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Article

Effects of Escherichia coli Alkaline Phosphatase PhoA on the Mineralization of Dissolved Organic Phosphorus

by
Yanwen Zhou
1,5,†,
Tingxi Zhang
2,3,*,†,
Shengyan Jin
3,
Siyu Chen
4 and
Yinlong Zhang
1,*
1
Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Longpan Road 159, Nanjing 210037, China
2
Jiangsu Center for Collaborative Innovation in Geographical Information Resource Development and Application, School of Geography Science, Nanjing Normal University, Nanjing 210023, China
3
School of Environment, Nanjing Normal University, Nanjing 210023, China
4
School of Life Sciences, Nanjing Normal University, Nanjing 210023, China
5
Ecological and Environmental Protection Research Institute of Nanjing, Huju Road 175, Nanjing 210019, China
*
Authors to whom correspondence should be addressed.
†
Tingxi Zhang and Yanwen Zhou and are co-first authors of the article.
Water 2021, 13(23), 3315; https://doi.org/10.3390/w13233315
Submission received: 27 September 2021 / Revised: 9 November 2021 / Accepted: 15 November 2021 / Published: 23 November 2021
(This article belongs to the Special Issue Nutrient Biogeochemical Cycles in Eutrophic Inland Waters and Eutrophication Control)
Browse Figures
Versions Notes

Abstract

:
Alkaline phosphatases, which play the key role in the mineralization of organic phosphorus, have been grouped into three distinct families, PhoA, PhoX, and PhoD. PhoA is still an important component of the Pho regulon for many microbes although its distribution is not as wide as that of PhoX and PhoD. However, several questions remain unclear about the effect of PhoA mineralization of dissolved organic phosphorus. In this study, the role of Escherichia coli alkaline phosphatase PhoA (hereinafter referred to as PhoA) in the mineralization of different organic phosphorus including phosphate monoesters, phosphate diesters, and phytic acids was investigated. The influence of the reaction time, organic phosphorus concentration, and L-amino acid on PhoA mineralization was examined. The results show that PhoA specifically hydrolyzes phosphate monoesters except for phytic acid and the optimal reaction time is around 12 h. The PhoA mineralization rate of glucose 6-phosphate disodium (G6P), 5′-adenosine monophosphate (AMP), and sodium glycerophosphate (BGP) significantly decreased by 38.01%, 55.31%, and 57.08%, respectively (p < 0.01), while the concentration of organic phosphorus increased from 0.50 to 5.00 mg/L. Overall, L-amino acids inhibited PhoA mineralization in a concentration-independent manner. The inhibitory effect of neutral amino acids serine (L-Ser) and tyrosine (L-Tyr) was significantly higher than that of basic amino acids arginine (L-Arg), lysine (L-Lys), and histidine (L-His). All the five amino acids can inhibit PhoA mineralization of AMP, with the highest inhibition rate observed for L-Tyr (23.77%), the lowest—for L-Arg (1.54%). Compared with other L-amino acids, L-Tyr has the highest G6P and BGP mineralization inhibition rate, with the average inhibition rates of 12.89% and 11.65%, respectively. This study provides meaningful information to better understand PhoA mineralization.

1. Introduction

The growth of phytoplankton is limited by nutrients such as nitrogen and phosphorus [1]. The role of phosphorus limitation in the primary productivity of eutrophic lakes has received extensive attention since 1970s [2,3,4]. The capabilities to utilize different forms of phosphorus are varied among phytoplankton. When the most readily available dissolved inorganic phosphorus (Pi) is in short supply, microorganisms satisfy the demand of phosphorus for growth and reproduction by releasing alkaline phosphatases (APases) to mineralize organic phosphorus [5]. To date, at least three distinct prokaryotic alkaline phosphatases (PhoA, PhoX, and PhoD) have been identified based on their sequence similarities and substrate specificity [6]. PhoA, initially discovered to be expressed and synthesized by Escherichia coli [7], was the first alkaline phosphatase to be characterized [8]. While PhoX and PhoD have recently been shown to be more abundant than PhoA in marine and terrestrial ecosystems [8,9,10], PhoA is still an important component of the Pho regulon for many microbes. Subcellular localizations of APases showed that Bacteroidetes, an important source for APases in plant/soil ecosystems [11], were overrepresented in PhoA [6]. E. coli or purified PhoA played an important role in Ca phosphate precipitation [12]. PhoX was found to be more widely distributed in cyanobacteria than PhoA and PhoD [9,13,14]. However, confocal microscopy studies on aggregates of microalgae from turbid and phosphate-limited Lake Markermeer revealed that microbes, rather than algae, were responsible for the extracellular alkaline phosphatase activity [15]. This indicates that microbes play a vital role in P cycling in lakes [16] and it can be inferred that the Pi required for algae growth in freshwater ecosystems may partly come from the mineralization of organic phosphorus by APases PhoA or PhoD produced by microbes.
PhoA can be secreted to the extracellular or periplasmic space through the Sec protein channel in the bacterial membrane [17]. The E. coli phoA gene has been widely used in the study of protein localization and membrane topology because its product is active only when outside the plasma membrane [18]. Currently, studies on alkaline phosphatases are mainly focused on gene sequences, protein spatial structures, optimal pH values, and the influence of metal ions on gene expression and enzyme activity [19,20,21,22]. Generally, microorganisms contain multiple alkaline phosphatase genes, while the mechanism and impact of each alkaline phosphatase remain unclear.
The cycling of nutrients (carbon, nitrogen, phosphorus, etc.) in lakes is closely related to organic matters. Amino acids, widely present in lake water and sediments [23,24,25], are the main form of organic nitrogen [23] and play the essential role in the nitrogen cycle. The changes in concentration and composition of amino acids can be used to evaluate biodegradability of dissolved organic matter (DOM) [26,27,28], which reflects the dynamic changes of DOM in lake water. The increase in the concentration of amino acids can increase the risk of lake eutrophication to a certain extent [25]. It can be hypothesized that in a given environment where Pi is limited and a certain type of amino acids prevails, APases might be more efficient (or not) in mineralizing organic phosphorus. However, the research on the effect of amino acids on the mineralization of organic phosphorus is scarce [29].
In this study, the effects of E. coli alkaline phosphatase PhoA (hereafter PhoA) on the mineralization of dissolved organic phosphorus were investigated, and the impact of PhoA on the mineralization of dissolved organic phosphorus in the presence of amino acids was examined. The results are helpful in exploring the mechanism of mineralization of organic phosphorus in microorganisms.

2. Materials and Methods

2.1. Experimental Materials

2.1.1. Alkaline Phosphatase PhoA

Purified E. coli PhoA (lot No. DB13BD0318) was purchased from Shanghai Biological Company, with the optimal temperature of 25 °C and the optimal pH of 8.00.

2.1.2. Organic Phosphorus

Sodium phytate (IP6-Na; Sigma-Aldrich), sodium glycerophosphate (BGP; Sigma-Aldrich), glucose 6-phosphate disodium (G6P; MP Biomedicals, LLC), and 5′ adenosine monophosphate (AMP; Sigma-Aldrich) were chosen as representative phosphate monoesters. Ribonucleic acid from baker’s yeast (RNA; Sigma-Aldrich) was selected as the representative of phosphate diesters. The properties of each organic phosphorus are shown in Table 1.

2.1.3. Amino Acids

Soluble amino acids including histidine (L-His, CAS: 71-00-1, lot No. E315BA0034; Sigma-Aldrich), arginine (L-Arg, CAS: 74-79-3, lot No. E108BA0028; Sigma-Aldrich), lysine (L-Lys, CAS: 56-87-1, lot No. E115BA0029; Sigma-Aldrich), serine (L-Ser, CAS: 56-45-1, lot No. E125BA0116; Sigma-Aldrich), and tyrosine (L-Tyr, CAS: 60-18-4, lot No. E110BA0009; Sigma-Aldrich), were used to explore the effect of amino acids on the mineralization of phosphate monoesters.

2.2. Experimental Methods

2.2.1. Specificity of PhoA to Hydrolyze Phosphate Monoesters

G6P, RNA, and IP6-Na, which are representative monoester, diester, and special monoester, respectively, were used to study the hydrolysis specificity of PhoA. In the experimental group, the solution with G6P, RNA, and IP6-Na at the initial concentration (calculated on P) of 2.00 mg/L and PhoA at the concentration of 0.44 U/mL was prepared, and pH was adjusted to 8.00. Two control groups were set up. One control group contained G6P, RNA, and IP6-Na at the same initial concentrations as those in the experimental group but without PhoA, whereas the other control group contained only PhoA and high-purity water. The experiments were conducted in triplicates for each group. Both the experimental group and the control groups oscillated in the dark at the constant temperature of 25 °C for 180 min. The molybdenum antimony anti-spectrophotometry method [30] was used to determine the amount of orthophosphate produced by organic phosphorus mineralization.

2.2.2. Reaction Time of PhoA to Completely Mineralize Phosphate Monoesters

Three phosphate monoesters, G6P, BGP, and AMP, were selected as organic phosphorus substrates to study the effect of reaction time on mineralization. The initial concentrations of total phosphorus in the experimental group were set as 2.00 mg/L and 5.00 mg/L, and pH was adjusted to 8.00; then, 0.44 U/mL PhoA was added to the solution. The setup of the control groups was the same as described in Section 2.2.1. Both the experimental and the control groups oscillated in the dark at the constant temperature of 25 °C. Samples were collected after 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 20 h, respectively, to determine the amount of orthophosphate [30]. The reaction times of PhoA to completely mineralize phosphate monoesters were thus obtained. The experiments were conducted in triplicates for each group.

2.2.3. Influence of Organic Phosphorus Concentration on PhoA Mineralization

Phosphate monoesters, G6P, BGP, and AMP, were selected as organic phosphorus substrates to study the effect of different organic phosphorus concentrations on PhoA mineralization. In the experimental group, solutions of each phosphate monoester at concentrations of 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 4.00, and 5.00 mg/L were prepared, and pH was adjusted to 8.00; then, PhoA at the concentration of 0.44 U/mL was added to each solution. The setting of the control groups was the same as described in Section 2.2.1. Both the experimental and the control groups oscillated in the dark at the constant temperature of 25 °C for 12 h. The molybdenum antimony anti-spectrophotometry method [30] was used for orthophosphate determination to reveal the effect of organic phosphorus concentration on PhoA mineralization. The experiments were conducted in triplicates for each group.

2.2.4. Effects of Soluble Amino Acids on PhoA Mineralization

Three phosphate monoesters, G6P, BGP, and AMP, were chosen as organic phosphorus substrates to study the influence of L-His, L-Arg, L-Lys, L-Ser, and L-Tyr on PhoA mineralization of organic phosphorus. Amino acid solutions with concentrations of 1.60, 8.00, 40.00, 200.00, and 1000.00 nmol/L were prepared, and pH was set to 8.00. Solutions with the concentration of 4.00 mg/L were prepared for G6P, BGP, and AMP, and pH was set to 8.00 as well. In the experimental group, the organic phosphorus solution and the amino acid solution were mixed at the ratio of 1:1 (v/v); then, PhoA with the concentration of 0.44 U/mL was added. In the control group, the organic phosphorus solution and high-purity water were mixed at the ratio of 1:1 (v/v); other conditions were the same as those of the experimental group. Both the experimental and the control groups oscillated in the dark at the constant temperature of 25 °C for 12 h. The experiments were conducted in triplicates for each group. The molybdenum antimony anti-spectrophotometry method [30] was used to determine orthophosphate to find out the impact of different concentrations of soluble amino acids on PhoA mineralization of three organophosphate monoesters.

2.3. Data Processing and Analysis

Origin 9.0 was used to create graphs to illustrate the specificity of PhoA to hydrolyze phosphate monoesters and the influence of the reaction time, organic phosphorus concentration, and soluble amino acid on PhoA mineralization. The effect of organic phosphorus concentration on PhoA mineralization was analyzed with a paired t-test using SPSS 19.0. Pearson correlation analysis was performed to analyze the effect of different concentrations of soluble amino acids on PhoA mineralization.
Enzymatic hydrolysis rate = (the amount of orthophosphate in the experimental group − the amount of orthophosphate in the control group)/the amount of orthophosphate in the experimental group—100%.
Inhibition rate = (the amount of orthophosphate in the control group − the amount of orthophosphate in the experimental group)/the amount of orthophosphate in the experimental group—100%.

3. Results and Discussion

3.1. The Specificity of PhoA to Hydrolyze Phosphate Monoesters

The hydrolysis rate of G6P by PhoA reached 56.36% within 3 h, while the hydrolysis rates of RNA and IP6-Na were only 0.36% and 0.16%, respectively (Figure 1).
The results showed that PhoA can hydrolyze neither phytic acids (monoesters) nor RNA (diesters). Being the first and well-studied alkaline phosphatase, E. coli PhoA is highly specific for the monophosphate ester bond but does not show specificity for the organic part and thus is able to hydrolyze a wide range of compounds [19]. PhoD and PhoX are monomeric enzymes that can hydrolyze both phosphomonoesters and phosphodiesters [18,31,32,33], which indicates that the well-documented monoesterase should be referred to only as PhoA [34]. Phytic acid or phytate (salt form), also known as inositol hexakisphosphate (IP6), is the main storage form of P in many soils [35], plant tissues [36], and lake sediments [37]. Phytic acid is a phosphate monoester and can change in binding sites between one and six phosphate groups, resulting in the formation of isomeric forms [36]. Under environmental conditions with pH between 6 and 9, five phosphate groups are positioned in the symmetry plane, and the remaining one phosphate group is in the vertical axial position [36,38], which is relatively stable. In addition, phytic acid is a highly negatively charged substrate, and the corresponding enzymes must be able to adapt to its characteristics in order to bind and react. That is, the binding site needs to be positively charged under acidic conditions to hydrolyze phytic acid molecules. Our results showed that PhoA could not mineralize RNA and IP6-Na in the organic phosphorus solution with pH 8.00. As a monoesterase, PhoA cannot mineralize phytic acid, a kind of phosphate monoester, which further reflects the highly negatively charged characteristics of phytic acid.

3.2. The Reaction Time of PhoA to Completely Mineralize Phosphate Monoesters

With excess enzymes, when the initial concentration of total phosphorus was 2.00 mg/L and 5.00 mg/L, the enzymatic hydrolysis rate of each phosphate monoester showed a similar trend over time. With the extension of reaction time, the enzymatic hydrolysis rate first increased and then tended to be stable (Figure 2). When the initial phosphorus concentration was 2.00 mg/L, G6P and AMP reached the maximal enzymatic hydrolysis rate at 12 h, while BGP reached the maximal enzymatic hydrolysis rate at 6 h. When the initial phosphorus concentration was 5.00 mg/L, all three phosphate monoesters reached their maximal enzymatic hydrolysis rates at 12 h. In addition, it is clear that the maximal enzymatic hydrolysis rate of phosphate monoester at a high initial phosphorus concentration (48.47%) was lower than that at a low initial phosphorus concentration (85.80%).

3.3. Effects of Organic Phosphorus Concentration on PhoA Mineralization

With excess enzymes, the enzymatic hydrolysis rate of PhoA to mineralize the three phosphate monoesters decreased with the increase in the phosphate monoester concentration. The enzymatic hydrolysis rate initially decreased rapidly and then slowed down. The enzymatic hydrolysis rate significantly decreased (p < 0.05) at the organic phosphorus concentration of 0.50–3.00 mg/L, whereas no significant difference was observed at the organic phosphorus concentration of 3.00–5.00 mg/L. This indicated that a high concentration of phosphate monoester (>3.00 mg/L) could inhibit the mineralization of PhoA, and the inhibitory trends of these three phosphate monoesters were highly similar.
As shown in Figure 3, with excess enzymes, when the concentration of organic phosphorus initially increased from 0.50 mg/L to 1.50 mg/L, the PhoA mineralization rate of G6P, AMP, and BGP decreased rapidly from 88.17% to 62.44%, from 88.50% to 54.39%, and from 93.17% to 59.39%, respectively. As the concentration of organic phosphorus continued to increase, the mineralization rate of PhoA decreased slowly, and then slowed down until stabilization. As shown in Figure 3, when the concentration of organic phosphorus increased from 4.00 mg/L to 5.00 mg/L, the mineralization rate of PhoA decreased slowly from 58.00% to 54.64% for G6P, from 42.90% to 38.63% for AMP, and from 41.96% to 39.98% for BGP. E. coli PhoA is regulated by the external phosphate concentration and is induced several hundred-fold when phosphate is in low supply [19]. Previous studies showed that the activity of alkaline phosphatases in algal cells with G6P or PNPP as the sole phosphorus source were 75% and 70% of those in algal cells under phosphorus starvation [39], indicating an inhibitory effect of organic phosphorus on enzymatic activities. Similar results were obtained in studies of Li [40] and Ericab [41]. In addition, Lu et al. investigated the factors affecting alkaline phosphatases in various areas of Lake Taihu and found that the activity of alkaline phosphatases was inhibited when the concentration of orthophosphate in the water body was high, resulting in a decrease in the enzymatic hydrolysis rate of organic phosphorus [42]. The results obtained in this study showed that high concentrations of organic phosphorus inhibited the mineralization efficiency of PhoA, which further confirmed this conclusion. Different types of organic phosphorus showed different inhibitory effects on PhoA mineralization. For example, when the concentration of organic phosphorus was 5.00 mg/L, the PhoA mineralization rate of GP, BGP, and AMP was 54.65%, 39.98%, and 38.65%, respectively, indicating the organic phosphorus sources have an effect on PhoA mineralization. In addition, APase PhoA is predominantly cytoplasmic, periplasmic, extracellular [6]. It was shown that PhoA localization influenced the pattern of Ca phosphate nucleation and growth [12]. To fully understand their specific relationship, further studies in the presence of different strains of E. coli PhoA as well as purified PhoA are needed.

3.4. Effects of Different Soluble Amino Acids on PhoA Mineralization of Organic Phosphorus

The results showed that each L-amino acid inhibited PhoA mineralization in a concentration-independent manner (Figure 4, Table 2). L-Tyr and L-Lys inhibited PhoA mineralization of three phosphate monoesters (G6P, AMP, and BGP) to a different degree. The strongest inhibitory effect of L-Tyr on PhoA mineralization was observed in AMP (mean inhibition rate = 23.77%), followed by G6P (12.89%) and BGP (11.65%), whereas the strongest inhibitory effect of L-Lys on PhoA mineralization was in G6P (7.56%), followed by AMP (4.54%) and BGP (2.14%). L-His significantly inhibited PhoA mineralization of AMP (20.27%) but showed no inhibitory effect in BGP and G6P. L-Arg had an inhibitory effect on PhoA mineralization of G6P (9.50%) and BGP (2.93%). L-Ser was able to inhibit the PhoA mineralization of G6P (5.10%) and AMP (8.90%).
The inhibition of L-amino acid on APases has been reported in mammalian APases. L-phenylalanine and L-leucine strongly inhibit intestinal, placental, and germ cell ALPs (tissue-specific ALPs) [29]. Fernandes [29] pointed out that, unlike mammalian APases, bacterial APases are not stereospecifically inhibited by L-amino acids through a noncompetitive mechanism. In the study of Saccharomyces cerevisiae APases coded for by the PHO8 and PHO13 genes, the activation of L-amino acids (L-homoarginine, L-leucine, and L-phenylalanine) on APases was observed [29].
Under the experimental conditions with pH 8.00, L-amino acids dissociated negatively charged amino acid carboxyl groups. Carboxyl groups reversibly bound to the positively charged group near the active site of the enzyme, which prevented substrates from binding to the active center of the enzyme. As a result, the enzyme activity was inhibited [43]. In addition, due to the combination of amino acids, the molecular conformation of the enzyme changed, resulting in the change of the catalytic function of alkaline phosphatases [23]. Therefore, the L-amino acid had an inhibitory effect on PhoA mineralization of phosphate monoesters. Meanwhile, different amino acids showed different degrees of inhibition (Figure 4), which was related to the structure and property of amino acids. Different amino acids exhibit differences in polarity, acidity, and basicity, the side chain group. The molecular conformation formed by binding to alkaline phosphatases is also different. All these aspects affect mineralization of organic phosphorus by alkaline phosphatases [18]. Among the five amino acids examined in this study, L-Ser and L-Tyr are neutral amino acids, and the other three are basic amino acids. This indicated that for different phosphate monoesters, the inhibitory effect of neutral amino acids on PhoA mineralization of organic phosphorus was higher than that of basic amino acids.

4. Conclusions

The main findings in this study provide additional information to our understanding of the mechanism of PhoA mineralization. E. coli PhoA is indeed responsible for mineralization of phosphate monoesters rather than of phosphate diesters or phytic acids. L-amino acid inhibited PhoA mineralization of phosphate monoesters in a concentration-independent manner. The inhibitory effect of neutral amino acids (L-Ser and L-Tyr) on PhoA mineralization was higher than that of basic amino acids (L-Arg, L-Lys, and L-His). L-Tyr had the highest rate of inhibition of the PhoA mineralization of G6P, BGP, and AMP. Further studies should be carried out in the presence of different strains of E. coli as well as purified PhoA.

Author Contributions

Conceptualization, T.Z. and Y.Z. (Yinlong Zhang); methodology, T.Z. and S.J.; formal analysis, S.J. and Y.Z. (Yanwen Zhou); investigation, S.J. and Y.Z. (Yanwen Zhou); writing—original draft preparation, Y.Z. (Yanwen Zhou), S.J., T.Z., and S.C.; writing—review and editing, T.Z.; supervision, T.Z. and Y.Z. (Yinlong Zhang); funding acquisition, T.Z. and Y.Z. (Yinlong Zhang). All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Natural Science Foundation of China (41303058, 41877336, 31971561, 31370217) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data, models, and code generated or used during the study appear in the submitted article.

Conflicts of Interest

The authors declare no conflict of interest.

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Water 13 03315 g001 550
Figure 1. Mineralization of different organic phosphorus substrates (G6P, RNA, and IP6-Na) by PhoA.
Figure 1. Mineralization of different organic phosphorus substrates (G6P, RNA, and IP6-Na) by PhoA.
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Figure 2. The reaction time of PhoA to mineralize organic phosphorus at the initial concentrations of 2.00 mg/L and 5.00 mg/L. Lines in the figures refer to the fitting curves of different reactive time.
Figure 2. The reaction time of PhoA to mineralize organic phosphorus at the initial concentrations of 2.00 mg/L and 5.00 mg/L. Lines in the figures refer to the fitting curves of different reactive time.
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Figure 3. Effects of organic phosphorus concentration on PhoA mineralization.
Figure 3. Effects of organic phosphorus concentration on PhoA mineralization.
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Figure 4. Effects of amino acids L-Tyr, L-His, L-Arg, L-Lys, and L-Ser on PhoA mineralization of organic phosphorus.
Figure 4. Effects of amino acids L-Tyr, L-His, L-Arg, L-Lys, and L-Ser on PhoA mineralization of organic phosphorus.
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Table
Table 1. The properties of organic phosphorus used in this study.
Table 1. The properties of organic phosphorus used in this study.
NameEster BondSolubilityMolecular WeightpH
G6PMonoesterSoluble304.107.8
RNADiesterSoluble20,000–30,0007.2
IP6-NaMonoesterSoluble923.829.5–11.5
BGPMonoesterSoluble306.117.6
AMPMonoesterSoluble365.243.5
Table
Table 2. Pearson correlation coefficient of the concentrations of amino acids and the inhibition rates.
Table 2. Pearson correlation coefficient of the concentrations of amino acids and the inhibition rates.
G6PAMPBGP
L-Tyr0.6420.3250.707
L-His0.3840.014−0.554
L-Lys−0.653−0.508−0.685
L-Arg−0.715−0.323−0.743
L-Ser0.2820.1570.426
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Zhou, Y.; Zhang, T.; Jin, S.; Chen, S.; Zhang, Y. Effects of Escherichia coli Alkaline Phosphatase PhoA on the Mineralization of Dissolved Organic Phosphorus. Water 2021, 13, 3315. https://doi.org/10.3390/w13233315

AMA Style

Zhou Y, Zhang T, Jin S, Chen S, Zhang Y. Effects of Escherichia coli Alkaline Phosphatase PhoA on the Mineralization of Dissolved Organic Phosphorus. Water. 2021; 13(23):3315. https://doi.org/10.3390/w13233315

Chicago/Turabian Style

Zhou, Yanwen, Tingxi Zhang, Shengyan Jin, Siyu Chen, and Yinlong Zhang. 2021. "Effects of Escherichia coli Alkaline Phosphatase PhoA on the Mineralization of Dissolved Organic Phosphorus" Water 13, no. 23: 3315. https://doi.org/10.3390/w13233315

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