Rhodotorula mucilaginosa YR29 is able to

accumulate Pb2+ in vacuoles: a yeast with

bioremediation potential
Gabriela Angeles de Paz
Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional
Hugo Martínez Gutiérrez
Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional
Adrián Ramírez Granillo
Escuela Nacional de Ciencias Biológicas (ENCB)
Edgar Oliver López Villegas
Laboratorio Central de Microscopía, Escuela Nacional de Ciencias Biológicas (ENCB)
María Gabriela Medina-Canales
Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional
Aida Verónica Rodríguez-Tovar (  [email protected] )
Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional

Research Article

Keywords: Rhodotorula mucilaginosa, Bioremediation, Heavy metals, Biosorption, PGP compounds

Posted Date: May 11th, 2023

DOI: https://doi.org/10.21203/rs.3.rs-2905294/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
In response to pollution, microorganisms showed unique mechanisms to resist and detoxify harmful
metals. This study shows the relationship between presence of heavy metals and plant growth regulator
compounds. Additionally, the responses of Rhodotorula mucilaginosa YR29 isolated from the rhizosphere
of Prosopis sp. growing in a polluted mine jal in Mexico are presented. This research carries out a
phenotypic characterization of R. mucilaginosa to identify response mechanisms to metals and confirm
its potential as a bioremediation agent. Firstly, Plant Growth-Promoting (PGP) compounds were assayed
using the Chrome Azurol S (CAS) medium and the Salkowski method. Then, to clarify its heavy metal
tolerance mechanisms, several techniques were performed, such as optical microscopy, scanning
electron microscopy (SEM) and transmission electron microscopy (TEM) supplemented with assorted
detectors. Likewise, scanning transmission electron microscopy (STEM) was used for elementary
mapping of the cell. Further, yeast viability after all treatments was confirmed by confocal laser scanning
microscopy (CLSM).

The results have suggested that R. mucilaginosa could be a PGP yeast capable of triggering Pb2+
biosorption (representing 22.93% of the total cell surface area, the heavy metal is encapsulated between
the cell wall and the microcapsule), and Pb2+ bioaccumulation (representing 11% of the total weight
located in the vacuole). Based on these results, R. mucilaginosa as a bioremediation agent and its wide
range of useful mechanisms for ecological purposes are highlighted.

Introduction
Contamination by heavy metals, pesticides, and phenolic compounds due to natural and anthropogenic
sources is presently a global environmental concern. Metals such as lead (Pb), cadmium (Cd), mercury
(Hg) and arsenic (As) are considered threats to public health and ecosystems due to their persistence,
biomagnification, and accumulation (Chibuike et al. 2014).

The removal from the environment of many potentially toxic compounds is complicated. It often involves
several disadvantages, such as the excavation and removal of soils to “secured” landfills, the requirement
of expensive technology and the performance of in situ restoration (Glick 2003). One alternative to this
trend is the use of bioremediation techniques, biological strategies to repair damaged environments that
are more economical than traditional methods (Kumar et al. 2011).

Besides, in response to metal pollution many microorganisms have developed unique mechanisms to
resist detoxifying harmful metals; such mechanisms can be specific to a particular metal or a variety of
these chemical elements (Wasi et al. 2008). Similarly, some plants have also developed different
mechanisms, such as absorption and accumulation of metals in their cells, including the formation of
metal-bound compounds. An integrated approach of plants and microorganisms in bioremediation can
provide effective cleanup of heavy metals in contaminated soils (Krishnamoorthy et al. 2017), coupled
with a low cost, high efficiency, and environmentally friendly technology (Rajendran et al. 2003).

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Plant growth promoting fungi (PGPF) are a heterogeneous group of nonpathogenic microorganisms, that
are associated and mediate improvements in plant growth and health (Bent 2006). These microbial
communities have an impact on plant growth promotion and disease suppression. Mechanisms involved
in these beneficial effects are related to the synthesis of chemical compounds such as phytohormones,
organic acids, and siderophores. These improve seed germination, exert an effect on root development
inducing greater size and thickness. Furthermore, the increase in crop yield is triggered by supporting the
improvement in photosynthetic efficiency, which will stimulate the production of secondary metabolites
of the host, and lead to a protective response for the plants known as induction of systemic resistance
(ISR) (Arshad and Frankenberger 1997; Shoresh et al. 2010). PGPFs which can manifest multiple plant-
supportive mechanisms could also be employed as potential bio-remediators to detoxify pollutants such
as heavy metals and pesticides (Prathap et al. 2015). In addition, these microorganisms have developed
several mechanisms that are classified as active and passive. The active mechanisms include the
outflow of metal ions out of the cells, accumulation, and complexation of metal ions inside the cells,
chemical transformation and metallothionein production (Spain and Alm 2003). Passive mechanisms
involve the binding of metals to the microbial cell wall and extracellular polymeric substances. On the
other hand, the selection of microorganisms, with appropriate characteristics such as tolerance to metals
and efficient production of plant growth regulator (PGR) compounds, could be enhancing the
recolonization of the rhizosphere of plants in contaminated soils (Rathaur et al. 2012). Microbial
attributes are shared by Rhodotorula genus, which has numerous species with heavy metal tolerance
mechanisms and could behave as PGPF (Cho and Kim 2003; Xin et al. 2009; Cho et al. 2011; Ilyas et al.
2016). Previously, Ramos-Garza et al. (2016) isolated R. mucilaginosa YR29 from the rhizosphere of
Prosopis sp. in a highly contaminated mining jal. The aim of this work is to perform a phenotypic
characterization of R. mucilaginosa YR29 to identify some of its mechanisms of resistance to heavy
metals and to confirm their potential as a bioremediation agent.

Material and Methods

Microorganism and Culture Methods
The yeast R. mucilaginosa YR29 was previously isolated and identified by Ramos-Garza et al. (2016)
from the rhizosphere of Prosopis laevigata. This plant species was growing in soils contaminated with
heavy metals, and were in Villa de la Paz, San Luis Potosí in northern Mexico (23.7 N, 178.7 W). This
yeast was selected for its positive production of indole acetic acid (IAA) and siderophores.

The typical growth curve of R. mucilaginosa is usually obtained on Yeast Extract–Peptone–Dextrose

(YPD)-rich culture medium (DIFCO®, Wickerham, USA). However, considering the limitations for testing
sensitivity to heavy metals (Kumar et al. 2013; Rathnayake et al. 2013), a Yeast Nitrogen Base (YNB)
minimal medium (DIFCO®, Wickerham, USA) was used.

Two different flasks (YPD and YNB) were inoculated using a 1 mL aliquot from an 18 h culture (1×108
yeast cells/mL) and incubated for 48 h. Then, each hour a culture sample was harvested by

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centrifugation and washed three times with 1 mL of 1X sterile physiological solution phosphate buffered
saline (PBS) (8 g/L NaCl (JT Baker®, Estado de Mexico, Mexico), 0.2 g/L KCl (JT Baker®, Estado de
Mexico, Mexico), 1.37 g/L NaH2PO4 (JT Baker®, Estado de Mexico, Mexico) and 2.4 g/L KH2PO4 (JT
Baker®, Estado de Mexico, Mexico). The yeast growth was determined by measuring the optical density
at a wavelength of 600 nm (OD 600).

Determination of the Minimal Inhibitory Concentration (MIC) to Heavy

Metals
The assay was performed in a 96-well microplate (Costar®, Kennebunk, USA). Each well was filled with
YNB minimal medium, yeast biomass (living or dead) and a heavy metal suspension from stock
solutions. Stock solutions were prepared using analytical-grade salts of lead nitrate (Pb (NO3)2) (Merck®,
Darmstadt, Germany), zinc chloride (ZnCl2) (CQM®, Mexico), copper sulfate (CuSO4) (JT Baker®, Estado
de Mexico, Mexico) and sodium arsenite (NaAsO2) (JT Baker®, Estado de Mexico, Mexico) at several
concentrations (0.2–1.6 mM) of each heavy metal. The treatments included a positive control (living
yeast biomass and YNB medium), a negative control (dead yeast biomass and YNB medium), the reagent
control and the test solutions. Fungal growth was evaluated after a 24 h incubation period at 28°C using
a microplate reader (UT-2100C) with a 600 nm filter (MRC®, Jolon, Israel). The MIC was considered the
lowest concentration of each heavy metal that completely inhibited the detectable growth of the test
strain in the wells.

Production of siderophores
Siderophore determination was performed using Chrome Azurol S (CAS) medium (5% chrome CAS
reagent, Merck®, Darmstadt, Germany; 1% heavy metal stock in HCl; 4% CTAB, AMRESCO®, Texas, USA;
90% YNB medium) modified from the original protocol described by Schwyn and Neilands (1987). The
plates were inoculated with 2.5 µL from an 18 h culture (1×108 yeast cells/mL) and incubated for 24 h at
28°C. A pink halo around the colony was considered a positive result of siderophore production. For
semiquantitative determination, we considered the difference calculated between the chelation halo
diameter and the colony diameter; these results were used for statistical analysis. In addition, the
siderophore production index (PI) was calculated [PI = (colony diameter + halo diameter)/colony
diameter].

Effect of the Heavy Metals on the Production of Indol Acetic Acid

Test flasks were inoculated with 100 µL from an 18 h culture (1×108 yeast cells/mL). Each flask
corresponded to one of the following treatments: 1) Control, YNB medium; 2) YNB medium supplemented
with 1% Tryptophan (Trp) (Merck®, Darmstadt, Germany), added 24 h after inoculation; 3) YNB medium
supplemented with 1 mM each heavy metal analyzed and 4) YNB medium supplemented with 1% Trp,
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added 24 h after inoculation and 1 mM each metal. All treatments were incubated at 28°C, and each day
a 1 mL aliquot of supernatant was sampled and centrifuged at 13,800 x g for 5 minutes. Then, IAA was
determined by using the Salkowski reagent as described from Gordon and Weber (1951). The IAA
calibration curve was prepared with pure IAA reagent (JT Baker®, Estado de México, Mexico) as a
standard solution.

Determination of Possible Biosorption or Bioaccumulation Heavy Metal Mechanisms of R. mucilaginosa

Microcapsule Measurement by Optical Microscopy to Examine the

Biosorption Mechanism
Yeast cells from an 18 h culture (1×108 yeast cells/mL) were incubated in YNB medium supplemented
with 1 mM of each heavy metal that was assessed for 48 h, except for Zn. Subsequently, 1 mL aliquots
were sampled each day and microcapsule was observed by negative staining with India ink. The yeast
growth in the absence of metals was included as a control. The microcapsule of fungal yeast was
observed with an optical microscope (Olympus CX31 microscope, Allentown, USA) and measured with
Infinity Analyze software (Luminera Co., Denver, USA).

Ultrastructure Analysis by Scanning Electron Microscopy to Examine the Biosorption Mechanism and
Transmission Electron Microscopy to Examine the Bioaccumulation Mechanism

Starting from a 48 h incubation fungal yeast culture from YNB medium supplemented with 1 mM of each
heavy metal assayed, blastoconidia were obtained for analysis by Scanning Electron Microscopy (SEM).
Two samples were collected daily from each treatment. Both samples were fixed and postfixed for 2 h
with 2% glutaraldehyde (Electron Microscopy Sciences®, Washington, USA), further fixed for 2 h with 1%
osmium tetroxide (Electron Microscopy Sciences®, Washington, USA). Subsequently, all samples were
dehydrated with an ethanol series (10–100%) (Bozzola and Russel 1999; Vazquez-Nin and Echeverria
2000). One of the samples was dried by a critical point drying (CPD) process using CO2 as a transition
fluid in an automated CPD equipment model K850 (Quorum technologies, Lewes, UK). Another sample
obtained was dried with 1 µL of reagent grade hexamethyldisilazane (Merck®, Darmstadt, Germany).
Afterwards, the samples were coated with carbon and observed in a SEM model JSM 7800 (Jeol Inc.,
Tokyo, Japan). Afterwards, the samples were examined with a bottom electron detector (LED) and a
backscattered electron detector (BED). An energy-dispersive X-ray spectroscopy detector (EDS) was used
to obtain the elemental microanalysis.

Otherwise, samples for Transmission Electron Microscopy (TEM) were acquired as described above.
According to the protocol, the samples were dehydrated with an ethanol series (10–100%) and embedded
in Spurr resins. Thin sections (70 nm) were collected using a microtome Ultracut UTC (Leica AG®,
Wetzlar, Germany) and stained with uranyl acetate and lead citrate at lower concentrations under the
detectable limit of the microscope (< 2000 ppm) (Bozzola and Russel 1999; Vazquez-Nin and Echeverria
2000). The samples were observed with a TEM model JEM-1010 (Jeol Inc., Tokyo, Japan). Elemental
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microanalysis was performed on an EDS spectroscope integrated into an Atomic Resolution Analytical
Microscope model JEM-ARM200F (Jeol Inc., Tokyo, Japan) in the Scanning Transmission Electron
Microscope (STEM) mode. Additionally, an elementary mapping was performed in STEM mode in an
SEM microscope (data not shown).

Viability of R. mucilaginosa in Heavy Metal-Supplemented Media Analyzed by Confocal Laser Scanning

Microscopy

Similarly, from a yeast fungal culture of 18 h incubation under conditions previously described (see
microcapsule assays), cells were harvested by centrifugation and washed with 1X PBS. Subsequently,
cell samples are placed in a fluorochrome mixture containing 10 mg/L propidium iodide (PI) (AbD Serotec
Raleigh, NC, USA) to detect nucleic acids and 1 g/L calcofluor white (CW) (Merck®, Darmstadt, Germany)
to label chitin. Samples were observed on a Confocal Laser Scanning Microscope (CLSM) model 710
(Carl Zeiss, Oberkochen, Germany) with filters of 560 nm for IP and 440 nm for CW.

Statistical Analysis
All experimental designs of the quantitative assays performed were completely randomized. Culture
method testing consisted of three replicates and a one-way ANOVA with a Duncan's multiple range test
was performed. MIC culture tests were consisted of eight replicates and a two-way ANOVA with a
Duncan's multiple range test was performed. Production of siderophores assays were consisted of three
replicates and a one-way ANOVA with a Duncan's multiple range test was performed. IAA production
assays were consisted of three replicates and a two-way ANOVA with Tukey's multiple range test was
performed. Additionally, a linear regression analysis was applied for the IAA standard curve,
corresponding to three replicates. Data for all statistical analyses were performed with a significant
difference set at a p-value < 0.05 and using the SigmaPlot 12.0 statistical analysis program (Systat
Software Inc., San Jose, CA, USA).

Results
Determination of the optimal culture medium for heavy metal analysis

Growth tests of R. mucilaginosa YR29 on the evaluation media (YNB and YPD) showed typical sigmoidal
forms and no significant difference in lag phase. Notably, although there were no differences in the lag
phase, the time and growth rate in YNB medium were lower from early in the growth kinetics compared to
YPD medium (Fig. 1). This difference did not change the right growth of the yeast in the minimal medium
(Fig. 1). Finally, this difference did not change successful yeast growth on YNB minimal medium, and
remained in use in future assays, as described earlier in the methodology.

Determination of MIC of Pb, Zn, Cu and As for R. mucilaginosa YR29.

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The minimum inhibitory concentration (MIC) in the culture media was determined by increasing the
concentration of heavy metals. A significant variability in the assayed metals was observed for the YPD
medium compared to the YNB medium. MIC rates of the yeast in YPD medium were reported to be higher.
The yeast growth in YNB medium, are significantly decreased against heavy metals. At concentrations
from 0.2 mM to 1.4 mM for Pb, and 1.6 mM for Zn, Cu and As, respectively. The values determined for the
MIC of the yeast are shown in Table 1.

Table 1

Comparison of minimal inhibitory concentrations (MIC) of heavy metals in

Rhodotorula mucilaginosa
Heavy metals MIC (mM)

YNB YPD

Pb Pb(NO3)2 1.4 a >9 c

Zn ZnCl2 1.6 b > 22.01 d

As NaAsO2 1.6 b > 23.09 d

Cu CuSO4 1.6 b > 18.78

Different letters besides the MIC´s means indicate statistical significance (p < 0.05)

Productions Siderophore by R. mucilaginosa YR29

R. mucilaginosa YR29 produced siderophores when grown with As, Cu and Zn but not with Pb. Chelation
halos were observed in CAS medium and negative controls are shown in Fig. 2A. Highest siderophore
production index (SI) was obtained in the presence of Cu, whereas least SI occurred on media
supplemented with Zn and As. There is no significant difference between SI of Zn and As (Fig. 2B).

Effect of heavy metals on IAA production by R. mucilaginosa YR29

Yeast growth in YNB medium with and without Trp showed no significant difference in the level of IAA
production. However, when a heavy metal was added, IAA production was significantly higher compared
to a metal-free control; further improvement of this result was observed with the addition of Trp. Such
behavior was reproducible with every metal used, except in media supplemented with Zn and Cu. Thus,
the addition of Zn did not modify IAA levels, even when Trp was added, but maximum production was
obtained at different incubation periods. Likewise, Cu was observed to behave similarly to the control.
The best IAA production was obtained in YNB with Pb and Trp after ten days of incubation. The
maximum IAA production time was different for each metal (Fig. 3). Zn and As (without Trp) were able to
induce IAA production in less time, but did not reach the highest production compared to the other metals.
Each heavy metal led to a decrease in IAA production at a specific time after inoculation (Table S1). The

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standard curve (Fig. S1) had a significant regression coefficient R2 = 0.986 (p < 0.001), so it was used for
IAA quantitative determination.

Determination of possible biosorption or bioaccumulation heavy metal mechanisms of R. mucilaginosa

YR29

Yeast cell morphology by optical microscopy in the presence of heavy metals, effects on microcapsule.

The typical morphology of R. mucilaginosa YR29 was observed as ovoid cells 6–10 µm in size and
surrounded by a thin microcapsule in all samples. Nevertheless, cells were treated with heavy metals
showed several vacuoles at different incubation times (Fig. 4A). Pb, Cu and Zn were found to induce
vacuole formation at 24 h of incubation, while As was induced at a slower rate, at 48 h. The cell size did
not change when the cells were treated, in contrast to the microcapsules whose proportion significantly
decreased with respect to the total cell size (Fig. 4B).
Yeast ultrastructure alterations in the presence of heavy metals by SEM and TEM

Cells treated with hexamethyldisilazane had shown certain cell surface damage regardless of the metal
used in the fungal culture. Although this could be due to the drying process, which also causes loss of the
microcapsule (Fig. 5a-d). Under this drying process, the treated cells exhibited no change in their topology.
And only on lower electron detector (LED) imaging for Pb treatment, an atypical three-dimensionality
leakage was revealed (Fig. 5b). Subsequently, after treatment with Pb, As, Cu and Zn, a few floccules
could be observed on the cell surface (Fig. 5b-d). Otherwise, from Fig. 5 of the e-p graph, it is observed
that the yeasts maintain their microcapsules, the samples were dried by a critical point process (CPD).
The aspect of the microcapsules was a thin layer that surrounded the entire cell. In addition, this layer
was seen to cover small bodies of the cell wall, whose differences in chemical nature with the rest of the
cellular components were detected by a backscattered electron detector (BED-C) (Fig. 5i-l). No changes
were detected in the topology of LED-treated and control R. mucilaginosa cells (Fig. 5e-h). In BED-C/LED
fusion, heavy metals were shown to be present in the cell (covered by the microcapsule) and were not
present on the surface (Fig. 5m-p).

Additionally, the chemical nature of these floccules was confirmed by elemental microanalysis by X-ray
spectroscopy EDS spectra in which Pb, Zn, As and Cu peaks appeared at different kiloelectronvolts
(KeVs) (Data not shown). EDS spectra, on the dried cells examined by CPD, was shown peaks of Pb, Cu
and As at different KeVs in the orange areas (Fig. 5). Therefore, elemental microanalysis was not possible
due to the location of the microcapsule on the metals. Nevertheless, a remarkable result was found in
elemental microanalysis on dry hexamethyldisilazane-treated samples (Table 2), where untreated cells
had a Pb content (W%) of 0%, whereas treated cells had a Pb content of 22.93%. In addition, the
percentage of phosphate was also increased in the treated cells. All these results would demonstrate that
biosorption of Pb, Zn, Cu and As occurred in R. mucilaginosa cells.

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 Table 2
Element content semiquantitative analysis of the Rhodotorula mucilaginosa cell
surface.
Element % Total

C O Au S Si Pb P

Treated cells 4.59 4.26 62.66 0.002 - 22.93 5.55 100.1

Control cells 8.36 5.87 45 - 1.34 - - 100

Likewise, the typical structure of R. mucilaginosa was shown in the control micrograph with a
homogeneous electrodensity in the cell wall, a single nucleus, a well-defined cytoplasmic membrane,
some mitochondria surrounding the nucleus, and multiple vacuoles with a high electron density in the
cytoplasm (Fig. 6A, a). Yeast cells cultured in Pb-supplemented medium (Fig. 6C, c) were not altered in
their organelle organization. Thus also, blastoconidia were maintained with normal shape of their cell
wall, but this structure appeared slightly thicker, less electronically dense, and their membrane lacked
integrity. Also, multiple electron-dense bodies appeared evenly distributed throughout the cytoplasm, and
electrodense vacuoles were still visible. Besides, there were also sequential effects of As on the cell
ultrastructure, producing a deformation of the cell wall that presented a similar thickness and appearance
to the other treatments (Fig. 6D, d). There were any differences between the control cell ultrastructure and
that of the Zn-treated cells (data not shown). Energy dispersive X-ray spectroscopy could only be
displayed in the vacuoles of Pb-treated cells. Pb atoms were detected inside the vacuoles by EDS
analysis (Fig. 7). In both cases, control and treated cells, Cu peaks were shown as a product of the
mounting grid.

Finally, elemental analysis of the intracellular composition by STEM and EDS, which was performed on
control and Pb-treated cells (Fig. 8), showed the general composition of the yeast including elements
common to all living organisms (C and O) and the element tested (Pb). The semiquantitative analysis
was corrected by ZAF (ZAF® correction software) and this showed that Pb represents 16.01% of the total
weight of the studied zone, while in the control cells it corresponds to 16.70%.

The following statement is included to clarify the detection of Pb in the control cells. Normally, the
samples used in electron microscopy go through a stain process with lead citrate to create the contrast
between the organelles shown on the micrograph. The amount of the lead citrate could vary between the
samples, and it is considered as a basal threshold. Nevertheless, under any circumstance the lead could
be considered as a part of a biological structure if it was never treated or grown with this element before.
Thus, together with the fact that the lead in control cells was homogeneously distributed because of the
stain process, lead us to conclude that the Pb amount in the control cells is merely a basal amount. In
this way, R. mucilaginosa accumulated Pb in its vacuoles with a 16.01% of the total weight of the studied
area.

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Evaluation of the viability of R. mucilaginosa YR29 in presence of heavy metals by CLSM

In CLSM micrographs, no differences were shown between the number of dead yeast cells (labeled by
propidium iodide) in the control and treatments with each heavy metal analyzed. Cell wall and membrane
damage due to Pb exposure did not lead to unviability of R. mucilaginosa. Live yeast cells were observed
as green halos by calcofluor white in all assays performed (Fig. 9).

Discussion
In this work, the optimal medium for the growth of R. mucilaginosa was established and identified. A
sigmoid kinetics of yeast growth was observed in both YNB and YPD media. Both media were shown to
have three typical growth phases, demonstrating the correct development of R. mucilaginosa YR29 on
YNB. The use of a medium with a reduced heavy metal availability composition has been a common
mistake in most bioremediation studies and often leads to increased tolerance levels of various
microorganisms (Dary et al. 2010; Anahid et al. 2011; Hipoal 2014; Lampis et al. 2015). Nutritious broth
and rich media are reported to be the most used media for these assays. Nevertheless, these media could
alter the actual MIC values of heavy metals (Mandal and Suzuhi 2002; Rathnayake et al. 2013). Thus, in
terms of heavy metal assays, the evaluation and deployment of a minimal composition that allows the
microorganism development are essential.

R. mucilaginosa YR29 resistance to different heavy metals was confirmed by measurements of the MIC
values in both media used. Overall, MICs obtained were higher than reported values for several groups of
microorganisms with bioremediation potential, e.g., Cystobasidium capitatum and R. glutinis, which can
tolerate 10 mM of Zn and Cu (in YPD broth), and R. mucilaginosa RCL-11, which has Cu removal
capability and tolerates 1 mM Cu in YNB broth (Vadkertiová and Sláviková 2006; Villegas et al. 2009).
Among ascomycetes, Candida spp. and Saccharomyces spp. (12 mM of Cu in YPD broth) (Adamo et al.
2012); and several plant growth-promoting rhizobacterias (PGPRs), such as Bradyrhizobium sp.,
Ochrobactrum ciceri and Pseudomonas sp. (2 mM, 8 mM and 4 mM to As; 1.5 mM, 3.5 mM and 4.5 mM
to Cu; 2 mM, 6 mM and 5 mM to Pb and < 1 mM, 10 mM and 3 mM; respectively) (Dary et al. 2010).

Although the results appeared unrepresentative (compared to those of other authors) for a heavy metal
tolerant yeast, we cannot underestimate its usefulness in bioremediation techniques. Popular culture
media used for metal tolerance by fungi include YPD, PDA and others. Among their components such as
nutrient broth, peptone, tryptone and yeast extract are associated with very high levels of tolerance to
metals by microorganisms (Dary et al. 2010; Anahid et al. 2011; Li et al. 2012; Hipol 2014; Lampis et al.
2015). These molecules could cause a complexation or chelation of metals to the unspecified organic
constituents in the medium and the lack of stability constants of metal organic complexes, as
Rathnayake et al. (2013) and Kumar et al. (2013) have previously mentioned. When the assay was
repeated in rich media, the MICs greatly increased, confirming the interference caused by the YPD
medium.

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Several microorganisms highly resistant to heavy metals have different plant growth-promoting
characteristics such as siderophore production and, have received interest in recent years due to their
potential roles and applications in various areas of environmental research (Ahmed and Holmström
2014). Ramos-Garza et al. (2017) were reported the ability of R. mucilaginosa to produce Fe-
siderophores. Screening with various heavy metals was performed in this study. The results
demonstrated the potential of R. mucilaginosa to synthesize several types of siderophores, which may
have high affinity or selectivity for specific metal ions (As3+ and Zn2+). Nonetheless, it did not have an
affinity for Pb2+ ions. Likewise, siderophore (SI) production rates reported by Rhodotorula sp. were higher
than a lower threshold produced by other fungi. (Rajendran et al. 2003; Wasi et al. 2008; Khan et al. 2009;
Krishnamoorthy et al. 2017). Rhodotorulic acid (RA) could be one of these specific siderophores since it
was reported in other species of Rhodotorula growing in iron-limited conditions (Atkin et al. 1970;
Andersen et al. 2003). However, there was no evidence of its specificity. There are some reports of specific
siderophore production; although, these reports highlight growth conditions rather than the presence of
heavy metals as a possible cause of specificity (Braud et al. 2009; Kraepiel et al. 2009; Hong et al. 2010).
The ability of R. mucilaginosa for attaching to a variety of metals certainly has a potential role to play in
several areas of environmental research (Ahmed and Holmström 2014).

The production of IAA by yeast is another important characteristic for a PGPF, thus this ability was tested
with different conditions related to the presence of heavy metals. The results obtained indicate that no
significant differences were found between IAA production with and without the addition of Trp,
suggesting that R. mucilaginosa YR29 was able to activate the two known pathways for IAA synthesis
(tryptophan-dependent and tryptophan-independent pathways). One of these pathways was reported in
several basidiomycetes, including Rhodosporidium paludigenum, R. graminis and R. mucilaginosa PTD2
and PTD3, which produced IAA only in the presence of exogenous conditions and Trp-supplemented (Xin
et al. 2009; Nutaratat et al. 2016), while certain ascomycetes were also enabled to activate both
pathways (Rao et al. 2010). Although, statistical studies have shown that Trp must be present in culture
media for several yeast species to produce IAA (Nassar et al. 2005), an effect that was not evident for R.
mucilaginosa YR29. Therefore, a growth factor such as Trp-supplemented, should be considered as an
interspecific feature rather than a rule. This suggests that it could represent an advantage in promoting
plant growth in hostile nutrient-starved habitats.

The maximum IAA production was reached at different incubation times, depending on the heavy metal
supplied. These incubation times are reported from strain to strain, according to the maximum level of
production. The minimum incubation time is at least 7 days, as prolonged incubation time leads to
increased production of other indole derivatives (Xin et al. 2009; Nutaratat et al. 2016). A considerable
increase in IAA production was observed in the results when heavy metals were added to the medium,
which may be related to the cytotoxic effect triggered by the metals. In general, heavy metals can produce
reactive oxygen species (ROS) (Mostofa et al. 2015; Huang et al. 2017; Wang et al. 2017). Historically, it
has been reported that auxin, indole-3-acetic acid (IAA), is degraded by spontaneous oxidation with
hydrogen peroxide (H2O2). IAA could be disrupting the auxin signaling pathway and decreasing auxin

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concentration in root tissue, a process of auxin down-regulation (Omran 1977). Several recent studies
have proposed theoretical models of the IAA signaling pathway that are closely related to the actual
pathway of auxin synthesis in microorganisms. A specific model has been proposed for the extracellular
activation of ROS signaling, together with the activation of IAA and transcription factors for auxins, which
lead to growth by elongation (Kawano 2003; Spaepen et al. 2007; Tognetti et al. 2012; Wang et al. 2017).

Another remarkable result of the present work was the significant decrease in the concentration of IAA
after maximum production was reached. The origin of this behavior could be related to the degradation
of the IAA by enzymatic activity or oxidation effect by the cells, with the purpose of ensuring cell survival
against free IAA toxicity (Tromas and Perrot-Rechenmann 2010); and also, to produce a new source of
carbon and nitrogen such as skatole, a product derived from indole (Bau 1981; Deslandes et al. 2001;
Leveau and Lindow 2005; Mujahid et al. 2011). Thus, the protection of the conjugated form of IAA could
be implemented to avoid this anti-productive effect due to enzymatic degradation. It should be
considered that IAA free may be oxidized by ROS which interferes with the detection by Salkowski's
method (Cohen and Bandurski 1982; Woodward and Bartel 2005; Seidel et al. 2006).

Hence, the cytotoxic effects of heavy metals involve an important role in auxin production. According to
the results and references cited above, a directly proportional behavior is shown between the toxicity of
the heavy metal and the auxin production synthesized by the yeast. Yet, this effect is considered more
dependent of the microorganism than heavy metal, triggering possible variations in auxin production
among different strains (Mendoza-Hernández et al. 2016).

As reported by several authors, metal uptake takes place at the cell surface as a mechanism that reduces
the damage caused by heavy metals, which means that metal recruitment probably occurs in the outer
layer of the cell. R. mucilaginosa is known to possess a polysaccharide-rich microcapsule containing
compounds such as carotenoids that confer a high resistance against environmental stress due to the
presence of lead. (Moliné et al. 2010; Wang et al. 2021). During microscopy assays performed, there was
no significant increase in size or microcapsule leakage. One possible rationale is that, despite the
damage caused by heavy metals, microcapsule covered the whole metal particle and retained it on the
fungal cell wall, because of primary sites of metal complexation from various functional groups on the
surface (Cho et al. 2011).

The adsorption of heavy metals in R. mucilaginosa was reported and confirmed previously by Li et al.
(2019) with Pb+ and Wang et al. (2020) with Cu2+ and it is confirmed in this study.

The adsorption of heavy metals on R. mucilaginosa was previously reported by Li et al. (2019) with Pb+
and confirmed by Wang et al. (2020) with Cu2+, as confirmed by these studies in this work. Such as the
adsorption effect demonstrated in this study of R. mucilaginosa to other heavy metals including As and
Zn. The heavy metals were localized under the microcapsule between the cell wall, as described by Wang
et al. (2020).

Page 12/28
Similarly, bioaccumulation of heavy metals in the cell was evidenced by observations of ultrastructural
alterations. There was no apparent difference between a Cu micrograph and the assay control, probably
due to the decreased Cu2+ concentration. In studies by Wang et al. (2020), we also observed alterations of
the cell ultrastructure with high concentration of this metal at 200 mg/L.

On the other hand, several changes appeared in yeast cells cultured in Pb-supplemented medium, such as
depletion of cell wall electronic density, and lack of cell membrane integrity. Once again, Wang et al.
(2020) reported something similar with high concentrations of Cu2+, this metal could be interacting with
free radicals and ROS. This effect was only observed with Pb, which is the most toxic metal tested in this
research. The Pb bioaccumulation was into the vacuole and corresponding at 16% of the weight cell, the
bioaccumulation location is not reported previously.

Another finding of interest was related to the relatively small number of electrodense bodies inside the
cell, possibly due to the interaction with Pb and As. In the case of these heavy metals, there are not many
studies that are related to changes in the yeast cell ultrastructure. Few studies have highlighted on the
appearance of distinct dark bodies in different genus of yeasts, mainly located in vacuoles corresponding
to different heavy metals (Ramsay and Gadd 1997; Ksheminska et al. 2005; Villegas et al. 2009; Li et al.
2019; Wang et al. 2020). Thus, EDS analysis represents a viable method to confirm this speculation,
which has not actually been performed before. On one hand, the cells exposed to As were not evaluated
by EDS because electron acceleration from the microscope caused the sample to immediately
disintegrate. On the other hand, the bioaccumulation phenomenon was conclusively confirmed by energy-
dispersive X-ray spectroscopy for Pb inside vacuoles.

Microbial viability assays by CLSM confirmed the effect to determine the toxic damage of Pb. In addition,
such assays have suggested that cell wall damage did not represent a cause of cell death. Samples
grown on medium supplemented with Zn, As and Cu were still viable after treatment with each heavy
metal. On labeling the microcapsule with CW and IP, it was evident that this type of signal is not
commonly observed with IP. Clearly, the composition of the microcapsule represents an important target
of IP in the basidiomycota group (Zhang et al. 2018).

According to the results, Rhodotorula mucilaginosa YR29 is a highly tolerant yeast to different heavy
metals, which also has typical characteristics of the PGPF group (production of IAA, specific siderophores
and growth promotion). Moreover, this strain is also capable of biosorption (at the cell wall level), coated
(inside the microcapsule) and bioaccumulate (at the vacuolar level) the Pb(NO3)2. Moreover, our results
indicate that metal uptake mechanisms act together and depend more on metabolic activity than on
surface binding mechanisms, which contradicts previous studies.

Declarations
Acknowledgments

Page 13/28
We thank M. en C. Héctor Francisco-Mendoza Leon (Laboratory of Ultra-High-Resolution Scanning
Electron Microscopy, CNMN-IPN), Dra. María de Jesús Perea-Flores (Laboratory of Confocal Laser
Scanning Microscopy, CNMN-IPN), and Dr. Raúl Borja Urbi (Laboratory of Atomic Resolution
Transmission Electron Microscopy, CNMN-IPN) for their support with the SEM, TEM, CSLM and STEM
micrographs.

AVRT and MGMC were the tutors of GAP. GAP is a CONACyT and BEIFI fellow; AVRT, MGMC and EOLV are
COFAA, EDI and SNI fellows. This work was edited by American Journal Experts.

Funding.

Part of this work was supported by the “Secretaria de Investigación y Posgrado, del Instituto Politécnico
Nacional” projects SIP-IPN 20211222, 20221010 and 20220564.

Competing interests.

The authors have no relevant financial, declare no competing interests.

Author contribution.

GAP carried out the experimentation and drafted the first draft of the manuscript. GAP and EOLV
processed and interpreted yeast samples for TEM. GAP, MGMC and HMG processed and interpreted yeast
samples for SEM and STEM. AVRT and MGMC conceived the original idea, supervised the project, and
contributed analyze, write and edit the final manuscript, ARG write and edit the final manuscript.

Data availability.

The data material is real, available, and transparent.

Ethics approval.

No human or animal participants were involved in this study. This is an observational study.

Consent to publish.

All authors have read and approved the final manuscript.

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Figures

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Figure 1

Comparison of growth kinetic curve of Rhodotorula mucilaginosa YR29 in YPD (•) and YNB (o) medium.
Black arrows indicate stationary phase initiation. Bar = 1 standard error of the mean. Different letters
above the bars indicate statistical significance (p < 0.05)

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Figure 2

Specific siderophore production on chrome azurol S (CAS) medium supplemented with heavy metals. A.
Qualitative assays: Experimental group for R. mucilaginosa inoculated in CAS medium supplemented
with a) Pb(NO3)2, b) ZnCl2, c) NaAsO2 and d) CuSO4. Control plates for CAS medium supplemented with
e) Pb(NO3)2, f) ZnCl2, g) NaAsO2 and h) CuSO4. Black arrows highlight the chelation halo, and white
arrows indicate the colonies growth. B. Semiquantitative assays. Different letters above the bars indicate
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statistical significance (p < 0.05). Numbers inside the bars indicate the siderophore production indexes.
Black horizontal line indicates the lower threshold for fungal production reported

Figure 3

Production of Indole Acetic Acid (IAA µg/mL) by R. mucilaginosa. Assays were performed in broth
supplemented with different heavy metals Pb(NO3)2, ZnCl2, NaAsO2 and CuSO4 with and without
tryptophan. White arrows indicate those treatments where the highest production was detected. Bar = 1
standard error of the mean

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Figure 4

Effect of heavy metals on R. mucilaginosa microcapsule structure. A. Yeast stained with India Ink and
observed using an optical microscope at 60X after growing in YNB supplemented with different heavy
metals at 24, 48 and 72 h. Single white arrows indicate the microcapsule. Double white arrows indicate
the vacuoles. B. Relative capsule size (%). Bar = 1 standard error of the mean

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Figure 5

Ultrastructure of R. mucilaginosa observed on SEM with BED-C, LED and BED-C/LED detectors at 5,000X
and 10,000X. a, e, i, m) Yeast growth in YNB. b, f, j, n) Yeast growth in YNB supplemented with Pb(NO3)2.
c, g, k, o). Yeast growth in YNB supplemented with NaAsO2. d, h, l, p) Yeast growth in YNB supplemented
with CuSO4. For treatments with Pb, As, Cu and Zn, floccules were observed on the cell surface (b-d).
Single white arrows indicate microcapsule formation

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Figure 6

Ultrastructure of R. mucilaginosa observed in TEM at 25,000X and 50,000X. A-a) Control. Yeast grown in
YNB, B-b) CuSO4, C-c) Pb(NO3)2, D-d) NaAsO2. CM: Cell Membrane, CW: Cell Wall, M: Mitochondria, N:
Nucleus, V: Vacuole. White arrows indicate the probable metal location

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Figure 7

Elemental microanalysis of R. mucilaginosa vacuoles observed and detected by High Resolution

Transmission Electron Microscopy (HR TEM). A) Control, B) Cells treated with Pb(NO3)2. Black arrows
indicate Pb peaks. White arrows indicate the site of EDS analysis was performed. Cupper signal was
related with the grid use to support the samples

Figure 8

SEM image and elemental mapping of Rhodotorula mucilaginosa cell surface growth in YNB broth, with
and without Pb(NO₃)₂, analyzed and observed by EDS and BED-C detector at 25, 000 X. A) Carbon
location, B) Oxygen distribution on the cells, C) Lead location on cells, D) Merge of C, O and Pb detection.
White arrows highlight the site of EDS analysis was performed

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Figure 9

Viability of Rhodotorula mucilaginosa grown in YNB broth supplemented with different heavy metals
(CLSM). White arrowheads indicate colocalization of cell signals for propidium iodide (IP) and calcofluor
white (CFW), corresponding to death cells

Supplementary Files

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This is a list of supplementary files associated with this preprint. Click to download.

FigureS1.tiff
TableS1.docx

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