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However, to date, the engineered polyP synthesis using genetically accessible environmental

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bacteria remains a challenge. This study develops a simple solo medium-copy plasmid based

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polyphosphate kinase (PPK1) overexpression strategy for achieving maximum intracellular

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polyphosphate accumulation by environmental bacteria. The polyP content of the subsequently

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engineered Citrobacter freundii (CPP) could reach as high as 12.7% of its dry weight. The

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biomass yield of CPP was also guaranteed because of negligible metabolic burden effects

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resulting from the medium plasmid copy number. Consequently, substantial phosphate (Pi)

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removal from the ambient environment was achieved simultaneously. Due to the need of

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exogenous Pi for in vivo ATP regeneration, CPP could thoroughly remove Pi from synthetic

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municipal wastewater when it was applied for the “one-step” removal of Pi with a bench-scale

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sequence batch membrane reactor. Almost all the phosphorus except for that assimilated by CPP

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for cellular growth could be recovered in the form of more concentrated Pi. Overall, engineering

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environmental bacteria to overexpress PPK1 via a solo medium-copy plasmid strategy may

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represent a valuable general option for not only biotechnological research based on sufficient

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intracellular polyP production but also Pi removal from wastewater and Pi enrichment.

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INTRODUCTION

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Inorganic polyphosphate (polyP) is present in almost all organisms, where it is considered to be a

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reserve of phosphate (Pi) and high-energy phosphoanhydride bonds.1,2 PolyP of prokaryotic

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origin is primarily synthesized by polyphosphate kinase (PPK1), which reversibly catalyzes the

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transfer of terminal Pi from ATP to a growing polyP chain.3,4 Microbial polyP is central to

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enhanced biological phosphorus removal (EBPR) from municipal wastewater, in which

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polyphosphate accumulating organisms (PAOs) take up Pi beyond their growth requirements and

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concentrate it in biomass as polyP.5 However, because of the unavailability of pure cultures of

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PAOs and groundless deterioration of the EBPR system,6 studies on removing Pi from

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wastewater based on engineered production of intracellular polyP using genetically accessible

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bacteria are of important environmental significance.

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To date, research on this issue has been mainly conducted in Escherichia coli via two kinds

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of genetic engineering strategies: (1) the enhancement of Pi transport capability (including

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increasing the dosage of genes that encode Pi-specific transport systems7,8 and mutation of the

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phoU gene that encodes a negative regulator of the Pi regulon9-11) and (2) plasmid-borne PPK1

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overexpression. As far as the latter is concerned, these strategies can be divided into three

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categories according to the copy number of the plasmid and the number of plasmids that occur in

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one host cell: (1) solo high-copy plasmid strategies; (2) solo low-copy plasmid strategies; and (3)

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dual plasmid strategies (i.e., one host cell harboring two plasmids, in which one plasmid

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overexpresses PPK1 and the other one overexpresses auxiliary enzymes to increase the amount 2 ACS Paragon Plus Environment

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of intermediates related to ATP generation and/or regeneration).7,8,12,13 Benefiting from these

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strategies, a certain amount of intracellular polyP accumulation has been achieved. However, in

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most cases, the polyP yields have a poor ratio of achieved versus theoretically estimated

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accumulation (< 10-20% of the cell dry weight).6 Obviously, both PPK1 overexpression and

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polyP synthesis require a metabolite, ATP, which is vital and limited at any life stage of the host

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cell. From the metabolic engineering perspective, to maximize polyP productivity, researchers

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must weigh the trade-offs between the ATP budget put into PPK1 overexpression and that

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reserved as the substrate for polyP synthesis.14,15 In this regard, it should be evident that neither

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the high-copy plasmid strategy (including dual plasmid strategy) nor the low-copy plasmid

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strategy is an optimal option. For the former, the presence of one high-copy plasmid can divert a

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substantial amount of ATP from polyP synthesis toward plasmid DNA replication and

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plasmid-borne gene translation,16,17 which may cause a poor polyP yield because of the lack of

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substrate. For the latter, inadequate PPK1 dosage resulting from the low copy number of the

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plasmid may also cause a poor polyP yield because of the lack of enzymes. Therefore, a genetic

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engineering strategy for highly effective polyP production based on a more balanced distribution

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of ATP between PPK1 overexpression and its substrate reservation warrants further exploitation.

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Based on these valuable findings, we now speculate that a solo medium-copy plasmid

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strategy (i.e., overexpression of PPK1 from one medium-copy plasmid, which is only harbored

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in the host cell) may be a better option for genetically engineered polyP production. The strategy

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was assessed using an environmental bacterium, Citrobacter freundii ATCC 8090, and its 3 ACS Paragon Plus Environment


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superiority was validated by comparison with all three types of strategies adopted by those

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studies with E. coli. Moreover, to determine how the second plasmid itself in the dual plasmid

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strategy affected the genetically manipulated polyP production, we constructed a high-copy

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plasmid harboring a reporter gene that is not directly related to the polyP synthetic pathway and

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introduced it into the engineered C. freundii (which already harbored a medium-copy plasmid) to

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mimic a dual plasmid strategy. Via this strategy, the potential negative effects imposed by the

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second plasmid were comprehensively demonstrated at different levels, including the biomass

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yield, polyP production, ppk1 transcription, host cell morphology, and polyP synthesis kinetics.

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In addition, we also performed Pi removal and concentration from synthetic municipal

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wastewater using a sequence batch membrane bioreactor (SBMBR) to illustrate the feasibility of

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the application of the environmental bacterium engineered based on the solo medium-copy

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plasmid strategy.

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EXPERIMENTAL

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Plasmids and Bacterial Strains. The bacterial strains, plasmids and primers used in this study

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are all listed in Table S1 (Supporting Information, SI). Wild-type Citrobacter freundii ATCC

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8090 was purchased from China Center of Industrial Culture Collection (CICC, China) and

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designated CWT. A broad-host-range medium-copy plasmid, pBBR1MCS2,18 harboring the ppk1

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of CWT was constructed and then transformed into CWT, and the resulting recombinant was

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designated CPP. Before introducing a second plasmid into CPP, the following three 4 ACS Paragon Plus Environment

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characteristics of the plasmid were considered: (1) a high-copy plasmid that could impose

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significant metabolic burden upon the host cell; (2) a plasmid that possesses the same leaky

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expression system as pBBR1MCS2; and (3) a plasmid that can express a fluorescent reporter to

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serve as an indication of whether the leaky expression from the lac promoter could be achieved.

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To meet these conditions, the gene rfp, which encodes red fluorescent protein (RFP),19 was

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cloned into the high-copy plasmid pMD19-Simple (TaKaRa, Japan). CPP transformed with

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pMD19-rfp was designated CPR. The specific gene cloning, plasmid construction and

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transformant selection procedures are detailed in Text S1.

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Culture Conditions. To obtain uniform polyP-free inocula, CWT and its derivatives were

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cultured overnight at 30°C in LB medium supplemented with antibiotics as required. Under such

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nutrient-rich conditions no significant intracellular polyP formed.20 PolyP-free cells were

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harvested by centrifugation (8000 rpm, 5 min) and washed twice with HEPES buffer (20 mM,

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pH 7.0). For practical applications, nutrient-poor synthetic wastewater (without chemical inducer

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and antibiotics) that mimics municipal sewage was adopted to evaluate the strains constructed in

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this study. Synthetic municipal wastewater (SMW) was prepared from inorganic and organic

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components with deionized water according to Bassin et al.21 and contained per liter 300 mg

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glucose (300 mg-COD/L), 100 mg tryptone (50 mg-COD/L), 50 mg NaCl, 226 mg MgSO4·7H2O,

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180 mg NH4Cl, and 1 mg yeast extract. The Pi concentration in SMW was set at 30 mg-P/L by

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the addition of 220 mg KH2PO4·3H2O per liter to prevent Pi from becoming a growth-limiting

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factor unless otherwise indicated. For all experiments, the polyP-free culture volume to be 5 ACS Paragon Plus Environment


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inoculated into the SMW was calculated beforehand to attain an initial OD600 of 0.15. After

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inoculation, 500 mL Erlenmeyer flasks containing 200 mL SMW were shaken at 200 rpm on a

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rotary shaker at 30°C. Liquid samples of 4 mL were taken at the indicated time points for

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analysis. The OD600 was monitored using a UV1800 spectrophotometer (Shimadzu, Japan),

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whereas the Pi, total phosphorous (TP), chemical oxygen demand (COD), volatile suspended

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solids (VSS) and cell dry weight (DW) were measured via standard methods.22

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Intracellular PolyP Assay. Intracellular polyP was quantified according to the direct

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4’-6-diamidino-2-phenylindole (DAPI)-based protocol described by Kulakova et al.23 using Type

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45 polyP (Sigma, USA) as a standard and expressed as milligrams phosphorus per gram VSS

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(mg-P/g-VSS). The VSS was correlated to OD600 using the equation: VSS (mg/L) = 161.25 ×

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OD600 + 48.51 (R2 = 0.9937), which was determined for C. freundii in SMW. The VSS data were

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converted to DW using the conversion factor 0.80 mg-VSS/mg-DW for the convenience of

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comparison whenever needed. In vivo ATP measurements of each strain were performed with a

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modified protocol described by Gray et al.24 Briefly, at the indicated OD600, 100 µL cell cultures

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were added to 900 µL 20 mM HEPES (pH 7.8) and incubated at 99°C on a ThermoMixer

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(Eppendorf, USA) for 5 min. After that, the samples were cooled on ice and the total cellular

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ATP was assayed with the Luminescent ATP Detection Assay Kit (Abcam, UK).

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Quantitative Real-time PCR. After harvesting bacteria from either LB medium or SMW,

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purification of total RNA was performed using RNAiso Plus reagent (TaKaRa, Japan) following

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the protocol described by the manufacturer. One microgram of qualified total RNA was subjected 6 ACS Paragon Plus Environment

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to reverse transcription with a PrimeScript RT reagent Kit with gDNA Eraser per the

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manufacturer’s instructions (TaKaRa, Japan). qRT-PCR of the resulting cDNA was performed

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with gene-specific primers (Table S1) on a CFX Connect Real-Time PCR Detection System

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(Bio-Rad, USA) with a SYBR Premix Ex Taq (Tli RNaseH Plus) Kit (TaKaRa, Japan). Standard

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curves of cDNA dilutions were used to determine the PCR efficiency. An expression data

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analysis was performed by the Pfaffl method of relative quantification using CFX Manager 3.1

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software (Bio-Rad, USA).

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Microscopic Examination and Live Cell Confocal Imaging. The presence of intracellular

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polyP granules was examined by light microscopy after staining the cells according to the

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method of Albert.25 To further display the morphology of each strain, a Nikon A1 confocal laser

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scanning microscope (Nikon, Japan) equipped with different filter sets was used to acquire

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images after the cells were incubated with 10 µM DAPI in darkness for 1 h.26

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PolyP Synthesis Kinetics. Intracellular polyP content measured for CPP and CPR during the

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polyP synthesis stage was used for data fitting to depict their respective polyP synthesis kinetic

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behavior. For convenient operation, short term polyP synthesis experiments performed with CPP

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were conducted in triplicate Corning 50 mL Mini Bioreactors (Corning, USA) containing 10 mL

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SMW with different Pi concentrations (4, 8, 20, 40 and 80 mg-P/L). The intracellular polyP

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content was determined at times 0.5, 1, 1.5, 2, 3, 4, and 5 h and then plotted with time.

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SBMBR. To achieve “one-step” removal of Pi and facilitate the following Pi enrichment, a

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bench-scale MBR with a working volume of 22 L was employed in this study (Figure 6a, 7 ACS Paragon Plus Environment


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detailed information on its configuration is provided in Text S2). SMW with 8 mg-P/L Pi freshly

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prepared from tap water was used to feed the reactor. The MBR was conducted in a batch regime

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with a cycle duration of 18 h by sequencing through three steps: (1) filling and preculture (4 h 15

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min); (2) continuous feeding and permeate filtration (12 h); and (3) biomass concentrating and

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retentate discharge (1 h 45 min). In the first step, the reactor was filled with 22 L SMW and

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inoculated with polyP-free CPP afterwards. Aerobic preculturing was initiated to remove the

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in-reactor Pi. When the in-reactor Pi was depleted, step two was introduced to achieve

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continuous Pi removal from the feeding SMW. Once Pi (≥ 0.01 mg-P/L) could be detected in the

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permeate fluid, water feeding ceased and permeate filtration carried on till the liquid level was

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below the membrane module. At the time of step three, the concentrated retentate (i.e., enriched

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cell suspension) was withdrawn from the bottom of the reactor and added to two 5 L glass

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vessels (4.5 L cell suspension per vessel, Figure 6c inset), after which the vessels were sealed by

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tightening the lids. The subsequent Pi release assessment was carried out under anaerobic

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conditions without the addition of glucose or other organic substrates to save the consumption of

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both aeration and carbon source. Throughout the experiment, the pH and dissolved oxygen (DO)

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were monitored using HQ 30d portable meters (Hach, USA) but not controlled (Figure S5a).

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Mixed liquor and effluent were regularly taken from the reactor or the outlet of the permeate

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pump, respectively, for analysis. The Pi concentration of the enriched cell suspension supernatant

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was monitored to determine the end point of the Pi release stage. Details of the global evaluation

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of Pi recovery efficiency using such a treatment process is provided in Text S3. 8 ACS Paragon Plus Environment

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RESULTS AND DISCUSSION

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Intracellular PolyP Production and Exogenous Pi Removal. Prior to the beginning of this

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study, an environmental bacterium other than E. coli was selected to serve as the starting strain

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because E. coli is not suitable for practical wastewater treatment. In the E. coli chromosome,

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there is one native ppk1 gene copy located on an operon that also includes the

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exopolyphosphatase gene ppx.3 Considering the comparability of results, those bacteria that are

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of the same genotype as E. coli were especially desired by this study. After querying the

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GenBank database in NCBI, one bacterium termed C. freundii ATCC 8090 (accession number

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NZ_ANAV01000007), which is broadly distributed in soil, water, and sewage,27 was picked and

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genetically engineered for further study.

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To test whether the C. freundii derivative that was constructed based on the solo

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medium-copy strategy could perform well for polyP synthesis in SMW, the polyP content of CPP

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was intensively measured during the polyP synthesis stage. Both CPR and CWT served as

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controls. As shown in Figure 1a, the polyP content of CPP increased rapidly and reached up to

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159 mg-P/g-VSS (127 mg-P/g-DW) at 15 h. Associated with this process was the continuous

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uptake of exogenous Pi from the SMW, and maximal Pi removal of 19 mg-P/L was then

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achieved at 15 h (Figure 1b). By contrast, profiles of polyP content versus time obtained with

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CPR showed a relatively slow increase to its maximal level by approximately 11 h, and the

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amount of polyP produced (51 mg-P/g-VSS) and hence Pi removed (7 mg-P/L) were both 9 ACS Paragon Plus Environment


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significantly (t test, p < 0.05) lower than those obtained with CPP. For CWT, at no time during

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the course of the experiment was intracellular polyP formed. The small amount of Pi removal

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could be attributed to stoichiometric incorporation into its cellular growth, which only formed a

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background TP content of approximately 13 mg-P/g-DW, which is similar to most reported

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bacteria.7,28 These results indicated that a polyP content equal to 10-fold over the background TP

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content of CWT can be easily achieved through solo medium-copy plasmid strategy. In addition,

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decreases in both polyP production and Pi removal approaching 70% of CPR relative to CPP

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suggested that the cooccurrence of a second high-copy plasmid impacted the genetically

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engineered enhancement of polyP biosynthesis.

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However, consistent with a previously observed phenomenon in PPK1 overexpressing E.

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coli,13,29 polyP degradation accompanied by Pi secretion occurred in both CPP and CPR between

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15 and 16 h. Since then, they entered the stage of Pi release until their intracellular polyP pools

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were totally depleted at 92 h (Figure 1b, subsequent data not shown). This result implied that we

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can recycle phosphorus from CPP in the form of Pi, which will be discussed later. At this point,

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we asked whether the polyP content of CPP could be further elevated. It is well known that the

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intracellular polyP of heterotrophic bacteria is essentially derived from exogenous Pi and carbon

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source (represented as COD).30 In the transition phase of intracellular polyP synthesis and

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degradation, a certain amount of Pi (11 mg-P/L) still remained in the supernatant, whereas COD

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was almost depleted (Figures 1b and S1). Therefore, a lack of available COD and not Pi likely

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accounted for further intracellular polyP accumulation in CPP. To directly test this idea and avoid 10 ACS Paragon Plus Environment

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growth dilution effects,12 filter-sterilized glucose was added to a final concentration of 10

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mg-COD/L (a value calculated based on Figure S1) per hour. Nevertheless, the polyP content of

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CPP was not further elevated and Pi release continued (data not shown). This result indicated that

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the inability of CPP to accumulate more polyP is not because of the depletion of COD but rather

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a result of another limiting factor or point of regulation in the system. Therefore, we confirmed

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that 127 mg-P/g-DW was the maximal polyP content that CPP can achieve in the present study.

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As far as TP content is concerned, the maximal cellular phosphorus level of CPP could reach 140

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mg-P/g-DW. This value surpassed almost all those achieved in E. coli strains engineered via

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various plasmid-borne PPK1 overexpression strategies (Table 1) except one derivative termed

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MV1184 (pBC29 and pEP02.2).7 However, because of metabolic burden effects resulting from

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sustaining two plasmids in one cell, the cell yield (expressed as maximal OD600) of this strain

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was severely limited (OD600 ≤ 0.20). Such a difference in biomass ultimately resulted in lower

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overall Pi removal for this E. coli derivative (8 mg-P/L) compared to CPP (19 mg-P/L).

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Therefore, our strategy is more advantageous because it achieved highly effective polyP

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production and substantial Pi removal simultaneously. In addition, the ratio of COD to removed

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phosphorus (COD : P, 350 : 19) determined from CPP was 18, which is only 60% of that

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generally required (30 or above) to achieve the high-level removal of phosphorus.31

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Indeed, cell yield is an important factor for enhanced biological Pi removal, which together

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with intracellular polyP content determine the overall amount of Pi that can be removed from the

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wastewater. In addition to MV1184 (pBC29 and pEP02.2), the remaining engineered E. coli 11 ACS Paragon Plus Environment


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strains showed cell yields that were more or less decreased compared with their wild type, which

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was attributed to the metabolic burden imposed by exogenous plasmids. Unexpectedly, however,

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the cell yields of CPP and CPR were virtually identical (OD600 of 0.52 versus 0.54, Figure 1c),

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and both significantly (t test, p < 0.05) surpassed that of CWT (OD600 of 0.40). Kuroda et al.

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demonstrated that polyP can promote protease Lon mediated ribosomal protein subunit

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degradation, thereby supplying the host cell with amino acids (additional endogenous nutrients)

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needed for vigorous growth under nutrient-poor conditions.32 In such cases, the true metabolic

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burden imposed by exogenous plasmids upon cell yield can only be clearly elicited via

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nutrient-rich cultivation, where intracellular polyP does not form and exogenous nutrients are in

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surplus. Cell yield determination performed with LB medium confirmed our speculation and

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demonstrated that the medium-copy plasmid, similar to the low-copy plasmid,12 caused only a

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slight but not significant decrease in cell yield, whereas the coexistence of a second high-copy

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plasmid resulted in a significant decline in cell yield (Figure 1c inset).

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Transcriptional Analysis of Plasmid-borne ppk1. As an indicator of leaky expression, the

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substantial over-production of RFP by CPR in SMW implied that the lac promoter was strong

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enough to drive the expression of plasmid-borne genes (Figure 2 inset). To demonstrate that

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polyP accumulation was a consequence of the elevated transcription of ppk1 mediated by the

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medium-copy plasmid and assess whether the high-copy plasmid would affect such

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plasmid-borne ppk1 transcription, the transcription profile of ppk1 in CPP and CPR was

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investigated. Because there is one copy of native ppk1 present in the chromosome of the host cell 12 ACS Paragon Plus Environment

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and it is generally transcribed in coordination with ppx (i.e., transcription levels equal to each

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other) (Figure S2a), to distinguish the expression of chromosomal ppk1 from that of

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plasmid-borne ppk1, native ppk1 expression levels in CPP and CPR were first evaluated

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indirectly via a ppx analysis. As shown in Figure S2b, virtually identical expression profiles of

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ppx depicted in CPP and CPR relative to CWT indicated that the native ppk1 expression levels

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stayed unaltered and no imbalance in transcription between endogenous ppk1 and ppx (i.e., ppx

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downregulation) contributed to polyP production. Thus, elevated ppk1 expression and polyP

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accumulation detected in the engineered strains were entirely from the exogenous medium-copy

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plasmid. qRT-PCR revealed that highly elevated ppk1 expression was achieved in both CPP and

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CPR via the medium-copy plasmid (Figure 2). In addition, at any given time point,

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plasmid-borne ppk1 transcription in CPR was significantly (t test, p < 0.05) lower than that in

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CPP. This result indicated that the high-copy plasmid significantly affected ppk1 transcription

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from the medium-copy plasmid, which could be attributed to the occupation of limited cellular

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transcription resources by the former. Furthermore, for either CPP or CPR, the host cell

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continuously downregulated plasmid-borne ppk1 expression as growth progressed until only

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approximately 30% of the original transcription level remained in the stationary phase (Figure 2).

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From this perspective (i.e., to sustain a certain ppk1 dosage), relative to low-copy plasmids,

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medium-copy plasmids are recommended.

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Diversion of in vivo ATP. As noted above, introducing a second high-copy plasmid into CPP

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significantly reduced its polyP content. The diversion of substantial amounts of in vivo ATP 13 ACS Paragon Plus Environment


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toward futile high-copy plasmid DNA replication and RFP synthesis likely accounts for this

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decline in intracellular polyP. To directly test this scenario, we measured ATP levels in each

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strain and compared the absolute ATP decreases in CPP and CPR. As cellular growth proceeded,

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CWT, which was not capable of forming polyP, maintained most of its ATP, whereas both CPP

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and CPR were shown to experience a very rapid decline in their in vivo ATP levels (Figure 3).

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Within the same OD600 range (OD600 0.20 to 0.40), nearly the same quantities of ATP were

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over-consumed by both strains compared with CWT (Figure 3), whereas the absolute polyP

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increase in CPR (12 mg-P/g-DW, corresponding to a time frame 2 to 5 h, Figures 1a and 1c) was

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only approximately 50% of that in CPP (23 mg-P/g-DW, corresponding to a time frame of 1 to 3

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h, Figures 1a and 1c). These results strongly suggested that CPR actively redirected a substantial

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proportion of such over-consumed ATP to sustain the high-copy plasmid relevant metabolism

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rather than polyP synthesis and therefore provided a poor polyP yield.

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PolyP Granules and Cell Morphology Display. As a routine test, we performed Albert

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staining to intuitively display the intracellular polyP granules in CPP and CPR cells. During the

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polyP synthesis stage, almost synchronous formation and augmentation of polyP granules in the

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CPP population could be easily visualized with an optical microscope (Figure 4a, dashed frame).

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Consistent with quantitative polyP assays, polyP granules observed in CPP at 15 h were

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significantly larger than those found in CPR (Figure 4a, solid-line frame). Remarkably, the

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cellular morphology of CPP even became hard to distinguish because of excess intracellular

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polyP accumulation. To surmount this obstacle, we applied confocal microscopy to further 14 ACS Paragon Plus Environment

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display the living cells stained with DAPI. As shown in Figure 4b, each CPP cell possessed a

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huge bright yellow DAPI-polyP fluorescent focus at each pole. Because these two major

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granules are close to each other and the diameter of both significantly surpasses that of the

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rod-shaped cell, a unique dumbbell-like bacterium was formed. In contrast, CPP retained its

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original rod shape because the size of its polyP granules is much smaller. Based on current

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observation and the indisputable fact that the intracellular storage space of any given bacterium

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is finite, it is reasonable to envisage that the excessive polyP accumulated in CPP exhausts its

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cellular storage space and pushes the product level within the crowded environment above the

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equilibrium concentration, triggering the reaction toward polyP degradation. Therefore, finite

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cellular storage space might be the bottleneck for genetically engineered polyP production and

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could explain why the polyP content of CPP cannot be elevated any further.

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Intracellular PolyP Synthesis Kinetics. Intracellular polyP assays conducted at the polyP

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synthesis stage revealed that CPP accumulated polyP with a dynamic different from that of CPR.

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To better understand this difference, we performed data fitting during this time frame and found

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that intracellular polyP synthesis by CPP followed a zero-order kinetic model (R2 = 0.9928),

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whereas the same process proceeding in CPR showed a better fit with first-order kinetics (R2 =

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0.9973) (Figure 5a). Apparently, the high-order kinetic behavior of CPR resulted from an

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increasing shortage of ATP as it progressed to stationary phase. For CPP, we interpret ATP

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regeneration as the rate-limiting step rather than saturation of intracellular PPK1’s enzymatic

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capacity leading to the zero-order kinetic behavior. This interpretation is supported by the 15 ACS Paragon Plus Environment


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observation that IPTG induction of CPP inoculum (which harbored more PPK1) did not change

299

its kinetic behavior or the initial polyP synthesis rate (Figure S3a). This rate, 10 mg-P/g-VSS/h,

300

although approximately one order of magnitude higher than that determined in Lampropedia

301

spp.,33 is only approximately 1/3 of that reported for Microlunatus phosphovoru 34 and that found

302

in PAOs.35 Consequently, to achieve a polyP content as high as that achieved by PAOs, CPP

303

requires a much longer time. Next, to test how CPP would respond to exogenous Pi, the kinetics

304

of initial polyP synthesis as a function of ambient Pi concentration were investigated. As shown

305

in Figure 5b, at all tested Pi concentrations, the initial rate did not vary, indicative of unanimous

306

zero order kinetic behavior. This result further demonstrated that polyP synthesis in CPP directly

307

depended on in vivo ATP flux through the polyP synthetic pathway rather than exogenous Pi.

308

However, given that one phosphoric acid radical of ATP is deprived by PPK1 for polyP synthesis,

309

regeneration of in vivo ATP necessitated the uptake of exogenous Pi. For this reason, when

310

exogenous Pi was insufficient, CPP would totally deplete exogenous Pi and even could

311

experience an abrupt cessation of polyP synthesis (Figures S3b and 3c) and cellular growth

312

(Figure S4). This characteristic of CPP is very attractive, and it raises the intriguing application

313

perspective that it can achieve thorough Pi removal from wastewater at COD: P ≥ 18.

314

Pi Removal and Enrichment with Bench-scale SBMBR. Increasing concerns over global

315

phosphorus resource depletion together with more stringent phosphorus discharge limits make

316

the recovery of phosphorus from wastewater sensible and attractive.31 However, economically

317

feasible recovery generally requires a liquid phase with phosphorus concentrations of > 50 mg/L, 16 ACS Paragon Plus Environment

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318

which is not suitable for municipal wastewater (which usually has phosphorus concentrations of

319

< 10 mg/L). Thus, the enrichment of phosphorus from such inherently diluted waste streams is

320

essential for the subsequent phosphorus recovery.36 Therefore, as the last part of this study, we

321

sought to leverage the strengths of CPP to remove and concentrate Pi from SMW. Consistent

322

with the results from shake-flask tests, the in-reactor Pi was completely depleted within 4 h

323

(Figure 6b). Thereafter, practically Pi-free water was consistently obtained through continuous

324

withdrawal of the reactor supernatant until 0.02 mg/L Pi was detected in the permeate stream at

325

16 h. During this period, the Pi from 82 L of SMW was fully taken up by CPP and mainly

326

converted to a concentrated form (i.e., intracellular polyP) (Figure S5b). After membrane

327

concentration, 9 L of enriched CPP cell suspension was obtained and then subjected to Pi release

328

(Figure 6c inset). At the end of the Pi release phase, a cell suspension with supernatant Pi

329

concentrations of up to 62 mg-P/L was formed (Figure 6c); namely, Pi was effectively enriched

330

up to 8-fold. As confirmed by recovery efficiency calculation, almost all the Pi was successfully

331

concentrated apart from the portion assimilated by CPP (Text S3). These results suggest that

332

full-scale Pi removal and enrichment using CPP is technologically feasible.

333

Yet full exploitation of the environmental application of CPP will only be achieved if we can

334

better understand its potential advantages and disadvantages. Compared with conventional EBPR

335

processes, its advantages are: (1) no requirement of anaerobic pretreatment (efficient Pi removal

336

could be achieved fully aerobically in a single phase); 2) a favorable rate of throughput;37 and (3)

337

no dependence upon volatile fatty acid (VFA) concentrations (polyP accumulation in CPP is not 17 ACS Paragon Plus Environment


338

VFA-dependent based on the bioavailability of different carbon sources, Text S4 and Table S3).

339

The major disadvantages relative to PAO organisms that can use endogenous carbon sources

340

[mainly polyhydroxyalkanoates (PHAs)] for biomass growth and polyP accumulation is that Pi

341

removal and polyP production in CPP depends mainly on exogenous organic substrates. Under

342

these circumstances, CPP may face considerable competition with other aerobic microorganisms

343

for bio-assimilable substrates in a realistic wastewater treatment process setting where organic

344

matter type and concentrations are highly variable. Thus, these dynamics and carbon

345

requirements would affect the performance of CPP. Our ongoing research program is

346

investigating the possibility of these limitations and mechanisms to overcome them. More

347

specifically, we wish to initiate a pilot-scale trial of real municipal wastewater treatment to assess

348

Pi removal performance and economic viability because a carbon source, such as crude glycerol,

349

may need to be added and leverage the high phosphorus processing capacity of CPP to remove

350

and “refine” Pi from some wastewaters that generally contain high concentrations of mixed

351

organic matters. Examples of such situations are abattoir wastewater, poultry wastewater, and

352

soybean protein wastewater, where Pi is difficult to strip by chemical precipitation and

353

coagulation. Lastly, the biosafety aspects of CPP need to be further assessed because C. freundii

354

is an enterobacterium and may contribute to the propagation of environmental antimicrobial

355

resistance.38,39 Thus, more potentially safe environmental bacteria, such as Acinetobacter

356

calcoaceticus and Pseudomonas putida, should be further investigated using our framework, in

357

addition to other cultivars, as long as they are Gram-negative, culturable under aerobic 18 ACS Paragon Plus Environment

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358


conditions, genetically accessible, and relatively harmless to human health.

359

Overall, our study demonstrates that practically optimum intracellular polyP production can

360

be easily achieved with C. freundii engineered based on a solo medium-copy plasmid strategy.

361

Moreover, this strategy takes into account cell yield at the same time because the medium-copy

362

plasmid would not impose appreciable metabolic burden upon the host cell, thanks to the

363

guidelines of metabolic engineering. More importantly, the engineered CPP constructed based on

364

this strategy could work for the first time in SMW, and exogenous Pi of low concentration could

365

be thoroughly removed and subsequently enriched, which raises the practical application

366

potential of this strategy. The strategy presented here is so simple and effective that it might be

367

extended to other environmental bacteria with different environment adaptabilities as long as

368

enhanced biological Pi removal and/or enrichment is desired in various wastewater treatment

369

processes.

370 371

ASSOCIATED CONTENT

372

Supporting Information

373

Additional materials and methods, Pi recovery efficiency calculation and experimental data

374

(Texts S1-S4, Tables S1-S2 and Figures S1-S5). This material is available free of charge via the

375

Internet at http://pubs.acs.org.

376 19 ACS Paragon Plus Environment


377

AUTHOR INFORMATION

378

Corresponding Author

379

*

380

Notes

381

The authors declare no competing financial interests.

Phone: +86 25 8968 0257; E-mail: [email protected]

382 383

ACKNOWLEDGMENTS

384

This work was supported by the National Special Program of Water Environment

385

(2017ZX07204).

386 387

REFERENCES

388

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389 390

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Brown, M. R.; Kornberg, A. The long and short of it–polyphosphate, PPK and bacterial survival. Trends Biochem. Sci. 2008, 33 (6), 284-290.

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4′-6-diamidino-2-phenylindole (DAPI). Environ. Sci. Technol. 2011, 45 (18), 7799-7803.

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N. T.; Krieger, A. G.; Smith, E. M.; Bender, R. A.; Bardwell, J. C. Polyphosphate is a

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Pavlov, E. High sensitivity, quantitative measurements of polyphosphate using a new

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sequence of the type species of the genus Citrobacter, Citrobacter freundii MTCC 1658.

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Kornberg, A. Role of inorganic polyphosphate in promoting ribosomal protein degradation

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Rabaey, K.; Meesschaert, B. Global phosphorus scarcity and full-scale P-recovery

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the application of low pH-inducible polyphosphate accumulation to the biological removal

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of phosphate from wastewaters. Environ. Sci. Technol. 2006, 40 (1), 296-301.

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495



496

Figure and Table Captions

497

Figure 1. Intracellular polyP content (a), supernatant Pi concentration (b), and optical density (c)

498

of CPP grown in SMW compared with CPR or CWT. The left segment of the time axes

499

represents the polyP synthesis stage (also called Pi uptake stage), whereas the right

500

segment represents the polyP degradation stage (also called Pi release stage). The inset

501

in Figure 1c indicates the maximal optical density of each strain cultured using LB

502

medium. The values of the bars with different letters on the top are significantly

503

different (t test, p < 0.05) from each other. Data are representative of three independent

504

experiments, and error bars correspond to the standard deviation (i.e., mean ± s.d., n =

505

3).

506

Figure 2. qRT-PCR analysis of plasmid-borne ppk1 expression in CPP and CPR sampled from

507

either LB medium or SMW; results are presented relative to the average expression

508

level of native ppk1 determined from CWT, set as 1. In the inoculum, ppk1 expression

509

levels determined from LB samples at 12 h also represent the initial (0 h) ppk1

510

expression levels of each strain in SMW. The inset is a photograph of concentrated

511

cells (harvested by centrifugation from 200 mL SMW and resuspended in 4 mL

512

deionized water) of each strain. RFP was significantly expressed and can be visualized

513

by the naked eye. The values of the bars with different letters on the top are

514

significantly different (t test, p < 0.05) from each other. Data are the mean ± s.d. (n =

515

3). 26 ACS Paragon Plus Environment

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Page 29 of 36

516 517


Figure 3. In vivo ATP levels of each strain grown in SMW as a function of OD600. Data are the mean ± s.d. (n = 3).

518

Figure 4. (a) Light microscopy images of Albert stained cells (malachite green and toluidine blue;

519

polyP granules appear purple-black and polyP free cells appear blue-green). Dashed

520

frame: gradually increasing intracellular polyP inclusions in CPP with time. Solid-line

521

frame: comparison of intracellular polyP inclusions in three strains sampled at 15 h.

522

Scale bars are 5 µm. (b) Corresponding to the light microscopy images in the solid-line

523

frame. Confocal laser scanning microscopy images of three strains as obtained via

524

different channels (DAPI-DNA: 403 nm laser, filter bandpass = 425-475 nm;

525

DAPI-polyP: 403 nm laser, filter bandpass = 552-617 nm; RFP: 543 nm laser, filter

526

bandpass = 552-617 nm; Overlay: combined images of three channels). Scale bars are

527

2 µm.

528

Figure 5. (a) Variation of intracellular polyP content in CPR and CPP with time during the Pi

529

uptake stage in SMW. (b) Relationship between intracellular polyP synthesis rate of

530

CPP and exogenous Pi concentration in SMW. See also Figure S3. Data are the mean ±

531

s.d. (n = 3).

532

Figure 6. (a) Photograph of the MBR. (b) Variation of Pi concentration and in-reactor cell

533

density with operation time. The arrow indicates the time period of feeding and

534

withdrawal. Pi concentrations before 4 h were determined from the supernatant of the

535

in-reactor cell suspension. Pi concentrations after 4 h were determined from the 27 ACS Paragon Plus Environment


536

effluent. (c) Variation of supernatant Pi concentration of enriched sludge with time.

537

The inset shows the sludge withdrawal vessel for Pi concentration. Data shown are the

538

averages of two measurements from a single cycle and are representative of three

539

cycles conducted similarly. Error bars are the standard deviation of the two

540

measurements.

541 542 543

Table 1. Comparison of TP content and Pi removal of different strains engineered based on a solo medium-copy plasmid strategy versus three other strategies.

544


Page 30 of 36

Page 31 of 36


545

546 547

Figure 1

548

549 550

Figure 2

551 552



553 554

Figure 3

555


Page 32 of 36

Page 33 of 36


556 557

Figure 4

558



559 560

Figure 5

561

562 563

Figure 6


Page 34 of 36

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564

Table 1

Host cell E. coli MV1184 C. freundii ATCC 8090 E. coli MV1184 C. freundii ATCC 8090 E. coli BL21(DE3) E. coli MV1184 E. coli DH10B

TP content

Pi removal

(mg-P/g-DW)

(mg-P/L)

OD600

COD

Plasmid

(mg/L)

(copy number)

b

160

8

0.20

2400

140

19

0.52

350

67a

12

0.60

2400b

54 a

7

0.54

pBC29c (500-700)d pEP02.2 (10-12)d pBBR1MCS2c (15-20)d pEP03c (500-700)d pBBR1MCS2c (15-20)d

350

simple19T (500-700)d b

pML001c (15-20)d

38

9

0.80

3000

26a

6

0.78

2400b

pBC29c (500-700)d

25

/

2.50

3600b

pF12c (1-2)d

d

pLysS (10-12)

Strategy category dual plasmid (high & medium)

Ref

7

solo

this

medium-copy

study

solo high-copy

7

dual plasmid

this

(high & medium)

study

dual plasmid (dual medium) solo high-copy solo low-copy

13

7

12

565 566

a

567

equals 0.3 g-DW/L.12

Data were calculated based on the values of Pi removal and OD600 using the equation TP = (Pi removal × 1L)/(OD600 × 0.3× 1L), where OD600 ~ 1



568

b

569

the original references.

570

c

Plasmids that were used to express PPK1.

571

d

Copy number of each plasmid, which was estimated based on Qiagen literature resource: Origin of replication and copy number of various plasmids and

572

cosmids (https://www.qiagen.com).

Values are the starting COD in the medium rather than that consumed by the bacteria because the absolute consumption values were not available from


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