Phosphorus Cycling in Eutrophic Lake Sediments: A Research Study

Journal of Environmental Management 320 (2022) 115906
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Journal of Environmental Management
journal homepage: www.elsevier.com/locate/jenvman
Research article
Spatiotemporal distributions and relationships of phosphorus content,
phosphomonoesterase activity, and bacterial phosphomonoesterase genes
in sediments from a eutrophic brackish water lake in Chile
Marco Campos a, b, Jacquelinne J. Acuña a, b, Joaquin I. Rilling a, b, Susett González–González a, b,
Fernando Peña‒Cortés c, Deb P. Jaisi d, Anthony Hollenback d, Andrew Ogram e, Junhong Bai f,
Ling Zhang f, Rong Xiao g, Milko A. Jorquera a, b, *
a
Laboratorio de Ecología Microbiana Aplicada (EMALAB), Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Temuco, Chile
Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco, Chile
c
Laboratorio de Planificación Territorial, Escuela de Ciencias Ambientales, Facultad de Recursos Naturales, Universidad Católica de Temuco, Temuco, Chile
d
Department of Plant and Soil Sciences, University of Delaware, Newark, USA
e
Soil and Water Science Department, University of Florida, Gainesville, FL, USA
f
School of Environment, Beijing Normal University, Beijing, China
g
College of Environment and Safety Engineering, Fuzhou University, Fuzhou, 350108, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bacterial community
Eutrophication
Lake sediments
Phosphomonoesterase
Phosphorus cycling
Phosphorus (P) cycling by microbial activity is highly relevant in the eutrophication of lakes. In this context, the
contents of organic (Po) and inorganic (Pi) phosphorus, the activity of acid (ACP) and alkaline (ALP) phospho­
monoesterase (Pase), and the abundances of bacterial Pase genes (phoD, phoC, and phoX) were studied in sedi­
ments from Budi Lake, a eutrophic coastal brackish water lake in Chile. Our results showed spatiotemporal
variations in P fractions, Pase activities, and Pase gene abundances. In general, our results showed higher
contents of Pi (110–144 mg kg− 1), Po (512–576 mg kg− 1), and total P (647–721 mg kg− 1) in sediments from the
more anthropogenized sampling sites in summer compared with those values of Pi (86–127 mg kg− 1), Po
(363–491 mg kg− 1) and total P (449–618 mg kg− 1) in less anthropogenized sampling sites in winter. In
concordance, sediments showed higher Pase activities (μg nitrophenyl phosphate g− 1 h− 1) in sediments from the
more anthropogenized sampling sites (9.7–22.7 for ACP and 5.9 to 9.6 for ALP) compared with those observed in
less anthropogenized sampling sites in winter (4.2–12.9 for ACP and 0.3 to 6.7 for ALP). Higher abundances
(gene copy g− 1 sediment) of phoC (8.5–19 × 108), phoD (9.2–47 × 106), and phoX (8.5–26 × 106) genes were also
found in sediments from the more anthropogenized sampling sites in summer compared with those values of
phoC (0.1–1.1 × 108), phoD (1.4–2.4 × 106) and phoX (0.7–1.2 × 106) genes in the less anthropogenized sites in
winter. Our results also showed a positive correlation between P contents, Pase activities, and abundances of
bacterial Pase genes, independent of seasonality. The present study provided information on the microbial ac­
tivity involved in P cycling in sediments of Budi Lake, which may be used in further research as indicators for the
monitoring of eutrophication of lakes.
1. Introduction
2007; Sinha et al., 2017; Cao et al., 2018). Under this scenario, it is
highly relevant to investigate the fates and ecological risks involved in
the contamination and eutrophication of lakes, particularly how mi­
croorganisms may increase the bioavailability of nutrients such as
phosphorus (P) and nitrogen (N), favoring the eutrophication process.
Therefore, recent studies have determined that more attention should be
In recent decades, as a result of increased anthropogenic activities (e.
g., agriculture and aquaculture), the contamination and eutrophication
of lakes by pollutant and nutrient inputs have adversely impacted the
ecosystem services (ES) provided by these water bodies (Søndergaard,
* Corresponding author. Laboratorio de Ecología Microbiana Aplicada (EMALAB), Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La
Frontera, Ave. Francisco Salazar 01145, Temuco, Chile.
E-mail address: [email protected] (M.A. Jorquera).
https://doi.org/10.1016/j.jenvman.2022.115906
Received 17 February 2022; Received in revised form 21 July 2022; Accepted 28 July 2022
Available online 12 August 2022
0301-4797/© 2022 Elsevier Ltd. All rights reserved.
M. Campos et al.
Journal of Environmental Management 320 (2022) 115906
given to monitoring some microbial groups as indicators of eutrophi­
cation in lake sediments (Kuang et al., 2022) and developing normative
management policies to control the pollution of lakes (Zhang et al.,
2022).
In lake sediments, P can be present as diverse inorganic (Pi) and
organic (Po) forms or complexed with metal ions and fulvic and humic
fractions into the organic matter (OM) of sediments (Tiessen et al., 1984;
Goedkoop and Pettersson, 2000). Spatial and temporal changes in the
biogeochemistry of lake sediments can occur, where less labile P forms,
such as phosphomonoesters (PMEs) and phosphodiesters (PDEs), can be
mobilized to more labile ones (inorganic phosphates) by microbial
enzymatic activity (Goedkoop and Pettersson, 2000; Torres et al., 2014).
In this context, the Po pool may represent more than 70% of the total P
(TP) in lake sediment (Lü et al., 2016; Fraser et al., 2017; Ni et al.,
2019a), with bacterial phosphomonoesterases (Pases) under their
alkaline (ALP; EC 3.1.3.1) and acidic (ACP; EC 3.1.3.2) isotypes, critical
enzymes in hydrolyzing PDEs and PMEs from dissolved Po (DOP) into
readily labile dissolved Pi forms (DIP) (Worsfold et al., 2008; Zhou et al.,
2011; Ma et al., 2019). The gene regulating the expression of these
enzymes is sensitive to DIP concentration, and the prevalence of the Pase
isotype depends on the environmental pH (Torres et al., 2017; Ma et al.,
2019). In freshwater ecosystems, the occurrence of ALP (phoD or phoX)and ACP (phoC)-encoding genes in bacterial communities has been re­
ported over the past years (Sebastian and Ammerman, 2009; Dai et al.,
2018; Campos et al., 2021), and studies have shown that the activities
and expression of these bacterial Pases are frequently related to higher
concentrations of Po fractions (Fraser et al., 2017). Nevertheless, the
spatiotemporal distribution of P forms and their associated microor­
ganisms are still unknown in most lakes worldwide, particularly in Chile
(Pandey and Yadav, 2017; Zhang et al., 2019).
Budi Lake is considered a unique coastal brackish water lake in South
America that provides a wide diversity of ESs for the surrounding
Chilean population (Bertrán et al., 2010; Peña-Cortés et al., 2020).
However, during the last few decades, intensive tourism, agriculture,
and forestry activities in the catchment have increased the sediment and
soil‒P loads into the lake, which has accelerated its eutrophication
(Stuardo et al., 1989; Hauenstein et al., 1999; Valdovinos et al., 2005).
In this context, a recent study described the noticeable activity of
Fig. 1. Schematic distribution of sampling sites in Budi Lake, which were coded as Puaucho (Pu), Puerto Dominguez (PD), Comue Stream (EsC), Temo (Te), and
Temo Stream (EsT). Asterisks (*) denote the more anthropogenized sampling sites of this study.
2
M. Campos et al.
Journal of Environmental Management 320 (2022) 115906
microbial Pases and abundances of bacterial Pase genes in two rivers of
southern Chile (Campos et al., 2021); however, a study focused on P
cycling and P‒mobilizing microbes in Chilean eutrophic lakes has not
been conducted thus far. Therefore, our understanding of the fates and
ecological risks of P inputs and the role of microbial communities in the
P cycling and eutrophication of lakes is urgently required to provide
valuable information for the design of efficient countermeasures and
policies for protecting ESs provided by eutrophic lakes in southern Chile.
Therefore, the present study investigated the spatial and temporal
distributions and relationships of content, bacterial Pase activity, and
bacterial Pase genes in surface sediments of Budi Lake.
nuclear magnetic resonance (NMR) analysis, and the third set was frozen
at − 80 ◦ C for genomic DNA (gDNA) extraction.
2.4. Measurement of total carbon and total nitrogen
2. Materials and methods
For the estimations of TC and TN, sieved (150 μm pore size) and
homogenized freeze‒dried sediment aliquots were loaded on an EA
3000 automated elemental analyzer (Eurovector, Milano, IT) following
the recommendations of Yang et al. (2010). Then, the elemental
composition of TC and TN was calculated by interpolation into a cali­
bration curve (r2 = 0.98) using EDTA as a standard (99.4% purity;
LECO®, USA) and expressed as mg of C or N per kilogram of dried
weight (dw) of sediment (mg kg− 1).
2.1. Budi Lake and its watershed
2.5. Extraction of phosphorus pools
Budi Lake (8◦ 53′ 00′′ S, 73◦ 17′ 00” W) is a tidal brackish water lake
located near the Pacific Ocean coast in southern Chile. Geo­
morphologically, the catchment landscape is influenced by hydric
erosion, which is worsened by the scarcity of vegetation and barren
lands. The high erodibility of soil leads to OM‒ and P‒rich soil particle
transport to the lake (Bertrán et al., 2010; Peña-Cortés et al., 2014). The
predominant land use is farmland (Fig. 1). In the northeast region of the
lake lies the Temo Stream watershed, with more than 75% of land used
for intensive agriculture. This watershed supplies the majority of
freshwater input to Budi Lake. In the central-eastern region, the Comue
Stream watershed has more diverse land use with a combination of
agriculture, forestry, and livestock. Puerto Dominguez city, located at
the bank of the Comue Stream, is a large urban environment and a
source of nutrient pollution. Important land use in the Puaucho River
watershed, located in the southwest, includes livestock pastures and
natural meadows (Peña-Cortés et al., 2014, 2020).
The labile, moderately labile, and nonlabile P fractions in each
sediment sample were sequentially extracted following the method
described by Ivanoff et al. (1998). Briefly, the labile Pi (NaHCO3‒Pi) and
Po (NaHCO3‒Po) fractions were extracted by suspending an aliquot of
sieved freeze‒dried sediment into a solution of NaHCO3 0.5 M pH 8.5
(1:50 v/v) and incubated by shaking (100 r.p.m.) at room temperature
for 16 h. Following centrifugation (4427×g), the supernatant was
filtered through Whatman filter paper (11 μm pore size) and collected.
This procedure was performed for each subsequent fractionation. For
the microbial Po (MPo) extraction, an extra freeze‒dried sediment
aliquot was fumigated with 2 mL of P‒free CHCl3, evaporated at 37 ◦ C
for 24 h, and subjected to the same treatment described for extraction of
the labile fraction. For extraction of the moderately labile Pi bound to
Ca, Mg, Fe, and Al cations (HCl‒Pi), the sediment residue resulting after
extraction of readily labile fractions was resuspended in HCl 1 M (1:50
v/v) and incubated for 3 h. For the extraction of the moderately labile Po
associated with the fulvic acid (FA‒Po) and nonlabile Po associated with
humic acid (HA‒Po) fractions, the sediment residue was resuspended in
NaOH 0.5 M (1:50 v/v) and incubated for 16 h. Finally, for extraction of
the nonlabile residual Po (Res‒Po), the sediment residue was incinerated
with 1 mL H2SO4 3.0 M HCl at 550 ◦ C for 1 h and diluted with double
distilled water (ddH2O). Then, dissolved Pi in the supernatants was
determined directly by the molybdenum blue method (Murphy and
Riley, 1962), and its concentration was calculated by absorbance
interpolation into a standard curve (r2 = 0.99) and expressed as mg per
kg− 1 of dried weight of sediment (mg kg− 1). For the dissolved Po esti­
mation, a supernatant aliquot was first subjected to Po hydrolysis
(Rowland and Haygarth, 1997), measured as Pi by the molybdenum blue
method, and finally calculated as the arithmetic difference between the
dissolved Pi and hydrolyzed Pi concentrations. Furthermore, total Pi
(TPi) and total Po (TPo) were calculated from the sum of each Pi and Po
fraction, while total P (TP) was the sum of TPi and TPo.
2.2. Sampling sites
The present study was spatially conducted by considering five sam­
pling sites with “less” and “more” anthropogenic impacts (Fig. 1). The
sampling sites with less anthropogenic impacts were the Puaucho (Pu)
site, which is highly affected by waters from the Pacific Ocean, and the
mouth of the Comue Stream (EsC) site, which receives waters from the
less impacted Comue Stream. The sampling sites with more significant
anthropogenic impacts were the Puerto Dominguez (PD) site, which
receives sewage discharges from Puerto Dominguez City, and the Temo
(Te) and mouth of Temo Stream (EsT) sites, which are influenced by
highly polluted waters from Temo Stream.
2.3. Sediment sampling and procedure
Sediment samples were collected in triplicate to a depth of 10 cm
with a Petersen‒like grab in winter (July) 2018 and summer (February)
2019. Briefly, nine sediment subsamples were randomly collected
within a 10 m radius at each sampling site. Later, sets of three sub­
samples were chosen and homogeneously mixed to form three
composited samples and displayed at a volume of 500 mL in sterile
plastic flasks. Immediately, each composited sample was subjected to in
situ measurements of pH, temperature (Temp), dissolved oxygen (DO),
and electrical conductivity (EC) using an HI 9829 multiparameter sensor
(Hanna Instruments, Inc., Rhode Island, USA) (Supplementary
Table ST1). After the sampling procedure, a total of 30 sediment samples
(5 sites × 2 seasons × 3 composite samples) were kept cooled at 4 ◦ C and
immediately transported to the Applied Microbial Ecology Laboratory
(EMALAB) in the Universidad de La Frontera (Temuco, Chile) for pro­
cessing. Each composited sample was separated into three different
sample sets. The first set was immediately analyzed for Pase activity
(ACP and ALP), the second set was freeze-dried for chemical determi­
nation of total carbon (TC), total nitrogen (TN), P fractionation, and 31P
2.6. 31P NMR analysis of phosphorus species
Composed freeze‒dried sediment samples (equal volumes of each
replicate) collected in each season were mixed and used for 31P NMR
analysis using the method established by Cade-Menun et al. (2005). In
brief, 1.5 g of freeze‒dried sediment was subjected to Po extraction by
mixing the sample aliquots with 30 mL of 0.25 M EDTA and 0.05 M
solution (1:20) and shaken for 4 h at 22 ◦ C. Next, the extracted Po
compounds were separated by centrifugation for 30 min at 10,000 g.
Subsequently, the supernatant was frozen at − 80 ◦ C, freeze‒dried for Po
preconcentration and stored in a freezer until analysis.
For 31P NMR analysis, 125 mg of freeze‒dried supernatant was
redissolved with 0.9 mL of 1 M NaOH and 0.1 mL of ddHD2O. The
mixture was vortexed for 1 min and then centrifuged at 10,000×g for 10
min. For each analysis, 0.6 mL solubilized supernatant was used in a 5
mm diameter NMR tube. Proton decoupled 31P NMR spectra were
collected on a NEO600 NMR spectrometer (Bruker Co., Billerica, MA,
3
M. Campos et al.
Journal of Environmental Management 320 (2022) 115906
USA) at an operating frequency of 202.5 MHz for 31P. The NMR pa­
rameters were 30◦ pulse (i.e., 9.22 μs pulse width), 0.88 s acquisition
time, 3.7 s pulse delay, 25 ◦ C, and 7000 scans. The raw spectra were
processed using TopSpin software (Bruker Co., Billerica, MA, USA). The
31
P chemical shifts (δP-31) were reported relative to an external 85%
H3PO4 solution set to 0 ppm. Spectra were plotted with a 5 Hz line
broadening. Each region was integrated from one edge to the other.
Edges were defined based on distance from the orthophosphate (ortho‒
P) peak as defined in Cade-Menun et al. (2017). Then, the peaks were
compared to those included in the developed library by Cade-Menun
(2015) to identify free or grouped P compounds such as ortho‒P, PMEs,
PDEs, and pyrophosphates (pyro‒P).
(RQ) after dividing them by the 16 S rRNA gene abundance (Acuña
et al., 2016).
2.9. Statistical analyses
Data were spatiotemporally contrasted in triplicate by one‒way
ANOVA with Tukey’s honestly significant difference (HSD) test. In
addition, a principal component analysis (PCA) was constructed in the
stats v3.6.2 package and visualized with the ggbiplot function in the R
tool to explore the structure and relationship among all data in this
study. Complementarily, the significance (P‒value) of correlations
among variables was determined by Pearson’s correlation coefficients
using the BSDA function in R. All the above tests were performed with a
confidence interval at a 99% level, and values are shown as the means of
three replicates (means ± standard deviations).
2.7. Acid and alkaline phosphomonoesterase activity
The ACP and ALP enzymatic activities were estimated following the
procedure described by Tabatabai and Bremner (1969). Briefly, 0.2 g
wet weight (ww) of each fresh sediment sample was mixed with 1 mL of
modified universal buffer (MUB) stock solution containing 0.05 M para‒
nitrophenyl phosphate (p‒NPP) (Sigma–Aldrich, Merck KGaA, Bur­
lington, MA, USA) as the substrate in glass flasks. The pH values of MUB
for ACP and ALP estimations were 6.5 and 11.0, respectively. Then,
flasks were incubated by shaking for 1 h at 37 ◦ C, and the reaction was
quickly stopped by adding 1 mL CaCl (0.5 M) and 4 mL NaOH (0.5 M). In
parallel, to avoid overestimations resulting from adsorption of p‒NPP
into the sediment samples, blanks received the p‒NPP solution promptly
after incubation. Finally, the formation of p‒nitrophenol (p‒NP) in the
supernatant of samples and blanks was measured at 420 nm using a
microplate spectrophotometer (MultiskanGO, Thermo Fisher Scientific
Inc., MA, USA), and the absorbances were interpolated into a calibration
curve (r2 = 0.97) and expressed as mg of p‒NP released per 1 g of dw of
sediment per hour (mg p‒NP g− 1 h− 1).
3. Results and discussion
3.1. Spatiotemporal distribution of sediment phosphorus fractions and
species
The contents of the P fractions showed a spatiotemporal distribution
in the sediment samples of Budi Lake (Table 1 and Table 2). Spatially,
sediments from the more anthropogenized sampling sites contained
significantly higher (P < 0.01) contents of labile (12.3–94.3 mg kg− 1),
moderately labile (85.1–359.2 mg kg− 1), and nonlabile (61.5–230.5 mg
kg− 1) P fractions than those from the less anthropogenized sampling
sites with the values of the labile (3.1–44.4 mg kg− 1), moderately labile
(69.0–188.4 mg kg− 1), and nonlabile (32.5–168.3 mg kg− 1) P fractions.
Similarly, significantly higher (P < 0.01) TPo (512.5–773.1 mg kg− 1),
TPi (110.9–156.0 mg kg− 1), and TP (647.4–929.7 mg kg− 1) in sediments
from the more anthropogenized sampling sites were observed compared
with TPo (263.1–491.1 mg kg− 1), TPi (86.1–127.4 mg kg− 1), and TP
(354.3–618.5 mg kg− 1) in sediments from the less anthropogenized
sampling sites. These results are in accordance with studies that have
reported that peaks of P fractions occurred in lakes with elevated
anthropogenic intervention and troublesome eutrophic status (Zhang
et al., 2008; Torres et al., 2014; Wan et al., 2020a).
Individually, our results of labile NaHCO3‒Pi (12.3–35.2 mg kg− 1)
and ‒Po (14.2–94.3 mg kg− 1) fractions were similar to those reported in
sediments of Chinese (2.0–43.2 mg kg− 1) and American lakes (1.6–15.0
mg kg− 1) (Huo et al., 2011; Lü et al., 2018; Torres et al., 2014). In a
similar context, the moderately labile HCl‒Pi fraction is expected to be
abundant in sediments with elevated primary productivity (Zhu et al.,
2013); however, our HCl‒Pi contents (85.1–139.5 mg kg− 1) were lower
than those reported for Chinese (357.9–909.5 mg kg− 1) and American
lakes (367–670 mg kg− 1) (Torres et al., 2014; Zhang et al., 2008). These
differences could be attributed to the different mineral compositions of
sediments, drainage regimes, and sources of pollution between Budi
Lake and the mentioned lakes (Ting and Appan, 1996; Zhang et al.,
2008; Condron and Newman, 2011; Lü et al., 2016).
Notably, the moderately labile FA‒Po (204.7–359.2 mg kg− 1) and
the nonlabile HA‒Po (111.5–230.5 mg kg− 1) fractions represented more
than 50% of TP in the more anthropogenized sampling sites of Budi
Lake. Both fractions are considered indicators of P inputs from agricul­
tural lands in eutrophic lakes (Zhang et al., 2008; Zafar et al., 2017; Gao
et al., 2019). Similar FA‒Po and HA‒Po have also been reported in
sediments of eutrophic Chinese lakes (Zhang et al., 2008; Huo et al.,
2011; Ni et al., 2019a; Wan et al., 2020a) and American wetlands
(Florida Everglades; Morrison et al., 2016). In these studies, the high
FA‒Po and HA‒Po contents were related to an enriched sedimentary OM
with fulvic and humic acids (Stone and English, 1993).
As observed in other P fractions, similar contents of the Res‒Po
(61.5–119.3 mg kg− 1) fraction have been found in sediments of Chinese
lakes (46.2–185.7 mg kg− 1) (Zhang et al., 2008). Nevertheless, high
proportions of Res‒Po (>40%) have also been found in oligotrophic
2.8. Quantification of bacterial phosphomonoesterase genes
Thawed sediment aliquots (~250 mg ww) were subjected to genomic
DNA (gDNA) extraction using a DNeasy® PowerBiofilm Kit (QIAGEN,
Carlsbad, CA, USA) according to the manufacturer’s instructions. The
extracted gDNA was quantified in a Qubit4™ Fluorometer (Thermo
Fisher Scientific Inc.) with the broad range Quant-iT™ dsDNA Assay Kit
(Thermo Fisher Scientific, Inc.) and further frozen at − 20 ◦ C for bacte­
rial gene analysis.
The absolute abundance of the total sediment bacterial community
based on the 16 S rRNA gene and bacterial Pase genes (phoC, phoD, and
phoX) was assessed by quantitative polymerase chain reaction (qPCR)
using ~25 ng μL− 1 of gDNA and PowerUp™ SYBR™ Green Master Mix
(Applied Biosystems™, Foster City, CA, USA) in a StepOne Real‒Time
PCR System (ThermoFisher Scientific, Inc.). The primer sets and qPCR
conditions are summarized in Supplementary Table ST2.
The copy numbers of targeted genes were determined via interpo­
lation into external standard curves, which were constructed as follows:
Template gene sequences were synthesized by in silico PCRs using
FastPCR® software version 6.7.01 (PrimerDigital Ltd., Helsinki, FI),
resulting in amplicons between 203 and 717 bp in size. Amplicons were
later built as dsDNA gBlock® Gene Fragments (Integrated DNA Tech­
nologies, Inc. Iowa, USA) and rehydrated. Later, DNA quality and con­
centrations were confirmed, and the copy number of each gene was
calculated according to the following equation: [concentration of the
dsDNA gBlock® Gene Fragment in ng μl− 1] × [molecular weight in fmol
ng− 1] × [Avogadro’s number] = copy number, as described by Whelan
et al. (2003). Then, standards were serially ten‒fold diluted and loaded
in triplicate for qPCR, and the efficiencies (from 86 to 103%), r2 (from
0.992 to 0.997), and slopes (from − 3.71 to − 3.25) of the curves were
calculated. Thus, absolute quantification (AQ) of bacterial genes was
expressed as copy number per gram of dw of sediment (gene copy g− 1),
with the values of Pase genes transformed into relative quantification
4
M. Campos et al.
Journal of Environmental Management 320 (2022) 115906
Table 1
Phosphorus fractionation of sediments from Budi Lake collected during the winter and summer seasons.
Season
Sample
Phosphorus fractions (mg per kg− 1 of dried weight of sediment)
Labile Pi and Po
Winter
Pu_W
EsC_W
PD_W
Te_W
c
c
EsT_Wc
Summer
Pu_S
EsC_S
PD_Sc
Te_Sc
EsT_Sc
MPo
NaHCO3‒Pi
NaHCO3‒Po
3.1 ± 0.2a
Dfb
9.7 ± 0.1 Ce
7.4 ± 0.6 Ce
12.3 ± 0.3
Bde
16.5 ± 0.7
Acd
13.8 ± 0.4
Bde
22.3 ± 2.9
Bbc
13.1 ± 1.8
Cde
27.7 ± 2.9
ABb
35.2 ± 1.9
Aa
25.9 ± 1.8
Bb
46.9 ± 2.0
Ac
14.2 ± 0.7
Ce
28.8 ± 2.6
Bd
54.9 ± 3.6
Ac
19.2 ± 1.8
Dde
78.9 ± 4.7
ABb
60.5 ± 7.3
BCc
48.9 ± 2.9
Cc
94.3 ± 7.0
Aa
9.2 ± 1.3
Cf
14.9 ± 1.4
BCef
24.3 ± 0.5
Bde
35.8 ± 3.1
Ac
34.3 ± 3.8
Acd
21.8 ± 3.4
De
44.4 ± 3.9
Cc
76.1 ± 4.3
Bb
82.8 ± 3.2
ABab
92.1 ± 4.6
Aa
Moderately labile Pi and Po
Non‒labile Po
HCl‒Pi
FA‒Po
HA‒Po
Res‒Po
83.0 ± 1.5
Cd
117.6 ±
1.2 Bb
137.0 ±
1.4 Aa
139.5 ±
1.0 Aa
138.4 ±
1.3 Aa
69.0 ± 2.9
Ce
91.9 ± 3.6
Bd
107.1 ±
2.9 Ac
109.5 ±
5.2 Abc
85.1 ± 3.5
Bd
159.2 ± 7.5
Dde
188.4 ± 1.3
Ccd
284.6 ± 4.3
Bb
359.2 ± 3.7
Aa
273.2 ± 2.8
Bb
114.5 ± 8.8
Cf
156.7 ±
15.5 Bc
193.8 ± 5.6
ABc
199.8 ±
16.0 Ac
204.7 ± 6.0
Ac
130.1 ± 4.6
Dc
168.3 ± 6.3
Cb
202.3 ± 4.5
Ba
230.5 ± 7.0
Aa
222.4 ± 3.0
Aa
75.0 ± 5.6
Ce
91.9 ± 6.1
BCde
120.8 ± 3.9
Bc
170.0 ±
20.7 Ab
111.5 ± 3.9
Bcd
57.2 ± 3.5
Def
72.6 ± 1.9
Ccd
91.3 ± 2.3
Bb
119.3 ±
2.9 Aa
92.4 ± 3.0
Bb
32.5 ± 2.9
Bg
44.5 ± 4.2
Bfg
61.5 ± 3.5
Ade
74.9 ± 3.9
Ac
66.7 ± 6.7
Acde
TPi
TPo
TP
86.1 ± 1.8
Cf
127.4 ± 1.3
Bc
149.3 ± 1.7
Aab
156.0 ± 1.7
Aa
152.2 ± 1.5
Aa
91.2 ± 5.6
Bef
104.9 ± 5.4
Bde
134.8 ± 5.3
Abc
144.8 ± 7.0
Bab
110.9 ± 5.4
Bd
363.1 ± 3.9
Ef
491.1 ± 9.8
Dd
616.8 ±
11.7 Cc
773.7 ±
11.4 Aa
677.2 ±
10.1 Bb
263.1 ±
14.4 Cg
416.5 ±
20.4 Be
512.5 ±
17.7 Ad
576.4 ±
18.8 Ac
569.4 ±
22.6 Ac
449.2 ± 5.4
Dde
618.5 ±
11.1 Cbc
766.1 ±
78.1 Bcb
929.7 ±
12.4 Aa
829.4 ±
11.5 Aba
354.3 ±
19.8 Ce
521.4 ±
24.3 Bcd
647.4 ±
22.0 Ab
721.2 ±
25.5 Ab
680.3 ±
27.9 Ab
NaHCO3‒Pi: labile inorganic phosphorus extracted with NaHCO3; NaHCO3‒Po: labile organic phosphorus extracted with NaHCO3; MPo: microbial organic phos­
phorus; HCl‒Pi: moderately labile inorganic phosphorus extracted with HCl associated with Ca, Fe and Al cations; FA‒Po: moderately labile organic phosphorus
extracted with NaOH associated with fulvic acids; HA‒Po: non‒labile organic phosphorus extracted with NaOH associated with humic acid fraction; Res‒Po: non‒
labile residual organic phosphorus. TPi: total inorganic phosphorus = Σ (NaHCO3‒Pi + HCl‒Pi); TPo: total organic phosphorus = Σ (NaHCO3‒Po + MPo + FA‒Po +
HA‒Po + Res‒Po); TP: total phosphorus = Σ (TPi + TPo).
a
Values represent the mean (n = 3) ± standard deviation.
b
Capital letters in the same column represent spatially significant differences (P < 0.01) among samples inside an individual, while lowercase letters in the same
column represent temporally significant differences (P < 0.01) among samples from both seasons.
c
Asterisk denotes the more anthropogenized sampling sites of this study.
Table 2
Relative contributions of phosphorus fractions measured from sediments of Budi Lake.
Season
Sample
Relative contribution (%) of P fractions in relation to TP
Labile
Winter
Summer
a
Pu_W
EsC_W
PD_Wa
Te_Wa
EsT_Wa
Pu_S
EsC_S
PD_Sa
Te_Sa
EsT_Sa
MPo
NaHCO3‒Pi
NaHCO3‒Po
0.7
1.6
1.6
1.8
1.7
6.3
2.5
4.3
4.9
3.8
1.6
7.6
1.9
3.1
6.6
5.4
15.1
9.3
6.8
13.9
2.0
2.4
3.2
3.9
4.1
6.1
8.5
11.8
11.5
13.5
Moderately labile
Non‒labile
HCl‒Pi
FA‒Po
HA‒Po
Res‒Po
18.5
19.0
17.9
15.0
16.7
19.5
17.6
16.6
15.2
12.5
35.4
30.5
37.2
38.6
32.9
32.3
30.0
29.9
27.7
30.1
29.0
27.2
26.4
24.8
26.8
21.2
17.6
18.7
23.6
16.4
12.7
11.7
11.9
12.8
11.1
9.2
8.5
9.5
10.4
9.8
TPi
TPo
19.2
20.6
19.5
16.8
18.4
25.7
20.1
20.8
20.1
16.3
80.8
79.4
80.5
83.2
81.6
74.3
79.9
79.2
79.9
83.7
Asterisk denotes the more anthropogenized sampling sites of this study.
aquatic environments in Singapore (Ting and Appan, 1996) and China
(Huo et al., 2011), demonstrating that Res‒Po accumulation also de­
pends on the contents of clay, cation contents, and secondary P minerals
in sediments (Tiessen et al., 1984; Condron and Newman, 2011).
Our results also showed temporal variations in the P fractions (Ta­
bles 1 and 2). The labile P fractions (13.1–94.3 mg kg− 1) showed
significantly higher (P < 0.01) contents in summer than in winter
(3.1–54.9 mg kg− 1). Conversely, the contents of the moderately labile
(83.0–359.2 mg kg− 1) and nonlabile (57.2–230.5 mg kg− 1) fractions in
winter were significantly higher (P < 0.01) than the values of the
moderately labile (69.0–204.7 mg kg− 1) and nonlabile (32.5–170.5 mg
kg− 1) fractions in summer. Similarly, significantly higher (P < 0.01) TPi
(86.1–156.0 mg kg− 1) and TPo (491.1–773.7 mg kg− 1) contents were
observed in winter than TPi (91.2–144.8 mg kg− 1) and TPo
(263.1–576.4 mg kg− 1) in summer. The TP values did not show apparent
significant differences between the two sampling seasons.
Although studies on the contents and natures of P fractions of lake
sediments at the temporal scale are scarce, it is widely accepted that
abiotic and biotic changes derived from the transition between colder
and warmer seasons may result in significant variations (Søndergaard,
2007). In this context, higher temperatures in summer than in winter
(Supplementary table ST1) increased the contents of MPo (from 9.2 to
92.1 mg kg− 1). It has been observed that higher water temperatures
commonly stimulate the growth of microorganisms as well as the con­
centrations of phosphate biomolecules forming part of the sediment
biomass (Upreti et al., 2015). Consequently, we observed higher values
of labile P forms in summer (13.1–94.3 mg kg− 1), possibly related to
their greater mobilization from less labile P forms by growing sedi­
mentary biomass (Yiyong et al., 2002; Zhang et al., 2008; Huo et al.,
2011). This behavior is similar to that described in several anthro­
pogenized lakes in Sweden (Goedkoop and Pettersson, 2000), India
(Gireeshkumar et al., 2013), Scotland (Spears et al., 2007), and China
5
M. Campos et al.
Journal of Environmental Management 320 (2022) 115906
(Yiyong et al., 2002; Yuan et al., 2020; Zhao et al., 2012).
Regarding TP in sediments, the results showed from 74.25 to 83.69%
(263.1 and 773.7 mg kg− 1, respectively) of TPo (Table 2), where
moderately labile FA‒Po (114.5–359.2 mg kg− 1) and non‒labile HA‒Po
(75.0–230.5 mg kg− 1) together represented 46.5–64.4% of TP. The
dominance of Po has also been described in sediments of eutrophic
Chinese lakes (Huo et al., 2011) and American wetlands (Morrison et al.,
2016). In this context, it has been postulated that high Po contents in
Budi Lake are due to the considerable runoff of eroded soil particles as a
result of intensive regional agriculture established on acidic soils, which
are recognized by their high content of fixed Po (Bertrán et al., 2010;
Peña-Cortés et al., 2014).
The 31P NMR results for the sediment lake are shown in Fig. 2 and
Table 3. In general, terms, ortho‒P was the major P species present in all
samples, followed by PMEs, PDEs, and pyro‒P. Polyphosphate (poly‒P)
was not detected in the analyzed sediments. Spatially, the percentages of
ortho‒P were higher in less anthropogenized sites (83.22–84.94%) than
in the more anthropogenized sites (62.47–81.62%). In contrast, the
more anthropogenized sites presented higher percentages of PMEs
(13.23–26.58%) and DNA‒P (4.74–10.12%) than those observed for
PMEs (11.90–13.80%) and DNA‒P (2.97–3.16%) in the less anthro­
pogenized sites. Similar trends have been found in sediments from
polluted lakes in Sweden (Ahlgren et al., 2006) and China (Bai et al.,
2009; Dong et al., 2012; Ni et al., 2019b; Yuan et al., 2020; Zhang et al.,
2013a, 2015).
The 31P NMR analysis also showed a spatiotemporal distribution of P
species. The high presence of ortho‒P and biogenic species (such as
PMEs, DNA‒P, and pyro‒P) is typical of anthropogenized sediments
(Cade-Menun, 2005). In this context, studies have reported similar
values in lake sediments in Sweden (35% of PMEs, 35% ortho‒P, and
12–17% of DNA‒;P; Ahlgren et al., 2006) and the US (22–36% of PMEs
and 7–31% of DNA‒;P; Torres et al., 2014). Similarly, Zhang et al.
(2013a) found greater values of ortho‒P (12.1–27.1%), PMEs
(2.4–5.2%), DNA‒P (1.0–3.4%), and pyro‒P (0.1–0.4%) in sediments of
highly polluted lakes compared with those values of ortho‒P
(14.1–14.4%) PMEs (1.4–1.7%), DNA‒P (0.8–0.9%), and pyro‒P
(~0.1%) of less polluted lakes in China.
Temporally, the values of ortho‒P were quite similar in winter
(74.93–83.22%) and summer (62.47–84.94%). However, the values of
PMEs (11.90–26.58%), DNA‒P (3.16–10.12%) and pyro‒P
(0.83–1.24%) were higher in summer sediments than those values of
PMEs (13.80–19.44%), DNA‒P (2.97–6.18%), and pyro‒P (not detec­
ted) in the winter sediments. The temporal comparison of P species
detected by 31P NMR in sediments of eutrophic lakes has been a topic
scarcely approached. However, since these biogenic molecules are
related to autochthonous OM sedimentation and further microbial
decomposition (Ahlgren et al., 2006; Torres et al., 2014; Zhang et al.,
2015), their sedimentations are expected to be more significant during
warmer seasons. Accordingly, Reitzel et al. (2006) determined that
sedimentary PMEs and DNA‒P were the most significant contributors to
DIP (primarily as ortho‒P form) in Danish lakes during the decay of
phytoplankton biomass in warmer periods. Higher contents of DNA‒P,
PMEs, PDEs and pyro‒P fractions were also observed in sediments of a
Polish artificial water reservoir (Młynarczyk et al., 2013) and a New
Zealander lake (Özkundakci et al., 2014) during summer compared with
colder seasons. In addition, PMEs and DNA‒P have been correlated with
MPo, FA‒Po, and HA‒Po fractions in the sediments of several lakes
(Reitzel et al., 2006; Torres et al., 2014; Ni et al., 2019b; Yuan et al.,
2020). Therefore, considering that MPo, FA‒Po, and HA‒Po were the
main contributors of Po to Budi Lake sediments in summer, this temporal
relationship with PMEs and DNA‒P species could be applicable.
Interestingly, ortho‒P was the dominant P species detected in the 31P
NMR solution, resulting in an overestimation of Pi species and an un­
derestimation of Po molecules compared with the findings of the
sequential P fractionation for Budi Lake. Differences between P frac­
tionation and 31P NMR methods are typical and well described in lake
sediments by various studies (Dong et al., 2012; Hezhong et al., 2017; Ni
et al., 2019b). In this context, Po overestimation by P fractionation is
possible since the extraction of FA‒Po and HA‒Po with NaOH solution
commonly coextracts several non‒reactive Pi species (e.g., poly‒P and
pyro‒P) bound to colloid minerals and organic macromolecules (Turner
et al., 2006). Additionally, extractant pH values, types of sediments
(organic‒rich, siliceous, or calcareous), readsorption of extracted P onto
Fe (OOH) and/or CaCO3 of the residual phase, and pretreatment of
samples (freeze‒drying, drying, or fresh) are additional limitations of P
fractionation (Wang et al., 2013). Regarding 31P NMR, extraction and
preconcentration of P with NaOH‒EDTA solution could tend to over­
estimate ortho‒P, as this molecule can be a breakdown product of the
alkaline reduction and metal ion catalysis of poly‒P (Zhang et al.,
2013b), a molecule not detected in our study and possibly degraded
during the process. While poly‒P can be readily degraded (Cade-Menun
et al., 2006), including by reaction with reagents, our experimental
approach tried its best to avoid experimental artifacts. We used EDTA
during extraction, which is reported to decrease the decomposition of
poly‒P (Cade-Menun, 2005), and NMR spectra were obtained immedi­
ately after extraction (Turner et al., 2006). Some earlier reports have
pointed out that poly‒P is often found in soils with low microbial ac­
tivity or OM content (Dai et al., 1996; Makarov, 1997). This means that
poly‒P concentrations are likely not detectable in these sediments.
Additionally, vacuolar ortho‒P cannot be detected by the molybdenum
blue method but is suitably measurable by 31P NMR, constituting other
aspects contributing to the ortho‒P overestimation (Reitzel et al., 2006).
Then, since the extraction solution mostly includes Pi species, sediments
with lower TP values, such as the Pu site of Budi Lake, are prone to show
poor 31P NMR signals for Po species, thereby hindering the integration of
peaks for their interpretation and making the interpretation of results
impossible (Fig. 2 and Table 3).
3.2. Acid and alkaline phosphomonoesterase activity
The activities of the bacterial ACP and ALP enzymes showed
spatiotemporal differences in the present study (Fig. 3). Spatially, sed­
iments in the more anthropogenized sites exhibited significantly higher
(P < 0.01) levels of ACP (9.7–22.7 μg PNP g− 1 h− 1) and ALP (5.9–9.6 μg
PNP g− 1 h− 1) than those in less anthropogenized sampling sites with
levels of ACP from 4.2 to 8.5 μg PNP g− 1 h− 1 and ALP from 0.3 to 6.4 μg
PNP g− 1 h− 1. Temporally, significantly higher (P < 0.01) levels of ACP
(from 5.6 to 22.7 μg PNP g− 1 h− 1) and ALP (2.6–9.6 μg PNP g− 1 h− 1)
were observed in summer compared with the levels of ACP (4.2–12.9 μg
PNP g− 1 h− 1) and ALP (0.3–6.7 μg PNP g− 1 h− 1) for winter samples.
Fig. 2. 31P NMR spectra for sediment samples of Budi Lake collected during
winter (W) and summer (S) seasons. Samples: Puerto Dominguez (PD), Comue
Stream (EsC), Temo (Te), Temo Stream (EsT).
6
M. Campos et al.
Journal of Environmental Management 320 (2022) 115906
Table 3
Composition of different P species and their proportions in sediments of Budi Lake detected by the31P NMR method.
Season
Winter
Summer
Sample
EsC_W
PD_Wa
Te_Wa
EsT_Wa
EsC_S
PD_Sa
Te_Sa
EsT_Sa
Integrated area
8.35
12.86
11.95
10.48
8.84
8.70
11.78
10.26
Phosphate groups (%)
ortho‒P
pyro‒P
PMEs
83.22
81.62
80.62
74.93
84.94
76.36
75.27
62.47
‒
‒
‒
‒
‒
1.20
1.24
0.83
13.80
13.23
13.20
19.44
11.90
17.70
17.22
26.58
PDEs
DNA‒P
Non‒DNA‒P
2.97
5.15
6.18
5.63
3.16
4.74
6.26
10.12
‒
1.03
‒
‒
‒
‒
0.30
‒
‒: Not detected.
a
Asterisk denotes the more anthropogenized sampling sites of this study.
In each of these reports, increasing P mobilization was accompanied by
increasing Pase activities, an association promoted during the warmer
seasons when environmental conditions were suitable for microbial
replication (Huang and Morris, 2005).
As mentioned above, the ALP and ACP activities in sediments of Budi
Lake were higher in summer than in winter. Increased Pase activity in­
dicates an intense transformation of DOP (Ma et al., 2019), a suitable
pool to support bacterial growth (Cao et al., 2018). Then, since PMEs
and PDEs coming from the moderately and nonlabile Po are significant
components of DOP (Torres et al., 2014; Lü et al., 2016; Ma et al., 2019),
the elevated Pase activities registered in Budi Lake in summer may be
explained by intensive biotransformation of these molecules by micro­
bial activity. An additional factor regarding the higher Pase activities
during summer may be that these enzymes can be reversibly complexed
with sediment cations and humic‒rich Po substances that constitute an
enzymatic pool prone to release after physicochemical changes within
sediments (Zhao et al., 2019a). Therefore, typical summer features, such
as enhanced microbial OM consumption and a decreasing DO concen­
tration, constitute extra factors for the liberation of immobilized ACP
and ALP enzymes (Huang and Morris, 2005; Zheng et al., 2019b).
Interestingly, the ACP activities were generally higher (4.2–22.7 μg
PNP g− 1 h− 1) than ALP (0.3–9.6 μg PNP g− 1 h− 1) in each of the sedi­
ments analyzed (Fig. 3). Traditionally, the majority of investigations
assessing Pase activities in sediments have mainly focused on evaluating
ALP activities, minimizing the contribution of bacterial ACP to the
recycling of P in these environments (Yiyong et al., 2002; Huang and
Morris, 2005; Ni et al., 2019a; Yuan et al., 2020). Similar results have
been reported for sediments from rivers (Campos et al., 2021; Huang and
Morris, 2003, 2005) and estuaries (Jiang et al., 2018). Thus, although
the pH values of our sediment samples ranged close to neutrality (from
6.4 to 7.3) (Supplementary Table ST1), the predominance of ACP ac­
tivities is justified, as its measurement was carried out at its optimum pH
condition (6.5), a value that is relatively close to the pH values regis­
tered for Budi Lake.
Fig. 3. Quantification of acid (ACP) and alkaline (ALP) phosphomonoesterase
activity in sediments of Budi Lake collected during the winter (W) and summer
(S) seasons. Capital letters represent spatially significant differences (P < 0.01)
among samples in each season, while small letters represent temporally sig­
nificant differences (P < 0.01) among seasons. Samples: Puaucho (Pu), Puerto
Dominguez (PD), Comue Stream (EsC), Temo (Te), Temo Stream (EsT). Aster­
isks (*) denote the more anthropogenized sampling sites of this study.
3.3. Quantification of bacterial phosphomonoesterase genes
Similar to the P fractions and Pase activities, the absolute abun­
dances of the total bacterial community and Pase genes in sediment
showed a spatiotemporal distribution (Table 4). Spatially, absolute
abundances of the 16 S rRNA (2.3 × 1010 to 1.8 × 1011 copies g− 1), phoC
(1.6 × 106 to 1.9 × 109 copies g− 1), phoD (1.5 × 106 to 4.7 × 107 copies
g− 1) and phoX genes (6.7 × 104 to 2.6 × 106 copies g− 1) were signifi­
cantly higher (P < 0.01) in sediments from the more anthropogenized
sampling sites than the values for 16 S rRNA (1.8 × 109 to 1.2 × 109
copies g− 1), phoC (1.2 × 106 to 2.1 × 107 copies g− 1), phoD (1.4 × 105 to
6.3 × 106 copies g− 1) and phoX (4.0 × 104 to 6.9 × 105 copies g− 1) in
those from the less anthropogenized ones. However, concerning the
total bacteria (16 S rRNA genes), the relative abundances of phoC (7.6 ×
Spatially, it is known that sediments containing elevated levels of OM
facilitate the deposition of less labile Po species and hence promote their
mineralization and mobilization into more labile forms by the action of
the Pase‒harboring bacterial populations (Yiyong et al., 2002; Zhang
et al., 2015). This behavior has commonly been described in sediments
from eutrophic freshwater bodies in Spain (López et al., 2006) and China
(Bai et al., 2020; Luo and Gu, 2015; Ni et al., 2019a; Yiyong et al., 2002)
and, more recently, in sediments of Chilean rivers (Campos et al., 2021).
7
M. Campos et al.
Journal of Environmental Management 320 (2022) 115906
Table 4
Absolute and relative quantification of bacterial genes from sediment samples collected from Budi Lake during the winter and summer seasons.
Absolute Quantification (gene copy g− 1 of dried weight of sediment)c
Season
Winter
Summer
9
Sample
16 S rRNA ( × 10 )
Pu_W
EsC_W
PD_Wd
Te_Wd
EsT_Wd
Pu_S
EsC_S
PD_Sd
Te_Sd
EsT_Sd
a
b
4.4 ± 1.0 Cd
18 ± 8.2 BCd
34 ± 2.5 Bcd
71 ± 11 Abc
23 ± 3.9 BCcd
12 ± 1.8 Cd
1.8 ± 0.5 Cd
110 ± 26 Bb
110 ± 29 Bb
180 ± 16 Aa
phoC ( × 10
8c
0.12 ± 0.03 Bc
1.1 ± 0.6 ABc
1.2 ± 0.2 Ac
0.62 ± 0.5 ABc
0.16 ± 0.04 ABc
2.1 ± 0.65 CDc
0.26 ± 0.09 Dc
19 ± 4.5 Aa
8.5 ± 0.9 BCb
15 ± 1.6 Aba
6
phoD ( × 10 )
1.4 ± 0.18 Acd
2.4 ± 1.6 Acd
2.5 ± 0.3 Acd
1.5 ± 1.3 Acd
3.3 ± 0.23 Acd
6.3 ± 2.3 Ccd
0.62 ± 0.17 Cd
21 ± 3.9 Bb
9.2 ± 1.5 Cc
47 ± 4.9 Aa
Relative Quantification
6
phoX ( × 10 )
1.2 ± 0.2 Ac
0.69 ± 0.46 Ac
0.67 ± 0.05 Ac
0.56 ± 0.32 Ac
0.51 ± 0.3 Ac
12 ± 0.6 BCb
0.4 ± 0.08 Dc
26 ± 3 Aa
8.5 ± 0.9 CDb
21 ± 5.4 Aba
phoC ( × 10− 3)
2.8 ± 0.5 Ad
3.9 ± 2.1 Acd
3.5 ± 0.3d Acd
1.3 ± 0.7d Ad
0.76 d 0.29 Ad
18 ± 2.9 Aa
14 ± 3.2 ABab
17 ± 3.2 Aa
8 ± 2.5 Bbc
8 ± 1.1 Bbc
phoD ( × 10− 5)
a
b
0.33 4. Aab
0.12 ± 4.5 BCcde
7.5 ± 1.2 BCde
3.0 ± 2.6 Ce
0.15 ± 3.9 ABcd
0.53 ± 0.12 Aa
0.27 ± 2.2 ABabc
0.19 ±d1.3 BCbcd
8.9 ± 3.9 Cde
0.26d± 3.1 Bbc
phoX ( × 10− 5)
0.28 ± 0.11 Ab
3.5 ± 1.2 Bde
2 ± 29 Bde
82 ± 55 Be
2.4 ± 1.6 Bde
0.01 ± 0.12 Aa
0.25 ± 0.11 Bbc
0.24 ± 3.2 Bbc
7.9 ± 1.6 Bcde
0.11 ± 1.9 Bbcd
a
Values represent the mean (n = 3) ± standard deviation.
Capital etters in the same column represent spatially significat differences (P < 0.01) among samples inside an individual, while lowercase letters in the same column
represent temporally significant differences (P < 0.01) among samples from both seasons.
c
Quantification by qPCR with an efficiency from 86% to 103%.
d
Asteisk denotes the more anthropogenized sampling sites of this tudy.
b
10− 4 to 1.7 × 10− 2), phoD (3.0 × 10− 5 to 2.6 × 10− 4), and phoX (8.2 ×
10− 6 to 2.4 × 10− 4) in the more anthropogenized sampling sites did not
show a clear pattern of differentiation from the relative abundances of
phoC (2.8 × 10− 3 to 1.8 × 10− 2), phoD (1.2 × 10− 5 to 5.3 × 10− 4) and
phoX (3.5 × 10− 5 to 1.0 × 10− 3) in the less anthropogenized sampling
sites.
Temporally, the absolute abundances of 16 S rRNA (1.8 × 109 to 1.8
× 1011 copies g− 1), phoC (2.6 × 107 to 1.9 × 109 copies g− 1), phoD (6.2
× 105 to 4.7 × 107 copies g− 1), and phoX (4.0 × 105 to 2.6 × 107 copies
g− 1) were significantly higher (P < 0.01) in sediments in summer than
the values of 16 S rRNA (4.4 × 109 to 7.1 × 1010 copies g− 1), phoC (1.2
× 107 to 1.1 × 108 copies g− 1), phoD (1.4 × 106 to 3.3 × 106 copies g− 1)
and phoX (5.1 × 105 to 1.2 × 106 copies g− 1) observed in sediments in
winter. Similarly, the relative abundances of phoC (8.0 × 10− 3 to 1.8 ×
10− 2) and phoX (7.9 × 10− 5 to 1.0 × 10− 3) from the samples collected in
summer were significantly higher (P < 0.01) than the values of phoC
(7.6 × 10− 4 to 3.9 × 10− 3) and phoX (8.2 × 10− 6 to 2.8 × 10− 4) obtained
from sediments in winter. Exceptionally, the relative abundances of
phoD (8.9 × 10− 5 to 5.3 × 10− 4) in sediments in summer were not
significantly (P < 0.01) different from those phoD values (7.5 × 10− 5 to
3.3 × 10− 4) in winter.
Studies assessing the distribution of bacterial Pase genes in fresh­
water environments, such as lakes, are relatively limited. Regarding our
results of absolute and relative abundances of phoD (105 to 107 copies
g− 1 and rates of 10− 5 to 10− 3, respectively), higher values have been
reported for sediments from eutrophic American wetlands (1011 to 1012
copies g− 1; Morrison et al., 2016) and Chinese lakes (2.2 × 107 to 3.1 ×
107 copies g− 1, 10− 3 to 10− 2) (Chen et al., 2019; Fan et al., 2019).
However, our results were similar to those reported for suspended
sediment particles from Chinese Lake (105 to 106 copies g− 1; Zhang
et al., 2020) and Chilean rivers (106 to 107 copies g− 1; Campos et al.,
2021). Regarding phoX, the absolute and relative abundances (104 to
106 copies g− 1 and 10− 5 to 10− 3, respectively) were lower than those
reported for American wetlands (1011 to 1012 copies g− 1; Morrison et al.,
2016) but similar to those reported for sediments from Chilean rivers
(105 to 106 gene copies g− 1; Campos et al., 2021) and higher than those
reported for sediments from Chinese lakes (10− 5 to 10− 4; Fan et al.,
2019).
Comparing both ALP-encoding genes, our results demonstrated that
phoD was generally more abundant than phoX. Therefore, considering
that Budi Lake is a coastal waterbody highly influenced by the Pacific
Ocean, this distribution is possible because phoD is considered to be the
most abundant microbial Pase gene in marine environments (Luo et al.,
2009), while phoX predominates in freshwater ecosystems (Sebastian
and Ammerman, 2009). In addition, soil particles transported by agri­
cultural runoff and sewage can significantly increase phoD abundance
and diversity, as has been reported for the river moth zones of Taihu
Lake (Zhang et al., 2020) and Min River (Hu et al., 2020).
Concerning the general distribution of bacterial Pase genes, phoC
showed the highest absolute values and relative abundances, followed
by phoD and phoX. This result indicates that the phoC gene is the most
abundant of the three evaluated Pase genes, showing a similar distri­
bution as described for sediments of the Chilean rivers (107 to 108 copies
g− 1; Campos et al., 2021). The phoC has been reported to range primarily
from 106 to 107 copies g− 1 soil for soil systems (Fraser et al., 2017; Zheng
et al., 2019b). These abundances are lower than the values reported for
Budi Lake, indicating the active participation of phoC in recycling the
sedimentary Po, a finding supported by the predominance of ACP over
ALP (Fig. 3). However, these affirmations should be considered with
prudence because the primer sets employed in the present study were
mainly designed and used for soil Proteobacteria, and better coverage of
bacterial Pase genes from aquatic environments is still required (Sakurai
et al., 2008; Ragot et al., 2015; Fraser et al., 2017).
The higher absolute quantifications of the total bacterial community
and the Pase genes scored spatially with the higher values for the more
anthropogenized samples and temporally for the summer season
(Table 3). This observation supports the higher bacterial activities in
these sampling sites and seasons, which was previously based on the
MPo and Pase activity values. The inputs of nutrient‒rich pollutants can
rapidly stimulate the activities of Pase‒harboring bacterial populations
and the growth of the total bacterial community in soils and aquatic
systems (Liu et al., 2015; Acuña et al., 2016; Luo et al., 2017; Chen et al.,
2019). However, low P bioavailability is the strongest factor in
increasing bacterial Pase activities after upregulation of the expression
of functional Pase genes (Tiessen et al., 1984; Vershinina and Zna­
menskaya, 2002; Nannipieri et al., 2011).
Consequently, the normalization of absolute Pase gene quantifica­
tion to the total 16 S rRNA genes revealed that the Pase‒harboring
bacterial populations predominated in sampling sites with lower
anthropogenic impact in Budi Lake (Table 4). Valdespino-Castillo et al.
(2014) reported that high mineralization rates of DOP particles present
in waters of the P poor Mexican of Lake Alchichica were accompanied by
higher concentrations of phoD and phoX. Morrison et al. (2016) found
high phoD abundances in sites with low‒P in the Florida Everglades,
while high phoX abundances were present in sites with a very high
abundance of Po forms. Wan et al. (2020b) showed a significant decrease
in Pase activities and Pase gene abundances after dredging TC and P‒
rich sediments of Nanhu Lake. Contrary to the spatial distribution,
similar temporal shifts to those observed for Budi Lake have been more
consistently reported. Zhang et al. (2019) accounted for higher phoD
abundances in sampling sites with elevated influences of nutrient‒rich
waters of shallow Taihu Lake, especially in summer. Later, Zhang et al.
8
M. Campos et al.
9
‒0.91**
‒0.92**
‒0.37
‒0.70**
‒0.86**
‒0.73**
‒0.87**
‒0.73**
‒0.84**
‒0.68**
‒0.92**
‒0.91**
‒0.70**
‒0.86**
0.68**
0.79**
1
‒0.71**
‒0.77**
‒0.68**
‒0.23
‒0.76**
‒0.82**
‒0.75**
‒0.90**
‒0.85**
‒0.87**
‒0.81**
‒0.85**
‒0.58*
‒0.81**
0.62*
1
0.52*
‒0.70**
‒0.84**
‒0.47
‒0.45
‒0.69**
‒0.30
‒0.60*
‒0.66**
‒0.72**
‒0.40
‒0.71**
‒0.69**
‒0.44
‒0.44
1
0.34
0.37
0.84**
0.79**
0.52*
0.49
0.82**
0.89**
0.88**
0.79**
0.85**
0.86**
0.89**
0.91**
0.74**
1
‒0.12
‒0.31
‒0.74**
0.84**
0.65**
0.53*
0.61*
0.92**
0.51
0.89**
0.49
0.80**
0.58*
0.85**
0.84**
1
0.86**
0.15
‒0.09
‒0.58*
0.94**
0.90**
0.63*
0.58*
0.96**
0.81**
0.96**
0.83**
0.98**
0.84**
0.99**
1
0.47
0.65**
‒0.64*
‒0.39
‒0.83**
0.96**
0.91**
0.59*
0.64*
0.97**
0.76**
0.97**
0.79**
0.97**
0.79**
1
0.98**
0.53*
0.76**
‒0.61*
‒0.41
‒0.84**
0.66**
0.68**
0.77**
0.13
0.73**
0.95**
0.76**
0.90**
0.86**
1
0.94**
0.91**
0.68**
0.82**
‒0.42
‒0.33
‒0.93**
0.90**
0.90**
0.75**
0.45
0.95**
0.77**
0.93**
0.86**
1
0.97**
0.99**
0.96**
0.58*
0.76**
‒0.58*
‒0.37
‒0.87**
0.69**
0.79**
0.78**
0.12
0.73**
0.81**
0.68**
1
0.92**
0.87**
0.97**
0.93**
0.53*
0.77**
‒0.55*
‒0.45
‒0.75**
* Asterisk denotes a P < 0.05 significant difference in the correlations among parameters.
** Asterisks denote a P < 0.01 significant difference in the correlations among parameters.
0.95**
0.85**
0.57*
0.63**
0.94**
0.72**
1
0.85**
0.96**
0.99**
0.92**
0.90**
0.70**
0.82**
‒0.39
‒0.32
‒0.93**
0.63*
0.63*
0.53*
0.27
0.66**
1
0.87**
0.90**
0.97**
0.89**
0.96**
0.95**
0.50
0.69**
‒0.69**
‒0.35
‒0.76**
0.95**
0.86**
0.62*
0.61*
1
1**
0.87**
0.90**
0.97**
0.89**
0.96**
0.95**
0.50
0.69**
‒0.69**
‒0.35
‒0.76**
phoD
phoC
ALP
ACP
TP
TPo
TPi
Res‒Po
HA‒Po
FA‒Po
HCl‒Pi
MPo
NaHCO3‒Po
0.69**
0.55*
‒0.20
1
0.42
0.42
0.48
0.14
0.45
0.48
0.38
0.45
0.09
0.11
‒0.35
0.12
‒0.52*
0.50
0.54*
1
0.48
0.92**
0.92**
0.96**
0.92**
0.98**
0.97**
0.98**
0.96**
0.56*
0.78**
‒0.52*
‒0.39
‒0.91**
0.92**
1
0.94**
0.47
0.97**
0.97**
0.86**
0.91**
0.96**
0.88**
0.97**
0.98**
0.42
0.62*
‒0.70**
‒0.36
‒0.78**
1
0.97**
0.90**
0.60*
0.92**
0.92**
0.81**
0.82**
0.91**
0.83**
0.92**
0.94**
0.31
0.52*
‒0.74**
‒0.28
‒0.74**
TN
TC
NaHCO3‒Pi
NaHCO3‒Po
MPo
HCl‒Pi
FA‒Po
HA‒Po
Res‒Po
TPi
TPo
TP
ACP
ALP
phoC
phoD
phoX
Fig. 4. Principal component analysis (PCA) of chemical properties, phospho­
monoesterase activity, and bacterial phosphomonoesterase genes (phoC, phoD,
phoX) in sediments of Budi Lake collected during winter (W; blue) and summer
(S; red) seasons. Samples: Puaucho (Pu), Puerto Dominguez (PD), Comue
Stream (EsC), Temo (Te), Temo Stream (EsT). Asterisks (*) denote the more
anthropogenized sampling sites of this study. (For interpretation of the refer­
ences to colour in this figure legend, the reader is referred to the Web version of
this article.)
NaHCO3‒Pi
The PCA visualization confirmed the spatiotemporal distribution
with a clear separation between summer and winter and between the
less (Pu, and EsC) and more (PD, Te, and EsT) anthropogenized sampling
sites (Fig. 4). In summer, labile P forms (NaHCO3‒Pi, NaHCO3‒Po, and
MPo), Pase activities (ACP and ALP), and total bacteria (16 S rRNA gene)
showed a positive correlation with sediments from more anthro­
pogenized sampling sites. However, in winter, sediments from more
anthropogenized sampling sites were positively correlated with
moderately (FA‒Po, HCl‒Pi) and nonlabile (HA‒Po, Res‒Po) P fractions,
TPi, TPo, TP, TC, and TN. For sediments from less anthropogenically
impacted sampling sites, a positive correlation was observed with bac­
terial Pase genes in summer. No positive correlation was observed be­
tween the studied parameters at the less anthropogenic sampling sites in
winter.
In this context, an increased coaccumulation of P fractions in clay
and OM complexes in lake sediments has been observed during periods
of low microbial activity (Zhang et al., 2008; Huo et al., 2011; Joshi
et al., 2015; Wan et al., 2020a). In contrast, the observed positive cor­
relations of the labile P forms, Pase activity, 16 S rRNA genes, and
bacterial Pase genes with the more anthropogenized sampling sites in
summer have also been evidenced by higher microbial activities and
increasing upward mobilization of P from less labile forms in this season
(Yuan et al., 2020). These different relationships for the more anthro­
pogenized sampling sites suggested that the process of deposition,
mineralization, and mobilizations of P fractions from the sediments
determined the microbial uptake (Lü et al., 2016; Wan et al., 2020a). On
the other hand, the presence of positive correlations of phoC, phoD, and
phoX copy numbers with the less anthropogenized samples in winter
suggests that Pase‒harboring bacterial populations were considerably
higher in these low‒P enriched sediments, as observed by Ragot et al.
(2015) and Fraser et al. (2017) in soils.
The Pearson correlation analysis (Table 5) showed consistently
TC
3.4. Relationship among the P‒related variables
TN
Table 5
Pearson’s correlation among chemical properties (TN, TC, NaHCO3‒Pi, NaHCO3‒Po, MPo, HCl‒Pi, FA‒Po, HA‒Po, Res‒Po, TPi, TPo, and TP), phosphomonoesterase activity (ACP and ALP), and bacterial phospho­
monoesterase gene abundances (phoC, phoD and phoX) in sediment samples from Budi Lake during winter (white cells) and summer (gray cells) seasons.
(2020) reported higher abundances and diversities of phoD in the par­
ticulate DOP of this Chinese lake during summer. Similarly, Dai et al.
(2018) reported significant increases in phoX abundances during warm
incubation of eutrophic water samples from Taihu Lake. Fan et al.
(2019) reported higher abundances of phoD and phoX in sediments of
Chaohu Lake during warmer periods than during colder periods.
phoX
Journal of Environmental Management 320 (2022) 115906
M. Campos et al.
Journal of Environmental Management 320 (2022) 115906
significant (P < 0.01) positive correlations among the labile NaHCO3‒
Pi, moderately and nonlabile P fraction, TPi, TPo, TP, TC, and TN pa­
rameters in both sampling seasons. The NaHCO3‒Po fraction only pre­
sented a significant (P < 0.01) positive correlation with TN in winter and
with TN, TC, MPo, FA‒Po, TPo, and TP in summer. Interestingly, almost
all determined P fractions, TC and TN, were significantly (P < 0.01)
positively correlated with Pase enzymes in both sampling seasons,
except ACP, which showed a significant positive correlation with
NaHCO3‒Pi, FA‒Po, HA‒Po, Res‒Po, TPi, and TPo. Conversely, negative
correlations among most bacterial Pase genes with TC, TN, P fractions,
and Pase activities in both sampling seasons were observed. Similar
relationships were observed for Chinese lakes (Huo et al., 2011; Yuan
et al., 2020; Zhang et al., 2008; Zhu et al., 2013). The colloidal TC
represented the most critical factor controlling the sedimentation of P
and N in the mentioned lakes (Zhang et al., 2008; Huo et al., 2011; Joshi
et al., 2015; Wan et al., 2020a), pointing to a reasonable explanation for
the cosedimentation and mobilization of these molecules in sediments of
Budi Lake. Likewise, the significant positive correlations observed for
ACP and ALP with almost all studied P fractions and nutrients (TN and
TC) suggest an increase in the biotransformation from moderately and
nonlabile Po forms to more labile forms (such as NaHCO3‒Po) in sedi­
ments of Budi Lake. Similar behavior was observed in American and
Chinese lakes (Zhang et al., 2008; Huo et al., 2011; Torres et al., 2017).
Then, because ACP and ALP are mineralizing enzymes targeting PMEs
and PDEs, molecules accumulate into the moderately and nonlabile Po
fractions (Torres et al., 2014; Lü et al., 2016; Ma et al., 2019; Yuan et al.,
2020). The generality of this relationship is likely, considering that both
Po fractions were the most abundant in the sediments of Budi Lake.
However, this statement cannot be generalized due to the limited
capability of Pases to hydrolyze molecules such as Myo‒inositol hex­
akisphosphate, β‒glycerophosphate, phosphocholine, DNA‒P, and
phosphonate, which are significant in PMEs and PDEs (Zhao et al.,
2019b).
Particularly concerning bacterial Pase genes, the phoX gene pre­
sented significantly (P < 0.01) negative correlations with almost all the
studied parameters, except for its positive correlations with the copy
number of phoC and phoD genes. Furthermore, the phoD copy numbers
were not significantly correlated with any measured parameter during
winter; however, they presented significant negative correlations with
most of them in summer, except for their positive correlation with the
phoC gene. Finally, the correlations between phoC copy numbers and the
remaining measured parameters were less consistent than those
observed for the other two Pase genes. Then, the phoC copy numbers
were significantly negatively correlated with the labile (NaHCO3‒Pi and
MPo), moderately labile (HCl‒Pi), nonlabile Po, TPo, TP, TN, and TC
during winter and summer, while the same relationship with the
moderately labile FA‒Po, TP, phoD, and phoX was only observed during
the summer season.
A negative relationship between the consumption and biotransfor­
mation of Po molecules to supply the P necessities of the growing Pase‒
harboring bacterial population is likely (Huang and Morris, 2003;
2005). Then, high biotransformation rates of nonlabile Po form more
labile Po, as observed for Budi Lake, which plays a part in the negative
feedback over the induction of phoC and phoD for soil and freshwater
sediment systems (Fraser et al., 2017; Luo et al., 2017; Yang et al.,
2010). Contrary to our findings, various reports indicated a positive
correlation between bacterial phoD, phoC, and phoX and the activities of
ALP and ACP enzymes in aquatic and terrestrial ecosystems (Fraser
et al., 2015; Morrison et al., 2016; Dai et al., 2018; Zheng et al., 2019a;
Yuan et al., 2020). Nevertheless, negative relationships are plausible due
to the potential complexities of interactions among the P contents, Pase
activities, and Pase gene copy numbers. Little is known at this time
about the spatiotemporal heterogeneity of Pase‒harboring bacterial
communities of sediments from freshwater ecosystems (Pandey and
Yadav, 2017; Zhang et al., 2019). Similarly, a negative relationship
between the abundance and diversity of phoD and the bacterial
community was observed by Wang et al. (2021) in organically fertilized
soils, where the introduction of carbonated substrates lacking P may
stimulate the growth of antagonistic bacterial populations over the
phoD-harboring ones. Finally, it is necessary to mention that our current
knowledge about the abundances, activities, and diversities of bacterial
Pase genes is still considered a black box in many freshwater ecosystems
worldwide. In this sense, high‒throughput DNA sequencing and prote­
omic techniques are considered valuable tools for better understanding
the composition of bacterial communities involved in P recycling in the
sediments of lakes.
4. Conclusions
The sediment samples of Budi Lake presented a significant spatio­
temporal distribution of phosphorus (P) content, phosphomonoesterase
(Pase) activities, and bacterial Pase gene (phoC, phoD, and phoX)
abundances. In general, terms, our results showed higher total inorganic
P (TPi), total organic P (TPo), and total P (TP) contents in sediments from
the more anthropogenized sampling sites in summer than in sediments
from the less anthropogenized sampling sites in winter. The 31P NMR
analysis also revealed ortho‒phosphate (ortho‒P) as the primary group
of TP, followed by phosphomonoesters (PMEs) and phosphodiesters
(PDEs). In concordance, sediments showed higher Pase activities and
higher copy numbers of bacterial Pase genes (phoC, phoD, and phoX) in
the more anthropogenized sites in summer than those observed in less
anthropogenized sites in winter. Finally, our results showed a positive
correlation between P contents, Pase activities, and bacterial Pase genes,
independent of season. Although the present study provides valuable
information about bacterial communities involved in P cycling in the
sediments of Budi Lake, further studies are still needed to analyze indepth the role of microbial Pase activities and Pase genes, which may
be used for designing new public policies and norms to address the
environmental protection and conservation of lakes (and their related
ecosystem services) in southern Chile.
Author contributions statement
Marco Campos: Conceptualization, Formal analysis, Investigation,
Writing – original draft, Writing – review & editing. Jacquelinne J.
Acuña: Conceptualization, Formal analysis, Investigation, Writing –
review & editing. Joaquin I. Rilling: Formal analysis, Investigation,
Writing – review & editing. Susett Gonzalez: Investigation, Writing –
review & editing. Fernando Peña-Cortés: Writing – review & editing.
Deb P. Jaisi: Formal analysis, Writing – review & editing. Anthony
Hollenback: Investigation, Writing – review & editing. Andrew
Ogram: Formal analysis, Investigation, Writing – review & editing.
Junhong Bai: Formal analysis, Writing – review & editing. Ling Zhang:
Writing – review & editing. Rong Xiao: Writing – review & editing.
Milko A. Jorquera: Conceptualization, Formal analysis, Investigation,
Writing – original draft, Writing – review & editing.
Funding
This study was funded by The National Fund for Scientific and
Technological Development (FONDECYT) postdoc fellowship no.
3180198 (to M.A.C.), by the China‒Chile Joint Projects on Water Re­
sources Management NSFC‒ANID (Grant No. 51961125201 in China
and NSFC190012 in Chile) (to M.A.J., J.J.A., M.A.C., and R.X.), by IN­
TERNATIONAL COOPERATION Project Chile‒USA from National
Research and Development Agency of Chile (ANID) code REDES190079
(to M.A.J., M.A.C., A. O, and D.J.), by FONDECYT project no. 1201386
and 1221228 (to M.A.J. and J.J.A.), and by Science and Technology
Research Partnership for Sustainable Development (SATREPS JICA/
JST) project code JPMJSA1705 (to I.R., J.J.A, and M.A.J.).)
10
M. Campos et al.
Journal of Environmental Management 320 (2022) 115906
Declaration of competing interest
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
The authors acknowledge (i) the Scientific and Technological Bio­
resource Nucleus (BIOREN) for the availability of Illumina MiSeq
equipment (Fondequip, code EQM150126), (ii) Eng. Vitalia Araya for
their help during the collection and transport of samples and (iii) the
Editor and the anonymous reviewer for their constructive commentaries
during the publishing processes of this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jenvman.2022.115906.
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